Dentritic cells (DC) reside in an immature state in most tissues and organs and are critical in detecting invading pathogens (21). Dentritic cells can be characterized as either immature or mature. Immature DCs have a high endocytotic capacity and low expression of MHC and costimulatory molecules on their surface, and mature DCs have a high endocytotic capacity and an enhanced surface expression of costimulatory molecules. In addition, mature DCs have the capacity to secrete cytokines and chemokines. The function of dentritic cells is not primarily to destroy pathogens but to present pathogen-associated antigens to T cells thereby activating the adaptive immune response (19). Activation and maturation of immature dentritic cells is triggered by microbial pathogens. Upon activation, the DC expresses pathogen peptides with MHC on its surface and traffics from the site of infection to regional lymph nodes where it produces T cell activation. The interaction of TLRs and PAMPs is essential to dentritic cell activation and maturation (3). TLR-mediated recognition of pathogen-associated molecular patterns and induction of co-stimulation of DCs is required to direct T cell responses against pathogen-derived antigens in a cognate interaction between T cells and APC cells. Production of IL-6 by DCs in response to TLR ligation during infection is critical for T cell activation because it allows pathogen-specific T cells to overcome the suppressive effect of CD4+CD25+ regulatory T cells (22). Therefore, TLRs are important in both the innate immune response of pathogen pattern recognition and in the activation of the adaptive immune response. A recent observation has suggested that submucosal immature dendritic cells can extend an appendage through enterocyte tight junctions and sample pathogens within the lumen. This interaction mediated by TLRs results in maturation of the dendritic cell, antigen presentation and release of cytokines, which determines the T-helper cell phenotype (either Th1 or Th2) (Fig. 3).
The development of immune tolerance to innocuous foreign antigens and luminal commensal microorganisms crossing the mucosal surface is a critical protective mechanism of mucosal defense. This response appears to be mediated by TLRs. Naturally occurring mutations have been identified in the TLR4 gene in C3H/HeJ and C57BL/10ScCr mouse strains (23,24). Complete deletion of TLR4 function results in increased susceptibility to Gram-negative infections. The genetic variations or polymorphisms associated with TLR4 are important in the variations in susceptibility to sepsis across and within species (25–27). TLR4 mutation renders C3H/HeJ mice hyperresponsive to oral peanut antigen with impaired immune tolerance in a murine peanut allergy model (28). In human asthma patients, missense mutations in the extracellular domain of the TLR4 result in a blunted airway response to inhaled LPS (29). Polymorphisms in TLR2 may be associated with diminished in vitro responses to bacterial lipoprotein, a known TLR2 ligand (30). These mutations, if identified in patients, might help explain the increased incidence of allergic conditions or certain infectious disease over the last several decades. Additional research on TLRs and similar receptors may help to define susceptibility to clinical disease and design preventive therapy.
Although genetic factors have long been thought to play a role in the pathogenesis of Crohn's disease, there had been no confirmation of this consideration until the recent identification of NOD2/CARD15, an intracellular receptor for bacterial endotoxin (lipopolysaccaride, LPS). Using a positional cloning strategy, the nucleotide oligomerization domain 2 (NOD2) gene was identified on the human chromosome 16q12 within the area of strongest linkage to Crohn's disease (31). NOD2, recently renamed as caspase activating recruitment domain (CARD) 15, belongs to a family of NOD1/apoptotic protease activating factors (Apaf-1) proteins that regulate apoptosis. Some of them also activate nuclear factor κB (NFκB) (32,33). Structurally, NOD2/CARD15 encodes a cytoplasmic protein with at least three domains: two amino terminal CARDs linked to a nucleotide-binding domain (NBD) and a carboxy terminus component containing leucine rich repeats (LRR) (Fig. 4). This LRR domain can confer responsiveness to bacterial products (e.g., microbial molecular patterns such as LPS, flagellin, etc.) (Table 1).
NOD2/CARD15 was initially thought to be involved in the lipopolysaccharide (LPS) response pathway (34). A recent study suggests, however, that bacterial peptidoglycan, a component of both Gram negative and positive bacteria cell walls, is the main pathogen associated molecular pattern that induces NFκB activation through CARD15 (35). NOD2/CARD15 acts as an antibacterial factor in Caco-2 intestinal epithelial cells by preventing invasion of Salmonella typhimurium, whereas mutant forms of CARD15 lose the protective effect (36). Initially, NOD2/CARD15 expression was considered to primarily exist in monocytes (33), but recent data have shown that its expression can be identified in both dentritic cells and intestinal epithelial cells (37,38). In analyses of ileal and colonic tissues from IBD patients, CARD15 expression was found to be localized to Paneth cells within the ileum or in metaplastic Paneth cells within the colon (39,40). NOD2/CARD15 expression is enriched in crypts compared with villi. Cells expressing NOD2/CARD15 also strongly express and secrete TNF-α, a potent stimulus to induced NOD2/CARD15 expression (40). It has also been suggested that NOD2/CARD15 may be involved in the degranulation of Paneth cells, an important innate immune response in the intestinal lumen (41).
More than 60 sequence variants have been identified in the NOD2/CARD15 gene. Three main variants (R702W, G908R, and 1007fs) seem to be associated with susceptibility to Crohn's disease (31,42–46) (Fig. 5). The majority of Crohn's disease associated mutations directly affect the LRR domain, which is a motif common to bacterial resistance R protein in plants and mammals, notably the TLR family which enables recognition of PAMPs. These data suggest that a defect in the host response to peptidoglycan may have some role in Crohn's disease susceptibility. It is possible that mutations in the LRR domain affect its sensing function to intraluminal bacteria leading to aberrant activation of the NFκB/Rel pathways and the production of inflammatory cytokines. In Crohn's disease, mutations of the NOD2/CARD15 gene are most tightly associated with ileal involvement, young age of onset, more extensive granuloma formation and perhaps also with fistulizing and/or fibrostenotic phenotypes (46–48). Inheritance of Crohn's disease occurring as a result of NOD2/CARD15 polymorphisms is manifested somewhere between a gene dosage and an autosomal recessive phenotypic disease expression (49). Heterozygotes have a relatively small increase in risk (of the order of that of smokers) of developing Crohn's disease, whereas homozygotes and compound heterozygotes have a much greater relative risk (49). In a recent study of CARD15 mutations in 55 pediatric patients with Crohn's disease in Saxony, Germany (50), 14 different polymorphic and/or disease-related nucleotide alterations were identified and 65% of the genomic DNA samples harbored at least one of 6 mutations in the CARD15 gene. The cytosine insertion mutation 3020insC was significantly more common in children with Crohn's disease than healthy controls (36% vs. 4%). Patients with at lease one of the 6 CARD 15 disease-associated mutations demonstrated a high risk of inflammation located in the terminal ileum and ascending colon, and in 10 of 19 patients with two mutations, intestinal resection was necessary because of stricturing (50). The genotype-phenotype correlation suggests that it may be possible to predict the outcome in a child with Crohn's disease based on the CARD 15 mutation(s) carried by the patient. Analysis of disease-associated CARD 15 mutations may facilitate more prompt and accurate diagnosis in children with equivocal clinical presentations. Finally, in high risk populations, it may be helpful to identify children carrying the mutations even before the onset of clinical manifestations of disease and especially if new therapeutic interventions become available to prevent the occurrence of Crohn's disease.
Antimicrobial peptides are a group of host defense effector molecules in the innate immune system. They can be divided into general categories: (1) peptides released into internal fluids such as lymph; (2) peptides localized within circulating phagocytic cells and (3) peptides released onto mucosal surfaces (51). These antimicrobial peptides generally function by disrupting the bacterial cell membrane. Interaction of positively charged (cationic) antimicrobial peptides with negatively charged (anionic) elements of the bacterial membrane (including LPS in gram-negative bacteria, polysaccharides such as teichoic acid in gram-positive bacteria and phospholipids such as phosphotidyl-glycerol) results in insertion of these peptides into the membrane and creation of membrane pores. These pores then lead to disruption of energy and ionic gradients across the membrane with subsequent cell lysis (52,53).
Paneth cells are specialized epithelial cells located in clusters at the base of crypts of Lieberkühn in the small intestine and in close proximity to epithelial stem cells (54). Paneth cells are most numerous in the terminal ileum and are recognized by unusually large apical eosinophilic secretory granules. These secretory granules are rich in antibiotic peptides, including lysozyme (55), secretory phospholipase A2 (56,57), α-defensins (58–60), trypsin (54) and angiogenins (61). Paneth cells release secretory granules into the crypt lumen when stimulated by cholinergic agonists, such as pilocarpine and nethanochol (62,63) and bacterial stimuli (60,64). The anatomically close proximity of Paneth cells to epithelial stem cells in the crypts led to an initial hypothesis that Paneth cells protect stem cells by releasing antimicrobial peptides to destroy luminal pathogens which might injure the stem cell. In a recent study, NOD2/CARD15 expression was noted in the Paneth cell in close proximity to its secretory granules (39). This close proximity prompted speculation that NOD2/CARD15 might be involved in degranulation and peptide release by Paneth cells in response to bacterial molecular patterns. Therefore, Paneth cells are critical contributors to innate immune defense in the intestine (65).
Defensins are a family of evolutionarily related vertebrate antimicrobial peptides (3–4 kDa, positively charged, arginine-rich) with a characteristic β-sheet-rich fold and a framework of six disulfide-linked cysteines forming three disulfide bridges (52). There are two main defensin subfamilies, α- and β-defensins, which differ in the length of peptide segments between the six cysteines and the pairing of the cysteines connected by disulphide bonds (52). Defensins are encoded by single genes as pre-propeptides that are then processed to their active forms by enzymetic cleavage. α-defensins consist of 29 to 35 amino acid residues and are shorter than β-defensins which consist of 38 to 42 residues. Defensins are abundant in cells and tissues involved in innate host defense against microbial infections, including leukocytes, macrophages and intestinal Paneth cells (Table 2). α-defensins are produced and stored as pre-propeptides in mature neutrophils and Paneth cells whereas β-defensins are constitutively expressed in the epithelial compartment and can be induced to higher levels of expression upon infection or inflammation. Therefore, Paneth cell α-defensins and epithelial cell β-defensins exert their antibacterial effect in the intestinal lumen whereas the neutrophil α-defensins serve as an intracellular antimicrobial peptide.
α-defensins are abundant constituents of mouse and human Paneth cell granules. Human Paneth cells have two α-defensin peptides, human defensin 5 and 6, whereas mice express numerous Paneth cell α-defensin isoforms termed “cryptdins” (66). Human defensin 5 is stored in Paneth cell granules in its precursor form. The process of activation to its mature form likely occurs in the crypt lumen upon granule secretion. Trypsin released from Paneth cells after microbial or inflammatory stimuli acts as a critical prodefensin convertase that mediates the conversion of precursor human defensin 5 into its mature form (67). Defensin gene expression is developmentally regulated and exhibits species-specific tissue distributions. For example, human α-defensins are found in neutrophils and Paneth cells but not in macrophages, whereas rabbit α-defensins are expressed in both neutrophils and macrophages and mouse α-defensins are present in Paneth cells and testes (1). Human α-defensin (HD) 5 and 6 are expressed in the developing fetus as early as the 13th week of gestation (detected by RT-PCR) with HD-5 present in both the small intestine and colon and HD-6 present only in the small intestine (68). By week 17 of gestation, both HD-5 and HD-6 expression is limited to the small intestine. By week 24, enteric defensin expression reaches levels detectable by Northern blot mRNA analysis. In the late second trimester of gestation, both the number of Paneth cells and the level of enteric α-defensin mRNA are significantly lower than in the adult. In contrast, mouse α-defensins of the small intestine (cryptdins) are present at low levels before birth but rapidly increase during weaning (69–71).
Paneth cell α-defensins have anti-microbicidal activity against a variety of microbes. Recombinant Paneth cell human defensin 5 is active against Listeria monocytogenes, Escherichia coli, Salmonella typhimurium, and Candida albicans (72). A developmental profile of the expression of human enteric defensins 5 and 6 demonstrated that enteric defensin mRNAs were expressed at extremely low levels during fetal life compared with term newborns and adults (68). This low level expression may contribute to the immaturity of intestinal innate defense and predispose to the development of necrotizing enterocolitis (NEC). A study of small intestinal samples from infants with NEC demonstrated that human defensin 5 and 6 mRNA was increased threefold in the NEC infant as compared with age matched controls suggesting a possible involvement of defensins in the pathogenesis of NEC (73). Enhanced expression of human defensins 5 and 6 has also been found in colonic specimens from patients with inflammatory bowel disease suggesting a role of human enteric defensins in controlling microbial invasion of the colon in IBD. The expression of enteric defensins may also be increased by inflammation or inflammatory cytokines (74).
In animal studies, mice with a homozygous disruption of the metalloproteinase matrilysin (MMP7) enzyme failed to hydrolyse intestinal prodefensins to defensins and were more susceptible to infection with Salmonella typhimurium. Mice with the mutation required an eightfold lower oral innoculum for a 50% mortality (75). It also took a longer time for these mice to clear infection with an enteropathogenic strain of Escherichia coli (75). Transgenic expression of human defensin 5 in mice at physiologic levels protects them against lethal intestinal infection with Salmonella tryphymurium (76). Taken together, these animal studies provide crucial evidence for the protective role of defensins in enteric infections.
Increasing evidence has also underscored the role of defensins in the activation and recruitment of cells important in the adaptive immune response. Human α-defensins are chemotactic for monocytes, dendritic cells and T lymphocytes at concentrations 10- and 100-fold below those required to produce direct bactericidal damage (77). Human β-defensins have chemotactic activity for immature dentritic cells and memory T cells through the chemokine receptor CCR6 (78). Human α-defensins enhance the expression of TNF-α and IL-1β in activated human monocytes and reduce the expression of VCAM-1 in human endothelial cells activated by TNF-α (79). Collectively, these data indicate that human defensins are not only critical in innate host immune defense, but also are an active participant in regulating adaptive immunity.
Scavenger receptors are a group of functionally related but structurally unrelated molecules expressed by myeloid cells (macrophages and dentritic cells) and certain endothelial cells. They play an important role in the uptake and clearance of effete components such as modified host molecules and apoptotic cells (80). They recognize bacterial molecular patterns such as gram-positive bacteria (lipoteichoic acid) and bacterial CpG DNA and thus serve as pattern recognition receptors similar to TLRs and CARD 15/NOD2 (80).
The gene for DMBT 1 (human deleted in malignant brain tumor) encodes a large cysteine rich B protein scavenger receptor (81). It was initially thought to be a possible tumor suppressor gene because of the high frequency of its homozygous deletion and the lack of expression in a variety of cancers. Identification of two mucosal defense-related molecules, gp-340 and salivary agglutinin (alternatively spliced products of DMBT1) suggests that DMBT1 is likely to be another pattern recognition receptor in the innate immune system (82–84).
The DMBT 1 gene is expressed in the crypts of the small intestine and the neck region of human gastric mucosa (85–87). Through the cysteine rich B domain, the DMBT1 product binds to a variety of bacteria including Streptococcus mutans, Escherichia coli, Lactobacillus casei, Helicobacter pylori and Prevotella intermedia (88). In the gastrointestinal tract, DMBT1 is secreted from the apical membrane of epithelia onto the mucosal surface where binding to invading pathogens such as S. mutans and H. pylori (83), and host defense components such as surfactant protein (SP)-D (89) and secretory IgA (90) can occur. All these ligands are involved in local inflammation in gastric mucosa (91). Studies on airway epithelia have demonstrated that DMBT1 expression is upregulated by stimulation with IL-8 and IL-6, known inflammatory mediators, suggesting that DMBT 1 plays a role in local epithelial inflammatory responses (92). Furthermore, gp-340, an alternatively spliced form of DMBT1, has stimulatory effects on alveolar macrophage chemokinesis suggesting that it directly regulates tissue macrophage protective function (93). Data suggest that DMBT1 acts as a typical pattern recognition receptor in the innate anti-microbial defense system through its interaction with invading pathogens and other local defense components (94). Because of the extensive expression of DMBT1 transcripts in immune organ systems, including spleen, thymus, alveolar macrophages and mucosal T and B cell lines, it has been suggested that DMBT1 may also be involved in the regulation of the adaptive immune response (82,85,95,96). Detected by immunostaining, enhanced DMBT1 expression has been shown in crypt cells of the small intestine and along the neck region of normal human gastric mucosa where intestinal epithelial stem cells are located, implying a possible role in the physiological renewing process of gastrointestinal epithelia.
Loss or reduction in the expression or inactivation of DMBT1 may be involved in tumor formation in the central nervous system, gastrointestinal tract and lung (81,97–99). In vitro studies have demonstrated inflammation-related upregulation of DMBT1 in airway epithelia and differentiation-related downregulation in gastric epithelia (87). The complex interactions of DMBT1 with microbial pathogens, mucosa defense components and possibly trefoil proteins suggest that DMBT1 may serve as a unique, integral factor promoting the regeneration of mucosal epithelia following damage due to pathogen invasion and/or local inflammation (94). Also, impaired gastrointestinal healing after infection and/or inflammation may result from loss of function of the DMBT1 receptor because of its potential role in epithelial regeneration, a common defense mechanism in the innate host defense against microbial invasion. Therefore, it is possible that loss of DMBT1 could potentially lead to impaired mucosal defense against microbial pathogens and a dysregulated adaptive mucosal immune response.
The innate immune system in the gut is comprised of a complex cellular network, multiple pattern recognition receptors and many biologically active secretory peptides or proteins. It plays a critical role in the first line host immune defense provided by the intestinal mucosa. The innate immune system not only serves as the initial step in the ability of the intestinal mucosa to maintain homeostasis but also serves as an important bridge to the activation and regulation of adaptive immune responses. Dysfunction of the innate immune response of the intestinal mucosa may lead to devastating consequences for the host caused by invasion of microbial pathogens, impaired epithelial regeneration and dysreguation of mucosa inflammatory responses, which are thought to play a role in the pathogenesis of inflammatory bowel disease, NEC, enteric microbial infection and malignances.
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