Necroptosis has emerged as an important pathway of programmed cell death in embryonic development, tissue homeostasis, immunity and inflammation. RIPK1 is implicated in inflammatory and cell death ...signalling and its kinase activity is believed to drive RIPK3-mediated necroptosis. Here we show that kinase-independent scaffolding RIPK1 functions regulate homeostasis and prevent inflammation in barrier tissues by inhibiting epithelial cell apoptosis and necroptosis. Intestinal epithelial cell (IEC)-specific RIPK1 knockout caused IEC apoptosis, villus atrophy, loss of goblet and Paneth cells and premature death in mice. This pathology developed independently of the microbiota and of MyD88 signalling but was partly rescued by TNFR1 (also known as TNFRSF1A) deficiency. Epithelial FADD ablation inhibited IEC apoptosis and prevented the premature death of mice with IEC-specific RIPK1 knockout. However, mice lacking both RIPK1 and FADD in IECs displayed RIPK3-dependent IEC necroptosis, Paneth cell loss and focal erosive inflammatory lesions in the colon. Moreover, a RIPK1 kinase inactive knock-in delayed but did not prevent inflammation caused by FADD deficiency in IECs or keratinocytes, showing that RIPK3-dependent necroptosis of FADD-deficient epithelial cells only partly requires RIPK1 kinase activity. Epidermis-specific RIPK1 knockout triggered keratinocyte apoptosis and necroptosis and caused severe skin inflammation that was prevented by RIPK3 but not FADD deficiency. These findings revealed that RIPK1 inhibits RIPK3-mediated necroptosis in keratinocytes in vivo and identified necroptosis as a more potent trigger of inflammation compared with apoptosis. Therefore, RIPK1 is a master regulator of epithelial cell survival, homeostasis and inflammation in the intestine and the skin.
Gut microbial dysbiosis is associated with the development of autoimmune disease, but the mechanisms by which microbial dysbiosis affects the transition from asymptomatic autoimmunity to inflammatory ...disease are incompletely characterized. Here, we identify intestinal barrier integrity as an important checkpoint in translating autoimmunity to inflammation. Zonulin family peptide (zonulin), a potent regulator for intestinal tight junctions, is highly expressed in autoimmune mice and humans and can be used to predict transition from autoimmunity to inflammatory arthritis. Increased serum zonulin levels are accompanied by a leaky intestinal barrier, dysbiosis and inflammation. Restoration of the intestinal barrier in the pre-phase of arthritis using butyrate or a cannabinoid type 1 receptor agonist inhibits the development of arthritis. Moreover, treatment with the zonulin antagonist larazotide acetate, which specifically increases intestinal barrier integrity, effectively reduces arthritis onset. These data identify a preventive approach for the onset of autoimmune disease by specifically targeting impaired intestinal barrier function.
Epithelial barrier defects are implicated in the pathogenesis of inflammatory bowel disease (IBD); however, the role of microbiome dysbiosis and the cytokine networks orchestrating chronic intestinal ...inflammation in response to barrier impairment remain poorly understood. Here, we showed that altered Schaedler flora (ASF), a benign minimal microbiota, was sufficient to trigger colitis in a mouse model of intestinal barrier impairment. Colitis development required myeloid-cell-specific adaptor protein MyD88 signaling and was orchestrated by the cytokines IL-12, IL-23, and IFN-γ. Colon inflammation was driven by IL-12 during the early stages of the disease, but as the mice aged, the pathology shifted toward an IL-23-dependent inflammatory response driving disease chronicity. These findings reveal that IL-12 and IL-23 act in a temporally distinct, biphasic manner to induce microbiota-driven chronic intestinal inflammation. Similar mechanisms might contribute to the pathogenesis of IBD particularly in patients with underlying intestinal barrier defects.
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•ASF microbiota is sufficient to induce colitis upon intestinal barrier disruption•MyD88 signaling in myeloid cells mediates microbiota-induced colon inflammation•The cytokines IL-12, IL-23, and IFN-γ dynamically regulate colitis pathogenesis•IL-12 triggers colitis initiation, whereas IL-23 drives the chronic disease phase
The mechanisms triggering and sustaining intestinal inflammation remain unclear. Eftychi et al. show that a benign minimal microbiota is sufficient to elicit colitis in mice with impaired intestinal barrier. MyD88 signaling in myeloid cells and the cytokines IL-12, IL-23, and IFN-γ were required for colitis development. IL-12 and IL-23 acted sequentially to dynamically regulate disease initiation and chronicity respectively.
Alterations in enteric microbiota are associated with several highly prevalent immune-mediated and metabolic diseases
, and experiments involving faecal transplants have indicated that such ...alterations have a causal role in at least some such conditions
. The postnatal period is particularly critical for the development of microbiota composition, host-microbe interactions and immune homeostasis
. However, the underlying molecular mechanisms of this neonatal priming period have not been defined. Here we report the identification of a host-mediated regulatory circuit of bacterial colonization that acts solely during the early neonatal period but influences life-long microbiota composition. We demonstrate age-dependent expression of the flagellin receptor Toll-like receptor 5 (TLR5) in the gut epithelium of neonate mice. Using competitive colonization experiments, we demonstrate that epithelial TLR5-mediated REG3γ production is critical for the counter-selection of colonizing flagellated bacteria. Comparative microbiota transfer experiments in neonate and adult wild-type and Tlr5-deficient germ-free mice reveal that neonatal TLR5 expression strongly influences the composition of the microbiota throughout life. Thus, the beneficial microbiota in the adult host is shaped during early infancy. This might explain why environmental factors that disturb the establishment of the microbiota during early life can affect immune homeostasis and health in adulthood.
The intestinal microbiota conveys significant benefits to host physiology. Although multiple chronic disorders have been associated with alterations in the intestinal microbiota composition and ...function, it is still unclear whether these changes are a cause or a consequence. Hence, to translate microbiome research into clinical application, it is necessary to provide a proof of causality of host–microbiota interactions. This is hampered by the complexity of the gut microbiome and many confounding factors. The application of gnotobiotic animal models associated with synthetic communities allows us to address the cause–effect relationship between the host and intestinal microbiota by reducing the microbiome complexity on a manageable level. In recent years, diverse bacterial communities were assembled to analyze the role of microorganisms in infectious, inflammatory, and metabolic diseases. In this review, we outline their application and features. Furthermore, we discuss the differences between human-derived and model-specific communities. Lastly, we highlight the necessity of generating novel synthetic communities to unravel the microbial role associated with specific health outcomes and disease phenotypes. This understanding is essential for the development of novel non-invasive targeted therapeutic strategies to control and modulate intestinal microbiota in health and disease.
The composition of intrinsic microbial communities determines if invading pathogens will find a suitable niche for colonization and cause infection or be eliminated. Here, we investigate how ...commensal E. coli mediate colonization resistance (CR) against Salmonella Typhimurium (S. Tm). Using synthetic bacterial communities, we show that the capacity of E. coli Mt1B1 to block S. Tm colonization depends on the microbial context. In an infection-permissive context, E. coli utilized a high diversity of carbon sources and was unable to block S. Tm invasion. In mice that were stably colonized by twelve phylogenetically diverse murine gut bacteria (OMM12), establishing a protective context, E. coli depleted galactitol, a substrate otherwise fueling S. Tm colonization. Here, Lachnospiraceae, capable of consuming C5 and C6 sugars, critically contributed to CR. We propose that E. coli provides CR by depleting a limited carbon source when in a microbial community adept at removing simple sugars from the intestine.
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•E. coli mediates colonization resistance against Salmonella in a specific microbiota•E. coli can prevent Salmonella invasion by depleting galactitol•Lachnospiraceae consume free sugars and contribute to colonization resistance
Eberl et al. show that E. coli provides colonization resistance (CR) against Salmonella by competing for the limiting carbon source galactitol in a specific microbial context. In this protective context, Lachnospiraceae that consume C5 and C6 sugars play an essential role.
The Oligo-Mouse-Microbiota (OMM
) is a recently developed synthetic bacterial community for functional microbiome research in mouse models (Brugiroux et al., 2016). To date, the OMM
model has been ...established in several germ-free mouse facilities world-wide and is employed to address a growing variety of research questions related to infection biology, mucosal immunology, microbial ecology and host-microbiome metabolic cross-talk. The OMM
consists of 12 sequenced and publically available strains isolated from mice, representing five bacterial phyla that are naturally abundant in the murine gastrointestinal tract (Lagkouvardos et al., 2016). Under germ-free conditions, the OMM
colonizes mice stably over multiple generations. Here, we investigated whether stably colonized OMM
mouse lines could be reproducibly established in different animal facilities. Germ-free C57Bl/6J mice were inoculated with a frozen mixture of the OMM
strains. Within 2 weeks after application, the OMM
community reached the same stable composition in all facilities, as determined by fecal microbiome analysis. We show that a second application of the OMM
strains after 72 h leads to a more stable community composition than a single application. The availability of such protocols for reliable
generation of gnotobiotic rodents will certainly contribute to increasing experimental reproducibility in biomedical research.
The role of the spleen in the induction of an immune response to orally administered antigens is still under discussion. Although it is well known that after oral antigen administration specific ...germinal centres are not only formed in the Peyers patches (PP) and the mesenteric lymph nodes (mLN) but also in the spleen, there is still a lack of functional data showing a direct involvement of splenic B cells in an IgA immune response in the gut. In addition, after removal of mLN a high level of IgA+ B cells was observed in the gut. Therefore, in this study we analysed the role of the spleen in the induction of IgA+ B cells in the gut after mice were orally challenged with antigens. Here we have shown that antigen specific splenic IgM+ B cells after in vitro antigen stimulation as well as oral immunisation of donor mice were able to migrate into the gut of recipient mice, where they predominantly switch to IgA+ plasma cells. Furthermore, stimulation of recipient mice by orally administered antigens enhanced the migration of the splenic B cells into the gut as well as their switch to IgA+ plasma cells. Removal of the mLN led to a higher activation level of the splenic B cells. Altogether, our results imply that splenic IgM+ B cells migrate in the intestinal lamina propria, where they differentiate into IgA+ plasma cells and subsequently proliferate. In conclusion, we demonstrated that the spleen plays a major role in the gut immune response serving as a reservoir of immune cells that migrate to the site of antigen entrance.
Obesity has emerged as a major global health problem and is associated with various diseases, such as metabolic syndrome, type 2 diabetes mellitus, and cardiovascular diseases. The inbred C57BL/6 ...mouse strain is often used for various experimental investigations, such as metabolic research. However, over time, genetically distinguishable C57BL/6 substrains have evolved. The manifestation of genetic alterations has resulted in behavioral and metabolic differences. In this study, a comparison of diet-induced obesity in C57BL/6JHanZtm, C57BL/6NCrl and C57BL/6 J mice revealed several metabolic and immunological differences such as blood glucose level and cytokine expression, respectively, among these C57BL/6 substrains. For example, C57BL/6NCrl mice developed the most pronounced adiposity, whereas C57BL/6 J mice showed the highest impairment in glucose tolerance. Moreover, our results indicated that the immunological phenotype depends on the intestinal microbiota, as the cell subset composition of the colon was similar in obese ex-GF B6NRj
and obese B6JHanZtm mice. Phenotypic differences between C57BL/6 substrains are caused by a complex combination of genetic and microbial alterations. Therefore, in performing metabolic research, considering substrain-specific characteristics, which can influence the course of study, is important. Moreover, for unbiased comparison of data, the entire strain name should be shared with the scientific community.
Gut-draining mesenteric lymph nodes (mLNs) are important for inducing peripheral tolerance towards food and commensal antigens by providing an optimal microenvironment for de novo generation of Foxp3
...regulatory T cells (Tregs). We previously identified microbiota-imprinted mLN stromal cells as a critical component in tolerance induction. Here we show that this imprinting process already takes place in the neonatal phase, and renders the mLN stromal cell compartment resistant to inflammatory perturbations later in life. LN transplantation and single-cell RNA-seq uncover stably imprinted expression signatures in mLN fibroblastic stromal cells. Subsetting common stromal cells across gut-draining mLNs and skin-draining LNs further refine their location-specific immunomodulatory functions, such as subset-specific expression of Aldh1a2/3. Finally, we demonstrate that mLN stromal cells shape resident dendritic cells to attain high Treg-inducing capacity in a Bmp2-dependent manner. Thus, crosstalk between mLN stromal and resident dendritic cells provides a robust regulatory mechanism for the maintenance of intestinal tolerance.
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