Paneth Cells Of Small Intestine Research Articles

WESTERN DIET DEPLETES SMALL INTESTINAL INTRAEPITHELIAL LYMPHOCYTES VIA FXR-INTERFERON PATHWAY

Abstract BACKGROUND The prevalence of obesity has continued to increase over the past several decades. Understanding how diet-induced obesity modulates mucosal immunity is of clinical relevance. We previously showed that consumption of a high fat, high sugar “Western” diet (WD) reduces the density and function of small intestinal Paneth cells, a small intestinal epithelial cell type with innate immune function. We hypothesized that obesity could also result in repressed adaptive immunity. METHODS Small intestine samples from non-inflammatory bowel disease (IBD) subjects who underwent resection were used for CD3 immunohistochemistry (IHC), and the CD3+ intraepithelial lymphocytes (IEL) densities were correlated with the patients’ body mass index (BMI). Mice were fed with either standard diet (SD) or WD for various durations, and IELs were isolated for flow cytometry analysis. RESULTS In non-IBD subjects, high BMI correlated with reduced IEL density. We recapitulated this in wild type (WT) mice fed with WD. A 4-week WD consumption was able to deplete IEL (including major subsets CD4+, CD8+, CD8α+8β-CD4+, and γδCD8+ T cells) but not splenic lymphocytes, and the effect was reversible after another 4 weeks of SD washout. ob/ob mice that spontaneously developed obesity while fed with SD did not show IEL depletion, suggesting that the quality, not the quantity of the diet is key to this phenotype. Importantly, WD-associated IEL depletion was not dependent on the presence of gut microbiota, as WD-fed germ-free mice also showed IEL depletion. We previously showed that a key molecular pathway activated by WD in the ileum is the FXR pathway. Phagocyte-specific Fxr conditional knockout (cKO) mice were protected from WD-mediated IEL depletion, suggesting that FXR activation in the phagocytes is critical in mediating this phenotype. Activated FXR signaling stimulated phagocytes to secrete type I interferon (IFN), and inhibition of type I IFN signaling within the phagocytes (type I IFN monoclonal antibody treatment and Stat1 cKO mice) prevented WD-mediated IEL loss. CONCLUSION WD consumption depletes IEL by a FXR-IFN pathway mediated by gut phagocytes. These findings have significant clinical implications in understanding the pathogenesis of gut inflammatory disorders such as IBD.

NAD+ precursors and bile acid sequestration treat preclinical refractory environmental enteric dysfunction.

Environmental enteric dysfunction (EED) is a diffuse small bowel disorder associated with poor growth, inadequate responses to oral vaccines, and nutrient malabsorption in millions of children worldwide. We identify loss of the small intestinal Paneth and goblet cells that are critical for innate immunity, reduced villous height, increased bile acids, and dysregulated nicotinamide adenine dinucleotide (NAD+) synthesis signaling as potential mechanisms underlying EED and which also correlated with diminished length-for-age z score. Isocaloric low-protein diet (LPD) consumption in mice recapitulated EED histopathology and transcriptomic changes in a microbiota-independent manner, as well as increases in serum and fecal bile acids. Children with refractory EED harbor single-nucleotide polymorphisms in key enzymes involved in NAD+ synthesis. In mice, deletion of Nampt, the gene encoding the rate-limiting enzyme in the NAD+ salvage pathway, from intestinal epithelium also reduced Paneth cell function, a deficiency that was further aggravated by LPD. Separate supplementation with NAD+ precursors or bile acid sequestrant partially restored LPD-associated Paneth cell defects and, when combined, fully restored all histopathology defects in LPD-fed mice. Therapeutic regimens that increase protein and NAD+ contents while reducing excessive bile acids may benefit children with refractory EED.

Intestinal TLR9 deficiency exacerbates hepatic IR injury via altered intestinal inflammation and short-chain fatty acid synthesis.

Mice deficient in intestinal epithelial TLR9 develop small intestinal Paneth cell hyperplasia and higher Paneth cell IL-17A levels. Since small intestinal Paneth cells and IL-17A play critical roles in hepatic ischemia reperfusion (IR) injury, we tested whether mice lacking intestinal TLR9 have increased hepatic IR injury. Mice lacking intestinal TLR9 had profoundly increased liver injury after hepatic IR compared to WT mice with exacerbated hepatocyte necrosis, apoptosis, neutrophil infiltration, and inflammatory cytokine generation. Moreover, we observed increased small intestinal inflammation and apoptosis after hepatic IR in intestinal TLR9 deficient mice. As a potential explanation for increased hepatic IR injury, fecal short-chain fatty acids butyrate and propionate levels were lower in intestinal TLR9 deficient mice. Suggesting a potential therapy for hepatic IR, exogenous administration of butyrate or propionate protected against hepatic IR injury in intestinal TLR9 deficient mice. Mechanistically, butyrate induced small intestinal IL-10 expression and downregulated the claudin-2 expression. Finally, IL-10 neutralization abolished the protective effects of butyrate against hepatic IR injury. Our studies show intestinal TLR9 deficiency results in exacerbated hepatic IR injury with increased small intestinal apoptosis and inflammation. Furthermore, short-chain fatty acids butyrate and propionate protect against hepatic IR injury and intestinal apoptosis/inflammation in intestinal TLR9 deficient mice.

DOP86 An IFN-STAT1-MLKL axis drives programmed necrosis of Paneth cells in Crohn’s ileitis

Abstract Background Interferons (IFNs) are immune-modulatory cytokines expressed by epithelial and mucosal cells in response to viral and bacterial infection. Just recently, we discovered a correlation between IFN-λ expression and disease activity, including small intestinal inflammation and Paneth cell dysfunction, in human Crohn’s disease patients. On a molecular level, we uncovered that IFN-λ mediates epithelial cell death, in particular, Paneth cell death by a programmed necrosis, dependent on STAT1 activation and controlled by caspase-8. These results suggested that IFN-λ can be considered as a pathogenic cytokine in Crohn′s ileitis and should be considered as a new and promising target for future therapeutic intervention for this particular subtype of IBD. Our central question is now by which pathways interferon-regulated programmed necrosis of epithelial cells contributes to intestinal inflammation and how these mechanisms could be targeted for future therapeutic intervention. Methods We use a mouse model for Crohn’s Disease like inflammation and Paneth cell death that has a specific deletion of Caspase-8 in intestinal epithelial cells (Casp8∆IEC). We stimulate small intestinal organoids derived from Casp8∆IEC mice with IFNs in vitro and we overexpress IFN-λ in these mice in vivo by hydrodynamic tail vein injection of an IFN-λ expression vector. Furthermore, we use JAK-inhibitors to impede pharmacologically cell death pathways in the pathogenesis of intestinal inflammation in vitro and in vivo. Results We uncovered that gene expression of the cell death mediators Mlkl and Caspase-8 is dependent on IFN-λ-mediated JAK-STAT1 signalling. The non-specific pan JAK-inhibitor Tofacitinib is able to attenuate gene expression of Mlkl and Caspase-8 in vitro as well as in vivo. It prevents non-apoptotic as well as apoptotic cell death of small intestinal organoids stimulated with IFN-λ and is sufficient to prevent small intestinal tissue destruction in Casp8∆IEC mice challenged with IFN-λ. Additionally, we use the selective JAK1-inhibitor Filgotinib to limit the targeted JAK-STAT signalling pathways to only JAK1-STAT1 signalling and thus reduce side effects of the inhibitor on other signalling pathways. This had a similar effect as Tofacitinib suggesting that IFN controls MLKL-mediated cell death via JAK1. Conclusion In summary, our results indicate that targeting IFN-λ-mediated JAK-STAT1 signalling by the small-molecules Tofacitinib and Filgotinib impedes induction of Mlkl and Caspase-8-mediated cell death pathways. Therefore, JAK1 inhibitors such as Filgotinib might represent a promising novel therapy that may be sufficient to achieve efficacy particularly in Crohn′s ileitis patients who display elevated IFN-l serum levels.

Norepinephrine released by intestinal Paneth cells exacerbates ischemic AKI.

Small intestinal Paneth cells play a critical role in acute kidney injury (AKI) and remote organ dysfunction by synthesizing and releasing IL-17A. In addition, intestine-derived norepinephrine is a major mediator of hepatic injury and systemic inflammation in sepsis. We tested the hypothesis that small intestinal Paneth cells synthesize and release norepinephrine to exacerbate ischemic AKI. After ischemic AKI, we demonstrated larger increases in portal venous norepinephrine levels compared with plasma norepinephrine in mice, consistent with an intestinal source of norepinephrine release after renal ischemia and reperfusion. We demonstrated that murine small intestinal Paneth cells express tyrosine hydroxylase mRNA and protein, a critical rate-limiting enzyme for the synthesis of norepinephrine. We also demonstrated mRNA expression for tyrosine hydroxylase in human small intestinal Paneth cells. Moreover, freshly isolated small intestinal crypts expressed significantly higher norepinephrine levels after ischemic AKI compared with sham-operated mice. Suggesting a critical role of IL-17A in Paneth cell-mediated release of norepinephrine, recombinant IL-17A induced norepinephrine release in the small intestine of mice. Furthermore, mice deficient in Paneth cells (SOX9 villin Cre mice) have reduced plasma norepinephrine levels after ischemic AKI. Finally, supporting a critical role for norepinephrine in generating ischemic AKI, treatment with the selective α-adrenergic antagonists yohimbine and phentolamine protected against murine ischemic AKI with significantly reduced renal tubular necrosis, inflammation, and apoptosis and less hepatic dysfunction. Taken together, we identify Paneth cells as a critical source of norepinephrine release that may lead to intestinal and liver injury and systemic inflammation after AKI.

Butyric Acid and Leucine Induce α-Defensin Secretion from Small Intestinal Paneth Cells.

The intestine not only plays a role in fundamental processes in digestion and nutrient absorption, but it also has a role in eliminating ingested pathogenic bacteria and viruses. Paneth cells, which reside at the base of small intestinal crypts, secrete α-defensins and contribute to enteric innate immunity through potent microbicidal activities. However, the relationship between food factors and the innate immune functions of Paneth cells remains unknown. Here, we examined whether short-chain fatty acids and amino acids induce α-defensin secretion from Paneth cells in the isolated crypts of small intestine. Butyric acid and leucine elicit α-defensin secretion by Paneth cells, which kills Salmonella typhimurium. We further measured Paneth cell secretion in response to butyric acid and leucine using enteroids, a three-dimensional ex vivo culture system of small intestinal epithelial cells. Paneth cells expressed short-chain fatty acid receptors, Gpr41, Gpr43, and Gpr109a mRNAs for butyric acid, and amino acid transporter Slc7a8 mRNA for leucine. Antagonists of Gpr41 and Slc7a8 inhibited granule secretion by Paneth cells, indicating that these receptor and transporter on Paneth cells induce granule secretion. Our findings suggest that Paneth cells may contribute to intestinal homeostasis by secreting α-defensins in response to certain nutrients or metabolites.

Impact of Maternal Malnutrition on Gut Barrier Defense: Implications for Pregnancy Health and Fetal Development.

Small intestinal Paneth cells, enteric glial cells (EGC), and goblet cells maintain gut mucosal integrity, homeostasis, and influence host physiology locally and through the gut-brain axis. Little is known about their roles during pregnancy, or how maternal malnutrition impacts these cells and their development. Pregnant mice were fed a control diet (CON), undernourished by 30% vs. control (UN), or fed a high fat diet (HF). At day 18.5 (term = 19), gut integrity and function were assessed by immunohistochemistry and qPCR. UN mothers displayed reduced mRNA expression of Paneth cell antimicrobial peptides (AMP; Lyz2, Reg3g) and an accumulation of villi goblet cells, while HF had reduced Reg3g and mucin (Muc2) mRNA and increased lysozyme protein. UN fetuses had increased mRNA expression of gut transcription factor Sox9, associated with reduced expression of maturation markers (Cdx2, Muc2), and increased expression of tight junctions (TJ; Cldn-7). HF fetuses had increased mRNA expression of EGC markers (S100b, Bfabp, Plp1), AMP (Lyz1, Defa1, Reg3g), and TJ (Cldn-3, Cldn-7), and reduced expression of an AMP-activator (Tlr4). Maternal malnutrition altered expression of genes that maintain maternal gut homeostasis, and altered fetal gut permeability, function, and development. This may have long-term implications for host-microbe interactions, immunity, and offspring gut-brain axis function.

Mist1 Expression Is Required for Paneth Cell Maturation.

BackgroundPaneth cells are professional secretory cells found within the small intestinal crypt epithelium. Although their role as part of the innate immune complex providing antimicrobial secretory products is well-known, the mechanisms that control secretory capacity are not well-understood. MIST1 is a scaling factor that is thought to control secretory capacity of exocrine cells.MethodsMist1+/+ and Mist1–/– mice were used to evaluate the function of MIST1 in small intestinal Paneth cells. We used histologic and immunofluorescence staining to evaluate small intestinal tissue for proliferation and lineage allocation. Total RNA was isolated to evaluate gene expression. Enteroid culture was used to evaluate the impact of the absence of MIST1 expression on intestinal stem cell function.ResultsAbsence of MIST1 resulted in increased numbers of Paneth cells exhibiting an intermediate cell phenotype but otherwise did not alter overall epithelial cell lineage allocation. Muc2 and lysozyme staining confirmed the presence of intermediate cells at the crypt base of Mist1–/– mice. These changes were not associated with changes in mRNA expression of transcription factors associated with lineage allocation, and they were not abrogated by inhibition of Notch signaling. However, the absence of MIST1 expression was associated with alterations in Paneth cell morphology including decreased granule size and distended rough endoplasmic reticulum. Absence of MIST1 was associated with increased budding of enteroid cultures; however, there was no evidence of increased intestinal stem cell numbers in vivo.ConclusionsMIST1 plays an important role in organization of the Paneth cell secretory apparatus and managing endoplasmic reticulum stress. This role occurs downstream of Paneth cell lineage allocation.

Healthy hosts rule within: ecological forces shaping the gut microbiota.

A balanced gut microbiota is important for human health, but the mechanisms that maintain homeostasis are incompletely understood. Recent insights suggest the host plays a key role in shaping its gut microbiota to be beneficial. While host control in the small intestine curbs bacterial numbers to avoid competition for simple sugars and amino acids, the host limits oxygen availability in the large intestine to obtain microbial fermentation products from fiber. Epithelial cells are major players in imposing ecological control mechanisms, which involves the release of antimicrobial peptides by small-intestinal Paneth cells and maintenance of luminal anaerobiosis by epithelial hypoxia in the colon. Harnessing these epithelial control mechanisms for therapeutic means could provide a novel lynchpin for strategies to remediate dysbiosis.

Erratum to: Synergistic effect of aluminum and ionizing radiation upon ultrastructure, oxidative stress and apoptotic alterations in Paneth cells of rat intestine

Environmental and occupational exposure to aluminum along with ionizing radiation results in serious health problems. This study was planned to investigate the impact of oxidative stress provoked by exposure to ionizing radiation with aluminum administration upon cellular ultra structure and apoptotic changes in Paneth cells of rat small intestine . Animals received daily aluminum chloride by gastric gavage at a dose 0.5 mg/Kg BW for 4 weeks. Whole body gamma irradiation was applied at a dose 2 Gy/week up to 8 Gy. Ileum malondialdehyde, advanced oxidation protein products, and protein carbonyl were assessed as biomarkers of lipid peroxidation along with superoxide dismutase, catalase, and glutathione peroxidase activities as enzyme antioxidants. Moreover, analyses of cell cycle division and apoptotic changes were evaluated by flow cytometry. Intestinal cellular ultra structure was investigated using transmission electron microscope. Oxidative stress assessment in the ileum of rats revealed that aluminum and ionizing radiation exposure either alone or in combination exhibits a significant effect upon the increase in biomarkers of lipid peroxidation along with tumor necrosis factor-α with concomitant significant decrease of the antioxidant enzyme activities. Flow cytometric analyses showed significant alterations in the percentage of cells during cell cycle division phases along with significant increase in apoptotic cells. Ultra structurally, intestinal cellular alterations with marked injury in Paneth cells at the sites of bacterial translocation in the crypt of lumens were recorded. The results of this study have clearly suggested that aluminum exposure and ionizing either alone or in combination induced apoptosis and oxidative stress in the Paneth cells of rat intestine, which appeared to play a major role in the pathogenesis of cellular damage. Furthermore, the interaction of these two intestinal toxic routes was found to be synergistic.

Human α-Defensin 6: A Small Peptide That Self-Assembles and Protects the Host by Entangling Microbes.

Human α-defensin 6 (HD6) is a 32-residue cysteine-rich peptide that contributes to innate immunity by protecting the host at mucosal sites. This peptide is produced in small intestinal Paneth cells, stored as an 81-residue precursor peptide named proHD6 in granules, and released into the lumen. One unusual feature of HD6 is that it lacks the broad-spectrum antimicrobial activity observed for other human α-defensins, including the Paneth cell peptide human α-defensin 5 (HD5). HD6 exhibits unprecedented self-assembly properties, which confer an unusual host-defense function. HD6 monomers self-assemble into higher-order oligomers termed "nanonets", which entrap microbes and prevent invasive gastrointestinal pathogens such as Salmonella enterica serovar Typhimurium and Listeria monocytogenes from entering host cells. One possible advantage of this host-defense mechanism is that HD6 helps to keep microbes in the lumen such that they can be excreted or attacked by other components of the immune system, such as recruited neutrophils. In this Account, we report our current understanding of HD6 and focus on work published since 2012 when Bevins and co-workers described the discovery of HD6 nanonets in the literature. First, we present studies that address the biosynthesis, storage, and maturation of HD6, which demonstrate that nature uses a propeptide strategy to spatially and temporally control the formation of HD6 nanonets in the small intestine. The propeptide is stored in Paneth cell granules, and proteolysis occurs during or following release into the lumen, which affords the 32-residue mature peptide that self-assembles. We subsequently highlight structure-function studies that provide a foundation for understanding the molecular basis for why HD6 exhibits unusual self-assembly properties compared with other characterized defensins. The disposition of hydrophobic residues in the HD6 primary structure differs from that of other human α-defensins and is an important structural determinant for oligomerization. Lastly, we consider functional studies that illuminate how HD6 contributes to mucosal immunity. We recently discovered that in addition to blocking bacterial invasion into host epithelial cells by Gram-negative and Gram-positive gastrointestinal pathogens, HD6 suppresses virulence traits displayed by the opportunistic human fungal pathogen Candida albicans. In particular, we found that C. albicans biofilm formation, which causes complications in the treatment of candidiasis, is inhibited by HD6. This observation suggests that HD6 may contribute to intestinal homeostasis by helping to keep C. albicans in its commensal state. We intend for this Account to inspire further biochemical, biophysical, and biological investigations that will advance our understanding of HD6 in mucosal immunity and the host-microbe interaction.

Elucidating the Bacterial Membrane Disruption Mechanism of Human α-Defensin 5: A Theoretical Study.

Human α-defensin 5 (HD5) is a broad-spectrum antibacterial peptide produced by small intestinal Paneth cells. Despite considerable experimental evidence for the correlation between bacterial membrane destruction and the antibacterial activity of HD5, its membrane disruption mechanism remains unclear. Using all-atom molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area analysis, we demonstrate the membrane disruption mechanism of HD5 based on the intrinsic binding of HD5 to Gram-negative (GN) bacterial inner membrane. It was found that both monomer and dimer forms of HD5 bind to the surface of the GN membrane rather than embedding in the hydrophobic core region of the bilayer. Regardless of the form of HD5, the peptide orientated itself similarly on the membrane surface with an inward-pointing electric dipole moment and an outward-pointing hydrophobic dipole moment. We investigated its possible membrane disruption mechanisms and determined that anionic lipid clustering is a plausible mode of action for HD5. Relative binding free energy analysis revealed that electrostatic interactions play a major role in this mechanism. Our findings shed light on the biophysical phenomena of HD5-GN membrane binding and suggest a possible membrane disruption mechanism for HD5. This analysis of the fundamental binding properties of the monomeric HD5-GN membrane complex provides a useful guide for defensin-derived antibiotic design.

Synergistic effect of aluminum and ionizing radiation upon ultrastructure, oxidative stress and apoptotic alterations in Paneth cells of rat intestine.

Environmental and occupational exposure to aluminum along with ionizing radiation results in serious health problems. This study was planned to investigate the impact of oxidative stress provoked by exposure to ionizing radiation with aluminum administration upon cellular ultra structure and apoptotic changes in Paneth cells of rat small intestine . Animals received daily aluminum chloride by gastric gavage at a dose 0.5mg/Kg BW for 4weeks. Whole body gamma irradiation was applied at a dose 2Gy/week up to 8Gy. Ileum malondialdehyde, advanced oxidative protein products, protein carbonyl and tumor necrosis factor-alpha were assessed as biomarkers of lipid peroxidation, protein oxidation and inflammation respectively along with superoxide dismutase, catalase, and glutathione peroxidase activities as enzymatic antioxidants. Moreover, analyses of cell cycle division and apoptotic changes were evaluated by flow cytometry. Intestinal cellular ultra structure was investigated using transmission electron microscope.Oxidative and inflammatory stresses assessment in the ileum of rats revealed that aluminum and ionizing radiation exposures exhibited a significant effect upon the increase in oxidative stress biomarkers along with the inflammatory marker tumor necrosis factor-α accompanied by a significant decreases in the antioxidant enzyme activities. Flow cytometric analyses showed significant alterations in the percentage of cells during cell cycle division phases along with significant increase in apoptotic cells. Ultra structurally, intestinal cellular alterations with marked injury in Paneth cells at the sites of bacterial translocation in the crypt of lumens were recorded. The results of this study have clearly showed that aluminum and ionizing radiation exposures induced apoptosis with oxidative and inflammatory disturbance in the Paneth cells of rat intestine, which appeared to play a major role in the pathogenesis of cellular damage. Furthermore, the interaction of these two intestinal toxic routes was found to be synergistic.

O-011 Paneth Cell Phenotypes Define a Subtype of Pediatric Crohnʼs Disease Through Alterations in Host-Microbial Interactions

To identify and develop novel targeted therapies for complex diseases such as Crohn's disease (CD), functional subtypes rooted in pathogenesis must be defined. One candidate method to subtype CD is to define the small intestinal Paneth cell phenotypes based on the intracellular distribution of antimicrobial proteins. We have previously shown that in CD patients, Paneth cell phenotype correlates with the patients' genotype, distinct gene expression signature, presence of granuloma (pathologic hallmark), and time to recurrence after surgery. However, the mechanism by which abnormal Paneth cells contribute to pathogenesis and sub-classify disease in the context of host-microbial interaction is unclear. We analyzed Paneth cell phenotype and its correlation with mucosal microbiome and transcriptome in a cohort of pediatric CD and non-inflammatory bowel disease (IBD) patients.We first retrospectively analyzed Paneth cell phenotypes using archived resection specimens from adult (n = 531) and pediatric (n = 73) CD patients. We next analyzed a prospectively recruited pediatric cohort, including CD (n = 44) and non-IBD (n = 62) patients aged 4 to 18. These patients were recruited at the time of routine endoscopy. Ileal mucosal biopsy samples were collected and analyzed for Paneth cell phenotype (lysozyme/defensin 5 immunofluorescence), mucosal microbiome (16S rRNA sequencing), and transcriptome (RNA-sequencing). Paneth cell phenotype was determined by the percentage of normal Paneth cells in each sample. Type I Paneth cell phenotype was defined as <80% normal Paneth cells, whereas Type II Paneth cell phenotype was defined as ≥80% normal Paneth cells.The prevalence of type I Paneth cell phenotype in pediatric CD patients was higher than in adult CD cohorts (47% versus 18%; P < 0.0001). In pediatric CD patients, the type I Paneth cell phenotype was associated with significant changes in the ileal mucosal microbiome, characterized by reduced abundance of barrier-associated microbes (Faecalibacterium, Blautia, Ruminococcaceae, Porphyromonas, Lachnospira, Peptostreptococcus, Anaerostipes, and Odoribacteraceae) and enrichment of potentially pro-inflammatory microbes (Corynebacterium and Erysipelotrichaceae). In addition, pediatric CD patients with type I Paneth cell phenotype also displayed an altered epithelial gene expression profile, with significant reduction in oxidative phosphorylation gene cluster. Furthermore, the down regulation of oxidative phosphorylation gene cluster in CD was in turn associated with reduced abundance of Faecalibacterium, suggesting a complex network between Paneth cell function, epithelial energy/metabolism, and microbiome homeostasis. The connections between Paneth cell phenotypes, microbiome, and transcriptome profiles were not observed in non-IBD patients.These data support a functional role for Paneth cells in subtyping CD that is based on a specific pattern of metabolic dysregulation and mucosal dysbiosis.

Paneth cell α-defensin 6 (HD-6) is an antimicrobial peptide.

Defensins protect human barriers from commensal and pathogenic microorganisms. Human α-defensin 6 (HD-6) is produced exclusively by small intestinal Paneth cells but, in contrast to other antimicrobial peptides (AMPs) for HD-6, no direct antibacterial killing activity has been detected so far. Herein, we systematically tested how environmental factors, like pH and reducing conditions, affect antimicrobial activity of different defensins against anaerobic bacteria of the human intestinal microbiota. Remarkably, by mimicking the intestinal milieu we detected for the first time antibacterial activity of HD-6. Activity was observed against anaerobic gut commensals but not against some pathogenic strains. Antibiotic activity was attributable to the reduced peptide and independent of free cysteines or a conserved histidine residue. Furthermore, the oxidoreductase thioredoxin, which is also expressed in Paneth cells, is able to reduce a truncated physiological variant of HD-6. Ultrastructural analyses revealed that reduced HD-6 causes disintegration of cytoplasmic structures and alterations in the bacterial cell envelope, while maintaining extracellular net-like structures. We conclude that HD-6 is an antimicrobial peptide. Our data suggest two distinct antimicrobial mechanisms by one peptide: HD-6 kills specific microbes depending on the local environmental conditions, whereas known microbial trapping by extracellular net structures is independent of the reducing milieu.

Differential Susceptibility of Bacteria to Mouse Paneth Cell α-Defensins under Anaerobic Conditions.

Small intestinal Paneth cells secrete α-defensin peptides, termed cryptdins (Crps) in mice, into the intestinal lumen, where they confer immunity to oral infections and define the composition of the ileal microbiota. In these studies, facultative bacteria maintained under aerobic or anaerobic conditions displayed differential sensitivities to mouse α-defensins under in vitro assay conditions. Regardless of oxygenation, Crps 2 and 3 had robust and similar bactericidal activities against S. typhimurium and S. flexneri, but Crp4 activity against S. flexneri was attenuated in the absence of oxygen. Anaerobic bacteria varied in their susceptibility to Crps 2-4, with Crp4 showing less activity than Crps 2 and 3 against Enterococcus faecalis, and Bacteroides fragilis in anaerobic assays, but Fusobacterium necrophorum was killed only by Crp4 and not by Crps 2 and 3. The influence of anaerobiosis in modulating Crp bactericidal activities in vitro suggests that α-defensin effects on the enteric microbiota may be subject to regulation by local oxygen tension.

TCF-1-mediated Wnt signaling regulates Paneth cell innate immune defense effectors HD-5 and -6: implications for Crohn's disease

Wnt signaling regulates small intestinal stem cell maintenance and Paneth cell differentiation. In patients with ileal Crohn's disease (CD), a decrease of Paneth cell α-defensins has been observed that is partially caused by impaired TCF-4 and LRP6 function. Here we show reduced expression of the Wnt signaling effector TCF-1 (also known as TCF-7) in patients with ileal CD. Reporter gene assays and in vitro promoter binding analysis revealed that TCF-1 activates α-defensin HD-5 and HD-6 transcription in cooperation with β-catenin and that activation is mediated by three distinct TCF binding sites. EMSA analysis showed binding of TCF-1 to the respective motifs. In ileal CD patients, TCF-1 mRNA expression levels were significantly reduced. Moreover, we found specifically reduced expression of active TCF-1 mRNA isoforms. Tcf-1 knockout mice exhibited reduced cryptdin expression in the jejunum, which was not consistently seen at other small intestinal locations. Our data provide evidence that TCF-1-mediated Wnt signaling is disturbed in small intestinal CD, which might contribute to the observed barrier dysfunction in the disease.

Expression of group XIIA phospholipase A2 in human digestive organs.

Cellular distribution of group XIIA phospholipase A2 (GXIIA PLA2) was studied in human digestive organs by immunohistochemistry. GXIIA PLA2 protein was detected in epithelial cells of normal gastrointestinal tract, gallbladder and pancreatic acinar cells. The GXIIA PLA2 protein was evenly distributed in the cytoplasm in contrast to secretory granular distribution of GIB PLA2 and GIIA PLA2 in pancreatic acinar cells and small intestinal Paneth cells respectively. Epithelial cells of intestinal glands in Crohn's disease and ulcerative colitis expressed abundant GXIIA PLA2 , whereas inflammatory cells were devoid of the enzyme protein. Tumour cells in colonic adenomas and carcinomas and pancreatic ductogenic carcinomas expressed GXIIA PLA2 protein at varying intensity levels. The putative functions of GXIIA PLA2 remain to be investigated and its role in healthy and diseased digestive organs can only be speculated on at present.

Sa1982 High Fat Diet Induced Upregulation of EP2 Receptor in Small Intestine Paneth Cells Is Responsible for the Activation of Unfolded Protein Response and Reduction of, α Defensins in Rats

Sa1982 High Fat Diet Induced Upregulation of EP2 Receptor in Small Intestine Paneth Cells Is Responsible for the Activation of Unfolded Protein Response and Reduction of, α Defensins in Rats

Hydrophobic Determinants of α-Defensin Bactericidal Activity

Mammalian α-defensins are approximately 4- to 5-kDa broad-spectrum antimicrobial peptides and abundant granule constituents of neutrophils and small intestinal Paneth cells. The bactericidal activities of amphipathic α-defensins depend in part on electropositive charge and on hydrophobic amino acids that enable membrane disruption by interactions with phospholipid acyl chains. Alignment of α-defensin primary structures identified conserved hydrophobic residues in the loop formed by the Cys(III)-Cys(V) disulfide bond, and we have studied their role by testing the effects of mutagenesis on bactericidal activities. Mouse α-defensin 4 (Crp-4) and rhesus myeloid α-defensin 4 (RMAD-4) were selected for these studies, because they are highly bactericidal in vitro and have the same overall electropositive charge. Elimination of hydrophobicity by site-directed mutagenesis at those positions in Crp-4 attenuated bactericidal activity markedly. In contrast to native Crp-4, the (I23/F25/L26/G)-Crp-4 variant lacked bactericidal activity against Salmonella enterica serovar Typhimurium and did not permeabilize Escherichia coli ML35 cells as a result of removing aliphatic side chains by Gly substitutions. Ala replacements in (I23/F25/L26/A)-Crp-4 restored activity, evidence that hydrophobicity contributed by Ala methyl R-groups was sufficient for activity. In macaques, neutrophil α-defensin RMAD-6 is identical to RMAD-4, except for a F28S difference, and (F28S)-RMAD-4 mutagenesis attenuated RMAD-4 bactericidal activity and E. coli permeabilization. Interestingly, (R31/32D)-Crp-4 lacks activity in these assays despite the presence of the Ile23, Phe25, and Leu26 hydrophobic patch. We infer that electrostatic interactions between cationic α-defensin residues and negative charge on bacteria precede interactions between critical hydrophobic residue positions that mediate membrane disruption and bacterial cell killing.