**2. Histology and physiology of the gastro-intestinal tract**

Gallus species have villi which decrease in length from 1.5 mm in the duodenum to 0.4–0.6 mm in the ileum and rectum. The number of villi decreases from 1 to 10 days of age, but thereafter remains constant. Genetic selection for growth has altered villi morphology [2]. The villi of broilers are larger than White Leghorns, and show more epithelial cell protrusions from the apical surface of the duodenal villi. However, the villi from both types of chickens consist of a zig-zag arrangement which is thought to slow the passage rate. The intestinal wall contains four layers as including the mucosal, submucosal, muscle tunic, and the serosal layer. The mucosal layer consists of the muscularis mucosa, lamina propria, and epithelium. However, the muscularis mucosa and lamina propria are poorly developed in chickens, possibly because of the absence of a central lacteal. Although Brunner's glands, common to mammals, are absent [3] tubular glands possibly homologous to Brunner's glands, are present in some birds [4]. The epithelium has chief cells, goblet cells, and endocrine cells. The crypts of Lieberkühn are the source of epithelial cells lining the villi. The crypts contain undifferentiated cells, goblet cells, endocrine cells, and lymphocytes. Globular leukocytes and Paneth cells appear near the base of the crypts. The intestine contains extensive innervation from both the sympathetic and parasympathetic nervous system. As described [5], innervation is both cholinergic and adrenergic. Contraction of the rectum appears to be mediated by noncholinergic, non-adrenergic nerves [6, 7].

The mucosa of the GIT is a functional interface between the environment and the internal physiological compartments of the organism. As such, the mucosal and associated cells constitute a dynamic and metabolically active barrier possessing selective permeability [8]. This barrier has multiple functions that involve the digestion, transport and uptake of specific substances and nutrients and exclusion of microorganisms and toxins. The processes of digestion and absorption occur in a micro-environment modified by the intestinal mucosa, its secretions, and the ancillary organs (pancreas, liver). The importance of 'the intestinal barrier' as it relates to gut function and gut health in poultry has been reviewed [9, 10]. Optimal digestive and absorptive functions are essential for growth, development and health of the animal. In addition, the intestine must act as a physical barrier to pathogenic organisms and toxins and play a role in both innate and acquired immunity. The integration of the digestive, absorptive and immune function of the GIT and the genetic regulation of these processes are central to animal production and health.

### **3. Innate immunity of the GIT**

The epithelial cell physical barrier in the GIT represents a vast surface area that is very vulnerable to intraluminal impacts. Continual confrontation by direct

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*Secretory Defense Response in the Bird's Gastro-Intestinal Tract and Nutritional Strategies…*

many of these responses through interaction with microbial ligands [12].

and physiological alterations of mucus and its major components [14].

carbohydrates accounting for 80% of the mass [16].

extent of negative charges on each mucin molecule [17].

The protective functions of mucus are attributable to mucus glycoproteins, the major macromolecules present in the mucus gel. Mucus glycoproteins, now widely known as mucins, are defined as a class of high-molecular-weight proteins that are heavily glycosylated with complex oligosaccharide chains [15]. The molecular weight of mucins has been estimated from early studies of ~1000 kDa with attached

According to cellular localization and distribution, mucins are broadly classified

into secretory and membrane-associated proteins [17]. Structurally mucins are comprised of a linear protein backbone in the center and a large number of carbohydrate chains attached around it. The carbohydrate components, usually heterosaccharides, are bound covalently to the peptide chains and terminated with sialic acid (sialoglycoproteins) or with both sialic acid and sulphate ester (sialosulphoglycoproteins) or with neutral ends (neutral glycoproteins). These ends determine the

Intestinal secretory mucins are synthesized and secreted by goblet cells, a specialized wine-goblet-shaped epithelial cell lineage dispersed along the intestinal lining. The dimerization and/or polymerization of mucin molecules and the

The intestine is protected by that substance, which forms a tightly adherent layer along the epithelial surface, followed by a more loosely adherent, partially hydrolyzed layer. It is also part of an integral process, and is secreted, forming and "unstirred" water gel layer covering the epithelial surface. This gelatinous molecular "coat" is subjected to continuous erosion by luminal fluid flow and rapid replenishment from epithelial secretion. The dynamics of mucus gel turnover contributes to a complex milieu where digestive events occur, nutrients approach epithelial cells, microbes build ecological niches, exfoliated enterocytes break down and immunological molecules (defensins, IgA, etc.) carry out surveillance. Consequently, the mucin layer, which encompasses all the of these components, constructs a gel-like biological barrier that shields the underlying tissue compartments, and eventually serves as an important component of the innate arm of the host system in the GIT [11]. In the small intestine the mucus layer is penetrable, but the bacteria are kept away from the epithelium by antibacterial mediators. In the large intestine, the inner mucus layer is impenetrable to bacteria whereas the outer mucus layer is expanded and serves as the habitat for bacteria (esp. mucolytic bacteria) [13]. Serving not only as a lubricant but also a protective barrier, the mucus gel layer(s) in the GIT is the largest area and of critical importance to the body both physiologically and nutritionally. Compromised mucin function is associated with many gastro-enteric disorders and nutritional insufficiencies. Particularly, many functional modulations of the GIT are closely related to expressional, structural,

contact with foreign substances, the mucosal system is tightly regulated in order to allow selective entry of macromolecules necessary for mucosal defense [11]. The cells and molecules that comprise the innate immune responses encompass both physical and chemical barrier mechanisms. For example, epithelial cells are tightly connected by multi-protein junctional complexes which regulate passage of solutes while providing an obstacle to luminal microbes and the lamina propria. Mucosal epithelial cells also produce non-specific macro-molecules (such as defensins) with antimicrobial action. Inflammatory and anti-viral responses are produced by specific mucosal cell types, which include: dendritic cells (DC), macrophages, and innate lymphoid cells (ILC). Pattern recognition receptors on these cells regulate

*DOI: http://dx.doi.org/10.5772/intechopen.95952*

**3.1 Mucus and mucins**

*Secretory Defense Response in the Bird's Gastro-Intestinal Tract and Nutritional Strategies… DOI: http://dx.doi.org/10.5772/intechopen.95952*

contact with foreign substances, the mucosal system is tightly regulated in order to allow selective entry of macromolecules necessary for mucosal defense [11]. The cells and molecules that comprise the innate immune responses encompass both physical and chemical barrier mechanisms. For example, epithelial cells are tightly connected by multi-protein junctional complexes which regulate passage of solutes while providing an obstacle to luminal microbes and the lamina propria. Mucosal epithelial cells also produce non-specific macro-molecules (such as defensins) with antimicrobial action. Inflammatory and anti-viral responses are produced by specific mucosal cell types, which include: dendritic cells (DC), macrophages, and innate lymphoid cells (ILC). Pattern recognition receptors on these cells regulate many of these responses through interaction with microbial ligands [12].

#### **3.1 Mucus and mucins**

*Advances in Poultry Nutrition Research*

in part through secretory antibodies and other mucosal defense mechanisms. Consistent with these functions, the epithelial surface of the GIT is lubricated and protected by mucus secretion and by a highly specialized immune system underlying the epithelium which exports immunoglobulins into the intestinal mucosa. Secretory defenses are some of the most important means to protect the intestinal epithelium from enteric pathogens and toxins. Secretory IgA (sIgA) production, Goblet, Paneth, M cells and GALT tissues are the key cells in this defense. The objective of this review is to describe a variety of secretory immune responses against pathogens in GIT and the role of nutrients in immunomodulation.

**2. Histology and physiology of the gastro-intestinal tract**

by noncholinergic, non-adrenergic nerves [6, 7].

**3. Innate immunity of the GIT**

Gallus species have villi which decrease in length from 1.5 mm in the duodenum to 0.4–0.6 mm in the ileum and rectum. The number of villi decreases from 1 to 10 days of age, but thereafter remains constant. Genetic selection for growth has altered villi morphology [2]. The villi of broilers are larger than White Leghorns, and show more epithelial cell protrusions from the apical surface of the duodenal villi. However, the villi from both types of chickens consist of a zig-zag arrangement which is thought to slow the passage rate. The intestinal wall contains four layers as including the mucosal, submucosal, muscle tunic, and the serosal layer. The mucosal layer consists of the muscularis mucosa, lamina propria, and epithelium. However, the muscularis mucosa and lamina propria are poorly developed in chickens, possibly because of the absence of a central lacteal. Although Brunner's glands, common to mammals, are absent [3] tubular glands possibly homologous to Brunner's glands, are present in some birds [4]. The epithelium has chief cells, goblet cells, and endocrine cells. The crypts of Lieberkühn are the source of epithelial cells lining the villi. The crypts contain undifferentiated cells, goblet cells, endocrine cells, and lymphocytes. Globular leukocytes and Paneth cells appear near the base of the crypts. The intestine contains extensive innervation from both the sympathetic and parasympathetic nervous system. As described [5], innervation is both cholinergic and adrenergic. Contraction of the rectum appears to be mediated

The mucosa of the GIT is a functional interface between the environment and the internal physiological compartments of the organism. As such, the mucosal and associated cells constitute a dynamic and metabolically active barrier possessing selective permeability [8]. This barrier has multiple functions that involve the digestion, transport and uptake of specific substances and nutrients and exclusion of microorganisms and toxins. The processes of digestion and absorption occur in a micro-environment modified by the intestinal mucosa, its secretions, and the ancillary organs (pancreas, liver). The importance of 'the intestinal barrier' as it relates to gut function and gut health in poultry has been reviewed [9, 10]. Optimal digestive and absorptive functions are essential for growth, development and health of the animal. In addition, the intestine must act as a physical barrier to pathogenic organisms and toxins and play a role in both innate and acquired immunity. The integration of the digestive, absorptive and immune function of the GIT and the genetic regulation of these processes are central to animal production and health.

The epithelial cell physical barrier in the GIT represents a vast surface area that is very vulnerable to intraluminal impacts. Continual confrontation by direct

**120**

The intestine is protected by that substance, which forms a tightly adherent layer along the epithelial surface, followed by a more loosely adherent, partially hydrolyzed layer. It is also part of an integral process, and is secreted, forming and "unstirred" water gel layer covering the epithelial surface. This gelatinous molecular "coat" is subjected to continuous erosion by luminal fluid flow and rapid replenishment from epithelial secretion. The dynamics of mucus gel turnover contributes to a complex milieu where digestive events occur, nutrients approach epithelial cells, microbes build ecological niches, exfoliated enterocytes break down and immunological molecules (defensins, IgA, etc.) carry out surveillance. Consequently, the mucin layer, which encompasses all the of these components, constructs a gel-like biological barrier that shields the underlying tissue compartments, and eventually serves as an important component of the innate arm of the host system in the GIT [11]. In the small intestine the mucus layer is penetrable, but the bacteria are kept away from the epithelium by antibacterial mediators. In the large intestine, the inner mucus layer is impenetrable to bacteria whereas the outer mucus layer is expanded and serves as the habitat for bacteria (esp. mucolytic bacteria) [13]. Serving not only as a lubricant but also a protective barrier, the mucus gel layer(s) in the GIT is the largest area and of critical importance to the body both physiologically and nutritionally. Compromised mucin function is associated with many gastro-enteric disorders and nutritional insufficiencies. Particularly, many functional modulations of the GIT are closely related to expressional, structural, and physiological alterations of mucus and its major components [14].

The protective functions of mucus are attributable to mucus glycoproteins, the major macromolecules present in the mucus gel. Mucus glycoproteins, now widely known as mucins, are defined as a class of high-molecular-weight proteins that are heavily glycosylated with complex oligosaccharide chains [15]. The molecular weight of mucins has been estimated from early studies of ~1000 kDa with attached carbohydrates accounting for 80% of the mass [16].

According to cellular localization and distribution, mucins are broadly classified into secretory and membrane-associated proteins [17]. Structurally mucins are comprised of a linear protein backbone in the center and a large number of carbohydrate chains attached around it. The carbohydrate components, usually heterosaccharides, are bound covalently to the peptide chains and terminated with sialic acid (sialoglycoproteins) or with both sialic acid and sulphate ester (sialosulphoglycoproteins) or with neutral ends (neutral glycoproteins). These ends determine the extent of negative charges on each mucin molecule [17].

Intestinal secretory mucins are synthesized and secreted by goblet cells, a specialized wine-goblet-shaped epithelial cell lineage dispersed along the intestinal lining. The dimerization and/or polymerization of mucin molecules and the electrochemical properties of mucopolysaccharides are believed to determine the chemical and biophysical characteristics of mucus along the GIT [18].

Mucins have a key role in avoiding potential damage from microbes. The mechanism by which mucus controls microflora colonization is referred to as part of innate epithelial cells [19]. The role of mucin on microbe colonization is manifested in at least two distinct ways. First, some microbes are mucolytic, including Bacteroidetes, and use mucin glycoproteins and carbohydrates as an energy source and provide physical support for intestinal colonization. Moreover, these bacteria provide substrates for other bacteria in the outer mucus layer by degrading the mucins [20, 21]. Second, mucins are generally "toxic" to the proliferation of certain microbes. Mucus gel inhibits proliferation by entrapping microbes that are starved or killed by antimicrobial peptides, and/or expulsed by the luminal flow. Mucus also provides a physicochemical barrier to prevent microbes from direct contact with epithelial cells.

Moreover, the mucus gel provides a matrix for antimicrobial molecules, which are mainly produced by Paneth cells. Direct interactions with mucins can facilitate the diffusion of these antimicrobial molecules [22]. Taken together, mucins have been proposed to play an important role in shaping microbial communities at the intestinal mucosa. Recent studies suggest the correlation between changes in mucin glycosylation profile and deviations of overall microbial community ecology as well as altered abundances of specific microbes [23, 24].

#### **3.2 Trefoil factors**

Co-expressed with mucin-secreting cells and in close relation with mucus, trefoil factors (TFF) demonstrate an interesting group of mucus molecules. Trefoil factors were initially discovered in the pig pancreas [25] and further characterization of this family has strikingly observed their abundant expression in the GIT and their efficacy as therapeutics especially for preventing and treating various GIT conditions [26, 27]. They are named as trefoil by their "three-leaf" structure and are a family of small (7-12 kDa in mammals) protease resistant peptides whose common unit is the trefoil motif [25].

It is now clear that TFF participate in the healing of mucosal injury in disease conditions by promoting cell migration over damaged areas (rather than promoting cell division), and inhibiting cell death, and are also believed to be involved in physiological repair of epithelia from daily apical sloughing against frequent luminal insults [25, 28, 29].

TFF have recently been found to participate in immune responses. It was showed that TFF2 deficiency or administration of recombinant TFF2 altered the expression of immune associated genes including defensin genes in Paneth cells [30]. The presence of TFF in immune organs, including spleen, thymus, lymph nodes and bone marrow [31], may suggest possible regulatory role(s) played there. TFF can be a potent mitogen by regulating chemotaxis, stimulating the migration of immune cells. The molecular basis of such may be supported by the recent in vitro evidence that recombinant TFF2 activates CXCR4 chemokine receptors and attenuates CXCR4 mediated chemotaxis [32]. This finding also highlights a molecular linkage between TFF and the immune system.

TFF are thought to cooperatively interact with mucins in the lumen to enhance the protective barrier properties of the adherent mucus layer against bacterial and toxic insults [25, 28]. Thim et al. [33] observed significant increase in the viscosity and elasticity of gastric mucin solutions because of TFF2 addition [33]. Increased viscosity could help prevent antigens from approaching the epithelium surface, especially in healing epithelia, which eventually benefits epithelium restitution and

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*Secretory Defense Response in the Bird's Gastro-Intestinal Tract and Nutritional Strategies…*

alleviates immune system burden. In this scenario, TFF are predicted to be involved

Goblet cells together with absorptive enterocytes, Paneth cells (secreting antimicrobial peptides etc.) and enteroendocrine cells, represent the four principal cell types that are continuously renewed in the epithelium of the small intestine. During intestinal epithelial cell regeneration, pluripotent stem cells that reside at the bottom of the crypt divide to generate multiple cell lineages which migrate from the proliferative crypts to the villus tip [34]. While migrating along the crypt-to-villus axis, goblet cells are terminally differentiated from secretory cell lineage derived from a common Math1-expresing progenitor cell [35]. Goblet cell differentiation is controlled by winged helix transcription factors Foxa1/a2 which can also transacti-

It is generally believed that goblet cells producing neutral mucins contain little sialic acid and represent an immature state; while goblet cells containing acidic mucins are more likely resistant to infections because they are normally "upregulated" in response to bacterial infection. In addition to mucins, several other molecules are co-expressed within the intestine such as ingobsin (localized in human and rat goblet cells) with endoproteolytic activity in the presence of both epidermal growth factor and cobalamin-binding protein haptocorrin [37]. TFFs are

M cells or Microfold cells (because of uneven microvilli) are classified as epithelial cells with large fenestrations in their membranes. These features enhancing the uptake of antigens from the gut lumen [38]. They have a capability for capturing luminal antigens and transporting them across the epithelium ("transcytosis"). They are placed in the gut epithelium called follicle associated epithelium overlying the domes of Peyer's patches and other lymphoid organs. M cells are not professional antigen-presenting cells because they do not have the ability to process and present antigens to the major histocompatibility complex (MHC) molecules. Instead, they serve as antigen delivery cells, that is, as a functional equivalent to lymphoid nodes because they provide antigens to professional antigen-presenting cells, such as dendritic cells (DCs), macrophages as well as B lymphocytes. Indeed, many pathogens take advantage of their transport efficacy to invade the body [39–41]. M cells subsequently transfer these antigens to underlying DCs enabling the transfer of captured molecules through transcytosis mechanism (which remain to be elucidated) as well as intracellular material through microvesicles to underlying DCs [42]. In conclusion, M cells provide specialized full-service immune

Paneth cells are physiologically found at the distal small intestinal crypts of Lieberkühn and contain abundant secretory granules. Their unique histomorphological features implicate special functions in cellular homeostasis as well as in the establishment and configuration of the mucosal barrier as a physical and highly organized immune interface [43]. Previous studies suggesting the existence of Paneth cells in the chicken remained controversial. However, recent research has supported Paneth cells existence in the small intestine of the chicken by electron

(specifically TFF3) along with mucins biomarkers of goblet cells.

*DOI: http://dx.doi.org/10.5772/intechopen.95952*

in mucus polymerization.

vate Muc2 promoters [36].

surveillance capabilities.

**3.5 Paneth cells**

**3.4 M cells**

**3.3 Goblet cells**

alleviates immune system burden. In this scenario, TFF are predicted to be involved in mucus polymerization.
