Marijana Basic

RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis

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.

Dysbiotic gut microbiota causes transmissible Crohn's disease-like ileitis independent of failure in antimicrobial defence

Objectives Dysbiosis of the intestinal microbiota is associated with Crohns disease (CD). Functional evidence for a causal role of bacteria in the development of chronic small intestinal inflammation is lacking. Similar to human pathology, TNFdeltaARE mice develop a tumour necrosis factor (TNF)-driven CD-like transmural inflammation with predominant ileal involvement. Design Heterozygous TNFdeltaARE mice and wildtype (WT) littermates were housed under conventional (CONV), specific pathogen-free (SPF) and germ-free (GF) conditions. Microbial communities were analysed by high-throughput 16S ribosomal RNA gene sequencing. Metaproteomes were measured using LC-MS. Temporal and spatial resolution of disease development was followed after antibiotic treatment and transfer of microbial communities into GF mice. Granulocyte infiltration and Paneth cell function was assessed by immunofluorescence and gene expression analysis. Results GF-TNFdeltaARE mice were free of inflammation in the gut and antibiotic treatment of CONV-TNFdeltaARE mice attenuated ileitis but not colitis, demonstrating that disease severity and location are microbiota-dependent. SPF-TNFdeltaARE mice developed distinct ileitis-phenotypes associated with gradual loss of antimicrobial defence. 16S analysis and metaproteomics revealed specific compositional and functional alterations of bacterial communities in inflamed mice. Transplantation of disease-associated but not healthy microbiota transmitted CD-like ileitis to GF-TNFdeltaARE recipients and triggered loss of lysozyme and cryptdin-2 expression. Monoassociation of GF-TNFdeltaARE mice with the human CD-related Escherichia coli LF82 did not induce ileitis. Conclusions We provide clear experimental evidence for the causal role of gut bacterial dysbiosis in the development of chronic ileal inflammation with subsequent failure of Paneth cell function.

Salt-responsive gut commensal modulates TH17 axis and disease

A Western lifestyle with high salt consumption can lead to hypertension and cardiovascular disease. High salt may additionally drive autoimmunity by inducing T helper 17 (TH17) cells, which can also contribute to hypertension. Induction of TH17 cells depends on gut microbiota; however, the effect of salt on the gut microbiome is unknown. Here we show that high salt intake affects the gut microbiome in mice, particularly by depleting Lactobacillus murinus. Consequently, treatment of mice with L. murinus prevented salt-induced aggravation of actively induced experimental autoimmune encephalomyelitis and salt-sensitive hypertension by modulating TH17 cells. In line with these findings, a moderate high-salt challenge in a pilot study in humans reduced intestinal survival of Lactobacillus spp., increased TH17 cells and increased blood pressure. Our results connect high salt intake to the gut–immune axis and highlight the gut microbiome as a potential therapeutic target to counteract salt-sensitive conditions.

Norovirus triggered microbiota-driven mucosal inflammation in interleukin 10-deficient mice.

Background:Infection may trigger clinically overt mucosal inflammation in patients with predisposition for inflammatory bowel disease. However, the impact of particular enteropathogenic microorganisms is ill-defined. In this study, the influence of murine norovirus (MNV) infection on clinical, histopathological, and immunological features of mucosal inflammation in the IL10-deficient (Il10−/−) mouse model of inflammatory bowel disease was examined. Methods:C57BL/6J and C3H/HeJBir wild-type and Il10−/− mice kept under special pathogen-free conditions and devoid of clinical and histopathological signs of mucosal inflammation were monitored after MNV infection for structural and functional intestinal barrier changes by in situ MNV reverse transcription PCR, transgene reporter gene technology, histology, flux measurements, quantitative real-time PCR, immunohistology, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. In addition, the influence of the enteric microbiota was analyzed in MNV-infected germfree Il10−/− mice. Results:Although MNV-infected wild-type mice remained asymptomatic, mucosal inflammation was noted in previously healthy Il10−/− mice 2 to 4 weeks after infection. MNV-induced changes in Il10−/− mice included increased paracellular permeability indicated by increased mucosal mannitol flux, reduced gene expression of tight junction molecules, and an enhanced rate of epithelial apoptosis. MNV-induced reduction of tight junction protein expression and inflammatory lesions were absent in germfree Il10−/− mice, whereas epithelial apoptosis was still observed. Conclusions:Despite its subclinical course in wild-type animals, MNV causes epithelial barrier disruption in Il10−/− animals representing a potent colitogenic stimulus that largely depends on the presence of the enteric microbiota. MNV might thus trigger overt clinical disease in individuals with a nonsymptomatic predisposition for inflammatory bowel disease by impairment of the intestinal mucosa.

Caspase-8 controls the gut response to microbial challenges by Tnf-α-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.

Active suppression of intestinal CD4 + TCRαβ + T-lymphocyte maturation during the postnatal period

Priming of the mucosal immune system during the postnatal period substantially influences host–microbial interaction and susceptibility to immune-mediated diseases in adult life. The underlying mechanisms are ill defined. Here we show that shortly after birth, CD4 T cells populate preformed lymphoid structures in the small intestine and quickly acquire a distinct transcriptional profile. T-cell recruitment is independent of microbial colonization and innate or adaptive immune stimulation but requires β7 integrin expression. Surprisingly, neonatal CD4 T cells remain immature throughout the postnatal period under homeostatic conditions but undergo maturation and gain effector function on barrier disruption. Maternal SIgA and regulatory T cells act in concert to prevent immune stimulation and maintain the immature phenotype of CD4 T cells in the postnatal intestine during homeostasis. Active suppression of CD4 T-cell maturation during the postnatal period might contribute to prevent auto-reactivity, sustain a broad TCR repertoire and establish life-long immune homeostasis.

Age-Dependent Susceptibility to Enteropathogenic Escherichia coli (EPEC) Infection in Mice

Enteropathogenic Escherichia coli (EPEC) represents a major causative agent of infant diarrhea associated with significant morbidity and mortality in developing countries. Although studied extensively in vitro, the investigation of the host-pathogen interaction in vivo has been hampered by the lack of a suitable small animal model. Using RT-PCR and global transcriptome analysis, high throughput 16S rDNA sequencing as well as immunofluorescence and electron microscopy, we characterize the EPEC-host interaction following oral challenge of newborn mice. Spontaneous colonization of the small intestine and colon of neonate mice that lasted until weaning was observed. Intimate attachment to the epithelial plasma membrane and microcolony formation were visualized only in the presence of a functional bundle forming pili (BFP) and type III secretion system (T3SS). Similarly, a T3SS-dependent EPEC-induced innate immune response, mediated via MyD88, TLR5 and TLR9 led to the induction of a distinct set of genes in infected intestinal epithelial cells. Infection-induced alterations of the microbiota composition remained restricted to the postnatal period. Although EPEC colonized the adult intestine in the absence of a competing microbiota, no microcolonies were observed at the small intestinal epithelium. Here, we introduce the first suitable mouse infection model and describe an age-dependent, virulence factor-dependent attachment of EPEC to enterocytes in vivo.

Expression of the Blood-Group-Related Gene B4galnt2 Alters Susceptibility to Salmonella Infection

Glycans play important roles in host-microbe interactions. Tissue-specific expression patterns of the blood group glycosyltransferase β-1,4-N-acetylgalactosaminyltransferase 2 (B4galnt2) are variable in wild mouse populations, and loss of B4galnt2 expression is associated with altered intestinal microbiota. We hypothesized that variation in B4galnt2 expression alters susceptibility to intestinal pathogens. To test this, we challenged mice genetically engineered to express different B4galnt2 tissue-specific patterns with a Salmonella Typhimurium infection model. We found B4galnt2 intestinal expression was strongly associated with bacterial community composition and increased Salmonella susceptibility as evidenced by increased intestinal inflammatory cytokines and infiltrating immune cells. Fecal transfer experiments demonstrated a crucial role of the B4galnt2-dependent microbiota in conferring susceptibility to intestinal inflammation, while epithelial B4galnt2 expression facilitated epithelial invasion of S. Typhimurium. These data support a critical role for B4galnt2 in gastrointestinal infections. We speculate that B4galnt2-specific differences in host susceptibility to intestinal pathogens underlie the strong signatures of balancing selection observed at the B4galnt2 locus in wild mouse populations.

Regulation of epithelial cell expressed C3 in the intestine: Relevance for the pathophysiology of inflammatory bowel disease?

The complement system not only plays a critical role in efficient detection and clearance of bacteria, but also in intestinal immune homeostasis as mice deficient for key complement components display enhanced intestinal inflammation upon experimental colitis. Because underlying molecular mechanisms for this observation are unclear, we investigated the crosstalk between intestinal epithelial cells (IEC), bacteria and the complement system in the course of chronic colitis. Surprisingly, mouse intestinal epithelial cell lines constitutively express high mRNA levels of complement component 3 (C3), Toll-like receptor 2 (Tlr2) and Tlr4. Stimulation of these cells with lipopolysaccharide (LPS), but not with flagellin, LD-muramyldipeptide or peptidoglycan, triggered increased C3 expression, secretion and activation. Stimulation of the C3aR on these cell lines with C3a resulted in an increase of LPS-triggered pro-inflammatory response. Tissue biopsies from C57BL/6J mice revealed higher expression of C3, Tlr1, Tlr2 and Tlr4 in colonic primary IECs (pIECs) compared to ileal pIECs, while in germ-free mice no differences in C3 protein expression was observed. In DSS-induced chronic colitis mouse models, C3 mRNA expression was upregulated in colonic biopsies and ileal pIECs with elevated C3 protein in the lamina propria, IECs and the mucus. Notably, increased C3b opsonization of mucosa-attached bacteria and decreased fecal full-length C3 protein was observed in DSS-treated compared to untreated mice. Of significant interest, non-inflamed and inflamed colonic biopsy samples from CD but not UC patients displayed exacerbated C3 expression compared to controls. These findings suggest that a novel TLR4-C3 axis could control the intestinal immune response during chronic colitis.