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Jan Wehkamp - One of the best experts on this subject based on the ideXlab platform.
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crohn s disease derived monocytes fail to induce Paneth Cell defensins
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: L Courth, Maureen J Ostaff, Daniela Mailandersanchez, Nisar P Malek, Eduard F Stange, Jan WehkampAbstract:Crohn’s disease (CD) is associated with a multitude of genetic defects, many of which likely affect Paneth Cell function. Paneth Cells reside in the small intestine and produce antimicrobial peptides essential for the host barrier, principally human α-defensin 5 (HD5) and HD6. Patients with CD of the ileum are characterized by reduced constitutive expression of these peptides and, accordingly, compromised antimicrobial barrier function. Here, we present a previously unidentified regulatory mechanism of Paneth Cell defensins. Using cultures of human ileal tissue, we showed that the secretome of peripheral blood mononuclear Cells (PBMCs) from healthy controls restored the attenuated Paneth Cell α-defensin expression characteristic of patients with ileal CD. Analysis of the Wnt pathway in both cultured biopsies and intestinal epithelial Cells implicated Wnt ligands driving the PBMC effect, whereas various tested cytokines were ineffective. We further detected another defect in patients with ileal CD, because the PBMC secretomes derived from patients with CD were unable to restore the reduced HD5/HD6 expression. Accordingly, analysis of PBMC subtypes showed that monocytes of patients with CD express significantly lower levels of canonical Wnt ligands, including Wnt3, Wnt3a, Wnt1, and wntless Wnt ligand secretion mediator (Evi/Wls). These studies reveal an important cross-talk between bone marrow-derived Cells and epithelial secretory Paneth Cells. Defective Paneth Cell-mediated innate immunity due to inadequate Wnt ligand stimulation by monocytes provides an additional mechanism in CD. Because defects of Paneth Cell function stemming from various etiologies are overcome by Wnt ligands, this mechanism is a potential therapeutic target for this disease.
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intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth Cell antimicrobial host defense
Hepatology, 2012Co-Authors: Zora Teltschik, Charles L Bevins, Eduard F Stange, Jan Wehkamp, Reiner Wiest, Julia Beisner, Sabine Nuding, C Hofmann, Juergen SchoelmerichAbstract:Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl(4)-induced ascitic cirrhosis and 2-day portal vein-ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth Cell α-cryptdins and β-defensins was determined by real-time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence-activated Cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth Cell α-cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth Cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Conclusion: Compromised Paneth Cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver-gut axis including the underlying mechanisms could help us to find new treatment avenues.
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defective Paneth Cell mediated host defense in pediatric ileal crohn s disease
The American Journal of Gastroenterology, 2010Co-Authors: Gori Perminow, Eduard F Stange, Julia Beisner, Maureen J Koslowski, Lars Gustav Lyckander, Morten H Vatn, Jan WehkampAbstract:OBJECTIVES: Adult ileal Crohn's disease (CD) is characterized by a specific decrease in ileal Paneth Cell alpha-defensins. In addition to NOD2, we previously identified a disturbance of the Wnt-signaling transcription factor TCF-4 as a major mechanism for this deficiency. The aim of this study was to evaluate human alpha-defensin-5 (HD-5) and TCF-4 in an independent cohort of pediatric CD patients. METHODS: Expression levels of HD-5 and TCF-4 mRNA were quantified by real-time PCR in biopsies from newly diagnosed untreated pediatric CD patients (<18 years, n=36) and age-matched symptomatic non-inflammatory bowel disease controls with a histologically normal gut (n=29). To assess the influence of current inflammation, mucosal interleukin-8 (IL-8) and fecal calprotectin levels were determined. RESULTS: Small intestinal HD-5 and TCF-4 mRNA were significantly reduced in pediatric ileal CD (L1+L3) (P=0.022 and P=0.0005, respectively) and were significantly correlated (r=0.499; P=0.0001). In ileal but not colonic CD, TCF-4 was also reduced in the colon (P=0.005). Importantly, both HD-5 and TCF-4 were independent of inflammation, as measured by IL-8 expression or fecal calprotectin. In contrast to the small intestine, colonic Paneth Cell HD-5 mRNA was significantly elevated in colonic CD (L2) (P=0.026) and was correlated with fecal calprotectin levels (r=0.481; P=0.020). CONCLUSIONS: In this study, we describe a specific decrease in HD-5 and TCF-4 mRNA expression levels in children with ileal CD. In the small intestine, this decrease was independent of current inflammation, whereas inflammation seems to induce Paneth Cell metaplasia in the colon. Our data extend the hypothesis of an important role of antimicrobial host defense in pediatric CD patients.
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decreased Paneth Cell defensin expression in ileal crohn s disease is independent of inflammation but linked to the nod2 1007fs genotype
Gut, 2009Co-Authors: Charles L Bevins, Eduard F Stange, Jan WehkampAbstract:We read with interest the recent research article by Lisa Simms and her colleagues ( Gut 2008; 57 :903–10). While we agree with Simms et al that the NOD2 genotype is an important variable in the study of ileal Crohn’s disease (CD), we disagree with her conclusion that α-defensin expression is related to inflammation rather than NOD2 status. This genotype is especially important in the analysis of Paneth Cell antimicrobial expression, given the prominent expression of NOD2 in Paneth Cells.1 In a previous investigation, we reported a significant association of the NOD2 genotype with HD5 mRNA expression levels (Wehkamp et al ,2 fig 1D), but, importantly, the effect was found only with the 1007fs mutation (SNP13). The decrease of HD5 levels with the 1007fs NOD2 genotype was confirmed at the protein level by western blot analysis (Wehkamp et al ,2 fig 1E). The significance of the western blot data was emphasised by our observation that the same 1007fs samples had no decrease in levels of either sPLA2, lysozyme or α-1-antiprotease, three other Paneth Cell proteins (Wehkamp et al ,2 fig 1G). Of special note, we saw no association of either the R702W or G908R genotypes with HD5 mRNA levels (Wehkamp et al ,2 fig 1D). In their recent paper, Simms et al did not stratify their genotype data to investigate if there was a difference in HD5 expression levels associated with any particular NOD2 genotype. Since eight patients in their cohort had a 1007fs mutation, we …
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the Paneth Cell α defensin deficiency of ileal crohn s disease is linked to wnt tcf 4
Journal of Immunology, 2007Co-Authors: Jan Wehkamp, Sabine Nuding, Guoxing Wang, Irmgard Kubler, Alex Gregorieff, Anke Schnabel, Robert J Kays, Klaus Fellermann, Oliver BurkAbstract:Ileal Crohn′s disease (CD), a chronic mucosal inflammation, is characterized by two pertinent features: a specific decrease of Paneth Cell-produced antimicrobial α-defensins and the presence of mucosal-adherent bacteria. A mutation in NOD2, the muramyl dipeptide recognition receptor, is found in some patients, which leads to an even more pronounced α-defensin decrease. However, the underlying mechanism remains unclear for the majority of patients. In this study, we report a reduced expression in ileal CD of the Wnt-signaling pathway transcription factor Tcf-4, a known regulator of Paneth Cell differentiation and α-defensin expression. Within specimens, the levels of Tcf-4 mRNA showed a high degree of correlation with both HD5 and HD6 mRNA. The levels of Tcf-4 mRNA were decreased in patients with ileal disease irrespective of degree of inflammation, but were not decreased in colonic CD or ulcerative colitis. As a functional indicator of Tcf-4 protein, quantitative binding analysis with nuclear extracts from small intestine biopsies to a Tcf-4 high-affinity binding site in the HD-5 and HD-6 promoters showed significantly reduced activity in ileal CD. Furthermore, a causal link was shown in a murine Tcf-4 knockout model, where the comparably reduced expression of Tcf-4 in heterozygous (+/−) mice was sufficient to cause a significant decrease of both Paneth Cell α-defensin levels and bacterial killing activity. Finally, the association between Paneth Cell α-defensins and Tcf-4 was found to be independent of the NOD2 genotype. This new link established between a human inflammatory bowel disease and the Wnt pathway/Tcf-4 provides a novel mechanism for pathogenesis in patients with ileal CD.
Andre J Ouellette - One of the best experts on this subject based on the ideXlab platform.
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entamoeba histolytica alters ileal Paneth Cell functions in intact and muc2 mucin deficiency
Infection and Immunity, 2018Co-Authors: Eduardo R Cobo, Kiminori Nakamura, Tokiyoshi Ayabe, Andre J Ouellette, Yoshihiro Eriguchi, Jennifer R Mastroianni, Ravi Holani, Kris ChadeeAbstract:: Enteric α-defensins, termed cryptdins (Crps) in mice, and lysozymes secreted by Paneth Cells contribute to innate host defense in the ileum. Antimicrobial factors, including lysozymes and β-defensins, are often embedded in luminal glycosylated colonic Muc2 mucin secreted by goblet Cells that form the protective mucus layer critical for gut homeostasis and pathogen invasion. In this study, we investigated ileal innate immunity against Entamoeba histolytica, the causative agent of intestinal amebiasis, by inoculating parasites in closed ileal loops in Muc2+/+ and Muc2-/- littermates and quantifying Paneth Cell localization (lysozyme expression) and function (Crp secretion). Relative to Muc2+/+ littermates, Muc2-/- littermates showed a disorganized mislocalization of Paneth Cells that was diffusely distributed, with elevated lysozyme secretion in the crypts and on villi in response to E. histolytica Inhibition of E. histolytica Gal/GalNAc lectin (Gal-lectin) binding with exogenous galactose and Entamoeba histolytica cysteine proteinase 5 (EhCP5)-negative E. histolytica had no effect on parasite-induced erratic Paneth Cell lysozyme synthesis. Although the basal ileal expression of Crp genes was unaffected in Muc2-/- mice in response to E. histolytica, there was a robust release of proinflammatory cytokines and Crp peptide secretions in luminal exudates that was also present in the colon. Interestingly, E. histolytica-secreted cysteine proteinases cleaved the proregion of Crp4 but not the active form. These findings define Muc2 mucin as an essential component of ileal barrier function that regulates the localization and function of Paneth Cells critical for host defense against microbes.
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Paneth Cell mediated multiorgan dysfunction after acute kidney injury
Journal of Immunology, 2012Co-Authors: Sang Won Park, Kevin M Brown, Andre J Ouellette, Yuko Moriakiyama, Vivette D DagatiAbstract:Acute kidney injury (AKI) is frequently complicated by extrarenal multiorgan injury, including intestinal and hepatic dysfunction. In this study, we hypothesized that a discrete intestinal source of proinflammatory mediators drives multiorgan injury in response to AKI. After induction of AKI in mice by renal ischemia-reperfusion or bilateral nephrectomy, small intestinal Paneth Cells increased the synthesis and release of IL-17A in conjunction with severe intestinal apoptosis and inflammation. We also detected significantly increased IL-17A in portal and systemic circulation after AKI. Intestinal macrophages appear to transport released Paneth Cell granule constituents induced by AKI, away from the base of the crypts into the liver. Genetic or pharmacologic depletion of Paneth Cells decreased small intestinal IL-17A secretion and plasma IL-17A levels significantly and attenuated intestinal, hepatic, and renal injury after AKI. Similarly, portal delivery of IL-17A in macrophage-depleted mice decreased markedly. In addition, intestinal, hepatic, and renal injury following AKI was attenuated without affecting intestinal IL-17A generation. In conclusion, AKI induces IL-17A synthesis and secretion by Paneth Cells to initiate intestinal and hepatic injury by hepatic and systemic delivery of IL-17A by macrophages. Modulation of Paneth Cell dysregulation may have therapeutic implications by reducing systemic complications arising from AKI.
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alternative luminal activation mechanisms for Paneth Cell α defensins
Journal of Biological Chemistry, 2012Co-Authors: Jennifer R Mastroianni, Nita H Salzman, Jessica K Costales, Jennifer Zaksheske, Michael E Selsted, Andre J OuelletteAbstract:Abstract Paneth Cell α-defensins mediate host defense and homeostasis at the intestinal mucosal surface. In mice, matrix metalloproteinase-7 (MMP7) converts inactive pro-α-defensins (proCrps) to bactericidal forms by proteolysis at specific proregion cleavage sites. MMP7(−/−) mice lack mature α-defensins in Paneth Cells, accumulating unprocessed precursors for secretion. To test for activation of secreted pro-α-defensins by host and microbial proteinases in the absence of MMP7, we characterized colonic luminal α-defensins. Protein extracts of complete (organ plus luminal contents) ileum, cecum, and colon of MMP7-null and wild-type mice were analyzed by sequential gel permeation chromatography/acid-urea polyacrylamide gel analyses. Mature α-defensins were identified by N-terminal sequencing and mass spectrometry and characterized in bactericidal assays. Abundance of specific bacterial groups was measured by qPCR using group specific 16 S rDNA primers. Intact, native α-defensins, N-terminally truncated α-defensins, and α-defensin variants with novel N termini due to alternative processing were identified in MMP7(−/−) cecum and colon, and proteinases of host and microbial origin catalyzed proCrp4 activation in vitro. Although Paneth Cell α-defensin deficiency is associated with ileal microbiota alterations, the cecal and colonic microbiota of MMP7(−/−) and wild-type mice were not significantly different. Thus, despite the absence of MMP7, mature α-defensins are abundant in MMP7(−/−) cecum and colon due to luminal proteolytic activation by alternative host and microbial proteinases. MMP7(−/−) mice only lack processed α-defensins in the small intestine, and the model is not appropriate for studying effects of α-defensin deficiency in cecal or colonic infection or disease.
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α defensins in enteric innate immunity functional Paneth Cell α defensins in mouse colonic lumen
Journal of Biological Chemistry, 2009Co-Authors: Jennifer R Mastroianni, Andre J OuelletteAbstract:Abstract Paneth Cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth Cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth Cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth Cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.
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mouse Paneth Cell secretory responses to Cell surface glycolipids of virulent and attenuated pathogenic bacteria
Infection and Immunity, 2005Co-Authors: Hiroki Tanabe, Tokiyoshi Ayabe, Samuel I Miller, Brian W Bainbridge, Tina Guina, Robert K Ernst, Richard P Darveau, Andre J OuelletteAbstract:Mouse Paneth Cells respond to bacteria and bacterial Cell surface antigens by discharging secretory granules into the lumen of small intestinal crypts (T. Ayabe et al., Nat. Immunol. 1:113-118, 2000). To investigate mechanisms regulating these responses, purified surface glycolipid molecules with known acyl chain modifications and attenuated properties were tested for the ability to stimulate Paneth Cell secretion. The antigens included lipopolysaccharide (LPS) from wild-type and msbB-null Escherichia coli and phoP-null and phoP-constitutive Salmonella enterica serovar Typhimurium strains, as well as LPS, lipid A, and lipoteichoic acid from Pseudomonas aeruginosa and Listeria monocytogenes grown in Mg2+-limited media. Measurements of total secreted protein, secreted lysozyme, and the bactericidal peptide activities of collected secretions showed that the purified antigens elicited similar secretory responses from Paneth Cells in mouse crypts ex vivo, regardless of glycolipid acyl chain modification. Despite their impaired Tlr4 pathway, Paneth Cells in ex vivo C3H/HeJ mouse crypts released equivalent amounts of bactericidal peptide activity in response to purified bacterial antigens, including lipid A. Thus, mouse Paneth Cells respond equivalently to purified bacterial Cell envelope glycolipids, regardless of functional Tlr4, the structural properties of glycolipid acyl chains, or their association with virulence in humans.
Charles L Bevins - One of the best experts on this subject based on the ideXlab platform.
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dysbiosis a consequence of Paneth Cell dysfunction
Seminars in Immunology, 2013Co-Authors: Nita H Salzman, Charles L BevinsAbstract:Abstract The complex community of colonizing microbes inhabiting the mucosal surfaces of mammals is vital to homeostasis and normal physiology in the host. When the composition of this microbiota is unfavorably altered, termed dysbiosis, the host is rendered more susceptible to a variety of chronic diseases. In the mammalian small intestine, specialized secretory epithelial Cells, named Paneth Cells, produce a variety of secreted antimicrobial peptides that fundamentally influence the composition of the microbiota. Recent investigations have identified numerous genetic and environmental factors that can disrupt normal Paneth Cell function, resulting in compromised antimicrobial peptide secretion and consequent dysbiosis. These findings suggest that Paneth Cell dysfunction should be considered a common cause of dysbiosis.
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intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth Cell antimicrobial host defense
Hepatology, 2012Co-Authors: Zora Teltschik, Charles L Bevins, Eduard F Stange, Jan Wehkamp, Reiner Wiest, Julia Beisner, Sabine Nuding, C Hofmann, Juergen SchoelmerichAbstract:Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl(4)-induced ascitic cirrhosis and 2-day portal vein-ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth Cell α-cryptdins and β-defensins was determined by real-time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence-activated Cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth Cell α-cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth Cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Conclusion: Compromised Paneth Cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver-gut axis including the underlying mechanisms could help us to find new treatment avenues.
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decreased Paneth Cell defensin expression in ileal crohn s disease is independent of inflammation but linked to the nod2 1007fs genotype
Gut, 2009Co-Authors: Charles L Bevins, Eduard F Stange, Jan WehkampAbstract:We read with interest the recent research article by Lisa Simms and her colleagues ( Gut 2008; 57 :903–10). While we agree with Simms et al that the NOD2 genotype is an important variable in the study of ileal Crohn’s disease (CD), we disagree with her conclusion that α-defensin expression is related to inflammation rather than NOD2 status. This genotype is especially important in the analysis of Paneth Cell antimicrobial expression, given the prominent expression of NOD2 in Paneth Cells.1 In a previous investigation, we reported a significant association of the NOD2 genotype with HD5 mRNA expression levels (Wehkamp et al ,2 fig 1D), but, importantly, the effect was found only with the 1007fs mutation (SNP13). The decrease of HD5 levels with the 1007fs NOD2 genotype was confirmed at the protein level by western blot analysis (Wehkamp et al ,2 fig 1E). The significance of the western blot data was emphasised by our observation that the same 1007fs samples had no decrease in levels of either sPLA2, lysozyme or α-1-antiprotease, three other Paneth Cell proteins (Wehkamp et al ,2 fig 1G). Of special note, we saw no association of either the R702W or G908R genotypes with HD5 mRNA levels (Wehkamp et al ,2 fig 1D). In their recent paper, Simms et al did not stratify their genotype data to investigate if there was a difference in HD5 expression levels associated with any particular NOD2 genotype. Since eight patients in their cohort had a 1007fs mutation, we …
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regional variations in Paneth Cell antimicrobial peptide expression along the mouse intestinal tract
BMC Immunology, 2008Co-Authors: Jenny Karlsson, Charles L Bevins, Robert J Kays, Katrin Putsep, Mats AnderssonAbstract:Background Enteric antimicrobial peptides secreted from Paneth Cells, including α-defensins (in mice named cryptdins), are key effector molecules of innate immunity in the small intestine. The importance of Paneth Cells α-defensins emerged from studies of enteric bacterial infection in genetically modified mice, as well as from recent studies linking reduced levels of these α-defensins to Crohn's disease localized to the ileum. However, analysis of expression of Paneth Cell α-defensins is incomplete. We therefore performed a comprehensive evaluation of the distribution of antimicrobial molecules along the mouse small intestinal tract to identify potential variations in regional expression.
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Paneth Cell defensins key effector molecules of innate immunity
Biochemical Society Transactions, 2006Co-Authors: Charles L BevinsAbstract:Antimicrobial peptides are fundamental effector molecules of innate immunity, utilized in host defence by virtually all organisms studied. These gene-encoded peptides have direct antibiotic activity against a wide range of bacteria and other microbes. In humans and other mammals, defensins are a predominant class of such peptides. In the mammalian small intestine, Paneth Cells, specialized secretory epithelial Cells located at the base of the crypt invaginations lining the intestinal wall, produce defensins and other antibiotic proteins. Recent investigations in murine models provide compelling support for the hypothesis that enteric defensins play a pivotal role in defence from food- and water-borne pathogens in the intestinal lumen. Investigations by others indicate that intestinal commensal bacteria are key factors in the pathogenesis of IBD (inflammatory bowel disease) in genetically susceptible humans. Recent studies provide evidence that reduced expression of Paneth Cell defensins may be a key factor in the pathogenesis of ileal Crohn9s disease, a subgroup of IBD. Future studies to further define the function and regulation of Paneth Cell defensins will enhance our understanding of normal small bowel physiology, and probably contribute to a better understanding of the pathogenesis of inflammatory and infectious diseases of the bowel. Such knowledge may provide new therapeutic targets and strategies.
Tokiyoshi Ayabe - One of the best experts on this subject based on the ideXlab platform.
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Paneth Cell granule dynamics on secretory responses to bacterial stimuli in enteroids.
Scientific Reports, 2019Co-Authors: Yuki Yokoi, Kiminori Nakamura, Tsukasa Yoneda, Mani Kikuchi, Rina Sugimoto, Yu Shimizu, Tokiyoshi AyabeAbstract:Paneth Cells at the base of small intestinal crypts secrete granules containing α-defensins in response to bacteria and maintain the intestinal environment by clearing enteric pathogens and regulating the composition of the intestinal microbiota. However, Paneth Cell secretory responses remain debatable and the mechanisms that regulate the secretion are not well understood. Although enteroids, three-dimensional cultures of small intestinal epithelial Cells, have proven useful for analyzing intestinal epithelial Cell functions including ion transport, their closed structures have imposed limitations to investigating interactions between Paneth Cells and the intestinal microbiota. Here, we report that microinjection of bacteria or lipopolysaccharide (LPS) into the enteroid lumen provides an ex vivo system for studying Paneth Cell secretion in real-time. The results show that Paneth Cells released granules immediately when the apical surfaces of enteroid epithelial Cells were exposed to LPS or live bacteria by microinjection. However, Paneth Cells did not respond to LPS delivered in culture media to enteroid exterior basolateral surface, although they responded to basolateral carbamyl choline. In addition, Paneth Cells replenished their granules after secretion, enabling responses to second stimulation. These findings provide new insight for apically-induced Paneth Cell secretory responses in regulating the intestinal environment.
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entamoeba histolytica alters ileal Paneth Cell functions in intact and muc2 mucin deficiency
Infection and Immunity, 2018Co-Authors: Eduardo R Cobo, Kiminori Nakamura, Tokiyoshi Ayabe, Andre J Ouellette, Yoshihiro Eriguchi, Jennifer R Mastroianni, Ravi Holani, Kris ChadeeAbstract:: Enteric α-defensins, termed cryptdins (Crps) in mice, and lysozymes secreted by Paneth Cells contribute to innate host defense in the ileum. Antimicrobial factors, including lysozymes and β-defensins, are often embedded in luminal glycosylated colonic Muc2 mucin secreted by goblet Cells that form the protective mucus layer critical for gut homeostasis and pathogen invasion. In this study, we investigated ileal innate immunity against Entamoeba histolytica, the causative agent of intestinal amebiasis, by inoculating parasites in closed ileal loops in Muc2+/+ and Muc2-/- littermates and quantifying Paneth Cell localization (lysozyme expression) and function (Crp secretion). Relative to Muc2+/+ littermates, Muc2-/- littermates showed a disorganized mislocalization of Paneth Cells that was diffusely distributed, with elevated lysozyme secretion in the crypts and on villi in response to E. histolytica Inhibition of E. histolytica Gal/GalNAc lectin (Gal-lectin) binding with exogenous galactose and Entamoeba histolytica cysteine proteinase 5 (EhCP5)-negative E. histolytica had no effect on parasite-induced erratic Paneth Cell lysozyme synthesis. Although the basal ileal expression of Crp genes was unaffected in Muc2-/- mice in response to E. histolytica, there was a robust release of proinflammatory cytokines and Crp peptide secretions in luminal exudates that was also present in the colon. Interestingly, E. histolytica-secreted cysteine proteinases cleaved the proregion of Crp4 but not the active form. These findings define Muc2 mucin as an essential component of ileal barrier function that regulates the localization and function of Paneth Cells critical for host defense against microbes.
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Paneth Cell α defensins and enteric microbiota in health and disease
Bioscience and microflora, 2016Co-Authors: Kiminori Nakamura, Naoya Sakuragi, Akiko Takakuwa, Tokiyoshi AyabeAbstract:Antimicrobial peptides are major effectors of innate immunity of multiCellular organisms including humans and play a critical role in host defense, and their importance is widely recognized. The epithelium of the intestine is the largest surface area exposed to the outer environment, including pathogens, toxins and foods. The Paneth Cell lineage of intestinal epithelial Cells produces and secretes α-defensin antimicrobial peptides and functions in innate enteric immunity by removing pathogens and living symbiotically with commensal microbiota to contribute to intestinal homeostasis. Paneth Cells secrete α-defensins, HD5 and HD6 in humans and cryptdins in mice, in response to bacterial, cholinergic and other stimuli. The α-defensins have selective activities against bacteria, eliciting potent microbicidal activities against pathogenic bacteria but minimal or no bactericidal activity against commensal bacteria. Therefore, α-defensins regulate the composition of the intestinal microbiota in vivo and play a role in homeostasis of the entire intestine. Recently, relationships between dysbiosis, or abnormal composition of the intestinal microbiota, and diseases such as inflammatory bowel disease and lifestyle diseases including obesity and atherosclerosis have been reported. Because α-defensins regulate the composition of the intestinal microbiota, Paneth Cells and their α-defensins may have a key role as one mechanism linking the microbiota and disease.
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decreased secretion of Paneth Cell α defensins in graft versus host disease
Transplant Infectious Disease, 2015Co-Authors: Yoshihiro Eriguchi, Kiminori Nakamura, Tokiyoshi Ayabe, Daigo Hashimoto, Shinji Shimoda, Nobuyuki Shimono, Koichi Akashi, Takanori TeshimaAbstract:Background Intestinal microbial ecology is actively regulated by Paneth Cell-derived antimicrobial peptides, α-defensins. Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem Cell transplantation (SCT). We previously demonstrated that Paneth Cells are targeted by GVHD, and their expression of antimicrobial peptide α-defensins is impaired, leading to a loss of physiological diversity among the microflora and development of bloodstream infection. Herein, we evaluated whether fecal levels of α-defensins could be surrogate marker of intestinal dysbiosis. Methods We directly measured α-defensin cryptdin-1 (Crp1) in fecal pellets of mice with GVHD by using a novel enzyme-linked immunosorbent assay. Results Fecal levels of Crp1 were significantly decreased in mice with GVHD but unchanged in mice without GVHD after SCT. These were correlated with intestinal flora diversity. Conclusion We demonstrate a link between reduced secretion of Paneth Cell α-defensins and dysbiosis of intestinal flora in GVHD. Fecal levels of α-defensins could be surrogate markers for intestinal microbial homeostasis.
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mouse Paneth Cell secretory responses to Cell surface glycolipids of virulent and attenuated pathogenic bacteria
Infection and Immunity, 2005Co-Authors: Hiroki Tanabe, Tokiyoshi Ayabe, Samuel I Miller, Brian W Bainbridge, Tina Guina, Robert K Ernst, Richard P Darveau, Andre J OuelletteAbstract:Mouse Paneth Cells respond to bacteria and bacterial Cell surface antigens by discharging secretory granules into the lumen of small intestinal crypts (T. Ayabe et al., Nat. Immunol. 1:113-118, 2000). To investigate mechanisms regulating these responses, purified surface glycolipid molecules with known acyl chain modifications and attenuated properties were tested for the ability to stimulate Paneth Cell secretion. The antigens included lipopolysaccharide (LPS) from wild-type and msbB-null Escherichia coli and phoP-null and phoP-constitutive Salmonella enterica serovar Typhimurium strains, as well as LPS, lipid A, and lipoteichoic acid from Pseudomonas aeruginosa and Listeria monocytogenes grown in Mg2+-limited media. Measurements of total secreted protein, secreted lysozyme, and the bactericidal peptide activities of collected secretions showed that the purified antigens elicited similar secretory responses from Paneth Cells in mouse crypts ex vivo, regardless of glycolipid acyl chain modification. Despite their impaired Tlr4 pathway, Paneth Cells in ex vivo C3H/HeJ mouse crypts released equivalent amounts of bactericidal peptide activity in response to purified bacterial antigens, including lipid A. Thus, mouse Paneth Cells respond equivalently to purified bacterial Cell envelope glycolipids, regardless of functional Tlr4, the structural properties of glycolipid acyl chains, or their association with virulence in humans.
Kaatje Lenaerts - One of the best experts on this subject based on the ideXlab platform.
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total parenteral nutrition induces a shift in the firmicutes to bacteroidetes ratio in association with Paneth Cell activation in rats
Journal of Nutrition, 2012Co-Authors: Caroline M Hodin, Kaatje Lenaerts, Sander S Rensen, Ruben G J Visschers, Bas Boonen, Steven Olde W M Damink, Wim A BuurmanAbstract:The use of total parenteral nutrition (TPN) in the treatment of critically ill patients has been the subject of debate because it has been associated with disturbances in intestinal homeostasis. Important factors in maintaining intestinal homeostasis are the intestinal microbiota and Paneth Cells, which exist in a mutually amendable relationship. We hypothesized that the disturbed intestinal homeostasis in TPN-fed individuals results from an interplay between a shift in microbiota composition and alterations in Paneth Cells. We studied the microbiota composition and expressionof Paneth Cell antimicrobial proteins in rats receiving TPN or a control diet for 3, 7, or 14 d. qPCR analysis of DNA extracts from small intestinal luminal contents of TPN-fed rats showed a shift in the Firmicutes:Bacteroidetes ratio in favor of Bacteroidetes after 14 d (P < 0.05) compared with the control group. This finding coincided with greater staining intensity for lysozyme and significantly greater mRNA expression of the Paneth Cell antimicrobial proteins lysozyme (P < 0.05), rat a-defensin 5 (P < 0.01), and rat a-defensin 8 (P < 0.01). Finally, 14 d of TPN resulted in greater circulating ileal lipid-binding protein concentrations (P < 0.05) and greater leakage of horseradish peroxidase (P < 0.01), which is indicative of enterocyte damage and a breached intestinal barrier. Our findings show a shift in intestinal microbiota in TPN-fed rats that correlated with changes in Paneth Cell lysozyme expression (rs = 20.75, P < 0.01). Further studies that include interventions with microbiota or nutrients that modulate them may yield information on the involvement of the microbiota and Paneth Cells in TPN-associated intestinal compromise. J. Nutr. 142: 2141‐2147, 2012.
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reduced Paneth Cell antimicrobial protein levels correlate with activation of the unfolded protein response in the gut of obese individuals
The Journal of Pathology, 2011Co-Authors: Caroline M Hodin, Joep Grootjans, Fons Verheyen, Cornelis H C Dejong, Wim A Buurman, Froukje J Verdam, Sander S Rensen, Jan Willem M Greve, Kaatje LenaertsAbstract:The intestinal microbiota is increasingly acknowledged to play a crucial role in the development of obesity. A shift in intestinal microbiota composition favouring the presence of Firmicutes over Bacteroidetes has been observed in obese subjects. A similar shift has been reported in mice with deficiency of active Paneth Cell alpha-defensins. We aimed at investigating changes in Paneth Cell antimicrobial levels in the gut of obese subjects. Next, we studied activation of the unfolded protein response (UPR) as a possible mechanism involved in altered Paneth Cell function. Paneth Cell numbers were counted in jejunal sections of 15 severely obese (BMI > 35) and 15 normal weight subjects. Expression of Paneth Cell antimicrobials human alpha-defensin 5 (HD5) and lysozyme were investigated using immunohistochemistry, qPCR, and western blot. Activation of the UPR was assessed with western blot. Severely obese subjects showed decreased protein levels of both HD5 and lysozyme, while Paneth Cell numbers were unchanged. Lysozyme protein levels correlated inversely with BMI. Increased expression of HD5 (DEFA5) and lysozyme (LYZ) transcripts in the intestine of obese subjects prompted us to investigate a possible translational block caused by UPR activation. Binding protein (BiP) and activating transcription factor 4 (ATF4) levels were increased, confirming activation of the UPR in the gut of obese subjects. Furthermore, levels of both proteins correlated with BMI. Involvement of the UPR in the lowered antimicrobial protein levels in obese subjects was strongly suggested by a negative correlation between BiP levels and lysozyme levels. Additionally, indications of ER stress were apparent in Paneth Cells of obese subjects. Our findings provide the first evidence for altered Paneth Cell function in obesity, which may have important implications for the obesity-associated shift in microbiota composition. In addition, we show activation of the UPR in the intestine of obese subjects, which may underlie the observed Paneth Cell compromise. Copyright (c) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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level of activation of the unfolded protein response correlates with Paneth Cell apoptosis in human small intestine exposed to ischemia reperfusion
Gastroenterology, 2011Co-Authors: Joep Grootjans, Caroline M Hodin, Jacco J De Haan, Joep P M Derikx, Kasper M A Rouschop, Fons Verheyen, Cornelis H C Dejong, Wim A Buurman, Kaatje LenaertsAbstract:Background & Aims In the intestine, Paneth Cells participate in the innate immune response. Their highly secretory function makes them susceptible to environmental conditions that cause endoplasmic reticulum (ER) stress. We investigated whether intestinal ischemia/reperfusion (I/R) induces ER stress, thereby activating the unfolded protein response (UPR), and whether excessive UPR activation affects Paneth Cells. In addition, we investigated the consequences of Paneth Cell compromise during physical barrier damage. Methods Jejunal I/R was studied using a human experimental model (n = 30 patients). Activation of the UPR was assessed using immunofluorescence for binding protein and quantitative polymerase chain reaction analyses for C/EBP homologous protein (CHOP), growth arrest and DNA-damage inducible protein 34 (GADD34), and X-box binding protein 1 (XBP1) splicing. Paneth Cell apoptosis was assessed by double staining for lysozyme and M30. Male Sprague–Dawley rats underwent either intestinal I/R to investigate UPR activation and Paneth Cell apoptosis, or hemorrhagic shock with or without intraperitoneal administration of dithizone, to study consequences of Paneth Cell compromise during physical intestinal damage. In these animals, bacterial translocation and circulating tumor necrosis factor-α and interleukin-6 levels were assessed. Results In jejunum samples from humans and rats, I/R activated the UPR and resulted in Paneth Cell apoptosis. Apoptotic Paneth Cells showed signs of ER stress, and Paneth Cell apoptosis correlated with the extent of ER stress. Apoptotic Paneth Cells were shed into the crypt lumen, significantly lowering their numbers. In rats, Paneth Cell compromise increased bacterial translocation and inflammation during physical intestinal damage. Conclusions ER stress-induced Paneth Cell apoptosis contributes to intestinal I/R-induced bacterial translocation and systemic inflammation.
