Caspase-8 controls the gut response to microbial challenges by Tnf-alpha-dependent and independent pathways

Objectives: Intestinal epithelial cells (IEC) express toll-like receptors (TLR) that facilitate microbial recognition. Stimulation of TLR ligands induces a transient increase in epithelial cell shedding, a mechanism that serves the antibacterial and antiviral host defence of the epithelium and promotes elimination of intracellular pathogens. Although activation of the extrinsic apoptosis pathway has been described during inflammatory shedding, its functional involvement is currently unclear. Design: We investigated the functional involvement of caspase-8 signalling in microbial-induced intestinal cell shedding by injecting Lipopolysaccharide (LPS) to mimic bacterial pathogens and poly(I:C) as a probe for RNA viruses in vivo. Results: TLR stimulation of IEC was associated with a rapid activation of caspase-8 and increased epithelial cell shedding. In mice with an epithelial cell-specific deletion of caspase-8 TLR stimulation caused Rip3-dependent epithelial necroptosis instead of apoptosis. Mortality and tissue damage were more severe in mice in which IECs died by necroptosis than apoptosis. Inhibition of receptor-interacting protein (Rip) kinases rescued the epithelium from TLR-induced gut damage. TLR3-induced necroptosis was directly mediated via TRIF-dependent pathways, independent of Tnf-α and type III interferons, whereas TLR4-induced tissue damage was critically dependent on Tnf-α. Conclusions: Together, our data demonstrate an essential role for caspase-8 in maintaining the gut barrier in response to mucosal pathogens by permitting inflammatory shedding and preventing necroptosis of infected cells. These data suggest that therapeutic strategies targeting the cell death machinery represent a promising new option for the treatment of inflammatory and infective enteropathies

Epithelial calcineurin controls microbiota-dependent intestinal tumor development.

Inflammation-associated pathways are active in intestinal epithelial cells (IECs) and contribute to the pathogenesis of colorectal cancer (CRC). Calcineurin, a phosphatase required for the activation of the nuclear factor of activated T cells (NFAT) family of transcription factors, shows increased expression in CRC. We therefore investigated the role of calcineurin in intestinal tumor development. We demonstrate that calcineurin and NFAT factors are constitutively expressed by primary IECs and selectively activated in intestinal tumors as a result of impaired stratification of the tumor-associated microbiota and toll-like receptor signaling. Epithelial calcineurin supports the survival and proliferation of cancer stem cells in an NFAT-dependent manner and promotes the development of intestinal tumors in mice. Moreover, somatic mutations that have been identified in human CRC are associated with constitutive activation of calcineurin, whereas nuclear translocation of NFAT is associated with increased death from CRC. These findings highlight an epithelial cell-intrinsic pathway that integrates signals derived from the commensal microbiota to promote intestinal tumor development.This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants ZE814/5-1 (S.Z.), BA2863/5-1 (J.F.B.) and CH279/5-1 (T.C.), the European Research Council (ERC) starting grant 336528 (S.Z.), a Postdoctoral Fellowship Award from the Crohn's and Colitis Foundation of America (S.Z.), the European Commission (Marie Curie International Reintegration grant 256363; S.Z.), the DFG Excellence Cluster 'Inflammation at Interfaces' (S.Z. and J.F.B.), the DFG Excellence Cluster 'Center for Regenerative Therapies' (S.Z.); the US National Institutes of Health grants DK044319 (R.S.B.), DK051362 (R.S.B.), DK053056 (R.S.B.) and DK088199 (R.S.B.), the Harvard Digestive Diseases Center (HDDC) grant DK0034854 (R.S.B.), and the AIRC grant IG-14233 (M.E.B.).This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nm.407

Composition of the Intestinal Microbiota Determines the Outcome of Virus-Triggered Colitis in Mice

The intestinal microbiota is a complex ecosystem implicated in host health and disease. Inflammatory bowel disease (IBD) is a multifactorial chronic disorder of the gastrointestinal mucosa. Even though the exact mechanisms are still unknown, the intestinal microbiota is crucial in IBD development. We previously showed that murine norovirus (MNV) induces colitis in the Il10-deficient (Il10(-/-)) mouse model of IBD in a microbiota-dependent manner. Thus, in this study we analyzed whether distinct minimal bacterial consortia influence the outcome of MNV-triggered colitis in Il10(-/-) mice. Gnotobiotic Il10(-/-) mice associated with Oligo-Mouse-Microbiota 12 (OMM12) or Altered Schaedler Flora (ASF) developed little to no inflammatory lesions in the colon and cecum. MNV infection exacerbated colitis severity only in ASF-colonized mice, but not in those associated with OMM12. Four weeks after MNV infection, inflammatory lesions in ASF-colonized Il10(-/-) mice were characterized by epithelial hyperplasia, infiltration of inflammatory cells, and increased barrier permeability. Co-colonization of ASF-colonized Il10(-/-) mice with segmented filamentous bacteria (SFB) abolished MNV-induced colitis, whereas histopathological scores in SFB-OMM12-co-colonized mice stayed unchanged. Moreover, SFB only colonized mice associated with ASF. The SFB-mediated protective effects in ASF-colonized mice involved enhanced activation of intestinal barrier defense mechanisms and mucosal immune responses in the chronic and acute phase of MNV infection. SFB colonization strengthened intestinal barrier function by increasing expression of tight junction proteins, antimicrobial peptides and mucus. Furthermore, SFB colonization enhanced the expression of pro-inflammatory cytokines such as Tnf alpha, Il1 beta, and Il12 alpha, as well as the expression of the regulatory cytokine Tgf beta. Altogether, our results showed that MNV-triggered colitis depends on the microbial context

Age-related impairment of intestinal inflammation resolution through an eicosanoid-immune-microbiota axis

Aging manifests a decline of immune function, induces microbiome dysbiosis, drives organ inflammation, and impedes the resolution of inflammation. However, the mechanisms underlying age-related intestinal inflammation remain poorly described. Here, we find that the resolution of T cell-initiated intestinal inflammation is impaired with aging. This impairment is mediated by disrupting the immune-microbiota interplay, controlled by intestinal eicosanoid metabolism. Pharmacologically inhibiting eicosanoid biosynthesis, blocking the prostaglandin E receptor subtype 4 (EP4), or genetically ablating EP4 diminishes age-related impairment of intestinal inflammation resolution. Mechanistically, mononuclear phagocyte-intrinsic eicosanoid-EP4 signaling impedes the resolution of intestinal inflammation through fostering gut microbial dysbiosis and, more importantly, interrupting segmented filamentous bacterial adhesion to the intestinal epithelium. Colonization with EP4-ablated mouse microbiota or segmented filamentous bacteria improves the resolution of intestinal inflammation. These findings reveal that eicosanoid-dependent immune-microbiota interactions impair inflammation resolution in the aged intestine, highlighting potential intervention strategies for improving age-related gut health

Gnotobiotics: Past, present and future

Gnotobiotics or gnotobiology is a research field exploring organisms with a known microbiological state. In animal research, the development of gnotobiotics started in the late 19th century with the rederivation of germ-free guinea pigs. Cutting-edge achievements were accomplished by scientists in the Laboratories of Bacteriology at the University of Notre Dame (LOBUND). The primary goals of gnotobiotics were not only the development of the equipment required for long-term husbandry but also phenotypic characterization of germ-free animals. The first isolators were designed by Reynolds and Gustafsson as rigid-wall stainless steel autoclave-like chambers, which were subsequently replaced by Trexler’s flexible-film polyvinyl plastic isolators. Flexible-film or semi-rigid isolators are commonly used today. The long-term maintenance of gnotobiotic rodents is performed in positive-pressure isolators. However, to facilitate gnotobiotic experimental procedures, short-term husbandry systems have been developed. Gnotobiotic animal husbandry is laborious and requires experienced staff. Germ-free animals can be rederived from existing rodent colonies by hysterectomy or embryo transfer. The physiology and anatomy of germ-free rodents are different from those of specified pathogen-free (SPF) rodents. Furthermore, to guarantee gnotobiotic status, the colonies need to be regularly microbiologically monitored. Today, gnotobiotics provides a powerful tool to analyse functional effects of host-microbe interactions, especially in complex disease models. Gnotobiotic models combined with ‘omics’ approaches will be indispensable for future advances in microbiome research. Furthermore, these approaches will contribute to the development of novel therapeutic targets. In addition, regional or national gnotobiotic core facilities should be established in the future to support further applications of gnotobiotic models. </jats:p

Synthetic Microbiomes on the Rise—Application in Deciphering the Role of Microbes in Host Health and Disease

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

Synthetic Microbiomes on the Rise—Application in Deciphering the Role of Microbes in Host Health and Disease

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.</jats:p