Antimicrobial
Size (kDa)
Antibacterial activity
Cellular source
α-Defensin 1–4
3.4
Gm+ and Gm−
Neutrophils
α-Defensin 5
3.6
Gm+ and Gm−
Paneth cells
α-Defensin 6
3.7
Self-assembles
Forms protective nets
Paneth cells
β-Defensin 1–3
4
Gm+ and Gm−
Colonocytes
Lysozyme
14
Gm+ > Gm−
Paneth cells, mØ
sPLA2
16
Gm+ > Gm−
Paneth cells, colonocytes, mØ
Reg3A/HIP/PAP
16
Lectin, Gm+
Paneth cells, enterocytes
Lipocalin 2
24
Selectively bacteriostatic
(Sequesters chelated iron)
Colonocytes
Defensins are a major family of antimicrobial peptide of mammals [71–75], and in vivo models have provided compelling support for their innate immune functions in the intestinal tract [64, 76–78]. Characteristically, defensins are 18–40 amino acids in length and contain three intramolecular disulfide bonds [71, 72]. In the course of evolution, the genes encoding defensins have undergone duplication and diversity, accompanied by relatively rapid changes in primary sequence attributed to host’s interactions with microbes in the internal and external environment [79]. Together these sequence changes have resulted in much diversity in the defensin family between species. However, a conserved sequence feature is the alignment of the six cysteines that participate in the three-disulfide bonds, which helps define the two subfamilies of defensins expressed in humans. One of these subfamilies, α(alpha)-defensins , is expressed most highly in neutrophils and in Paneth cells. In both of these cell types, α(alpha)-defensin expression is constitutive and part of the cellular differentiation of these cells as they mature. While this expression pattern is observed in marsupials, glires (including rats) and some other mammals, mice are a striking exception, where defensins are expressed in Paneth cells (where they are called “cryptdins ,” to emphasize crypt defensins), but not neutrophils [71, 73]. Paneth cells granules contain proteolytic enzymes that mediate processing of α(alpha)-defensin precursors [76, 80]. In humans, Paneth cell trypsin is the processing enzyme of pro-α(alpha)-defensins , while in mice matrix metalloproteinase (MMP)-7 , also called matrilysin, serves this function [76, 80]. The β(beta)-defensin sub-family is expressed in many epithelial cells, including colonocytes. In the intestine, epithelial expression of β(beta)-defensin is inducible, except for the apparent constitutive expression of HBD-1. In summary, in the human intestine, α(alpha)-defensins are highly expressed in Paneth cells of the small intestine , and β(beta)-defensins are expressed in the colon. At both locations, infiltrating neutrophils also carry an abundant stockpile of α(alpha)-defensins.
Both α(alpha)- and β(beta)-defensins typically have bactericidal activity against both Gm− and Gm+ bacteria. Some defensins also have activity against fungi, viruses, and protozoa [72, 81]. Many defensins kill their target microbes by disrupting membrane integrity, but recent studies find that some defensin peptides interfere specifically with lipid II function and thereby block bacterial wall biosynthesis [82–84]. Curiously, while Paneth cell HD5 has potent bactericidal activity, the second Paneth cell α(alpha)-defensin , HD6 , does not have cidal activity against bacteria [78, 85] (at least while its tri-disulfide bonds are intact [86]). Nevertheless, HD6 has significant mucosal protective function via a novel mechanism. HD6 can block the ability of enteric bacterial pathogens to invade cultured epithelial cells in vitro [78, 87], and HD6 expressed in Paneth cells at physiological levels can protect mice from oral challenge by S. typhimurium in vivo [78]. The protection was attributable to unique binding and self-association properties of HD6, resulting in formation of nanofibrils and nanonets, which can surround and entangle the targeted microbes. This unique mechanism helps explain the sequence conservation of HD6 throughout primate evolution and suggests a key role for HD6 in protecting the human small intestine against invasion by diverse enteric pathogens [78].
Besides their antimicrobial activities, some defensins have additional activities that suggest their capacity to help coordinate host defense responses in the intestine [72, 73]. For example, human β(beta)-defensin -1 and -2 have chemoattractant activity for cells expressing the chemokine receptor CCR-6, including dendritic cells [88, 89]. Human α(alpha)-defensin 5 is a potent lectin [90] and can neutralize bacterial exotoxins [90]. Certain other α(alpha)-defensins promote ion fluxes in epithelial cells [91, 92]. These and other activities of intestinal defensins suggest a broad role where these peptides likely contribute in several ways to innate immunity.
Lysozyme is an enzyme that specifically hydrolyzes peptidoglycan. This enzyme is very abundant in the surface-lining fluid of many organs, including the intestine where it is chiefly made by Paneth cells. In addition, lysozyme is abundant in macrophages and neutrophils. Its high concentration in these cells and at mucosal surfaces supports that lysozyme has a substantial role as an antimicrobial agent in vivo, even though specific activity in vitro is modest. In addition to its role as an antimicrobial agent, analysis of knockout mice points to another key in vivo role for lysozyme—an ability to impede peptidoglycan accumulation in tissue. By enzymatic degradation of peptidoglycan, lysozyme activity appears to help avoid prolonged inflammatory responses that otherwise would result from persistence of bacterial cell wall antigens [93]. Thus, whether eliminating peptidoglycan from crypts and intestinal tissue, and/or antibacterial activity against lumenal microbes is the chief role for intestinal lysozyme remains an open question.
Phosholipase A2 enzymes are a large family of catalytic molecules that hydrolyze the fatty acid ester bond at position sn-2 of membrane phosphotriglycerides. Paneth cells granules contain abundant quantities of one specific member of this family that has selective activity on bacterial membranes [94, 95]. Colonic epithelial cells also express this enzyme, which is named group IIA secretory phospholipase A2 (PLA2G2A, or simply sPLA2 ) [96]. Like lysozyme , sPLA2 is not only expressed in epithelial cells, but also in macrophages [95]. Bacterial membranes , rich in phosphatidylglycerol and phosphatidylethanolamine, are the key targets of sPLA2 , but the enzyme can cleave other phosphotriglyceride substrates [95, 97]. The enzyme is bactericidal, with preferential activity against Gm+ bacteria [97, 98]. Interestingly, genetic evidence found that the gene encoding sPLA2 is a key modifier gene in mouse lines that spontaneously form intestinal adenomas and tumors [99, 100], but the mechanism leading to increased neoplastic growth is unknown [101].
An interesting C-type lectin with a single carbohydrate recognition domain is expressed in the human intestine, named REG3A (also called hepatocarcinoma-intestine-pancreas (HIP) or pancreatitis-associated protein (PAP ) ). Like its mouse ortholog, Reg3γ(gamma) , this lectin binds peptidoglycan and is bactericidal against Gm+ bacteria [102–105]. This lectin is abundantly expressed in Paneth cells and enterocytes [102, 106, 107]. Another lectin abundantly expressed in Paneth cells (although without direct antimicrobial activity) is intelectin-1 [108, 109], which is encoded by INTL1, a gene that was identified in genetic screens for IBD-susceptibility loci [110].
Lipocalin-2 binds bacterial siderophores, which are the catechol-related iron chelators secreted by bacteria to acquire iron from their environment. Sequestering these iron chelators prevents this mode of bacterial iron acquisition and inhibits bacterial growth [111]. Many cells express lipocalin-2 inducibly, including liver, macrophages and epithelial cells of the lung and intestine [68, 112, 113]. In the intestine, lipocalin-2 may selectively inhibit growth of siderocalin-dependent bacteria at the mucosal surface.
Mouse Paneth cells express certain antimicrobial peptides that have not been identified in humans, including a group of peptides called cryptdin-related sequences (CRS) [114, 115]. The genes encoding CRS peptides are homologs of α(alpha)-defensins [116]. The CRS peptides are highly expressed, cationic and cysteine-rich, like α(alpha)-defensins, and they have similar antimicrobial activity, but differ in many structural features [73]. Angiogenin-4 is another antimicrobial peptide that does not have a clear human ortholog. Originally identified as a Paneth cell product induced when germ-free mice were colonized [117], angiogenin-4 belongs to a subfamily of ribonuclease enzymes that have antibacterial and antiviral activities [118].
Before leaving the topic of intestinal antimicrobial peptides, it must be mentioned that the host is not the sole source of antimicrobial peptides in the intestinal lumen. While antimicrobial peptides in the intestinal tract are chiefly derived from the host epithelium, some Gm+ and Gm− bacteria also secrete ribosome-synthesized antimicrobial peptides called bacteriocins [119–123]. Bacteria seem to use these peptides to establish or protect an environmental niche. The bacterial strains producing bacteriocins will affect, like their host-derived counterparts, the composition of complex bacterial communities, adding considerable complexity to the dynamics of the intestinal microbiota [119, 121–123].
Paneth Cells , An Antimicrobial Peptide Factory
Paneth cells are epithelial cells with intensive secretory activity. Their large secretory granules contain massive concentrations of antimicrobial peptides [26, 124–126]. The cells reside in small clusters at the base of the small intestinal crypts. Although difficult to maintain in tissue culture with conventional approaches, new “organoid” models hold promise for facilitating in vitro studies of Paneth cell function [127, 128]. In the crypt, Paneth cells discharge their granules into the crypt lumen, and their contents diffuse from the crypt to target microbes at the intestinal surface and in the lumen, including both resident and newly acquired microbes. In so doing, Paneth cells antimicrobials can maintain host sovereignty at its surface interface with the intestinal lumen, provide protection from food and water-borne pathogens and help shape the composition of the endogenous microbiota (Fig. 5.1) [26, 126]. In vivo studies in mouse models have provided strong support for these proposed antimicrobial functions [64, 76–78, 104, 129, 130]. The biological activities of Paneth cell antimicrobial peptides may be evident not only in the small intestine, but also in the colon, as lumenal contents transit via peristalsis [131].
Fig. 5.1
Proposed roles for enteric antimicrobial peptides. The two principal roles for antimicrobial peptides in the intestine, such as Paneth cell α-defensins, are to protect the host from ingested pathogens and to help shape the composition of the colonizing microbiota. Depicted here in cartoon form is the release of secretory granules, chock-full of antimicrobial peptides, from Paneth cells in the crypts of the small intestine
A direct role of Paneth cell antimicrobials in regulating the small intestinal microbiome was investigated using complementary mouse models [64]. In α(alpha)-defensin-deficient mice (via MMP7 gene knockout, encoding the processing enzyme for α(alpha)-defensins ) , there was decreased abundance of Bacteroidetes and increased Firmicutes, as compared to wild-type littermate controls. In HD5 transgenic mice, the abundance of Bacteroides species increased and the Firmicutes dropped inversely. Thus, Paneth cell α(alpha)-defensin expression had significant impact on the colonizing microbiota under baseline conditions. In addition to these shifts in the dominant phyla of the small intestinal microbiota, transgenic expression of HD5 also eliminated colonization by Candidatus Arthromitus , also referred to as segmented filamentous bacteria (SFB) [64]. SFB is a bacterium that directly contacts the surface of the small intestinal epithelium. The loss of SFB in the HD5 transgenic mice resulted in a significant decrease of Th17 cells in the small intestinal lamina propria, highlighting that Paneth cell antimicrobials can indirectly affect lymphocyte populations in intestinal tissues [64]. Interestingly, a converse SFB increase was reported in a mouse model that had marked Paneth cell dysfunction [132]. Together these data support that Paneth cell antimicrobials have an important homeostatic role in regulating the small intestinal microbiome. Furthermore, in their capacity to regulate the composition of the microbiome, Paneth cell antimicrobials can indirectly alter the abundance of subsets of lymphocytes in the lamina propria. Taken together, it seems clear that genetic or acquired abnormalities in Paneth cell function may have significant impact on host–microbe homeostasis.
While antimicrobial peptides are the most abundant component of the secretory granules, other secretory peptides and proteins impart additional biological roles for Paneth cells [124, 126]. Many molecules that have likely roles in innate immunity, or bridge between innate and adaptive immunity, are expressed in Paneth cells [124, 125, 133–136]. For example, Paneth cells express NOD2 , the gene encoding nuclear oligomerization domain 2 protein, an intracellular receptor for muramyl dipeptide of bacterial cell walls [133]. New data reveals that Paneth cells also express several molecules that serves as ligands for the adjacent intestinal stem cells, providing them with vital trophic factors [137]. It is likely that these cells have a much more fundamental and sophisticated role in intestinal homeostasis than currently acknowledged [126], although caveats have been highlighted in experimental approaches to study these intriguing cells [138].
Paneth Cells and Crohn’s Disease
CD is often involves the distal portions of the small intestine where Paneth cells are abundant [1, 139, 140]. CD is associated with dysbiosis and abnormal bacterial adherence to the intestinal mucosal surface [5], consistent with impaired innate antimicrobial defenses. The identification of several CD susceptibility genes has placed a focus of CD pathogenesis squarely on Paneth cells and their antimicrobial products [140]. Indeed, Paneth cell structural abnormalities detected by routine histology are associated with inherited CD-susceptibility alleles [141–143]. Although the risk-alleles in these susceptibility genes are found in only a subset of patients with CD, and all of these genes are expressed in other cells in the body, a compelling thread connects them to suggest that Paneth cell dysfunction [144] leads to an antimicrobial peptide deficiency [107], which underlies disease pathogenesis in predisposed hosts (Fig. 5.2). The name “Paneth Disease ” has been suggested to describe CD of the ileum [140].
Fig. 5.2
Genetic associations implicating Paneth cells in Crohn’s disease pathogenesis . Paneth cells are quintessential secretory cells of the small intestinal epithelium. Their functions contribute to intestinal homeostasis. Several genetic susceptibility factors for IBD may have their phenotypic underpinnings in Paneth cell dysfunction. These include KCNN4 (encoding KCa3.1 ) [145], NOD2 [146, 147], ATG16L1 [148], XBP1 [149], IRGM [150], TCF7L2 (encoding TCF7L2, formerly TCF4) [151], and LRP6 [152]
Wehkamp and colleagues found reduced Paneth cell α(alpha)-defensin expression in ileal CD compared to controls [107, 153]. Similar reduced expression of defensins was not observed in either UC or CD limited to the colon (L2) [107]. The decrease in Paneth cell α(alpha)-defensin was independent of intestinal inflammation [107]. This suggests that reduced α(alpha)-defensin expression is not the result of inflammation, but rather an intrinsic and early event in the pathophysiology of CD. Individuals with L1007fs (SNP13) NOD2 mutations, have especially low α(alpha)-defensin levels, which are significantly lower than others with ileal CD but without this specific genetic variant [107]. This specific genetic polymorphism in NOD2 is also linked to increased disease severity and more significant ileal involvement [2, 154, 155], Additional studies show that deficiency of TCF7L2 (formerly TCF4) is associated with ileal CD [156]. TCF7L2 is an integral transcription factor that drives both Paneth cell differentiation and α(alpha)-defensin expression [157]. The decrease in TCF7L2 expression was not dependent on an abnormal NOD2 genotype, and was independent of the degree of tissue inflammation, again suggesting that α(alpha)-defensin-deficiency may be a common inciting event in CD pathogenesis. A genetic association of TCF7L2 with ileal CD provides evidence that a decrease in Paneth cell α(alpha)-defensins is a primary factor in disease pathogenesis for some individuals [151]. Despite the aggregate of supportive data, the role of reduced α(alpha)-defensin expression is a subject of some residual controversy [158, 159].
NOD2 was the first susceptibility gene identified for IBD and is especially linked to CD [146, 147]. Epithelial expression of NOD2 in the small intestine is restricted to Paneth cells [160]. Nod2 −/− mice have reduced Paneth cell α(alpha)-defensin expression [133] and are deficient in immune response to enteric pathogens, including Listeria monocytogenes [133] and Helicobacter hepaticus [38]. Other investigations of Nod2 −/− mice have found conflicting results [161]. Interestingly, Nod2 −/− mice develop granulomatous lesions in the ileal mucosa when challenged with H. hepaticus, a phenotype that is rescued by transgenic expression of HD5 in the mouse Paneth cells [38]. In addition, Nod2 −/− mice have alterations in their small intestinal microbial microbiota [162].
Other CD susceptibility genes are associated with dysfunction of Paneth cell secretory pathways, rather than antimicrobial expression. One such susceptibility gene is ATG16L1 , which is homologous to the yeast autophagy gene ATG16 [148]. Autophagy is an essential cellular process for renewal and homeostasis, whereby organelles and other components (including secretory granules) are recycled following targeting to lysosomes for degradation [163, 164]. Mouse strains with reduced expression of the gene encoding Atg16l1 resulted in abnormalities in Paneth cell granule form and function [144, 165–167]. CD patients with the ATG16L1 (T300A) mutation have similar abnormalities of their Paneth cell granules [143, 165, 168], and have both alterations in their microbiota [169] and increases in mucosal-adherent E. coli [168]. The mouse phenotype in Atg16l1 hypomorphic mice is triggered by viral infection [170]. Interestingly, viral triggers have been implicated in human IBD [171].
Studies have linked CD to polymorphisms in another gene encoding a protein involved in autophagy, the immune-related GTPase M (IRGM) [150, 172, 173]. Irgm1 k/o mice are susceptible to ileal inflammation upon ingestion of dextran sodium sulfate [174]. Abnormalities in Paneth cells of Irgm1 k/o mice, including abnormalities in Paneth cell granule size and histology, support the purported perturbations in autophagy processes [174]. In patients with CD, there was also evidence of autophagy in Paneth cells with or without disease-associated variants of IRGM and ATG16L1 , where a significant decrease in number and morphology of secretory granules was noted [141]. Finally, like with ATG16L1, there is a tantalizing connection of IRGM with viral infection [175] that potentially could have relevance in CD [141].
Variants of the transcription factor X-box binding protein 1 (XBP1) have been associated with increased risk for CD [149]. XBP1 is involved in maintaining endoplasmic reticulum (ER) function, especially important in intensive secretory cells, such as Paneth cells [176]. Selectively deletion of Xbp1 in intestinal epithelial cells leads to Paneth cell (and goblet cell) dysfunction and results in intestinal inflammation in mice [149]. Genetic variations in the gene encoding XBP1 are associated with IBD susceptibility, and some uncommon variants may also predispose to CD [149]. A recently reported striking link between Xbp1 and Atg16L1 function further advances a mechanistic model for Paneth cell dysfunction in CD pathogenesis [144]. Mice with lineage-specific Xbp1 gene knockout in Paneth cells showed induced autophagy in these epithelial cells and had aberrant secretory granules. A majority of these mice developed spontaneous enteritis. Conversely, lineage-specific Atg16l1 gene knockout in intestinal epithelial cells increases susceptibility to ER stress. Mice with the genetic deficits in both pathways developed severe spontaneous transmural ileitis [144], supporting the notion that small intestinal CD is a specific disorder of Paneth cell function [140].
Another gene implicated in CD susceptibility may have pathogenic roots in Paneth cell secretion. KCNN4 , which encodes for the calcium-activated potassium channel KCa3.1 , was identified in a CD susceptibility genome-wide association study [145]. KCa3.1 is expressed in Paneth cells where it is involved in granule secretion [177]. One might speculate that disruption of the KCa3.1 channel would disrupt Paneth cell granule secretion, and result in deficiency of α(alpha)-defensins and other Paneth cell antimicrobials.
Finally, two genes in the WNT signaling pathway , TCF7L2 and LRP6 , are implicated in CD pathogenesis [151, 152, 156]. Wnt signaling is a key regulatory circuit for Paneth cell differentiation and α-defensin expression [126]. Levels of TCF7L2 mRNA showed a high degree of correlation with both HD5 and HD6 mRNA [156]. The levels of TCF7L2 mRNA and TCF7L2 α(alpha)-defensin gene-promoter binding activity were decreased in CD patients with ileal disease, but were not decreased in colonic CD (L2) or UC [156]. Reduced expression of Tcf7l2 in heterozygous (+/−) mice caused a significant decrease of both Paneth cell α(alpha)-defensin levels and bacterial killing activity [156]. Furthermore, genetic study identified an association of sequence variants in the TCF7L2 promoter region with ileal CD [151]. The co-receptor named low-density lipoprotein receptor related protein 6 (LRP6) is a crucial WNT signaling pathway factor [178]. A rare sequence variant of LRP6 (I1062V) was linked with early onset ileal CD and with penetrating ileal CD behavior, but not to adult onset ileal CD, colonic CD, or UC [152]. This variant was also linked to particularly low defensin levels in ileal CD patients who were carrying this genetic alteration [152].
Taken together, these puzzle pieces merge to implicate Paneth cells, and their arsenal of antimicrobial peptides in the pathogenesis of CD (Fig. 5.3) [140]. It seems likely that the aforementioned susceptibility genes may manifest part of their phenotypic effects through compromise of Paneth cell function. The aberrant function may lead to chronic alterations in microbiota, establishing dysbiosis [179], or limitations in the ability of these cells to successfully cope with acute microbial challenges.
Fig. 5.3
Hypothesis on the possible link of Paneth cell α(alpha)-defensins and ileal Crohn’s disease (CD). Reduced expression of Paneth cell α(alpha)-defensins is characteristic of CD of the ileum (bottom), but not ulcerative colitis or CD of the colon [107]. Genetic and environmental factors reduce expression of Paneth cell α(alpha)-defensins (solid arrows). The resulting α(alpha)-defensin deficit is thought to contribute to dysbiosis (solid arrow). In turn, the dysbiosis (solid arrow), when combined with perhaps additional independent genetic susceptibility factors and environmental triggers (gray arrows), leads to CD. Mucosal pathology may further promote dysbiosis and further impairment of Paneth cell function (dashed arrows)
Conclusions
The human intestine is colonized by a diverse, abundant, and dynamic microbial ecosystem. These microbes are key to physiological homeostasis and proper balance of the immune system. The interactions between antimicrobial peptides and this intestinal microbiota may have a significant impact on both health and disease, and many aspects of this dynamic interplay are areas of current investigation. Paneth cell antimicrobial peptides, including α-defensins, are likely fundamental host factors that determine the composition of the colonizing microbiota. Perturbations of Paneth cell function, and α-defensin expression in particular, may be a fundamental factor in the pathogenesis of ileal CD. A vital link could be aberrant interactions with the intestinal microbiota that initiate and/or propagate ileal inflammation. Future studies to further define the function(s) and regulation of enteric antimicrobial peptides will enhance our understanding of normal intestinal physiology and homeostasis. This knowledge may provide new therapeutic targets for treating inflammatory diseases in the intestine.
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