Commensal bacteria weaken the intestinal barrier by suppressing epithelial neuropilin-1 and Hedgehog signaling.

The gut microbiota influences intestinal barrier integrity through mechanisms that are incompletely understood. Here we show that the commensal microbiota weakens the intestinal barrier by suppressing epithelial neuropilin-1 (NRP1) and Hedgehog (Hh) signaling. Microbial colonization of germ-free mice dampens signaling of the intestinal Hh pathway through epithelial Toll-like receptor (TLR)-2, resulting in decreased epithelial NRP1 protein levels. Following activation via TLR2/TLR6, epithelial NRP1, a positive-feedback regulator of Hh signaling, is lysosomally degraded. Conversely, elevated epithelial NRP1 levels in germ-free mice are associated with a strengthened gut barrier. Functionally, intestinal epithelial cell-specific Nrp1 deficiency (Nrp1ΔIEC) results in decreased Hh pathway activity and a weakened gut barrier. In addition, Nrp1ΔIEC mice have a reduced density of capillary networks in their small intestinal villus structures. Collectively, our results reveal a role for the commensal microbiota and epithelial NRP1 signaling in the regulation of intestinal barrier function through postnatal control of Hh signaling.

α-Linolenic Acid-Rich Diet Influences Microbiota Composition and Villus Morphology of the Mouse Small Intestine.

α-Linolenic acid (ALA) is well-known for its anti-inflammatory activity. In contrast, the influence of an ALA-rich diet on intestinal microbiota composition and its impact on small intestine morphology are not fully understood. In the current study, we kept adult C57BL/6J mice for 4 weeks on an ALA-rich or control diet. Characterization of the microbial composition of the small intestine revealed that the ALA diet was associated with an enrichment in Prevotella and Parabacteroides. In contrast, taxa belonging to the Firmicutes phylum, including Lactobacillus, Clostridium cluster XIVa, Lachnospiraceae and Streptococcus, had significantly lower abundance compared to control diet. Metagenome prediction indicated an enrichment in functional pathways such as bacterial secretion system in the ALA group, whereas the two-component system and ALA metabolism pathways were downregulated. We also observed increased levels of ALA and its metabolites eicosapentanoic and docosahexanoic acid, but reduced levels of arachidonic acid in the intestinal tissue of ALA-fed mice. Furthermore, intestinal morphology in the ALA group was characterized by elongated villus structures with increased counts of epithelial cells and reduced epithelial proliferation rate. Interestingly, the ALA diet reduced relative goblet and Paneth cell counts. Of note, high-fat Western-type diet feeding resulted in a comparable adaptation of the small intestine. Collectively, our study demonstrates the impact of ALA on the gut microbiome and reveals the nutritional regulation of gut morphology.

The Microbiota Promotes Arterial Thrombosis in Low-Density Lipoprotein Receptor-Deficient Mice.

Atherosclerotic plaque development depends on chronic inflammation of the arterial wall. A dysbiotic gut microbiota can cause low-grade inflammation, and microbiota composition was linked to cardiovascular disease risk. However, the role of this environmental factor in atherothrombosis remains undefined. To analyze the impact of gut microbiota on atherothrombosis, we rederived low-density lipoprotein receptor-deficient (Ldlr-/- ) mice as germfree (GF) and kept these mice for 16 weeks on an atherogenic high-fat Western diet (HFD) under GF isolator conditions and under conventionally raised specific-pathogen-free conditions (CONV-R). In spite of reduced diversity of the cecal gut microbiome, caused by atherogenic HFD, GF Ldlr-/- mice and CONV-R Ldlr-/- mice exhibited atherosclerotic lesions of comparable sizes in the common carotid artery. In contrast to HFD-fed mice, showing no difference in total cholesterol levels, CONV-R Ldlr-/- mice fed control diet (CD) had significantly reduced total plasma cholesterol, very-low-density lipoprotein (VLDL), and LDL levels compared with GF Ldlr-/- mice. Myeloid cell counts in blood as well as leukocyte adhesion to the vessel wall at the common carotid artery of GF Ldlr-/- mice on HFD were diminished compared to CONV-R Ldlr-/- controls. Plasma cytokine profiling revealed reduced levels of the proinflammatory chemokines CCL7 and CXCL1 in GF Ldlr-/- mice, whereas the T-cell-related interleukin 9 (IL-9) and IL-27 were elevated. In the atherothrombosis model of ultrasound-induced rupture of the common carotid artery plaque, thrombus area was significantly reduced in GF Ldlr-/- mice relative to CONV-R Ldlr-/- mice. Ex vivo, this atherothrombotic phenotype was explained by decreased adhesion-dependent platelet activation and thrombus growth of HFD-fed GF Ldlr-/- mice on type III collagen.IMPORTANCE Our results demonstrate a functional role for the commensal microbiota in atherothrombosis. In a ferric chloride injury model of the carotid artery, GF C57BL/6J mice had increased occlusion times compared to colonized controls. Interestingly, in late atherosclerosis, HFD-fed GF Ldlr-/- mice had reduced plaque rupture-induced thrombus growth in the carotid artery and diminished ex vivo thrombus formation under arterial flow conditions.

Coagulation factor 9-deficient mice are protected against dextran sulfate sodium-induced colitis.

Patients with inflammatory bowel disease (IBD) are susceptible to thromboembolism. Interestingly, IBD occurs less frequently in patients with inherited bleeding disorders. Therefore, we analyzed whether F9-deficiency is protective against the onset of acute colitis in a genetic hemophilia B mouse model. In the 3.5% dextran sulfate sodium (DSS)-induced colitis model, F9-deficient mice were protected from body-weight loss and had a reduced disease activity score. We detected decreased colonic myeloperoxidase activity and decreased CXCL1 levels in DSS-treated F9-deficient mice compared with wild-type (WT) littermate controls, indicating decreased neutrophil infiltration. Remarkably, we identified expression of coagulation factor IX (FIX) protein in small intestinal epithelial cells (MODE-K). In epithelial cell cultures, cellular FIX protein expression was increased following stimulation with the bacterial Toll-like receptor agonists lipopolysaccharide, macrophage-activating lipopeptide-2 and Pam3CSK4. Thus, we revealed a protective role of F9-deficiency in DSS-induced colitis and identified the intestinal epithelium as a site of ectopic FIX.This article has an associated First Person interview with the first author of the paper.

Gut microbiota regulate hepatic von Willebrand factor synthesis and arterial thrombus formation via Toll-like receptor-2.

The symbiotic gut microbiota play pivotal roles in host physiology and the development of cardiovascular diseases, but the microbiota-triggered pattern recognition signaling mechanisms that impact thrombosis are poorly defined. In this article, we show that germ-free (GF) and Toll-like receptor-2 (Tlr2)-deficient mice have reduced thrombus growth after carotid artery injury relative to conventionally raised controls. GF Tlr2-/- and wild-type (WT) mice were indistinguishable, but colonization with microbiota restored a significant difference in thrombus growth between the genotypes. We identify reduced plasma levels of von Willebrand factor (VWF) and reduced VWF synthesis, specifically in hepatic endothelial cells, as a critical factor that is regulated by gut microbiota and determines thrombus growth in Tlr2-/- mice. Static platelet aggregate formation on extracellular matrix was similarly reduced in GF WT, Tlr2-/- , and heterozygous Vwf+/- mice that are all characterized by a modest reduction in plasma VWF levels. Defective platelet matrix interaction can be restored by exposure to WT plasma or to purified VWF depending on the VWF integrin binding site. Moreover, administration of VWF rescues defective thrombus growth in Tlr2-/- mice in vivo. These experiments delineate an unexpected pathway in which microbiota-triggered TLR2 signaling alters the synthesis of proadhesive VWF by the liver endothelium and favors platelet integrin-dependent thrombus growth.