Effects of Influenza Infection on Murine Alveolar Type II Cell Function
THESIS
Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in
the Graduate School of The Ohio State University
By
Christian Carlisle Hofer
Graduate Program in Veterinary Biosciences
The Ohio State University
2014
Master's Examination Committee:
Ian C. Davis, DVM, PhD – Advisor
Matthew Allen, DVM, PhD
Valerie K. Bergdall, DVM, ACLAM
Estelle Cormet-Boyaka, PhD
Copyrighted by
Christian Carlisle Hofer
2014
Abstract
Influenza A virus infections result in 250,000 to 500,000 deaths annually during seasonal
epidemics and pandemic outbreaks have historically killed millions. The ability of the
influenza A virus genome to undergo minor changes through “antigenic drift” and major
changes through “antigenic shift” poses significant challenges in developing effective
annual vaccines. More importantly, these genetic alterations may yield novel strains
leading to the next global pandemic. Severe respiratory disease from influenza A viruses
leads to acute respiratory distress syndrome where viral replication occurs in the
epithelial cells lining the alveolus. Damage to the alveolar type I (ATI) and type II (ATII)
cells leads to flooding of the alveolus with edematous fluid, fibrin, erythrocytes, and
other inflammatory mediators. This study investigated the effects of influenza A virus
infection on those alveolar epithelial cells in a mouse model. We demonstrated that
influenza infection reduces the number of ATII cells as well as dramatically alters their
production of surfactant proteins. We observed a transition of ATII cells into a ATI cell
phenotype by measuring the production of at ATI cell specific marker T1α/Podoplanin by
ATII cells isolated from mice post infection. Continued examination and characterization
of ex-vivo isolated ATII cells can provide important information into the role of ATII
cells in restoring the normal epithelial lining of a damaged alveolus. Restoration of an
intact respiratory epithelium is essential in the recovery from acute lung injury.
ii
Acknowledgments
I would like to thank Matthew Hogan, Tara Martin, Parker Woods, Famke Aeffner, and
Lisa Joseph for their assistance and technical expertise while working together in the
Davis Lab and at OSU throughout my residency. Your knowledge and friendship has
been a great source of support to me over the last several years. I would also like to thank
all the members of the University Laboratory Animal Resources department. Your
dedication to research animal welfare and promotion of high quality science is often
overlooked, but never unappreciated by me. Finally, I would like to thank the members of
my MS committee: Ian Davis, Matthew Allen, Valerie Bergdall, and Estelle CormetBoyaka. Your professional advice and guidance has helped develop my critical thinking
and scientific writing/presentation skills which will no doubt serve me well as my career
progresses.
iii
Vita
June 1987 .......................................................Whitewater Sr. High School
May 2001 .......................................................B.S. Animal Science & Zoology, University
of Florida
May 2005 .......................................................D.V.M., University of Florida
August 2011 to present .................................Resident
Veterinarian,
University
Laboratory Animal Resources, The Ohio
State University
Publications
Bowman AS, Nelson SW, Edwards JL, Hofer CC, Nolting JM, Davis IC, Slemmons RD.
2013. Comparative effectiveness of isolation techniques for contemporary Influenza A
virus strains circulating in exhibition swine. J Vet Diagn Invest 25(1): 82-90.
Melendez P, Hofer CC, Donovan GA. 2006. Risk factors for udder edema and its
association with lactation performance on primiparous Holstein cows in a large Florida
herd, U.S.A. Prev Vet Med 76(3-4):211-21.
iv
Landolt GA, Karasin AI, Hofer C, Mahaney J, Svaren J, Olsen CW. 2005. Use of realtime reverse transcriptase polymerase chain reaction assay and cell culture methods for
detection of swine influenza A viruses. Am J Vet Res 66(1):119-24.
Fields of Study
Major Field: Veterinary Biosciences
v
Table of Contents
Abstract ............................................................................................................................... ii
Vita..................................................................................................................................... iv
Table of Contents ............................................................................................................... vi
List of Figures .................................................................................................................... ix
Chapter 1: Literature Review .............................................................................................. 1
Influenza .......................................................................................................................... 1
Influenza Epidemiology .............................................................................................. 1
Influenza Virology....................................................................................................... 2
Influenza Clinical Disease and Pathophysiology ........................................................ 3
Alveolus Anatomy and Physiology ................................................................................. 4
The Alveolus................................................................................................................ 4
Alveolar Type I Epithelial Cells (ATI Cells) .............................................................. 5
Alveolar Type II Epithelial Cells (ATII Cells) ............................................................ 5
Alveolar Epithelial Cell Markers ................................................................................. 6
Pulmonary Surfactant ...................................................................................................... 6
Effects of Influenza A Virus on Alveolar Epithelial Cells.............................................. 7
vi
Chapter 2: Study Design ..................................................................................................... 9
Chapter 3: Materials and Methods .................................................................................... 10
Mice............................................................................................................................... 10
Influenza Virus Inoculation ........................................................................................... 10
Alveolar Type II Cell Isolation ..................................................................................... 10
In-Vivo Imaging System (IVIS) .................................................................................... 11
Flow Cytometry............................................................................................................. 12
Protein Analysis by Western Blot ................................................................................. 12
Gene Expresion Analysis by rtPCR .............................................................................. 13
Other Methods ............................................................................................................... 13
Statistical Analysis ........................................................................................................ 13
Chapter 4: Results ............................................................................................................. 14
Effects of influenza A virus infection on C57BL/6 mice.............................................. 14
Effects of influenza A virus infection on SP-C expression at the whole lung level ..... 14
Effects of influenza A virus infection on ATII cells ..................................................... 14
Gene expression in isolated ATII cells following influenza A virus infection ............. 15
Protein expression in isolated ATII cells following influenza A virus infection .......... 15
Chapter 5: Discussion ....................................................................................................... 17
References ......................................................................................................................... 21
vii
Appendix A: Figures ......................................................................................................... 32
viii
List of Figures
Figure 1. Effects of influenza A virus infection on C57BL/6 mice. ............................ 33
Figure 2. Effects of influenza A virus infection on SP-C expression at the whole lung
level. .................................................................................................................................. 35
Figure 3. Effects of influenza A virus infection on ATII cells. .................................... 36
Figure 4. Gene expression in isolated ATII cells following influenza A virus
infection............................................................................................................................ 38
Figure 5. Protein expression in isolated ATII cells following influenza A virus
infection............................................................................................................................ 39
ix
Chapter 1: Literature Review
Influenza
Influenza Epidemiology
Influenza A virus is a respiratory pathogen that causes significant disease and deaths
worldwide. Seasonal epidemics result in 3 to 5 million cases of severe illness, resulting in
250,000 to 500,000 deaths annually1. The economic impact of seasonal epidemics to the
United States alone exceeds an estimated $87 billion per year when considering direct
medical costs, indirect loss of earnings, productivity, and lives2. During the twentieth
century 3 pandemics have occurred: the global outbreak in 1918 to 1919 (Spanish Flu,
H1N1) which killed more than 50 million people, the Asian Flu (H2N2) in 1957 to 1958
which claimed 1 to 1.5 million lives, and the Hong Kong Flu (H3N2) in 1968 which
killed over 750,000 individuals3,4. The first global pandemic of the 21st century started in
the spring of 2009 and was caused by a novel swine-origin influenza A virus (designated
as A (H1N1) pdm09). A recent comprehensive model estimated that fatalities from
respiratory disease was 201,200 and 83,300 additional deaths were due to cardiovascular
disease. In contrast to traditional seasonal epidemics, 80% of these deaths occurring in
people less than 65 years of age5.
1
Influenza Virology
Influenza A, B, and C viruses belong to the family Orthomyxoviridae which encompasses
enveloped, negative-sense, segmented RNA viruses. Epidemic influenza disease
outbreaks are caused by type A and B viruses whereas type C viruses cause sporadic
influenza-like disease in young children6. Type A and B genomes consist of 8 segments
encoding 11 different proteins. These proteins include three transcriptases (PB1, PB2,
and PA), two matrix proteins (M1 and M2), one nucleocapsid protein (NP), two nonstructural proteins (NS-1 and NS-2), and two surface glycoproteins hemagglutinin (HA)
and neuraminidase (NA)7. 17 different HA and 10 different NA serotypes have been
identified with the recent discovery of novel influenza A viruses in South American bats8.
It is the two surface glycoproteins (HA and NA) that form the basis of strain
differentiation using H and N designations (i.e. H3N2). The combination of any HA and
NA serotype is theoretically possible, however human disease is generally caused by
infection with H1, H2, and H3 combined with N1 or N2 serotypes. Newly emerging
strains of avian origin such as H5N1 and H7N9, have the potential to cause significant
human pandemics with only minor genetic changes of the virus increasing
transmissibility between people9-11.
The polymerase complex of the influenza virus has poor proofreading ability and thus
point mutations often arise during replication. Consequently, minor changes can occur
within the antigenic epitopes of the HA or NA glycoproteins causing “antigenic drift”.
These minor changes necessitate annual reformulation of vaccines against seasonal
influenza strains. A more significant change in the genome is “antigenic shift”, which
2
occurs when whole genome segments are exchanged between two or more influenza A
virus strains infecting the same cell. This occurs as a result of reassortment during viral
packaging and can lead to devastating pandemics because of the novelty of the virus
being exposed to naïve populations with no pre-existing immunity. Pigs have been
described as being mixing vessels for avian and mammalian influenza viruses because
they possess cellular receptors for both virus types potentially allowing infection with
multiple strains of influenza A virus12. Genetic analysis of the viruses that caused the
1957 H2N2, the 1968 H1N1, and the 2009 H1N1 pandemics demonstrated that antigenic
shift occurred between avian and human viruses yielding completely new strains.
Specifically, the 2009 H1N1 was derived from two previously unrelated swine-origin
viruses, one of which was a derivative of the human 1918 H1N1 virus. Moreover, there
appears to be consistent contributions of one or more genes from avian influenza viruses
to pandemic viruses13,14.
Influenza Clinical Disease and Pathophysiology
Clinical manifestations of influenza in humans can vary from asymptomatic infection to
serious illness requiring hospitalization, or death. The Centers for Disease Control and
Prevention defines influenza-like illness to include cases with one or more of the
following signs and symptoms: fever >100°F (>37.8°C), muscle aches, headaches, lack
of energy, dry cough, sore throat, or runny nose. Diagnosis of suspected influenza virus
infection is confirmed through viral culture, serology, and several different rapid
influenza diagnostic tests. The timing of accurate diagnosis of influenza A virus infection
can directly impact the prognosis in some patients, as most available antiviral drugs have
3
shown to have limited efficacy late in the course of the disease. Therefore, it is essential
that a timely and accurate diagnosis be achieved in those severe cases of influenza
requiring hospitalization.
Influenza infections are usually more severe in children and the elderly15; however this
varies by strain as the pandemics in 1918 and 2009 primarily affected people outside
those typical age groups. Additional risk factors for contracting serious disease include
underlying cardiac or other pulmonary disease, obesity, and pregnancy16-19. The primary
complication of serious disease is viral pneumonia which is sometimes followed by a
secondary bacterial pneumonia. Severe cases may progress to acute respiratory distress
syndrome (ARDS), which is clinically characterized by cyanosis, hypoxemia, and
pulmonary edema10,20-22. In influenza infections leading to ARDS, the virus extends deep
into the respiratory tract and replicates within the cells lining the alveolus 23. Damage to
the alveolar epithelial cells can result in respiratory failure as a consequence of flooding
of the alveolus with edematous fluid, fibrin, erythrocytes and other inflammatory cells
preventing adequate gas exchange24. Left untreated, ARDS can be followed by multiorgan failure and ultimately death in many cases.
Alveolus Anatomy and Physiology
The Alveolus
The alveolus is the terminal portion of the mammalian lung that serves as the site of
pulmonary gas exchange. Two distinct types of alveolar epithelial cells line the gas filled
lumen of the alveolus. These pneumocytes lie on the basement membrane which is in
contact with the endothelial cells of the pulmonary capillary beds. Pulmonary gas
4
exchange occurs across this epithelial-endothelial membrane. Some normal alveoli also
contain resident alveolar macrophages.
Alveolar Type I Epithelial Cells (ATI Cells)
ATI cells are large, flat cells that cover approximately 95% of the surface area of the
alveolus, but only account for approximately 8-15% of the total alveolar epithelia cell
numbers25,26. ATI cells are terminally differentiated and are have a life span of
approximately 120 days in the normal lung27.Their primary function is to allow easy gas
diffusion across the epithelial-endothelial membrane yet they also have a role in fluid
exchange and ion balance. There is little known about other functions of ATI cells;
however, they have been shown to have a limited role in the innate immune response in
bacterial pneumonia in mice28. Because they cover the majority of the lining of the
alveolus, ATI cells are the primary target of many lung insults. Exposure to viral,
bacterial, toxic, and even physical agents can result in ATI cell injury and death.
Alveolar Type II Epithelial Cells (ATII Cells)
ATII cells are small cuboidal cells which cover approximately 5% of the alveolar surface
area not covered by the ATI cells and account for 15% of the cell numbers within the
alveolus10,25. ATII cells are found in the corners of alveoli and are attached via tight
junctions. They contain characteristic lamellar bodies; secretory organelles found in ATII
cells and keratinocytes, and have apical microvilli. ATII cells have been extensively
studied and have multiple functions. ATII cells produce, secrete, and recycle pulmonary
surfactant proteins and are the primary player in fluid clearance and ion balance within
5
the alveoli29. Additionally, ATII cells have many innate immune functions30 and are
known to differentiate into ATI cells following ATI cell injury or death31,32.
Alveolar Epithelial Cell Markers
Cellular markers exist that are either common to or unique for alveolar type 1 or type 2
cells. Use of these markers allows for specific identification of these cells types to further
examine cellular function and physiology. RTI40/T1α/Podoplanin33,34 and HTI56
35
have
been identified as antigens that are unique to ATI cells. Several other antigens that are not
unique to ATI cells (i.e. are expressed in other cells in the body) are not expressed in
ATII cells. Hence, they can be used to identify ATI cells within lung tissues. These ATI
cell selective markers include Caveolin -1/-2
36,37
, Na+/K+ - ATPase α2-isoform38,
Aquaporin 539, Cytochrome P450 2B140, and Carboxypeptidase M
41
. ATII cell specific
markers include: CD44v6, Thomsen-Friedenreich antigen, thyroid transcription factor 1,
MUC 1
42
, and CD208 (DC-LAMP)
43
. Additionally, ATII cells produce surfactant
associated proteins SP-A, -B, -C, and –D. These proteins are selective for ATII cells;
however, only SP-C is ATII cells specific30,44.
Pulmonary Surfactant
Pulmonary surfactant covers the alveolar epithelium, and is a complex mixture of
phospholipids (80-85%), neutral lipids (5-10%), and a minor constituent of proteins. The
primary role of surfactant is to maintain proper alveolar surface tension, which facilitates
alveolar inflation during inspiration. Surfactant also provides protection against oxidants
and infectious agents by regulating host defenses and immune responses45. There are four
different surfactant-associated proteins: SP-A, SP-B, SP-C, and SP-D. SP-B and SP-C are
6
small hydrophobic proteins which are closely associated with the surfactant film. SP-B is
considered to be the most important surfactant-associated protein for normal surfactant
structure and function, and is essential for final packaging of surfactant within the
lamellar bodies of the ATII cells46. It also regulates formation of tubular myelin47, and
SP-B deficiency is lethal in mice48,49. SP-C is the smallest surfactant-associated protein
and has a transmembrane orientation. This allows SP-C to interact with the acylated
phospholipid side chains which stabilizes the surfactant film50-52. SP-A and SP-D are
hydrophobic molecules that belong to the family of collectins and their role is
predominantly associated with host defenses and immune response53. SP-D has also been
shown to influence the uptake of surfactant by ATII cells during the surfactant recycling
process, which can impact the overall surfactant pool54. SP-B and SP-C are associated
with and exocytosed in the lamellar bodies of ATII cells, whereas SP-A and SP-D are
secreted by the ATII cells independent of lamellar body exocytosis55.
Effects of Influenza A Virus on Alveolar Epithelial Cells
Initial exposure to influenza virus in humans is typically through the upper respiratory
tract and replication begins in the nasopharynx, peaking approximately 48 hours after
inoculation23. In the absence of complete clearance, viral infection extends to the lower
respiratory tract where the primary targets for infection and replication are the ATI and
ATII cells56. Particular Influenza virus strains may have tropism for either ATI or ATII
cells, possibly as a result of differences in cell surface expression of sialosaccharides.
ATI cells generally express α-2,6-linked sialosaccharides, whereas ATII cells
predominately express α-2,3-linked sialosaccharides10. Influenza A virus binds to the host
7
cell by attachment of the viral HA to these sialosaccharides. Autopsies from patients with
fatal H5N1 infections have demonstrated that viral antigens are most prevalent in ATII
cells57,58. In contrast, patients who succumbed to the 2009 pandemic H1N1 virus had
viral antigens in both ATI and ATII cells. Such dual tropism is consistent with that virus
being of both mammal and avian origin59. H5N1 and human-adapted H1N1 and H3N2
viruses could all replicate efficiently in human lung tissue cultures, whereas classic
swine-origin viruses and low-pathology avian viruses did not and the viruses were
detected nearly exclusively in ATII cells60. Although there is evidence that some
influenza viruses have direct cytopathic effects on ATI cells, it is likely that cytokinemediated destruction of these cells also contributes to the diffuse alveolar damage
associated with severe influenza virus infections61. ATII cells are also targets of direct
viral infection and may undergo lysis and/or apoptosis through both extrinsic and
intrinsic pathways56. As previously discussed, one of the main roles of ATII cells is to
serve as replacement cells for ATI cells. The mechanisms initiating and regulating this
transition between phenotypes is not clearly defined. Recovery from severe influenza A
virus infection starts with the regeneration of the airway epithelial cell populations and
repair of the epithelial-endothelial membrane thereby restoring normal alveolar function.
Several growth factors have been shown to increase ATII cell numbers including
keratinocyte growth factor (KGF), hepatocyte growth factor, and fibroblast growth
factor-1062.
8
Chapter 2: Study Design
Gaining a better understanding of the effects and outcomes of influenza virus infection in
alveolar epithelial cells may provide insight into potential therapeutic measures for
treating influenza infections. Severe influenza infections lead to acute respiratory distress
syndrome stemming from a breakdown of the normal alveolar epithelium and subsequent
edema filling the lumen resulting in the inability to conduct normal gas exchange. The
goal of this study is to isolate ATII cells from normal and influenza-infected mice in
order to study the cellular changes due to influenza infection. This ex vivo approach has
several benefits when compared to studies conducted using cultured ATII cells or other
cell lines that do not effectively mimic the physiology of these cells in their natural
environment. ATII cells isolated from mice will be characterized using multiple
molecular and biochemical assays to determine how infection with influenza virus affects
the function and phenotype of these cells. We hypothesize that as a consequence of the
acute lung injury induced by influenza virus infection, the cellular structure and function
of ATII cells will be altered and that there will be transition from the ATII cell phenotype
to the ATI cell phenotype indicating an attempt to restore the normal alveolar epithelium.
9
Chapter 3: Materials and Methods
Mice
C57BL/6 mice were purchased from NCI (Frederick, MD) and SP-C/GFP mice on a
C57BL/6 background were bred in-house following an initial stock provided by Joe Rae
Wright (Duke University Medical Center, Durham, NC). SP-C/GFP mice express the
GFP transgene under control of the surfactant protein C (SP-C) promoter (Lo, 2008).
Mice were maintained in standard ventilated racks and provided with ad libitum food and
water, as well as appropriate environmental enrichment with Nestlets™ (Ancare,
Bellmore, NY). All animal procedures were performed in compliance with local and
National Institutes of Health guidelines and were approved by The Ohio State University
Institutional Animal Care and Use Committee.
Influenza Virus Inoculation
Mice (8-12 week old) were inoculated intranasally with 10,000 plaque-forming units
(pfu) of H1N1 influenza A/WSN/33 in 50μL PBS with 0.1% BSA under ketaminexylazine anesthesia63,64. Mice were individually marked and weighed before infection and
at time of sacrifice to confirm infection status.
Alveolar Type II Cell Isolation
ATII cells were isolated from SP-C/GFP and C57BL/6 mice using a procedure previously
described65. In brief, the lungs were perfused and lavaged with saline via a tracheal
10
catheter and allowed to deflate. Lungs were then inflated with approximately 2-2.5 mL of
Dispase (5 units/mL in PBS, BD Biosciences). Without allowing the lungs to completely
deflate, warmed low melting point agarose (1%) was quickly instilled into the trachea
(approximately 0.3mL in volume) and the lungs were gently packed on ice for 5 minutes.
The trachea was removed at the hilus and the lungs were incubated in 5 mL of Dispase at
room temperature on a rocker. Occasional mechanical agitation (shaking) of the lungs
enhanced digestion of the tissues and incubation continued for 45-60 minutes until nearly
all lung tissue had broken down to a slurry. DNase (0.01% in DMEM) was added to the
slurry and incubated an additional 5 minutes at room temperature. The tissue slurry was
passed through sequential nylon mesh (100 um, 40 um, and 21 um) and the filtrate was
centrifuged. The cell pellet was re-suspended in 10mL of DMEM + 10% newborn calf
serum and panned on antibody treated (anti-CD45 and anti-CD16/32, BD biosciences)
petri dishes for 2 hours at 37°C and 5% CO2. Nonadherent cells were collected and resuspended for cell counts and cytospin preps to determine purity by modified
Papanicolaou staining66.
In-Vivo Imaging System (IVIS)
SP-C/GFP mice were sacrificed using an intraperitoneal overdose of ketamine/xylazine
anesthesia and their lungs were removed for determination of GFP fluorescence
corresponding to SP-C production. The lungs were imaged on the IVIS 100
bioluminescent imaging workstation (Xenogen, Caliper Life Sciences). Individual lungs
were imaged to measure expressed GFP fluorescence (BIN:HR(4), FOV10, f16, 2 s).
11
Flow Cytometry
ATII cell suspensions were fixed, permeabilized, and stained with various fluorochrome
conjugated antibodies to alveolar type I cell markers (T1-α), ATII cell markers (SP-A,
SP-C, EpCAM-326), and viability dyes (Annexin-V, 7-AAD) for FACS analysis (BD
FACSCalibur and BD LSRII). ATII cell suspensions from SP-C/GFP mice were FACS
sorted (FACSAria, Becton Dickson Biosciences, San Jose, CA) for high GFP signal
yielding a highly enriched population of SP-C+ cells. Flow cytometry data was analyzed
using FlowJo (v.10.0) software (TreeStar, Inc. Ashland, OR).
Protein Analysis by Western Blot
Whole lung homogenates were prepared in Cell Lysis Buffer (CTS #9803) using the
FastPrep®-24 bead beater instrument. Pelleted ATII cells were lysed in a small volume
of lysis buffer and passed through a 30g needle to ensure complete membrane disruption.
Lysate protein concentration was determined via the BCA method (Pierce). Equal
amounts of protein were loaded onto 4-12% gels and subjected to SDS-PAGE. Proteins
were then transferred to pvdf membranes for antibody probing. All primary antibody
incubations were carried out overnight at 4oC {Rb anti-proSPC (Millipore ab3786); Rb
anti-SPA (abcam 115791); Rb anti-SPD (abcam 168366); Rb anti-H1N1 NP (GenScript
01506); Rat anti-T1α/Podoplanin (Imgenex 6743A-100); Rb anti-active Caspase3
(abcam2302); Rb anti-GAPDH (SantaCruz 25778)}. Subsequently, membranes were
exposed to species-specific HRP-conjugated secondary antibodies. SuperSignal West
Pico Chemiluminescent Substrate (Pierce) allowed for the visualization of proteins.
12
Gene Expresion Analysis by rtPCR
RNA was isolated from ATII cell preparations using Qiagen’s RNAeasy system. Purified
RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit
(Applied Biosystems). Quantitative real-time PCR reactions were prepared using cDNA,
TaqMan® Gene Expression Master Mix, and predesigned TaqMan® probes. (sftpc Assay
ID: Mm00488144_m1; T1α/Podoplanin Assay ID: Mm01348911_g1; rn18s Assay ID:
Mm03928990_g1). Reactions were carried out on the StepOnePlus™ Real-Time PCR
System (Applied Biosystems). Relative fold-change was calculated based on the
comparative CT (2−ΔΔCT) method.
Other Methods
Carotid arterial blood oxygen saturation, pulmonary edema by lung wet to dry weight
ratio, lung homogenate viral titers, and pulmonary resistance and compliance were
performed as described in earlier studies67,68.
Statistical Analysis
Descriptive statistics were calculated using Instat 3.05 (GraphPad Software, San Diego,
CA). Gaussian data distribution was verified by the method of Kolmogorov and Smirnov.
Differences between group means were analyzed by analysis of variance, with TukeyKramer multiple comparison post-tests. All data are presented as mean ± SEM.
13
Chapter 4: Results
Effects of influenza A virus infection on C57BL/6 mice
Body weight decreases significantly by 2 days post infection (DPI) and by 6 DPI the
mice have lost over 30% of their body weight (Fig. 1A). Oxygen saturation in arterial
blood is significantly decreased at 6 DPI (Fig. 1B) and significant pulmonary edema is
noted at both 2 and 6 DPI (Fig. 1C). Influenza viral titers from infected lungs verify
influenza A virus infection without any significant alterations in the viral load between 2,
4, and 6 DPI (Fig. 1D). Additionally, total lung resistance increased significantly at 2 and
6 DPI (Fig. 1E) while compliance significantly decreased at 2 and 6 DPI (Fig. 1F).
Effects of influenza A virus infection on SP-C expression at the whole lung level
Expression of SP-C promoter genes induces expression of the GFP thereby indicating
that changes in GFP correlate to changes in SP-C production. Measurement of GFP
fluorescence using the IVIS demonstrates that there is a significant decrease in the level
of GFP expression in whole lungs at 2 and 6 DPI when compared to uninfected lungs
(Fig. 2A & 2B).
Effects of influenza A virus infection on ATII cells
ATII cell isolates from uninfected mice demonstrate purities between 90-95% (data not
shown) using Papanicolau-staining defining characteristic darkly stained granules in the
cytoplasm of ATII cells (Fig. 3A). Overall yield of ATII cells is reduced at 6 DPI
14
compared to uninfected and at 2 DPI (Fig 3B). Freshly isolated ATII cells were analyzed
by flow cytometry using the gating scheme described to select ATII cells based on
FSC/SSC properties and GFP fluorescence (Fig 3C). The use of a fluorochrome
conjugated anti-SP-C antibody demonstrated the validity of using GFP fluorescence as a
selection tool for SP-C producing ATII cells (Fig. 3D). Influenza A virus infection of SPC/GFP mice demonstrated a significant reduction in the percentage of freshly isolated
ATII cells expressing GFP (Fig. 3E) as well as a significant reduction in the mean
channel fluorescence of GFP at 2 and 6 DPI (Fig. 3F).
Gene expression in isolated ATII cells following influenza A virus infection
rtPCR analysis of lysates from freshly isolated ATII cells from C57BL/6 mice at 6 DPI
demonstrated a significant reduction in the expression of the SP-C gene (Fig. 4A) and a
significant increase in the expression of the T1α/Podoplanin gene (Fig. 4B) when
compared to the expression of those genes in uninfected mice.
Protein expression in isolated ATII cells following influenza A virus infection
Expression of proSP-C proteins is notably reduced in freshly isolated ATII cells at 2 and
6 DPI as demonstrated by western blot analysis (Fig. 5A) and the ATI cell specific
protein T1α/Podoplanin expression is increased at 6 DPI (Fig. 5B). Further examination
of ATII cell protein expression demonstrates that the decline in production of surfactant
associated proteins in not limited to SP-C, but can also be seen in SP-A and SP-D by 6
DPI. Influenza A virus NP protein was visualized in freshly isolated ATII cells at 2 and 6
DPI (Fig 5C). Freshly isolated ATII cells sorted based on GFP expression (+ or -)
15
demonstrate that there is a slightly higher incidence of infection in those ATII cells that
are expressing SP-C as indicated by GFP fluorescence (Fig. 5D).
16
Chapter 5: Discussion
The use of annual vaccines and antivirals for the prevention and treatment of seasonal
influenza infection fails to provide complete protection or cure disease. It is hard to
predict the total impact that a significant influenza pandemic would have on society, but
scenarios yielding millions of human deaths and breakdown of essential services and
infrastructure are within the realm of possibility. Basic research investigating the
pathogenesis of influenza viruses and the host’s cellular and immunological response to
infection is critical to determine effective prophylaxis and treatment interventions. Our
investigation into the effects of influenza A virus infection on murine ATII cells has
demonstrated significant changes in cellular structure and function and their contribution
to the pathogenesis of ARDS in severe influenza infections. Since alveolar epithelial cells
are the prime target of influenza virus infection and damage to those cells result in key
features of ARDS, a better understanding of the cellular mechanisms that either induce or
are prompted by these changes in ATII cells populations within the diseased alveoli may
yield significant advances in the development of effective influenza therapies.
We have demonstrated that ATII cells become infected with influenza A virus (FACS
sort WB data) and some undergo apoptosis (Caspase WB) as a result of infection.
Cellular necrosis is another well described and accepted outcome. Our isolation method
is likely too mechanically harsh to effectively capture any cells that have started to
17
undergo necrosis. We have also demonstrated that ATII cells undergo differentiation and
begin to differentiate into ATI cells. This transition is marked by the ATII cellular
expression of the T1α/Podoplanin antigen, an antigen specific to ATI cells; and the loss
of SP-C or EpCAM which are both ATII cellular markers. This finding is remarkable as
EpCAM has traditionally been thought to be an antigen found on all cells of epithelial
origin. Additionally, our findings have demonstrated that once a cell begins the
differentiation process towards the ATI cell phenotype, the cell suspends production of
SP-C and other surfactant associated proteins. This indicates that there are likely limited
cells of intermediate state that possess functional traits of both ATI and ATII cells.
Demonstration that infection with influenza A virus causes changes in surfactant protein
levels has been previously described as either reducing69 or increasing70 levels of all or
some surfactant associated protein levels. In fact, supplementation with artificial
surfactant has been described as a potential therapeutic intervention to accompany a
traditional antiviral regimen71. We have consistently demonstrated that within freshly
isolated ATII cells populations SP-A, SP-C, and SP-D protein levels are reduced as a
result of influenza A virus infection. Additionally, the gene transcripts for SP-C are
reduced over the course of infection. Because of the varied and complex functions of
ATII in normal lung physiology, supplementation of surfactant alone is unlikely to have
significant impact on the disease progression because that pathology of ARDS includes
significant pulmonary edema and inflammation along with the breakdown of the normal
epithelial-endothelial barrier and dysregulation of surfactant production and catabolism.
The reduction in the absolute number of ATII cells within the lung over the course of
18
influenza virus infection likely contributes to the breakdown in normal alveolar function
and architecture. Without an adequate number of ATII cells, there is likely a reduced
capability to eliminate edematous fluid from the alveoli and maintain an adequate
surfactant layer required for normal alveolus inflation. Grossly, these changes can be seen
in the severely congested and atelectatic lungs of mice.
Our ATII cell isolation protocol has been shown to consistently yield large number of
cells of high purity. In contrast to several other studies which utilized ATII cell culture or
immortalized cell lines (A549), our methodology yields cells that maintain their normal
cellular structure and function. ATII cells in culture have been shown to take on nontypical shapes and display varied surface antigens that are not consistent with normal
ATII cell characteristics72. A549 cells were originally isolated and cultured from a
primary lung tumor and no longer display normal ATII function. Our ex vivo isolation of
ATII cells over the course of a severe influenza A virus infection in mice uniquely
demonstrated the significant changes in alveolar epithelial cell populations.
Keratinocyte growth factor (KGF) has been reported to be effective the treatment of acute
lung injury from multiple causes62. It is suggested that KGF therapy has the ability to
induce ATII cell hyperplasia and increase ATII cell numbers73-75. It is reasonable to
deduce that KGF therapy may provide some improvement to lung pathology in influenza
virus infections. We have demonstrated that in our mouse model there is a significant
reduction in KGF gene expression at the whole lung level, which would seem to indicate
that supplementation of KGF might have some impact; however, there is also significant
reductions in the KGF receptor gene expression in ATII cells. This finding suggests that
19
supplementation with KGF might be ineffective; however, further scrutiny of the cellular
mechanisms that could up regulate the KGF receptors may prove more productive.
Ultimately, influenza A virus infection in the mouse reduces the overall number of ATII
cells within the lung. There is also a reduction in the levels of surfactant associated
proteins (SP-A, SP-C, and SP-D) produced by the ATII cells and at least a fraction of the
remaining ATII cells undergo differentiation to an ATI cell phenotype. A percentage of
ATII cells also exhibited signs of apoptosis. It is evident that ATII cells play a key role in
the restoration of normal alveolar function. Thus, further investigation into the
mechanisms that drive cellular differentiation and/or proliferation of ATII cells in the
alveoli may help identify improved therapeutics for severe influenza infections.
20
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Appendix A: Figures
32
Figure 1. Effects of influenza A virus infection on C57BL/6 mice.
33
Figure 1. Effects of influenza A virus infection on C57BL/6 mice. (A) Changes in
body weight over course of infection with influenza A virus; (B) Carotid arterial oxygen
saturation; (C) Measurement of pulmonary edema by lung wet:dry ratio; (D) Lung
homogenate viral titers; (E) Total lung resistance; (F) Static lung compliance. (n= 6-8
mice per group, # = P < 0.005).
34
Figure 2. Effects of influenza A virus infection on SP-C expression at the whole lung
level. (A) Representative IVIS images of whole lungs isolated from C57BL/6
(demonstrating minimal auto fluorescence in tracheal cartilage) and SPC/GFP mice (GFP
fluorescence reported in photons/sec/cm2/steradian); (B) significant decreased in GFP
fluorescence intensity noted at 6 DPI (n = 10/timepoint; # = P<0.0005).
35
Figure 3. Effects of influenza A virus infection on ATII cells.
36
Figure 3. Effects of influenza A virus infection on ATII cells. (A) Photomicrograph of
Papanicolau-stained ATII cells from an uninfected C57BL/6 mouse showing
characteristic dark stained granules (arrows). (B) Yield of ATII cells per isolation over
the course of influenza A virus infection. (C) Identification of the FSC/SSC gate used to
identify ATII cells. (D) Flow cytometric scatter plot validating the use of GFP
fluorescence as an indicator of SP-C production through the use of an anti-SP-C antibody
conjugated with an APC flourochrome. (E) % of freshly isolated ATII cells expressing
GFP fluorescence and (F) the mean channel fluorescence (MCF) of GFP for those
isolated ATII cells. (n = 4 mice / group; # = P < 0.005; ** = P < 0.0005).
37
Figure 4. Gene expression in isolated ATII cells following influenza A virus
infection. rtPCR analysis of ATII cell lysates evaluating gene expression of (A) SP-C
and (B) T1α/Podoplanin. (n = 6 mice / time point).
38
Figure 5. Protein expression in isolated ATII cells following influenza A virus
infection.
39
Figure 5. Protein expression in isolated ATII cells following influenza A virus
infection. WB analysis for (A) proSP-C protein; (B) T1α/T1α/Podoplanin; (C) SP-A, SPD, and H1N1 NP; (D) H1N1 NP on FACS GFP sorted and non-sorted ATII cells. β-Actin
and GAPDH used as loading controls. (n = 3 mice / time point).
40