Innate Immunity at Mucosal Surfaces

C H A P T E R

6 Innate Immunity at Mucosal Surfaces Kosuke Fujimoto1,2 and Satoshi Uematsu1,2 1

Department of Immunology and Genomics, Osaka City University Graduate School of Medicine, Osaka, Japan 2Division of Innate Immune Regulation, International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

I. INTRODUCTION The mucosal immune system, which comprises the gastrointestinal tract, the respiratory tract, and the urogenital tract, protects the internal surfaces of the body from damage. Among these tracts, the gastrointestinal tract, which consists of the oral cavity, esophagus, stomach, and intestines, is unique because its lumen is constantly exposed to food particles and pathogenic microorganisms. The large surface area (almost 400 m2) of the intestine is, in fact, much larger than the surface area of the skin or lungs in humans [1,2]. Hosts have developed a multidefense system for impeding pathogen invasion [3]. For instance, the intestinal epithelium and its mucus layer play pivotal roles in the frontline defenses of the mucosal surfaces, and as such they are regarded as constituents of the innate immune system [4,5]. A wide variety of epithelial cells, including absorptive epithelial cells, endocrine cells, goblet cells, tuft cells, Paneth cells, stem cells, transit-amplifying cells, and M cells, make up a thin layer of the epithelium and create a physiological barrier against external antigens (Fig. 6.1) [6] (Chapter 3: Mucosal Antigen Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00006-7

Sampling Across the Villus Epithelium by Epithelial and Myeloid Cells and Chapter 28: M Cell-Targeted Vaccines). Innate immunity also protects the gastrointestinal mucosa from damage by effectively recognizing and eliminating pathogens from hosts [7 9]. Many cell types, such as dendritic cells, macrophages, and epithelial cells, play important roles in the innate immune response initiated by the germline-encoded pattern recognition receptors (PRRs), which recognize specific patterns in the microbial components expressed by microorganisms [10]. Recently, communication between the innate immune system and the intestine via disturbed intestinal function has been shown to be involved in a multitude of diseases [9,11], such as rheumatoid arthritis [12 15], ankylosing spondylitis [16,17], type 1 diabetes [18,19], inflammatory bowel diseases (IBDs) [20 26], allergic diseases [27], nonalcoholic fatty liver disease [28], carcinogenesis [29,30], obesity [31 33], and atherosclerosis [34 37] (Fig. 6.2). This indicates that innate immune responses provide a solid causal link between the disease-associated alteration of the intestinal environment known as dysbiosis and the pathophysiological functions of the host.

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FIGURE 6.1 Intestinal epithelial cells. Diagrammatic representation of intestinal epithelial cells in the gut. There are many different types of intestinal epithelial cells: absorptive epithelial cells, endocrine cells, goblet cells, tuft cells, Paneth cells, stem cells, transit-amplifying cells, and M cells. These cells play crucial roles in regulating intestinal homeostasis.

FIGURE 6.2 Relationships between intestinal mucosal dysfunction and human diseases. Alterations in innate mucosal immunity, such as disruption of the intestinal barrier system and innate immune function in the gut, contribute to the induction and/or exacerbation of many inflammatory disorders, including rheumatoid arthritis, ankylosing spondylitis, type 1 diabetes, inflammatory bowel diseases, allergic diseases, nonalcoholic fatty liver disease, carcinogenesis, obesity, and atherosclerosis (Chapter 52: Mucosal Immunity for Inflammation).

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In addition, several lines of evidence have shown that dysfunctional PRRs are involved in the induction of intestinal inflammation [5,38]. This chapter describes the current knowledge on intestinal mucosal immunity, with special focus placed on the innate immune responses occurring at the mucosal surfaces of the gut.

II. INNATE MUCOSAL BARRIERS IN THE GUT A. Structures of the Mucosal Barriers There are two main types of mucosal barriers in the small and large intestines: physical barriers and chemical barriers [5]. They separate a plethora of foreign antigens in the form of commensal microorganisms, pathogenic microbes, and food antigens on the luminal side and superbly regulate gut homeostasis. Several types of intestinal epithelial cells, such as goblet cells, Paneth cells, and absorptive epithelial cells, are present in the two barriers. Physical barriers are represented mostly by cell cell junctions and the mucus layers produced by intestinal secretary cells. The biophysical features of these barriers are central to protecting the mucosal surfaces from various intestinal components. Cell cell junctions such as tight junctions are critically important to the maintenance of the intestinal barrier system [39 41]. The huge diversity of microbiota and food antigens present in the intestines makes effective regulation of junctional integrity and paracellular permeability essential for maintaining innate immune mucosal homeostasis. Dysfunctional intestinal permeability has been shown to induce various intestinal inflammation conditions [see 1,2]. It should be noted that the mucus layers in the large intestine differ from those in the small intestine. To be more precise, in the small intestine, a variety of antimicrobial peptides (AMPs) such as defensins, which are major contributors to the chemical

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barriers in this organ, are crucially important for the segregation of commensal bacteria and intestinal epithelial cells [42,43]. In contrast, the mucus layers of the large intestine are thick (10 700 μm). They are divided into an inner firm mucus layer and an outer loose mucus layer and are organized in this fashion by gelforming MUC2 (Fig. 6.3). The sterile inner mucus layer acts as an efficient physical barrier against bacteria [44]. Conversely, huge numbers of commensal microbiota exist in the outer mucus layer [44,45], suggesting that the outer mucus layer in the large intestine forms a unique microbial niche. In fact, the number of goblet cells in the large intestine is much higher than that in the small intestine; hence the mucus layer in the large intestine is thought to be fundamentally thick. This thick mucus layer is able to establish a concentration gradient, resulting in retention of AMPs and immunoglobulins. Germ-free animals have decreased numbers of goblet cells and reduced goblet cell thecae sizes. However, under germ-free conditions, goblet cells are constantly produced despite being unnecessary for protection against enteropathogens [46]. These findings suggest that intestinal microorganisms are only partially essential for the induction of goblet cells. Chemical barriers include various types of AMPs that mainly segregate intestinal epithelial cells from intestinal microorganisms [47,48]. As antimicrobial molecules, AMPs such as the defensins produced by Paneth cells in the small intestine are gene-encoded natural antibiotics that regulate the intestinal microbiota [49,50]. Defensins are active against Gram-negative and Gram-positive bacteria, as well as against fungi, viruses, and protozoa [4,51], and are thought to be key factors in innate mucosal immunity in the intestine. The 17 defensins found at various bodily sites in humans fall into two major categories: α-defensins and β-defensins [52]. Panethcell-related dysbiosis has revealed the role played by defensins in the host mucosal immune system [53]. Indeed, studies of several mouse

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FIGURE 6.3 Distinct intestinal barriers in the small and large intestines. Diagrammatic representation of the secreted mucosal barrier in (A) the small intestine and (B) the large intestine. Specialized secretory cells such as goblet cells and Paneth cells are shown. In the small intestine, chemical barriers represented by the AMPs produced by Paneth cells are central factors for the segregation of intestinal microorganisms and intestinal epithelial cells. In the large intestine, however, the enormous number of intestinal microorganisms and intestinal epithelial cells are separated by an inner mucus layer, which is a special feature of the intestinal mucus of this organ.

strains with genetically modified α-defensin production by their Paneth cells showed that the gut microbiome composition in the small intestine is influenced by the α-defensins secreted by Paneth cells [53,54]. In HD5 transgenic mice, which express human Paneth cell defensin (DEFA5) in their own Paneth cells, the ratio of Bacteroides to Firmicutes was higher than that in their wildtype littermate controls [55]. In contrast, in matrix metalloproteinase-7-deficient mice, which lack mature α-defensin in their Paneth cells, the ratio of Bacteroides to Firmicutes decreased in comparison with that of their wild-type littermate controls [55]. These findings indicate that Paneth cell defensins can regulate the commensal microbiome and may be involved in the

pathophysiology of dysbiosis related-diseases such as IBDs. However, interestingly, there are no Paneth cells in the large intestine, and the level of antimicrobial peptides is lower than that in the small intestine [56]. A recent study has clearly shown that Ly6/Plaur domain-containing 8 (Lypd8) is essential for segregating the intestinal bacteria and intestinal epithelial cells in the large intestine [57]. Lypd8 can bind to flagellated bacteria (e.g., Escherichia, Proteus, and Helicobacter) and inhibit bacterial invasion of colonic epithelial cells [57]. Lypd8-deficient mice also have an absence of bacterial free space [57], suggesting that Lypd8 regulates intestinal homeostasis by suppressing bacterial invasion of the colonic mucosa.

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B. Innate Barrier Dysfunction and Disease Pathogenesis The innate mucosal barrier system is indispensible for the regulation of gut homeostasis. As well as disruption of the epithelial barrier, access of luminal contents to the lamina propria and the abnormal immune response that follows are key factors for the progression of intestinal diseases. A variety of intestinal diseases, including IBDs [58 60], Clostridioidesdifficile (C. difficile)-induced colitis [61], enteropathogenic Escherichia coli (EPEC) infection [62], and graftversus-host disease (GVHD) [63], are associated with a dysfunctional mucosal barrier in the gut. Recent genome-wide association studies have identified more than 200 susceptible loci for the IBDs represented by Crohn’s disease (CD) and ulcerative colitis (UC) [64,65]. Interestingly, these genetic analyses have strongly linked an increased intestinal permeability with such diseases. For example, a frameshift insertion at position 3020 of the gene encoding the cytoplasmic sensor nucleotidebinding oligomerization domain-containing 2 (NOD2) protein induces barrier defects in patients with CD [66]. MUC19 is one of the gelforming mucins expressed in epithelial cells along with MUC2, MUC5AC, MUC5B, and MUC6 [67]. Genetic variants of MUC19 in patients with IBD cause quantitative changes in mucus production or structural changes in the glycoprotein core, resulting in mucosal barrier dysfunction [68]. In addition to being associated with UC, polymorphism in the RING finger protein 186, which is a member of the RING finger protein family, induces increased intestinal permeability and an increased risk of intestinal inflammation with enhanced endoplasmic reticulum stress in colonic epithelial cells [69]. Furthermore, nonsense polymorphisms in the gene encoding fucosyltransferase 2 (FUT2) are correlated with various pathophysiological conditions in humans [3]. Loss-of-

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function mutations in FUT2 are associated with microbiome changes and an increased risk of developing IBDs [70,71]. Antibiotic use has increased the number of infectious diseases that are curable. However, the extensive use of antibiotics has caused an increase in the number of infections with C. difficile, which is a spore-forming, toxinproducing, Gram-positive anaerobic bacterium that normally colonizes healthy individuals. C. difficile produces toxins A and B, which are major virulence factors for C. difficile-induced colitis [72 74]. Release of these toxins at disease onset disrupts actomyosin and induces glycotransferase toxin expression in this bacterium. Subsequent inactivation of RHO or RAC GTPases causes dysfunctional tight junctions and disrupts the epithelial integrity of the gut [61,75]. In developing countries, EPEC infections are among the leading causes of childhood mortality [76]. Both the architecture and the barrier function of intestinal tight junctions are altered by EPEC, and the apical basal polarity of intestinal cells is also perturbed by such infections [77]. The type III secretion system, which is a special protein-export apparatus in Gramnegative bacteria, is involved in this intestinal barrier defect [78]. GVHD develops after transplantation with bone marrow or hematopoietic stem cells. Recently, it has been found that intestinal barrier dysfunction plays a role in the pathogenesis of GVHD [79 81]. For example, altered expression and localization of occludin, which is a tight junction molecule, contribute to increased intestinal permeability and disruption of tight junctions [82,83]. Structural epithelial alterations occur when increased claudin-2 expression occurs [84] and epithelial cells undergo apoptosis [85]. Pertinently, the resulting intestinal barrier loss is thought to be associated with alterations in the gut microbiota in GVHD as well as in IBDs [86 91].

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III. INNATE IMMUNE REGULATION IN THE GUT A. Pattern Recognition Receptors The immune system is divided into two major branches: the innate immune system and the adaptive immune system. After pathogenic microorganisms invade beyond the structural barriers in the host, the first line of defense against pathogens is innate immunity, and the second line is adaptive immunity. A key function of innate immunity is to recognize invading pathogens and quickly mount defensive responses against them. For a long time, innate immunity has been considered to be nonspecific in its actions, its main functions being to digest pathogens and to present antigens to the cells involved in adaptive immunity. However, recent studies have shown that innate immunity is not necessarily nonspecific but is specific enough to distinguish between self and pathogens using evolutionarily preserved receptors, such as toll-like receptors (TLRs) [10,92 94]. Germline-encoded PRRs can sense microorganisms. Currently, four different PRR family classes have been identified: TLRs, C-type lectin receptors (CLRs), retinoic-acid-inducible gene-I-like receptors (RLRs), and NOD-like receptors (NLRs) [95]. PRRs recognize the highly conserved pathogen-associated molecular patterns (PAMPs) expressed by microorganisms but not by mammalian host cells. After PAMP recognition, PRRs immediately induce a series of signaling systems that execute the first line of host defensive responses. PRR signaling also introduces the maturation of particular immune cells, such as dendritic cells (DCs), which subsequently leads to activation of antigen-specific acquired immunity. The intestinal barrier system uses many different mechanisms to protect mucosal surfaces from pathogens. For example, intestinal epithelial cells express PRRs and can recognize intestinal microorganisms through secretion of

AMPs and cytokines [96]. A deficiency of PRRrelated genes in the intestinal epithelial cells of mice has revealed the crucial role they play in mucosal immune homeostasis [97,98], indicating that the function of PRRs in the mucosal barrier is a fundamental aspect of innate mucosal immunity.

B. Associations Between Individual TLRs and Intestinal Inflammation TLRs were identified as the first PRRs. To date, the mammalian TLR family comprises 13 members, and 10 and 12 functional TLRs have been characterized in humans (TLR1 TLR10) and mice (TLR1 TLR9, TLR11 TLR13), respectively. Each TLR recognizes distinct PAMPs derived from bacteria, viruses, mycobacteria, fungi, and parasites (Table 6.1). In common with TABLE 6.1 TLR Ligands TLR family Ligands TLR1

Triacyl lipopeptides

TLR2

Peptidoglycan, lipoprotein, lipopeptides, lipoteichoic acid, zymosan, glycolipids, GPI anchored

TLR3

dsRNA, Poly(I:C)

TLR4

LPS, endogenous ligands (HSPS, fibronectin, hyaluronic acid)

TLR5

Flagellin

TLR6

Diacyl lipopeptides

TLR7

ssRNA, imiquimod

TLR8

ssRNA, imiquimod

TLR9

CpG DNA, hemozin

TLR10

Unknown

TLR11

Profilin, flagellin

TLR12

Profilin

TLR13

Bacterial 23S ribosomal RNA

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other organs, intestinal TLRs play an essential role in the induction of host defenses (Chapter 10: Innate Immunity based Mucosal Modulator and Adjuvant). However, their distribution and effects on the gut are noteworthy because of the presence of commensal microflora in this organ (Chapter 9: Influence of Commensal Miro-biota and metabolite for mucosal immunity). TLR1, which is functionally associated with TLR2 [99], is involved in the recognition of triacyl lipopeptides derived from bacteria [100]. It has been reported that none of the singlenucleotide polymorphisms (SNPs) in TLR1 are associated with a susceptibility to IBDs; however, a nonsynonymous variant in TLR1 (R80T) is linked to the pancolitis of UC [101]. The involvement of TLR2 in the recognition of components from a variety of microbial pathogens, such as peptidoglycan, lipopeptides, lipoteichoic acid, zymosan, and glycolipids, has been established [10]. TLR2 cooperates with TLR1 and TLR6, resulting in the ability to detect various microbial components. TLR2 constantly senses commensal microorganisms and regulates intestinal inflammation by controlling intestinal barrier functions [102 104]. In the inflamed colonic mucosa of IBD patients, TLR2 levels were higher than those in the controls in one study [105]. Additionally, polymorphisms in TLR2 are associated with the risk of developing IBDs [106]. TLR3 is involved in recognizing the dsRNA produced by viruses during their replication. TLR3 is constitutively expressed on the intestinal epithelial cells of healthy individuals [107]. However, in active CD, TLR3 expression was found to be highly downregulated in intestinal epithelial cells [107]. TLR3 is a potential mediator of CCL20 production, the levels of which can be used to predict the risk of IBD. In fact, TLR3 silencing is believed to have an effect on intestinal inflammation [108]. TLR3 also plays a critical role in the pathogenesis of radiationinduced intestinal inflammation and the

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subsequent gastrointestinal syndrome associated with p53-dependent crypt cell death [109], indicating that targeting it may offer a therapeutic strategy for reducing intestinal inflammation. TLR4 mainly recognizes the lipid A component of lipopolysaccharide (LPS) from Gramnegative bacteria. LPS is a crucial mediator of the AMPs produced by intestinal epithelial cells and Paneth cells [42]. Several lines of evidence show that TLR4 is involved in the pathogenesis of IBDs. TLR4 expression is increased in both CD and UC [107]. TLR4-deficient mice and Myd88-deficient mice are highly susceptible to dextran sodium sulfate (DSS)-induced intestinal inflammation [38]. SNPs in TLR4, such as A299G in IBD patients, are associated with IBDs, and they carry an increased risk of infection occurring with Gram-negative bacteria [110 113]. The T399I polymorphism in TLR4 has also been reported in UC patients [114]. TLR4 rs4986790A . G and rs4986791C . T genetic polymorphisms are also correlated with an increased risk of IBDs [115]. TLR5, which recognizes bacterial flagellin, is expressed on the basolateral surface of intestinal epithelial cells, indicating that it performs a crucial role in detecting invasive flagellated bacteria at the mucosal surface [116,117]. When exposed to flagellin, interleukin 8 (IL-8) was found to be produced by human intestinal epithelial cell lines, and this resulted in the induction of neutrophil and immature DC migration [118]. Intestinal epithelial cell TLR5 also maintains the intestinal microbiome by preventing intestinal inflammation [119]. However, in the intestinal lamina propria, TLR5 is expressed mainly on CD11c1 DCs [120]. Low-density cells in the small intestinal lamina propria can be divided into four subsets based on their CD11c and CD11b expression patterns: CD11chiCD11blo DCs, CD11chiCD11bhi DCs, CD11cintCD11bint macrophages, and CD11cintCD11bhi eosinophils [121,122]. Among them, the CD11chiCD11bhi DCs, which have also been characterized as

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FIGURE 6.4 The role of TLR5 in the small intestine. TLR5 is expressed on the basolateral surface of intestinal epithelial cells. TLR5 is highly expressed by CD11chiCD11bhi lamina propria DCs in the small intestine. These specialized DCs recognize invasive flagellated bacteria through TLR5 and induce the expansion of IgA1 plasma cells, Th1 cells, and Th17 cells after TLR5 stimulation.

CD1031 conventional DCs, specifically express TLR5, and contribute not only to the differentiation of naı¨ve B cells into immunoglobulin A positive (IgA1) plasma cells, but also to the differentiation of antigen-specific T helper type 1 (Th1) and Th17 cells in response to flagellin (Fig. 6.4) [121,122]. Interestingly, the polymorphisms in TLR5 are correlated with UC in North Indian patients, in whom they have been found to induce decreased levels of inflammatory cytokines [123]. Functional SNPs in TLR5 have also been seen to be involved in the response to anti-tumor necrosis factor therapy in IBD patients [124]. In common with TLR1, TLR6 has been shown to functionally cooperate with TLR2. TLR6 recognizes the diacyl lipopeptides derived from bacteria [125]. TLR6 levels in the inflamed colon were higher than those in the noninflamed colon in one study [126]. TLR6 controls Th1 and Th17 responses in gastrointestinal-associated lymphoid tissue, and TLR6-deficient mice are protected against DSS-induced intestinal inflammation [126].

TLR7 is another important innate immune response regulator. It recognizes singlestranded (ss) RNA and synthetic compounds such as imiquimod, a medicinal immune response modifier. Imiquimod administration can ameliorate DSS-induced intestinal inflammation [127]. TLR7 stimulation induces type I interferon and antimicrobial molecules, indicating a potential role for TLR7 as an agonistic therapy for IBDs. TLR8, the gene for which is located on the X chromosome, is involved in recognizing ssRNA and imidazoquinolines in humans. However, mouse TLR8 is known to be nonfunctional [10]. TLR8 expressed on regulatory T cells plays an important role in regulating immune responses to cancer [128]. Furthermore, TLR8 haplotypes are correlated with IBDs in females, where they have also been shown to be predisposing and protective factors for these diseases [129]. TLR9 mediates the recognition of bacterial DNA containing unmethylated CpG motifs (otherwise known as CpG DNA) [130]. Expression of TLR9 on the apical side of

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intestinal epithelial cells contributes to the maintenance of colonic homeostasis when activated by bacterial DNA. TLR9 has a protective function against intestinal inflammation via the induction of type I interferon [131]. Increased levels of it in memory B cells from patients with IBD are correlated with disease severity [132]. Administration of a Western-style diet altered the small intestinal mucosa in TLR9 knockout mice but not in TLR2-, TLR4-, or NOD2deficient mice, indicating that TLR9 signaling is crucial for regulating the small intestinal mucosa [133]. Despite thousands of reports about TLRs in the scientific literature, a role for TLR10 remains elusive. Very recently, it has been shown that TLR10 is expressed on human B cells and is involved in B cell activity [134]. TLR10 can also regulate the differentiation of primary human monocytes into DCs [135]. However, the association between TLR10 and intestinal inflammation remains unclear. TLR11 recognizes flagellin from Salmonella typhimurium [136]. Both TLR11 and TLR12 are receptors for profilin derived from Toxoplasma gondii [137]. TLR13 is associated with the recognition of bacterial 23S ribosomal RNA [137,138]. However, there are no reports that TLR11, TLR12, and TLR13 are associated with the pathogenesis of intestinal inflammation.

C. Association Between CLRs and Intestinal Inflammation CLRs are an emerging family of receptors that recognize various carbohydrate structures, such as mannose, fucose, sialic acid, and β-glucan [139]. CLRs, such as dectin-1, dectin-2, DC-specific intercellular adhesion molecule-3grabbing nonintegrin, and CLR-specific intracellular adhesion molecule-3-grabbing nonintegrin homolog-related 3 (SIGNR3), are expressed by myeloid cells, in particular by macrophages and dendritic cells [140]. CLR recognition of microorganisms on antigen-presenting cells leads to

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their internalization and subsequent antigen processing and presentation [141]. Previous studies have shown that CLRs are responsible for regulating intestinal inflammation. Dectin-1deficient mice are susceptible to DSS-induced intestinal inflammation with alteration of their commensal fungi [26]. In addition, a polymorphism in the human dectin-1 gene (CLEC7A) is associated with a severe form of UC [26]. Mice that are deficient in SIGNR1 are resistant to experimental colitis [142], and SIGNR3 is an important regulator of mucosal immunity against commensal fungi in the colon [143]. Macrophage-restricted C-type lectin and the dendritic cell immunoreceptor also contribute to the pathogenesis of DSS-induced intestinal inflammation [140].

D. Association Between RLRs and Intestinal Inflammation RLRs are cytoplasmic viral RNA receptors that recognize viral double-stranded (ds) RNA and trigger a signal that induces the innate immune response, whereas TLRs recognize only viral components at the cell surface or in endosomes [144 147]. Retinoic-acid-inducible gene I (RIG-I) and melanoma differentiationassociated gene 5 (MDA5) encode RLRs, and each protein encodes a caspase activation domain and a recruitment domain [148]. Because RIG-I recognizes comparatively short dsRNA and MDA5 recognizes long dsRNA, different RNA viruses are recognized by each of them [149 152]. While RIG-I is essential for immune responses against Sendai virus, influenza A virus, vesicular stomatitis virus, and hepatitis C virus, MDA5 is involved in the detection of encephalomyocarditis virus, poliovirus, and picornaviruses [144,147,153 155]. However, both RIG-I and MDA5 recognize West Nile virus and Japanese encephalitis virus [144,147]. RIG-I-deficient mice are highly susceptible to DSS-induced intestinal inflammation and have Peyer’s patches of decreased size

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and number, abnormal activation of peripheral T cells, and downregulated Gai2, which is one of a number of candidate genes for IBD [156]. In addition, RIG-I was found to be predominantly expressed on the apical side of intestinal epithelial cells, and RIG-I expression was downregulated in the intestinal tissues of patients with CD [157].

E. Association Between NLRs and Intestinal Inflammation That NLRs recognize cytoplasmic pathogens indicates that they are essential sensors for immune responses against intracellular bacteria. NLRs are multidomain proteins, and structurally and functionally, they represent the most diverse family among the PRRs [158]. Recently, it has been shown that NLR function is crucial for autophagy, antigen presentation, and embryonic development as well as for pattern recognition [159,160]. Among the NLRs, NOD1 and NOD2, which are regarded as noninflamasomeforming NLRs, are well characterized members of the NLR family. NOD2 dysfunction is strongly linked with susceptibility to CD, but NOD2 normally plays a pivotal role in regulating intestinal homeostasis [161]. Polymorphisms in NOD2, such as those encoding R702W, G908R, and L1007insC, are widely known to be genetic risk factors for CD [22,162,163], but the common NOD2 variants found in Western individuals are not correlated with predisposition to CD in the Japanese [164]. Nevertheless, it is obvious that NOD2 is involved in the pathogenesis of CD. However, interestingly, NOD2deficient mice and NOD1-deficient mice do not develop spontaneous intestinal inflammation [165,166]. These findings indicate that more scientific and clinical knowledge is needed to determine the pathophysiological role of NOD2 in CD. In contrast, NLRP3, which is one of the inflammasome-forming NLRs, is also a wellknown NLR. In humans, polymorphisms in NLRP3 are correlated with increased

susceptibility to CD [167]. NLRP3-deficient mice show severe intestinal inflammation in experimental colitis models, with dysregulation of IL1β and/or IL-18 production [168,169].

IV. CONCLUDING REMARKS The mucosal surface is the main bodily interface between the host and external antigens such as microorganisms. In the intestines, the barrier system plays a fundamental role in mucosal protection from antigens. In this organ, the monolayer epithelial cells form physical and chemical barriers consisting of nonstructural components such as the AMPs produced by Paneth cells and the mucus produced by goblet cells. Both barriers provide protection against enteropathogenic microorganisms. Intestinal epithelial cell dysfunction leads to disruption of the mucosal barrier in the gut and subsequent induction of intestinal disorders. On the other hand, a series of cells relevant to innate immunity constitutively express gene-encoded PRRs, including TLRs, CLRs, RLRs, and NLRs. When the mucosal surface is exposed to pathogens, the innate immune responses mediated by the PRRs provide key functions that regulate mucosal homeostasis. To date, a large number of molecules involved in mucosal innate immunity have been identified, and defective innate immune functioning in the gut is thought to contribute to intestinal inflammation. However, the need to elucidate the mechanisms of pathophysiology in human disorders with the goal of developing new therapeutic agents remains because a large number of patients with intractable diseases have been waiting for them.

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