Project description:BACKGROUND: The gut microbiome and the host immune system interact synergistically to maintain intestinal health and defend against infections. Toll-like receptor 5 (TLR5), known for recognizing bacterial flagellin, plays a pivotal role in this regulatory network. Despite significant advances in understanding the gut microbiome and TLR5, the precise role of TLR5 in gut health and infection resistance remains unclear. This study aimed to elucidate the role of TLR5 in regulating gut microbiota and metabolites and their impact on resistance to Salmonella infection using a TLR5 intestinal-specific overexpression mouse model. METHODS: Fecal microbiota and metabolite transplantation experiments were conducted using feces from TLR5-overexpressing mice (TLR5+/+ group) and wild-type mice (WT group) into C57 mice, creating four experimental groups: Flora-TLR5-C57, Metabolite-TLR5-C57, Flora-WT-C57 and Metabolite-WT-C57. Mice were infected with Salmonella, and various parameters including survival time, fecal Salmonella load, and alterations in gut microbiota and metabolites were assessed. RESULTS: Despite notable shifts in gut microbiota composition and metabolite profiles induced by TLR5 overexpression, no significant differences in fecal Salmonella load or survival time were observed across the experimental groups. TLR5-overexpressing mice exhibited an enrichment of beneficial bacterial taxa and significant changes in metabolites; however, these alterations did not correlate with enhanced resistance to Salmonella infection. CONCLUSION: Our findings suggest that while TLR5 overexpression significantly modifies gut microbiota and metabolite profiles, these changes do not directly enhance resistance to Salmonella infection. The immune response to Salmonella is multifaceted and involves various components beyond the gut microbiota and metabolites. Further research is required to elucidate the detailed molecular pathways through which TLR5 influences infection outcomes and to explore potential combined therapeutic strategies for enhancing host defenses against enteric pathogens.

Project description:The gastrointestinal tract is colonized by trillions of microorganisms collectively known as the gut microbiota. These microbes provide essential signals to support healthy gut function. The microbiota is separated from internal tissue by a single layer of intestinal epithelial cells that not only provides a physical barrier but also relays luminal signals to underlying gut immune cells. Altered microbiota composition including loss of anti-inflammatory microbes or outgrowth of mucosa-associated bacteria such as adherent-invasive E. coli (AIEC) are hallmarks of inflammatory disease including inflammatory bowel disease (IBD). In contrast to their hypothesized role in pathology, we recently identified select AIEC isolates that improve outcomes in mouse colitis models. These AIEC induce macrophage production of the anti-inflammatory cytokine IL-10 which limits gut inflammation and supports barrier repair. These benefits were lost if the AIEC was unable to attach to epithelial cells. However, the epithelial signaling underlying this protection remained unclear. To understand if intestinal epithelial cells signaled to immune cells after microbial attachment, we utilized human colonic organoid monolayers and found co-culture with a subset of AIEC isolates upregulated immune regulatory genes including CCL2, a macrophage recruiting chemokine. This effect was only observed in undifferenced epithelial cells, indicating epithelial stem cell recognition of microbes leads to macrophage recruitment. In vivo, antibody blockade of CCR2 abrogated the protective effect of AIEC colonization. Using bacterial transcriptome analysis, we identified high flagellin expression in AIEC isolates that activated epithelial signaling, with lost signaling in organoids deficient for TLR5, the receptor for flagellin. Together our findings suggest intestinal epithelial cells recognize microbial signals to coordinate macrophage recruitment that support intestinal repair, protecting from colitis.

Project description:The healthy gut microbiota is composed of three different functional subgroups, symbionts, commensals and pathobionts. Additionally, infection with pathogenic bacteria can occur. Microbe Associated Molecular Patterns (MAMPs) are expressed by most members of both, microbiota and pathogens and interact with Pattern Recognition Receptors (PRRs), resulting in activation of the innate immune system. It is still unclear whether the interaction of PRRs with MAMPs of different origin, leads to a differential activation of innate immunity and whether thereby, distinction can be made between symbionts, commensals, pathobionts and pathogens. Here we addressed the question whether the interaction of flagellin with Toll-like receptor 5 (TLR5) might be a possible mechanism of the innate immune system to distinguish between the different microbial subgroups and initiate the respective immune response. To characterize distinct cellular immune responses to symbiont or pathogen derived flagellin we run transcriptomics, phosphoproteomics and proteomics of TLR5 overexpressing cells. Responses to different flagellins were characterized by differential kinetics and regulation of protein phosphorylation as well as variant protein expression patterns. In vivo experiments revealed that commensal derived flagellin mediated an inert TLR5 signal, thus not affecting Dextran-Sodium-Sulfate (DSS) induced intestinal inflammation. In vitro pathogenic derived flagellin induced a strong pro-inflammatory TLR5 response and did not ameliorate intestinal inflammation. In contrast, symbiont derived flagellin revealed an intermediate TLR5 activation and clearly ameliorated DSS induced intestinal inflammation thus shifting the host immune response in favor of mucosal immune balance. Bone marrow chimeric mice proofed that CD11c+TLR5+ intestinal lamina propria cells mediate the anti-inflammatory symbiotic effect of flagellin. We suggest that the quality of flagellin-induced TL signal might determine the balance between homeostasis and intestinal inflammation and may help the innate immune system to distinguish between symbionts, commensals, pathobionts and pathogenic microbes. We used microarrays to analyze differential expression of genes between treatments with flagellins of E. coli Nissle (EcN) and Salmonella Typhimurium SL1344 (ST).

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Cytosolic flagellin receptor NLRC4 protects mice against mucosal and systemic challenges

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