THE ROLE OF HYALURONAN IN INNATE INTESTINAL DEFENSE
by
DAVID RICHARD HILL
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy
Dissertation Adviser: Dr. Carol A. de la Motte
Department of Molecular Medicine
CASE WESTERN RESERVE UNIVERSITY
May, 2013
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
DAVID RICHARD HILL
Candidate for the
DOCTOR OF PHILOSOPHY
(signed)
degree*.
Edward Greenfield, PhD, Committee Chair
Carol de la Motte, PhD, Advisor
Jean-Paul Achkar, MD
Vince Hascall, PhD
Edward Maytin, MD, PhD
(date)
March 14, 2013
.
* We also certify that written approval has been obtained for any proprietary material
contained therein.
2
DEDICATION
To my parents, David and Kathleen Hill, my unswerving foundation. Your tireless words
of tender support, quiet confidence, and loving guidance have sustained and encouraged
me in the pursuit of every aspiration I have ever strived towards. I am overjoyed to share
in my every success with you.
To my grandparents, Richard and Janet Petit and Vernon and Juan Hill, who instilled in
all their family the value of education, the fortitude to persevere, and the wisdom to
appreciate not only the destination, but also the path that leads there.
To my wife Michelle and our child soon to be born, who fill my days with laughter and
joy. All my challenges were easy to bear with a happy and kind woman, a warm cup of
tea, a merry little cat, some gentle music, and an abundance of treasured memories and
memories yet unmade awaiting my arrival at the end of the day.
3
TABLE OF CONTENTS
LIST OF TABLES …………………………….…………...………………………..…..9
LIST OF FIGURES.…………………………….…………...…………..……........…..10
PREFACE…………………………………..….………...…………….....………....…..12
ACKNOWLEDGMENTS.……………………………….…………………………….13
LIST OF ABBREVIATIONS………………….…………...……………..……..…….15
ABSTRACT…………………………….…………………..……………..………...…..16
CHAPTER 1: INTRODUCTION…………………………….…………..…….….…..18
1.1 BIOLOGY OF THE GASTROINTESTINAL TRACT……..…………...….18
1.1.1 Structure and Function of the Digestive System………………….…..18
1.1.2 The Intestinal Microbiota……………………………..……………..….22
1.1.3 Mucosal Barrier Function…………………………………………...….25
1.1.4 Innate Epithelial Defense…………………………..…………….….….29
1.1.5 Toll-like Receptors……………..…………………..……………..….….32
1.1.6 Defensin Structure, Function, and Expression…..………….…….….35
1.1.7 Inflammatory Bowel Disease…..………………………….………...….38
1.1.8 Milk in Gastrointestinal Health and Disease…..……….…….…..….42
1.2 GLYCOSAMINOGLYCAN STRUCTURE AND FUNCTION………...….46
1.2.1 Biochemistry of Hyaluronan …………………………………….….….50
1.2.2 Hyaluronan Synthesis……………………………….…………….….….51
1.2.3 Hyaluronan Catalysis ……………………………….…………….…….53
1.2.4 Hyaluronan in Inflammation……………………….……………….….56
1.2.5 Hyaluronan in Intestinal Defense……………….……………….…….59
4
CHAPTER 2: SPECIFIC-SIZED HYALURONAN FRAGMENTS PROMOTE
EXPRESSION OF HUMAN β-DEFENSIN 2 IN INTESTINAL EPITHELIUM.....60
2.1 INTRODUCTION……………………………………………………….…..60
2.2 EXPERIMENTAL PROCEDURES………………………………………....64
2.2.1 Cell Culture………………………………….…….…………………..….64
2.2.2 Experimental Cultures ……………………..…….……………….…….64
2.2.3 HA Fragment Sizing ……………………..…….………………………..65
2.2.4 Detection of HβD2 by Immunoblot Analysis…………………….……65
2.2.5 Detection of HβD2 by Fluorescence Histochemistry……………..…66
2.2.6 Detection of HβD2 by Enzyme-Linked Immunosorbent Assay…..…67
2.2.7 Specificity of HA-35 Induction of HβD2 in HT29 cells ………....…68
2.2.8 In Vivo Induction of Murine HβD2 Ortholog by HA-35………….....69
2.2.9 Evaluation of Size-Specific Induction of Murine HβD2 Ortholog...69
2.2.10 Detection of TLR4 by Fluorescence Histochemistry…………......70
2.2.11 Evaluating TLR4 and CD44 in HβD2 Expression In Vivo …..…..70
2.2.12 Quantification of Histological Observations…………………..…71
2.2.13 Statistical Analysis…………………………………..………..…..72
2.3 RESULTS………………………………………………………………..…..72
2.3.1 HA-Specific Induction of HβD2 Expression in HT-29 Colonic
Epithelial Cells Occurs in a Time Dependent Manner……………….…...72
2.3.2 Induction of HβD2 Expression in HT29 Colonic Epithelial Cells is
Specific to HA ……………………………………………………….…..74
5
2.3.3 Induction of HβD2 in HT29 Cells is Dependent on HA Size and
Concentration……………………………………………………….………..…74
2.3.4 Orally Administered HA-35 Induces the Expression of the HβD2
Ortholog in Mouse Intestinal Epithelium In Vivo……………………....…..77
2.3.5 Induction of HβD2 Ortholog in Mouse Intestinal Epithelium by HA
Fragments is Highly Size-Specific……………………………………..……..78
2.3.6 Specific-Sized HA Fragment Mediated Induction of HβD2 ortholog
Expression in Mice is Dependent on TLR4………………………………….79
2.4 DISCUSSION…………………………………………………………….….81
CHAPTER 3: HUMAN MILK HYALURONAN ENHANCES INNATE DEFENSE
OF THE INTESTINAL EPITHELIUM………………………………………..…...100
3.1 INTRODUCTION……………………………………………………..…...100
3.2 EXPERIMENTAL PROCEDURES……………………………………..…104
3.2.1 Human Milk Sample Collection ……………………………….…..…104
3.2.2 Isolation of HA from Milk………………………………………..……104
3.2.3 Quantification of HA by Enzyme-linked Sorbent Assay…………...105
3.2.4 Fluorophore-assisted Carbohydrate Electrophoresis....................106
3.2.5 Cell Culture …………………………………………………………….108
3.2.6 Experimental Cultures…………………………………………………108
3.2.7 Hylauronidase Digestion of Human Milk HA Preparations……..108
3.2.8 Detection of HβD2 by Immunoblot Analysis………………….……109
3.2.9 Real-time Quantitative PCR Analysis of DEFB4 Expression…....110
6
3.2.10 Detection of Murine HβD2 Ortholog by Fluorescence
Histochemistry……………………………………………….…………..……111
3.2.11 Evaluation of Murine HβD2 Ortholog Expression in Nursing
Mice……………………………………….………………………………….…112
3.2.12 In Vivo Induction of Murine HβD2 Ortholog by
Human Milk HA………………………………………………………….…….112
3.2.13 Evaluating the role of TLR4 and CD44 in HA-35 induced HβD2
expression in vivo……………………….……………………………….…….113
3.2.14 Quantization of Histological Observations………………………..113
3.2.15 Salmonella enterica Infection of Cultured Epithelium.................114
3.2.16 Statistical Analysis…………………………………………….……..115
3.3. RESULTS………………………………………………………………....115
3.3.1 Human Milk HA Concentrations are Greatest in the First 60 Days
Postpartum……………………………………………………………………..115
3.3.2 Milk HA Specifically Induces HβD2 Expression………………...116
3.3.3 Oral Administration of Human Milk HA Promotes In Vivo
Expression of the Murine HβD2 Ortholog in Intestinal Epithelium……119
3.3.4 The Intestinal Epithelium of Nursing Mice Expresses the HβD2
Ortholog...................................................................................................119
3.3.5 In vivo induction of murine HβD2 by milk HA is both CD44 and
TLR4 dependent.......................................................................................120
3.3.6 Milk HA Enhances Resistance to Intracellular Salmonella Infection
in Intestinal Epithelium ...........................................................................121
7
3.4 DISCUSSION…………………………………………………………...….123
CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS…………………..….141
4.1 SUMMARY OF FINDINGS…………………………………………….…141
4.2 A PROPOSED MODEL FOR THE FUNCTION OF HUMAN MILK HA
IN INTESTINAL HOMEOSTASIS……………………………………………142
4.2.1 Synthesis of HA During Lactation……………………………………142
4.2.2 Passage of HA through the Gastrointestinal Tract………………...145
4.2.3 Epithelial Cell Surface Receptor Engagement and Size-Specific
Signal Transduction ……….…………………………………………..….….147
4.2.4 Induction of HβD2 Protein Expression………………………….…..152
4.2.5 Functional Resistance to Intracellular Infection…………………...157
4.2.6 Milk HA Shapes Intestinal Microbiota Development……………....162
4.3 MILK COMPONENTS AS A SOURCE OF NOVEL
GASTROINTESTINAL BARRIER THERAPEUTICS…………………….…163
CHAPTER 5: APPENDIX............................................................................................166
BIBLIOGRAPHY…………………………………………………………………….184
8
LIST OF TABLES
Table 5-1. Carbohydrate Constituents of Milk HA Preparations…………………..…..182
9
LIST OF FIGURES
Figure 2-1. HA-Specific Induction of HβD2 Expression in HT-29 Colonic Epithelial
Cells Occurs in a Time Dependent Manner……………………………………………...88
Figure 2-2. Induction of HβD2 Expression in HT29 Colonic Epithelial Cells is Specific to
HA ………………………………………………………………………...……………..89
Figure 2-3. Induction of HβD2 in HT29 Cells is Dependent on HA Size and
Concentration………………………………………………………...………………..…92
Figure 2-4. Orally Administered HA-35 Induces the Expression of the HβD2 Ortholog in
Mouse Intestinal Epithelium In Vivo……………………..………………………...…....96
Figure 2-5. Induction of HβD2 Ortholog in Mouse Intestinal Epithelium by HA
Fragments is Highly Size-Specific…………………………………………………...…..97
Figure 2-6. Specific-Sized HA Fragment Mediated Induction of HβD2 ortholog
Expression in Mice is Dependent on TLR4…………………………………………..….98
Figure 3-1. Human Milk HA Concentrations are Greatest in the First 60 Days
Postpartum……………………………………………………………………….……..132
Figure 3-2. Milk HA Specifically Induces HβD2 Expression……………………..…...133
Figure 3-3. Oral Administration of Human Milk HA Promotes In Vivo Expression of the
Murine HβD2 Ortholog in Intestinal Epithelium……………………………………….137
Figure 3-4. In Vivo Induction of Murine HβD2 by Milk HA is both CD44 and TLR4
Dependent…………………………………………………………………………...….138
Figure 3-5. Milk HA Enhances Resistance to Intracellular Salmonella Infection in
Intestinal Epithelium .......................................................................................................140
10
Figure 5-1. Representative Images for the Quantification of MuβD3
staining intensity…………………………………………………………………….….166
Figure 5-2. HA-28 Promotes Expression of Intracellular HβD2 Protein
in HT-29 Cells…………………………………………………………………….…….167
Figure 5-3. TLR4 Protein Expression is Minimal in the Colonic Epithelium of TLR4-/Mice Relative to Wild-type or CD44-/- Mice……………………………………..…….168
Figure 5-4. Functional TLR4 Antibody Inhibits HβD2 Induction in HT-29 Intestinal
Epithelial Cells Following HA-35 Treatment…………………………………………..169
Figure 5-5. Induction of HβD2 in Caco-2 Intestinal Epithelial cells Following HA-35
Treatment…………………………………………………………….…………………170
Figure 5-6. Induction of HβD2 in MA-104E Kidney Epithelial Cells Following HA-35
Treatment………………………………………………………………………….……171
Figure 5-7. HA-35 Enhances Resistance to Infection by Salmonella enterica serotype
Typhimurium SL1344 in Cultured HT-29 Cells……………………………..…………172
Figure 5-8. Milk HA Confers Rapid Protection from Salmonella Infection………...…180
Figure 5-9. Milk HA Promotes Expression of MuβD3 in Distal Colonic Mucosa of Adult,
Wild-type Mice………………………………………………………………….……...181
11
PREFACE
“The certain truth there is no man who knows, nor ever shall be, about the gods or in
regard to all things. Yea, even if a man should chance to say something utterly true, still
he himself knows it not- there is nowhere anything but guessing. Nonetheless, guesses
are allotted to all.”
- Xenophanes (Ξενοφάνης), 6th century BCE
12
ACKNOWLEDGEMENTS
The work presented in this document was accomplished through the efforts of numerous
highly skilled and thoroughly dedicated scientists, clinicians, administrators, students,
and volunteers, each of whom participated with the earnest desire to contribute to the
advancement of medical knowledge for the betterment of man and society. The author
would like to express his most sincere and humble gratitude to these individuals.
Dr. Carol A. de la Motte served as the author’s dissertation advisor and the Principal
Investogator for the project, guiding the entirety of the work presented herein by directing
the refinement and execution of the scientific approach, and contributed experimental
findings. Dr. Sean P. Kessler assisted in the design and execution of all animal studies,
contributed experimental findings, and maintained the mouse colony. Hyunjin Rho, MS
coordinated the collection of human milk samples, conducted the analysis of milk
hyaluronan content, and contributed other experimental findings. Dr. Mary K. Cowman
and Ripal Amin, MS designed and optomized the protocol for isolation of milk
hyaluronan and contributed to hyaluronan molecular sizing analyses. Craig Homer, MS
and Dr. Christine McDonald contributed extensive technical expertise and experimental
findings in the analysis of functional infection resistance following hyaluronan treatment.
The staff of the Cleveland Clinic Children’s Hospital, particularly Shirley Leaser, RN,
IBCLC, assisted in the recruitment of new mothers for the milk hyaluronan study cohort.
Drs. Judy Drazba and John Peterson contributed microscopy assistance and Dr. Mark
Lauer assisted with carbohydrate analysis. Artin Soroosh, Melissa Michaud, and Ryan
13
Verbic diligently documented and processed numerous donated human milk samples.
Drs. Vince Hascall, Jean-Paul Achkar, Ed Maytin, and Ed Greenfield served on the
doctoral thesis committee, guiding and directing the progess of the research. Drs. Vince
Hascall, Ed Maytin, and Jean-Paul Achkar contributed to revision of the dissertation
manuscript and Dr. Jean-Paul Achkar served as the author’s clinical mentor.
The author would like to thank the educators of the Department of Molecular Medicine
for their enthusiastic encouragement and dedication to graduate education, particularly
Drs. Martha Cathcart and Jonathan Smith. Finally, the author thanks the administrators of
the Department of Molecular Medicine, Dr. Marcia Jarrett and Erica Healey-Pavlik, and
the Depatrment of Pathobiology, Mary Ann Verbic and Nicole Fennell, who efficiently
and effortlessly attended to a seemingly endless deluge of financial, regulatory, and
academic paperwork.
14
LIST OF ABBREVIATIONS
18S
CFU
CpG
CS
DAMP
DEFB4
DS
DSS
ECM
ELISA
FACE
GAG
GALT
GAPDH
GI
HA
HA-35
Has
HβD2
HMO
HMW
HPLC
HS
Hyal
IBD
IL
KS
LMW
LPS
M cells
MuβD3
NF-κB
NLR
NOD
PAMP
PI3K
PRR
PSA
TCR
TIR
TLR
TNF-β
18S subunit of ribosomal RNA
colony-forming units
unmethylated 2’-deoxyribo-cytosine-phosphate-guanidine
chondroitin sulfate
damage-associated molecular pattern
defensin β-4 (gene encoding HβD2)
dermatan sulfate
dextran sulfate sodium
extracellular matrix
Enzyme-linked Immunosorbent Assay
Fluorophore-assisted Carbohydrate Electrophoresis
glycosaminoglycan
gut-associated lymphoid tissue
glyceraldehyde 3-phosphate dehydrogenase
Gastrointestinal
hyaluronan
35kDa hyaluronan fragment preparation
hyaluronan synthase
human β-defensin 2
human milk oligosaccharide
high molecular weight
High Performance Liquid Chromatography
heparan sulfate
hyaluronidase
Inflammatory bowel disease
Interleukin
keratan sulfate
low molecular weight
lipopolysaccharide
intestinal epithelium microfold cells
murine β-defensin 3
nuclear factor κ-light-chain-enhancer of activated B cells
NOD-like receptor
Nucleotide-binding oligomerization domain
pathogen-associated molecular pattern
phosphoinositide-3-kinase
pattern recognition receptor
polysaccharide A
T-cell receptor
Common Toll/Interleukin receptor domain
Toll-like receptor
Tumor Necrosis Factor-α
15
The Role of Hyaluronan in Innate Defense of the Intestine
ABSTRACT
by
DAVID RICHARD HILL
Hyaluronan (HA) is a glycosaminoglycan polymer found in the extracellular matrix of all
mammalian tissues and a component of human milk. Breast-feeding is associated with
enhanced protection from gastrointestinal infection in infants. However, the function of
naturally produced milk hyaluronan relevant to antimicrobial function was unknown.
Recent work has suggested a role for small, fragmented HA polymers in initiating innate
defense responses through interaction with innate molecular pattern recognition receptors
such as TLR4. It was hypothesized that hyaluronan from human breast milk enhances
innate intestinal epithelial antimicrobial defense. Synthetically produced HA fragments
promote expression of the innate antimicrobial peptide human β-defensin 2 (HβD2) in
intestinal epithelial cells. Treatment of HT-29 colonic epithelial cells with HA fragment
preparations resulted in time- and dose-dependent upregulated expression of HβD2
protein in a fragment size-specific manner, with 35 kDa HA fragment preparations
emerging as the most potent inducers of intracellular HβD2. Furthermore, oral
administration of specific-sized HA fragments promotes the expression of an HβD2
ortholog in the colonic epithelium of both wild-type and CD44-deficient mice, but not in
TLR4-deficient mice. Thus, a highly size-specific, TLR4-dependent, innate defense
response to fragmented HA contributes to intestinal epithelium antimicrobial defense
through the induction of intracellular HβD2 protein.
16
Having examined the induction of antimicrobial defense by synthetically produced HA,
functional antimicrobial defense was evaluated following treatment of epithelium with
HA isolated from human milk. The expression of milk HA was defined during the first 6
months postpartum among a cohort of 44 healthy donors. Treatment of HT-29 epithelium
with preparations of human milk HA at physiologic concentrations resulted in a time- and
dose-dependent induction of the antimicrobial peptide HβD2 that was abrogated by
digestion of milk HA with a specific hyaluronidase. In addition, oral administration of
human milk-derived HA to adult, wild-type mice resulted in induction of murine HβD2
ortholog in intestinal mucosa. Finally, treatment of cultured colonic epithelium with
human milk HA preparations significantly enhanced resistance to infection by the enteric
pathogen Salmonella enterica. Together, these observations suggest that maternally
provided HA mediates one of the multiple protective antimicrobial defense mechanisms
delivered through milk to the newborn.
17
CHAPTER 1: INTRODUCTION
1.1. BIOLOGY OF THE GASTROINTESTINAL TRACT
Approximately half of all Eukaryotic taxa are, perhaps regrettably, entirely incapable of
photosynthesis and therefore must obtain sustenance through the consumption of organic
material derived from other living organisms. Among multicellular organisms, or at least
those of us more phylogenetically sophisticated than the Cnidaria, a billion years of
evolution has resulted in the emergence of an endless variety of internalized digestive
systems, each more or less ideally suited to the dietary needs and lifecycle of the
organism it supports. The phylum Nematoda is generally credited with the convenient
innovation of separate entry and exit points for the passage of organic material through
the digestive tract, and from there a progressive series of evolutionary developments has
resulted in the emergence of the stomach, the large and small bowel, and the critical
secretory and metabolic functions of the pancreas, liver and gallbladder found in all
mammals. The unique diversity of viable dietary and lifestyle practices among Homo
sapiens is made possible by a highly sophisticated and adaptable gastrointestinal system
capable of absorbing the nutrients, water and electrolytes required to sustain every cell of
the body while simultaneously supporting an immense population of metabolically active
microbes and maintaining selective protection from pathogenic organisms.
1.1.1 Structure and Function of the Digestive System
Although the microanatomy of the gastrointestinal wall varies along its length, general
organizational themes are present. A single layer of columnar epithelial cells lines the
18
lumen of the GI tract, and this layer of epithelium together with the underlying
extracellular matrix, or lamina propria, and a thin layer of structural smooth muscle
termed the lamina muscularis mucosae is referred to as the mucosa. The epithelial cells
of the intestinal mucosa are continuously renewed through the division of epithelial stem
cells residing at the base of the intestinal villi. The lamina propria contains capillaries,
enteric neurons, and immune cells, which interact with a variety of diffusible luminal
nutrients, cellular signals, and microbial products. The outer submucosa consists of
additional collagen-rich connective tissue as well as intermittent glandular follicles. The
muscularis externa is highly innervated and generates the peristaltic action of the
esophagus, stomach, and bowel through the contraction of both circular and longitudinal
smooth muscle fibers. An enveloping layer of squamous epithelial cells is referred to as
the serosa (1).
Digestion of complex macromolecules and their absorption in readily utilized molecular
form is the primary and essential function of the human gastrointestinal tract, and a
process that is best understood when followed from its initiation to conclusion along the 9
meters of digestive tube that connects the open ends of the digestive system. The process
of digestion involves both mechanical and chemical digestion of ingested food as well as
absorption. Regulation of the initial impulse to feed is generally attributed to the central
nervous system, though more recently the influence of gut hormones on the balance
between hunger and satiety is gaining appreciation (2, 3). Regardless of the reason for
feeding, digestion begins in earnest as food enters through the mouth, except in cases of
disease or injury when simple nutritional substances may be directly deposited into the
esophagus or stomach by means of various medical devices. The process of mastication
19
physically disrupts the macrostructure of ingested food and is greatly enhanced in
efficiency by the presence of and array of healthy teeth. Enzymes, such as lingual lipase
and salivary amylase, are secreted by the salivary glands during mastication, initiating the
breakdown of complex lipids and carbohydrates at the point of ingestion. After
swallowing, the food bolus is transported to the stomach by gravitational momentum and
the peristaltic action of the esophagus, a somewhat digestively inert process regulated by
the contraction and relaxation of the upper and lower esophageal sphincters. In the
stomach, the food bolus encounters a wholly different environment from that of the upper
digestive tract. The lining of the stomach actively secretes free hydrogen ions and an
array of acid-active proteases including pepsin generate polypeptides from complex
dietary protein sources. The stomach expands tremendously to accommodate food, from
an empty volume of 50-70 ml up to a maximum volume of 1L or more following a
particularly indulgent meal. Peristalsis of the stomach ensures efficient mixing of gastiric
contents prior to the release of chyme into the duodenum by the relaxation of the pyloric
sphincter. The small intestine, including the duodenum, jejunum, and ileum, accounts for
the majority of the length of the GI tract at 7 meters. Hormonal signals including secretin
and cholycystokinin generated in the duodenum regulate the opening of the pyloric
sphincter, subsequently triggering the release of bile from the liver and gallbladder and
the release of bicarbonate and digestive enzymes such as trypsin, colipase, lipase, and
amylase from the pancreas. Bicarbonate neutralizes the highly acidic stomach contents
and enhances intestinal enzymatic activity, while bile salt emulsifies dietary triglycerides,
increasing susceptibility to digestion by pancreatic lipases and the subsequent generation
of monoglycerides and free fatty acids, absorbed by the enterocytes lining the intestinal
20
lumen. Small, finger-like protrusions of mucosal tissue called villi line the entire bowel,
resulting in a digestive surface area of approximately 200 m2. Additionally, the surface
area of individual enterocytes, or epithelial cells, is increased by the presence of
microvilli protrusions in the plasma membrane. Specific transporters expressed in the
apical epithelial plasma membrane microvilli (or ‘brush border’) actively mediate the
uptake of luminal sugars, amino acids, free fatty acids, electrolytes, water, and other
small molecules. Co-regulated transporters at the basolateral membrane mediate the
transfer of digestive products into the capillaries of the venous system, or the lymphatic
system in the case of dietary fats. Nutrient-rich venous blood empties into the hepatic
portal vein where nutrients are further processed and extracted by the liver for glycogen
and plasma protein synthesis. Fats transported in the lymphatic system eventually enter
the circulatory system through the thoracic duct. Liproprotein lipase expressed by
capillary endothelium liberates free fatty acids from circulating triglyceride micelles,
which may be used directly as an energy substrate or stored in adipose tissue as resynthesized triglyceride in response to systemic hormonal control (4). With the bulk of
nutrient absorption completed in the small intestine, the primary function of the large
intestine is the re-absorption of water and electrolytes from the fecal material. The large
intestine, approximately 1.5 meters in length, supports the largest population of
microorganisms present in or on the human body, as discussed below. The catalytic
activity of the large intestinal microbiota results in the release of additional nutrients and
the synthesis of vitamins, which are absorbed by the enterocytes of the large intestinal
mucosa along with any nutrients escaping absorption by the small intestine. The anatomy
of the human large intestine is subdivided according to location. The pouch-like cecum
21
connects the large bowel to the terminal ileum, with digestive contents flowing upwards
through the ascending colon before bending through the hepatic colic flexure to the
transverse colon, which extends across the abdomen, and descending at the splenic colic
flexure and into the sigmoid colon. The sigmoid colon terminates in the rectum with the
anal sphincter, which is composed of a double layer of both smooth and striated muscle
under voluntary control (1).
1.1.2 The Intestinal Microbiota
Current estimates place the number of living microorganisms in the average human
gastrointestinal tract at somewhere on the order of 1012 to 1014 (5, 6, 7), or roughly ten
times the number of individual cells that make up a human being. The persistence of
multicellular life in the presence of vastly more numerous single cell organisms suggests
that a mutually beneficial relationship between the Kingdoms Bacteria and Animalia
must be obtainable. Indeed, such a balance is more the standard of active mammalian life
rather than the exception, and the microbiome, or the complete complement of coexistent microorganisms present on or within the body, is increasingly appreciated as a
major component of the function of both the digestive and immune systems. The only
portion of the human life cycle that is generally not thought to include constitutive direct
contact with microorganisms is the period of gestation, with the first critical contact with
microorganisms occurring during passage through the birth canal. The process of
intestinal microbiome ontology begins with ingestion of maternally derived and
environmental microorganism, and multiple factors including the mode of delivery and
maternal microbiome composition influence neonatal microbial development with
22
potentially long term implications for development of disease (8, 9). In addition to
providing critical nutritional components, breast milk includes numerous factors,
including glycans and oligosaccharides, which powerfully impact the development and
composition of the developing gastrointestinal microbiome (10). The infant microbiome
is enriched with species specialized in the utilization of milk substrates such as lactate,
with additional microbial metabolic diversity coinciding with the introduction of solid
foods (11). By adulthood, the microbiome may include more than 1,200 unique species,
predominated by the phyla Bacteroides and Firmicutes with common Genera in the gut
including Faecalibacterium, Roseburia, and Bacteroides (12, 13, 14). Additional archea,
eukaryotes, and phage viruses contribute to the adult microbiota (15). However, as data
sets expand to include a broader range of human diversity, the elucidation of a core set of
human microbiome species appears increasingly unlikely (16). Rather, individuals are
likely to contain unique microbial communities whose composition has been influenced
by the interaction of many complex factors. The application of ecological theory to the
human gut microbiome predicts that the composition of the gut microbial community is
dependent upon environmental selection and evidence is beginning to accumulate to
indicate that genetic, dietary, cultural, and anatomical factors define gut microbial
community development (17, 18). In addition, the timing of microbial exposure,
community disruption caused by antibiotic exposure, or the dispersal patterns conferred
by specific microbial biology or human cultural practices could result in dramatically
different microbial composition even among genetically similar human hosts (16, 18).
Despite the overwhelming complexity of microbial community development, functional
relationships between microbes and the human gastrointestinal tract have become
23
apparent. The gut microbiome participates in the process of digestion and metabolism in
a highly dynamic manner, with multiple bacterial genera acting sequentially or
independently in concert with human host epithelium to generate bile acids (19), shortchain fatty acids (20), choline (21), vitamins (11, 22), and additional energy substrates
such as glucose, succinate, and lactate (23, 24). Microbial metabolism may have
profound implications in the etiology of obesity (23, 25), cardiovascular disease (21),
inflammatory bowel disease (6), and diabetes (26). Colonization of mucosal surfaces with
bacteria provides enhanced immune protection through numerous mechanisms. This
includes exclusion of pathogenic organisms from access to epithelial membrane heavily
populated by commensal species (5), but also by direct immunomodulation of epithelial
cells and gut lymphoid cells by colonizing bacteria (6, 27). One example of this is the
production of polysaccharide A (PSA) by the gut symbiont Bacteroides fragilis.
Colonization of germ-free mice with B. fragilis or dietary supplementation with PSA
results in the expansion of gut T-cells in a manner that enhances homeostatic balance by
suppressing inflammatory T-cell subtypes while expanding regulatory, anti-inflammatory
T-cell differentiation and suppressing pro-inflammatory cytokine expression (28). The
mutualist gut microbiota interacts with the epithelial surface to generate an ideal substrate
for growth. For example, microbial colonization results in a dramatic FUT2-dependent
increase in fucoslyation of the apical surface of colonic epithelium, facilitating interaction
and growth of microbial symbionts (29). Additionally, the interaction of gut microbiota
with epithelial cells through the Toll-like receptors (discussed below) results in the
specific expression of antimicrobial peptides, such as β-defensins and RegIIIγ, and the
induction of immunoregulatory cytokines (27, 30). Antimicrobial peptides and cytokine
24
signals act in a combinatorial manner to confer substrate selectivity and
immunomodulation in a manner that is determined by the pattern of Toll-like receptor
engagement produced by specific microorganisms (27, 30-32). Investigations into the gut
microbiota have resulted in paradigm-shifting findings regarding the nature of
metabolism and immunity, and additional studies aiming to clarify central themes of
human-microbial interactions are proceeding at a rapid pace. Despite the seemingly
chaotic nature of our current understanding, it has become clear that the microbial
inhabitants of the digestive tract are an essential component of both the metabolic and
immune functions of the gastrointestinal system.
1.1.3 Mucosal Barrier Function
At the forefront of both the digestive and immune functions of the gastrointestinal tract is
a single-cell layer of epithelium. This thin lining of epithelial cells simultaneously
functions as the primary site of nutrient and waste exchange, as well as the physical and
immunological barrier between the external environment and the sterile internal tissues of
the body. The combined area of the mucosal surfaces of the gastrointestinal tract is
enormous, many times greater than the surface of the skin. The integrity of this barrier is
of paramount importance to the continued function of the digestive and immune systems,
and thus to the organism as a whole. Numerous processes, both innate and adaptive,
contribute to the continuous maintenance and renewal of the intestinal epithelial barrier
(33). Mucosal barrier defense is maintained by both immune and non-immune
components. Immune components include both cellular and soluble elements while the
25
non-immune, or innate, components include physical and chemical barriers generated
through a wide range of physiologic and cellular processes.
The intestinal mucosa and submucosa supports a vast population of specialized immune
cells that compose the gut-associated lymphoid tissue (GALT). This includes lymphoid
cells, (T, B cells, and dendritic cells) as well as myeloid cells (macrophages, neutrophils,
eosinophils and mast cells). In addition specialized epithelial cells, known as M cells
populate the epithelial barrier and interact with lymphoid cells to generate antigen
specific responses (30). Dietary, microbial, or self-antigens, abundant in the gut lumen,
traverse the epithelial barrier either through a paracellular or transcellular pathway.
Paracellular transport involves passive movement of material through the space between
adjacent epithelial cells, or may occur at sites of epithelial damage or infection. The
transcellular pathway occurs through two classes of specialized antigen-presenting cells
(33). M cells, or microfold cells, are restricted to the epithelium directly overlying areas
of lymphoid tissue present in the small intestine known as Peyer’s patches. Unlike typical
enterocytes, M cells do not secrete mucus or digestive enzymes. Rather, M cells express
numerous cytoplasmic vesicles, facilitating the transport of luminal antigens across the
epithelial barrier and presentation at the basal epithelium to the underlying lymphoid
tissue (34). The remaining epithelial surface of the intestine is sampled intermittently by
dendritic cells, specialized antigen presenting cells of the lymphoid lineage populating
the lamina propria. Dendritic cells form cytoplasmic extensions that protrude across the
epithelial barrier through the paracellular space. These extensions sample luminal
antigens, which are then presented via MHC class-specific carriers in the context of
costimulatory signals to generate antigen-specific T-cell mediated responses, either
26
locally or following migration to the lymphatic tissues. Intestinal antigens traversing the
epithelial barrier by paracellular means are similarly detected by dendritic cells residing
in the lamina propria (35). T-cells are initially generated in early development and a
unique process of gene rearrangement produces an endlessly variable array of specific
antigen receptors, with each T-cell recognizing a unique T-cell receptor. Following a
process of selection to eliminate self-recognizing or ineffective T-cells, these cells
migrate through the circulatory and lymphatic systems to populate the tissues of the body
and are particularly prevalent in the mucosal lymphoid tissues. Dormant T-cells reside in
tissues or circulate throughout the lymphatic system in the absence of antigen
corresponding to the unique TCR. Upon interaction with cognate antigen presented by
dendtritic cells, T-cells respond by proliferation and generation of distinct cytokine
signals. Therefore, exposure to cognate antigen results in the expansion of T-cells
expressing specific complementary TCRs, enhancing recognition of the antigen
subsequently (36). T-cells can be classified according to several distinct functional types.
Th1 cells secrete IL-2 and IFN-γ cytokines, enhancing cellular immunity and promoting
T-cell and macrophage activation and B-cell development, whereas Th2 cells express
cytokines (IL-4, IL-5, IL-6, IL-13) dedicated to the expansion of B-cell humoral
responses, predominantly the secretion of IgG and IgE (30, 36, 37). Th1 and Th2 cells are
commonly referred to as effector T-cells, while Th3 and Treg1 cells are associated with
suppression of the adaptive immune response through the secretion of TGFβ and IL-10,
respectively (38). Another class of T-cell, the Th17 lineage, may be dedicated to the
direct opposition of Treg1 cells through the expression of proinflammatory IL-17 (39).
The balance between proinflammatory Th1/Th2/Th17 responses and Treg1 responses to a
27
given antigen ultimately determines the adaptive response, with tolerance to the great
majority of commensal and dietary antigens predominating in healthy individuals. The
context in which a given antigen is presented, including homeostatic or inflammatory
innate defense signals as well as microbial signals and extracellular matrix cues, may aid
in determining the subsequent T-cell response (28, 38, 40,41).
The major phagocytic cell of the gut is the macrophage, derived from the myeloid lineage
and residing in the lamina propria and lymphoid patches. The release of cytokine or
chemokine signals from epithelium or lymphoid cells subsequent to epitheilial barrier
breach activates resident tissue macrophages and increases the extravasation of
circulating monocytes and their differentiation into additional macrophages (37, 42). The
phagocytic activity of macrophages increases dramatically in response to local signals,
resulting in the engulfment of pathogens, foreign bodies, and necrotic and apoptotic
debris alike, targeted broadly by the presence of conserved microbial- or damageassociated molecular patterns and their interaction with a variety of pattern recognition
receptors present on the macrophage plasma membrane (43). Interestingly, intestinal
macrophages are far less sensitive to cytokine activation signals in comparison to
macrophages found in other tissues, consistent with the need to tolerate the presence of a
vast population of luminal bacterial (44). Mast cells are specialized for the response to
intestinal parasites through the IgE-mediated allergic response, the release of TNF-α and
other cytokines, and neutrophil recruitment (45, 46). Circulating neutrophils are recruited
to sites of inflammatory activity, participate in the phagocytosis of microbes, secrete
potent granule contents including soluble antimicrobial peptides, such as defensins and
cathelicidins, and amplify inflammatory reactions through the release of cytokines (47).
28
Natural-killer T-cells are primary effectors of cellular immunity, participating in the
removal of damaged or infected epithelium and contributing to the inflammatory
cytokine milieu (48). Together, the many cell types that populate the gut-associated
lymphoid tissue and the lamina propria represent a means of amplifying or selectively
limiting intestinal defense following a significant challenge to epithelial barrier integrity.
The majority of intestinal barrier defense is generated through innate functions that have
evolved to limit the bulk of microbial intrusions across the epithelial barrier in a nonspecific manner.
1.1.4 Innate epithelial defense
Innate immunity is the predominant form of defense against infection among all
multicellular life, and many features of innate defense are highly conserved among
plants, invertebrates, and chordates. Adaptive immunity is a relatively recent
development in the evolutionary history of life on earth, first appearing in a primordial
form in jawless fishes (subphylum Vertebrata, class Agnatha) some 500 million years
ago and arising independently in upper vertebrates perhaps 50 million years later (49). In
contrast, many features of innate defense were firmly established at the time of
divergence between Kingdoms Animalia and Plantae, and the mechanisms of innate
immunity still expressed in modern Homo sapiens have been refined over more than 1.6
billion years by the unremitting selective pressure imposed by infectious organisms (50,
51). The primary function of innate immunity is the prevention, control, or elimination of
host infection. As previously alluded, a secondary function in mammals is the stimulation
and refinement of the adaptive immune response. The maintenance of the intestinal
29
epithelial barrier in the presence of continuous microbial challenges is dependent upon a
wide range of physical, chemical, molecular, and cellular innate defense mechanisms.
Intact epithelial surfaces form a physical barrier between microorganisms in the external
environment and host tissue. In the intestinal mucosa, this barrier is selectively
permeable, a necessary feature to support the exchange of fluid and dietary nutrients.
Extracellular components of the mucosal barrier confer a degree of selective permeability
to the epithelial membrane by physically slowing or preventing the translocation of
bacteria and macromolecules while permitting free diffusion of ions and small solutes.
Limited flow of solutes contributes to the development of an unstirred layer at the apical
epithelial surface that is protected from the convective mixing of intestinal contents,
reducing the rate of nutrient loss to diffusion. This extracellular mucosal barrier is
primarily composed of mucins, a family of heavily glycosylated proteins secreted by
specialized epithelial goblet cells in the intestine (33). The small intestine is continuously
coated by a gel-like layer of mucins, with two mucin layers found in the large intestine.
While the outer mucin layer is populated by gut bacteria, the inner mucin layer is
apparently impermeable to luminal bacteria under homeostatic circumstances (52).
Mucins may have a direct role in inhibition of bacterial infection, perhaps binding
bacterial surface receptors through glycan epitopes and preventing interaction with host
epithelium (53). Furthermore, glycans present on mucins may generate environmental
selectivity, encouraging the growth of bacteria able to utilize the presented substrate
while excluding potentially pathogenic organisms (54). Embedded within this mucus
lining are anti-microbial peptides, including defensins, discussed below. While generally
understudied in relation to disease, the importance of mucins is emphasized by the
30
observation that mucin-deficient mice develop spontaneous colitis, or inflammation of
the bowel (55, 56).
Despite the robust protection conferred by mucins secreted at the apical epithelial border,
the primary responsibility for mucosal barrier function resides with the function of
epithelial cells, and particularly the epithelial cell plasma membrane. Translocation
across the paracellular pathway is mediated by the apical junctional complex, composed
of the tight junction and adherens junction. Adherens junctions are formed by a family of
transmembrane proteins called cadherins, which form homotypic interactions with
complementary cadherins on adjacent epithelial cells (33). Transmembrane cadherins
interact with intracellular regulatory systems to influence actin assembly, which in turn
regulates epithelial cell polarization, differentiation, and apoptosis (57). Tight junctions
are apical to the adherens junction along the border between epithelial cells and are the
primary regulatory site of paracellular diffusion. Composed of multi-protein complexes,
tight junctions bind adjacent epithelial cells through tissue-specific transmembrane
caludin proteins to intracellular regulatory molecules, kinases and cytoskeleton
components to directly intertwine the regulation of physical, chemical, and cellular
behavior of the continuous epithelial membrane. Flux across the tight junction is
regulated by hormonal, dietary, and inflammatory signals (58, 59). Thus, through the
combined action of the apical mucus lining and the formation of selectively permeable
paracellular junctions, microorganisms are physically excluded from passive
translocation across the intestinal epithelial barrier. However, disruptions in barrier
integrity occur spontaneously, during injury, or through the action of invasive organisms
on a routine basis. For this, a system of bacterial pattern recognition receptors has
31
evolved which initiates a cascade of cellular responses aimed at limiting the influx of
bacteria and amplifying immune defense.
1.1.5 Toll-like Receptors
While the receptors of the adaptive immune system are capable of recognizing a virtually
limitless variety of molecular structures, the pattern recognition receptors (PRRs) of the
innate immune system have evolved to recognize a much more limited collection of
molecular patterns that are consistently associated with microbial activity. Pathogenassociated molecular patterns (PAMPs) are typically microbial products that are essential
for microbial survival and therefore are highly conserved across bacterial, fungal,
protozoan, and viral genera. Examples of PAMPs include double stranded RNA (dsRNA)
found only in replicating viruses, unmethylated CpG DNA, a characteristic of bacterial
genomes, flagellin components commonly expressed by motile bacteria, and
lipopolysaccharides (LPS), peptidoglycan, teichoic acid and mannose-rich
oligosaccharides, all requisite components of the bacterial cell wall or membrane.
Importantly, none of these structures are produced by human cells, eschewing selfrecognition by the PRRs of the innate immune system (36). Various PRRs are expressed
by nearly all mammalian cells. While some PRRs may be limited to phagocytic cells,
including C-type lectins, scavenger receptors such as CD36, and N-formyl Met-Leu-Phe
receptors, others are more widely expressed by non-immune cells including the NOD
(Nucleotide-binding oligomerization domain)-like receptors and Toll-like receptors
(TLR) (60). The NOD-like receptors are generally expressed within the cytoplasm and
mediate the response to intracellular infection. TLRs are embedded within the plasma
32
membrane or vesicular membranes by hydrophobic transmembrane domains. Originally
identified by similarity to a Drosophila gene (Toll) involved in detection of fungal
infection (61), the earliest evidence that Toll-like proteins functioned in the recognition
of pathogens in mammals resulted from the observation that TLR4-deficient mice are
unresponsive to bacterial LPS, the inciting factor in septic shock (62, 63). Subsequent
experiments have identified ligands for each of the 10 human (11 in mice) Toll-like
receptors using genetically modified “knockout” mice and in vitro gene expression
techniques. TLR2 is involved in the detection of several bacterial products, including
peptidoglycan, lipopeptides, and zymosan (64, 65), and hyaluronan (66). TLR3 is
expressed in the lysosomal membrane, generating responses to ligands within the
lysosome including dsRNA (67). TLR4 detects a wide variety of bacterial and
endogenous ligands, most notably LPS (63), hyaluronan (68) and heparan sulfate (80).
TLR5 mediates the innate response to flagellin, a 55 kDa monomer derived from
polymeric appendages extending from Gram-negative bacteria (69). TLR7 and TLR8 are
involved in the recognition of free single stranded RNA (70, 71), with TLR7 also known
to mediate reponses to several pharmaceutical compounds (72). TLR9 recognizes
unmethylated 2’-deoxyribo-cytosine-phosphate-guanidine (CpG) common in bacterial
and viral genomic DNA (73) and TLR11 is involved in recognition of various structures
associated with uropathogenic bacteria (74). TLRs always function as dimers in the cell
membrane, and TLR heterodimers often have ligand specificity that is distinct from the
activity of homodimers (31). For example, TLR1/2 recognizes diacyl lipopeptide, while
TLR2/6 recognized MALP-2, expressed by Mycoplasma (74, 75). The list of
characterized Toll-like receptor ligands grows with each passing year, and now includes
33
numerous endogenous molecular patterns associated with tissue injury, or damageassociated molecular patterns (DAMPs). Examples of DAMPs include intracellular
components such as heat-shock proteins and chromatin-associated HMGB1 proteins (76),
ATP (77), uric acid (78), and DNA (79), as well as fragmented heparan sulfate (80) and
hyaluronan (66, 68) generated through the destruction of extracellular matrix. Signal
transduction occurs subsequent to engagement of TLR ligands with their complementary
receptors through the interaction of the intracellular TIR domain with adapter protein
MyD88 (81), the exception being TLR3, which is MyD88 independent (31). A cascade of
MAP kinase family members activates a common signaling pathway that ultimately
results in the nuclear translocation of NF-κB, a transcription factor consistently
implicated in regulatory control of cytokine expression (31). While TLRs have been
principally described relative to their induction of various pro-inflammatory cytokine
responses in other tissues, reciprocal anti-inflammatory pathways selectively attenuate
inflammation in response to TLR ligands in the intestine (82). Indeed, TNF-α and IL-1β
expression in gut epithelium is highly responsive to TLR stimulation prior to birth, but
notably hyporesponsive subsequent to intestinal colonization (10, 83-85). In addition,
TLR ligands have been evaluated experimentally in isolation, while physiologic settings
frequently involve the simultaneous engagement of multiple TLRs (86, 87), with
potentially unique signaling events occurring as a result of the activation of multiple
intracellular pathways. The consequence of the interactions between an array of unique
PAMPs and DAMPs and specific cell surface receptors on epithelium is the expression of
an innate response finely tuned to a distinct challenge. The intestinal epithelial barrier
34
regulates the expression of an array of effectors in response to TLR-mediated sampling of
the epithelial surface and lumen, including antimicrobial peptides.
1.1.6 Defensin Structure, Function, and Expression
Among the effectors of the innate PRR system, small cationic antimicrobial peptides play
an essential role in the preservation of epithelial integrity against persistent microbial
challenges. By definition (88), antimicrobial peptides contain fewer than 100 amino acids
and exhibit antimirobial activity locally under the typical physiological conditions of the
tissue of origin. There are two evolutionarily and structurally distinct families of
antimicrobial peptides in mammals: defensins and cathelicidins (88). Defensin-like
proteins are found in plants, insects, and vertebrates, but only mammalian defensins have
clearly arisen from a common evolutionary ancestor (88). The two major mammalian
defensin subfamilies, α- and β-defensins, share a characteristic triple-stranded β-sheet
fold and scaffold of six disulfide-linked cysteines, but differ in amino acid sequence in
segments between cysteines and in the disulfide pairing arrangements of the six cysteine
residues (88). A third structurally unique defensin subfamily, the θ-defensins, has been
identified in leukocytes of Macaca mulatta (89), but is inactivated by premature stop
codons in humans (90). Cathelicidins, including LL-37, have a comparable distribution,
expression, and activity to the defensins (91) and several additional antimicrobial
peptides are known that do not clearly belong to either the defensins or cathelicinins,
including histatins, dermcidin, RegIIIγ and various other ‘anionic peptides’ (27, 88, 92).
The structure of defensins is the essential determinant of antimicrobial activity. The
amino acid composition, amphipathicity, cationic charge, and molecular size of defensin
35
peptides facilitates their spontaneous incorporation into membrane bilayers and the
formation of pores, resulting in lethal lysis of microbes (92, 93). Additional observations
support the possibility that defensins and other antimicrobial peptides may inhibit cellwall synthesis, nucleic-acid synthesis, protein synthesis and other enzymatic activities in
bacteria (92).
There are at least 12 known human defensins that have been characterized in detail,
forming a potent barrier to invasive bacteria in places where epithelium interacts directly
with an abundance of microorganisms, including the gastrointestinal, urogenital, and
ocular mucus membranes and in the skin (94, 95). Human defensins are found in the
greatest concentrations within leukocyte granules, and function in the elimination of
phagocytized microorganisms (96), and in the secretory granules of Paneth cells found at
the base of villi of the small intestine (97). Paneth cells are specialized epithelial subtypes
that secrete large quantities of both α- and β-defensins into the lumen of the intestine,
maintaining microbial populations under homeostatic conditions and responding to
inflammatory signals through the upregulation of antimicrobial secretions (97). While βdefensin 1 is constitutively expressed in intestinal epithelial cells (98, 99), β-defensins 2,
3 and 4 are inducible by bacterial stimuli (98, 100-102), cytokine signals (32, 98, 103),
and dietary components (104-106). HβD2 has strong antimicrobial effects against several
common opportunistic pathogens, including E. coli (107, 108), P. aeruginosa (107, 109),
and C. albicans (109, 110). In the intestine, pathogen-associated molecular patterns
(PAMPs) act as critical local activators of HβD2 expression through specific interactions
with Toll-like receptors (TLRs) and other recognition molecules expressed by epithelial
cells (32). The consequence of the interactions between an array of unique PAMPs
36
expressed by a given microbe and specific cell surface receptors on epithelium is the
expression of an innate response finely tuned to a distinct microbial challenge. Increased
transcription of the gene encoding the HβD2 peptide, DEFB4, has been reported in some
intestinal epithelial cell lines following treatment with lipopolysaccharide (LPS) derived
from cell membrane of Gram-negative bacteria and by peptidoglycan, a component of the
cell wall of Gram-positive bacteria, through TLR4- and TLR2-dependent mechanisms,
respectively (102). Salmonella enteritidis flagellin also promotes HβD2 expression (101)
via TLR5 (111). Additional PAMPs have been shown to upregulate HβD2 expression in
respiratory epithelial cells including dsRNA, which binds via TLR3 (31), and CpG
signaling through TLR9 (112). Fragments of peptidoglycan, muramyl dipeptide, found in
the cell wall of intracellular microbes induce NOD2-dependent HβD2 upregulation in
HEK293 cells (113), enhancing host defenses against invasive microorganisms. Two key
pro-inflammatory cytokines are known to directly promote HβD2 expression in epithelial
cells: TNF-α (109) and IL-1β (103). Expression of defensins is therefore a critical
component of the homeostatic maintenance of the mucosal barrier, as well as an innate
mechanism of responding to injury or infection and limiting the intrusion of opportunistic
or pathogenic organisms in damaged epithelium.
Together, both innate and adaptive immune processes function in the continuous
maintenance of the intestinal epithelial barrier, resulting in long-term intestinal
homeostasis despite the presence of immense microbial and dietary challenges. However,
dysfunction in mucosal barrier maintenance has significant consequences for human
health, including the development of chronic inflammation of the gastrointestinal tract.
37
1.1.7 Inflammatory Bowel Disease
Inflammatory bowel disease is a highly heterogeneous chronic and progressive
inflammatory disorder of the gastrointestinal tract. The etiology of inflammatory bowel
disease is unclear, with both genetic and environmental factors clearly contributing.
Inflammatory bowel disease affects approximately 0.5% of the U.S. population, or about
1.4 million individuals, with similar incidence in Western Europe. While primarily
associated with a Westernized lifestyle, the number of cases of inflammatory bowel
disease has been increasing globally since at least 1970 (114, 115).
Clinical symptoms of inflammatory bowel disease typically manifest before the age of
40, and often earlier, and the initial presentation of patients may include diarrhea,
vomiting, nausea, severe abdominal pain, weight loss, and bloody stool. Diagnosis based
on symptoms or responsiveness to initial treatment is confirmed by examination of the
intestinal mucosal surface by colonoscopy, often with histological examination of lesion
biopsies. Inflammatory bowel disease is typically classified as Ulcerative colitis or
Crohn’s disease based primarily on the nature and location of inflammatory lesions.
Ulcerative colitis is defined by lesions restricted to the large bowel and rectum, with
inflammatory lesions limited to the bowel mucosa. Crohn’s disease may affect any
portion of the gastrointestinal tract from the mouth to anus, with transmural inflammation
extending to the bowel wall. Extra-intestinal manifestations are usually associated with
Crohn’s disease, but may occasionally occur in cases of Ulcerative colitis, including
arthritis, pyoderma gangrenosum, and sclerosing cholangitis. Over time, chronic
inflammation of the bowel results in a net loss of functioning epithelium and the
conversion of chronically inflammed mucosa into abnormally functioning fibrotic tissue,
38
resulting in malnutrition, severe abdominal pain, and poor bowel control. Both the
intestinal and extraintestinal manifestations of inflammatory bowel disease (IBD) result
in a high degree of disability among affected patients, with as many as 58% of IBD
patients reporting disabling disease within 5 years of diagnosis (116). In addition, the
early age of disease onset (15-25 years of age in Crohn’s disease patients, 25-35 years of
age among Ulcerative colitis patients) often requires the active management of disease
symptoms for decades, placing significant demands on health-care resources. Medical
treatment of inflammatory bowel disease typically follows a conservative or “bottom-up”
approach, in which initial medications include antibiotics or mild anti-inflammatory
drugs such as 5-aminosalicylic acid, with patients progressing to the use of
corticosteroids (prednisone) or anti-proliferative agents such as methotrexate or
mercaptopurine if the symptoms are not adequately managed by the use of
aminosalicylates (117). More recently, the systemic application of humanized
monoclonal antibodies against the proinflammatory cytokine TNF-α has shown great
promise in management of a subpopulation of cases refractory to other medical
intervention, with as many as 83% of treatment responders achieving disease remission
(117). Still, some cases of inflammatory bowel disease are unresponsive to anti-TNF
therapy, often due to acquired resistance to treatment, and the treatment itself is
associated with significant adverse effects including increased infection and colorectal
cancer risk. Thus, anti-TNF agents are not ideal agents for long-term management of
inflammatory bowel disease. The primary objective of medical treatment is the
management of symptoms and delay of surgical intervention.
39
Even with available medical treatments, as many as 60% of Crohn’s disease patients may
progress to stenosing or penetrating disease within 10 years of diagnosis (118). Late
clinical stage disease is characterized by significant loss of functional bowel, often
accompanied by fibrostenotic changes in the bowel wall that necessitate surgical
intervention, often in the form of bowel resection and anastomosis. Penetrating disease is
characterized by the formation of fistulas, spontaneous fibrotic connections between
portions of the bowel or between the bowel and the skin, urogenital tract, or other organ
systems. The need for surgical intervention results in significantly increased morbidity
and mortality among Crohn’s disease patients (119), with a relatively low rate of disease
resolution following surgical treatment among most patients (117). Disease may be a
primary cause of death in very severe cases due to significant loss of functional bowel,
liver failure resulting from nutritional deficiencies, or sepsis resulting from bacterial
translocation through the bowel. While Crohn’s disease and ulcerative colitis are rarely
directly lethal with medical and surgical treatment, recent epidemiological studies have
demonstrated that affected individuals suffer higher all-cause mortality, as well as
increased risk of colorectal cancer, pulmonary disease, and nonalchoholic liver disease
relative to non-IBD patients (120). In some cases, prolonged disease remission can be
obtained through medical treatment alone or medical treatment with surgical intervention.
Ulcerative colitis can in fact be definitively treated through complete colectomy.
However, it is important to emphasize that none of the available treatments for Crohn’s
disease or Ulcerative colitis appear to resolve the underlying pathological processes that
cause the disease, but instead are thought to limit symptoms resulting from secondary
events in disease pathobiology.
40
While the cause of inflammatory bowel disease remains unknown, both genetic and
environmental components are thought to contribute to disease etiology. Recent evidence
supports a critical role for the intestinal mucosal barrier in the pathology of inflammatory
bowel disease as the site at which genetic predisposition to barrier dysfunction may be
challenged by the intestinal microbiota (121,122, 123), a component of the intestine that
is highly influenced by environmental factors (17, 18). Longitudinal studies have shown
that regardless of treatment course, mucosal healing is the clinical parameter that is most
predictive of positive disease outcome. For example, mucosal healing was associated
with delayed bowel resection surgery in a Norwegian cohort of Crohn’s disease patients
(124). Genome-wide association studies have identified numerous gene polymorphisms
associated with Crohn’s disease and Ulcerative colitis, and have consistently implicated
pathways regulating host-microbial interaction (125). Among these, the single gene
consistently demonstrating the strongest association with Crohn’s disease is the
cytoplasmic PRR NOD2 (126), known to regulate expression of human β-defensin 2
(HβD2) in response to the presence of cytoplasmic peptidoglycan (113). Changes in
HβD2 expression have been associated with epithelial inflammation in vivo, first in the
epidermis of psoriasis patients (127), and later in the colon of individuals with ulcerative
colitis (103) where induction of HβD2 is correlated with disease severity and increased
pro-inflammatory cytokine concentrations (128). Conversely, in Crohn’s disease deficient
induction of HβD2 in the colonic epithelium (128,129) may contribute to the loss of
barrier integrity and vulnerability to invasive, and pro-inflammatory, mucosal flora (123).
Copy number variation may (130) or may not (131) play a role in dysregulated HβD2
expression in these patients. It is unclear whether changes in HβD2 expression contribute
41
to or are a result of IBD pathogenesis. According to one proposed model for the function
of intestinal bacteria and host defensins in the pathogenesis of IBD, the healthy
gastrointestinal tract is characterized by a homeostatic balance of host antimicrobial
peptides and intestinal microbes. However, in inflammatory bowel disease this balance is
disturbed and is correlated with insufficient expression of antimicrobial defensin
molecules (129). Antimicrobial deficiency facilitates the translocation of intestinal
microbes across the host mucosa. In addition, inflammatory cytokines expressed by
epithelium and lamina propria myeloid cells subsequent to TLR stimulation by bacterial
PAMPS increases flux across tight junctions (33). When this occurs chronically, loss of
tolerance to healthy mucosal flora may occur as a result of the increased influence of proinflammatory Th1/Th2/Th17 relative to anti-inflammatory Treg subsequent to T-cell
expansion during mucosal bacterial influx, resulting in an abnormal and extended
inflammatory response (123). Application of this model predicts that therapeutic
approaches aimed at enhancing antimicrobial peptide expression in intestinal epithelium
may be effective in the treatment of inflammatory bowel disease.
1.1.8 Milk in Gastrointestinal Health and Disease
Milk production and consumption, a defining characteristic of all mammalian life,
intimately connects the nutritional (132), immunological (133), and cognitive
development (134) of parent and offspring in the postnatal period. Triglyceride, protein,
and carbohydrate-rich milk is the first nourishment to enter the neonatal digestive tract,
providing complete nutrition in early life and shaping critical developmental processes
(135). Numerous positive health outcomes are associated with breast-feeding in infants
42
(10, 134, 136, 137), particularly regarding gastrointestinal infection. Grulee et al. (138)
conducted the first major evaluation of morbidity and mortality among 20,061 breast-fed
and artificially-fed infants in 1934, reporting as much as 50% reduction in
gastrointestinal infection incidence among breast-fed infants at a time when artificial
feeding was becoming increasingly prevalent among the general population. Modern
epidemiologic studies reinforced and expanded upon these findings (136, 139), indicating
that breast-feeding confers remarkably enhanced protection from both gastrointestinal
and respiratory infections, including Salmonella infection (140). Early reports suggest
that this protection endures well beyond the period of breast milk consumption (136), and
breast-feeding in infancy is now associated with reduced lifetime risk of several chronic
diseases including inflammatory bowel disease (137), obesity (141, 142), and allergic
diseases (143, 144). More recently, specific molecular mechanisms of infectious disease
protection among breast-fed infants have been revealed.
In addition to the essential caloric content of milk, breast-feeding supplies a wide array of
bioactive components that enhance both innate and adaptive immunity in the neonatal
gastrointestinal tract. The transition from the sterile uterine environment into ex utero life
results in abrupt and continuous exposure to novel microbial challenges. However, the
adaptive immune response is underdeveloped at birth, requiring months of gradual
education through exposure to environmental, dietary, and microbial antigens to
adequately provide targeted protection from pathogenic organisms while regulating
tolerance to commensal microorganisms and self ligands. Milk components act as a
critical stimulus in the ontology of intestinal immune education and microflora
development (10), supplying passive defense mediators (145, 146), growth hormones
43
(147), prebiotics (148-151), and immunomodulators (133). The best characterized of
these immunity enhancing milk components is soluble IgA. Antigens presented to
dentritic cells residing in the maternal mucosa stimulate B lymphocyte activation via
interaction with circulating antigen-specific T lymphocytes, ultimately resulting in
secretion of soluble IgA by plasma cells residing in the mammary gland (145, 152). Thus,
breast-feeding infants consume high concentrations (as much as 1g/L) of antigen-specific
IgA generated through the interaction of the maternal mucosa with enteric pathogens, and
the interaction of sIgA with cognate pathogenic antigens introduced to the infant gut
reduces the infectious capacity of many such organisms (145, 152). However there are
limitations to the protection provided by sIgA. For example, pIgR(-/-) mice, completely
deficient in secretory immunoglobulins, exhibit resistance to oral Salmonella
typhimurium infection equivalent to wild-type mice (153). Despite the elegant integration
of maternal and infant adaptive immunity through the transfer of sIgA in milk, this
mechanism cannot account for the full range of pathogenic protection conferred by
breast-feeding.
Innate immune mechanisms contribute significant, broad-ranging protection from enteric
disease in breast-feeding infants. Numerous milk-secreted proteins have bactericidal
activity, such as iron-chelating lactoferrin (146, 154) and the peptidoglycan degrading
lysozyme (155). Free-fatty acids, released by the digestion of triglycerides, act as potent
detergents at endogenous concentration, with demonstrated inhibition of viral, bacterial,
and protozoan pathogens (156, 157). Milk also contains an abundant and extraordinarily
diverse array of glycans, a term encompassing oligosaccharides, glycolipids,
glycoproteins, mucins, glycosaminoglycans and other complex carbohydrates, which
44
constitute a major component of human milk immunity (10, 158, 159). The functions of
human milk glycans in shaping innate gastrointestinal defense are multifactorial (158,
159), including prebiotic function (148-151), anti-adhesive antimicrobial activity (53,
160, 161), and intestinal epithelial cell modulation (162-164). Induction of altered gene
expression in intestinal epithelium by human milk oligosaccharides (HMOs) results in
enhanced protection from pathogenic E. coli infection through modulation of epithelial
cell surface glycans (162) and milk lactose induces the expression of antimicrobial
peptide LL-37 in cultured epithelium (165). These observations suggest that direct effects
of human milk glycans on intestinal epithelial cells may contribute significantly to the
protection from gastrointestinal infection associated with breast-feeding.
Among the non-nutrient glycan components of both human and bovine milk are abundant
glycosaminoglycans (GAGs), large linear polysaccharide polymers containing amino
sugars. Classified among the GAGs is hyaluronan (HA), usually found as a high
molecular weight polymer consisting of disaccharides of N-acetylglucosamine and βglucuronic acid. Unlike other GAGs, HA does not typically form covalent bonds with
protein and is not sulfated, nitrosylated, or phosphorylated in vivo (166). A recent study
has evaluated the GAG composition of human and bovine milk using specific enzymatic
digestion, gel electrophoresis, and HPLC, determining that among other GAGs, milk
contains HA (167). Milk GAGs may play a significant role in enhancing intestinal
defense against pathogens, as suggested by inhibition of HIV engagement with host
receptor CD4 by chondroitin sulfate derived from human milk (168). While the function
of HA in milk has not been previously studied, a rapidly accumulating literature has
identified roles for HA in contexts as diverse as fertilization, development, tumor
45
metastasis and angiogenesis, wound repair, and regulation of the inflammatory response
(166).
1.2 GLYCOSAMINOGLYCAN STRUCTURE AND FUNCTION
Among the vast complement of human glycans is a unique class of structures referred to
as glycosaminoglycans (GAGs). Glycosaminoglycans are classically defined as linear
polysaccharides composed of an amino sugar (various N-substituted glucosamines, Nacetyglucosamine, or N-acetylgalactosamine) and galactose or a uronic acid (glucuronic
acid or iduronic acid). Glycosaminoglycans are produced by all mammalian cells in
various configurations and play a particularly essential role in the composition of the
extracellular matrix, determining the physical characteristics of tissues and profoundly
altering the biological function of the cells embedded within. In addition,
glycosaminoglycans organize secretory vesicles, modify protein function, and act as
endogenous cell signaling agents among other functions. In vivo, most
glycosaminoglycans, with the exception of hyaluronan, discussed below, are found
covalently attached to a core protein, making up a structure known as a proteoglycan.
Proteoglycans, a major component of all tissues, exhibit tremendous structural variation.
A large number of core proteins have been identified over the years, and each may be
covalently linked with up to two types of glycosaminoglycan. GAG chains present on
proteoglycans are many times larger than the glycan modifications found on
glycoproteins (e.g., 10-100 fold larger in mass) and the chemical properties of GAG
chains often define the properties of proteolycans. A single protein core may be
substituted with more than 100 GAG chains (e.g., aggrecan) and a population of
46
proteoglycans with a shared protein core may be highly heterogeneous due to varying
GAG substitution stoichiometry and differences in length and sufation patterns among
individual GAG chains. Unlike nucleic acid or protein synthesis, GAG synthesis is not
template-driven, resulting in high heterogeneity. Most GAGs, again with the exception of
the perennial outlier hyaluronan, are synthesized from single sugar substrates in the golgi,
facilitating the covalent attachment to proteins, through the action of a wide variety of
epimerases, deacetylases, sulfotransferases, gluco-, galactosaminyl-, and glucuronosyltransferases, often with redundant or multiple enzymatic activities. This seemingly
chaotic process results in a wide variety of GAGs with unique sugar composition,
sulfation patterns, chain length, and biological functions, broadly defined as keratan
sufate, heparan sulfate/heparin, dermatan sulfate, chondroitin sulfate, and of course
hyaluronan (169).
Keratan sulfate (KS) is defined as a chain of sulfated poly-N-acetyllactosamine,
distinguished from the N-acetyllactosamine found on glycoproteins and mucins by
consisting of multiple repeating structural units. Karatan sulfate structures are divided
into two categories according to their linkage to protein. KS I is linked to asparagine
found in core proteins through an N-glycan core whereas KS II is linked to serine or
threonine via N-acetylgalactosamine. KS polymers up to 25 kD are known and can
contain many combinations of nonsulfated, monosulfated, and disulfated dissacharide
units (169). Of all GAGs, KS has been perhaps the least studied, with its best-defined
function being the maintenance of type I collagen arrangement within the cornea of the
eye to permit the passage of light. Mutations in corneal GlcNAc-6-O-sulfotransferase
47
result in macular corneal dystrophy, presumably through the disordering of corneal
collagen in the absence of proper KS sulfation (170).
Both heparan sulfate and chondroitin sulfate are linked to serine residues found in core
proteins by xylose. Following serine substitution with xylose by a xylosyltransferase
within the golgi, a linkage tetrasaccharide consisting of two galactose residues is
assembled through the action of unique β1-4 galactosyl-, β1-3 galactosyl-, andβ1-3
glucuronosyltransferase enzymes. Linkage of either α1-4 N-acetylglucosamine by
EXTL3 or the addition of β1-4 N-acetlygalactosamine by GalNAc transferase 1 to the
linkage tetrasacchardie commits the growing carbohydrate polymer as either a heparan
sulfate (HS) or chondroitin sulfate (CS) chain, respectively. Both heparin and heparan
sulfate are made up variously sulfated and epimerized dissacharide chains of α1-4 Nacetylglucosamine and β1-4 Glucuronic acid or iduronic acid. Unlike chondroitin
sulfation, heparan sulfate modifications occur in clusters along the length of the growing
HS polymer, referred to as N-acetylated, N-sulfated, or mixed domains. Heparin is
generally more highly sulfated that heparan sulfate and is synthesized solely in mast cells
as the GAG component of the proteoglycan serglycin, whereas a variety of heparan
sulfate proteoglycans have been identified and are synthesized by virtually all cell types
(169). Heparin is probably the most widely recognized of all the glycosaminoglycans due
to its ubiquitous clinical use as an anticoagulant. Heparin binds antithrombin with high
affinity, resulting in its activation through conformational change and the subsequent
inactivation of the clotting factor thrombin and other proteases involved in the clotting
cascade (171). Heparin is isolated for clinical use from porcine and bovine entrails
extracted in industrial slaughterhouses (172), however the role of heparin produced as
48
serglycin by mast cells in vivo is subject to debate. Heparan sulfate proteoglycans are an
important component of the plasma membrane of all mammalian cells as well as a major
component of the extracellular matrix (173). Hydrophobic residues within the protein
core embed HS proteoglycans in the plasma membrane, and proteolytic cleavage can
result in release of HS proteoglycans with a wide variety of physiologic effects.
Alternately, endocytosis of membrane containing HS proteoglycans results in their
degradation in lysosomes and the generation of specific heparan sulfate oligosaccharides
(174). Heparan sulfate proteoglycans have been demonstrated to function as regulators of
cell adhesion, cytokine and growth factor signaling, and secretory vesicle composition
and make up an important component of the basement membranes that organize epithelial
surfaces (173).
Chondroitin sulfate (CS) is composed of repeating dissacharides of β4 Nacetylgalactosamine and β3 glucuronic acid, with a related structure substituting α3
iduronic acid in place of glucuronic acid known as dermatan sulfate (DS). Nacetylgalactosamine residues of CS are subject to 4-O and 6-O sulfation, whereas
iduronic acid may be sulfated at the C-2 position. Polymerization of CS in the golgi is
catalyzed by multiple chondroitin synthases, which have both glucuronosyltransferase
and N-acetygalactosaminyltransferase activity, and multiple sulfotransferases regulate the
degree of sulfate group substitution (169). Chondroitin is also found in a nonsulfated
form, particularly in invertebrates such as C. elegans but also in mammals, and when
dissociated from a protein core nonsulfated chondroitin is nearly identical to hyaluronan
in chemical structure with the exception of the stereochemistry of the hydroxyl group
linked to C-4 of N-acetyl-galactosamine (175). Chondroitin sulfates make up the GAG
49
component of a wide variety of proteoglycans, including aggrecan, versican, decorin and
biglycan (176), and modify membrane bound receptors including CD44 (177).
Chondroitin sulfate is best appreciated as an important structural component of cartilage,
capable of generating tremendous resistance to compressive forces in the joints.
Chondroitin sulfates may play a protective compensatory role in the pathophysiology of
bone and joint disease (178) due to anti-inflammatory and immunomodulatory activity,
which may apply to other physiological circumstances such as inflammatory bowel
disease (179). As the name may suggest, dermatan sulfate is the most abundant
glycosaminoglycan present in the skin. Though understudied in comparison to heparan
sulfate and chondroitin sulfate, a growing literature implicates dermatan sulfate in diverse
processes including wound repair, fibrosis, cardiovascular disease, and tumorigenesis
(180).
1.2.1 Biochemistry of Hyaluronan
The rapid advancements in organic chemistry that followed Frederick Wöhler’s
serendipitous synthesis of urea in 1824 (181) facilitated the isolation and description of
sulfated glycosaminoglycans in the late nineteenth century. However, it was not until
1934 that Karl Meyer and John Palmer isolated from the bovine vitreous humor a
“polysaccharide acid of high molecular weight”, subsequently named hyaluronic acid,
from the Greek “hualos” or glass, referring to the vitreous nature of the substance, and
uronic acid (182). While at the time Drs. Meyer and Palmer speculated that the structure
might also include a pentose, we now know that this substance, referred to as hyaluronan
in more recent work, is composed solely of repeating disaccharide units of β4 N-
50
acetylglucosamine and β3 glucuronic acid. Despite its exceedingly simple structure,
hyaluronan (HA) is a recent evolutionary development coinciding with the emergence of
the notocord in the phylum Vertebrata (175). Interestingly, HA appears to occur
independently in at least one member of phylum Mollusca, the marine bivalve Mytilus
galloprovincialis. The physiologic function of HA in this organism has not been studied
(183). Pathogenic bacteria of the genera Streptococus and Pasteurella also express
hyaluronan as a component of the extracellular capsule, a function that has been proposed to
act as a form of molecular mimicry in the human host facilitating immune evasion or
modulation (184).
Regardless of the source, hyaluronan is not modified by sulfation, nitroslyation, or
phosphorylation nor is its glucuronic acid epimerized to form iduronic acid. Unlike all other
GAGs, hyaluronan is not synthesized attached to a core protein; rather numerous
hyaluronan binding proteins have emerged which interact through a variety of non-covalent
binding mechanisms. A potentially limitless degree of polymerization is possible for
hyaluronan, with 106 – 107 Da polymers typical in many tissues, individually reaching endto-end lengths of up to 10 µm. At physiologic pH, hyaluronan is polyanionic due to the
influence exerted by the carboxyl groups of glucuronic acid (pKa~4.5) and this confers a
relatively inflexible random coil structure in many typical biologic settings. Hyaluronan is
highly hygroscopic, occupying large hydrodynamic volumes with high viscosity. Perhaps
because of these unique properties, hyaluronan has been an incredibly useful innovation, to
take the teleologic point of view, with essential roles in an ever-expanding array of diverse
biological processes (169), all of which begin with synthesis.
1.2.2 Hyaluronan Synthesis
Virtually all mammalian cells are capable of hyaluronan synthesis. Unlike the synthesis of
all other GAGs, hyaluronan synthesis does not occur in the golgi. Instead, hyaluronan is
51
synthesized by hyaluronan synthases (HAS) embedded in the plasma membrane, freeing
polymer size from the limitations of intracellular golgi and permitting extrusion of very high
molecular weight disaccharide polymers directly into the extracellular matrix. The three
mammalian HAS enzymes, Has 1-3, are composed of paralogous protein sequences
containing 5-6 membrane spanning hydrophobic domains and a central cytoplasmic dual
catalytic domain (185). Membrane-embedded HAS remains catalytically inactive following
protein synthesis in the endoplasmic reticulum and during transport in the golgi (186), with
HA synthesis only proceeding from HAS positioned in the outer plasma membrane. Has 13 catalyze both reactions required for the polymerization of hyaluronan from the
corresponding nucleotide sugars through a two-site mechanism. The reducing end of a
growing HA chain, either N-acetylglucosamine or β-glucuronic acid, is tethered to a terminal
carrier nucleotide, uridine diphosphate (UDP). The incoming UDP-sugar substrate
displaces the terminal UDP from the HA chain, releasing free UDP, catalyzing the
attachment of the UDP-bound substrate, and rotating the conformation of HAS to expose
an alternate catalytic site. Conformational cycling of HAS at the catalytic site alternately
exposes either the N-acetylglucosamine or β-glucuronic acid glycosyltransferase activity,
thus maintaining consistent disaccharide composition with high efficiency and fidelity
(187). The synthesis of hyaluronan from UDP-sugar substrates intimately links hyaluronan
to both sugar and nucleotide metabolism, with potential implications for hyaluronan
synthesis in the pathology of metabolic disease (188). Regulation of hyaluronan synthesis
has been primarily investigated at the level of enzyme expression, with a variety of stimuli,
including inflammatory cytokines (189, 190), viral stimulus (191, 192), growth factors (193195), prostaglandins (196), and hormonal signals (197) associated with altered Has gene
transcription. Hyaluronan synthesis also appears to be regulated at the posttranslational
level, through modification of HAS proteins by ubiquitination (198) or glycosylation (199).
In addition, UDP-sugar substrate limitation differently affects the activity of HAS 1-3 (200),
suggesting that the three mammalian Has genes serve specialized roles in regulating HA
52
synthesis in the context of various metabolic conditions. HA synthesis is essential to the
formation and repair of all mammalian tissues. In particular, Has2 plays an essential role in
embryonic development, with Has2-/- embryos exhibiting severe, lethal cardiac abnormalities
at mid-gestation (201). Has1- and Has3-deficient animals are phenotypically normal, even
when both genes are absent simultaneously, but appear to display differences in comparison
to wild-type animals when challenged by skin wounding (202). Assuming that the highly
conserved duplication of the Has gene family has resulted in the adaptation of each
hyaluronan synthase to unique functions, future studies of Has null animals will likely
identify additional specific roles for the mammalian hyaluronan synthases in various
pathologies or tissue-specific contexts. Hyaluronan is synthesized as a predominantly high
molecular weight polymer in vivo. It is another process entirely that generates the array of
polymer sizes which determine the biological function of hyaluronan.
1.2.3 Hyaluronan Catalysis
Of the estimated 15 g of hyaluronan present in an adult human, the combined action of
degradation and synthesis result in turnover of about 5 g HA each day. The rate at which
HA turnover proceeds varies by tissue, with HA in serum degraded within a few minutes
while the half-life of HA present in cartilage may be as long as several weeks (203).
Hyaluronan turnover occurs in both enzymatic and enzyme-independent fashion. Of the
known enzyme-independent means of HA degradation, both alkaline hydrolysis and
ultrasonic degradation can be excluded as inapplicable under physiologic conditions. HA
is stable at pH < 2, implying that acidic hydrolysis is limited even in specialized contexts,
such as the stomach lumen, or the lysosome in the absence of lysosomal hyaluronidases.
Thermal degradation of HA does not occur in vivo, with HA polymerization entirely
stable at temperatures up to 90°C and possibly greater (204). However, the generation of
53
reactive oxygen species (ROS), particularly prevalent during inflammation (205), can
result in fragmentation of high molecular weight hyaluronan. Generation of oxygen free
radicals is an important component of the phagocytic activity of macrophages in infection
or sterile injury, and inflammatory conditions can result in the degradation of hyaluronan
by leukocyte myeloperoxidase (206). The relative importance of non-enzymatic cleavage
of hyaluronan in generating inflammatory or other biological responses has been difficult
to ascertain due to the prevalence of enzymes with hyaluronidase activity in nearly all
tissues. This includes two lysosomal enzymes with non-specific hyaluronidase activity,
β-exoglucuronidase and β-N-acetylglucosaminidase, which generate tetra-and
hexasaccharides of hyaluronan (204). The human genome encodes six sequences with
hyaluronidase (endo-β-n-acetylhexosaminidase) homology. This includes Hyal-1, Hyal2, Hyal-3, Hyal-4, PH-20 and Phyal1. Phyal1 is a pseudogene, a kind of evolutionary
accident producing a transcript but not transcribed into protein. If this sequence has any
function, it has not been evaluated. Hyal-3 and Hyal-4 are understudied and do not appear
to account for a significant proportion of hyaluronidase activity in human tissues, but
may act as functional hyaluronidases under specialized circumstances (204). PH-20 is
only expressed by spermatocytes, but plays a critical role in fertilization by facilitating
penetration of the hyaluronan-rich cumulus mass (207). The enzymes Hyal-1 and Hyal-2
account for the majority of specific hyaluronidase activity in human somatic tissues
(166). Hyal-1 acts on high molecular weight HA, yielding numerous individual HA
tetrasaccharides (208) while Hyal-2 specifically degrades large HA polymers into
fragments of approximately 20 kDa (209). High molecular weight HA polymers interact
with cell surface receptors such as CD44, resulting in endocytosis and transport to the
54
lysosome containing Hyal-1 and, to a lesser extent, β-exoglucuronidase and β-Nacetylglucosaminidase (210). This process may be facilitated by Hyal-2, tethered to the
plasma membrane by a GPI anchor and acting in coordination with CD44 to bind
extracellular hyaluronan (210), generating hyaluronan polymers no smaller than 20 kDa
(209). Some degree of hydrolysis of hyaluronan is required to permit internalization of
high molecular weight polymers that would otherwise require impossibly large endocytic
vesicles and would dramatically alter lysosomal chemistry through hygroscopic
interactions. Complete absence of HA turnover is generally considered incompatible with
life. However, at least one patient with deficient serum hyaluronidase activity resulting
from a pair of mutations to the Hyal1 gene has been identified. This patient exhibited
relatively mild lysosomal storage disorder, subsequently deemed mucopolysaccharidosis
IX, with noticeable soft tissue masses and short stature and elevated serum HA, but no
neurological or visceral involvement (211). Thus, the process of endocytic HA turnover
may be more redundant than is currently appreciated, involving multiple hyaluronidases
or more involvement by non-specific hyaluronidases. Hyal-2 deficiency was initially
considered embryonic lethal (169, 212) but is now known to result in severe cardiac
hypertrophy and fibrosis coinciding with the accumulation of extracellular HA in outbred mice capable of surviving gestation (213). Conditional Hyal2-deficiency results in
the onset of physiologic symptoms resembling mucopolysaccharidosis IX including the
elevation of serum HA, indicating a function for Hyal2 in regulating serum HA
concentration in vivo (214). Hyaluronan expression is up-regulated in the microvascular
endothelium of inflamed tissues (191,192, 215). High molecular weight hyaluronan in the
endothelial lumen is converted to low molecular weight HA fragments by Hyal2-
55
expressing platelets circulating in blood, and platelet Hyal2 may serve an important role
in regulating localized or systemic HA polymer size and concentration within the blood
(216). Thus, in addition to facilitating the regular turnover of hyaluronan in the
extracellular matrix, Hyal1 and Hyal2 likely participate in the generation of fragmented
HA with signaling properties that are distinct from high molecular weight HA. The
shifting balance of HA synthesis and catalysis results in an “information-rich” spectrum
of HA polymer fragments (166).
1.2.4 Hyaluronan in Inflammation
Interpretation of the spectrum of HA polymers is mediated by hyaluronan-binding
proteins and HA receptors, including CD44 (217). CD44 is a cell surface glycoprotein
containing a cytoplsamic signaling domain, a hydrophobic transmembrane domain, and
an HA-binding extracellular domain (169). Binding to HA is conferred by a Link protein
motif expressed in the extracellular domain, and the strength of the non-covalent
interaction with HA increases as a function of HA size with HA polymers < 20
disaccharides binding reversibly while larger HA polymers are bound more tightly (218,
219). The CD44 cytoplasmic tail interacts with numerous regulatory molecules including
RHO GTPases, SRC kinases, phosphoinositide-3-kinase (PI3K), cytoskeleton
components, and cell survival pathways in response to HA binding to the extracellular
Link motif (169). This highly enigmatic protein is expressed by most mammalian cell
types in at least 8 isomer forms, encoded by alternate splicing of the Cd44 gene regulated
through a poorly understood mechanism (220-222). Early work by Noble et al. (223, 224)
provided the first indication that HA polymers of intermediate molecular weight (<105
56
Da) acted as immunomodulatory signals, activating NF-κB translocation to promote IL1β and TNF-α expression in macrophages through the HA cell surface receptor CD44.
Evidence has grown in support of the subsequent hypothesis that fragmented HA
polymers generated in damaged or inflammed tissue act as endogenous “danger signals”,
or DAMPs (66, 225-227) triggering localized innate defense responses. Many of the
effects of fragmented HA appear to be regulated via Toll –like receptors (TLR) 2 and 4
(66, 228, 229), with some cell types, including macrophages, exhibiting both CD44dependent (225, 228) and independent (66, 227) immunomodulatory responses to
fragmented HA. Endogenous fragmented HA may be recognized in much the same way
as the conserved pathogen-associate molecular patterns (PAMPs), such as LPS and
peptidoglycan, typically characterized as TLR ligands (66, 227). Low molecular weight
HA, injected intravenously or generated through sterile injury, acts through a TLR4dependent mechanism to promote expression of the chemokines IL-8 and MIP-2 in
endothelium and skin biopsies in both tissue culture and animal models (227, 230). Renal
tubular epithelial cells release the chemokine MCP-1 in response to fragmented HA
binding of CD44 (225). Nf-κB translocation and subsequent upregulation of TNF-α, IL1β, and IL-6 occurs in chondrocytes in a mechanism that is both TLR4- and CD44dependent, suggesting that fragmented HA generated during joint damage also acts to
enhance inflammation (231). HA may also signal through the TLR2 receptor. Selective
knockdown of MyD88 or TLR2 reverses the increased expression of inflammatory
mediators in vitro following treatment with HA polymers under 200 kDa and the T-cells
of wild type, but not TLR2null mice, are sensitized to Ag-specific activation by low
molecular weight HA (66). Furthermore, 6,000 kDa HA polymers suppress the in vitro
57
stimulatory activity of low molecular weight HA or non-HA TLR2 ligands when supplied
in combination (66).
HA deposition is increased in inflammed tissues including the lung (232), skin (227),
liver (233), and intestine (191, 192). In the intestine, HA cables are synthesized by
microvascular endothelial cells (191, 192), mesenchymal cells (190), and mucosal
smooth muscle cells in response to endoplasmic reticulm stress (234), viral infection
(215) and cytokine signals including TNF-α and IL-1β (235). HA produced by
microvascular endothelial cells extrudes into the lumen of the vessel and participates in
leukocyte recruitment (192). Leukocytes in the lumen of the blood vessel bind extruded
HA cables in a CD44 dependent manner, and HA binding facilitates leukocyte rolling and
extravasation (191, 215). The intestinal microvascular endothelial cells of IBD patients
have increased leukocyte binding capacity in comparison to healthy controls (236) and
HA deposition is increased in the mesenteric vasculature of patients with inflammatory
bowel disease (192). Time course experiments using the murine DSS model of
experimental colitis confirm that HA deposition precedes leukocyte infiltration from the
vasculature (192). Hyaluronan production by intestinal microvascular endothelial cells is
associated with an increase in Has3 mRNA transcription. Additionally, human intestinal
microvascular endothelial cells (HIMEC) isolated from IBD patients synthesize more HA
in response to TNF-α stimulation than HIMEC from control patients (192). Collectively,
the available data suggest that HA synthesis is increased in the intestinal microvascular
endothelium of IBD patients as a result of proinflammatory cytokine signaling, as well as
other stimuli, and increased HA deposition promotes recruitment of leukocytes from the
vasculature. Continued leukocyte extravasation may only exacerbate rampant
58
proinflammatory signals, furthering tissue damage and dysfunction in the setting of
inflammatory bowel disease.
1.2.5 Hyaluronan in Intestinal Defense
HA fragments also play a role in enhancing innate epithelial defense that appears to be
independent of the pro-inflammatory immunomodulation characteristic of macrophage
(224), chondrocyte (231), or endothelial activation (227, 237) by LMW HA, or the
stimulation of TLR2/4 by bacterial PAMPs (238). A polydispersed HA fragment
preparation of polymers less than 750 kDa protects wild-type mice, but not TLR4deficient animals, from a microflora mediated epithelial damage model of colitis (239), or
from the epithelium-depleting effects of radiation (240), through a mechanism that is
mediated in part by increased COX-2 expression (239, 240). Elevated expression of
antimicrobial defensin proteins may also contribute to enhanced epithelial barrier defense
following exposure to LMW HA (241, 242).
59
CHAPTER 2: SPECIFIC-SIZED HYALURONAN FRAGMENTS PROMOTE
EXPRESSION OF HUMAN β-DEFENSIN 2 IN INTESTINAL EPITHELIUM
2.1 INTRODUCTION
The mammalian gastrointestinal tract processes and absorbs the nutrients, water, and
electrolytes required to sustain every cell of the body. In addition, the intestine has the
added challenge of: 1) supporting a large and diverse beneficial bacterial population, the
intestinal ‘microbiome’; while 2) excluding potentially harmful microbes; and 3)
allowing education of the systemic immune system so that appropriate responses to
pathogenic organisms, commensal bacteria, and dietary antigens are developed. A
continuous single-cell layer of epithelial cells serves as the interface between host and an
intestinal environment containing high concentrations of microbes and ingested foreign
substances. Therefore, integrity of this barrier is of paramount importance to the
continued function of the digestive and immune systems, and thus to the organism as a
whole. Numerous processes, both innate and adaptive, contribute to the continuous
maintenance and renewal of the intestinal epithelial barrier (33). Among these, the
production of small cationic antimicrobial proteins, the defensins, has an essential role in
the preservation of epithelial integrity against persistent microbial challenges.
Hundreds of unique defensins are found throughout the animal kingdom in species as
evolutionarily distinct from man as the horseshoe crab (94), and at least twelve human
defensins have been characterized in detail (95). A common characteristic of human
defensins is a structure that contains six cysteine residues that form three disulfide bonds,
60
which facilitate folding of the peptide chains into the α-helix or β-sheets that define the
protein as an α- or β-defensin (243). Defensins have direct antimicrobial activity against a
broad range of human pathogens including gram-negative and gram-positive bacteria,
fungi, virus, and protozoa (93) and are expressed by gastrointestinal, urogenital, and
pulmonary mucus membranes as well as skin (244) and ocular surfaces (245). Thus, βdefensins contribute to a potent innate defense arsenal against potentially invasive
commensal bacteria in organs where the epithelium directly encounters the environment,
such as in the mammalian gastrointestinal tract (97).
While β-defensin 1 is constitutively expressed in intestinal epithelial cells (98,99), βdefensins 2, 3 and 4 are inducible by bacterial stimuli (98, 100-102), cytokine signals (32,
98, 103), and dietary components (104-105). HβD2 has strong antimicrobial effects
against several common opportunistic pathogens, including E. coli (107,108), P.
aeruginosa (107,109), and C. albicans (109,110). In the intestine, pathogen-associated
molecular patterns (PAMPs) act as critical local activators of HβD2 expression through
specific interactions with Toll-like receptors (TLRs) and other recognition molecules
expressed by epithelial cells (32). The consequence of the interactions between an array
of unique PAMPs expressed by a given microbe and specific cell surface receptors on
epithelium is the expression of an innate response finely tuned to a distinct microbial
challenge. Increased transcription of the gene encoding the HβD2 peptide, DEFB4, has
been reported in some intestinal epithelial cell lines following treatment with
lipopolysaccharide (LPS) derived from cell membrane of Gram-negative bacteria and by
peptidoglycan, a component of the cell wall of Gram-positive bacteria, through TLR4-
61
and TLR2-dependent mechanisms, respectively (102). Salmonella enteritidis flagellin
also promotes HβD2 expression (101) via TLR5 (111). Additional PAMPs have been
shown to upregulate HβD2 expression in respiratory epithelial cells including dsRNA,
which binds via TLR3 (31), and CpG (unmethylated C-G dinucleotides more common in
bacteria and viral DNA) signaling through TLR9 (112). Fragments of peptidoglycan,
muramyl dipeptide, found in the cell wall of intracellular microbes induce NOD2dependent HβD2 upregulation in HEK293 cells (113), enhancing host defenses against
invasive microorganisms.
Hyaluronan (HA) is a glycosaminoglycan polymer found in the extracellular matrix of
virtually all tissues of the body, and is composed of repeating disaccharides of βglucuronic acid and N-acetylglucosamine covalently bound end-to-end into a simple
linear glycosaminoglycan. HA is most commonly found in vivo as high molecular weight
polymers (up to 107 Da), but these large polymers can be broken down into fragments by
a number of enzymatic and non-enzymatic processes (204). A growing body of evidence
implicates HA as an “information-rich” molecule with diverse molecular weightdependent signaling functions reported in angiogenesis, inflammation, and tumorigenesis
among other fields of research (166). Low molecular weight HA is increasingly being
characterized as an endogenous danger signal that promotes the expression of immune
mediators. Noble et al. (223) were the first to report induction of IL-1β and TNF-α in
macrophages following stimulation with HA of indeterminate molecular weight below
105 Da. The same group demonstrated dependence of the response upon the cell surface
HA-receptor CD44 (223) and later implicated NF-κB in the HA-mediated inflammatory
62
response (224). Termeer et al. (246) demonstrated that HA oligosaccharides of 1.5-2
kDa, but not HA polymers greater than 80 kDa, promote maturation and cytokine release
in monocyte-derived dendritic cells (246) via Toll-like receptor 4 (TLR4) (68). Similarly,
HA oligosaccharides have been shown to promote TLR4-dependent immune activation of
endothelilal cells, both in vitro and in vivo (247).
Fragmented HA may also have a role in epithelial defense that is potentially independent
of the pro-inflammatory cytokine release observed in other tissues and cell types
following exposure to HA fragments. Intraperitoneal injection of a polydispersed HA
preparation less than 750 kDa suppresses the epithelial-damaging, pro-inflammatory
effects of DSS-induced murine colitis through a TLR4-dependent mechanism (239).
Additionally, intermediate molecular weight HA (<200 kDa), but not high molecular
weight HA (>1,000 kDa) promotes the expression of HβD2 peptide in human
keratinocytes partially through interaction with TLR4 without the accompanying
inflammatory cytokine response elicited by LPS (241). Thus endogenous HA may act as
a signal to increase innate epithelial defense without provocation of the potentially
damaging pro-inflammatory mechanisms of immune cells.
In this study, we demonstrate highly size-specific HA-dependent induction of the
antimicrobial protein HβD2 in intestinal epithelial cells grown in culture, as well as in the
colonic mucosa of HA fragment treated mice. The most potent HA preparation evaluated
is 35 kDa in average molecular weight, while a panel of both larger and smaller HA
fragment preparations have little apparent activity. Importantly, this process also occurs
63
in vivo, as oral administration of HA fragments to mice induces increased expression of
the murine ortholog of HβD2 in a size-specific manner in the colonic epithelium through
a mechanism that is TLR4-dependent.
2.2 EXPERIMENTAL PROCEDURES
2.2.1 Cell Culture
HT29 epithelial cells, a line initially derived from a human intestinal tumor, were
cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and
incubated at 37° C in a humidified environment containing 5% CO2. Stock cultures were
split at a ratio of 1:20 once per week.
2.2.2 Experimental Cultures
HT29 cells were released from stock cultures by dissociation with solution containing
0.05% trypsin and 0.53 mM EDTA in phosphate buffered saline for 1.5 m. Collected
cells were washed and plated at a 1:15 area:area ratio in 12-well plates (Becton
Dickinson, Franklin Lakes, NJ) and grown until 70-80% confluent (3 days). On the day
of the experiment, growth medium was removed, and the HT29 cells were treated with
fresh RPMI containing 10% FBS without or with specific molecular weight range HA
preparations at the concentrations (0- 100 µM) and times (0-24 h) specified in the figure
legends. Purified, lyophilized HA was purchased from Lifecore Biomedical, LLC,
Chaska, MN. The HA size designations were made on the basis of average molecular
weight: 4.7 kDa (HA-4.7), 16 kDa (HA-16), 28.6 kDa (HA-28), 35 kDa (HA-35), 74 kDa
(HA-74), and 2000 kDa (HA-2M) (see Figure 2-3 for full size spectrum) and suspended
64
in sterile H2O for concentrated stock solutions (10 mg/ml) prior to dilution in medium for
cell treatment.
2.2.3 HA Fragment Sizing
Highly purified HA fragment preparations (Lifecore Biomedical, LLC, Chaska, MN)
were electrophoretically separated on a 4-20% gradient polyacrylamide gel (HA-4.7, HA16), 5% agarose gel in TBE buffer (HA35, HA-74), or 0.5% agarose in TAE buffer (HA2M) along with HA standards of known molecular weight (Hyalose L.L.C., Oklahoma
City, OK) to determine the size range of each preparation. Polyacrylamide or agarose
gels were stained directly with Stains-All (Sigma-Aldrich Inc., St. Louis, MO) and
optical densitometry used to quantify the polydispersion of Lifecore HA fragments in
comparison to HA polymer standards (248).
2.2.4 Detection of HβD2 by Immunoblot (Western) Analysis
Whole cell lysates from HT29 cells were isolated for Western blotting in the following
lysis buffer: 300 mM NaCl, 50 mM Tris pH 8.0, 0.5% NP-40, 1 mM EDTA, 10%
glycerol, protease inhibitor for mammalian tissue P8340 (Sigma-Aldrich Inc., St. Louis,
MO). Cell lysate proteins were separated by SDS-PAGE using pre-cast Tricine based 420% gradient gels (Invitrogen, Carlsbad, CA). Separated proteins were transferred at 4° C
to PVDF membrane with an electroblotting apparatus (Bio-Rad Laboratories, Hercules,
CA) at 100 V for 45 m. In cases where analysis of numerous replicate samples or
multiple protein targets of differing molecular weight was required, the multistrip
Western blotting protocol developed by Aksamitiene et al. (249) was used to increase
65
quantitative output and improve signal consistency. PVDF membranes were air-dried at
room temperature for 60 m prior to blocking with Odyssey Blocking Buffer (LI-COR
Biosciences, Lincoln, NE) diluted to 50% concentration in Tris-buffered saline. The
membrane was incubated with rabbit polyclonal antibody against HβD2 at 1:750 (Abcam,
Cambridge, MA), and the primary antibody was followed by biotin conjugated anti-rabbit
IgG at 1:25,000 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA), and
finally horseradish peroxidase (HRP)-conjugated streptavidin was added at 1:100,000
(GE Healthcare, Piscataway, NJ). Membrane-bound GAPDH protein expression was
detected by incubation with rabbit polyclonal antibody against GAPDH at 1:5,000
(Abcam, Cambridge, MA) followed by HRP-conjugated donkey polyclonal anti-rabbit
IgG (1:20,000, GE Healthcare, Piscataway, NJ). All washing steps were conducted in
Tris-buffered saline with 0.1% Tween-20. Protein bands were visualized using ECL plus
chemiluminescent development (GE Healthcare, Piscataway, NJ) and detection by
BioMax XAR scientific imaging film (Carestream Health Inc., Rochester, NY).
Differences in chemiluminescent signal intensity were quantified using the NCBI ImageJ
software package (250).
2.2.5 Detection of HβD2 by Fluorescence Histochemistry
Descriptions of fluorescence histochemistry and confocal microscopy were provided
previously (191). Briefly, HT-29 cells, grown on cover slips were treated as described in
the figure legends, fixed in -20°C methanol for 5-10 m, and air dried. For staining, the
dry cover slips were incubated in a blocking solution of Hank’s Balanced Salt Solution
(HBSS) containing 2% FBS for >30 m. The coverslips were then transferred to a solution
66
containing rabbit polyclonal antibody against HβD2 (Abcam, Cambridge, MA) at 1:500
dilution in HBSS with 2% FBS overnight at 4°C. Coverslips were washed three times
with HBSS before incubation in a solution containing Alexa-568-tagged goat anti-rabbit
IgG (1:1000) (Invitrogen, Carlsbad, CA) in HBSS with 2% FBS for 1 h at 25° C. The
coverslips were washed an additional three times in HBSS, inverted and attached to glass
slides with Vectashield mounting medium (Vector Labs, Inc., Burlingame, CA)
containing 4',6-diamidino-2-phenylindole (DAPI), which fluorescently labels DNA. For
tissue section staining, fixed, paraffin embedded mouse colon sections were
deparafinized, and processed similarly to the cultured cells. Sections were incubated with
blocking buffer (HBSS with 2% FBS) for 30 m followed by overnight incubation at 4° C
in a solution of rabbit polyclonal antibody against MuβD3 (Santa Cruz Biotechnology
Inc., Santa Cruz, CA) at 1:100 dilution in HBSS with 2% FBS. Slides were washed three
times in HBSS and incubated in a solution containing Alexa-568-tagged goat anti-rabbit
IgG (1:1000) in HBSS with 2% FBS for 1 h at 25° C. After washing, Vectashield with
DAPI was used to adhere coverslips to antibody-labeled colon sections. Slides were
stored at -20° C until imaged. Confocal images were obtained using a Leica TCS-SP laser
scanning confocal microscope (Leica, Heidelberg, Germany).
2.2.6 Detection of HβD2 by Enzyme-Linked Immunosorbent Assay (ELISA)
HT-29 cells were cultured as described above and replicate wells treated with medium
alone or containing HA-35 (10 µM) for 9 h. Culture fluid was then harvested and stored
at -80° C. To remove high molecular weight media components and improve the
specificity of ELISA detection of the 7 kDa HβD2 protein, thawed media samples were
67
transferred to 10 kDa-cutoff Amicon Ultra-2 Centrifugal Filter devices (Millipore Corp.,
Billerica, MA) and centrifuged in a swinging bucket rotor at 4,000 x g for 45 m at 4° C.
HβD2 contents of filtered media samples were then quantified by ELISA kit according to
the manufacturer’s protocol (PeproTech, Rocky Hill, NJ).
2.2.7 Specificity of HA-35 Induction of HβD2 in HT29 cells
Two experimental strategies were employed to determine whether HA was the active
component of the HA-35 preparation for inducing HβD2 protein expression.
First: Hylauronidase digestion of HA-35 - The HA-35 fragment preparation was
specifically degraded to disaccharides by incubating 1.4 mM HA-35 fragment solution
with 0.025 U/ml Streptococcus dysgalactiae hyaluronidase (Seikagaku Corp., Japan) for
16 h at 37° C in PBS, and subsequently heat-inactivated, prior to using as a cell
treatment.
Second: Comparison of HA-35 and endotoxin effects on HβD2 expression - To
determine whether the increased production of HβD2 by HT29 cells could be due to
bacterial endotoxin contamination, the effects of HA-35 preparation were compared to
high levels of LPS (1 µg/ml) (Sigma-Aldrich Inc., St. Louis, MO) treatment in the in
vitro assays. The LPS preparation positive control activity was determined by its ability
to activate T-cells. HT-29 cells were treated with medium alone, medium containing
HA-35 (10 µM), or medium containing LPS (1 µg/ml,) for 9 h and harvested and
analyzed as described by described methods above.
68
2.2.8 In Vivo Induction of Murine HβD2 Ortholog by HA-35
Wild-type adult C57BL/6 mice were provided with conventional drinking water (5 mice),
or drinking water supplemented with HA-35 at a final concentration of 1.0 µM (5 mice).
Water bottles were changed each day, and the animals kept on this regimen for 7 days.
Mice were sacrificed on day 7, and 0.5 cm sections of the distal colon were harvested and
fixed in Histochoice (AMRESCO, Inc., Solon, OH) and paraffin embedded.
Representative cross-sections of the mouse distal colon were deparaffinized and stained
for MuβD3, the murine ortholog of HβD2, as described in histological methods.
2.2.9 Evaluation of Size-Specific Induction of Murine HβD2 Ortholog In Vivo
Male wild-type adult C57BL/6 mice were purchased from Jackson Laboratory (Bar
Harbor, ME) and housed by conventional methods. All treatments were conducted
according to Institutional Animal Care and Use Committee (IACUC)-approved protocols.
A total of 18 mice were divided equally among 6 treatment groups: Control, HA-4.7,
HA-16, HA-35, HA74, or HA-2M. All animals were gavage-fed 0.25 ml water (Control)
or 300 µg HA of the fragment preparation corresponding to the treatment group
designation (e.g. HA-4.7 animals received 300 µg of 4.7 kDa HA) suspended in 0.25 ml
water once daily for three consecutive days. Mice were sacrificed 16-18 h after the final
gavage treatment, and a 0.5 cm cross-section of the proximal colon descending from the
ileocecal junction was excised from each mouse, fixed in Histochoice (AMRESCO, Inc.,
Solon, OH), and paraffin embedded. These cross-sections of mouse proximal colon were
deparaffinized and stained for MuβD3, the murine ortholog of HβD2, as described in
69
histological methods. Replicate staining was quantified in a blinded manner by a
scientific panel according to the method described below.
2.2.10 Detection of TLR4 by Fluorescence Histochemistry
Fixed, paraffin embedded mouse proximal colon sections were deparafinized and
sections were incubated with blocking buffer (HBSS with 2% FBS) for 30 m followed by
overnight incubation at 4 °C in a solution of rabbit polyclonal antibody against murine
TLR4 (Abcam, Cambridge, MA) at 1:50 dilution in HBSS with 2% FBS. Slides were
washed three times in HBSS and incubated in a solution containing biotin conjugated
antirabbit IgG suspended in HBSS with 2% FBS at 1:1,000 (Jackson ImmunoResearch
Laboratories Inc., West Grove, PA) for 1 h at 25 °C. Slides were subsequently washed
three times in HBSS before incubation in a solution containing Alexa-488-tagged
streptavidin (Invitrogen, Carlsbad, CA) at 1:1,000 for 1 h at 25 °C. After final washing,
Vectashield with DAPI was used to adhere coverslips to antibody-labeled and nonspecificly stained control colon sections. Slides were stored at -20 °C until imaged.
2.2.11 Evaluating the Role of TLR4 and CD44 in HβD2 Expression In Vivo
Male C57/Bl6 (“wild-type”), B6.129(Cg)-CD44tm1Hbg/J (CD44-/-), and B6.B10ScNTlr4lps-del/JthJ (TLR4-/-) mice were purchased from Jackson Laboratory (Bar Harbor,
ME) and housed by conventional methods. All treatments were conducted according to
Institutional Animal Care and Use Committee (IACUC)-approved protocols. Five mice
of each of the three genotypes were fed once per day with tap water alone (250 µl) or a
solution containing HA fragments in tap water (300 µg in 250 µl) with an average
70
molecular weight of 28.6 kDa (HA-28, Lifecore Biomedical, LLC, Chaska, MN) or an
equivalent volume of tap water alone by oral gavage once daily for three consecutive
days. HA-28 was substituted for the HA-35 used in previous studies due to its nearly
identical molecular weight distribution and its availability in sufficient quantity for
practical use in animal studies. Mice were sacrificed 16-18 h after the final gavage
treatment, and a 2 cm section of the proximal colon descending from the ileocecal
junction was excised from each mouse, opened, pressed flat between two layers of paper
towel (251), fixed overnight in Histochoice (AMRESCO, Inc., Solon, OH), and paraffin
embedded. Representative longitudinal sections of the mouse proximal colon were
deparaffinized and stained for MuβD3 as described in histological methods.
2.2.12 Quantification of Histological Observations
Individual sections were labeled in a random fashion to shield the microscopists from
knowledge of mouse genotype or HA treatment status. Ideal fluorescent signal exposure
times for capturing the complete range of MuβD3 staining intensity were determined at
the outset of the experiment by a survey of ten random sections and were held constant
for subsequent image capture. Three MuβD3 stained fields of colonic epithelial
structures, as well as one unstained control from each animal, were digitally captured
from longitudinal sections of each of the mouse proximal colon samples. Capture fields
were selected on the basis of epithelial tissue morphology as determined by DAPI
staining. Each of the images (three stained fields and one unstained field from each
mouse) were graded on a 0-4 scale by a panel of four blinded researchers, with a grade of
0 indicating no MuβD3 staining and a grade of 4 corresponding to peak MuβD3 staining
71
intensity (Figure 5-1). The mean scores given by the evaluating panel for each of the
images were averaged for each mouse before treatment or genotype status were revealed
according to a key.
2.2.13 Statistical Analysis
The statistical difference between treatment groups was evaluated where appropriate by
unpaired one-tailed Student’s t-test and all error bars drawn to indicate the standard error
of the means (S.E.M.). Differences were considered significant when p < 0.05. Statistical
analysis was performed using R version 2.12.1 for Mac OS X. Graphing was completed
using R version 2.12.1 for Mac OS X or GraphPad Prism version 4.0c.
2.3 RESULTS
2.3.1 HA-Specific Induction of HβD2 Expression in HT-29 Colonic Epithelial Cells
Occurs in a Time Dependent Manner
HβD2 is upregulated in keratinocytes treated with a broadly polydispersed preparation of
HA fragments less than 200 kDa in size (241), and we hypothesized that fragmented HA
would induce HβD2 expression in intestinal epithelium. Therefore, we first tested the
ability of fragmented HA to promote expression of HβD2 protein in the human intestinal
epithelial tumor cell line HT-29. Cultured HT-29 cells were treated with or without a
polydispersed HA fragment preparation averaging 35 kDa (HA-35) at a concentration of
1 µM for 12 h, and fluorescently stained with a specific antibody against HβD2 and
observed using confocal microscopy. The micrographs (Figure 2-1) reveal substantially
72
increased intracellular HβD2 staining. Strikingly, HβD2 protein staining has a granular
cytoplasmic arrangement after HA-35 treatment (inset).
Defensins, including HβD2, have been principally characterized as secreted peptides
produced by epithelium, which participate in innate extracellular host defense (93).
Surprisingly, treatment of HT-29 cells with HA-35 at concentrations up to 10 µM for 9
hours resulted in no significant change in secreted HβD2 peptide concentration (P =
0.845 vs. Medium alone) compared to treatment with medium alone (Figure 2-1).
Induction of intracellular HβD2 protein in HT-29 cells by HA-35 is observed within
hours of the initial treatment. Figure 2-1 demonstrates that the ratio of HβD2 protein
relative to GAPDH protein in whole cell lysates increases with HA-35 treatment, peaking
9 hours after the addition of the HA-35 (p=0.0006 vs. 0 hours HA-35 treatment), and
returning to baseline expression by 24 hours. To test whether increases in intracellular
HβD2 protein expression were specifically induced by HA, HT-29 cells were treated with
medium alone, HA35 (10 µM), or HA-35 (10 µM) that was specifically degraded into
disaccharide components with no known activity by pre-digestion with Streptococcus
dysgalactiae hyaluronidase. Western blot analysis (Figure 2-1) of HT-29 whole cell
lystates reveals an increase in intracellular HβD2 protein expression relative to GAPDH
protein expression following treatment with 10 µM HA-35 that was entirely reversed by
specific destruction of HA-35 with hyaluronidase.
Together these findings strongly suggest that fragmented HA promotes expression of
intracellular HβD2 protein in colonic epithelium.
73
2.3.2 Induction of HβD2 Expression in HT29 Colonic Epithelial Cells is Specific to HA
Bacterial lipopolysaccharide (LPS), or endotoxin, is a component of the outer membrane
of Gram-negative bacteria that has long been understood to elicit essential innate immune
responses through numerous mechanisms (252), including promoting increased
transcription of the gene encoding the HβD2 peptide, DEFB4, in certain epithelial cell
lines (102). The potential for endotoxin contamination during reagent preparation has
long been a caveat to studies on the immune-stimulatory properties of hyaluronan
polymers (166), and with this in mind we compared the induction of HβD2 in HT-29
cells treated with LPS with those cultured in medium containing HA-35. As shown in
Figures 2-2, treatment of HT-29 cells with an LPS preparation with demonstrated
efficacy in T-cells (data not shown) does not result in increased intracellular HβD2
protein expression compared to treatment with medium alone, even at high
concentrations (1 µg/ml). Conversely, treatment with HA-35 (10 µM) consistently
resulted in up-regulation of intracellular HβD2 peptide, as evaluated by confocal
microscopy of immunostained cultures (Figure 2-2) as well as densitometric Western
blot analysis (Figure 2-2).
Collectively, our data (Figures 2-1 and 2-2) indicate that the induction of intracellular
HβD2 is HA-specific and is unlikely to be accounted for by inadvertent contamination of
HA fragment preparations with stimulatory agents.
2.3.3 Induction of HβD2 in HT29 Cells is Dependent on HA Size and Concentration
Specific signaling by HA is hypothesized to be polymer size-dependent (166), and
therefore we determined the optimal size of HA fragments for induction of HβD2 in
74
intestinal epithelial cells. HT-29 human intestinal epithelial cells were treated with
hyaluronan fragments with average molecular weight of 4.7 kD (HA-4.7), 16 kD (HA16), 35 kD (HA-35), 74 kD (HA-74) at equal-molar (Figure 2-3B) concentrations
ranging from 0.01-100 µM or at equal-mass concentration (Figure 2-3) for 9 h. Both
immunofluorescent staining (Figure 2-3) and Western blot analysis (Figure 2-3) of HT29 cells treated with HA polymers demonstrate specific induction of HβD2 peptide
following treatment with HA-35. The peak intracellular HβD2 peptide expression was
observed at 10 µM (350 µg/ml) concentration of HA-35 (Figure 2-3).
Immunofluorescence histochemistry confirmed that HT-29 cells treated with equal mass
doses (100 µg/ml) of HA fragments are also optimally induced to express HβD2 protein
by HA-35, as seen by red immunofluorescent staining with an HβD2-specific antibody
(Figure 2-3). Additionally, the HβD2-inducing properties of HA-35 were compared
against an equal-mass concentration of a large 2,000 kD HA polymer (HA-2M) or
medium alone. HA-2M had no effect on HβD2 protein expression when compared to
medium alone while HA-35 treatment doubled the relative abundance of intracellular
HβD2 protein (Figure 2-3). Whether used at equal-molar or equal-mass concentrations,
HA-35 was the most active inducer of HβD2 protein expression among the fragmented
HA preparations evaluated. Notably, cell growth rate, as determined by cell counting and
trypan blue dye exclusion (data not shown), was unchanged in HT-29 cells treated with
HA fragments at the concentrations evaluated (≤ 100 µM).
Finally, we wanted to evaluate whether the presence of smaller (HA-4.7) or larger (HA2M) HA fragments could alter the activity of HA35. HT-29 cells were incubated with
media containing equal-molar (10 µM) concentrations of HA-4.7 alone, HA-35 alone,
75
HA-35 and HA-4.7 in combination at 10 µM each, or medium alone for 8 h (Figure 2-3).
In addition, HT-29 cells were treated with equal-mass concentrations (350 µg/ml) of HA35 alone, HA-2M alone, HA-35 and HA-2M in combination at 350 µg/ml each, or
medium alone (Figure 2-3). In concordance with our prior experiments, quantitative
Western blot analysis of replicate samples revealed approximately 2.5-fold mean
induction of HβD2 protein expression in HT-29 cultures treated with media containing
HA-35 alone relative to control-treated cultures with neither HA4.7 alone nor HA-2M
alone promoting significant increases in HβD2 protein expression. While co-treatment of
HT-29 cells with HA-2M and HA-35 at equal-mass concentration resulted in HβD2
induction comparable to treatment with HA-35 alone, strikingly HT-29 cells treated with
an equal-molar combination of HA-4.7 and HA-35 exhibited no statistically significant
increase in HβD2 protein expression relative to control-treated cultures (Figure 2-3).
Thus, while the presence of HA-2M has no apparent effect on the activity of HA-35 when
HT-29 cells are treated in combination, equal-molar concentrations of HA-4.7 are
sufficient to interfere with the HβD2-inducing activity of HA-35 fragment preparations.
To further define and compare the distribution of HA fragment sizes in each of the
preparations, HA-4.7, HA-16, HA-35, HA-74, and HA-2M were electrophoretically
separated by polyacrylamide gel electrophoresis and the molecular weight distributions
compared against a series of HA polymer standards (Figure 2-3). Predictably, the HA
preparation of greatest molecular weight (HA-2M) contained an entirely distinct HA
polymer population, however HA-4.7, HA-16, HA-35, and HA-74 fragment preparations
share varying degrees of overlapping distribution with high similarity between HA-35
76
and HA-74. The HA-35 preparation nonetheless contains the greatest proportion of HA
fragments between 20 and 45 kDa of any HA preparation evaluated, and it is this HA
fragment population that most distinguishes HA-35 from HA-16 and HA-74 (Figure 23), neither of which promote increased HβD2 protein expression in HT-29 cells (Figures
2-3).
In summary, the experiments of Figure 2-3 demonstrate that the induction of intracellular
HβD2 protein expression by fragmented HA is highly size-specific, with HA-35
emerging as the most potent inducer of HβD2. Smaller (4.7 and 16 kDa) or larger (74 and
2000 kDa) HA fragments alone do not promote significant intracellular HβD2 protein
expression, and comparative electrophoretic polymer sizing analysis suggests that the
active HA fragments present in our polydispersed HA-35 preparation are between 20 and
45 kDa in size. Furthermore, the activity of HA-35 is partially inhibited in the presence of
equal-molar HA-4.7, but not HA-2M.
2.3.4 Orally Administered HA-35 Induces the Expression of the HβD2 Ortholog in Mouse
Intestinal Epithelium In Vivo
To test whether our in vitro observation, that HA-35 promotes expression of HβD2
protein in intestinal epithelial cells, also occurs with normal intestinal epithelium in vivo,
we enriched the drinking water of 5 adult wild-type C57BL/6 mice with HA-35 (1 µM)
and compared them to 5 age- and sex-matched controls supplied with standard drinking
water. Mice were sacrificed after 7 days of ad libitum consumption of HA-supplemented
drinking water. Distal colon tissue was excised in cross-sections for immunoflourescent
staining. Analysis of cross-sectioned intestinal tissue by confocal microscopy reveals
77
increased expression of murine β-defensin 3 (MuβD3, the mouse ortholog of HβD2
(253)) in the intestinal mucosa of mice consuming HA-supplemented water compared to
mice consuming standard water (Figure 2-4). Higher magnification reveals increased
MuβD3 protein expression localized to the epithelial layer, and in particular the base of
the intestinal crypts where putative epithelial stem cells reside (254), in the mucosal
tissue of mice consuming HA35-supplemented water (Figure 2-4).
2.3.5 Induction of HβD2 Ortholog in Mouse Intestinal Epithelium by HA Fragments is
Highly Size-Specific
Induction of HβD2 following HA fragment treatment in the HT-29 human intestinal
epithelial tumor cell line was found to be highly specific to an HA fragment preparation
enriched with HA polymers between 20 and 45 kDa with an average molecular weight of
35 kDa (Figure 2-3). Furthermore, supplementation of the drinking water of adult wildtype mice with HA-35 promoted increased MuβD3 protein expression in the colonic
mucosa relative to control-fed animals (Figure 2-4). However, it remained unclear
whether HA size-specific induction of defensin occurred in vivo in a manner similar to
our in vitro observations in HT-29 cells. In order to address this question, 18 age- and
sex-matched wild-type C57BL/6 mice were segregated equally into the following six
treatment groups: Control, HA-4.7, HA-16, HA-35, HA74, or HA-2M. All animals were
gavage-fed 0.25 ml water (Control) or 300 µg HA of the fragment preparation
corresponding to the treatment group designation (e.g. HA-4.7 animals received 300 µg
of 4.7 kDa HA) dissolved in 0.25 ml water once daily for three consecutive days. Mice
were sacrificed within 16-18 h of the final gavage treatment. Proximal colon tissue was
78
fixed and cut in cross-sections for immunoflourescent staining. Analysis of crosssectioned intestinal tissue by fluorescent microscopy revealed increased epithelial
MuβD3 protein expression only in the HA-35 treated animals relative to control-treated
animals (Figure 2-5). Figure 5A presents representative high magnification images of
murine colonic epithelium from each of the treatment groups immunostained for MuβD3
protein expression. Blinded analysis of fluorescent microscopy imaging revealed
significantly increased MuβD3 staining intensity in the epithelial cells of the proximal
large intestine of wild-type mice fed HA-35 compared to control-fed wild-type mice (P =
0.0184, Figure 2-5) while no significant difference relative to control animals was
observed in mice fed HA4.7, HA-16, HA-74, or HA-2M.
2.3.6 Specific-Sized HA Fragment Mediated Induction of HβD2 ortholog Expression in
Mice is Dependent on TLR4
A number of cell surface receptors have been associated with the activation of innate
defense responses by HA, including most prominently Toll-like receptor TLR4 (68, 227,
239, 241, 247) and CD44, the major HA receptor that is involved in inflammation as well
as homeostasis (215, 223, 227, 255, 256). Thus, both TLR4 and CD44 were evaluated as
candidate HA fragment receptors for the induction of murine HβD2 ortholog in vivo.
Adult wild-type, CD44-/-, and TLR4-/- C57BL/6 mice were gavage-fed a solution
containing 300 µg of fragmented HA averaging 28.6 kDa (HA-28) dissolved in 0.25 ml
water (≈100 µM) or 0.25 ml water alone once daily for three days. HA-28 was substituted
for the HA-35 used in previous studies due to its similar average molecular weight,
ability to promote HβD2 protein expression in vitro (Figure 5-2), and commercial
79
availability. Mice were sacrificed at 16-18 h after the final treatment and the colon tissue
extracted for histological analysis. Wild-type and CD44-/- mice exhibited comparable
TLR4 protein expression levels at baseline, and TLR4-/- mice did not express TLR4
protein in the intestinal epithelium (Figure 5-3). Figure 2-6 presents representative
images of colonic epithelium immunostained for MuβD3 protein from each of the
treatment groups. Blinded analysis by fluorescent microscopy revealed significantly
increased MuβD3 staining intensity in the epithelial cells of the proximal large intestine
of wild-type mice fed HA-28 compared to control-fed wild-type mice (p = 0.0003,
Figure 2-6). No increase in MuβD3 staining intensity was observed in the colonic
epithelium of TLR4-/- mice fed HA-28 relative to control-fed TLR4-/- mice. Increased
MµβD3 protein expression was observed in CD44-/- fed HA-28 relative to control-fed
CD44-/- (p=0.04). Unexpectedly, control-fed CD44-/- mice had significantly higher
levels of baseline MuβD3 protein expression in comparison to their wild-type
counterparts (Figure 2-6, p=0.005).
The experiments summarized in Figures 2-4, 2-5, and 2-6 provide compelling evidence
that our in vitro observations in HT-29 human intestinal epithelium tumor cells translate
to the healthy colonic mucosa of adult mice, with two HA fragment preparations (HA-35
and HA-28) supplied by two distinct methods (prolonged ad libitum consumption or
controlled oral administration) reproducibly promoting increased expression of the
murine HβD2 ortholog (MuβD3) in the epithelium of both the distal and proximal colon.
As with our in vitro studies, this response to fragmented HA is highly size-specific, with
only HA-35 and HA-28 promoting significantly increased expression of MuβD3 protein
in the colonic epithelium relative to control animals. Furthermore, through the use of
80
gene-specific knockout mice, we have identified TLR4 as a requisite cell surface receptor
for the induction of MuβD3 expression with fragmented HA.
2.4 DISCUSSION
Taken together, our data demonstrate that specific-sized HA fragments promote increased
intracellular expression of the key innate anti-microbial peptide HβD2 in intestinal
epithelial cells, both in vitro and in vivo through induction of murine HβD2 ortholog
MuβD3 (253) in both the proximal and distal large intestine. While activation of innate
defense responses by HA fragments has been shown using a variety of cell types (68,
223, 227), little was previously known regarding the effects of HA fragments on
intestinal epithelium. Furthermore, we have identified TLR4 as a requisite cell-surface
receptor for the induction of MuβD3 following oral administration of specific-sized HA
in vivo, thus contributing to the mounting evidence that TLR4 is a central mediator of
cellular responses to fragmented HA.
Size-specific HA signaling has been reported previously in other cell types (68, 223, 227,
247), often defining HA size broadly as either “high” or “low” molecular weight, with no
consensus in the literature on what size range defines either category. Induction of HβD2
in intestinal epithelium requires HA polymers within a relatively narrow molecular
weight range; ~35 kDa HA specifically promotes increased intracellular HβD2 protein
accumulation that is not observed following treatment with either smaller (HA-4.7, HA16) or larger (HA-74, HA-2M) HA polymers when compared at either equal-molar or
81
equal-mass concentrations (Figures 2-3 and 2-5). HA preparations with average
molecular weight of 28.6 kDa, also induce intracellular HβD2 peptide expression
comparable to HA-35 in HT-29 cells (Figure 5-2), and we have demonstrated that both
HA-28 and HA-35 upregulate MuβD3 expression in vivo (Figures 2-4, 2-5, and 2-6).
Equal-molar application of HA fragments presumes a ligand-receptor relationship in
which one HA fragment or polymer interacts in a specific manner with single receptors,
while an HA polysaccharide may be capable of interacting with multiple receptors due to
its extended molecular conformation and repeating structural motif (219). Therefore we
evaluated the size-specificity of HβD2 induction by HA in vitro using both equal-molar
and equal-mass dosing for the HA fragment preparations ≤ 74 kDa in average molecular
weight. Importantly, HA-35 is the most active inducer of HβD2 protein expression using
either method of comparison to other HA preparations. Thus the size range of
functionally active HA fragments is defined here as no less than 16 kDa and no greater
than 74 kDa. Gel electrophoresis analysis of the HA fragment preparations employed in
our studies suggests that the optimal HA fragment(s) for intracellular HβD2 induction is
likely in the range of 20-45 kDa (~53-132 disaccharides) (Figure 2-3). To our
knowledge, no signaling activity of any kind has been previously ascribed to an HA
polymer within this specific size range. HA found in living tissue assumes a widely
polydispersed distribution (166), and it is unclear how HA signaling systems respond to
HA fragments within this active range in the presence of larger or smaller HA fragments.
Our findings indicate that while high molecular weight HA (2000 kDa) has no impact on
the induction of HβD2 by HA-35, equal-molar HA-4.7 is sufficient to inhibit HβD2
protein expression during treatment of cultured HT-29 cells with HA-35 (Figure 2-3).
82
This could be accomplished through a number of different mechanisms, including direct
competition for receptor binding sites, or through an unknown alternate signaling
pathway acting counter to the TLR4-dependent pathway activated by HA-35. Conversely,
the TLR4-dependent mechanism of HβD2 induction by HA-35 is unaffected by the
presence of an equal-mass concentration of HA-2M, indicating selectivity among
chemically identical polymers of differing length. Our results contrast with the findings
of Campo et al. (231) in which it is proposed that high molecular weight HA displaces
small HA oligosaccharides at the cell surface, inhibiting the TLR4-dependent
inflammatory response to HA oligosaccharides (~1.5 kDa) in chondrocytes. Co-receptor
complexes incorporating a variety of HA-binding transmembrane proteins including
TLR4, CD44, and others, similar to those proposed by Taylor et al. (227), may confer the
size- and structural-specificity indicated by our experimental findings. However,
additional studies are required to clarify the molecular mechanisms of HA selectivity.
Bearing in mind that questions of purity have arisen to challenge several early reports of
innate responses to HA (166), we have tested the HA-35 preparation and compared its
effect to endotoxin (LPS). Pre-digestion of HA fragments with hyaluronidase fully
abrogated the effect of HA treatment in HT-29 cells (Figure 2-1), confirming that HA is
the specific stimulatory component of HA-35 required for induction of HβD2 protein
expression. Secondly, despite reports of increased defensin gene transcription following
treatment with LPS in certain epithelial cell lines transgenically expressing TLR4 (102),
we demonstrated that 35 kDa HA fragments promote intracellular HβD2 peptide
accumulation in HT-29 cells while high concentrations of LPS do not (Figure 2-2). Lee
83
et al. (257) have reported that sensitivity to LPS is decreased in HT-29 cells due to low
TLR4 expression. Induction of the murine HβD2 ortholog by fragmented HA is TLR4dependent as we report in Figure 2-5. Differing sensitivity of the TLR4 receptor to the
ligands HA and LPS or the presence of ligand-specific co-receptors could account for this
discrepancy. In either case, we offer compelling evidence to eliminate inadvertent
endotoxin contamination of HA-35 as a contributing factor in the induction of
intracellular HβD2 in HT-29 cells.
The effect of intermediate-sized HA on intestinal epithelium appears to be divergent from
its effect on skin keratinocytes grown in vitro (241) in that no significant change in
secreted HβD2 protein concentration is observed in HT-29 cells following treatment with
35kDa HA (Figure 2-1). While defensins are best characterized as secreted effector
molecules of innate defense (93), intracellular expression of HβD2 protein has been
suggested by positive immunostaining of mucosal tissue (103, 129, 245, 258,) and
epithelial cell cultures (102, 241) for some time. The function of intracellular HβD2
peptide has not been studied extensively, particularly in epithelium. A recent report by
Arnett et al. (259) describes a mechanism by which intracellular defensins inhibit
proliferation of the obligate intracellular pathogen Listeria monocytogenes in
macrophages. Given that 1) high concentrations of secreted HβD2 cause membrane
disruption in human epithelial cells (260), 2) that extraordinary numbers of beneficial
(30) and protective (261) microbes reside in direct proximity to the colonic epithelium
(33), and 3) that intracellular pathogens and their PAMPs are among the most potent
inducers of HβD2 expression (31, 101, 112, 113), it is possible that intracellular
84
compartmentalization of HβD2 may contribute to the process of epithelial barrier
maintenance without causing undue harm to epithelial integrity and commensal flora
populations, and without promoting unnecessary activation of immune cells (262).
An accumulating body of literature has implicated both TLR4 (68, 227, 241, 247) and
CD44 (215, 223, 227, 255, 256, 263) as major cell surface receptors in the activation of
innate defense responses, as well as homeostatic maintenance, upon exposure to HA of
various size ranges. In addition, two previous studies have demonstrated TLR4dependent mechanisms by which HA contributes to the integrity of the intestinal
epithelium in mice (239, 264). However, both of these studies utilized HA polymer
preparations predominantly >500 kDa in size. With these reports in mind, TLR4 and
CD44 were the natural choices for evaluation as candidate HA fragment receptors for the
induction of murine HβD2 ortholog MuβD3 (253) in vivo. Both HA-35 (Figures 2-4 and
2-5) and HA-28 (Figure 2-6) promote enhanced expression of MuβD3 in the colonic
epithelium of adult C57BL/6 mice, while no up-regulation of MuβD3 was observed in
HA-28 –fed mice deficient in TLR4 (Figure 2-6). CD44-/- mice exhibited greater
baseline MuβD3 expression in comparison to water-fed wild-type mice that was
increased further by HA-28 feeding (Figure 2-6). Thus it was evident that expression of
TLR4 is required for the in vivo induction of MuβD3 by HA-28, with CD44 possibly
having a regulatory role in baseline MuβD3 expression. We found no evidence of altered
TLR4 protein expression between wild-type and CD44-/- mice, suggesting that a
compensatory relationship between the HA receptors TLR4 and CD44 is unlikely to
account for the observed increase in baseline MuβD3 expression among CD44-deficient
85
animals (Figure 5-3). CD44 is thought to contribute to the maintenance of tissue
homeostasis (265), and CD44 deficient mice have been associated with a proinflammatory phenotype in some contexts, including LPS-induced septic shock (266). At
least one report places CD44 and TLR4 in opposing regulatory positions, with binding of
high molecular weight HA to CD44 attenuating TLR4 signaling in response to LPS
(267). One possible explanation for increased background MuβD3 expression in CD44-/mice could be a relative deficiency in pro-homeostatic signaling favoring release of
inflammatory cytokines (175, 266), several of which are associated with increased
defensin expression (32, 98, 103), in the presence of commensal microbes. Despite the
many open questions regarding the nature of specific-sized HA signaling in intestinal
epithelium, it is now clear that the induction of HβD2 ortholog MuβD3 by specific-sized
HA occurs in the intestinal mucosa of live animals and that TLR4 expression is a
necessary component of this innate defense response.
Our observations regarding induction of HβD2 by specific-sized HA must certainly
reflect an endogenous in vivo process maintained and enhanced by natural selection, yet
defining the larger physiologic role of fragmented HA in the digestive tract is difficult.
One analogy is that of butyrate and the histone deacetylase inhibitor sulforaphane, dietary
compounds found in vegetables of the Brassicaceae family such as broccoli (104), and
the F(ab’)2 fragments of immunoglobulin A secreted in human milk (105), all of which
have been demonstrated to promote enhanced HβD2 expression and secretion in
intestinal epithelium. HA is ubiquitous in vertebrate animals (268) and is almost certainly
consumed routinely in the Western world and, further, HA has recently been reported in
86
both human and bovine milk (167). Although digestive processing of fragmented HA has
not been fully evaluated, particularly with regard to alterations in polymer sizing, our
results suggest that orally-ingested HA-35 remains sufficiently intact within the digestive
tract to promote MuβD3 expression in the distal colon of mice (Figure 2-4). The GI tract
may have limited ability to absorb ingested HA; Balogh et al. (269) have demonstrated
that radioactivity is almost entirely recovered in the feces of rats fed radio-labeled high
molecular weight HA polymers. Induction of defensin expression by dietary HA could
conceivably contribute to shifts in microflora species distribution or quantity, as has been
shown to follow altered expression of α-defensins (270). Perhaps more importantly,
fragmented HA may have therapeutic potential for patients with diseases affecting the
integrity of the intestinal epithelial barrier (271). In Crohn’s disease, deficient induction
of HβD2 in the colonic epithelium (129) may contribute to the loss of barrier integrity
and vulnerability to invasive, and pro-inflammatory, mucosal flora (271).
87
Figure 2-1. HA-Specific Induction of HβD2 Expression in HT-29 Colonic
Epithelial Cells Occurs in a Time Dependent Manner
A
B
C
FIGURE 2-1. A. Representative confocal micrographs show HβD2 expression in
confluent cultures of HT-29 cells that were treated with medium alone or containing HA35 (1 µM) for 12 h. Cells were fluorescently immunostained for HβD2 protein (red) and
nuclei were stained with DAPI (blue). ‘NS’ indicates the immunostaining control in
which no α-HβD2 antibody was utilized. B. Mean secreted HβD2 as measured ELISA in
the culture media of HT-29 cells treated for 9 h with medium alone or containing HA-35
(10 µM). No significant difference in secreted HβD2 peptide was detected between
media or HA treated cultures. C. Average densitometric quantification of immunoblots
from four individual experiments in which the abundance of HβD2 protein relative to
GAPDH protein was evaluated in whole cell lysates of HT-29 cells. Replicate cultures
were treated with HA-35 (1 µM) at 3 h time intervals (0- 24 h) in each experiment.
Significance of differences in normalized HβD2 expression were evaluated by
comparison of each time point to control treatment using Student’s t-test, with ‘***’
indicating p < 0.001.
88
Figure 2-1 (cont.)
D
FIGURE 2-1 (cont.). D. Representative Western blots demonstrating HβD2 protein
expression relative to GAPDH in the whole cell lysates of HT-29 cells treated with
medium alone, medium containing HA-35 (10 µM) and medium containing HA-35 (10
µM) that was pre-digested with Streptococcus dysgalactiae hyaluronidase at 0.025 U/ml
for 16 h at 37° C.
89
Figure 2-2. Induction of HβD2 Expression in HT29 Colonic Epithelial Cells is
Specific to HA
A
FIGURE 2-2. A. Representative confocal micrographs of HT-29 cell cultures that were
treated with medium alone, medium containing HA-35 (10 µM), or medium containing
LPS (1 µg/ml) for 9 h. HβD2 protein is immunostained (red) and nuclei are stained with
DAPI (blue).
90
Figure 2-2 (cont.)
B
C
FIGURE 2-2 (cont.). B. Representative Western blot of HβD2 protein expression
relative to GAPDH protein expression in whole cell lysates of HT-29 cells treated with
media alone, media supplemented with HA-35 (10 µM), or media containing LPS (1
µg/ml). C. Average densitometric quantification of Western blot results of three
experiments in which HT-29 cultures were treated with media alone, HA-35 (10 µM), or
LPS (1 µg/ml) for 9 h. HβD2 protein expression is normalized to GAPDH protein in
whole cell lysates. Significance of differences in normalized HβD2 expression were
evaluated by comparison of each treatment to medium treatment using Student’s t-test,
with ‘*’ indicating p < 0.05.
91
Figure 2-3. Induction of HβD2 in HT29 Cells is Dependent on HA Size and
Concentration
A
B
FIGURE 2-3. A. Representative confocal micrographs of HT-29 cells treated with media
alone or equal-mass (100 µg/ml) concentrations of HA-4.7, HA-35, or HA-74 for 6 h.
Cells were fluorescently immunostained for HβD2 protein (red), and nuclei were stained
with DAPI (blue). B. Representative Western blot showing HβD2 protein relative to
GAPDH protein expression in whole cell lysates of HT-29 cells treated with HA-4.7,
HA-16, HA-35, or HA-74 at equal-molar concentrations (10 µM).
92
Figure 2-3 (cont.)
C
FIGURE 2-3 (cont.). C. Average densitometric quantification of immunoblots from four
separate experiments in which HβD2 protein expression was evaluated relative to
GAPDH protein expression in whole cell lysates of HT-29 cells. Confluent cultures of
HT-29 cells were treated for 9 h with medium alone, or a range of concentrations (0.01100 µM) of HA fragment preparations of different average molecular weight (HA-4.7;
HA-16; HA-35; HA-74). The solid black horizontal line indicates the mean HβD2 to
GAPDH ratio in medium-treated cells, and the surrounding grey shaded region denotes
the standard error of the means among replicate medium-treated samples. Significance of
differences in normalized HβD2 expression were evaluated by comparison of each
treatment and dosage to medium treatment using Student’s t-test, with ‘*’ indicating p <
0.05, and ‘**’ indicating p< 0.01.
93
Figure 2-3 (cont.)
D
E
Figure 2-3 (cont.). E. Average densitometric quantification of immunoblots from two
separate experiments, each with four replicates of each treatment group, in which HβD2
protein expression was evaluated relative to GAPDH protein expression in whole cell
lysates of HT-29 cells. Confluent cultures of HT-29 cells were treated for 9 h with
medium alone, medium containing equal-molar concentrations (10 µM) of HA4.7 alone
or HA-35 alone, a combination of HA-4.7 and HA35 (10 µM each), HA-2M at equalmass concentration (350 µg/ml) relative to HA-35 alone (350 µg/ml = 10 µM for HA35)
or a combination of HA-2M and HA-35 (350 µg/ml each) for 8 hours. Significance of
94
differences in normalized HβD2 expression were evaluated by comparison of each
treatment and dosage to medium treatment using Student’s t-test, with ‘*’ indicating p <
0.05, ‘**’ indicating p< 0.01, and ‘***’ indicating p<0.001.
Figure 2-3 (cont.)
F
FIGURE 2-3 (cont.). F. Molecular weight distribution and relative quantity of HA
polymers in commercial HA fragment preparations used for in vitro experiments. The
blue shaded region indicates the portion of HA-35 fragment distribution that is enriched
relative to other HA fragment preparations evaluated (approximately 20-45 kDa).
95
Figure 2-4. Orally Administered HA-35 Induces the Expression of the HβD2
Ortholog in Mouse Intestinal Epithelium In Vivo
FIGURE 2-4. Representative confocal micrographs of distal colon cross-sections from
adult mice provided with standard drinking water or drinking water supplemented with
HA-35 (1 µM) ad libitum for 7 days. The mouse ortholog of HβD2, MuβD3, is
fluorescently immunolabeled (red) and nuclei are stained with DAPI (blue). Lower panels
represent a more highly magnified portion of the lower power (upper) field (as indicated
by white boxes).
96
Figure 2-5. Induction of HβD2 Ortholog in Mouse Intestinal Epithelium by HA
Fragments is Highly Size-Specific
A
B
FIGURE 2-5. A. Representative fluorescent micrographs of epithelium of proximal
colon sections from adult C57BL/6 wild-type mice. The mice were fed single daily doses
of HA-4.7, HA-16, HA-35, HA-74, or HA-2M (300 µg/ 0.25 ml), and controls were
given an equivalent volume of water alone by oral gavage for three consecutive days.
MuβD3 is immunolabeled (shown in red) and nuclei are blue as a result of DAPI
staining. ‘NS’ indicates an immunostaining control in which no MuβD3 antibody was
utilized. B. Average scored MuβD3 staining intensity of proximal colon tissue sections
from wild-type mice fed single daily doses of HA-4.7, HA-16, HA-35, HA-74, or HA2M or an equivalent volume of water alone once daily for three consecutive days.
Average MuβD3 staining intensity score represents 3 mice per group, with 3 stained
sections per mouse, as judged by a blinded panel of 4 researchers on a scale of 0 to 4 with
a score of 4 corresponding to peak MuβD3 staining for this dataset. Significance of
differences in mean MuβD3 staining intensity was evaluated using Student’s t-test in a
pair-wise manner as indicated in the figure, with ‘*’ indicating p< 0.05.
97
Figure 2-6. Specific-Sized HA Fragment Mediated Induction of HβD2 ortholog
Expression in Mice is Dependent on TLR4
A
FIGURE 2-6. A. Representative fluorescent micrographs of epithelium of proximal
colon sections from adult C57BL/6 “Wild-type”, TLR4-/-, and CD44-/- mice. The mice
were fed single daily doses of HA-28 (300 µg/ 0.25 ml), and controls were given an
equivalent volume of water alone by gavage for three consecutive days. MuβD3 is
immunolabeled (red) and nuclei are blue.
98
Figure 2-6 (cont.)
B
FIGURE 2-6 (cont.). B. Average scored MuβD3 staining intensity of proximal colon tissue
sections from “Wild-type”, TLR4-/-, and CD44-/- mice fed HA-28 (300 µg) or an equivalent
volume of water alone by oral gavage once daily for three consecutive days. Average MuβD3
staining intensity score represents 5 mice per group, with 3 stained sections per mouse, as judged
by a blinded panel of 4 researchers on a scale of 0 to 4 with a score of 4 corresponding to peak
MuβD3 staining. Mean staining intensity score of non-specifically stained sections (1 per mouse)
was 0.24±0.09. Significance of differences in mean MuβD3 staining intensity was evaluated
using Student’s t-test in a pair-wise manner as indicated in the figure, with ‘*’ indicating p< 0.05,
‘**’ for p< 0.01, and ‘***’ for p<0.001.
99
CHAPTER 3: HUMAN MILK HYALURONAN ENHANCES INNATE DEFENSE
OF THE INTESTINAL EPITHELIUM
3.1 INTRODUCTION
Numerous positive health outcomes are associated with breast-feeding in infants (10,134,
136, 137), particularly regarding gastrointestinal infection. Grulee et al. (138) conducted
the first major evaluation of morbidity and mortality among 20,061 breast-fed and
artificially-fed infants in 1934, reporting as much as 50% reduction in gastrointestinal
infection incidence among breast-fed infants. Modern epidemiologic studies reinforced
and expanded upon these findings (136, 161), indicating that breast-feeding confers
remarkably enhanced protection from both gastrointestinal and respiratory infections,
including Salmonella infection (140).
In addition to nutrients, breast-feeding supplies a wide array of bioactive components that
enhance both innate and adaptive immunity in the neonatal gastrointestinal tract. Milk
components act as critical stimuli in the ontology of intestinal immune education and
microflora development (10), supplying passive defense mediators (145, 146), growth
hormones (147), prebiotics (148-151), and immunomodulators (133).
The best characterized of infant protective milk components is soluble IgA (133).
However, milk also contains an abundant and extraordinarily diverse array of glycans,
including oligosaccharides, glycolipids, glycoproteins, mucins, glycosaminoglycans and
other complex carbohydrates, which provide infant protection (10, 158, 159). The ways
in which human milk glycans shape innate gastrointestinal defense are diverse (158, 159),
and include prebiotic function (148-151), anti-adhesive antimicrobial activity (53, 160,
100
161), and intestinal epithelial cell modulation (162-165). Induction of altered gene
expression in intestinal epithelium by human milk oligosaccharides (HMOs) results in
enhanced protection from pathogenic E. coli infection through modulation of epithelial
cell surface glycans (162), and milk lactose induces the expression of antimicrobial
peptide LL-37 in cultured epithelium (165), suggesting that direct effects of human milk
glycans on intestinal epithelial cells may contribute significantly to the protection from
gastrointestinal infection associated with breast-feeding.
Among the known glycan components of both human and bovine milk are abundant
glycosaminoglycans (GAGs), large linear polysaccharide polymers containing amino
sugars. Hyaluronan (HA), is a GAG usually found as a high molecular weight polymer
and consists of repeating disaccharides of N-acetyl-β-D-glucosamine and β-D-glucuronic
acid. Unlike other GAGs, HA is synthesized without a protein core and is not sulfated,
nitroslylated, or phosphorylated in vivo (166). A recent study determined that HA is one
of the GAGs contained in milk (167). Milk GAGs may play a significant role in
enhancing intestinal defense against pathogens, as suggested by inhibition of HIV
engagement with host receptor CD4 by chondroitin sulfate derived from human milk
(168). However, the specific function of milk HA has not been previously reported.
HA is found in every tissue of the body, primarily in the form of high molecular weight
polymers (~107 Da), and plays a fundamental role in tissue homeostasis (272, 273).
Current evidence shows that fragmented HA polymers generated in damaged or
inflammed tissue act as endogenous “danger signals”, or “damage-associated molecular
patterns” (DAMPs) (225-227) triggering localized innate defense responses. Endogenous
fragmented HA may be recognized in much the same way as the conserved pathogen-
101
associate molecular patterns (PAMPs), such as LPS and peptidoglycan, typically
characterized as TLR ligands (66, 227).
HA fragments play a role in enhancing innate epithelial defense that appears to be
independent of the pro-inflammatory immunomodulation characteristic of macrophage
(224), chondrocyte (231), or endothelial cell activation (247) by low molecular weight
(LMW) HA, or the stimulation of TLR4 by bacterial PAMPs (238). A polydispersed HA
fragment preparation of polymers less than 750 kDa injected intraperitonelly protects
wild-type mice in a TLR4-dependent manner from a microflora mediated epithelial
damage model of colitis (239), or from the epithelium-depleting effects of radiation
(240). Low molecular weight HA has been also been shown to induce elevated
expression of antimicrobial defensin proteins that may contribute to enhanced epithelial
defense in the intestine (Chapter 2), skin (241) and reproductive epithelium (242).
Defensins, small cationic peptides that play a critical role in the preservation of epithelial
barrier integrity in the presence of continuous microbial challenges, are expressed by
gastrointestinal, urogenital, and pulmonary epithelium, skin, and the ocular surface (244,
245). Defensins have direct antimicrobial activity against a wide range of human
pathogens and commensals including both Gram-positive and Gram-negative bacteria,
virus, fungi, and protozoa (93). Interestingly, microbes also regulate the expression of the
inducible β-defensins 2, 3, and 4 in epithelium through the interaction of variety of
pathogen-associated molecular pattern molecules (PAMPs) with the Toll-like receptors
(32, 98, 100-102). TLR4 regulates the expression of human β-defensin 2 (HβD2) in
epithelium following stimulation with LPS (102). The same cell surface receptors, TLR4,
mediates the induction of HβD2, without an accompanying increase in inflammatory
102
cytokine production, in human keratinocytes exposed to LMW HA (241) and vaginal
epithelium (242). Our group has recently demonstrated the TLR4-dependent induction of
murine HβD2 ortholog in colonic epithelium in vivo following the administration of
specific sized HA (Chapter 2). Therefore, it is becoming increasingly clear that low to
intermediate molecular weight HA is an endogenous ligand capable of promoting
enhanced antimicrobial defense of epithelial barriers through TLR4-dependent pathways.
Despite the growing evidence of the significant role of HA in bolstering innate defense of
the intestine (239, 240, Chapter 2), an endogenous source of HA responsible for
mediating innate defense remains unknown. In light of the recent report that human milk
contains HA (167) and our finding that that HA promotes expression of the antimicrobial
peptide HβD2 in intestinal mucosa (Chapter 2), we hypothesized that innate epithelial
antimicrobial defense is enhanced in the intestinal epithelium by HA supplied in breast
milk.
We have determined the concentration range of HA in human breast milk collected from
a cohort of 44 mothers who provided multiple samples during the first 6 months after
delivery. Our data confirm the previous finding that human milk contains HA (167, 274),
and demonstrate that milk HA concentration is highest in the critical first weeks after
birth and decreases in the population to a steady state level over the next two months.
Accordingly, using physiologic levels of HA, we demonstrate two independent
parameters of enhanced epithelial antimicrobial defense that are specifically enhanced by
HA purified from human milk: 1) HA-dependent induction of the antimicrobial peptide
HβD2 in cultured human colonic epithelial cells and in murine colonic mucosa following
oral administration of a milk HA preparation; and 2) HA-dependent protection from
103
intracellular infection by Salmonella in cultured intestinal epithelial cells pre-treated with
milk
derived HA. Furthermore, the in vivo induction of murine HβD2 ortholog is
dependent upon expression of the cell surface receptors TLR4 and CD44.
3.2 EXPERIMENTAL PROCEDURES
3.2.1 Human Milk Sample Collection
Multiple, dated human breast milk samples were provided by 44 unique donors between
January 2011 and December 2012. All donors provided informed consent in accordance
with a protocol approved by the Cleveland Clinic Institutional Review Board, and
provided samples were de-identified and assigned a code number corresponding to
postpartum day of milk collection. All samples were stored at -20°C.
3.2.2 Isolation of HA from Milk
Donated human milk samples with a minimum volume of 50 ml were boiled for 10
minutes - to heat-inactivate endogenous milk enzymes. Digestion of milk protein content
was completed using Proteinase K (Roche, Indianapolis, IN) added to the sample at a
concentration of 0.6 mg/ml and incubated at 60° C for a minimum of 18 h. Following
proteolysis, milk samples were cooled at 4° C for 24 h to enhance physical separation of
milk lipid content. The bulk of milk fats were removed from the sample with a sterile
spatula before centrifugation at 35,000 x g for 10 minutes at 4° C to further precipitate
remaining lipid content, which was removed from the surface of the sample by suction.
104
Milk salts and small molecules, including the peptide products of proteolysis, were
removed by overnight dialysis against ultra-pure sterile H2O using dialysis membrane
cassettes with a maximum pore size of 2 kD (Thermo Scientific, Rockford, IL).
Following additional boiling to ensure sterility, NaCl was added to the dialyzed aqueous
milk fraction to a final concentration of 0.05 M NaCl. Amonium cation based anion
exchange maxi-columns (Thermo Scientific, Rockford, IL) were equilibrated to 0.05 M
NaCl before sample loading by centrifugation in a swinging bucket rotor at 500 x g for 5
m. Sample-loaded anion exchange columns were washed with 5 volumes 0.1 M NaCl to
remove weak cations, positively charged, or non-specifically bound content. Sample
elution was completed by loading the columns with 0.7 M NaCl elution buffer with
centrifugation at 500 x g for 5 m. Eluted content was again dialyzed for removal of NaCl
for downstream cell culture compatibility before slowly precipitating HA content in 95%
isopropanol overnight at 4°C. Following final HA precipitation by centrifugation at
30,000 x g for 30 m at 4°C, the supernatant was discarded and the sample was dehydrated
by centrifugation under vacuum. Precipitated HA isolates were re-hydrated in sterile H2O
and adjusted to 10µg/ml HA concentrated stock solutions following determination of HA
yield by Enzyme-Linked Sorbent Assay (ELSA). Wash and elution buffer salt
concentrations used in the HA isolation scheme were determined empirically for optimal
elution of purified, lyophilized HA with average molecular weight of 4.7 kDa, 35 kDa, or
2000 kDa (Lifecore Biomedical, LLC, Chaska, MN) as verified by HA ELSA.
3.2.3 Quantification of HA by Enzyme-linked Sorbent Assay (ELSA)
105
A 1 ml portion of each donated milk sample was stored separately from the time of
donation at -20°C and labeled to indicate the donor and sample number, indexed to the
postpartum day in a database. A total of 1710 unique milk samples were assayed
undiluted in triplicate and quantified by an ELISA kit according to the manufacturer’s
protocol (Echelon Biosciences Inc., Salt Lake City, UT). HA was similarly quantified in
human milk HA preparations prepared as described above, with appropriate dilutions
applied to account for the increased concentration of HA in the isolate. Mass-independent
detection of HA was verified in the ELSA using known quantities of purified, lyophilized
HA with average molecular weight of 4.7 kDa, 35 kDa, 74 kDa, or 2000 kDa (Lifecore
Biomedical, LLC, Chaska, MN). Structural specificity of the HA ELSA was verified
against nonsulfated chondroitin (Amsbio, LLC, UK).
3.2.4 Fluorophore-assisted Carbohydrate Electrophoresis
Fluorophore-assisted Carbohydrate Electrophoresis (FACE) was used to assess the purity
of milk HA preparations and to quantify carbohydrate constituents. A complete
description of this method has been previously published (275) and a protocol is available
online (http://pegnac.sdsc.edu/cleveland-clinic/protocols/). Briefly, glycosaminoglycans
were precipitated from milk HA preparations by overnight precipitation in 75% ethanol at
-20°C. The ethanol suspension was centrifuged at 14,000 x g for 10 m and the
supernatant was discarded. Precipitated GAGs were dehydrated at room temperature and
resuspended in 100 mM ammonium acetate. The GAG suspension was incubated with
0.7 mU/µl chondroitinase ABC (Seikagaku Corp., Japan) and 0.07 mU/µl Streptococcus
dysgalactiae hyaluronidase (Seikagaku Corp., Japan) at 37° C for > 18 h. Non-HA or CS
106
GAGs were precipitated in 75% ethanol at -20°C. The ethanol suspension was
centrifuged at 14,000 x g for 10 minutes and the supernatant was separated, dehydrated,
resuspended in100 mM ammonium acetate and stored at -20° C for analysis of HA and
CS species by FACE. Both precipitated and supernatant GAGs were heated at 100° C for
5 m to inactivate chondroitinase and hyaluronidase enzymes. Precipitated GAGs were
resuspended in 100 mM ammonium acetate and incubated with an equal mixture of
Heparinase, Heparinase I and Heparinase II derived from Flavobacterium heparinum
(Seikagaku Corp., Japan) at 0.05 mU/µl at 37° C for > 18 h. Heparinase digestion
products were precipitated in 75% ethanol, dehydrated, resuspended in 100 mM
ammonium acetate and heated at 100° C for 5 m. Both HA/CS and HS preparations were
then labeled in 0.3 mM 2-aminoacridone (AMAC, Invitrogen, Carlsbad, CA) in a dark
chamber at 37°C for 18 h. Gels for HA/CS electrophoresis consisted of 20% acrylamide,
40 mM Tris-Acetate, 2.5% glycerol, 0.05% ammonium persulfate, and 0.1% N,N,N’N’Tetra-methylethylenediamine (TEMED, Bio-Rad Laboratories, Hercules, CA). Gels for
HS electrophoresis consisted of 20% acrylamide, 0.5% Tris-Borate-EDTA (TBE), 2.5%
glycerol, 0.05% ammonium persulfate, and 0.1% TEMED. Enzyme digestion products
were separated in the appropriate gel solutions at 500 V for 1.25 h at 4° C. The gels were
visualized and stored as digital images by illumination with 365 nm UV light from and
Ultra Lum Transilluminator and imaged using a Quantix-cooled CCD camera (Roper
Scientific/Photometrics, Sarasota, FL). Digital images were quantified using the
ImageScanner III with the ImageQuantTL v7.0 software package (GE Healthcare,
Piscataway, NJ).
107
3.2.5 Cell Culture
HT-29 epithelial cells, a line initially derived from a human intestinal tumor, were
cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and
incubated at 37° C in a humidified environment containing 5% CO2. Stock cultures were
split at a ratio of 1:20 once per week.
3.2.6 Experimental Cultures
HT29 cells were released from stock cultures by dissociation with solution containing
0.05% trypsin and 0.53 mM EDTA in phosphate buffered saline for 1.5 m. Collected
cells were washed and plated at a 1:15 area:area ratio in 12-well plates (Becton
Dickinson, Franklin Lakes, NJ) and grown until 70-80% confluent (3 days). On the day
of the experiment, growth medium was removed, and the HT-29 cells were treated with
fresh RPMI containing 10% FBS without or with human milk HA preparations at the
concentrations (0.001-5 µg/ml HA) and times (0-48 h) specified in the figure legends.
3.2.7 Hylauronidase Digestion of Human Milk HA Preparations
HA content of human milk preparations was specifically degraded to tetra- and
hexaccharides (276, 277) by incubating 10 µg/ml milk HA preparation with 0.25 U/ml
Stremptomyces hyalurolyticus hyaluronidase (EC 4.2.2.1; Seikagaku Corp., Japan) for 16
h at 60° C and subsequently heat-inactivated prior to use as a cell treatment. Complete
108
enzymatic digestion of hyaluronan in human milk HA isolates using the above procedure
was verified by ELISA.
3.2.8 Detection of HβD2 by Immunoblot (Western) Analysis
Whole cell lysates from HT-29 cells were isolated for Western blotting in the following
lysis buffer: 300 mM NaCl, 50 mM Tris pH 8.0, 0.5% NP-40, 1 mM EDTA, 10%
glycerol, protease inhibitor for mammalian tissue P8340 (Sigma-Aldrich Inc., St. Louis,
MO). Cell lysate proteins were separated by SDS-PAGE using pre-cast Tricine based 1020% gradient gels (Invitrogen, Carlsbad, CA). Separated proteins were transferred at 4°C
to PVDF membrane (Millipore Corp., Billerica, MA) by electroblotting apparatus (BioRad Laboratories, Hercules, CA) at 110 V for 60 m. In cases where analysis of numerous
replicate samples or multiple protein targets of differing molecular weight was required, a
multistrip Western blotting protocol was used to increase quantitative output and improve
signal consistency (249). PVDF membranes were blocked in Odyssey Blocking Buffer
(LI-COR Biosciences, Lincoln, NE) diluted to 50% concentration in Tris-buffered saline
(TBS) for 60 m. The membrane was incubated with rabbit polyclonal antibody against
HβD2 (Abcam, Cambridge, MA) at 1:750 in the blocking buffer, and the primary
antibody was followed by biotin conjugated anti-rabbit IgG (Jackson ImmunoResearch
Laboratories Inc., West Grove, PA) at 1:30,000 in blocking buffer and finally by
horseradish peroxidase (HRP)-conjugated streptavidin (GE Healthcare, Piscataway, NJ)
at 1:100,000 in TBS alone. Membrane-bound GAPDH protein expression was detected
by incubation with rabbit polyclonal antibody against GAPDH at 1:5,000 (Abcam,
Cambridge, MA) followed by HRP-conjugated donkey polyclonal anti-rabbit IgG
109
(1:20,000, GE Healthcare, Piscataway, NJ). All washing steps were conducted in Trisbuffered saline with 0.1% Tween-20. Protein bands were visualized using ECL prime
chemiluminescent development (GE Healthcare, Piscataway, NJ) and detection by
BioMax XAR scientific imaging film (Carestream Health Inc., Rochester, NY).
Differences in chemiluminescent signal intensity were quantified using the ImageScanner
III with the ImageQuantTL v7.0 software package (GE Healthcare, Piscataway, NJ).
3.2.9 Real-time Quantitative PCR Analysis of DEFB4 Expression
HT-29 cells were cultured and treated with human milk HA preparations as indicated
above. Cell lysates were collected for real-time quantitative PCR analysis using the Cellsto-cDNA kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
Briefly, cultured epithelium were released by dissociation with solution containing 0.05%
trypsin and 0.53mM EDTA in phosphate buffered saline for 1.5 m before being
resuspended in 1 ml sterile PBS at 4°C. Trypsinized cells were centrifuged at 1200 x g
for 5 minutes at 4°C before removal of the supernatant and addition of lysis buffer.
Following digestion of genomic DNA by DNase, reverse transcription using oligo-d(T)
primers and M-MLV RTase was completed in accordance with the manufacturer’s
instructions. The cDNA product was stored at -20°C prior to quantitative PCR analysis.
Validated primers with associated 6-carboxyfluorescein (FAM) fluorogenic probes for
DEFB4 (Assay ID: Hs00175474_m1) and 18S rRNA (Assay ID: Hs99999901_s1) were
purchased from Applied Biosystems (Invitrogen, Carlsbad, CA). The real time PCR
amplifications were performed in 25 µl reaction volumes containing TaqMan gene
expression Master Mix, primers and fluorogenic probes (Invitrogen, Carlsbad, CA), and
110
cDNA generated by the Cells-to-cDNA kit. All reactions were performed with four
replicate reactions using a Bio-Rad C1000 Touch Thermal Cycler with attached CFX96
Real-Time System. The real time PCR reaction conditions were 50°C for 2 m and 95°C
for 10 min, followed by 50 cycles of 95°C for 15 s and 60°C for 60 s. Real time detection
data was analyzed using Bio-Rad CFX Manager 2.1 software.
3.2.10 Detection of Murine HβD2 Ortholog by Fluorescence Histochemistry
Descriptions of fluorescence histochemistry and confocal microscopy were provided
previously (191). For tissue section staining, fixed, paraffin embedded mouse colon
sections were deparafinized and incubated in a blocking solution of Hank’s Balanced Salt
Solution (HBSS) containing 2% FBS for >30 minutes followed by overnight incubation
at 4° C in a solution of rabbit polyclonal antibody against MuβD3 (Santa Cruz
Biotechnology Inc., Santa Cruz, CA) at 1:100 dilution in HBSS with 2% FBS. Coverslips
were washed three times with HBSS before incubation in a solution containing Alexa568-tagged goat anti-rabbit IgG (1:1000) (Invitrogen, Carlsbad, CA) in HBSS with 2%
FBS for 1 h at 25°C. The coverslips were washed an additional three times in HBSS.
After washing, Vectashield mounting medium (Vector Labs, Inc., Burlingame, CA)
containing 4',6-diamidino-2-phenylindole (DAPI), which fluorescently labels DNA, was
used to adhere coverslips to antibody-labeled colon sections. Slides were stored at -20° C
until imaged. Confocal images were obtained using a Leica TCS-SP laser scanning
confocal microscope (Leica, Heidelberg, Germany).
111
3.2.11 Evaluation of Murine HβD2 Ortholog Expression in Nursing Mice
Six pups of conventionally housed Wild-type adult C57BL/6 mice were euthanized on
the day of birth, 10 days postpartum (during nursing), or after weaning (>4 weeks). 0.5
cm cross-sections were collected at regular intervals along the entire length of the bowel,
fixed in Histochoice (AMRESCO, Inc., Solon, OH) and paraffin embedded.
Representative cross-sections of the mouse intestine were deparaffinized and stained for
MuβD3, the murine ortholog of HβD2 as described above.
3.2.12 In Vivo Induction of Murine HβD2 Ortholog by Human Milk HA
Age and sex-matched Wild-type adult C57BL/6 were purchased from Jackson Laboratory
(Bar Harbor, ME) and housed by conventional methods. All treatments were conducted
according to Institutional Animal Care and Use Committee (IACUC)-approved protocols.
Nine mice were gavage-fed once per day 0.25 ml water alone (control) or 1µg milk HA
or an equivalent, donor-matched quantity of hyaluronidase-treated milk HA preparation
suspended in 0.25 ml water once daily for three consecutive days. Mice were sacrificed
16-18 h after the final gavage treatment, and 0.5 cm cross-sections of the proximal colon
descending from the ileocecal junction, the transverse colon equidistant to the ileocecal
junction and rectum, and the last 0.5 cm of the distal colon were excised from each
mouse, fixed in Histochoice (AMRESCO, Inc., Solon, OH) and paraffin embedded.
Representative cross-sections of the mouse distal colon were deparaffinized and stained
for MuβD3, the murine ortholog of HβD2 as described above.
112
3.2.13 Evaluating the role of TLR4 and CD44 in HA-35 induced HβD2 expression in vivo
Age-matched male C57/Bl6 (“Wild-type”), B6.129(Cg)-CD44tm1Hbg/J (CD44-/-), and
B6.B10ScN-Tlr4lps-del/JthJ (TLR4-/-) mice were purchased from Jackson Laboratory (Bar
Harbor, ME) and housed by conventional methods. All treatments were conducted
according to Institutional Animal Care and Use Committee (IACUC)-approved protocols.
Five mice of each of the three genotypes were gavage-fed tap water alone (250 ml) or a
solution containing milk HA preparation in tap water (1 µg HA in 250 ml) once daily for
three consecutive days. Mice were sacrificed 16-18 h after the final gavage treatment and
a 2 cm section of the proximal colon descending from the ileocecal junction was excised
from each mouse, opened, pressed flat between two layers of paper towel (Peterson
2008), fixed overnight in Histochoice (AMRESCO, Inc., Solon, OH) and paraffin
embedded. Longitudinal sections of the mouse proximal colon were deparaffinized and
stained for MuβD3 as described in histological methods.
3.2.14 Quantization of Histological Observations
Individual sections were labeled in a random fashion so as to shield the microscopist
from knowledge of mouse genotype or treatment status. Ideal fluorescent signal exposure
times for capturing the complete range of MuβD3 staining intensity were determined at
the outset of the experiment by a survey of ten random sections and were held constant
for subsequent image capture. Three MuβD3 stained fields of colonic epithelial
structures, as well as one unstained control from each animal, were digitally captured
from longitudinal sections of each of the 40 mouse proximal colon samples. Capture
fields were selected on the basis of epithelial tissue morphology as determined by DAPI
113
staining. Each of the 160 images (three stained fields and one unstained field from each
of the 30 mice) were graded on a 0-4 scale by a panel of four blinded researchers, with a
grade of 0 indicating no MuβD3 staining and a grade of 4 corresponding to peak MuβD3
staining intensity (Chapter 2). The mean scores given by the evaluating panel for each of
the 140 images were averaged for each mouse before treatment and genotype status were
revealed according to a key.
3.2.15 Salmonella enterica Infection of Cultured Epithelium
HT-29 colonic epithelium were cultured in 48-well tissue culture plates (Corning Inc.,
Lowell, MA) according the protocol described above. 24 h prior to infection, HT-29 cells
were treated with new medium containing 0.5 µg/ml milk HA or an equivalent quantity
of donor-matched hyaluronidase-treated milk HA preparation in RPMI with 10% FBS, or
medium alone. Salmonella enterica serovar Typhimurium SL1344 (gift of Dr. Gabriel
Nuñe, University of Michigan) was cultured overnight in LB broth at 30°C with shaking
at 200 rpm before dilution at 1:7 in new LB broth the next day and further cultured until
A600=0.5 or approximately 3.5 x 108 cfu/ml. 24 h after the addition of milk HA or control
treatments, HT-29 epithelial cells were washed twice with fresh RPMI medium followed
by infection with new medium containing approximately 4.7x107 cfu/ml Salmonella
enterica serotype Typhimurium SL1344 taken from the 1:7 LB broth culture. Following a
30 min infection period, the cell culture medium containing Salmonella was removed and
HT-29 epithelium was washed twice with sterile phosphate-buffered saline (PBS) before
the addition of medium containing the host cell impermeable antibiotic gentamicin at 50
ng/ml (Sigma-Aldrich, St. Louis, MO) for an additional 1 h. HT-29 cells were then
114
washed twice and lysed in ice cold PBS containing 1% Triton X-100. Dilutions of this
lysis solution were spread on sterile LB agar plates and cultured overnight at 30°C.
Colony forming units were counted on the following day. The experimental design
included no less than 6 replicate culture wells per treatment, and 3 technical replicates
(LB Agar plates) per culture well and was separately replicated with three separate milk
HA isolates derived from unique donors.
3.2.16 Statistical Analysis
Statistical difference between treatment groups was evaluated where appropriate by
unpaired two-tailed Student’s t-test, and all error bars drawn to indicate the standard error
of the means (S.E.M.). A paired two-tailed Student’s t-test was used to evaluate the
difference between sigmoidal dose-response relationships. Differences were considered
significant when P < 0.05. Statistical analysis, including linear regression analysis of milk
HA concentration over days postpartum generated from analysis of donated milk samples
by ELISA, was performed using R version 2.12.1 for Mac OS X (R Foundation for
Statistical Computing; available on the World Wide Web). Graphing was completed
using R version 2.12.1 for Mac OS X or GraphPad Prism version 4.0c.
3.3 RESULTS
3.3.1 Human Milk HA Concentrations are Greatest in the First 60 Days Postpartum
While human milk is known to contain HA (167), variation in concentration between
individuals and over time after delivery is not known. We examined milk HA
115
concentration during the first 6 months postpartum among a group of 44 (41 Caucasian, 2
Asian, 1 African-American) mothers from the Cleveland metropolitan area who donated
milk samples at daily intervals starting in the first week after giving birth. The HA
concentration was determined in unprocessed milk samples using an competitive
enzyme-linked immunosorbent assay. As a whole, HA concentrations were greatest in the
immediate postpartum period, with a mean concentration of 755.01±94.15 ng/ml within
the first week after birth, and decreased at a linear rate over the first 60 days postpartum
(m= -10.9±1.2 ng/ml per day, R2=0.11, p< 1.0 x 10-16) (Figure 3-1). The mean HA
concentration in samples collected during the first 60 days postpartum was 452.34±19.58
ng/ml. Average HA concentrations were essentially static at 215.74±5.77 ng/ml at time
points greater than 60 days after birth (m= -0.4±0.1 ng/ml per day, R2=0.04, p= 1.4 x 109
), and persisted up to one year.
3.3.2 Milk HA Specifically Induces HβD2 Expression
Our group previously demonstrated that synthetically produced HA promotes enhanced
expression of HβD2 in intestinal mucosa (Chapter 2). However, the function of
hyaluronan contained in milk relevant to epithelial antimicrobial function remains
unknown. We hypothesized that hyaluronan from human breast milk would induce HβD2
expression in intestinal epithelium. To this end, we developed a method to purify HA
from the proteins, lipids and sugars and charged carbohydrate polymers (described in
methods) from the chemically complex milk. The concentration of HA in the milk HA
isolates was determined by ELSA, with an average HA yield of 55% relative to
unprocessed whole milk samples. Analysis of HA polymer size in milk preparations by
116
agarose gel electrophoresis (248) revealed broad polydispersity, between 104-106 Da
polymers (unpublished data). Our milk-HA preparations also contained chondroitin as a
contaminant (Table 5-1) that we were unable to specifically remove from our batch
preparations. This is consistent with the analysis of Coppa, et al., who reported an excess
of undersulfated chondoitin (167) in human milk.
We tested the ability of our milk HA preparations to promote expression of HβD2 protein
in the human intestinal epithelial cells. Cultured HT-29 cells were treated with 500 ng/ml
HA derived from pooled milk HA preparations obtained from three individual donors and
HβD2 protein was measured in cell lysates by immunoblot assay (Figure 3-2). Induction
of HβD2 protein expression relative to GAPDH occurs within 12 hours following
treatment with milk HA isolates. Maximal HβD2 protein expression in milk HA -treated
HT-29 cells was observed at 24 h after initial treatment (p= 0.006 vs. 0 h), with elevated
HβD2 protein levels still present at 48 h post-treatment. To confirm that increases in
HβD2 protein expression were specifically induced by HA in the milk preparations, HT29 cells were treated for 24 h with medium alone, milk HA isolates (0.5 µg/ml HA), or
donor-matched milk HA isolates that were predigested with a substrate-specific
hyaluronidase derived from Stremptomyces hyalurolyticus. Figure 3-2 demonstrates a
highly significant 4.5 fold increase in normalized HβD2 protein expression relative to
both treatment with medium alone and treatment with hyaluronidase-digested milk HA
isolates (p< 0.0001 and p= 0.0008, respectively). However, hyaluronidase-digested milk
HA preparations still retained the ability to induce a 2-fold increase in HβD2 protein
expression relative to treatment with medium alone (p= 0.0101). Furthermore, we
evaluated the effects of milk HA, on expression of the gene encoding the HβD2 peptide,
117
DEFB4 (Figure 3-2). Quantitative real-time PCR analysis revealed notable induction of
DEFB4 mRNA expression in HT-29 cells treated for 24 h with milk HA (500 ng/ml HA)
relative to treatment with medium alone (p= 0.0073). The effect of milk HA on DEFB4
expression was mostly dependent upon the presence of HA, as indicated by the
significant reduction in induced DEFB4 in HT-29 cell treated with hyaluronidasedigested milk HA (p= 0.04 vs. milk HA + HAse, Figure 3-2). The data presented in
Figure 3-2 represent the collective results of identical experiments conducted with three
separate milk HA isolates derived from unique donors. Given that milk HA concentration
varies over time and between individuals (Figure 3-1), we evaluated the induction of
HβD2 protein expression in vitro following milk HA isolate treatment over a dose range
of 0.001-5 µg/ml HA for 24 h (Figure 3-2). Matched aliquots of milk HA preparations
were pre-digested with Stremptomyces hyalurolyticus hyaluronidase and tested across the
same dilution range to specifically determine the contribution of HA to the dose-response
relationship of milk HA preparations to HβD2 protein induction in HT-29 intestinal
epithelium. Milk HA treatment resulted in significantly increased HβD2 protein
expression at a concentration range of 0.5-5.0 µg/ml HA. Importantly, HβD2 induction
was significantly reduced in cells treated with hyaluronidase treated milk isolates at doses
greater than 0.1 µg/ml HA (EC50=5.94 µg/ml vs. 0.03 µg/ml, respectively, with p=0.04),
although HβD2 protein expression was increased above background levels at doses
greater than 0.5 µg/ml HA (p= 0.01 vs. control).
Cumulatively, these findings (Figure 3-2) indicate that HA contained in human milk
specifically enhances induction of DEFB4 transcription and HβD2 protein expression in
118
HT-29 colonic epithelium. These effects of milk HA are both time- and concentrationdependent.
3.3.3 Oral Administration of Human Milk HA Promotes In Vivo Expression of the Murine
HβD2 Ortholog in Intestinal Epithelium
Since milk HA greatly enhances the induction of HβD2 protein expression in vitro, we
next tested whether isolated human milk HA could promote expression of murine HβD2
ortholog in colonic mucosa in adult mice. Nine age- and sex-matched adult wild-type
C57BL/6 mice were segregated equally into the following treatment groups: control, milk
HA, or donor-matched milk HA that was predigested Stremptomyces hyalurolyticus. All
animals were gavage-fed 0.25 ml water without (control), or containing isolated milk HA
(1 µg HA) or an equivalent mass of hyaluronidase-digested milk HA once daily for three
consecutive days. Fixed proximal colon tissue sections were analyzed for MuβD3
staining, the murine ortholog of HβD2 (253), using immunofluorescence microscopy.
Blinded microscopy analysis of intestinal tissue staining revealed enhanced epithelial
MuβD3 protein expression in colonic mucosa of mice fed milk HA compared to controltreated animals, or those receiving hyaluronidase degraded milk HA (Figure 3-3).
Representative images in Figure 3-3 show the median staining intensity of each of the
respective treatment groups.
3.3.4 The Intestinal Epithelium of Nursing Mice Expresses the HβD2 Ortholog
Since milk HA isolates specifically promote the expression of MuβD3 in adult mice, we
hypothesized that this observation may reflect a physiologic process that occurs in
119
nursing young. To test this in the mouse model, we examined expression of MuβD3 in
the intestinal mucosa of healthy, wild-type C57BL/6 mice at one and ten days after birth,
and five days after weaning (Figure 3-3). Analysis of immunofluorescently stained
MuβD3 protein in cross-sectioned intestinal mucosa revealed that expression was greatly
increased in the intestinal mucosa in the period between the first and tenth day after birth,
during which time the animals were deriving complete nutrition from the mother. After
weaning, baseline MuβD3 protein expression was relatively reduced in intestinal mucosal
tissue, suggesting a correlation between milk consumption and intestinal mucosal
MuβD3 expression.
3.3.5 In vivo induction of murine HβD2 by milk HA is both CD44 and TLR4 dependent
Among the known HA cell surface receptors, TLR4 has been implicated repeatedly in the
induction of augmented innate epithelial defense by HA in both in vitro and in vivo
experimental models (239-241, Chapter 2). In addition, CD44 is reported to mediate a
wide variety of HA-dependent signaling responses (224, 225, 228, 231, 232). Therefore,
CD44 and TLR4 were evaluated as candidate receptors mediating the HA-enhanced
induction of murine HβD2 ortholog following oral administration of human milk HA in
vivo. Adult wild-type, CD44-/-, and TLR4-/- C57BL/6 mice were gavage-fed 0.25 ml
water alone (Control), or containing milk HA (1µg HA) once daily for three consecutive
days, with 10 mice per genotype divided equally between the two treatment groups. Mice
were sacrificed 16-18 h after the final gavage treatment. Proximal colon tissue was fixed
and cut in longitudinal sections for MuβD3 immunofluorescent staining. Figure 3-4
presents representative images of MuβD3 protein expression in immunostained murine
120
proximal colonic epithelium of both control and milk HA-treated animals from each of
the three genotypes. Blinded analysis of fluoresent microscopy images utilizing an
immunostain scoring system (Materials and Methods) revealed significant induction of
MuβD3 protein expression occurred in the colonic epithelium of wild-type animals
following oral administration of the milk HA preparation relative to control-treated wildtype mice (p= 0.009; Figure 3-4). However, expression of MuβD3 in the colonic mucosa
of TLR4-/- or CD44-/- animals was unchanged after oral administration of milk HA
relative to control-treated animals of each genotype (p=0.387 and 0.311, respectively).
Significantly lower MuβD3 staining intensity was observed in TLR4-/- and CD44-/- mice
following oral administration of milk HA relative to milk HA-treated wild-type mice (p=
0.007 and 0.0002, respectively; Figure 3-4). Thus, genetic deletion of either TLR4 or
CD44 is sufficient to abrogate the induction of MuβD3 expression in the colonic
epithelium following oral administration of milk HA.
3.3.6 Milk HA Enhances Resistance to Intracellular Salmonella Infection in Intestinal
Epithelium
Inhibition of growth and infection by pathogenic organisms by direct interaction with
human milk glycans with varying structures and chemical properties, including GAGs,
have been previously reported (53, 158-161, 168). One previous report also suggests that
specific human milk oligosaccharides may directly protect intestinal epithelial cells from
gastrointestinal infection (162). Based on our data indicating that human milk HA
promotes induction of antimicrobial defense in intestinal mucosa (Figure 3-2, 3-3, and 34), we hypothesized that human milk HA also enhances functional resistance of human
121
colonic epithelium to the intracellular enteric pathogens. To test this hypothesis we
utilized an in vitro assay modified from Homer et al. (278), which employs Salmonella
enterica serovar Typhimurium, a significant source of morbidity in infants (140), as the
model organism. Confluent HT-29 colonic epithelial monolayers were treated for 24 h
with medium alone, medium containing 500 ng/ml of milk HA preparation, or donormatched milk HA that was pre-digested with Stremptomyces hyalurolyticus. After
incubation, cell culture medium was removed from all wells and replaced with new
medium containing live Salmonella enterica serovar Typhimurium SL1344. After a short
infection period, the antibiotic gentamicin was added in fresh culture medium to
eliminate extracellular Salmonella while preserving bacterium present within the host
epithelial cell cytoplasm. Epithelial cells were then lysed and intracellular bacteria
cultured to determine colony-forming units (CFU) per epithelial culture (Figure 3-5).
Pre-treatment of HT-29 cells with milk HA resulted in a highly significant decrease
(-24.6±7.5%) in CFU Salmonella recovered relative to epithelial cells pre-treated with
medium alone (p<0.0001) or hyaluronidase digested milk HA (p<0.0001, Figure 3-5).
Importantly, the data presented in Figure 3-5 represent the combined results of identical
experiments conducted with three distinct isolates of milk HA derived from unique
donors. These data indicate that milk HA enhances functional resistance to intracellular
Salmonella enterica Typhimurium infection in pre-treated host epithelium in vitro.
122
3.4 DISCUSSION
Our recent report indicating that HA promotes expression of the antimicrobial peptide
HβD2 in intestinal epithelium (Chapter 2), and the observation that human milk contains
HA (167) resulted in our hypothesis that HA supplied in human breast milk enhances
innate intestinal epithelial antimicrobial defense. Our data support the finding of Coppa et
al. (167) that human milk contains HA, while further defining the time-dependent change
in milk HA concentration from the start of lactation through the first 6 months
postpartum. In addressing our hypothesis, we have demonstrated two parameters of
epithelial antimicrobial defense that are greatly augmented by HA supplied in breastmilk. First, cultured human colonic epithelial cells or the intestinal mucosa of wild-type
mice treated with milk HA at human physiologically relevant concentrations exhibit
dramatic induction of HβD2 expression that is significantly reduced by pre-digestion
with a substrate-specific hyaluronidase. Similar induction of murine HβD2 ortholog is
observed in the intestinal mucosa of nursing mice relative to newborn or weaned animals.
The induction of murine HβD2 ortholog by milk HA is dependent upon both HA cell
surface receptors TLR4 and CD44 in vivo. Second, cultured monolayers of colonic
epithelium exhibit enhanced resistance to infection with the enteric pathogen Salmonella
enterica serovar Typhimurium SL1344 following pre-treatment with milk HA isolates, an
effect that is also abolished by hyaluronidase digestion. Taken together, our data indicate
that HA naturally supplied in human breast milk contributes significantly to the induction
of antimicrobial defense in intestinal epithelium.
123
Analysis of 1710 unique milk samples collected from a cohort of 44 mothers revealed
high concentrations of HA in the initial weeks of lactation (Figure 3-1). Despite wide
variation between individual donors, HA concentrations in milk consistently decreased
over the first 8 weeks postpartum, reaching a steady-state concentration by the third
month after birth. Importantly, all samples evaluated contained detectable levels of HA,
adding to the recent findings of Coppa et al. (167, 274), which utilized pooled milk
samples collected from a limited cohort, to suggest that HA is a universally expressed
component of human milk. The use of a competitive ELSA assay that was highly specific
to HA but independent of HA polymer size (data not shown) allowed for reproducible
quantitation of HA in whole milk samples without biochemical manipulation of the
sample. Numerous bioactive components of human milk are present in high
concentrations in the early stages of lactation, including other GAGs (274), sIgA (145)
and fatty acids (279). Given the critical nature of the immediate postnatal period and the
high susceptibility of newborns to infection (136, 140), it is perhaps unsurprising to find
that HA in milk is also present at higher concentrations in the first weeks after birth.
Maternal genetics and nutrition likely contribute to the intra-donor variation in HA
content we have observed.
Milk HA treatment exhibited both time-and dose-dependent induction of HβD2 protein
in cultured HT-29 epithelium (Figures 3-2), with maximal activity at 0.5 µg/ml, the
approximate mean concentration of HA present in milk within the first 30 days
postpartum (Figure 3-1). In addition, transcription of the gene encoding the HβD2
peptide, DEFB4, was specifically and significantly up-regulated by milk HA at
124
physiologic concentration (Figure 3-2), indicating that the increased expression of HβD2
peptide is mediated by transcriptional activation.
Numerous reports have indicated that the signaling properties of HA, including the
induction of HβD2 (Chapter 2), are highly size-specific (166). Size analysis of the HA
we isolated from milk indicates broad polydispersity, containing 104-106 Da polymers,
and with polymer size distribution varying widely between individuals (unpublished).
Future studies may be required to determine the size-specific bioactivity of naturally
occurring milk HA. Importantly, the milk HA preparations isolated from unique donors
independently exhibited similar HβD2 and DEFB4 inducing activity at similar
concentrations (Figure 3-2), indicating that the expression of bioactive HA in milk is
likely to be widespread among the general population. In addition, while previous work
suggests that HA acts as a specific inducer of HβD2 in intestinal epithelium independent
of other ligands (Chapter 2), we cannot exclude the possibility that additional
contaminant milk glycans contribute to or potentiate induction of HβD2 by milk HA.
Analysis of the milk HA preparations indicates that undersulfated chondoitin is the
predominant non-HA component in the isolates (Table 5-1). Indeed, hyaluronidasetreated milk preparations promoted some, albeit greatly reduced, induction of HβD2
protein and DEFB4 transcription at physiologically relevant concentrations (Figure 3-2).
However, substantially increased doses of hyaluronidase-treated milk HA isolates were
required to achieve effects comparable to treatment with milk preparations containing
intact HA. The observed EC50 of hyaluronidase-treated milk isolates is 200-fold higher
than the HA containing milk preparation (Figure 3-2). Clearly the presence of HA in
milk isolates contributes significantly to HβD2 induction. In addressing our hypothesis
125
that HA in milk enhances innate intestinal epithelial antimicrobial defense, evaluating
induction of antimicrobial peptide HβD2 by milk HA presented in context with other
milk carbohydrates has merit in that it perhaps more nearly replicates the physiologic
setting of milk HA presentation to the neonatal intestinal mucosa.
The intestinal mucosa of nursing mice expresses enhanced MuβD3 peptide relative to
newborn or weaned animals (Figure 3-3), suggesting that milk components contribute to
induction of MuβD3. We have previously detected HA in mouse milk (unpublished) and
together these observations indicate that mice are a relevant animal model for the
evaluation of the hypothesis that milk HA specifically enhances epithelial antimicrobial
MuβD3 expression in vivo. Oral administration of human milk HA to adult, wild-type
C57BL/6 mice resulted in substantial induction of the murine HβD2 ortholog in the
mucosa of the proximal colon relative to administration of control or hyaluronidase
degraded human milk preparations (Figure 3-3), recapitulating the induction of MuβD3
observed in nursing mice (Figure 3-3). Consistent with our in vitro observations,
induction of murine HβD2 ortholog in vivo by the human milk HA preparation is
substantially reduced in the absence of intact hyaluronan, suggesting that the presence of
HA in the milk contributes significantly to defensin induction. In addition, upregulated
expression of MuβD3 in the murine intestinal epithelium is seen distally in the transverse
colonic mucosa (Figure 5-9), suggesting that human milk HA retains bioactivity within
the digestive tract in the context of both murine and microflora-driven catabolic activity.
Induction of the murine HβD2 ortholog following oral administration of milk HA is
dependent upon expression of cell surface receptors TLR4 and CD44. Expression of
126
MuβD3 was significantly enhanced in the intestinal mucosa of wild-type animals
following oral administration of human milk HA. In contrast, MuβD3 expression was
similar to control treatment in both TLR4- and CD44-deficient animals following
administration of the milk HA preparation for 3 days (Figure 3-4). Prior work in animal
models has principally implicated TLR4 in HA-dependent modulation of intestinal
defense (239, 240), particularly following oral administration of HA (264, Chapter 2),
and the induction of HβD2 in cultured keratinocytes is dependent upon TLR4 (241). Our
results suggest that TLR4 is an essential cell surface receptor for the induction of murine
HβD2 ortholog by milk HA, a finding that is consistent with our previous report that
induction of MuβD3 by synthetic HA is TLR4-dependent (Chapter 2). However, CD44dependent induction of defense effectors has been reported in monocytes and
macrophages (227, 228, 232) and renal epithelium (225), and CD44 is required for the
induction of murine HβD2 ortholog following oral administration of milk HA (Figure 4).
To our knowledge, this is the first report to suggest a role for CD44 in the regulation of
defensin expression. Thus, both TLR4 and CD44 are required in the response to milk HA
in vivo, and genetic deletion of either putative HA receptor is sufficient to abrogate the
induction of MuβD3. Previous work by Taylor et al. (227) suggests a mechanism by
which TLR4 and CD44 complexes, colocalized in the cell membrane, cooperatively
regulate the macrophage response to HA fragments generated in sterile injury through a
shared signal transduction pathway. Recognition of milk HA and subsequent signal
transduction resulting in MuβD3 expression may utilize similar TLR4/CD44 receptor
complexes expressed on the intestinal epithelial surface. Alternately, separate signal
transduction pathways independently activated by TLR4 and CD44 may act in a
127
complementary manner to enhance defensin expression in the presence of broadly
polydispersed milk HA. While TLR4-dependent induction of HβD2 following
stimulation with bacterial PAMPs acts through the canonical TLR signal transduction
mediators IRAK, TRAF6, and JNK to promote translocation of the transcription factor
AP-1 and engagement of the DEFB4 promoter (32, 238), HβD2 expression is also
regulated by other ligands via the MAPK/ERK signal transduction pathway (32, 280).
Engagement of HA with CD44 has repeatedly been demonstrated to result in specific
MAPK/ERK activation (281, 282) in a mechanism that is potentially dependent on HA
size (283), and milk HA may induce HβD2 expression in a CD44-dependent manner
through this pathway. Future studies will be required to distinguish between cooperative
and complimentary signal transduction mechanisms regulating the TLR4- and CD44dependent response to milk HA.
Treatment of cultured colonic epithelium with human milk HA enhances resistance to
infection by the enteric pathogen Salmonella enterica servar Typhimurium SL1344
(Figure 3-5). Following infection, a significantly reduced quantity of viable Salmonella
was harvested from HT-29 epithelial cultures that had been pre-treated with human milk
HA relative to epithelium treated with medium alone or medium containing
hyaluronidase-digested human milk isolates, thus providing functional evidence in
support of the hypothesis that endogenous human milk HA enhances antimicrobial
defense of intestinal epithelium. Experimental conditions included the removal of
medium containing human milk HA prior to the introduction of Salmonella, suggesting
that the observed reduction in Salmonella infection occurs due to modification of host
128
epithelium. While numerous examples of direct pathogen inhibition through interaction
of human milk glycans with microbial surface receptors have been reported (53, 158,
160, 161, 168), infection resistance resulting from modulation of epithelium by human
milk glycans has not been extensively studied. Treatment of cultured Caco-2 epithelium
with sialyllactose, a human milk oligosaccharide, results in the down-regulated
expression of specific glycan epitopes on the surface of the host epithelium correlating
with a reduction in the binding of pathogenic E. coli (162). Milk HA may act similarly to
enhance resistance to Salmonella in HT-29 epithelium through the modulation of surface
receptor expression.
Intriguingly, peak milk HA-dependent expression of antimicrobial HβD2 peptide
occurred at same time point as peak milk HA-dependent protection against Salmonella.
This implies a potential role for HβD2 in the observed resistance to intracellular
infection. Accumulation of intracellular defensins has been demonstrated to inhibit
replication of the obligate intracellular pathogen Listeria monocytogenes in macrophages
(259), and a similar mechanistic link between HβD2 and resistance to intracellular
Salmonella may exist in milk HA-treated intestinal epithelium. While questions regarding
the mechanism of milk HA-mediated Salmonella infection resistance remain, our results
suggest that milk HA may contribute to the reduced incidence of enteric Salmonella
infection associated with breast-feeding in human infants (140). Numerous prior reports
have implicated HA in modulation of the immune response (166, 227, 229), however this
is the first report to our knowledge to suggest a role for endogenous HA in resistance to
enteric infection through direct effects on epithelium.
129
Our observations are consistent with prior publications indicating that human milk
contains HA (167, 274), while providing the first evidence that human milk HA has
biological function at physiologic concentrations. At least two previous reports have
indicated that oral administration of HA results in altered function of innate (Chapter 2)
or adaptive (264) components of intestinal defense, however it was previously unclear if
these observations reflected an endogenous physiological process. The role of human
milk glycans, including HA, in generating intestinal homeostasis in breast-fed infants in
the presence of diverse and persistent microbial challenges is incompletely understood.
Milk contains a diverse bacterial community (284), seeding the developing
gastrointestinal microbiome along with maternally and environmentally acquired species
and interacting with the antigen-naive adaptive mucosal immune system (152). The
combined effect of milk probiotics, prebiotics, antibiotics, immunomodulators, and
microbial transfer is evident in the observation that breast-fed infants exhibit dramatically
different gastrointestinal microbial communities in comparison to artificially-fed infants
(8, 9, 132). Given the robust correlation between breast-feeding and gastrointestinal
health (10, 136, 138, 158), the role of breast milk in establishing the microbial-epithelial
interface may be of critical importance to lifelong GI health (285), potentially accounting
in part for the reduced lifetime risk of inflammatory bowel disease (137), obesity (141,
142), and allergic disease (143, 144) associated with breast-feeding. The bioactivity of
milk components relative to the function of intestinal epithelium, such as the induction of
HβD2 in intestinal epithelium by milk HA, could have a profound effect in shaping
intestinal microbial ecology through the modulation of epithelial substrates and the
generation of selective niches (18). Altered expression of α-defensins in the intestine has
130
been shown to result in significant shifts in microbial species distribution (270), and our
data demonstrate that the effect of milk HA on intestinal epithelium alters the interaction
of host epithelium with at least one relevant component of the human microbiome,
Salmonella enterica. Future studies will likely enhance understanding of the complex
dynamic regulating host epithelium and microbial interactions and the role of dietary
components in shaping the relationship between enterocyte and bacterium. Of more
immediate practical relevance, the supplementation of artificial formulas with HA among
other glycan components of human milk may reduce the incidence of enteric infection
and diarrhea among susceptible infants (158).
131
Figure 3-1. Human Milk HA Concentrations are Greatest in the First 60 Days
Postpartum
FIGURE 3-1. Distribution of Milk HA concentration by postpartum day in 1710 human
milk samples collected from 44 unique donors during the first 6 months after delivery. A
fourth degree polynomial regression curve was fitted to the entire data set and is indicated
by the solid black line, with the 95% confidence interval of the curve represented by the
dashed blue lines (R2=0.17, p= 6.1 x 10-67).
132
Figure 3-2. Milk HA Specifically Induces HβD2 Expression
A
FIGURE 3-2. A. Average densitometric quantification of immunoblots from four
individual experiments in which the abundance of HβD2 protein relative to GAPDH
protein was evaluated in whole cell lysates of HT-29 cells. Replicate cultures were
treated with 0.5 µg/ml milk HA for the time intervals indicated (0- 48 h).
133
Figure 3-2 (cont.)
B
C
FIGURE 3-2. B. Representative Western blot demonstrating HβD2 protein expression
relative to GAPDH in the whole cell lysates of HT-29 cells treated for 24 h with medium
alone, medium containing Milk HA isolate containing 0.5µg/ml HA or medium
containing 0.5 µg/ml milk HA pre-digested with Stremptomyces hyalurolyticus
hyaluronidase at 0.25 U/ml for 16 h at 60° C. C. Average densitometric quantification of
immunoblots from four individual experiments each separately using three donor-unique
milk HA isolates in which the abundance of HβD2 protein relative to GAPDH protein
was evaluated in whole cell lysates of HT-29 cells. Replicate cultures were treated for 24
h with medium alone, 0.5 µg/ml milk HA or medium containing 0.5µg/ml milk HA predigested with S. hyalurolyticus hyaluronidase.
134
Figure 3-2 (cont.)
D
FIGURE 3-2. D. Combined real-time quantitative PCR results from four individual
experiments each separately using three donor-unique milk HA isolates in which the
abundance of DEFB4 mRNA relative to 18S rRNA was evaluated in whole cell lysates of
HT-29 cells. Replicate cultures were treated for 24 h with medium alone, 0.5µg/ml milk
HA or medium containing 0.5 µg/ml milk HA pre-digested with S. hyalurolyticus
hyaluronidase. Significance of differences in normalized HβD2 or DEFB4 expression
were evaluated by comparison of each time point to control treatment, unless otherwise
indicated by brackets, using Student’s t-test, with ‘*’ indicating P<0.05, and ‘**’
indicating P < 0.01.
135
Figure 3-2 (cont.)
E
FIGURE 3-2. E. Average densitometric quantification of immunoblots from six
individual experiments in which the abundance of HβD2 protein relative to GAPDH
protein was evaluated in whole cell lysates of HT-29 cells. Replicate cultures were
treated for 24 h with 0.001 – 5 µg/ml milk HA or medium containing 0.001 – 5 µg/ml
milk HA pre-digested with S. hyalurolyticus hyaluronidase. Sigmoidal dose-response
relationships are indicated, with the solid black curve corresponding to Milk HA isolate
treatment (EC50=0.03 µg/ml) and the dashed black line corresponding to hyaluronidasedigested Milk HA treatment (EC50=5.94 µg/ml), with the observed difference between
treatment responses being statistically significant by two-sided Student’s t-test analysis
(p=0.04).
136
Figure 3-3. Oral Administration of Human Milk HA Promotes In Vivo Expression
of the Murine HβD2 Ortholog in Intestinal Epithelium
A
B
FIGURE 3-3. A. Representative fluorescent micrographs of epithelium in proximal
colonic cross-sections from adult C57BL/6 wild-type mice. The mice were given single
daily doses of 1 µg milk HA in .25ml water (Milk HA) or 1 µg Milk HA that had been
pre-digested with S. hyalurolyticus hyaluronidase in 0.25 ml water (Milk HA + HAse) or
water alone (Control) for three consecutive days. MuβD3 is fluorescently immunolabeled
(red) and nuclei are stained with DAPI (blue). B. Representative fluorescent micrographs
of epithelium in intestinal cross-sections from newborn (day 1), nursing (day 10) or
weaned (28 days) mice. The mouse ortholog of HβD2, MuβD3, is fluorescently
immunolabeled (red) and nuclei are stained with DAPI (blue). ‘NS’ indicates an
immunostaining control in which no MuβD3 antibody was utilized.
137
Figure 3-4. In vivo induction of murine HβD2 by milk HA is both CD44 and TLR4
dependent
A
FIGURE 3-4. A. Representative fluorescent micrographs of epithelium in proximal
colon cross-sections from adult C57BL/6 wild-type, TLR4-/-, or CD44-/- mice. The mice
were given single daily doses of Milk HA containing 1 µg HA in 0.25ml water or water
alone (Control) by oral administration for three consecutive days. MuβD3 is fluorescently
immunolabeled (red) and nuclei are stained with DAPI (blue).
138
Figure 3-4 (cont.)
B
FIGURE 3-4 B. Average scored MuβD3 staining intensity of proximal colon tissue
sections from wild-type, TLR4-/-, or CD44-/- mice given single daily doses of 1µg milk
HA in 0.25ml water or water alone (Control) by oral administration for three consecutive
days. Average MuβD3 staining intensity score represents 5 mice per group, with 4
stained sections per mouse, as judged by a blinded panel of 5 researchers on a scale of 0
to 4 with a score of 4 corresponding to peak MuβD3 staining. Mean staining intensity
score (±S.E.) of non-specifically stained sections (1 per mouse) was 0.26±0.06.
Significance of differences in mean MuβD3 staining intensity were evaluated using a
two-tailed Student’s t-test with ‘**’ indicating P< 0.01 for the comparison indicated by
the bracket, and ‘##’ and “###” indicating P<0.01 or P<0.001, respectively, for the
comparison with wild-type mice receiving milk HA.
139
Figure 3-5. Milk HA Enhances Resistance to Intracellular Salmonella Infection in
Intestinal Epithelium
Figure 3-5. A box plot representing the number of colony forming units (CFU) of
Salmonella enterica serovar Typhimurium SL1344 collected by area of host HT-29
epithelium subject to pretreatment for 24 h with 0.5µg/ml milk HA or 0.5µg/ml milk HA
pre-digested with S. hyalurolyticus hyaluronidase, or medium alone. Horizontal lines
indicate median CFU/cm2 with inter-quartile range denoted by the shaded box and the
full range of observations represented by the error bars. Significance of differences in
CFU/cm2 were evaluated as indicated by brackets using Student’s t-test with ‘***’
indicating P<0.001.
140
CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS
4.1 SUMMARY OF FINDINGS
When viewed comprehensively, the data produced from the investigations presented in
Chapters II and III offer convincing evidence in support of the hypothesis that
hyaluronan promotes innate defense of the intestinal epithelial barrier. In summary, HA
fragments promote expression of the innate antimicrobial peptide human β-defensin 2
(HβD2) in intestinal epithelial cells. Treatment of HT-29 colonic epithelial cells with HA
fragment preparations resulted in time- and dose-dependent upregulated expression of
HβD2 protein in a fragment size-specific manner, with 35 kDa HA fragment preparations
(HA-35) emerging as the most potent inducers of intracellular HβD2. Furthermore, oral
administration of specific-sized HA fragments promotes the expression of an HβD2
ortholog in the colonic epithelium of both wild-type and CD44-deficient mice, but not in
TLR4-deficient mice.
Concurrent with these investigations was the report that human milk contains hyaluronan
(167, 274). While synthetic HA of a specific size range promotes the expression of
antimicrobial peptides in intestinal epithelium, the function of naturally produced milk
hyaluronan relevant to epithelial antimicrobial function was unknown. We hypothesized
that hyaluronan from human milk enhances innate intestinal epithelial antimicrobial
defense. Here we define the expression of milk HA concentration during the first 6
months postpartum among a cohort of 44 healthy donors. Treatment of HT-29 colonic
epithelium with preparations of human milk HA at physiologic concentrations resulted in
141
a time- and dose-dependent induction of the antimicrobial peptide human β-defensin 2
(HβD2) that was abrogated by digestion of milk HA with a specific hyaluronidase.
Finally, treatment of cultured colonic epithelium with human milk HA preparations
significantly enhanced resistance to infection by the enteric pathogen Salmonella
enterica. Together, our observations suggest that maternally provided HA mediates one
of the multiple protective antimicrobial defense mechanisms delivered through milk to
the newborn. Thus, human milk HA may enhance functional antimicrobial defense of the
intestinal epithelial barrier in the neonatal gastrointestinal tract and shapes the ontogeny
of gut microbial colonization in the nursing infant.
4.2 A PROPOSED MODEL FOR THE FUNCTION OF HUMAN MILK HA IN
INTESTINAL HOMEOSTASIS
Based on the data presented in Chapters 2 and 3, a model of the function of HA in
innate defense of the intestinal epithelium may be proposed. Construction of such a
model serves to summarize the current evidence. In addition, future studies will be
required to address numerous questions raised by the findings presented in Chapters 2
and 3. This model will highlight incomplete or uncertain aspects of the function of milk
HA in intestinal defense that could be clarified by future investigations.
4.2.1 Synthesis of HA During Lactation
While in the broadest sense many biological processes are best represented in a cyclical
nature, for this purpose the induction of innate defense by human milk HA begins with
142
HA synthesis during lactation. Human milk HA concentrations are greatest in the
immediate postpartum period, with a mean concentration of 755 ng/ml within the first
week after birth, and decrease at a linear rate over the first 60 days postpartum (Figure 31). However, HA concentrations are essentially static at 216 ng/ml at time points greater
than 60 days after birth. Importantly, all samples evaluated contained detectable levels of
HA, suggesting that HA is a universally expressed component of human milk. Previous
work corroborates the observation that human milk contains HA (167) and that milk
glycosaminoglycan content decreases over postpartum time (274), albeit with a
substantially smaller cohort and less quantitative methods of analysis. The timedependent expression of HA in human milk therefore appears analogous to
immunomodulatory milk components such as sIgA, expressed in greatest quantity in the
immediate postpartum period (152), rather than caloric milk sugars, such as lactose,
which becomes more abundant with postpartum time and correlating with the increasing
metabolic demands of the growing infant (286). Given the dose-dependent effect of
human milk HA (Figure 3-2) or synthetic HA-35 (Figure 2-3) in the induction of HβD2
and enhanced resistance to Salmonella infection in vitro (Figure 5-7), this pattern of milk
HA expression may confer maximal innate protection of the intestinal epithelium in the
immediate postpartum period when the infant is most vulnerable to gastrointestinal
infection (140, 287). One subsequent testable hypothesis is that milk hyaluronan content
is associated with incidence of gastrointestinal infection. While our study design did not
include collection of data regarding GI infection, future studies could seek to determine if
milk HA content confers GI infection risk reduction among breastfed infants. That human
milk contains about 0.1-1 µg/ml HA is all that can be definitively said based on current
143
evidence. Maternal genetics, nutrition, and hormonal factors likely contribute to variation
in milk HA content. The mechanism by which this HA synthesis is induced in the milk
ducts has not been studied. A recent investigation of cervical HA synthesis during
pregnancy in both humans and mice concluded that HA synthesis is dramatically increased
in the preterm period, and that HAS2 is specifically regulated by estrogen while HAS1 is
not (288). Systemic hormones associated with pregnancy could similarly regulate
production of hyaluronan in the mammary ducts. Milk HA polymer size distribution was
not evaluated among the cohort of human milk donors presented in Chapter 3, nor were
size-specific effects of human milk HA determined. The results of experiments with
synthetically produced HA presented in Chapter 2 suggest that milk HA, chemically
identical in structure, may exhibit size-specific induction of HβD2 expression in intestinal
epithelium. In addition, enhanced resistance to Salmonella enterica infection in vitro
following pre-treatment with synthetic HA appears to be at least somewhat size-selective
(Figure 5-7) indicating that functional epithelial defense is potentially size-dependent.
Importantly, milk HA preparations isolated from unique donors independently exhibited
similar HβD2 and DEFB4 inducing activity (Figure 3-2), indicating that the expression
of bioactive HA in milk is likely to be widespread among the general population.
Evaluation of HA polymer size distribution in human milk samples may reveal HA sizing
patterns that are predictive of protection from gastrointestinal disease among newborns.
Large-scale epidemiologic studies demonstrating an association between milk HA content
and prevention of neonatal infection would likely precipitate further investigation into the
mechanisms of HA synthesis and polymer size distribution during lactation. Additionally,
hyaluronan is highly hygroscopic, occupying large hydrodynamic volumes with high
viscosity as high molecular weight polymers (169), such as those present in milk (Chapter
3). This may contribute to the physical chemistry of milk, an emulsion of hydrophobic lipid
144
and hydrophilic protein and carbohydrates. Alternately, milk HA content may be
physiologically limited due to the need to limit milk viscosity.
4.2.2 Passage of HA through the Gastrointestinal Tract
Following consumption of milk HA during breast-feeding, or oral consumption of
synthetic HA, passage of HA through the digestive tract may play a significant role in
determining the properties of HA in intestinal defense. Oral gavage of HA-35 (Figures 24, 2-5, and 2-6) or human milk HA (Figures 3-3 and 3-4) results in induction of the
murine HβD2 ortholog, MuβD3, relative to control treatments throughout the length of
the large intestine. This may suggest that orally ingested HA remains sufficiently intact
within the digestive tract to promote defensin induction, even in the distal colon. While
previous studies have investigated the immunomodulatory effects of orally ingested HA
(264), little is known regarding hyaluronidase activity of the gastrointestinal tract. The GI
tract appears to have a limited ability to absorb ingested HA, with one study
demonstrating that radioactivity is almost entirely recovered in the feces (86.7-95.6%)
following ingestion of radiolabeled high molecular weight HA in rats (269). However,
this investigation did not evaluate alterations in HA chemical structure or polymer size,
potentially relevant to HA signaling properties (166). Passage of HA through the
gastrointestinal tract may result in catalysis or chemical modification through endogenous
or bacterial enzyme activity (289). This is not likely to include increases in HA polymer
size following ingestion as there are currently no known in vivo processes which could
extend an HA polymer with intact disaccharide composition after release from
hyaluronan synthase (187). Colonic tissue expresses hyaluronidase activity, primarily
derived from Hyal-1 and Hyal-2, however it is not clear if this applies to the epithelial
145
brush border or if the observed hyaluronidase activity is originating from myeloid cells in
the lamina propria (290). Airway epithelium expresses hyaluronidase activity originating
from intracellular Hyal-1, Hyal-2, and Hyal-3, but also Hyal-2 localized to the apical cell
surface and in soluble form in epithelial secretions (287), and it is possible that intestinal
epithelium may express apical Hyal-2 in a similar manner. The intestinal microbiota may
be an additional source of hyaluronidase activity, originating from prokaryotic or fungal
symbionts including Pasturella, Streptococcus, Candida (166, 169), and Enterococcus
(292) species, and variation in microbiota composition (17, 18) may alter catalysis of
ingested hyaluronan. However, it must be emphasized that there are currently no data to
indicate that catalytic processing of ingested HA occurs in vivo, and the findings
presented in Figure 2-5 may argue against this possibility. Oral administration of a panel
of HA preparations ranging from 4.7 kDa to 2,000 kDa in average molecular mass results
in significant defensin induction only among mice receiving the 35 kDa fragment
preparation with demonstrated in vitro size-specific activity (Figure 2-3). It is possible to
speculate that hyaluronidase activity, originating from endogenous or bacterial sources,
may result in the generation of active HA fragments from relatively high molecular mass
precursors. However, oral gavage of high molecular mass HA did not result in significant
induction of defensin expression in the murine intestinal mucosa, suggesting that such
fragments were not generated during passage through the digestive tract. Future studies
will be required to resolve this question, perhaps utilizing labeled HA probes to
investigate polymer size distribution following passage through the murine or human
gastrointestinal tract or evaluating hyaluronidase activity in cultured mucosal bacteria or
fecal extracts.
146
4.2.3 Epithelial Cell Surface Receptor Engagement and Size-Specific Signal
Transduction
Ingested milk-derived HA or synthetic HA passing through the digestive tract promotes
the expression of HβD2 protein through a TLR4-dependent mechanism. Oral
administration of synthetic HA fragments results in induction of murine HβD2 ortholog
in the intestinal mucosa of wild-type or CD44-/- mice, but not in the intestinal mucosa of
TLR4-/- mice (Figure 2-6). In addition, pre-treatment with functional antibodies against
TLR4 abrogates the induction of HβD2 protein expression by HA-35 in HT-29 colonic
epithelial cells while functional TLR2 antibody does not (Figure 5-8). These results are
consistent with numerous reports in the literature describing TLR4-dependent effects of
low molecular weight HA, including the induction of HβD2 in cultured epithelium (241,
242). Many of the effects of fragmented HA appear to be regulated via TLR2 and TLR4
(66, 229, 232), with some cell types, including macrophages, exhibiting both CD44dependent (225, 229) and independent (66, 227) immunomodulatory responses to
fragmented HA. Low molecular weight HA, injected intravenously or generated through
sterile injury, acts through a TLR4-dependent mechanism to promote expression of the
chemokines IL-8 and MIP-2 in endothelium and skin biopsies in both tissue culture and
animal models (227, 247). Nf-κB translocation and subsequent upregulation of TNF-α,
IL-1β, and IL-6 occurs in chondrocytes following treatment with low molecular weight
HA in a mechanism that is both TLR4- and CD44-dependent, suggesting that fragmented
HA generated during joint damage also acts to enhance inflammation (231). A
intraperitoneal injection of a polydispersed HA fragment preparation of polymers less
147
than 750 kDa protects wild-type mice, but not TLR4-deficient animals, from a microflora
mediated model of intestinal colitis (239), or from the epithelium-depleting effects of
radiation (240), through a mechanism that is mediated in part by increased COX-2
expression (239, 240). That many of the signaling functions of HA fragments require
TLR4 seems difficult to dispute, given the accumulating evidence from a variety of cell
types and animal models of disease. Less certain is the exact nature of the proposed
TLR4-HA interaction, which has not been demonstrated directly. Size-specific HA
signaling has been reported previously in other cell types (68, 223, 227, 247), often
defining HA size broadly as either “high” or “low” molecular weight, with no consensus
in the literature on what size range defines either category. Induction of HβD2 in
intestinal epithelium requires HA polymers within a relatively narrow molecular weight
range; ~35 kDa HA specifically promotes increased intracellular HβD2 protein
accumulation that is not observed following treatment with either smaller (HA-4.7, HA16) or larger (HA-74, HA-2M) HA polymers when compared at either equal-molar or
equal-mass concentrations (Figures 2-3 and 2-5). HA preparations with average
molecular weight of 28.6 kDa, also induce intracellular HβD2 peptide expression
comparable to HA-35 in HT-29 cells (Figure 5-2), and both HA-28 and HA-35
upregulate MuβD3 expression in vivo (Figures 2-4, 2-5 and 2-6). Equal-molar
application of HA fragments presumes a ligand-receptor relationship in which one HA
fragment or polymer interacts in a specific manner with single receptors, while an HA
polysaccharide may be capable of interacting with multiple receptors due to its extended
molecular conformation and repeating structural motif (219). Therefore the sizespecificity of HβD2 induction by HA in vitro was evaluated using both equal-molar and
148
equal-mass dosing for the HA fragment preparations ≤ 74 kDa in average molecular
weight. Importantly, HA-35 is the most active inducer of HβD2 protein expression using
either method of comparison to other HA preparations. Thus the size range of
functionally active HA fragments is defined here as no less than 16 kDa and no greater
than 74 kDa. Gel electrophoresis analysis of the HA fragment preparations suggests that
the optimal HA fragment(s) for intracellular HβD2 induction is likely in the range of 2045 kDa (~53-132 disaccharides) (Figure 2-3). No signaling activity of any kind has been
previously ascribed to an HA polymer within this specific size range. HA found in living
tissue assumes a widely polydispersed distribution (166), and it is unclear how HA
signaling systems respond to HA fragments within this active range in the presence of
larger or smaller HA fragments. Observations presented in Figure 2-3 indicate that while
high molecular weight HA (2000 kDa) has no impact on the induction of HβD2 by HA35, equal-molar HA-4.7 is sufficient to inhibit HβD2 protein expression during treatment
of cultured HT-29 cells with HA-35. This could be accomplished through a number of
different mechanisms, including direct competition for receptor binding sites, or through
an unknown alternate signaling pathway acting counter to the TLR4-dependent pathway
activated by HA-35. Conversely, the TLR4-dependent mechanism of HβD2 induction by
HA-35 is unaffected by the presence of an equal-mass concentration of HA-2M,
indicating selectivity among chemically identical polymers of differing length. Broadly
polydispersed human milk HA induces HβD2 protein expression (Figure 3-2) despite the
presence of high molecular weight HA polymers that data presented in Chapter 2
indicate have no independent activity. Given the results of evaluation of size-specific
induction of HβD2 using synthetic HA preparations, a subpopulation of milk HA
149
polymers 20-50 kDa in mass may account for the majority of HβD2 inducing activity.
This contrasts with the findings of Campo et al. (231), which propose that high molecular
weight HA displaces small HA oligosaccharides at the cell surface, inhibiting the TLR4dependent inflammatory response to HA oligosaccharides (~1.5 kDa) in chondrocytes. In
the absence of conclusive data regarding the mechanism of size-specific HA signaling,
numerous speculative explanations are possible. Co-receptor complexes incorporating a
variety of HA-binding transmembrane proteins including TLR4, CD44, and others,
similar to those proposed by Taylor et al. (227), may confer the size- and structuralspecificity indicated by the experimental findings. While the induction of HβD2
following treatment with synthetic HA (Figure 2-6) was independent of cell-surface HA
receptor CD44, the size-specific response to HA was not evaluated in receptor-deficient
animals. A recent mechanistic study of CD44 clustering in response to HA treatment
determined that high molecular weight HA binding to CD44 selectively induces CD44
clustering, which is inhibited by HA oligosaccharides (283). HA size-specific clustering
of CD44 could potentially influence TLR4-dependent HA signaling if CD44 and TLR4
are present as plasma membrane complexes similar to those proposed by Taylor et al.
(227). Given that HA polymers are composed of numerous identical repeating structural
motifs, intermediate to large HA polymers could potentially interact with multiple TLR4
receptor dimers simultaneously. The degree of aggregation of TLR4 receptors spatially
within the plasma membrane by an intermediate HA polymer could potentially result in
novel, size-specific signal transduction events. TLRs are known to form hetero- and
homotypic dimers (293, 294), and the formation of TLR multimers in the plasma
membrane has been proposed (295). TLR3 structural data suggest multimerization
150
concurrent with binding to a high molecular weight carbohydrate polymer ligand, dsRNA
(296), however there appears to be no data demonstrating the formation of TLR4
multimers directly. Additional studies are required to evaluate many proposed molecular
mechanisms regulating HA selectivity. This is currently among the most pressing
questions in the study of HA fragment signaling.
Induction of the murine HβD2 ortholog following oral administration of milk HA is
dependent upon expression of cell surface receptors TLR4 and CD44. Expression of
MuβD3 was significantly enhanced in the intestinal mucosa of wild-type animals
following oral administration of human milk HA. In contrast, MuβD3 expression was
similar to control treatment in both TLR4- and CD44-deficient animals following
administration of the milk HA preparation for 3 days (Figure 3-4). Prior work in animal
models has principally implicated TLR4 in HA-dependent modulation of intestinal
defense (239, 240), particularly following oral administration of HA (264, Chapter 2),
and the induction of HβD2 in cultured keratinocytes is dependent upon TLR4 (241). Our
results suggest that TLR4 is an essential cell surface receptor for the induction of murine
HβD2 ortholog by milk HA, a finding that is consistent with our previous report that
induction of MuβD3 by synthetic HA is TLR4-dependent (Chapter 2). However, CD44dependent induction of defense effectors has been reported in monocytes and
macrophages (227, 228, 232) and renal epithelium (225), and CD44 is required for the
induction of murine HβD2 ortholog following oral administration of milk HA (Figure 4).
To our knowledge, this is the first report to suggest a role for CD44 in the regulation of
defensin expression. Thus, both TLR4 and CD44 are required in the response to milk HA
in vivo, and genetic deletion of either putative HA receptor is sufficient to abrogate the
151
induction of MuβD3. Previous work by Taylor et al. (227) suggests a mechanism by
which TLR4 and CD44 complexes, colocalized in the cell membrane, cooperatively
regulate the macrophage response to HA fragments generated in sterile injury through a
shared signal transduction pathway. Recognition of milk HA and subsequent signal
transduction resulting in MuβD3 expression may utilize similar TLR4/CD44 receptor
complexes expressed on the intestinal epithelial surface. Alternately, separate signal
transduction pathways independently activated by TLR4 and CD44 may act in a
complementary manner to enhance defensin expression in the presence of broadly
polydispersed milk HA. While TLR4-dependent induction of HβD2 following
stimulation with bacterial PAMPs acts through the canonical TLR signal transduction
mediators IRAK, TRAF6, and JNK to promote translocation of the transcription factor
AP-1 and engagement of the DEFB4 promoter (32, 238), HβD2 expression is also
regulated by other ligands via the MAPK/ERK signal transduction pathway (32, 280).
Engagement of HA with CD44 has repeatedly been demonstrated to result in specific
MAPK/ERK activation (281, 282) in a mechanism that is potentially dependent on HA
size (283), and milk HA may induce HβD2 expression in a CD44-dependent manner
through this pathway. Future studies will be required to distinguish between cooperative
and complimentary signal transduction mechanisms regulating the TLR4- and CD44dependent response to milk HA.
4.2.4 Induction of HβD2 Protein Expression
Enhanced expression of HβD2 occurs in cultured intestinal epithelium following
treatment with medium containing HA-35 (Chapter 2) or human milk derived HA
152
(Chapter 3) in a time- and dose-dependent and hyaluronidase-sensitive manner.
Treatment of HT-29 cells with human milk HA promotes increased transcription of
DEFB4, the gene encoding HβD2, relative to treatment with medium alone or treatment
with hyaluronidase-digested human milk HA.
HT-29 colonic epithelium, a cell line derived from a human colonic tumor, was utilized
for the experimental induction of HβD2 protein and DEFB4 gene expression in cell
cultures. However, additional data indicate that induction of HβD2 protein expression
following HA-35 treatment is not specific to a single epithelial model system. Induction
of HβD2 protein expression was observed in human intestinal epithelial cell line Caco-2
(Figure 5-5) and kidney epithelium derived from Macaca mulatta (Figure 5-6) following
treatment with media containing 10 µM HA-35 relative to treatment with medium alone.
These findings, together with the observations made by other research groups
investigating the effect of LMW HA on keratinocytes (241) and vaginal epithelium (242),
and the observation that expression of murine HβD2 ortholog in intestinal mucosa is
enhanced by oral gavage of HA-35 (Chapter 2) or human milk HA (Chapter 3), suggest
that induction of HβD2 protein expression following exposure to HA is a universal
property of mammalian epithelium. The effect of intermediate-sized HA on intestinal
epithelium appears to be divergent from its effect on skin keratinocytes grown in vitro
(35) in that no significant change in secreted HβD2 protein concentration is observed in
HT-29 cells following treatment with 35 kDa HA (Figure 2-1). While defensins are best
characterized as secreted effector molecules of innate defense (93), intracellular
expression of HβD2 protein has been suggested by positive immunostaining of mucosal
tissue (103, 129, 245, 258) and epithelial cell cultures (102, 241) for some time. The
153
function of intracellular HβD2 peptide has not been studied extensively, particularly in
epithelium, though intracellular HβD2 may contribute to defense against intracellular
infection (259).
The work presented in Chapter 2 suggests that HA acts as a specific inducer of HβD2 in
intestinal epithelium independent of other ligands. However, maximal HβD2 protein
expression required a 350-700 fold increase in synthetic HA-35 concentration relative to
treatment with milk HA preparations (350 µg/ml HA-35 vs. 0.5 -1 µg/ml milk HA). In
addition, milk HA treatment resulted in a maximal 5-6 fold induction of HβD2 protein
expression relative to control treatment while the maximal HβD2 induction following
HA-35 treatment was approximately 2.5-3 fold. Additionally, the timing of maximal
HβD2 expression in milk HA-treated HT-29 cells (24 h) was extended relative to
treatment with HA-35 (9 h), while expression at 9 h may be comparable between the two
HA preparations. It is impossible to exclude the possibility that additional milk glycans or
other biochemical components present in the milk HA preparation may contribute to or
amplify the induction of HβD2 by milk HA in intestinal epithelium. Indeed,
hyaluronidase-treated milk HA preparations promoted significant induction of HβD2
protein and DEFB4 transcription at physiologically relevant concentrations (Figure 3-2).
However, substantially increased doses of hyaluronidase-treated milk HA isolate were
required to achieve effects comparable to treatment with milk HA containing intact HA,
with the observed EC50 of hyaluronidase-treated milk HA increased approximately 200fold relative to the untreated milk GAG preparation (Figure 3-2). Clearly the presence of
HA in milk isolates contributes significantly to HβD2 induction. In addressing the
hypothesis that HA in milk enhances innate intestinal epithelial antimicrobial defense,
154
evaluating induction of antimicrobial peptide HβD2 by endogenous milk HA presented in
context with other milk carbohydrates has merit in that it perhaps more nearly replicates
the physiologic setting of milk HA presentation to the neonatal intestinal mucosa.
Synergistic effects of the human milk carbohydrate lactose in combination with bacterial
fermentation products butyrate or phenlybutyrate result in enhanced induction of the gene
encoding human cathelicidin LL-37, CAMP in cultured HT-29 epithelium (165). Human
milk contains passive defense mediators (145, 146), growth hormones (147), prebiotics
(148-151), and immunomodulators (133), as well as a diverse and active microbial
community (284). Numerous compounds in milk could potentially influence the
induction of HβD2 expression. With the exception of the finding by Cedarlund et al.
(165) indicating that lactose promotes antimicrobial peptide expression in cultured
intestinal epithelium, milk derived compounds have not been evaluated for induction of
antimicrobial peptides. However, TLR ligands have been demonstrated to promote
expression of HβD2 in epithelium (32), including bacterial LPS (102), which is a TLR4
ligand (63). HA-35 fragments promote intracellular HβD2 peptide accumulation in HT29 cells while high concentrations of LPS do not (Figure 2-2). Lee et al. (257) have
reported that sensitivity to LPS is decreased in HT-29 cells due to low TLR4 expression,
however induction of the murine HβD2 ortholog by fragmented HA is TLR4-dependent
(Figure 2-6). Differing sensitivity of the TLR4 receptor to the ligands HA and LPS or the
presence of ligand-specific co-receptors could account for this discrepancy. Thus, while it
appears unlikely that bacterial LPS contributes significantly to the induction of HβD2
protein expression in hyaluronidase-digested human milk HA treated epithelium (Figure
3-2), the hypothesis that LPS or other bacterial PAMPs enhance induction of HβD2
155
protein expression by milk HA in a synergistic manner cannot be excluded. TLR ligands
have been evaluated experimentally in isolation, while physiologic settings frequently
involve the simultaneous engagement of multiple TLRs (86, 87), with potentially unique
signaling events occurring as a result of the activation of multiple intracellular pathways.
The consequence of the interactions between an array of milk immunomodulatory
components and bacterial PAMPs and specific cell surface receptors on epithelium is an
innate response finely tuned to a distinct physiologic circumstance, such as colonization
of the neonatal intestine during breast-feeding.
A complex signal transduction network may regulate the induction of HβD2 protein
expression by synthetically produced HA-35 or human milk HA. The intracellular events
occurring subsequent to presumed HA-TLR4 interaction and induction of DEFB4
transcription (Figure 3-2) have not been specifically evaluated. However, numerous
publications have addressed TLR-mediated signal transduction (31, 32), and clear
hypotheses can be formulated for the induction of HβD2 by HA. Canonical TLRmediated signal transduction occurs subsequent to engagement of TLR ligands with their
complementary receptors through the interaction of the intracellular TIR domain with
adapter protein MyD88 (81), the exception being TLR3, which is MyD88 independent
(31). A cascade of MAP kinase family members activates a common signaling pathway
that ultimately results the nuclear translocation of NF-κB, a transcription factor
consistently implicated in regulatory control of cytokine expression (31), but also in the
expression of HβD2 (32, 101, 102, 127). Induction of HβD2 in keratinocytes following
treatment with LMW HA is inhibited by pharmacologic Protein Kinase C (PKC)
inhibitors, but is unaffected by pharmacologic inhibition of NF-κB (241). Increased
156
binding of AP-1 subunit c-Fos was associated with induction of HβD2 in keratinicytes by
HA (241), consistent with descriptions of the HβD2 promoter that indicate the presence
of both AP-1 and NF-κB transcription factor binding sites (127). Inhibition of the
Akt/PI3 kinase signal transduction, an upstream regulatory step in NF-κB translocation
(32), has been shown to suppress HβD2 induction by HA in reproductive epithelium
(242). NF-κB independent signaling pathways may be required to generate increased
HβD2 protein expression without an accompanying pro-inflammatory cytokine response
in epithelium (241). Evaluation of the role of NF-κB is central to future investigation into
the signal transduction events regulating HβD2 induction by HA-35 or human milk HA,
with clear physiologic implications regarding the generation or suppression of
inflammatory responses following TLR4 activation by HA in epithelium. Histological
evaluation of intestinal mucosa of > 100 mice following oral gavage of synthetically
produced HA or human milk derived HA did not indicate the presence of inflammatory
tissue responses following HA ingestion, and additional studies of ingested (264) or
intraperitoneal (239, 240) HA suggest suppression of the inflammatory response in a
TLR4-dependent manner in HA-treated mice. The application of relatively non-specific
pharamacologic inhibitors, with confirmation by specific gene knockdown strategies, will
aid in understanding the role of PKC (241) or other signal transduction mediators in the
response to synthetically produced or human milk HA in intestinal epithelium.
4.2.5 Functional Resistance to Intracellular Infection
Treatment of cultured HT-29 colonic epithelium with human milk HA enhances
resistance to infection by the enteric pathogen Salmonella enterica serovar Typhimurium
157
SL1344 (Figure 3-5). While questions regarding the mechanism of milk HA-mediated
Salmonella resistance remain, our results suggest that milk HA may contribute to the
reduced incidence of enteric Salmonella infection associated with breast-feeding in
human infants (140). Numerous prior reports have implicated HA in modulation of the
immune response (166, 227, 229), however this is the first report to suggest a role for
naturally produced HA in resistance to enteric infection through direct effects on
epithelium. Following infection, a significantly reduced quantity of viable Salmonella
was harvested from HT-29 epithelial cultures that had been pre-treated with human milk
HA relative to epithelium treated with medium alone or medium containing
hyaluronidase-digested human milk HA, thus providing functional evidence in support of
the hypothesis that human milk HA enhances antimicrobial defense of intestinal
epithelium. Additional evidence supports the conclusion indicated in Chapter 3 that HA
in human milk enhances protection against Salmonella enterica infection in intestinal
epithelium. Pre-treatment of HT-29 cells with HA-35 resulted in a highly significant
decrease in Salmonella infection relative to pre-treatment with medium alone or pretreatment with medium containing HA-4.7 (Figure 5-7). These data indicate that HA-35
enhances functional resistance to intracellular Salmonella enterica Typhimurium
infection in pre-treated host epithelium in vitro in a mechanism that is size-specific.
Enhanced resistance to Salmonella infection following HA-35 treatment was also found
to be both time- and dose-dependent (Figure 5-7). The data presented in Chapter 3
implicated HA in enhanced resistance to epithelial infection following treatment with a
human milk HA preparation that was abrogated by digestion of the milk HA preparation
with substrate-specific hyaluronidase derived from Stremptomyces hyalurolyticus.
158
However, milk HA preparations contained numerous additional biological components,
including undersulfated chondroitin and other carbohydrate polymers. Thus, experimental
analysis which utilized hyaluronidase to demonstrate HA-specific activity in complex
milk HA preparations demonstrated that HA was necessary for innate epithelial
antimicrobial protection, whether induction of HβD2 or enhanced resistance to
intracellular Salmonella enterica infection. These findings are complemented by the
concurrent experimental data indicating that HA-35 is sufficient for the induction of both
HβD2 protein expression (Chapter 2) and enhanced intracellular infection protection
(Figure 5-7).
Experimental conditions included the removal of medium containing human milk HA
prior to introduction of Salmonella, suggesting that the observed reduction in Salmonella
infection occurs due to modification of host epithelium. While numerous examples of
direct pathogen inhibition through interaction of human milk glycans with microbial
surface receptors have been reported (53, 158, 160, 161, 168), infection resistance
resulting from modulation of epithelium by human milk glycans has not been extensively
studied. Treatment of cultured Caco-2 epithelium with sialyllactose, a human milk
oligosaccharide, results in the down-regulated expression of specific glycan epitopes on
the surface of the host epithelium correlating with a reduction in the binding of
pathogenic E. coli (162). Human milk HA may act similarly to enhance resistance to
Salmonella in HT-29 epithelium through the modulation of surface receptor expression.
Intriguingly, peak HA-dependent expression of antimicrobial HβD2 peptide was
observed in milk HA treated epithelium at the specific post-treatment time point at which
Salmonella infection was initiated in identically treated epithelium. This correlates
159
increased HβD2 expression with enhanced Salmonella resistance and implies a potential
role for HβD2 in the observed resistance to intracellular infection (Figures 3-2 and 3-4).
Accumulation of intracellular defensins has been demonstrated to inhibit replication of
the obligate intracellular pathogen Listeria monocytogenes in macrophages (259), and a
similar mechanistic link between HβD2 and resistance to intracellular Salmonella may
exist in milk HA treated intestinal epithelium. Additional findings indicate mechanism of
HA-induced Salmonella resistance appears to be independent of autophagy, a selfdegradation process in which intracellular contents are degraded in autophagosomallysosomal vesicles (297). HT-29 cells constitutively expressing transgenic inhibitory
shRNA against requisite autophagy-regulatory protein ATG16L1 have similar resistance
to intracellular Salmonella infection following HA-35 treatment in comparison to native
HT-29 cells (Figure 5-7). However, HT-29 cells constitutively expressing transgenic
inhibitory shRNA against intracellular peptidoglycan receptor NOD2 or expressing a
dominant-negative IκBα subunit of NF-κB, which inhibits the nuclear translocation of
inflammatory transcriptional regulator NF-κB, do not exhibit enhanced resistance to
Salmonella infection following HA-35 treatment (Figure 5-7).
These data indicate that HA-35 enhances functional resistance to intracellular Salmonella
enterica Typhimurium infection in pre-treated host epithelium in vitro through a
mechanism that is dependent upon NOD2 and NF-κB dependent and independent of
ATG16L1. This may be consistent with the hypothesis that accumulating intracellular
defensin mediates destruction and inhibition of cytoplasmic Salmonella as both NF-κB
(32, 101, 102, 127) and NOD2 (113) mediate induction of HβD2 in response to PAMP
and DAMP signals. Evaluation of HA-induced Salmonella in an HβD2-deficient cell
160
culture model would address this hypothesis. However, the induction of Salmonella
resistance following treatment with milk HA may not be entirely mediated through
modulation of epithelial cell behavior. Application of milk HA preparations
simultaneously with the addition of Salmonella in cell culture medium results in
significant inhibition of Salmonella infection (Figure 5-8). However, this did not occur
when HA-35 treatment was administered simultaneously with Salmonella infection
(Figure 5-7). These data indicate that milk HA enhances functional resistance to
intracellular Salmonella infection in vitro through a mechanism that is rapid and
potentially involving direct inhibition of Salmonella infection through physical
interaction with the pathogen or host. This observation is analogous to direct pathogen
inhibition occurring through interaction of human milk glycans with microbial surface
receptors (53, 158, 160, 161, 168). Interaction of milk HA with microbial or host cell
surface receptors may disrupt or inhibit the process of host entry during infection. That
HA-35 did not exhibit similar pathogen inhibition when administered simultaneously
may suggest that the mechanism of direct infection resistance has different sizespecificity than the induction of HβD2 or that non-HA components of human milk HA
preparations confer independent inhibitory activity in the immediate phase of infection
that was not demonstrated in epithelium pre-treated for 24 h with hyaluronidase-digested
milk HA preparations (Figure 3-5). The application of hyaluronidase-digested milk HA
preparations simultaneously with Salmonella infection would contribute significantly to
clarifying these two possible explanations.
161
4.2.6 Milk HA Shapes Intestinal Microbiota Development
The role of human milk HA, or any other dietary component for that matter, in generating
intestinal homeostasis in breast-fed infants in the presence of diverse and persistent
microbial challenges is only vaguely understood. The bioactivity of milk components
relative to the function of intestinal epithelium, such as the induction of HβD2 in
intestinal epithelium by milk HA, could have a profound effect in shaping intestinal
microbial ecology through the modulation of epithelial substrates and the generation of
selective niches (18). Altered expression of α-defensins in the intestine has been shown
to result in significant shifts in microbial species distribution (270) and data presented in
Chapter 3 demonstrates that the effect of milk HA on intestinal epithelium alters the
interaction of host epithelium with at least one relevant component of the human
microbiome, Salmonella. Milk contains a diverse bacterial community (284), seeding the
developing gastrointestinal microbiome along with maternally and environmentally
acquired species and interacting with the antigen-naive adaptive mucosal immune system
(152). The combined effect of milk probiotics, prebiotics, antibiotics,
immunomodulators, and microbial transfer is evident in the observation that breast-fed
infants exhibit dramatically different gastrointestinal microbial communities in
comparison to artificially-fed infants (8, 9, 132). Given the robust correlation between
breast-feeding and gastrointestinal health (10, 136, 138, 158), the role of breast milk in
establishing the microbial-epithelial interface may be of critical importance to lifelong GI
health (285), potentially accounting in part for the reduced lifetime risk of inflammatory
bowel disease (137), obesity (141, 142), and allergic disease (143, 144) associated with
breast-feeding. The application of ecological theory to the human intestinal microbiome
162
predicts that the composition of the gut microbial community is dependent upon
environmental selection resulting in part from dietary factors (17, 18). Numerous
variables regarding human milk HA including the timing and duration of breast-feeding,
maternal nutrition and hormonal status, and milk HA concentration and polymer size
distribution may shape gut microbial community development. Other molecular
mechanisms of protection are yet to be explored, and a recent study associating increased
dairy consumption with risk reduction in obesity, type-2 diabetes, heart disease, and
hypertension (298) implies that modulation of the intestinal microflora by milk
components may have important systemic effects on human health. The newly
appreciated interdependence of microbial and human life forms presents a profound
philosophical challenge to our conception of the individual with the potential to
fundamentally alter the paradigm of modern medicine. Future studies will likely enhance
understanding of the complex dynamic regulating host epithelium and microbial
interactions and the role of dietary components in shaping the relationship between
human host and the immense complement of microorganisms that populates the intestinal
lumen.
4.3 MILK COMPONENTS AS A SOURCE OF NOVEL GASTROINTESTINAL
BARRIER THERAPEUTICS
While a greater understanding of the molecular mechanisms mediating the reduction in
gastrointestinal disease incidence associated with breast-feeding (10, 136-138, 141-144,
158) is an exceptionally fascinating pursuit for the young and enthusiastic investigator,
163
human milk has tremendous practical potential as a source of novel therapeutics. The
most direct application for knowledge of the molecular mechanisms of milk immunity is
in the continual improvement of infant formulas (299, 300) aimed at reducing the
incidence of potentially life-threatening diarrhea (158) and necrotizing enterocolitis
among artificially-fed infants (301, 302). At a minimum, an understanding of the
functional benefits of milk in the prevention of gastrointestinal disease in infants provides
further evidence for the encouragement of breast-feeding. However, milk components
that enhance innate epithelial barrier defense may have application even in adult disease.
Bacterial translocation resulting from intestinal epithelial barrier insufficiency is a
significant source of sepsis, systemic inflammatory response syndrome, and multiorgan
failure (303, 304), major sources of morbidity and mortality among critically ill patients.
Deficiencies in epithelial barrier function are associated with numerous diseases,
including inflammatory bowel disease (123, 305), cancer (306, 307), AIDS (308), and
nonalcoholic steatohepatitis (309). Current treatment for gut barrier dysfunction relying
on broad-spectrum antibiotic therapy has limited efficacy in containing the vast and
diverse intestinal microbial complement and does not address physiologic epithelial
functional deficits directly (303). Breast-feeding is associated with accelerated maturation
of the gut epithelial barrier, while artificial feeding does not confer the same benefits
(310-312). Identification of the specific molecular components of milk, including
hyaluronan, which enhance resistance to intracellular infection and amplify expression of
antimicrobial peptides at the intestinal epithelial barrier, may precipitate the development
of new therapeutic approaches in the prevention of sepsis arising from gut microbial
translocation among critically ill patients. A defining feature of all mammalian life,
164
conserved across expansive evolutionary distance, it is certain that milk contains
substances that have enabled the survival of countless generations. Careful study of
natural phenomena has often led to the improvement of the human condition, and new
perspectives on the biological properties of human milk and their application to disease
may reveal novel therapeutic approaches.
165
CHAPTER 5: APPENDIX
Figure 5-1
Representative Images for the Quantification of MuβD3 staining intensity. Five
images were selected out of the complete dataset summarized in Figure 2-6 (90
individual stained fields representing a total of 30 mice) representing the complete range
of MuβD3 staining intensity. Selection of images for creation of the staining intensity
scale was completed without knowledge of mouse treatment or genotype. This scale was
provided to the panel of four blinded researchers to aid in the standardization of MuβD3
staining intensity scoring.
166
Figure 5-2
HA-28 Promotes Expression of Intracellular HβD2 Protein in HT-29 Cells. Western
blot showing HβD2 protein relative to GAPDH protein expression in whole cell lysates
of HT-29 cells treated with medium alone or HA-35 or HA-28, HA-35 at equal-molar
concentrations (10 µM) for 9 h. A. Representative Western blot of HβD2 protein
expression relative to GAPDH protein expression in whole cell lysates of HT-29 cells
treated with medium alone, medium supplemented with HA-35 or HA-28 (10 µM) B.
Average densitometric quantification of Western blot results of three experiments in
which HT-29 cultures were treated with medium alone, HA-35, or HA-28. HβD2 protein
expression is normalized to GAPDH protein in whole cell lysates. Significance of
167
differences in normalized HβD2 expression were evaluated by comparison of each
treatment to medium treatment using Student’s t-test, with ‘**’ indicating P < 0.01.
Figure 5-3
TLR4 Protein Expression is Minimal in the Colonic Epithelium of TLR4-/- Mice
Relative to Wild-type or CD44-/- Mice. Fluorescent micrographs of TLR4immunostained (green) murine proximal colon tissue. ‘NS’ indicates an immunostaining
control in which no TLR4 antibody was utilized. Images are representative of 3 animals
of each genotype, housed under standard conditions and not treated with exogenous HA.
No apparent difference in TLR4 protein expression was observed between wild-type and
CD44-/- animals. Residual TLR4 staining in the TLR4-/- colonic epithelium relative to
non-stained control may be accounted for by low affinity interactions between the
polyclonal TLR4 primary antibody and proteins with structural homology to TLR4, such
as other members of the Toll-like receptor family.
168
Figure 5-4
Functional TLR4 Antibody Inhibits HβD2 Induction in HT-29 Intestinal Epithelial
Cells Following HA-35 Treatment. Densitometric quantification of Western blot results
of one experiment in which HT-29 cultures were treated with medium alone, medium
containing HA-35, or medium containing TLR2 and/or TLR4 antibody (10 µg/ml) with
or without HA35 for 9 h is given in the upper panel. HT-29 intestinal epithelial cells were
cultured in RPMI medium containing 10% FBS and treated when confluence reached 7080%. The lower panel is a representative Western blot showing HβD2 protein relative to
GAPDH protein expression in whole cell lysates of HT-29 human intestinal epithelial
cells treated as indicated.
169
Figure 5-5
Induction of HβD2 in Caco-2 Intestinal Epithelial cells Following HA-35 Treatment.
Average densitometric quantification of Western blot results of three experiments in
which Caco-2 cultures were treated with medium alone or HA-35 is given in the upper
panel. Caco-2 intestinal epithelial cells were cultured in DMEM medium containing 20%
FBS and treated when confluence reached 70-80%. Significance of differences in
normalized HβD2 expression were evaluated by comparison of HA-35 treatment to
medium treatment using Student’s t-test, with ‘*’ indicating p < 0.05. The lower panel is
a representative Western blot showing HβD2 protein relative to GAPDH protein
expression in whole cell lysates of Caco-2 human intestinal epithelial cells treated with
medium alone of medium containing 10 µM HA-35 for 9 h.
170
Figure 5-6
Induction of HβD2 in MA-104E Kidney Epithelial Cells Following HA-35
Treatment. Representative Western blot showing HβD2 protein relative to GAPDH
protein expression in whole cell lysate of MA-104E kidney epithelial cells, originally
derived from rhesus macaque (Macaca mulatta), treated with medium alone, medium
containing 10 µM HA-4.7, or medium containing 10 µM HA-35 for 9 h. Densitometric
quantification of Western blot results is given in the upper panel. MA-104E canine
kidney epithelial cells were cultured in RPMI medium containing 10% FBS and treated
when confluence reached 70-80%.
171
Figure 5-7
A
HA-35 Enhances Resistance to Infection by Salmonella enterica serotype
Typhimurium SL1344 in Cultured HT-29 Cells. To test the hypothesis that HA-35
enhances functional antimicrobial defense, we utilized an in vitro assay modified from
Homer et al. (2010) exploiting the cell impermeable antibiotic gentamicin to specifically
assay the number of viable bacterium present within the cytoplasm of pre-treated host
epithelium. Confluent HT-29 colonic epithelial monolayers were treated for 24 h with
medium alone, medium containing 10 µM HA-4.7 or 10 µM HA-35. At 18 h cell culture
medium was removed and replaced with new medium containing live Salmonella
enterica serovar Typhimurium SL1344 for all pre-treatment groups, or medium
containing 25 µg/ml rapamycin, a positive control treatment demonstrated to induce
172
autophagy-mediated intracellular antibacterial activity, and Salmonella. After a short
infection period, the antibiotic gentamicin was added in fresh culture medium to
eliminate extracellular Salmonella while preserving bacterium present within the host
epithelium. Host epithelium was then lysed for bacterial culture to determine CFU per
area epithelial monolayer (Figure 5-7). Pre-treatment of HT-29 cells with HA-35 resulted
in a highly significant decrease of 38.2±4.8% (S.E.) in CFU/cm2 Salmonella relative to
pre-treatment with medium alone (P<0.0001) or pre-treatment with media containing
HA-4.7 (P<0.0001, Figure 5-7). Pre-treatment with HA-4.7 did not result in statistically
significant reduction in Salmonella relative to treatment with medium alone. These data
indicate that HA-35 enhances functional resistance to intracellular Salmonella enterica
Typhimurium infection in pre-treated host epithelium in vitro in a mechanism that is sizespecific.
173
Figure 5-7 (cont.)
B
HA-35 enhances epithelial resistance to infection by Salmonella in a time-dependent
manner. Confluent HT-29 colonic epithelial monolayers were treated with medium alone
(0 h) or medium containing 10 µM HA-35 for 4-24 h. At the end of the corresponding
HA-35 treatment time, cell culture medium was removed and replaced with new medium
containing live Salmonella for all pre-treatment groups. After a short infection period, the
antibiotic gentamicin was added in fresh culture medium to eliminate extracellular
Salmonella while preserving bacterium present within the host epithelium. Host
epithelium was then lysed for bacterial culture to determine CFU per well in a 48-well
plate. Pre-treatment of HT-29 cells with 10 µM HA35 resulted in a highly significant
decrease in CFU/well Salmonella at 24 h relative to pre-treatment with medium alone or
pre-treatment with media containing HA-35 for 4-8 h (Figure 5-7). Significance of
174
differences in CFU/well were evaluated in a pair-wise manner by comparison of HA-35
treatment time point to medium treatment using Student’s t-test, with ‘**’ indicating p <
0.01. These data indicate that HA-35 enhances functional resistance to intracellular
Salmonella enterica Typhimurium infection in pre-treated host epithelium in vitro
through a mechanism that is time-dependent.
175
Figure 5-7 (cont.)
C
HA-35 enhances epithelial resistance to infection by Salmonella in a dose-dependent
manner. Confluent HT-29 colonic epithelial monolayers were treated for 24 h with
medium alone (Control), or medium containing 0.1 – 10 µM HA-35. At 24 h cell culture
medium was removed and replaced with new medium containing live Salmonella for all
pre-treatment groups, or medium containing 25 µg/ml rapamycin and Salmonella (Rap.).
After a short infection period, the antibiotic gentamicin was added in fresh culture
medium. Host epithelium was then lysed for bacterial culture to determine CFU per area
epithelial monolayer, and differences are expressed as per cent CFU relative to the mean
CFU/area in control-treated HT-29. Pre-treatment of HT-29 cells with 1-10 µM HA35
resulted in a highly significant decrease of 18.5±5.8% (S.E.) CFU Salmonella at 24 h
176
relative to pre-treatment with medium alone (p=0.004 and 0.001, respectively; Figure 57). Treatment with rapamycin also resulted in a highly significant decrease in relative
CFU. These data indicate that HA-35 enhances functional resistance to intracellular
Salmonella enterica Typhimurium infection in pre-treated host epithelium in vitro
through a mechanism that is dose-dependent.
177
Figure 5-7 (cont.)
D
HA-35 induced resistance to Salmonella enterica is NOD2 and NF-κB dependent
and independent of ATG16L1. Native HT-29 cells, or HT-29 cells constitutively
expressing transgenic inhibitory shRNA against intracellular peptidoglycan receptor
NOD2, autophagy regulator ATG17L1, or expressing a dominant-negative IκBα subunit
of NF-κB, which inhibits the nuclear translocation of inflammatory transcriptional
regulator NF-κB (all gifts of Dr. Christine McDonald) were grown to 80% confluence
and treated for 24 h with medium alone (Control), or medium containing 10 µM HA-35.
At 24 h, cell culture medium was removed and replaced with new medium containing
live Salmonella for all pre-treatment groups. After a short infection period, the antibiotic
gentamicin was added in fresh culture medium. Host epithelium was then lysed for
178
bacterial culture to determine CFU per well in a 48-well plate. Pre-treatment of HT-29
cells with 10µM HA35 resulted in a highly significant decrease CFU Salmonella at 24 h
relative to pre-treatment with medium alone (p=0.002; Figure 5-7). Treatment with
10µM HA35 also resulted in a highly significant decrease in CFU in HT-29 expressing
ATG16L1 shRNA relative to cells expressing ATG16L1 shRNA treated with medum
alone (P=0.0006). Importantly, treatment with 10 µM HA35 did not result in significant
reduction in CFU Salmonella in HT-29 cells expressing NOD2 shRNA or dominantnegative IκBα relative to corresponding control treatments. These data indicate that HA35 enhances functional resistance to intracellular Salmonella enterica Typhimurium
infection in pre-treated host epithelium in vitro through a mechanism that is dependent
upon NOD2 and NF-κB and independent of ATG16L1.
179
Figure 5-8
Milk HA Confers Rapid Protection from Salmonella Infection. Confluent HT-29
colonic epithelial monolayers were treated with live Salmonella suspended in medium
alone, medium containing 25 µg/ml rapamycin, or medium containing milk GAG
preparation with 0.5 µg/ml HA. After a 30 m infection period, the antibiotic gentamicin
was added in fresh culture medium. Host epithelium was then lysed for bacterial culture
to determine CFU per area epithelial monolayer. Treatment of HT-29 cells with
rapamycin resulted in a highly significant decrease of 33.0% CFU/cm2 Salmonella
relative to treatment with medium alone (p<0.0001; Figure 5-8). Treatment with milk
HA preparation also resulted in a significant decrease in CFU/cm2 relative to treatment
with medium alone (p=0.0087). These data indicate that milk GAG rapidly enhances
functional resistance to intracellular Salmonella enterica Typhimurium infection in vitro
through a mechanism that is rapid and potentially involving direct inhibition of
Salmonella infection through physical interaction with the pathogen or host.
180
Figure 5-9
Milk HA Promotes Expression of MuβD3 in Distal Colonic Mucosa of Adult, Wildtype Mice. Representative fluorescent micrographs of epithelium in distal colonic crosssections from adult C57BL/6 wild-type mice. Distal colon is defined as the final 1/3length of bowel between the terminal ileum and rectum. The mice were given single daily
doses of 1µg milk HA in 0.25 ml water (Milk HA) or 1 µg Milk HA that had been predigested with S. hyalurolyticus hyaluronidase in 0.25 ml water (Milk HA + HAse) or
water alone (Control) for three consecutive days. MuβD3 is fluorescently immunolabeled
(red) and nuclei are stained with DAPI (blue).
181
Table 5-1
Saccharide species
Chondroitin
ΔDiOS
ΔDi4S
ΔDi6S
ΔDi2S
Other Chondroitin
ΔDi2,6S
ΔDi2,4,6S
Δ6S-Di2,4S
Heparan Sulfate
ΔDi-2S-U-6S-G-NS
ΔDi-U-G-NS
ΔDi-U-G-NAc
ΔDi-2S-U-G-NS
ΔDi-6S-G-NS
ΔDi-U-6S-G-NS
Other Sugars
Maltose
Maltotriose
Maltotetrose
GalNAc
6S-GalNAc
4S-GalNAc
Glucose
Relative Concentration
(µg/µg HA)
6.53
5.49
0.28
0.71
<0.01
0.04
N/A
N/A
N/A
0.94
0.33
0.26
0.22
0.08
0.06
<0.01
0.29
0.16
0.13
<0.01
<0.01
<0.01
<0.01
<0.01
S.E.
0.98
0.75
0.03
0.09
0.01
N/A
N/A
N/A
0.14
0.11
0.06
0.05
0.02
0.03
0.18
0.10
0.10
-
Carbohydrate constituents of milk HA preparations. Concentration of milk
carbohydrates present in milk HA isolates used for the presented biological assays as
measured by Fluorophore-assisted Carbohydrate Electrophoresis (FACE). Results
represent the mean concentration (± standard error) of each saccharide species relative to
1 µg/ml HA preparation (e.g. 0.5 µg/ml HA contains 3.27 µg/ml Chondroitin and 0.08
µg/ml Maltose, etc.) from five donor-unique milk HA preparations used in the biological
assays presented in Chapter 3. Note that three species of di- and tri-sulfated chondroitin
182
could not be individually resolved and are quantified under the term “Other
Chondroitins”.
183
BIBLIOGRAPHY
1.
Gray, H., Williams, P. L., and Warwick, R. (2008) Gray's anatomy, 40th Ed.,
Elsevier Saunders, Philadelphia, PA
2.
Bewick, G. A. (2012) Bowels control brain: gut hormones and obesity. Biochem
Med 22, 283-297
3.
Pimentel, G. D., Micheletti, T. O., Pace, F., Rosa, J. C., Santos, R. V. T., and Lira,
F. S. (2012) Gut-central nervous system axis is a target for nutritional therapies.
Nutr J 11, 22
4.
Boron, W. F., and Boulpaep, E. L. (2005) Medical Physiology, 2nd Ed., Elsevier
Saunders, Philadelphia, PA
5.
Xu, J., and Gordon, J. I. (2003) Honor thy symbionts. Proc Natl Acad Sci USA
100, 10452-10459
6.
Round, J. L., and Mazmanian, S. K. (2009) The gut microbiota shapes intestinal
immune responses during health and disease. Nat Rev Immunol 9, 313-323
7.
Kau, A. L., Ahern, P. P., Griffin, N. W., Goodman, A. L., and Gordon, J. I. (2011)
Human nutrition, the gut microbiome and the immune system. Nature 474, 327336
8.
Penders, J., Thijs, C., Vink, C., Stelma, F. F., Snijders, B., Kummeling, I., van
den Brandt, P. A., and Stobberingh, E. E. (2006) Factors influencing the
composition of the intestinal microbiota in early infancy. Pediatrics 118, 511-521
9.
Dominguez-Bello, M. G., Costello, E. K., Contreras, M., Magris, M., Hidalgo, G.,
Fierer, N., and Knight, R. (2010) Delivery mode shapes the acquisition and
structure of the initial microbiota across multiple body habitats in newborns. Proc
Natl Acad Sci USA 107, 11971-11975
10.
Newburg, D. S., and Walker, W. A. (2007) Protection of the neonate by the innate
immune system of developing gut and of human milk. Pediatr Res 61, 2-8
11.
Koenig, J. E., Spor, A., Scalfone, N., Fricker, A. D., Stombaugh, J., Knight, R.,
Angenent, L. T., and Ley, R. E. (2011) Succession of microbial consortia in the
developing infant gut microbiome. Proc Natl Acad Sci USA 108, 4578-4585
12.
Eckburg, P. B., Bik, E. M., Bernstein, C. N., Purdom, E., Dethlefsen, L., Sargent,
M., Gill, S. R., Nelson, K. E., and Relman, D. A. (2005) Diversity of the human
intestinal microbial flora. Science 308, 1635-1638
184
13.
Claesson, M. J., O'Sullivan, O., Wang, Q., Nikkilä, J., Marchesi, J. R., Smidt, H.,
de Vos, W. M., Ross, R. P., and O'Toole, P. W. (2009) Comparative analysis of
pyrosequencing and a phylogenetic microarray for exploring microbial
community structures in the human distal intestine. PLoS ONE 4, e6669
14.
Qin, J., Li, R., Raes, J., Arumugam, M., Burgdorf, K. S., Manichanh, C., Nielsen,
T., Pons, N., Levenez, F., Yamada, T., Mende, D. R., Li, J., Xu, J., Li, S., Li, D.,
Cao, J., Wang, B., Liang, H., Zheng, H., Xie, Y., Tap, J., Lepage, P., Bertalan,
M., Batto, J.-M., Hansen, T., Le Paslier, D., Linneberg, A., Nielsen, H. B.,
Pelletier, E., Renault, P., Sicheritz-Ponten, T., Turner, K., Zhu, H., Yu, C., Li, S.,
Jian, M., Zhou, Y., Li, Y., Zhang, X., Li, S., Qin, N., Yang, H., Wang, J., Brunak,
S., Doré, J., Guarner, F., Kristiansen, K., Pedersen, O., Parkhill, J., Weissenbach,
J., Consortium, M., Bork, P., Ehrlich, S. D., and Wang, J. (2010) A human gut
microbial gene catalogue established by metagenomic sequencing. Nature 464,
59-65
15.
Reyes, A., Haynes, M., Hanson, N., Angly, F. E., Heath, A. C., Rohwer, F., and
Gordon, J. I. (2010) Viruses in the faecal microbiota of monozygotic twins and
their mothers. Nature 466, 334-338
16.
Lozupone, C. A., Stombaugh, J. I., Gordon, J. I., Jansson, J. K., and Knight, R.
(2012) Diversity, stability and resilience of the human gut microbiota. Nature
489, 220-230
17.
Tremaroli, V., and Bäckhed, F. (2012) Functional interactions between the gut
microbiota and host metabolism. Nature 489, 242-249
18.
Costello, E. K., Stagaman, K., Dethlefsen, L., Bohannan, B. J. M., and Relman,
D. A. (2012) The application of ecological theory toward an understanding of the
human microbiome. Science 336, 1255-1262
19.
Swann, J. R., Want, E. J., Geier, F. M., Spagou, K., Wilson, I. D., Sidaway, J. E.,
Nicholson, J. K., and Holmes, E. (2011) Systemic gut microbial modulation of
bile acid metabolism in host tissue compartments. Proc Natl Acad Sci USA 108,
4523-4530
20.
Samuel, B. S., Shaito, A., Motoike, T., Rey, F. E., Backhed, F., Manchester, J. K.,
Hammer, R. E., Williams, S. C., Crowley, J., Yanagisawa, M., and Gordon, J. I.
(2008) Effects of the gut microbiota on host adiposity are modulated by the shortchain fatty-acid binding G protein-coupled receptor, Gpr41. Proc Natl Acad Sci
USA 105, 16767-16772
21.
Wang, Z., Klipfell, E., Bennett, B. J., Koeth, R., Levison, B. S., Dugar, B.,
Feldstein, A. E., Britt, E. B., Fu, X., Chung, Y.-M., Wu, Y., Schauer, P., Smith, J.
D., Allayee, H., Tang, W. H. W., DiDonato, J. A., Lusis, A. J., and Hazen, S. L.
185
(2011) Gut flora metabolism of phosphatidylcholine promotes cardiovascular
disease. Nature 472, 57-63
22.
Said, H. M. (2011) Intestinal absorption of water-soluble vitamins in health and
disease. Biochem J 437, 357-372
23.
Turnbaugh, P. J., Ley, R. E., Mahowald, M. A., Magrini, V., Mardis, E. R., and
Gordon, J. I. (2006) An obesity-associated gut microbiome with increased
capacity for energy harvest. Nature 444, 1027-1031
24.
Nicholson, J. K., Holmes, E., Kinross, J., Burcelin, R., Gibson, G., Jia, W., and
Pettersson, S. (2012) Host-gut microbiota metabolic interactions. Science 336,
1262-1267
25.
Ley, R. E., Turnbaugh, P. J., Klein, S., and Gordon, J. I. (2006) Microbial
ecology: human gut microbes associated with obesity. Nature 444, 1022-1023
26.
Vrieze, A., Van Nood, E., Holleman, F., Salojärvi, J., Kootte, R. S., Bartelsman,
J. F. W. M., Dallinga-Thie, G. M., Ackermans, M. T., Serlie, M. J., Oozeer, R.,
Derrien, M., Druesne, A., Van Hylckama Vlieg, J. E. T., Bloks, V. W., Groen, A.
K., Heilig, H. G. H. J., Zoetendal, E. G., Stroes, E. S., de Vos, W. M., Hoekstra, J.
B. L., and Nieuwdorp, M. (2012) Transfer of intestinal microbiota from lean
donors increases insulin sensitivity in individuals with metabolic syndrome.
Gastroenterology 143, 913-916
27.
Hooper, L. V., Littman, D. R., and Macpherson, A. J. (2012) Interactions between
the microbiota and the immune system. Science 336, 1268-1273
28.
Mazmanian, S. K., Round, J. L., and Kasper, D. L. (2008) A microbial symbiosis
factor prevents intestinal inflammatory disease. Nature 453, 620-625
29.
Meng, D., Newburg, D. S., Young, C., Baker, A., Tonkonogy, S. L., Sartor, R. B.,
Walker, W. A., and Nanthakumar, N. N. (2007) Bacterial symbionts induce a
FUT2-dependent fucosylated niche on colonic epithelium via ERK and JNK
signaling. Am J Physiol Gastrointest Liver Physiol 293, G780-787
30.
Sharma, R., Young, C., and Neu, J. (2010) Molecular modulation of intestinal
epithelial barrier: contribution of microbiota. J Biomed Biotechnol 2010, 305879
31.
Kawai, T., and Akira, S. (2005) Toll-like receptor downstream signaling. Arthritis
Res Ther 7, 12-19
32.
Froy, O. (2005) Regulation of mammalian defensin expression by Toll-like
receptor-dependent and independent signalling pathways. Cellular microbiology
7, 1387-1397
186
33.
Turner, J. R. (2009) Intestinal mucosal barrier function in health and disease. Nat
Rev Immunol 9, 799-809
34.
Kraehenbuhl, J. P., and Neutra, M. R. (2000) Epithelial M cells: differentiation
and function. Annu Rev Cell Dev Biol 16, 301-332
35.
Niess, J. H., and Reinecker, H.-C. (2006) Dendritic cells: the commanders-inchief of mucosal immune defenses. Curr Opin Gastroenterol 22, 354-360
36.
Abbas, A., Lichtman, A. H., and Pillai, S. (2007) Cellular and Molecular
Immunology, 6th Ed., Elsevier Saunders, Philadelphia, PA
37.
Neurath, M. F., Finotto, S., and Glimcher, L. H. (2002) The role of Th1/Th2
polarization in mucosal immunity. Nat Med 8, 567-573
38.
Roncarolo, M. G., Gregori, S., Battaglia, M., Bacchetta, R., Fleischhauer, K., and
Levings, M. K. (2006) Interleukin-10-secreting type 1 regulatory T cells in
rodents and humans. Immunol Rev 212, 28-50
39.
Miossec, P., and Kolls, J. K. (2012) Targeting IL-17 and TH17 cells in chronic
inflammation. Nat Rev Drug Discov 11, 763-776
40.
Bollyky, P. L., Evanko, S. P., Wu, R. P., Potter-Perigo, S., Long, S. A., Kinsella,
B., Reijonen, H., Guebtner, K., Teng, B., Chan, C. K., Braun, K. R., Gebe, J. A.,
Nepom, G. T., and Wight, T. N. (2010) Th1 cytokines promote T-cell binding to
antigen-presenting cells via enhanced hyaluronan production and accumulation at
the immune synapse. Cell Mol Immunol 7, 211-220
41.
Bollyky, P. L., Wu, R. P., Falk, B. A., Lord, J. D., Long, S. A., Preisinger, A.,
Teng, B., Holt, G. E., Standifer, N. E., Braun, K. R., Xie, C. F., Samuels, P. L.,
Vernon, R. B., Gebe, J. A., Wight, T. N., and Nepom, G. T. (2011) ECM
components guide IL-10 producing regulatory T-cell (TR1) induction from
effector memory T-cell precursors. Proc Natl Acad Sci USA 108, 7938-7943
42.
Luster, A. D., Alon, R., and von Andrian, U. H. (2005) Immune cell migration in
inflammation: present and future therapeutic targets. Nat Immunol 6, 1182-1190
43.
Weber, B., Saurer, L., and Mueller, C. (2009) Intestinal macrophages:
differentiation and involvement in intestinal immunopathologies. Semin
Immunopathol 31, 171-184
44.
Smythies, L. E., Sellers, M., Clements, R. H., Mosteller-Barnum, M., Meng, G.,
Benjamin, W. H., Orenstein, J. M., and Smith, P. D. (2005) Human intestinal
macrophages display profound inflammatory anergy despite avid phagocytic and
bacteriocidal activity. J Clin Invest 115, 66-75
187
45.
Furuta, G. T., Schmidt-Choudhury, A., Wang, M. Y., Wang, Z. S., Lu, L.,
Furlano, R. I., and Wershil, B. K. (1997) Mast cell-dependent tumor necrosis
factor α production participates in allergic gastric inflammation in mice.
Gastroenterology 113, 1560-1569
46.
Tete, S., Tripodi, D., Rosati, M., Conti, F., Maccauro, G., Saggini, A., Salini, V.,
Cianchetti, E., Caraffa, A., Antinolfi, P., Toniato, E., Castellani, M. L., Pandolfi,
F., Frydas, S., Conti, P., and Theoharides, T. C. (2012) Role of mast cells in
innate and adaptive immunity. J Biol Regul Homeost Agents 26, 193-201
47.
Hickey, M. J., and Kubes, P. (2009) Intravascular immunity: the host-pathogen
encounter in blood vessels. Nat Rev Immunol 9, 364-375
48.
Shi, F.-D., Ljunggren, H.-G., La Cava, A., and Van Kaer, L. (2011) Organspecific features of natural killer cells. Nat Rev Immunol 11, 658-671
49.
Boehm, T., Iwanami, N., and Hess, I. (2012) Evolution of the immune system in
the lower vertebrates. Annu Rev Genomics Hum Genet 13, 127-149
50.
Wang, D. Y., Kumar, S., and Hedges, S. B. (1999) Divergence time estimates for
the early history of animal phyla and the origin of plants, animals and fungi. Proc
Biol Sci 266, 163-171
51.
Janeway, C., Travers, P., and Walport, M. (2001) Immunobiology: The Immune
System in Health and Disease, 5th Ed. Garland Science, New York, NY
52.
Johansson, M. E. V., Ambort, D., Pelaseyed, T., Schütte, A., Gustafsson, J. K.,
Ermund, A., Subramani, D. B., Holmén-Larsson, J. M., Thomsson, K. A.,
Bergström, J. H., van der Post, S., Rodriguez-Piñeiro, A. M., Sjövall, H.,
Bäckström, M., and Hansson, G. C. (2011) Composition and functional role of the
mucus layers in the intestine. Cellular and molecular life sciences 68, 3635-3641
53.
Liu, B., Yu, Z., Chen, C., Kling, D. E., and Newburg, D. S. (2012) Human milk
mucin 1 and mucin 4 inhibit Salmonella enterica serovar Typhimurium invasion
of human intestinal epithelial cells in vitro. J Nutr 142, 1504-1509
54.
Johansson, M. E. V., Phillipson, M., Petersson, J., Velcich, A., Holm, L., and
Hansson, G. C. (2008) The inner of the two Muc2 mucin-dependent mucus layers
in colon is devoid of bacteria. Proc Natl Acad Sci USA 105, 15064-15069
55.
Van der Sluis, M., De Koning, B. A. E., De Bruijn, A. C. J. M., Velcich, A.,
Meijerink, J. P. P., Van Goudoever, J. B., Büller, H. A., Dekker, J., Van
Seuningen, I., Renes, I. B., and Einerhand, A. W. C. (2006) Muc2-deficient mice
spontaneously develop colitis, indicating that MUC2 is critical for colonic
protection. Gastroenterology 131, 117-129
188
56.
Heazlewood, C. K., Cook, M. C., Eri, R., Price, G. R., Tauro, S. B., Taupin, D.,
Thornton, D. J., Png, C. W., Crockford, T. L., Cornall, R. J., Adams, R., Kato, M.,
Nelms, K. A., Hong, N. A., Florin, T. H. J., Goodnow, C. C., and McGuckin, M.
A. (2008) Aberrant mucin assembly in mice causes endoplasmic reticulum stress
and spontaneous inflammation resembling ulcerative colitis. PLoS Med 5, e54
57.
Hermiston, M. L., and Gordon, J. I. (1995) In vivo analysis of cadherin function
in the mouse intestinal epithelium: essential roles in adhesion, maintenance of
differentiation, and regulation of programmed cell death. J Cell Biol 129, 489-506
58.
Laukoetter, M. G., Bruewer, M., and Nusrat, A. (2006) Regulation of the
intestinal epithelial barrier by the apical junctional complex. Curr Opin
Gastroenterol 22, 85-89
59.
Groschwitz, K. R., and Hogan, S. P. (2009) Intestinal barrier function: molecular
regulation and disease pathogenesis. J Allergy Clin Immunol 124, 3-20
60.
Kumar, H., Kawai, T., and Akira, S. (2011) Pathogen recognition by the innate
immune system. Int Rev Immunol 30, 16-34
61.
Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. M., and Hoffmann, J. A.
(1996) The dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the
potent antifungal response in Drosophila adults. Cell 86, 973-983
62.
Poltorak, A., He, X., Smirnova, I., Liu, M. Y., Van Huffel, C., Du, X., Birdwell,
D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P.,
Layton, B., and Beutler, B. (1998) Defective LPS signaling in C3H/HeJ and
C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085-2088
63.
Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda,
K., and Akira, S. (1999) Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice
are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene
product. J Immunol 162, 3749-3752
64.
Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T., Takeda,
K., and Akira, S. (1999) Differential roles of TLR2 and TLR4 in recognition of
gram-negative and gram-positive bacterial cell wall components. Immunity 11,
443-451
65.
Takeuchi, O., Kaufmann, A., Grote, K., Kawai, T., Hoshino, K., Morr, M.,
Mühlradt, P. F., and Akira, S. (2000) Cutting edge: preferentially the Rstereoisomer of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2
activates immune cells through a toll-like receptor 2- and MyD88-dependent
signaling pathway. J Immunol 164, 554-557
189
66.
Scheibner, K. A., Lutz, M. A., Boodoo, S., Fenton, M. J., Powell, J. D., and
Horton, M. R. (2006) Hyaluronan fragments act as an endogenous danger signal
by engaging TLR2. J Immunol 177, 1272-1281
67.
Alexopoulou, L., Holt, A. C., Medzhitov, R., and Flavell, R. A. (2001)
Recognition of double-stranded RNA and activation of NF-κB by Toll-like
receptor 3. Nature 413, 732-738
68.
Termeer, C., Benedix, F., Sleeman, J., Fieber, C., Voith, U., Ahrens, T., Miyake,
K., Freudenberg, M., Galanos, C., and Simon, J. C. (2002) Oligosaccharides of
Hyaluronan activate dendritic cells via toll-like receptor 4. J Exp Med 195, 99-111
69.
Hayashi, F., Smith, K. D., Ozinsky, A., Hawn, T. R., Yi, E. C., Goodlett, D. R.,
Eng, J. K., Akira, S., Underhill, D. M., and Aderem, A. (2001) The innate
immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature
410, 1099-1103
70.
Heil, F., Hemmi, H., Hochrein, H., Ampenberger, F., Kirschning, C., Akira, S.,
Lipford, G., Wagner, H., and Bauer, S. (2004) Species-specific recognition of
single-stranded RNA via toll-like receptor 7 and 8. Science 303, 1526-1529
71.
Diebold, S. S., Kaisho, T., Hemmi, H., Akira, S., and Reis e Sousa, C. (2004)
Innate antiviral responses by means of TLR7-mediated recognition of singlestranded RNA. Science 303, 1529-1531
72.
Hemmi, H., Kaisho, T., Takeuchi, O., Sato, S., Sanjo, H., Hoshino, K., Horiuchi,
T., Tomizawa, H., Takeda, K., and Akira, S. (2002) Small anti-viral compounds
activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat
Immunol 3, 196-200
73.
Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto,
M., Hoshino, K., Wagner, H., Takeda, K., and Akira, S. (2000) A Toll-like
receptor recognizes bacterial DNA. Nature 408, 740-745
74.
Zhang, D., Zhang, G., Hayden, M. S., Greenblatt, M. B., Bussey, C., Flavell, R.
A., and Ghosh, S. (2004) A toll-like receptor that prevents infection by
uropathogenic bacteria. Science 303, 1522-1526
75.
Takeuchi, O., Sato, S., Horiuchi, T., Hoshino, K., Takeda, K., Dong, Z., Modlin,
R. L., and Akira, S. (2002) Cutting edge: role of Toll-like receptor 1 in mediating
immune response to microbial lipoproteins. J Immunol 169, 10-14
76.
Scaffidi, P., Misteli, T., and Bianchi, M. E. (2002) Release of chromatin protein
HMGB1 by necrotic cells triggers inflammation. Nature 418, 191-195
190
77.
Bours, M. J. L., Swennen, E. L. R., Di Virgilio, F., Cronstein, B. N., and
Dagnelie, P. C. (2006) Adenosine 5'-triphosphate and adenosine as endogenous
signaling molecules in immunity and inflammation. Pharmacol Ther 112, 358404
78.
Shi, Y., Evans, J. E., and Rock, K. L. (2003) Molecular identification of a danger
signal that alerts the immune system to dying cells. Nature 425, 516-521
79.
Boeynaems, J.-M., and Communi, D. (2006) Modulation of inflammation by
extracellular nucleotides. J Invest Dermatol 126, 943-944
80.
Johnson, G. B., Brunn, G. J., Kodaira, Y., and Platt, J. L. (2002) Receptormediated monitoring of tissue well-being via detection of soluble heparan sulfate
by Toll-like receptor 4. J Immunol 168, 5233-5239
81.
Takeuchi, O., Takeda, K., Hoshino, K., Adachi, O., Ogawa, T., and Akira, S.
(2000) Cellular responses to bacterial cell wall components are mediated through
MyD88-dependent signaling cascades. Int Immunol 12, 113-117
82.
Murray, P. J., and Smale, S. T. (2012) Restraint of inflammatory signaling by
interdependent strata of negative regulatory pathways. Nat Immunol 13, 916-924
83.
Abreu, M. T., Vora, P., Faure, E., Thomas, L. S., Arnold, E. T., and Arditi, M.
(2001) Decreased expression of Toll-like receptor-4 and MD-2 correlates with
intestinal epithelial cell protection against dysregulated proinflammatory gene
expression in response to bacterial lipopolysaccharide. J Immunol 167, 1609-1616
84.
Savidge, T. C., Newman, P. G., Pan, W.-H., Weng, M.-Q., Shi, H. N.,
McCormick, B. A., Quaroni, A., and Walker, W. A. (2006) Lipopolysaccharideinduced human enterocyte tolerance to cytokine-mediated interleukin-8
production may occur independently of TLR-4/MD-2 signaling. Pediatr Res 59,
89-95
85.
Imani Fooladi, A. A., Mousavi, S. F., Seghatoleslami, S., Yazdani, S., and
Nourani, M. R. (2011) Toll-like receptors: role of inflammation and commensal
bacteria. Inflamm Allergy Drug Targets 10, 198-207
86.
Henneke, P., Takeuchi, O., van Strijp, J. A., Guttormsen, H. K., Smith, J. A.,
Schromm, A. B., Espevik, T. A., Akira, S., Nizet, V., Kasper, D. L., and
Golenbock, D. T. (2001) Novel engagement of CD14 and multiple toll-like
receptors by group B streptococci. J Immunol 167, 7069-7076
87.
Barton, G. M., and Medzhitov, R. (2002) Toll-like receptors and their ligands.
Curr Top Microbiol Immunol 270, 81-92
191
88.
Ganz, T. (1987) Extracellular release of antimicrobial defensins by human
polymorphonuclear leukocytes. Infect Immun 55, 568-571
89.
Tang, Y. Q., Yuan, J., Osapay, G., Osapay, K., Tran, D., Miller, C. J., Ouellette,
A. J., and Selsted, M. E. (1999) A cyclic antimicrobial peptide produced in
primate leukocytes by the ligation of two truncated α-defensins. Science 286,
498-502
90.
Cole, A. M., Hong, T., Boo, L. M., Nguyen, T., Zhao, C., Bristol, G., Zack, J. A.,
Waring, A. J., Yang, O. O., and Lehrer, R. I. (2002) Retrocyclin: a primate
peptide that protects cells from infection by T- and M-tropic strains of HIV-1.
Proc Natl Acad Sci USA 99, 1813-1818
91.
Zanetti, M. (2004) Cathelicidins, multifunctional peptides of the innate immunity.
J Leukoc Biol 75, 39-48
92.
Brogden, K. A. (2005) Antimicrobial peptides: pore formers or metabolic
inhibitors in bacteria? Nat Rev Microbiol 3, 238-250
93.
Zasloff, M. (2002) Antimicrobial peptides of multicellular organisms. Nature 415,
389-395
94.
Underwood, M. A., and Bevins, C. L. (2010) Defensin-barbed innate immunity:
clinical associations in the pediatric population. Pediatrics 125, 1237-1247
95.
Menendez, A., and Brett Finlay, B. (2007) Defensins in the immunology of
bacterial infections. Curr Opin Immunol 19, 385-391
96.
Ganz, T. (2003) Defensins: antimicrobial peptides of innate immunity. Nat Rev
Immunol 3, 710-720
97.
Bevins, C. L., and Salzman, N. H. (2011) Paneth cells, antimicrobial peptides and
maintenance of intestinal homeostasis. Nat Rev Microbiol 9, 356-368
98.
O'Neil, D. A. (2003) Regulation of expression of β-defensins: endogenous enteric
peptide antibiotics. Mol Immunol 40, 445-450
99.
Frye, M., Bargon, J., Lembcke, B., Wagner, T. O., and Gropp, R. (2000)
Differential expression of human α- and β-defensins mRNA in gastrointestinal
epithelia. Eur J Clin Invest 30, 695-701
100.
Wada, A., Mori, N., Oishi, K., Hojo, H., Nakahara, Y., Hamanaka, Y.,
Nagashima, M., Sekine, I., Ogushi, K., Niidome, T., Nagatake, T., Moss, J., and
Hirayama, T. (1999) Induction of human β-defensin-2 mRNA expression by
Helicobacter pylori in human gastric cell line MKN45 cells on cag pathogenicity
island. Biochem Biophys Res Commun 263, 770-774
192
101.
Ogushi , K., Wada, A., Niidome, T., Mori, N., Oishi, K., Nagatake, T., Takahashi,
A., Asakura, H., Makino , S., Hojo, H., Nakahara, Y., Ohsaki, M., Hatakeyama,
T., Aoyagi, H., Kurazono, H., Moss, J., and Hirayama, T. (2001) Salmonella
enteritidis FliC (flagella filament protein) induces human β-defensin-2 mRNA
production by Caco-2 cells. J Biol Chem 276, 30521-30526
102.
Vora, P., Youdim, A., Thomas, L. S., Fukata, M., Tesfay, S. Y., Lukasek, K.,
Michelsen, K. S., Wada, A., Hirayama, T., Arditi, M., and Abreu, M. T. (2004) βdefensin-2 expression is regulated by TLR signaling in intestinal epithelial cells. J
Immunol 173, 5398-5405
103.
O'Neil, D. A., Porter, E. M., Elewaut, D., Anderson, G. M., Eckmann, L., Ganz,
T., and Kagnoff, M. F. (1999) Expression and regulation of the human βdefensins hBD-1 and hBD-2 in intestinal epithelium. J Immunol 163, 6718-6724
104.
Schwab, M., Reynders, V., Loitsch, S., Steinhilber, D., Schröder, O., and Stein, J.
(2008) The dietary histone deacetylase inhibitor sulforaphane induces human βdefensin-2 in intestinal epithelial cells. Immunology 125, 241-251
105.
Barrera, G. J., Portillo, R., Mijares, A., Rocafull, M. A., del Castillo, J. R., and
Thomas, L. E. (2009) Immunoglobulin A with protease activity secreted in human
milk activates PAR-2 receptors, of intestinal epithelial cells HT-29, and promotes
β-defensin-2 expression. Immunol Lett 123, 52-59
106.
Schlee, M., Harder, J., Köten, B., Stange, E. F., Wehkamp, J., and Fellermann, K.
(2008) Probiotic lactobacilli and VSL#3 induce enterocyte β-defensin 2. Clin Exp
Immunol 151, 528-535
107.
Singh, P. K., Jia, H. P., Wiles, K., Hesselberth, J., Liu, L., Conway, B. A.,
Greenberg, E. P., Valore, E. V., Welsh, M. J., Ganz, T., Tack, B. F., and McCray,
P. B. (1998) Production of β-defensins by human airway epithelia. Proc Natl
Acad Sci USA 95, 14961-14966
108.
Antcheva, N., Boniotto, M., Zelezetsky, I., Pacor, S., Verga Falzacappa, M. V.,
Crovella, S., and Tossi, A. (2004) Effects of positively selected sequence
variations in human and Macaca fascicularis β-defensins 2 on antimicrobial
activity. Antimicrob Agents Chemother 48, 685-688
109.
Harder, J., Bartels, J., Christophers, E., and Schröder, J. M. (1997) A peptide
antibiotic from human skin. Nature 387, 861
110.
Feng, Z., Jiang, B., Chandra, J., Ghannoum, M., Nelson, S., and Weinberg, A.
(2005) Human β-defensins: differential activity against candidal species and
regulation by Candida albicans. J Dent Res 84, 445-450
193
111.
Ogushi, K.-i., Wada, A., Niidome, T., Okuda, T., Llanes, R., Nakayama, M.,
Nishi, Y., Kurazono, H., Smith, K. D., Aderem, A., Moss, J., and Hirayama, T.
(2004) Gangliosides act as co-receptors for Salmonella enteritidis FliC and
promote FliC induction of human β-defensin-2 expression in Caco-2 cells. J Biol
Chem 279, 12213-12219
112.
Platz, J., Beisswenger, C., Dalpke, A., Koczulla, R., Pinkenburg, O., Vogelmeier,
C., and Bals, R. (2004) Microbial DNA induces a host defense reaction of human
respiratory epithelial cells. J Immunol 173, 1219-1223
113.
Voss, E., Wehkamp, J., Wehkamp, K., Stange, E. F., Schröder, J. M., and Harder,
J. (2006) NOD2/CARD15 mediates induction of the antimicrobial peptide human
β-defensin-2. J Biol Chem 281, 2005-2011
114.
Molodecky, N. A., Soon, I. S., Rabi, D. M., Ghali, W. A., Ferris, M., Chernoff,
G., Benchimol, E. I., Panaccione, R., Ghosh, S., Barkema, H. W., and Kaplan, G.
G. (2012) Increasing incidence and prevalence of the inflammatory bowel
diseases with time, based on systematic review. Gastroenterology 142, 46-54
115.
Prideaux, L., Kamm, M. A., De Cruz, P. P., Chan, F. K. L., and Ng, S. C. (2012)
Inflammatory bowel disease in Asia: a systematic review. J Gastroenterol
Hepatol 27, 1266-1280
116.
Loly, C., Belaiche, J., and Louis, E. (2008) Predictors of severe Crohn's disease.
Scand J Gastroenterol 43, 948-954
117.
Vatn, M. H. (2009) Natural history and complications of IBD. Curr Gastroenterol
Rep 11, 481-487
118.
Cosnes, J., Cattan, S., Blain, A., Beaugerie, L., Carbonnel, F., Parc, R., and
Gendre, J.-P. (2002) Long-term evolution of disease behavior of Crohn's disease.
Inflamm Bowel Dis 8, 244-250
119.
Lu, K. C., and Hunt, S. R. (2013) Surgical management of Crohn's disease. Surg
Clin North Am 93, 167-185
120.
Bewtra, M., Kaiser, L. M., Tenhave, T., and Lewis, J. D. (2013) Crohn's Disease
and Ulcerative Colitis Are Associated With Elevated Standardized Mortality
Ratios: A Meta-Analysis. Inflamm Bowel Dis 19, 599-613
121.
Korzenik, J. R., and Dieckgraefe, B. K. (2000) Is Crohn's disease an
immunodeficiency? A hypothesis suggesting possible early events in the
pathogenesis of Crohn's disease. Dig Dis Sci 45, 1121-1129
122.
Cobrin, G. M., and Abreu, M. T. (2005) Defects in mucosal immunity leading to
Crohn's disease. Immunol Rev 206, 277-295
194
123.
Wehkamp, J., Koslowski, M., Wang, G., and Stange, E. F. (2008) Barrier
dysfunction due to distinct defensin deficiencies in small intestinal and colonic
Crohn's disease. Mucosal immunology Suppl 1, S67-74
124.
Frøslie, K. F., Jahnsen, J., Moum, B. A., Vatn, M. H., and Group, I. (2007)
Mucosal healing in inflammatory bowel disease: results from a Norwegian
population-based cohort. Gastroenterology 133, 412-422
125.
Jostins, L., Ripke, S., Weersma, R. K., Duerr, R. H., McGovern, D. P., Hui, K. Y.,
Lee, J. C., Schumm, L. P., Sharma, Y., Anderson, C. A., Essers, J., Mitrovic, M.,
Ning, K., Cleynen, I., Theatre, E., Spain, S. L., Raychaudhuri, S., Goyette, P.,
Wei, Z., Abraham, C., Achkar, J.-P., Ahmad, T., Amininejad, L.,
Ananthakrishnan, A. N., Andersen, V., Andrews, J. M., Baidoo, L., Balschun, T.,
Bampton, P. A., Bitton, A., Boucher, G., Brand, S., Büning, C., Cohain, A.,
Cichon, S., D'Amato, M., De Jong, D., Devaney, K. L., Dubinsky, M., Edwards,
C., Ellinghaus, D., Ferguson, L. R., Franchimont, D., Fransen, K., Gearry, R.,
Georges, M., Gieger, C., Glas, J., Haritunians, T., Hart, A., Hawkey, C., Hedl, M.,
Hu, X., Karlsen, T. H., Kupcinskas, L., Kugathasan, S., Latiano, A., Laukens, D.,
Lawrance, I. C., Lees, C. W., Louis, E., Mahy, G., Mansfield, J., Morgan, A. R.,
Mowat, C., Newman, W., Palmieri, O., Ponsioen, C. Y., Potocnik, U., Prescott, N.
J., Regueiro, M., Rotter, J. I., Russell, R. K., Sanderson, J. D., Sans, M., Satsangi,
J., Schreiber, S., Simms, L. A., Sventoraityte, J., Targan, S. R., Taylor, K. D.,
Tremelling, M., Verspaget, H. W., De Vos, M., Wijmenga, C., Wilson, D. C.,
Winkelmann, J., Xavier, R. J., Zeissig, S., Zhang, B., Zhang, C. K., Zhao, H.,
(IIBDGC), I. I. G. C., Silverberg, M. S., Annese, V., Hakonarson, H., Brant, S. R.,
Radford-Smith, G., Mathew, C. G., Rioux, J. D., Schadt, E. E., Daly, M. J.,
Franke, A., Parkes, M., Vermeire, S., Barrett, J. C., and Cho, J. H. (2012) Hostmicrobe interactions have shaped the genetic architecture of inflammatory bowel
disease. Nature 491, 119-124
126.
Ogura, Y., Bonen, D. K., Inohara, N., Nicolae, D. L., Chen, F. F., Ramos, R.,
Britton, H., Moran, T., Karaliuskas, R., Duerr, R. H., Achkar, J. P., Brant, S. R.,
Bayless, T. M., Kirschner, B. S., Hanauer, S. B., Nuñez, G., and Cho, J. H. (2001)
A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease.
Nature 411, 603-606
127.
Liu, L., Wang, L., Jia, H. P., Zhao, C., Heng, H. H., Schutte, B. C., McCray, P.
B., and Ganz, T. (1998) Structure and mapping of the human β-defensin HBD-2
gene and its expression at sites of inflammation. Gene 222, 237-244
128.
Aldhous, M. C., Abu Bakar, S., Prescott, N. J., Palla, R., Soo, K., Mansfield, J.
C., Mathew, C. G., Satsangi, J., and Armour, J. A. L. (2010) Measurement
methods and accuracy in copy number variation: failure to replicate associations
of β-defensin copy number with Crohn's disease. Hum Mol Genet 19, 4930-4938
195
129.
Wehkamp, J., Fellermann, K., Herrlinger, K. R., Baxmann, S., Schmidt, K.,
Schwind, B., Duchrow, M., Wohlschläger, C., Feller, A. C., and Stange, E. F.
(2002) Human β-defensin 2 but not β-defensin 1 is expressed preferentially in
colonic mucosa of inflammatory bowel disease. Eur J Gastroenterol Hepatol 14,
745-752
130.
Bentley, R., Pearson, J., Gearry, R., Barclay, M., McKinney, C., Merriman, T.,
and Roberts, R. (2009) Association of Higher DEFB4 Genomic Copy Number
With Crohn's Disease. The American journal of gastroenterology 105, 354-359
131.
Aldhous, M. C., Noble, C. L., and Satsangi, J. (2009) Dysregulation of human βdefensin-2 protein in inflammatory bowel disease. PLoS ONE 4, e6285
132.
Palma, G. D., Capilla, A., Nova, E., Castillejo, G., Varea, V., Pozo, T., Garrote, J.
A., Polanco, I., López, A., Ribes-Koninckx, C., Marcos, A., García-Novo, M. D.,
Calvo, C., Ortigosa, L., Peña-Quintana, L., Palau, F., and Sanz, Y. (2012)
Influence of milk-feeding type and genetic risk of developing coeliac disease on
intestinal microbiota of infants: the PROFICEL study. PLoS ONE 7, e30791
133.
M'Rabet, L., Vos, A. P., Boehm, G., and Garssen, J. (2008) Breast-feeding and its
role in early development of the immune system in infants: consequences for
health later in life. J Nutr 138, 1782S-1790S
134.
Anderson, J. W., Johnstone, B. M., and Remley, D. T. (1999) Breast-feeding and
cognitive development: a meta-analysis. Am J Clin Nutr 70, 525-535
135.
Gale, C., Logan, K. M., Santhakumaran, S., Parkinson, J. R. C., Hyde, M. J., and
Modi, N. (2012) Effect of breastfeeding compared with formula feeding on infant
body composition: a systematic review and meta-analysis. Am J Clin Nutr 95,
656-669
136.
Howie, P. W., Forsyth, J. S., Ogston, S. A., Clark, A., and Florey, C. D. (1990)
Protective effect of breast feeding against infection. BMJ 300, 11-16
137.
Klement, E., Cohen, R. V., Boxman, J., Joseph, A., and Reif, S. (2004)
Breastfeeding and risk of inflammatory bowel disease: a systematic review with
meta-analysis. Am J Clin Nutr 80, 1342-1352
138.
Grulee, C. G., Sanford, H. N., and Herron, P. H. (1934) Breast and artificial
feeding: Influence on morbidity and mortality of twenty thousand infants. JAMA
103, 735-738
139.
Morrow, A. L., Guerrero, M. L., Shults, J., Calva, J. J., Lutter, C., Bravo, J., RuizPalacios, G., Morrow, R. C., and Butterfoss, F. D. (1999) Efficacy of home-based
peer counselling to promote exclusive breastfeeding: a randomised controlled
trial. Lancet 353, 1226-1231
196
140.
Jones, T. F., Ingram, L. A., Fullerton, K. E., Marcus, R., Anderson, B. J.,
McCarthy, P. V., Vugia, D., Shiferaw, B., Haubert, N., Wedel, S., and Angulo, F.
J. (2006) A case-control study of the epidemiology of sporadic Salmonella
infection in infants. Pediatrics 118, 2380-2387
141.
Owen, C. G., Martin, R. M., Whincup, P. H., Smith, G. D., and Cook, D. G.
(2005) Effect of infant feeding on the risk of obesity across the life course: a
quantitative review of published evidence. Pediatrics 115, 1367-1377
142.
Oddy, W. H. (2012) Infant feeding and obesity risk in the child. Breastfeed Rev
20, 7-12
143.
Gdalevich, M., Mimouni, D., David, M., and Mimouni, M. (2001) Breast-feeding
and the onset of atopic dermatitis in childhood: a systematic review and metaanalysis of prospective studies. J Am Acad Dermatol 45, 520-527
144.
van Odijk, J., Kull, I., Borres, M. P., Brandtzaeg, P., Edberg, U., Hanson, L. A.,
Høst, A., Kuitunen, M., Olsen, S. F., Skerfving, S., Sundell, J., and Wille, S.
(2003) Breastfeeding and allergic disease: a multidisciplinary review of the
literature (1966-2001) on the mode of early feeding in infancy and its impact on
later atopic manifestations. Allergy 58, 833-843
145.
Brandtzaeg, P. (2003) Mucosal immunity: integration between mother and the
breast-fed infant. Vaccine 21, 3382-3388
146.
Ward, P. P., Uribe-Luna, S., and Conneely, O. M. (2002) Lactoferrin and host
defense. Biochem Cell Biol 80, 95-102
147.
Martin, L. J., Woo, J. G., Geraghty, S. R., Altaye, M., Davidson, B. S., Banach,
W., Dolan, L. M., Ruiz-Palacios, G. M., and Morrow, A. L. (2006) Adiponectin is
present in human milk and is associated with maternal factors. Am J Clin Nutr 83,
1106-1111
148.
Asakuma, S., Hatakeyama, E., Urashima, T., Yoshida, E., Katayama, T.,
Yamamoto, K., Kumagai, H., Ashida, H., Hirose, J., and Kitaoka, M. (2011)
Physiology of consumption of human milk oligosaccharides by infant gutassociated bifidobacteria. J Biol Chem 286, 34583-34592
149.
Marcobal, A., Barboza, M., Sonnenburg, E. D., Pudlo, N., Martens, E. C., Desai,
P., Lebrilla, C. B., Weimer, B. C., Mills, D. A., German, J. B., and Sonnenburg, J.
L. (2011) Bacteroides in the infant gut consume milk oligosaccharides via mucusutilization pathways. Cell Host Microbe 10, 507-514
197
150.
Sela, D. A., Li, Y., Lerno, L., Wu, S., Marcobal, A. M., German, J. B., Chen, X.,
Lebrilla, C. B., and Mills, D. A. (2011) An infant-associated bacterial commensal
utilizes breast milk sialyloligosaccharides. J Biol Chem 286, 11909-11918
151.
Yu, Z.-T., Chen, C., Kling, D. E., Liu, B., McCoy, J. M., Merighi, M., Heidtman,
M., and Newburg, D. S. (2012) The Principal Fucosylated Oligosaccharides of
Human Milk Exhibit Prebiotic Properties on Cultured Infant Microbiota.
Glycobiology 23,169-177
152.
Brandtzaeg, P. (2010) The mucosal immune system and its integration with the
mammary glands. J Pediatr 156, S8-15
153.
Uren, T. K., Wijburg, O. L. C., Simmons, C., Johansen, F.-E., Brandtzaeg, P., and
Strugnell, R. A. (2005) Vaccine-induced protection against gastrointestinal
bacterial infections in the absence of secretory antibodies. Eur J Immunol 35, 180188
154.
Jenssen, H., and Hancock, R. E. W. (2009) Antimicrobial properties of
lactoferrin. Biochimie 91, 19-29
155.
Ellison, R. T., and Giehl, T. J. (1991) Killing of gram-negative bacteria by
lactoferrin and lysozyme. J Clin Invest 88, 1080-1091
156.
Thormar, H., Isaacs, C. E., Brown, H. R., Barshatzky, M. R., and Pessolano, T.
(1987) Inactivation of enveloped viruses and killing of cells by fatty acids and
monoglycerides. Antimicrob Agents Chemother 31, 27-31
157.
Hamosh, M. (1998) Protective function of proteins and lipids in human milk. Biol
Neonate 74, 163-176
158.
Newburg, D. S. (2009) Neonatal protection by an innate immune system of
human milk consisting of oligosaccharides and glycans. J Anim Sci 87, 26-34
159.
Bode, L. (2012) Human milk oligosaccharides: every baby needs a sugar mama.
Glycobiology 22, 1147-1162
160.
Ruiz-Palacios, G. M., Cervantes, L. E., Ramos, P., Chavez-Munguia, B., and
Newburg, D. S. (2003) Campylobacter jejuni binds intestinal H(O) antigen (Fuc α
1, 2Gal β 1, 4GlcNAc), and fucosyloligosaccharides of human milk inhibit its
binding and infection. J Biol Chem 278, 14112-14120
161.
Morrow, A. L., Ruiz-Palacios, G. M., Altaye, M., Jiang, X., Guerrero, M. L.,
Meinzen-Derr, J. K., Farkas, T., Chaturvedi, P., Pickering, L. K., and Newburg,
D. S. (2004) Human milk oligosaccharides are associated with protection against
diarrhea in breast-fed infants. J Pediatr 145, 297-303
198
162.
Angeloni, S., Ridet, J. L., Kusy, N., Gao, H., Crevoisier, F., Guinchard, S.,
Kochhar, S., Sigrist, H., and Sprenger, N. (2005) Glycoprofiling with microarrays of glycoconjugates and lectins. Glycobiology 15, 31-41
163.
Kuntz, S., Rudloff, S., and Kunz, C. (2008) Oligosaccharides from human milk
influence growth-related characteristics of intestinally transformed and nontransformed intestinal cells. Br J Nutr 99, 462-471
164.
Kuntz, S., Kunz, C., and Rudloff, S. (2009) Oligosaccharides from human milk
induce growth arrest via G2/M by influencing growth-related cell cycle genes in
intestinal epithelial cells. Br J Nutr 101, 1306-1315
165.
Cederlund, A., Kai-Larsen, Y., Printz, G., Yoshio, H., Alvelius, G., Lagercrantz,
H., Strömberg, R., Jörnvall, H., Gudmundsson, G. H., and Agerberth, B. (2013)
Lactose in human breast milk an inducer of innate immunity with implications for
a role in intestinal homeostasis. PLoS ONE 8, e53876
166.
Stern, R., Asari, A. A., and Sugahara, K. N. (2006) Hyaluronan fragments: an
information-rich system. Eur J Cell Biol 85, 699-715
167.
Coppa, G. V., Gabrielli, O., Buzzega, D., Zampini, L., Galeazzi, T., Maccari, F.,
Bertino, E., and Volpi, N. (2011) Composition and structure elucidation of human
milk glycosaminoglycans. Glycobiology 21, 295-303
168.
Newburg, D. S., Linhardt, R. J., Ampofo, S. A., and Yolken, R. H. (1995) Human
milk glycosaminoglycans inhibit HIV glycoprotein gp120 binding to its host cell
CD4 receptor. J Nutr 125, 419-424
169.
Varki, A., Cummings, R., Esko, J. D., Doering, T. L., and Raetz CR, H. (2009)
Essentials of Glycobiology, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY
170.
Funderburgh, J. L. (2002) Keratan sulfate biosynthesis. IUBMB Life 54, 187-194
171.
Björk, I., and Lindahl, U. (1982) Mechanism of the anticoagulant action of
heparin. Mol Cell Biochem 48, 161-182
172.
Liu, H., Zhang, Z., and Linhardt, R. J. (2009) Lessons learned from the
contamination of heparin. Nat Prod Rep 26, 313-321
173.
Bishop, J. R., Schuksz, M., and Esko, J. D. (2007) Heparan sulphate
proteoglycans fine-tune mammalian physiology. Nature 446, 1030-1037
174.
Yanagishita, M., and Hascall, V. C. (1992) Cell surface heparan sulfate
proteoglycans. J Biol Chem 267, 9451-9454
199
175.
Yamada, S., Sugahara, K., and Ozbek, S. (2011) Evolution of
glycosaminoglycans: Comparative biochemical study. Commun Integr Biol 4,
150-158
176.
Handley, C. J., Samiric, T., and Ilic, M. Z. (2006) Structure, metabolism, and
tissue roles of chondroitin sulfate proteoglycans. Adv Pharmacol 53, 219-232
177.
Ruffell, B., Poon, G. F. T., Lee, S. S. M., Brown, K. L., Tjew, S.-L., Cooper, J.,
and Johnson, P. (2011) Differential use of chondroitin sulfate to regulate
hyaluronan binding by receptor CD44 in Inflammatory and Interleukin 4activated Macrophages. J Biol Chem 286, 19179-19190
178.
Martel-Pelletier, J., Kwan Tat, S., and Pelletier, J.-P. (2010) Effects of chondroitin
sulfate in the pathophysiology of the osteoarthritic joint: a narrative review.
Osteoarthr Cartil 18, S7-11
179.
du Souich, P., García, A. G., Vergés, J., and Montell, E. (2009)
Immunomodulatory and anti-inflammatory effects of chondroitin sulphate. J Cell
Mol Med 13, 1451-1463
180.
Trowbridge, J. M., and Gallo, R. L. (2002) Dermatan sulfate: new functions from
an old glycosaminoglycan. Glycobiology 12, 117R-125R
181.
Wöhler, F. (1828) Ueber künstliche Bildung des Harnstoffs. Annalen der Physik
88, 253-256
182.
Meyer, K., and Palmer, J. (1934) The polysaccharide of the vitreous humor. J Biol
Chem 107, 629-634
183.
Volpi, N., and Maccari, F. (2003) Purification and characterization of hyaluronic
acid from the mollusc bivalve Mytilus galloprovincialis. Biochimie 85, 619-625
184.
Liu, L., Liu, Y., Li, J., Du, G., and Chen, J. (2011) Microbial production of
hyaluronic acid: current state, challenges, and perspectives. Microb Cell Fact 10,
99
185.
McDonald, J. A., and Camenisch, T. D. (2002) Hyaluronan: genetic insights into
the complex biology of a simple polysaccharide. Glycoconj J 19, 331-339
186.
Tammi, R. H., Passi, A. G., Rilla, K., Karousou, E., Vigetti, D., Makkonen, K.,
and Tammi, M. I. (2011) Transcriptional and post-translational regulation of
hyaluronan synthesis. FEBS J 278, 1419-1428
187.
Weigel, P. H., and DeAngelis, P. L. (2007) Hyaluronan synthases: a decade-plus
of novel glycosyltransferases. J Biol Chem 282, 36777-36781
200
188.
Wang, A., de la Motte, C., Lauer, M., and Hascall, V. (2011) Hyaluronan matrices
in pathobiological processes. FEBS J 278, 1412-1418
189.
Oguchi, T., and Ishiguro, N. (2004) Differential stimulation of three forms of
hyaluronan synthase by TGF-β, IL-1β, and TNF-α. Connect Tissue Res 45, 197205
190.
Ducale, A. E., Ward, S. I., Dechert, T., and Yager, D. R. (2005) Regulation of
hyaluronan synthase-2 expression in human intestinal mesenchymal cells:
mechanisms of interleukin-1β-mediated induction. Am J Physiol Gastrointest
Liver Physiol 289, G462-470
191.
de la Motte, C. A., Hascall, V. C., Drazba, J., Bandyopadhyay, S. K., and Strong,
S. A. (2003) Mononuclear Leukocytes Bind to Specific Hyaluronan Structures on
Colon Mucosal Smooth Muscle Cells Treated with Polyinosinic Acid:
Polycytidylic Acid Inter-α-Trypsin Inhibitor Is Crucial to Structure and Function.
American Journal of Pathology 163, 121-133
192.
Kessler, S., Rho, H., West, G., Fiocchi, C., Drazba, J., and de la Motte, C. A.
(2008) Hyaluronan (HA) deposition precedes and promotes leukocyte recruitment
in intestinal inflammation. Clinical and Translational Science 1, 57-61
193.
Jacobson, A., Brinck, J., Briskin, M. J., Spicer, A. P., and Heldin, P. (2000)
Expression of human hyaluronan synthases in response to external stimuli.
Biochem J 348, 29-35
194.
Evanko, S. P., Johnson, P. Y., Braun, K. R., Underhill, C. B., Dudhia, J., and
Wight, T. N. (2001) Platelet-derived growth factor stimulates the formation of
versican-hyaluronan aggregates and pericellular matrix expansion in arterial
smooth muscle cells. Arch Biochem Biophys 394, 29-38
195.
Karvinen, S., Pasonen-Seppänen, S., Hyttinen, J. M. T., Pienimäki, J.-P.,
Törrönen, K., Jokela, T. A., Tammi, M. I., and Tammi, R. (2003) Keratinocyte
growth factor stimulates migration and hyaluronan synthesis in the epidermis by
activation of keratinocyte hyaluronan synthases 2 and 3. J Biol Chem 278, 4949549504
196.
Sussmann, M., Sarbia, M., Meyer-Kirchrath, J., Nüsing, R. M., Schrör, K., and
Fischer, J. W. (2004) Induction of hyaluronic acid synthase 2 (HAS2) in human
vascular smooth muscle cells by vasodilatory prostaglandins. Circ Res 94, 592600
197.
Zhang, W., Watson, C. E., Liu, C., Williams, K. J., and Werth, V. P. (2000)
Glucocorticoids induce a near-total suppression of hyaluronan synthase mRNA in
dermal fibroblasts and in osteoblasts: a molecular mechanism contributing to
organ atrophy. Biochem J 349, 91-97
201
198.
Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A.,
Yamashita, H., Hellman, U., Heldin, C.-H., and Heldin, P. (2010) The activity of
hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J Biol
Chem 285, 23647-23654
199.
Vigetti, D., Deleonibus, S., Moretto, P., Karousou, E., Viola, M., Bartolini, B.,
Hascall, V. C., Tammi, M., De Luca, G., and Passi, A. (2012) Role of UDP-Nacetylglucosamine (GlcNAc) and O-GlcNAcylation of hyaluronan synthase 2 in
the control of chondroitin sulfate and hyaluronan synthesis. J Biol Chem 287,
35544-35555
200.
Rilla, K., Oikari, S., Jokela, T. A., Hyttinen, J. M. T., Kärnä, R., Tammi, R. H.,
and Tammi, M. I. (2013) Hyaluronan synthase 1 (HAS1) requires higher cellular
UDP-GlcNAc concentration than HAS2 and HAS3. J Biol Chem 288, 5973-5983
201.
Camenisch, T. D., Spicer, A. P., Brehm-Gibson, T., Biesterfeldt, J., Augustine, M.
L., Calabro, A., Kubalak, S., Klewer, S. E., and McDonald, J. A. (2000)
Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and
hyaluronan-mediated transformation of epithelium to mesenchyme. J Clin Invest
106, 349-360
202.
Mack, J. A., Feldman, R. J., Itano, N., Kimata, K., Lauer, M., Hascall, V. C., and
Maytin, E. V. (2012) Enhanced inflammation and accelerated wound closure
following tetraphorbol ester application or full-thickness wounding in mice
lacking hyaluronan synthases Has1 and Has3. J Invest Dermatol 132, 198-207
203.
Stern, R. (2003) Devising a pathway for hyaluronan catabolism: are we there yet?
Glycobiology 13, 105R-115R
204.
Stern, R., Kogan, G., Jedrzejas, M. J., and Soltés, L. (2007) The many ways to
cleave hyaluronan. Biotechnol Adv 25, 537-557
205.
Halliwell, B. (1982) Production of superoxide, hydrogen peroxide and hydroxyl
radicals by phagocytic cells: a cause of chronic inflammatory disease? Cell Biol
Int Rep 6, 529-542
206.
Rees, M. D., Hawkins, C. L., and Davies, M. J. (2003) Hypochlorite-mediated
fragmentation of hyaluronan, chondroitin sulfates, and related N-acetyl
glycosamines: evidence for chloramide intermediates, free radical transfer
reactions, and site-specific fragmentation. J Am Chem Soc 125, 13719-13733
207.
Cherr, G. N., Yudin, A. I., and Overstreet, J. W. (2001) The dual functions of
GPI-anchored PH-20: hyaluronidase and intracellular signaling. Matrix Biol 20,
515-525
202
208.
Frost, G. I., Csóka, A. B., Wong, T., Stern, R., and Csóka, T. B. (1997)
Purification, cloning, and expression of human plasma hyaluronidase. Biochem
Biophys Res Commun 236, 10-15
209.
Lepperdinger, G., Strobl, B., and Kreil, G. (1998) HYAL2, a human gene
expressed in many cells, encodes a lysosomal hyaluronidase with a novel type of
specificity. J Biol Chem 273, 22466-22470
210.
Harada, H., and Takahashi, M. (2007) CD44-dependent intracellular and
extracellular catabolism of hyaluronic acid by hyaluronidase-1 and -2. J Biol
Chem 282, 5597-5607
211.
Triggs-Raine, B., Salo, T. J., Zhang, H., Wicklow, B. A., and Natowicz, M. R.
(1999) Mutations in HYAL1, a member of a tandemly distributed multigene
family encoding disparate hyaluronidase activities, cause a newly described
lysosomal disorder, mucopolysaccharidosis IX. Proc Natl Acad Sci USA 96,
6296-6300
212.
Lepperdinger, G., Müllegger, J., and Kreil, G. (2001) Hyal2--less active, but more
versatile? Matrix Biol 20, 509-514
213.
Chowdhury, B., Hemming, R., Hombach-Klonisch, S., Flamion, B., and TriggsRaine, B. (2013) Murine hyaluronidase 2 deficiency results in extracellular
hyaluronan accumulation and severe cardiopulmonary dysfunction. J Biol Chem
288, 520-528
214.
Jadin, L., Wu, X., Ding, H., Frost, G. I., Onclinx, C., Triggs-Raine, B., and
Flamion, B. (2008) Skeletal and hematological anomalies in HYAL2-deficient
mice: a second type of mucopolysaccharidosis IX? FASEB J 22, 4316-4326
215.
de la Motte, C. A., Hascall, V. C., Calabro, A., Yen-Lieberman, B., and Strong, S.
A. (1999) Mononuclear leukocytes preferentially bind via CD44 to hyaluronan on
human intestinal mucosal smooth muscle cells after virus infection or treatment
with poly(I:C). J Biol Chem 274, 30747-30755
216.
de la Motte, C. A., Nigro, J., Vasanji, A., Rho, H., Kessler, S. P., Bandyopadhyay,
S. K., Danese, S., Fiocchi, C., and Stern, R. (2009) Platelet-derived hyaluronidase
2 cleaves hyaluronan into fragments that trigger monocyte-mediated production
of proinflammatory cytokines. Am J Pathol 174, 2254-2264
217.
Miyake, K., Underhill, C. B., Lesley, J., and Kincade, P. W. (1990) Hyaluronate
can function as a cell adhesion molecule and CD44 participates in hyaluronate
recognition. J Exp Med 172, 69-75
218.
Lesley, J., Hascall, V. C., Tammi, M., and Hyman, R. (2000) Hyaluronan binding
by cell surface CD44. J Biol Chem 275, 26967-26975
203
219.
Wolny, P. M., Banerji, S., Gounou, C., Brisson, A. R., Day, A. J., Jackson, D. G.,
and Richter, R. P. (2010) Analysis of CD44-hyaluronan interactions in an
artificial membrane system: insights into the distinct binding properties of high
and low molecular weight hyaluronan. J Biol Chem 285, 30170-30180
220.
Zhang, T., Huang, X.-H., Dong, L., Hu, D., Ge, C., Zhan, Y.-Q., Xu, W.-X., Yu,
M., Li, W., Wang, X., Tang, L., Li, C.-Y., and Yang, X.-M. (2010) PCBP-1
regulates alternative splicing of the CD44 gene and inhibits invasion in human
hepatoma cell line HepG2 cells. Mol Cancer 9, 72
221.
Eberth, S., Schneider, B., Rosenwald, A., Hartmann, E. M., Romani, J., Zaborski,
M., Siebert, R., Drexler, H. G., and Quentmeier, H. (2010) Epigenetic regulation
of CD44 in Hodgkin and non-Hodgkin lymphoma. BMC Cancer 10, 517
222.
Brown, R. L., Reinke, L. M., Damerow, M. S., Perez, D., Chodosh, L. A., Yang,
J., and Cheng, C. (2011) CD44 splice isoform switching in human and mouse
epithelium is essential for epithelial-mesenchymal transition and breast cancer
progression. J Clin Invest 121, 1064-1074
223.
Noble, P. W., Lake, F. R., Henson, P. M., and Riches, D. W. (1993) Hyaluronate
activation of CD44 induces insulin-like growth factor-1 expression by a tumor
necrosis factor-α-dependent mechanism in murine macrophages. J Clin Invest 91,
2368-2377
224.
Noble, P. W., McKee, C. M., Cowman, M., and Shin, H. S. (1996) Hyaluronan
fragments activate an NF-κB/I-κB α autoregulatory loop in murine macrophages.
J Exp Med 183, 2373-2378
225.
Beck-Schimmer, B., Oertli, B., Pasch, T., and Wüthrich, R. P. (1998) Hyaluronan
induces monocyte chemoattractant protein-1 expression in renal tubular epithelial
cells. J Am Soc Nephrol 9, 2283-2290
226.
Chen, G. Y., and Nuñez, G. (2010) Sterile inflammation: sensing and reacting to
damage. Nat Rev Immunol 10, 826-837
227.
Taylor, K. R., Yamasaki, K., Radek, K. A., Di Nardo, A., Goodarzi, H.,
Golenbock, D., Beutler, B., and Gallo, R. L. (2007) Recognition of hyaluronan
released in sterile injury involves a unique receptor complex dependent on Tolllike receptor 4, CD44, and MD-2. J Biol Chem 282, 18265-18275
228.
Jiang, D., Liang, J., Fan, J., Yu, S., Chen, S., Luo, Y., Prestwich, G. D.,
Mascarenhas, M. M., Garg, H. G., Quinn, D. A., Homer, R. J., Goldstein, D. R.,
Bucala, R., Lee, P. J., Medzhitov, R., and Noble, P. W. (2005) Regulation of lung
injury and repair by Toll-like receptors and hyaluronan. Nat Med 11, 1173-1179
204
229.
Jiang, D., Liang, J., and Noble, P. W. (2007) Hyaluronan in tissue injury and
repair. Annu Rev Cell Dev Biol 23, 435-461
230.
Frey, H., Schroeder, N., Manon-Jensen, T., Iozzo, R. V., and Schaefer, L. (2013)
Biological interplay between proteoglycans and their innate immune receptors in
inflammation. FEBS J doi: 10.1111/febs.12145
231.
Campo, G. M., Avenoso, A., Campo, S., D'Ascola, A., Nastasi, G., and Calatroni,
A. (2010) Small hyaluronan oligosaccharides induce inflammation by engaging
both toll-like-4 and CD44 receptors in human chondrocytes. Biochem Pharmacol
80, 480-490
232.
Jiang, D., Liang, J., and Noble, P. W. (2011) Hyaluronan as an immune regulator
in human diseases. Physiol Rev 91, 221-264
233.
McDonald, B., McAvoy, E. F., Lam, F., Gill, V., de la Motte, C. A., Savani, R.
C., and Kubes, P. (2008) Interaction of CD44 and hyaluronan is the dominant
mechanism for neutrophil sequestration in inflamed liver sinusoids. J Exp Med
205, 915-927
234.
Majors, A. K., Austin, R. C., de la Motte, C. A., Pyeritz, R. E., Hascall, V. C.,
Kessler, S. P., Sen, G., and Strong, S. A. (2003) Endoplasmic reticulum stress
induces hyaluronan deposition and leukocyte adhesion. J Biol Chem 278, 4722347231
235.
Mohamadzadeh, M., DeGrendele, H., Arizpe, H., Estess, P., and Siegelman, M.
(1998) Proinflammatory stimuli regulate endothelial hyaluronan expression and
CD44/HA-dependent primary adhesion. J Clin Invest 101, 97-108
236.
Binion, D. G., West, G. A., Ina, K., Ziats, N. P., Emancipator, S. N., and Fiocchi,
C. (1997) Enhanced leukocyte binding by intestinal microvascular endothelial
cells in inflammatory bowel disease. Gastroenterology 112, 1895-1907
237.
Genasetti, A., Vigetti, D., Viola, M., Karousou, E., Moretto, P., Rizzi, M.,
Bartolini, B., Clerici, M., Pallotti, F., De Luca, G., and Passi, A. (2008)
Hyaluronan and human endothelial cell behavior. Connect Tissue Res 49, 120-123
238.
O'Neill, L. A. J. (2002) Signal transduction pathways activated by the IL-1
receptor/toll-like receptor superfamily. Curr Top Microbiol Immunol 270, 47-61
239.
Zheng, L., Riehl, T., and Stenson, W. (2009) Regulation of Colonic Epithelial
Repair in Mice by Toll-like Receptors and Hyaluronic Acid. Gastroenterology
137, 2041-2051
205
240.
Riehl, T. E., Foster, L., and Stenson, W. F. (2012) Hyaluronic acid is
radioprotective in the intestine through a TLR4 and COX-2-mediated mechanism.
Am J Physiol Gastrointest Liver Physiol 302, G309-316
241.
Gariboldi, S., Palazzo, M., Zanobbio, L., Selleri, S., Sommariva, M., Sfondrini,
L., Cavicchini, S., Balsari, A., and Rumio, C. (2008) Low molecular weight
hyaluronic acid increases the self-defense of skin epithelium by induction of βdefensin 2 via TLR2 and TLR4. J Immunol 181, 2103-2110
242.
Dusio, G. F., Cardani, D., Zanobbio, L., Mantovani, M., Luchini, P., Battini, L.,
Galli, V., Diana, A., Balsari, A., and Rumio, C. (2011) Stimulation of TLRs by
LMW-HA induces self-defense mechanisms in vaginal epithelium. Immunol Cell
Biol 89, 630-639
243.
Seo, E. S., Vargues, T., Clarke, D. J., Uhrín, D., and Campopiano, D. J. (2009)
Preparation of isotopically labelled recombinant β-defensin for NMR studies.
Protein Expression and Purification 65, 179-184
244.
Selsted, M. E., and Ouellette, A. J. (2005) Mammalian defensins in the
antimicrobial immune response. Nat Immunol 6, 551-557
245.
Garreis, F., Schlorf, T., Worlitzsch, D., Steven, P., Bräuer, L., Jäger, K., and
Paulsen, F. P. (2010) Roles of human β-defensins in innate immune defense at the
ocular surface: arming and alarming corneal and conjunctival epithelial cells.
Histochemistry and cell biology 134, 59-73.
246.
Termeer, C. C., Hennies, J., Voith, U., Ahrens, T., Weiss, J. M., Prehm, P., and
Simon, J. C. (2000) Oligosaccharides of hyaluronan are potent activators of
dendritic cells. J Immunol 165, 1863-1870
247.
Taylor, K. R., Trowbridge, J. M., Rudisill, J. A., Termeer, C. C., Simon, J. C., and
Gallo, R. L. (2004) Hyaluronan fragments stimulate endothelial recognition of
injury through TLR4. J Biol Chem 279, 17079-17084
248.
Bhilocha, S., Amin, R., Pandya, M., Yuan, H., Tank, M., LoBello, J., Shytuhina,
A., Wang, W., Wisniewski, H., de la Motte, C. A., and Cowman, M. (2011)
Agarose and Polyacrylamide Gel Electrophoresis Methods for Molecular Mass
Analysis of 5-500 kDa Hyaluronan. Anal Biochem 417, 41-9
249.
Aksamitiene, E., Hoek, J. B., Kholodenko, B., and Kiyatkin, A. (2007) Multistrip
Western blotting to increase quantitative data output. Electrophoresis 28, 31633173
250.
Abramoff, M., Magalhaes, P., and Ram, S. (2004) Image Processing with ImageJ.
Biophotonics International 11, 36-42
206
251.
Peterson, E., Miller, Y., Atchue, K., Margeson, L., and Yandl, E. (2008) Your
Paper Towels Can Do What? Histologic 41, 38-41
252.
Beutler, B., and Rietschel, E. T. (2003) Innate immune sensing and its roots: the
story of endotoxin. Nat Rev Immunol 3, 169-176
253.
Bals, R., Wang, X., Meegalla, R. L., Wattler, S., Weiner, D. J., Nehls, M. C., and
Wilson, J. M. (1999) Mouse β-defensin 3 is an inducible antimicrobial peptide
expressed in the epithelia of multiple organs. Infect Immun 67, 3542-3547
254.
Medema, J. P., and Vermeulen, L. (2011) Microenvironmental regulation of stem
cells in intestinal homeostasis and cancer. Nature 474, 318-326
255.
Siegelman, M. H., DeGrendele, H. C., and Estess, P. (1999) Activation and
interaction of CD44 and hyaluronan in immunological systems. J Leukoc Biol 66,
315-321
256.
Bourguignon, L. Y. W., Ramez, M., Gilad, E., Singleton, P. A., Man, M.-Q.,
Crumrine, D. A., Elias, P. M., and Feingold, K. R. (2006) Hyaluronan-CD44
interaction stimulates keratinocyte differentiation, lamellar body
formation/secretion, and permeability barrier homeostasis. J Invest Dermatol 126,
1356-1365
257.
Lee, S. K., Il Kim, T., Kim, Y. K., Choi, C. H., Yang, K. M., Chae, B., and Kim,
W. H. (2005) Cellular differentiation-induced attenuation of LPS response in HT29 cells is related to the down-regulation of TLR4 expression. Biochem Biophys
Res Commun 337, 457-463
258.
Lehmann, J., Retz, M., Harder, J., Krams, M., Kellner, U., Hartmann, J.,
Hohgräwe, K., Raffenberg, U., Gerber, M., Loch, T., Weichert-Jacobsen, K., and
Stöckle, M. (2002) Expression of human β-defensins 1 and 2 in kidneys with
chronic bacterial infection. BMC Infect Dis 2, 20
259.
Arnett, E., Lehrer, R. I., Pratikhya, P., Lu, W., and Seveau, S. (2011) Defensins
enable macrophages to inhibit the intracellular proliferation of Listeria
monocytogenes. Cellular microbiology 13, 635-651
260.
Zhuravel, E., Shestakova, T., Efanova, O., Yusefovich, Y., Lytvin, D., Soldatkina,
M., and Pogrebnoy, P. (2011) Human β-defensin-2 controls cell cycle in
malignant epithelial cells: in vitro study. Exp Oncol 33, 114-120
261.
Stöber, H., Maier, E., and Schmidt, H. (2010) Protective effects of Lactobacilli,
Bifidobacteria and Staphylococci on the infection of cultured HT29 cells with
different enterohemorrhagic Escherichia coli serotypes are strain-specific.
International Journal of Food Microbiology 144, 133-140
207
262.
Yang, D., Liu, Z.-h., Tewary, P., Chen, Q., de la Rosa, G., and Oppenheim, J. J.
(2007) Defensin participation in innate and adaptive immunity. Curr Pharm Des
13, 3131-3139
263.
Heldin, P., Karousou, E., Bernert, B., Porsch, H., Nishitsuka, K., and Skandalis,
S. S. (2008) Importance of hyaluronan-CD44 interactions in inflammation and
tumorigenesis. Connect Tissue Res 49, 215-218
264.
Asari, A., Kanemitsu, T., and Kurihara, H. (2010) Oral administration of high
molecular weight hyaluronan (900 kDa) controls immune system via toll-like
receptor 4 in the intestinal epithelium. J Biol Chem 285, 24751-24758
265.
Entwistle, J., Hall, C. L., and Turley, E. A. (1996) HA receptors: regulators of
signalling to the cytoskeleton. J Cell Biochem 61, 569-577
266.
Liang, J., Jiang, D., Griffith, J., Yu, S., Fan, J., Zhao, X., Bucala, R., and Noble,
P. W. (2007) CD44 is a negative regulator of acute pulmonary inflammation and
lipopolysaccharide-TLR signaling in mouse macrophages. J Immunol 178, 24692475
267.
Muto, J., Yamasaki, K., Taylor, K. R., and Gallo, R. L. (2009) Engagement of
CD44 by hyaluronan suppresses TLR4 signaling and the septic response to LPS.
Mol Immunol 47, 449-456
268.
Kawana, H., Karaki, H., Higashi, M., Miyazaki, M., Hilberg, F., Kitagawa, M.,
and Harigaya, K. (2008) CD44 suppresses TLR-mediated inflammation. J
Immunol 180, 4235-4245
269.
Balogh, L., Polyak, A., Mathe, D., Kiraly, R., Thuroczy, J., Terez, M., Janoki, G.,
Ting, Y., Bucci, L. R., and Schauss, A. G. (2008) Absorption, uptake and tissue
affinity of high-molecular-weight hyaluronan after oral administration in rats and
dogs. J Agric Food Chem 56, 10582-10593
270.
Salzman, N. H., Hung, K., Haribhai, D., Chu, H., Karlsson-Sjöberg, J., Amir, E.,
Teggatz, P., Barman, M., Hayward, M., Eastwood, D., Stoel, M., Zhou, Y.,
Sodergren, E., Weinstock, G. M., Bevins, C. L., Williams, C. B., and Bos, N. A.
(2010) Enteric defensins are essential regulators of intestinal microbial ecology.
Nat Immunol 11, 76-83
271.
O'Neill, L. A. J. (2009) A feed-forward loop involving hyaluronic acid and tolllike receptor-4 as a treatment for colitis? Gastroenterology 137, 1889-1891
272.
Lee JY, Spicer AP (2000) Hyaluronan: a multifunctional, megaDalton, stealth
molecule. Curr Opin Cell Biol 12, 581-6
208
273.
Laurent, T. C., Laurent, U. B., and Fraser, J. R. (1995) Functions of hyaluronan.
Ann Rheum Dis 54, 429-432
274.
Coppa, G. V., Gabrielli, O., Zampini, L., Galeazzi, T., Maccari, F., Buzzega, D.,
Galeotti, F., Bertino, E., and Volpi, N. (2012) Glycosaminoglycan content in term
and preterm milk during the first month of lactation. Neonatology 101, 74-76
275.
Calabro, A., Benavides, M., Tammi, M., Hascall, V. C., and Midura, R. J. (2000)
Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate
by fluorophore-assisted carbohydrate electrophoresis (FACE). Glycobiology 10,
273-281
276.
Ohya, T., and Kaneko, Y. (1970) Novel hyaluronidase from streptomyces.
Biochim Biophys Acta 198, 607-609
277.
Shimada, E., and Matsumura, G. (1980) Degradation process of hyaluronic acid
by Streptomyces hyaluronidase. J Biochem 88, 1015-1023
278.
Homer, C. R., Richmond, A. L., Rebert, N. A., Achkar, J.-P., and McDonald, C.
(2010) ATG16L1 and NOD2 interact in an autophagy-dependent antibacterial
pathway implicated in Crohn's disease pathogenesis. Gastroenterology 139, 16301641
279.
Bokor, S., Koletzko, B., and Decsi, T. (2007) Systematic review of fatty acid
composition of human milk from mothers of preterm compared to full-term
infants. Ann Nutr Metab 51, 550-556
280.
Moon, S.-K., Lee, H.-Y., Li, J.-D., Nagura, M., Kang, S.-H., Chun, Y.-M.,
Linthicum, F. H., Ganz, T., Andalibi, A., and Lim, D. J. (2002) Activation of a
Src-dependent Raf-MEK1/2-ERK signaling pathway is required for IL-1αinduced upregulation of β-defensin 2 in human middle ear epithelial cells.
Biochim Biophys Acta 1590, 41-51
281.
Meran, S., Luo, D. D., Simpson, R., Martin, J., Wells, A., Steadman, R., and
Phillips, A. O. (2011) Hyaluronan facilitates transforming growth factor-β1dependent proliferation via CD44 and epidermal growth factor receptor
interaction. J Biol Chem 286, 17618-17630
282.
Puré, E., and Assoian, R. K. (2009) Rheostatic signaling by CD44 and
hyaluronan. Cell Signal 21, 651-655
283.
Yang, C., Cao, M., Liu, H., He, Y., Xu, J., Du, Y., Liu, Y., Wang, W., Cui, L.,
Hu, J., and Gao, F. (2012) The high and low molecular weight forms of
hyaluronan have distinct effects on CD44 clustering. J Biol Chem 287, 4309443107
209
284.
Hunt, K. M., Foster, J. A., Forney, L. J., Schütte, U. M. E., Beck, D. L., Abdo, Z.,
Fox, L. K., Williams, J. E., McGuire, M. K., and McGuire, M. A. (2011)
Characterization of the diversity and temporal stability of bacterial communities
in human milk. PLoS ONE 6, e21313
285.
Le Huërou-Luron, I., Blat, S., and Boudry, G. (2010) Breast- v. formula-feeding:
impacts on the digestive tract and immediate and long-term health effects. Nutr
Res Rev 23, 23-36
286.
Saint, L., Smith, M., and Hartmann, P. E. (1984) The yield and nutrient content of
colostrum and milk of women from giving birth to 1 month post-partum. Br J
Nutr 52, 87-95
287.
Newburg, D. S. (2012) Prevention of rotavirus-induced diarrhea. J Pediatr
Gastroenterol Nutr 55, 2
288.
Akgul, Y., Holt, R., Mummert, M., Word, A., and Mahendroo, M. (2012)
Dynamic changes in cervical glycosaminoglycan composition during normal
pregnancy and preterm birth. Endocrinology 153, 3493-3503
289.
Stern, R., and Jedrzejas, M. J. (2006) Hyaluronidases: their genomics, structures,
and mechanisms of action. Chem Rev 106, 818-839
290.
Bouga, H., Tsouros, I., Bounias, D., Kyriakopoulou, D., Stavropoulos, M. S.,
Papageorgakopoulou, N., Theocharis, D. A., and Vynios, D. H. (2010)
Involvement of hyaluronidases in colorectal cancer. BMC Cancer 10, 499
291.
Monzón, M. E., Manzanares, D., Schmid, N., Casalino-Matsuda, S. M., and
Forteza, R. M. (2008) Hyaluronidase expression and activity is regulated by proinflammatory cytokines in human airway epithelial cells. Am J Respir Cell Mol
Biol 39, 289-295
292.
Domann, E., Hain, T., Ghai, R., Billion, A., Kuenne, C., Zimmermann, K., and
Chakraborty, T. (2007) Comparative genomic analysis for the presence of
potential enterococcal virulence factors in the probiotic Enterococcus faecalis
strain Symbioflor 1. Int J Med Microbiol 297, 533-539
293.
Godfroy, J. I., Roostan, M., Moroz, Y. S., Korendovych, I. V., and Yin, H. (2012)
Isolated Toll-like receptor transmembrane domains are capable of
oligomerization. PLoS ONE 7, e48875
294.
Triantafilou, M., Gamper, F. G. J., Haston, R. M., Mouratis, M. A., Morath, S.,
Hartung, T., and Triantafilou, K. (2006) Membrane sorting of toll-like receptor
(TLR)-2/6 and TLR2/1 heterodimers at the cell surface determines heterotypic
associations with CD36 and intracellular targeting. J Biol Chem 281, 3100231011
210
295.
Jin, M. S., and Lee, J.-O. (2008) Structures of the toll-like receptor family and its
ligand complexes. Immunity 29, 182-191
296.
Bell, J. K., Askins, J., Hall, P. R., Davies, D. R., and Segal, D. M. (2006) The
dsRNA binding site of human Toll-like receptor 3. Proc Natl Acad Sci USA 103,
8792-8797
297.
Pyo, J. O., Nah, J., and Jung, Y. K. (2012) Molecules and their functions in
autophagy. Exp Mol Med 44, 73-80
298.
Doidge, J. C., Segal, L., and Gospodarevskaya, E. (2012) Attributable risk
analysis reveals potential healthcare savings from increased consumption of dairy
products. J Nutr 142, 1772-1780
299.
Adamkin, D. H. (2012) Mother's milk, feeding strategies, and lactoferrin to
prevent necrotizing enterocolitis. JPEN J Parenter Enteral Nutr 36, 25S-29S
300.
De Curtis, M., and Rigo, J. (2012) The nutrition of preterm infants. Early Hum
Dev 88, S5-7
301.
Sherman, M. P. (2010) New concepts of microbial translocation in the neonatal
intestine: mechanisms and prevention. Clin Perinatol 37, 565-579
302.
Thomas, D. W., and Greer, F. R. (2010) Probiotics and Prebiotics in Pediatrics.
Pediatrics 126, 1217-1231
303.
Gatt, M., Reddy, B. S., and MacFie, J. (2007) Review article: bacterial
translocation in the critically ill--evidence and methods of prevention. Aliment
Pharmacol Ther 25, 741-757
304.
Puleo, F., Arvanitakis, M., Van Gossum, A., and Preiser, J.-C. (2011) Gut failure
in the ICU. Semin Respir Crit Care Med 32, 626-638
305.
McGuckin, M. A., Eri, R., Simms, L. A., Florin, T. H. J., and Radford-Smith, G.
(2009) Intestinal barrier dysfunction in inflammatory bowel diseases. Inflamm
Bowel Dis 15, 100-113
306.
Chin, K. F., Kallam, R., O'Boyle, C., and MacFie, J. (2007) Bacterial
translocation may influence the long-term survival in colorectal cancer patients.
Dis Colon Rectum 50, 323-330
307.
Mi, L., Zheng, H., Xu, X., Zhang, J., and Zhang, D. (2012) Bacterial
Translocation Contributes to Cachexia from Locally Advanced Gastric Cancer.
Hepatogastroenterology 59
211
308.
Sandler, N. G., and Douek, D. C. (2012) Microbial translocation in HIV infection:
causes, consequences and treatment opportunities. Nat Rev Microbiol 10, 655-666
309.
Ilan, Y. (2012) Leaky gut and the liver: a role for bacterial translocation in
nonalcoholic steatohepatitis. World J Gastroenterol 18, 2609-2618
310.
Teichberg, S., Isolauri, E., Wapnir, R. A., Roberts, B., and Lifshitz, F. (1990)
Development of the neonatal rat small intestinal barrier to nonspecific
macromolecular absorption: effect of early weaning to artificial diets. Pediatr Res
28, 31-37
311.
Boudry, G., Péron, V., Le Huërou-Luron, I., Lallès, J. P., and Sève, B. (2004)
Weaning induces both transient and long-lasting modifications of absorptive,
secretory, and barrier properties of piglet intestine. J Nutr 134, 2256-2262
312.
Hall, T., Dymock, D., Corfield, A. P., Weaver, G., Woodward, M., and Berry, M.
(2013) Bacterial invasion of HT29-MTX-E12 monolayers: Effects of human
breast milk. J Pediatr Surg 48, 353-358
212