Paneth cells directly sense gut commensals and maintain homeostasis at the intestinal host-microbial interface.
ABSTRACT: The intestinal epithelium is in direct contact with a vast microbiota, yet little is known about how epithelial cells defend the host against the heavy bacterial load. To address this question we studied Paneth cells, a key small intestinal epithelial lineage. We found that Paneth cells directly sense enteric bacteria through cell-autonomous MyD88-dependent toll-like receptor (TLR) activation, triggering expression of multiple antimicrobial factors. Paneth cells were essential for controlling intestinal barrier penetration by commensal and pathogenic bacteria. Furthermore, Paneth cell-intrinsic MyD88 signaling limited bacterial penetration of host tissues, revealing a role for epithelial MyD88 in maintaining intestinal homeostasis. Our findings establish that gut epithelia actively sense enteric bacteria and play an essential role in maintaining host-microbial homeostasis at the mucosal interface.
Project description: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.
Project description:Paneth cells reside at the base of crypts of the small intestine and secrete antimicrobial factors to control gut microbiota. Paneth cell loss is observed in the chronically inflamed intestine, which is often associated with increased reactive oxygen species (ROS). However, the relationship between Paneth cell loss and ROS is not yet clear. Intestinal epithelial-specific deletion of a protein kinase Tak1 depletes Paneth cells and highly upregulates ROS in the mouse model. We found that depletion of gut bacteria or myeloid differentiation factor 88 (Myd88), a mediator of bacteria-derived cell signaling, reduced ROS but did not block Paneth cell loss, suggesting that gut bacteria are the cause of ROS accumulation but bacteria-induced ROS are not the cause of Paneth cell loss. In contrast, deletion of the necroptotic cell death signaling intermediate, receptor-interacting protein kinase 3 (Ripk3), partially blocked Paneth cell loss. Thus, Tak1 deletion causes Paneth cell loss in part through necroptotic cell death. These results suggest that TAK1 participates in intestinal integrity through separately modulating bacteria-derived ROS and RIPK3-dependent Paneth cell loss.
Project description:Paneth cells are intestinal epithelial cells that release antimicrobial peptides, such as α-defensin as part of host defense. Together with mesenchymal cells, Paneth cells provide niche factors for epithelial stem cell homeostasis. Here, we report two subtypes of murine Paneth cells, differentiated by their production and utilization of fucosyltransferase 2 (Fut2), which regulates α(1,2)fucosylation to create cohabitation niches for commensal bacteria and prevent invasion of the intestine by pathogenic bacteria. The majority of Fut2<sup>-</sup> Paneth cells were localized in the duodenum, whereas the majority of Fut2<sup>+</sup> Paneth cells were in the ileum. Fut2<sup>+</sup> Paneth cells showed higher granularity and structural complexity than did Fut2<sup>-</sup> Paneth cells, suggesting that Fut2<sup>+</sup> Paneth cells are involved in host defense. Signaling by the commensal bacteria, together with interleukin 22 (IL-22), induced the development of Fut2<sup>+</sup> Paneth cells. IL-22 was found to affect the α-defensin secretion system via modulation of <i>Fut2</i> expression, and IL-17a was found to increase the production of α-defensin in the intestinal tract. Thus, these intestinal cytokines regulate the development and function of Fut2<sup>+</sup> Paneth cells as part of gut defense.
Project description: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 ?-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 ?-defensin secretion from Paneth cells in the isolated crypts of small intestine. Butyric acid and leucine elicit ?-defensin secretion by Paneth cells, which kills <i>Salmonella typhimurium</i>. 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, <i>Gpr41</i>, <i>Gpr43</i>, and <i>Gpr109a</i> mRNAs for butyric acid, and amino acid transporter <i>Slc7a8</i> 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 ?-defensins in response to certain nutrients or metabolites.
Project description: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 alpha-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 alpha-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 alpha-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 alpha-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 alpha-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.
Project description:Paneth cell-derived enteric antimicrobial peptides provide protection from intestinal infection and maintenance of enteric homeostasis. Paneth cells, however, evolve only after the neonatal period, and the antimicrobial mechanisms that protect the newborn intestine are ill defined. Using quantitative reverse transcription-polymerase chain reaction, immunohistology, reverse-phase high-performance liquid chromatography, and mass spectrometry, we analyzed the antimicrobial repertoire in intestinal epithelial cells during postnatal development. Surprisingly, constitutive expression of the cathelin-related antimicrobial peptide (CRAMP) was observed, and the processed, antimicrobially active form was identified in neonatal epithelium. Peptide synthesis was limited to the first two weeks after birth and gradually disappeared with the onset of increased stem cell proliferation and epithelial cell migration along the crypt-villus axis. CRAMP conferred significant protection from intestinal bacterial growth of the newborn enteric pathogen Listeria monocytogenes. Thus, we describe the first example of a complete developmental switch in innate immune effector expression and anatomical distribution. Epithelial CRAMP expression might contribute to bacterial colonization and the establishment of gut homeostasis, and provide protection from enteric infection during the postnatal period.
Project description:The development and severity of inflammatory bowel diseases and other chronic inflammatory conditions can be influenced by host genetic and environmental factors, including signals derived from commensal bacteria. However, the mechanisms that integrate these diverse cues remain undefined. Here we demonstrate that mice with an intestinal epithelial cell (IEC)-specific deletion of the epigenome-modifying enzyme histone deacetylase 3 (HDAC3(?IEC) mice) exhibited extensive dysregulation of IEC-intrinsic gene expression, including decreased basal expression of genes associated with antimicrobial defence. Critically, conventionally housed HDAC3(?IEC) mice demonstrated loss of Paneth cells, impaired IEC function and alterations in the composition of intestinal commensal bacteria. In addition, HDAC3(?IEC) mice showed significantly increased susceptibility to intestinal damage and inflammation, indicating that epithelial expression of HDAC3 has a central role in maintaining intestinal homeostasis. Re-derivation of HDAC3(?IEC) mice into germ-free conditions revealed that dysregulated IEC gene expression, Paneth cell homeostasis and intestinal barrier function were largely restored in the absence of commensal bacteria. Although the specific mechanisms through which IEC-intrinsic HDAC3 expression regulates these complex phenotypes remain to be determined, these data indicate that HDAC3 is a critical factor that integrates commensal-bacteria-derived signals to calibrate epithelial cell responses required to establish normal host-commensal relationships and maintain intestinal homeostasis.
Project description:Intestinal epithelial cells (IECs) form a physical and immunological barrier that separates the vast gut microbiota from host tissues. MyD88-dependent Toll-like receptor signaling is a key mediator of microbial-host cross-talk. We examined the role of epithelial MyD88 expression by generating mice with an IEC-targeted deletion of the Myd88 gene (MyD88(ΔIEC)). Loss of epithelial MyD88 signaling resulted in increased numbers of mucus-associated bacteria; translocation of bacteria, including the opportunistic pathogen Klebsiella pneumoniae, to mesenteric lymph nodes; reduced transmucosal electrical resistance; impaired mucus-associated antimicrobial activity; and downregulated expression of polymeric immunoglobulin receptor (the epithelial IgA transporter), mucin-2 (the major protein of intestinal mucus), and the antimicrobial peptides RegIIIγ and Defa-rs1. We further observed significant differences in the composition of the gut microbiota between MyD88(ΔIEC) mice and wild-type littermates. These physical, immunological, and microbial defects resulted in increased susceptibility of MyD88(ΔIEC) mice to experimental colitis. We conclude that MyD88 signaling in IECs is crucial for maintenance of gut homeostasis.
Project description:Paneth cell-derived enteric antimicrobial peptides significantly contribute to antibacterial host defense and host-microbial homeostasis. Regulation occurs by enzymatic processing and release into the small intestinal lumen, but the stimuli involved are incompletely understood. Here, the capacity of various microbial and immune stimuli to induce antimicrobial peptide release from small intestinal tissue was systematically evaluated using antibacterial activity testing, immunostaining for Paneth cell granules and mass spectrometry. We confirmed the stimulatory activity of the muscarinic receptor agonist carbachol and the nucleotide-binding oligomerization domain ligand muramyl dipeptide. In contrast, no release of antibacterial activity was noted after treatment with the Toll-like receptor ligands poly(I:C), lipopolysaccharide or CpG, and the cytokines interleukin (IL)-15, IL-22, IL-28 and interferon-?. Rapid Paneth cell degranulation and antimicrobial activity release, however, was observed after stimulation with the endogenous mediators IL-4 and IL-13. This process required phosphatidylinositol 3-kinase and was associated with protein kinase B phosphorylation in Paneth cells. Flow cytometric analysis confirmed expression of the IL-13 receptor ?1 on isolated Paneth cells. Our findings identify a novel role of IL-13 as inducer of Paneth cell degranulation and enteric antimicrobial peptide release. IL-13 may thus contribute to mucosal antimicrobial host defense and host microbial homeostasis.
Project description:Paneth cell ?-defensins are antimicrobial peptides involved in the control of the intestinal microbiota and immunological homeostasis. In mice, they are encoded by multiple, highly homologous genes (Defa). The transcriptional activity of ileal Defa genes was studied in response to pharmacological and genetic perturbations of the intestinal environment of C57BL/6 mice. Defa gene transcription was sensitive to oral antibiotic administration suggesting that commensal microbes regulate Defa expression. Ileal microbiota analysis showed that decreased transcription of Defa genes correlated with depletion of Lactobacillus. Defa expression was partially restored in vivo by lactobacillus administration to antibiotic-treated mice. Defa transcripts were less abundant in ex vivo, microbiota-free intestinal explants but recovered after explant exposure to UV-killed bacteria, Toll-like receptor (TLR)-2 or TLR4 agonists. Genetic deficiency of several TLRs or MyD88 led to dramatic drops in Defa transcription in vivo. These results show that Paneth cell Defa genes are regulated by commensal bacteria through TLR-MyD88 signaling and provide a further understanding of the dysregulation of intestinal homeostasis that occurs as a result of imbalances in the populations of commensal bacteria.