A small intestinal organoid model of non-invasive enteric pathogen-epithelial cell interactions.
ABSTRACT: Organoids mirror in vivo tissue organization and are powerful tools to investigate the development and cell biology of the small intestine. However, their application for the study of host-pathogen interactions has been largely unexplored. We have established a model using microinjection of organoids to mimic enteric infection, allowing for direct examination of pathogen interactions with primary epithelial cells in the absence of confounding variables introduced by immune cells or the commensal microbiota. We investigated the impact of Paneth cell ?-defensin antimicrobial peptides on bacterial growth. We demonstrate that organoids form a sealed lumen, which contains concentrations of ?-defensins capable of restricting growth of multiple strains of Salmonella enterica serovar Typhimurium for at least 20?h postinfection. Transgenic expression of human defensin 5 in mouse organoids lacking functional murine ?-defensins partially restored bacterial killing. We also found that organoids from NOD2(-/-) mice were not impaired in ?-defensin expression or antibacterial activity. This model is optimized for the study of non-invasive bacteria but can be extended to other enteric pathogens and is amenable to further genetic manipulation of both the host and microbe to dissect this critical interface of host defense.
Project description:Antimicrobial peptides are secreted by small intestinal Paneth cells as components of innate immunity. To investigate the role of alpha-defensins in enteric host defenses in nonhuman primates, alpha-defensin cDNAs were isolated, alpha-defensin peptides were purified from rhesus macaque small bowel, and the bactericidal activities of the peptides were measured. Six rhesus enteric alpha-defensin (RED) cDNAs, RED-1 to RED-6, were identified in a jejunum cDNA library; the deduced RED peptides exhibited extensive diversity relative to the primary structures of rhesus myeloid alpha-defensins. RED-4 was purified from monkey jejunum, and N-terminal peptide sequencing of putative RED-4 peptides identified two N termini, RTCYCRTGR. and TCYCRTGRC.; these corresponded to alternative N termini for the RED-4 molecules, as deduced from their molecular masses and RED cDNAs. In situ hybridization experiments localized RED mRNAs exclusively to small intestinal Paneth cells. Recombinant RED-1 to RED-4 were purified to homogeneity and shown to be microbicidal in the low micromolar range (</=10 micro g/ml) against gram-positive and gram-negative bacteria, with individual peptides exhibiting variable target cell specificities. Thus, compared to myeloid alpha-defensins from rhesus macaques, enteric alpha-defensin peptides are highly variable in both primary structure and activity. These studies should facilitate further analyses of the role of alpha-defensins in primate enteric immunity.
Project description:Antimicrobial peptides are important effectors of innate immunity throughout the plant and animal kingdoms. In the mammalian small intestine, Paneth cell alpha-defensins are antimicrobial peptides that contribute to host defense against enteric pathogens. To determine if alpha-defensins also govern intestinal microbial ecology, we analyzed the intestinal microbiota of mice expressing a human alpha-defensin gene (DEFA5) and in mice lacking an enzyme required for the processing of mouse alpha-defensins. In these complementary models, we detected significant alpha-defensin-dependent changes in microbiota composition, but not in total bacterial numbers. Furthermore, DEFA5-expressing mice had striking losses of segmented filamentous bacteria and fewer interleukin 17 (IL-17)-producing lamina propria T cells. Our data ascribe a new homeostatic role to alpha-defensins in regulating the makeup of the commensal microbiota.
Project description:Multicellular organisms utilize a battery of extracellular and cellular mechanisms to defend against microbial infiltration. Among the armamentarium used by the small intestine to defend against microbial invasion are antimicrobial peptides called defensins. We previously have shown that gut barrier function is impaired following hemorrhagic shock, resulting in translocation of bacteria or endotoxin. Using a rat model, we examined the effect of hemorrhagic shock on alpha-defensin expression. We utilized the anchored reverse transcriptase PCR strategy to isolate a rat enteric defensin cDNA. The cDNA is 406 bases in length and encodes a putative prepro-enteric defensin that we have named rat defensin 5 (RD-5). RD-5 expression is restricted to the small intestine and is specifically localized by in situ hybridization to the Paneth cells. A 10-fold increase in its steady state levels was observed in the distal intestine immediately after the termination of shock. This is the first study to show that enteric defensins are inducible following injury. We suggest that enteric defensins may contribute to the complex and integrated barrier function of the intestinal mucosal surface.
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 at the base of small intestinal crypts secrete microbicidal ?-defensins, termed cryptdins (Crps) in mice, as mediators of innate immunity. Proteomic studies show that five abundant Paneth cell ?-defensins in C57BL/6 mice are strain specific in that they have not been identified in other inbred strains of mice. Two C57BL/6-specific peptides are coded for by the Defcr20 and -21 genes evident in the NIH C57BL/6 genome but absent from the Celera mixed-strain assembly, which excludes C57BL/6 data and differs from the NIH build with respect to the organization of the ?-defensin gene locus. Conversely, C57BL/6 mice lack the Crp1, -2, -4, and -6 peptides and their corresponding Defcr1, -2, -4, and -6 genes, which are common to several mouse strains, including those of the Celera assembly. In C57BL/6 mice, ?-defensin gene diversification appears to have occurred by tandem duplication of a multigene cassette that was not found in the mixed-strain assembly. Both mouse genome assemblies contain conserved ?-defensin pseudogenes that are closely related to functional myeloid ?-defensin genes in the rat, suggesting that the neutrophil ?-defensin defect in mice resulted from progressive gene loss. Given the role of ?-defensins in shaping the composition of the enteric microflora, such polymorphisms may influence outcomes in mouse models of disease or infection.
Project description:The small intestinal epithelium produces numerous antimicrobial peptides and proteins, including abundant enteric ?-defensins. Although they most commonly function as potent antivirals in cell culture, enteric ?-defensins have also been shown to enhance some viral infections in vitro. Efforts to determine the physiologic relevance of enhanced infection have been limited by the absence of a suitable cell culture system. To address this issue, here we use primary stem cell-derived small intestinal enteroids to examine the impact of naturally secreted ?-defensins on infection by the enteric mouse pathogen, mouse adenovirus 2 (MAdV-2). MAdV-2 infection was increased when enteroids were inoculated across an ?-defensin gradient in a manner that mimics oral infection but not when ?-defensin levels were absent or bypassed through other routes of inoculation. This increased infection was a result of receptor-independent binding of virus to the cell surface. The enteroid experiments accurately predicted increased MAdV-2 shedding in the feces of wild type mice compared to mice lacking functional ?-defensins. Thus, our studies have shown that viral infection enhanced by enteric ?-defensins may reflect the evolution of some viruses to utilize these host proteins to promote their own infection.
Project description:The intestinal microbial ecosystem is actively regulated by Paneth cell-derived antimicrobial peptides such as ?-defensins. Various disorders, including graft-versus-host disease (GVHD), disrupt Paneth cell functions, resulting in unfavorably altered intestinal microbiota (dysbiosis), which further accelerates the underlying diseases. Current strategies to restore the gut ecosystem are bacteriotherapy such as fecal microbiota transplantation and probiotics, and no physiological approach has been developed so far. In this study, we demonstrate a novel approach to restore gut microbial ecology by Wnt agonist R-Spondin1 (R-Spo1) or recombinant ?-defensin in mice. R-Spo1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of ?-defensins. Administration of R-Spo1 or recombinant ?-defensin prevents GVHD-mediated dysbiosis, thus representing a novel and physiological approach at modifying the gut ecosystem to restore intestinal homeostasis and host-microbiota cross talk toward therapeutic benefits.
Project description:?-defensins are abundant antimicrobial peptides with broad, potent antibacterial, antifungal, and antiviral activities in vitro. Although their contribution to host defense against bacteria in vivo has been demonstrated, comparable studies of their antiviral activity in vivo are lacking. Using a mouse model deficient in activated ?-defensins in the small intestine, we show that Paneth cell ?-defensins protect mice from oral infection by a pathogenic virus, mouse adenovirus 1 (MAdV-1). Survival differences between mouse genotypes are lost upon parenteral MAdV-1 infection, strongly implicating a role for intestinal defenses in attenuating pathogenesis. Although differences in ?-defensin expression impact the composition of the ileal commensal bacterial population, depletion studies using broad-spectrum antibiotics revealed no effect of the microbiota on ?-defensin-dependent viral pathogenesis. Moreover, despite the sensitivity of MAdV-1 infection to ?-defensin neutralization in cell culture, we observed no barrier effect due to Paneth cell ?-defensin activation on the kinetics and magnitude of MAdV-1 dissemination to the brain. Rather, a protective neutralizing antibody response was delayed in the absence of ?-defensins. This effect was specific to oral viral infection, because antibody responses to parenteral or mucosal ovalbumin exposure were not affected by ?-defensin deficiency. Thus, ?-defensins play an important role as adjuvants in antiviral immunity in vivo that is distinct from their direct antiviral activity observed in cell culture.
Project description:Crohn's disease (CD) is an intractable inflammatory bowel disease, and dysbiosis, disruption of the intestinal microbiota, is associated with CD pathophysiology. ER stress, disruption of ER homeostasis in Paneth cells of the small intestine, and ?-defensin misfolding have been reported in CD patients. Because ?-defensins regulate the composition of the intestinal microbiota, their misfolding may cause dysbiosis. However, whether ER stress, ?-defensin misfolding, and dysbiosis contribute to the pathophysiology of CD remains unknown. Here, we show that abnormal Paneth cells with markers of ER stress appear in SAMP1/YitFc, a mouse model of CD, along with disease progression. Those mice secrete reduced-form ?-defensins that lack disulfide bonds into the intestinal lumen, a condition not found in normal mice, and reduced-form ?-defensins correlate with dysbiosis during disease progression. Moreover, administration of reduced-form ?-defensins to wild-type mice induces the dysbiosis. These data provide novel insights into CD pathogenesis induced by dysbiosis resulting from Paneth cell ?-defensin misfolding and they suggest further that Paneth cells may be potential therapeutic targets.
Project description:Defensins are antimicrobial peptides that contribute broadly to innate immunity, including protection of mucosal tissues. Human ?-defensin (HD) 6 is highly expressed by secretory Paneth cells of the small intestine. However, in contrast to the other defensins, it lacks appreciable bactericidal activity. Nevertheless, we report here that HD6 affords protection against invasion by enteric bacterial pathogens in vitro and in vivo. After stochastic binding to bacterial surface proteins, HD6 undergoes ordered self-assembly to form fibrils and nanonets that surround and entangle bacteria. This self-assembly mechanism occurs in vivo, requires histidine-27, and is consistent with x-ray crystallography data. These findings support a key role for HD6 in protecting the small intestine against invasion by diverse enteric pathogens and may explain the conservation of HD6 throughout Hominidae evolution.