L46 Intestinal epithelial barrier in poultry: function and nutritional modulation
Yuming Guo, Dan Liu, Bingkun Zhang
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
Corresponding author’s email address: [email protected]
Abstract
The intestinal epithelial barrier is the most critical element of maintaining an intact intestinal barrier
and made up of a layer of columnar epithelial cells and intercellular junctional complexes including
tight junctions, adherens junctions and desmosomes. Tight junctions(TJ), which are formed by proteins
including occludin, claudins, junctional adhesion molecule and zonula occludens(ZO), are primarily responsible for the permeability of the paracellular pathway. In poultry, only claudin-1, claudin-2, claudin3, claudin-5, claudin-16, ZO-1, ZO -2 and occludin are reported so far. The intestinal barrier function in
poultry is evaluated by measuring intestinal permeability, plasma LPS concentrations and bacterial translocation. Few studies have shown the developmental profile of intestinal barrier function and tight junction protein occludin, claudin-3, claudin-5, claudin-16 and ZO-2 in the intestinal epithelium of chicks in
embryonic phase or/and the early post-hatch period. Several feed additives, including nutrients (i.e. Zn),
probiotics, prebiotics, functional polysaccharide, epidermal growth factor and enzymes, were shown to
regulate intestinal barrier function by modifying expression and localization of TJ proteins, and in some
cases prevents or reverses the adverse effects of pathogens and heat stress in poultry.
Keywords: poultry, intestinal epithelial barrier, tight junction protein, nutritional regulation
Introduction
The animal intestine has the roles of absorbing nutrients and also acting as a barrier to prevent pathogens and toxins from entering into the body and potentially causing disease. Maintaining the integrity of
the intestinal barrier is fundamental to the proper functioning of the epithelial cells and to preventing the
entry of pathogenic bacteria. Injured intestinal barrier is characterized by increased intestinal permeability, which allows luminal antigenic agents (e.g., bacteria, toxins, and feed-associated antigens) to“leak”
across the epithelium to sub- epithelial tissues, resulting in inflammation, malabsorption, diarrhea, and
potentially systemic disease[1]。 Under environmental, nutritional and pathophysiological stress conditions, animals including poultry subject to barrier impairment[2]. In this review, the components of intestinal epithelial barrier, the primary measures of determining intestinal epithelial barrier function, and the
development and maturation of the intestinal epithelial barrier in poultry were summarized, and some nutritional solutions to modulate the intestinal epithelial barrier in poultry were discussed.
The intestinal epithelial barrier
The intestinal barrier is a complex structure made up of four main components: the intestinal epithelial, chemical, immunological and microbiological barriers[3]. The intestinal epithelium forms the largest
and most important barrier between internal and external environments of animals. The following sections describe the role of the intestinal epithelial barrier in maintaining intestinal barrier function.The intestinal epithelial barrier is made up of a layer of columnar epithelial cells that forms the first line of defense between the intestinal lumen and inner milieu. The intestinal epithelial cells are mainly absorptive
enterocytes (over 80%) but also include entero-endocrine, goblet, and Paneth cells[4]. The epithelium allows the absorption of nutrients while providing a physical barrier to the permeation of pro-inflammatory molecules, such as pathogens, toxins, and antigens, from the luminal environment into the mucosal
tissues and circulatory system. The epithelial selective permeability includes two pathways: the transcellular and the paracellular pathway. The transcellular pathway is involved in the absorption and transport
of nutrients, including sugars, amino acids, peptides, fatty acids, minerals, and vitamins. As the cell
membrane is impermeable, this process is predominantly mediated by specific transporters or channels
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located on the apical and basolateral membranes. The paracellular pathway is associated with transport
in the intercellular space between the adjacent epithelial cells. These epithelial cells are tightly bound together by intercellular junctional complexes that regulate the paracellular permeability and are crucial
for the integrity of the epithelial barrier。These junctions allow the passage of fluids, electrolytes, and
small macromolecules, but inhibit passage of larger molecules.
The junctional complexes consist of the tight junctions, gap junctions, adherens junctions, and desmosomes [7].Tight junctions are the most apical and are primarily responsible for controlling permeability of
the paracellular pathway [5]. Adherens junctions are located beneath the tight junctions and are involved
in cell-cell adhesion and intracellular signaling [6]. Both tight junctions and adherens junctions (together
known as the apical junctional complex) are associated to the actin cytoskeleton [6- 8]. Desmosomes and
gap junctions are involved in cell-cell adhesion and intracellular communication [9], respectively. The cytoskeleton is an intricate structure of protein filaments that extends throughout the cytosol that is essential for maintaining the structure of all eukaryotic cells. Disruption of the cytoskeleton is linked to the
loss of intestinal barrier integrity.
Tight junctions are formed by protein dimers that span the space between adjacent cell membranes
(see Fig. 2). There are over 50 proteins with well recognized roles in tight junction formation. These proteins comprise four integral transmembrane proteins (e.g., occludin, claudins, junctional adhesion molecules (JAM), and tricellulin), and cytosolic scaffold proteins, such as zonula occludens (ZO) proteins.
The extracellular domains of the transmembrane proteins form the selective barrier by hemophilic and
heterophilic interactions with the adjacent cells. The intracellular domains of these transmembrane proteins interact with ZO proteins [10], which in turn anchor the transmembrane proteins to the perijunctional
actomyosin ring [11]。. The interaction of TJ proteins with the actin cytoskeleton is vital to the maintenance of TJ structure and function. In addition, the interaction of the TJ complex with the actomyosin
ring permits the cytoskeletal regulation of TJ barrier integrity. Comprehensive reviews on the complex
molecular structure of TJ are available [see, e.g., (12)].
Occludin was the first integral membrane TJ protein identified in 1993. Occludin can be specifically
visualized at the tight junctions in the epithelia by confocal immunofluorescence microscopy, immunoelectron microscopy and freeze-fracture immuno-replica electron microscopy. The function of occludin
is not yet fully understood, but numerous studies using animals and cell cultures indicate that it is required for TJ assembly and barrier integrity in the intestinal epithelia. Occludin has been linked to the
regulation of intermembrane diffusion and paracellular diffusion of small molecules. The claudin proteins are considered to be the structural backbone of TJ. Claudins consist of at least 24 members in humans and mice, and each isoform shows a unique expression pattern in tissues and cell lines. In contrast
to their structural similarities, claudins perform different functions and can be roughly divided into two
types: those involved in barrier formation (decreasing paracellular permeability) and those playing a role
in channel pores (increasing paracellular permeability) In the intestines, claudin-1, -3, -4, -5, -8, -9, -11,
and -14 can be categorized as barrier-forming claudins, while claudin-2, -7, -12, and -15 are pore-forming claudins [for review, see (12)]. Several plaque proteins have been identified, including the zonula occludens (ZO) proteins, ZO-1, ZO-2, and ZO-3. ZO-1 interacts with the claudin proteins (36), other ZO
proteins to form dimers (37), and JAM-A (38). Plaque proteins potentially play a central role in TJ regulation, because they can cause reorganization of the cytoskeleton. To our knowledge, only claudin- 1、
claudin-2、
claudin-3、claudin-5, claudin-16, ZO-1, ZO -2 and occludin are reported in poultry so far[13-16]。
TJ are not static barriers but highly dynamic structures that are constantly being remodeled due to interactions with external stimuli, such as food residues and pathogenic and commensal bacteria[4]. Regulation of the assembly, disassembly, and maintenance of TJ structure is influenced by various physiological and pathological stimuli. Signaling pathways involved in TJ regulation, and interactions between
transmembrane proteins and the actomyosin ring are controlled by several signaling proteins, including
protein kinase C (PKC), mitogen- activated protein kinases (MAPK), myosin light chain kinase
(MLCK), and the Rho family of small GTPases[17].
Assessing of the intestinal epithelial barrier function in poultry
The intestinal epithelial barrier function in animals including poultry is evaluated by measuring intestinal permeability, plasma LPS concentrations and bacterial translocation.
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Intestinal permeability
Intestinal permeability is defined as the non-mediated diffusion of large (i.e., molecular weight >150
Da), normally restricted molecules from the intestinal lumen to the blood. The primary means of determining intestinal permeability in humans or animals is by measuring the passage of high molecular
weight probes across the gastrointestinal tract barrier[20- 22]. In humans, this involves ingestion of a solution containing nontoxic, non- metabolizable substances (such as sucrose, lactulose, sucralose and) [23, 24]
and assessing their excretion in the urine. The appearance of probes in the urine indicates loss of barrier
function in the gastrointestinal tract. But the process is inapplicable to poultry because separation
of urine and feces is difficult in poultry. In animal models including poultry, intestinal permeability is
frequently determined by infusing fluorescent probes, such as fluoroisothiocyanate (FITC)-dextrans, into the intestinal area of interest and measuring plasma concentrations over time[26]. Such probes come in
various molecular weights ranging from several hundred to several million. Thus, these probes provide
not only an index of intestinal permeability, but also provide an idea of how large the opening in the intestinal epithelium may be. Other commonly used probes, used in a similar manner in animal models,
are [51Cr]-EDTA [25]and horseradish peroxidase [27].The Ex vivo Ussing chamber is the most sensitive to
test the intestinal permeability by measuring transepithelial electrical resistance (TER) and paracellular
flux of probes to monitor intestinal permeability in most animals[2]. TER is considered to be the most sensitive measure of mucosal barrier function, since it reflects the opening of the tight junctions between epithelial cells and the paracellular permeability of the intestinal mucosa
Bacterial translocation
The disruption in barrier functions was associated with viral and bacterial translocation across the epithelial monolayers. Bacterial translocation is defined as the passage of viable bacteria from the intestinal
tract through the epithelial mucosa into extra-intestinal organs. Impaired mucosal surfaces can increase
vulnerability of the intestinal epithelium with an augmented risk of bacterial and viral penetration, or
bacterial overgrowth in the in the intestine [28].
Plasma LPS concentrations
Another index of intestinal barrier dysfunction is the plasma lipopolysaccharide (LPS) concentrations.
LPS is a highly pathogenic component of the walls of gram negative bacteria and is found in the intestinal tract in high concentrations. Its presence in the portal blood of animal models indicates passage from
the intestinal lumen to the circulation [29]. Increased LPS concentrations in the systemic circulation likely
indicate severe intestinal barrier dysfunction, in that its high permeability has overwhelmed the ability
of the liver to clear it from the blood [28, 30].
Development and maturation of the intestinal epithelial barrier
Many reports have described the development of the junctional complex in the epithelium of animals
during the embryonic phase or/and the early post- hatch period. In poultry, limited studies have shown
the developmental profile of intestinal epithelial barrier function and tight junction proteins in the intestinal epithelium of chicks in embryonic phase or/and the early post-hatch period, indicating that developmental patterns of different intestines and tight junction proteins are not coincident.
Okamoto and Ishimura (1978) reported that incomplete tight junctions in the duodenal epithelium of
chick embryos were already present after 6-7 days of incubation in the apical portion of the lateral plasma membrane, and the formation of the tight junction in chick duodenum might be complete by day 18
of incubation, suggesting that tight junctions begin to form in the gastrointestinal tract at an early developmental stage, thereafter, tight junctions develop rapidly and form complicated networks[31]. Kimura et
al. (1996) revealed that the structure of the tight junction was already apparent in intestinal samples
from chick embryos aged 13 days or more[32].
Kawasaki et al. (1998) [14. determined the developmental expression of occludin in the gastrointestinal
tract of 3- to 21-day-old chick embryos. Occludin mRNA was first detected by RT-PCR in the chick embryo on day 3 of incubation, by northern blot analysis on day 4, and by western blot analysis on day 5,
suggesting that synthesis of occludin begins in the chick embryo at a very early stage of development. In
addition, the immune- histochemical study revealed that occludin began to be weakly expressed only
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along the apical surface of the gastrointestinal epithelium of the 4-day-old chick embryo. As the embryo
developed, the immunoreactivity gradually became stronger and formed more complex networks near
the apical surface, which indicated that the developmental expression of occludin in the gastrointestinal
tract is closely correlated with the morphological as well as functional development of the tight junction.
The expression patterns of intestinal tight junctional proteins during embryogenesis and post-hatch period are not all alike. Earlier work on expression of claudins in developing intestine revealed that transcripts for claudins -1 and -3 are present in the epithelial lining of 5- to 8-day old chick embryos [15, 32, 33].
OZDEN et al （2010）
.
explored the developmental patterns of claudin-3, -5, and -16 proteins in the epithelium of embryonic chick intestine from 9 days prior to hatching through the early post-hatch period
[13]
. These claudin proteins either changed their cellular localization or first appeared around the time of
hatching. suggesting that in addition to their known barrier and fence functions within tight junctions,
these claudins may have additional roles in the differentiation and/or physiological function of chick intestine[13]. Conversely, transcript levels of ZO-2 decrease from 18 to 20 days and reach an even lower level by 2 days post-hatch. It may be significant that ZO-2 transcript levels are high 2 days before the increase of claudin transcripts and prior to the movement of claudin -3 and -5 proteins to the epithelial periphery. However, Roberts et al. (2005) thought that the intestinal epithelial barrier function is not fully
developed in chicks until d 11 of age for the jejunum and later than d 14 of age for the ileum.
Nutritional strategies to modulate the intestinal epithelial barrier in poultry
Numerous studies have shown that dietary factors and nutrients can regulate intestinal barrier structure and function in humans and animals [33- 36], and some of these could be developed as preventive and
therapeutic tools for defective barrier-associated diseases[34,36]. In contrast, limited studies in poultry have
reported that dietary factors and nutrients, such as minerals, probiotics, prebiotics, and dietary enzymes,
participate in intestinal epithelial barrier regulation.
Zn (Zinc)
The importance of Zn to intestinal development and function has been demonstrated in many studies,
such as increased intestinal crypt-cell production, reduced duration of mitosis[37], and improved epithelial
cell restitution[38], and maintaining the structure and function of the intestine barrier [39]. Zn as supplementation in diet reduced gut lesion scores, and reduced intestinal permeability and increased expression of
ZO-1 and occludin in mammals[36-39. . Zn deprivation induced a decrease of TER and altered tight and adherens junctions [40]。In poultry, several studies have demonstrated the beneficial effects of supplemental
Zn on the intestinal mucosal barriers. Zhang et al. (2012) reported that Zn (as ZnSO4) up-regulated occludin and claudin- 1 mRNA expression in the ileum and tended to reduce plasma endotoxin levels of
chickens challenged with Salmonella Typhimurium, suggesting that regulation of occludin and claudin1expression by Zn may be involved in ameliorating increased intestinal permeability induced by Salmonella Typhimurium challenge.[41]。However, Hu et al. (2013) showed that supplemental ZnO or ZnSO4
did not affect ileal and colonic barrier function and intestinal microflora in broiler chickens, but supplementing 60 mg of Zn/kg as ZnO-MMT(zinc oxide-montmorillonite hybrid) increased colonic TER values, and reduced colonic probe mannitol permeability as well as ileal or colonic inulin permeability of
chickens[42].
Probiotics
In human and animals, some probiotics have been shown to promote intestinal barrier integrity and to
prevent, and even reverse, the adverse effects of pathogens and stress on intestinal barrier function both
in vitro and in vivo[43-45]In poultry, several reports have showed probiotics could decrease intestinal barrier dysfunction induced by pathogens or stress.
The transmission electron microscopy confirmed that, compared to treatments with Saccharomyces
boulardii and Bacillus subtilis B10, the tight junctions of jejunum and ileum of broilers were comparatively loose in the control group, and Saccharomyces boulardii and Bacillus subtilis B10 also improved
the epithelial tight junctions through increasing occludin, cloudin2, and cloudin3 mRNA expression levels in broiler intestine[18]. The increased ocludin, claudin2, and claudin 3 gene expression might be due to
a direct response to probiotics or a secondary response to induced inflammatory cytokine secretions of
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IL-6 and TNF-α[18]. The addition of a microbial feed additive (L. salivarius and L. reuteri) to broiler diets increased glucose stimulated short-circuit current in both jejunum and colon in Ussing chamber, but
the conductivity of jejunal and colonic tissues remained unaffected by the dietary inclusion of Lactobacillus sp. , which support the concept that this microbial additive improves intestinal nutrient absorption
and enhances the maintenance and function of the epithelial barrier[43]. L. fermentum 1.2029 was able to
ameliorate the severity of necrotic enteritis lesions and inflammation and improve epithelial barrier
through increasing claudin-1 and occludin levels in necrotic enteritis -infected chickens [44].
In recent years, some reports have indicated that heat stress negatively affects intestinal mucosa and microbiota [45-47]. Heat stress also decreased jejunal TER，
increased jejunal paracellular permeability of FITCdextrans, and downregulated jejunal protein levels of occludin and ZO-1 in broilers[48]. Supplemental probiotic mixture containing Bacillus licheniformis, Bacillus subtilis and Lactobacillus plantarum increased
jejunal protein level of occludin in broilers, revealing that dietary addition of the probiotic mixture was effective in partially ameliorating intestinal barrier dysfunction induced by heat stress in broilers[48].
Prebiotics
Prebiotics were defined as non-digestible food (feed) ingredients that beneficially affect the host by
selectively stimulating the growth and/or activities of one or a limited number of bacteria in the gut,
thereby improving host health. Cello-oligosaccharide is a functional oligosaccharide obtained from plant
cellulose. As compared with heat stress group feeding basal diet, supplemental cello-oligosaccharide increased jejunal villus height and villus height to crypt depth ratio, as well as decreased jejunal paracellular permeability of fluorescein isothiocyanate dextran in broiler chickens, which demonstrated that cellooligosaccharide supplementation partially ameliorated the adverse effects caused by heat stress in broilers through improving intestinal microflora, morphology and barrier integrity [49].
Functional polysaccharides
β- 1,3/1,6- glucans from Saccharomyces cerevisiae have beneficial effects on both the innate and acquired immune systems in either non-challenged or challenged settings [51-55], and clearance of several important pathogens such as Salmonella, Escherichia coli and coccidiosis in broiler chickens [51,53,55]. In intestinal epithelial barrier, dietary β-1,3/1,6-glucan supplementation can reduce intestinal mucosal barrier
impairment of broiler chickens challenged with Salmonella Typhimurium, and the partial mechanism
might be related to the increased mRNA expression of claudin-1 and occludin, and increased goblet cell
numbers and sIgA level in the jejunum of broiler chickens [56].
Soluble NSP from plantain banana was able to block adhesion of various enteric gut pathogens to the
human intestinal epithelial cell or cell-line Caco2 [57- 59], and inhibit invasion of Escherichia coli into human intestinal epithelial cells [60. and translocation across specialised microfold (M)-cells of the follicle
associated epithelium cultured in vitro [61,62]. In chickens, In vivo dietary supplementation with plantain
NSP reduced invasion by S.Typhimurium, as reflected by viable bacterial counts from splenic tissue,
and in vitro plantain NSP inhibited adhesion of S.Typhimurium to a porcine epithelial cell- line and to
primary chick caecal crypts. Adherence inhibition was shown to be mediated via an effect on the epithelial cells and Ussing chamber experiments with ex-vivo human ileal mucosa showed that this effect was
associated with increased short circuit current but no change in electrical resistance [63]。
Epidermal growth factor (EGF)
EGF is a small amino acid peptide with a broad range of bioactivities on the intestinal epithelium, including the stimulation of cellular proliferation, differentiation, and intestinal maturation. Previous studies have shown that EGF administration plays a protective role in a variety of intestinal insults by either
reducing injury [65]or accelerating repair [66,67]. In chickens, EGF reduced jejunal C. jejuni colonization and
alleviated the dissemination of C. jejuni to the liver and spleen. In his in vitro study, the pretreatment
with EGF abolished the C. jejuni-induced intestinal epithelial abnormalities, such as disruption of tight
junctional claudin- 4, increasing of transepithelial permeability and the translocation of noninvasive
Escherichia coli C25. These findings highlight EGF’s ability to protect against pathogen-induced barrier defects[68]。
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Enzyme
C. perfringens challenge increased intestinal lesion score and also resulted in increased plasma endotoxin [69,70]., passive transcellular permeability[62], while dietary addition of xylanase[71. and enzyme complex containing xylanase, glucanase and mannanase as major components[72. could alleviate the alteration
caused by C. perfringens infection, indicating that dietary enzyme supplementation could benefit for gut
barrier integrity of C. perfringens challenged chickens.
Lysozyme as a natural antimicrobial protein occurs in a number of animal secretions and is considered an important component of the innate immune system. The addition of exogenous lysozyme significantly reduced the concentration of Clostridium perfringens in the ileum and the intestinal lesion scores,
and inhibited the overgrowth of E. coli and Lactobacillus in the ileum and intestinal bacteria translocation to the spleen of chickens challenged with Clostridium perfringens, suggesting that exogenous lysozyme could decrease Clostridium perfringens colonization and improve intestinal barrier function of
chickens [73].
Other dietary components such as glutamine[52,53,54,55], threonine[59. , fatty acids[64], and flavonoids[65. are also known to regulate intestinal epithelial barrier, but no reports were found in poultry. More nutritional
approaches for improving intestinal barrier function and the underlying molecular mechanisms are needed to be investigated.
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