Gut Microbiota Restricts NETosis in Acute Mesenteric Ischemia-Reperfusion Injury.

OBJECTIVE: Recruitment of neutrophils and formation of neutrophil extracellular traps (NETs) contribute to lethality in acute mesenteric infarction. To study the impact of the gut microbiota in acute mesenteric infarction, we used gnotobiotic mouse models to investigate whether gut commensals prime the reactivity of neutrophils towards formation of neutrophil extracellular traps (NETosis). Approach and Results: We applied a mesenteric ischemia-reperfusion (I/R) injury model to germ-free (GF) and colonized C57BL/6J mice. By intravital imaging, we quantified leukocyte adherence and NET formation in I/R-injured mesenteric venules. Colonization with gut microbiota or monocolonization with Escherichia coli augmented the adhesion of leukocytes, which was dependent on the TLR4 (Toll-like receptor-4)/TRIF (TIR-domain-containing adapter-inducing interferon-ß) pathway. Although neutrophil accumulation was decreased in I/R-injured venules of GF mice, NETosis following I/R injury was significantly enhanced compared with conventionally raised mice or mice colonized with the minimal microbial consortium altered Schaedler flora. Also ex vivo, neutrophils from GF and antibiotic-treated mice showed increased LPS (lipopolysaccharide)-induced NETosis. Enhanced TLR4 signaling in GF neutrophils was due to elevated TLR4 expression and augmented IRF3 (interferon regulatory factor-3) phosphorylation. Likewise, neutrophils from antibiotic-treated conventionally raised mice had increased NET formation before and after ischemia. Increased NETosis in I/R injury was abolished in conventionally raised mice deficient in the TLR adaptor TRIF. In support of the desensitizing influence of enteric LPS, treatment of GF mice with LPS via drinking water diminished LPS-induced NETosis in vitro and in the mesenteric I/R injury model. CONCLUSIONS: Collectively, our results identified that the gut microbiota suppresses NETing neutrophil hyperreactivity in mesenteric I/R injury, while ensuring immunovigilance by enhancing neutrophil recruitment.

The gut microbiota instructs the hepatic endothelial cell transcriptome.

The gut microbiota affects remote organ functions but its impact on organotypic endothelial cell (EC) transcriptomes remains unexplored. The liver endothelium encounters microbiota-derived signals and metabolites via the portal circulation. To pinpoint how gut commensals affect the hepatic sinusoidal endothelium, a magnetic cell sorting protocol, combined with fluorescence-activated cell sorting, was used to isolate hepatic sinusoidal ECs from germ-free (GF) and conventionally raised (CONV-R) mice for transcriptome analysis by RNA sequencing. This resulted in a comprehensive map of microbiota-regulated hepatic EC-specific transcriptome profiles. Gene Ontology analysis revealed that several functional processes in the hepatic endothelium were affected. The absence of microbiota influenced the expression of genes involved in cholesterol flux and angiogenesis. Specifically, genes functioning in hepatic endothelial sphingosine metabolism and the sphingosine-1-phosphate pathway showed drastically increased expression in the GF state. Our analyses reveal a prominent role for the microbiota in shaping the transcriptional landscape of the hepatic endothelium.

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 gut microbiota - a modulator of endothelial cell function and a contributing environmental factor to arterial thrombosis.

Introduction: There is emerging evidence linking the commensal gut microbiota with the development of cardiovascular disease and arterial thrombosis. In immunothrombosis, the host clotting system protects against the dissemination of invading microbes, not considering the huge number of microbes that interact with host physiology in a mutualistic fashion. Areas covered: Interestingly, recent research revealed that colonizing gut microbes profoundly influence host innate immune pathways that support arterial thrombus growth. The gut microbiota promotes arterial thrombus formation by enhancing the pro-adhesive capacity of the vascular endothelium, triggering hepatic von Willebrand factor synthesis and its release by Weibel-Palade body exocytosis, resulting in elevated von Willebrand factor levels and enhancing FVIII stability in plasma. Furthermore, the metabolic capacity of gut resident microbes promotes agonist-induced platelet activation and deposition. Here, we give an overview, with a focus on the vascular endothelium, on how this gut-resident microbial ecosystem contributes to arterial thrombus formation. Expert opinion: The gut microbiota and its metabolites not only act on agonist-induced platelet reactivity, but also influence the hepatic endothelial phenotype via remote signaling, facilitating arterial thrombus growth at the arterial injury site.

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.

Mice deficient in the anti-haemophilic coagulation factor VIII show increased von Willebrand factor plasma levels.

Von Willebrand factor (VWF) is the carrier protein of the anti-haemophilic Factor VIII (FVIII) in plasma. It has been reported that the infusion of FVIII concentrate in haemophilia A patients results in lowered VWF plasma levels. However, the impact of F8-deficiency on VWF plasma levels in F8-/y mice is unresolved. In order to avoid confounding variables, we back-crossed F8-deficient mice onto a pure C57BL/6J background and analysed VWF plasma concentrations relative to C57BL/6J WT (F8+/y) littermate controls. F8-/y mice showed strongly elevated VWF plasma concentrations and signs of hepatic inflammation, as indicated by increased TNF-α, CD45, and TLR4 transcripts and by elevated macrophage counts in the liver. Furthermore, immunohistochemistry showed that expression of VWF antigen was significantly enhanced in the hepatic endothelium of F8-/y mice, most likely resulting from increased macrophage recruitment. There were no signs of liver damage, as judged by glutamate-pyruvate-transaminase (GPT) and glutamate-oxalacetate-transaminase (GOT) in the plasma and no signs of systemic inflammation, as white blood cell subsets were unchanged. As expected, impaired haemostasis was reflected by joint bleeding, prolonged in vitro clotting time and decreased platelet-dependent thrombin generation. Our results point towards a novel role of FVIII, synthesized by the liver endothelium, in the control of hepatic low-grade inflammation and VWF plasma levels.