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.

Ethyl Hydroxyethyl Cellulose-A Biocompatible Polymer Carrier in Blood.

The biocompatibility of carrier nanomaterials in blood is largely hampered by their activating or inhibiting role on the clotting system, which in many cases prevents safe intravascular application. Here, we characterized an aqueous colloidal ethyl hydroxyethyl cellulose (EHEC) solution and tested its effect on ex vivo clot formation, platelet aggregation, and activation by thromboelastometry, aggregometry, and flow cytometry. We compared the impact of EHEC solution on platelet aggregation with biocompatible materials used in transfusion medicine (the plasma expanders gelatin polysuccinate and hydroxyethyl starch). We demonstrate that the EHEC solution, in contrast to commercial products exhibiting Newtonian flow behavior, resembles the shear-thinning behavior of human blood. Similar to established nanomaterials that are considered biocompatible when added to blood, the EHEC exposure of resting platelets in platelet-rich plasma does not enhance tissue thromboplastin- or ellagic acid-induced blood clotting, or platelet aggregation or activation, as measured by integrin αIIbß3 activation and P-selectin exposure. Furthermore, the addition of EHEC solution to adenosine diphosphate (ADP)-stimulated platelet-rich plasma does not affect the platelet aggregation induced by this agonist. Overall, our results suggest that EHEC may be suitable as a biocompatible carrier material in blood circulation and for applications in flow-dependent diagnostics.

The Interplay between Nutrition, Innate Immunity, and the Commensal Microbiota in Adaptive Intestinal Morphogenesis.

The gastrointestinal tract is a functionally and anatomically segmented organ that is colonized by microbial communities from birth. While the genetics of mouse gut development is increasingly understood, how nutritional factors and the commensal gut microbiota act in concert to shape tissue organization and morphology of this rapidly renewing organ remains enigmatic. Here, we provide an overview of embryonic mouse gut development, with a focus on the intestinal vasculature and the enteric nervous system. We review how nutrition and the gut microbiota affect the adaptation of cellular and morphologic properties of the intestine, and how these processes are interconnected with innate immunity. Furthermore, we discuss how nutritional and microbial factors impact the renewal and differentiation of the epithelial lineage, influence the adaptation of capillary networks organized in villus structures, and shape the enteric nervous system and the intestinal smooth muscle layers. Intriguingly, the anatomy of the gut shows remarkable flexibility to nutritional and microbial challenges in the adult organism.

The Commensal Microbiota Enhances ADP-Triggered Integrin αIIbß3 Activation and von Willebrand Factor-Mediated Platelet Deposition to Type I Collagen.

The commensal microbiota is a recognized enhancer of arterial thrombus growth. While several studies have demonstrated the prothrombotic role of the gut microbiota, the molecular mechanisms promoting arterial thrombus growth are still under debate. Here, we demonstrate that germ-free (GF) mice, which from birth lack colonization with a gut microbiota, show diminished static deposition of washed platelets to type I collagen compared with their conventionally raised (CONV-R) counterparts. Flow cytometry experiments revealed that platelets from GF mice show diminished activation of the integrin αIIbß3 (glycoprotein IIbIIIa) when activated by the platelet agonist adenosine diphosphate (ADP). Furthermore, washed platelets from Toll-like receptor-2 (Tlr2)-deficient mice likewise showed impaired static deposition to the subendothelial matrix component type I collagen compared with wild-type (WT) controls, a process that was unaffected by GPIbα-blockade but influenced by von Willebrand factor (VWF) plasma levels. Collectively, our results indicate that microbiota-triggered steady-state activation of innate immune pathways via TLR2 enhances platelet deposition to subendothelial matrix molecules. Our results link host colonization status with the ADP-triggered activation of integrin αIIbß3, a pathway promoting platelet deposition to the growing thrombus.

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.

Synergistic effect of peptide inhibitors derived from the extracellular and intracellular domain of αIIb subunit of integrin αIIbß3 on platelet activation and aggregation.

αIIbß3, the major platelet integrin, plays a central role in hemostasis and thrombosis. Upon platelet activation, conformation of αIIbß3 changes and allows fibrinogen binding and, subsequently, platelet aggregation. It was previously shown that a lipid-modified platelet permeable peptide, which corresponds to the intracellular acidic membrane distal sequence 1000LEEDDEEGE1008 of αIIb (pal-K-LEEDDEEGE or pal-K-1000-1008), inhibits thrombin-induced human platelet aggregation, by inhibiting talin association with the integrin. YMESRADR, a peptide corresponding to the extracellular sequence 313-320 of αIIb, is also a potent platelet aggregation inhibitor by mimicking the effect of a clasp between the head domains of αIIb and ß3. The aim of the present study was to investigate the synergistic effect of the intra- and extracellular- peptide inhibitors on platelet aggregation, as well as on the phosphorylation of two signaling proteins, focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Platelet preincubation with Pal-K-LEEDDEGE followed by YMESRADR showed a synergistic inhibitory activity on platelet aggregation. Platelet incubation with threshold inhibitory concentrations of both peptides provoked almost the total inhibition of aggregation, PAC-1 binding, and fibrinogen binding, but not P-selectin exposure on activated platelets' surface. Like RGDS peptide, this mixture inhibits FAK phosphorylation whose phosphorylation is well known to be consecutive to the aggregation (postoccupancy events). However, in contrast to RGDS peptide that enhances ERK phosphorylation and activation, the mixture of threshold inhibitory concentrations of Pal-K-LEEDDEEGE and YMESRADR inhibits ERK phosphorylation. We suggest that the use of the intracellular in combination with the extracellular peptide inhibitor, acting with a non-RGD-like mechanism, may provide an alternative way to antagonize integrin αIIbß3 activation.

Hypoxia impairs agonist-induced integrin αIIbß3 activation and platelet aggregation.

Under ischemic conditions, tissues are exposed to hypoxia. Although human physiology, to a certain extent, can adapt to hypoxic conditions, the impact of low oxygen levels on platelet function is unresolved. Therefore, we explored how reduction of atmospheric oxygen levels to 1% might affect agonist-induced aggregation and static adhesion of isolated human platelets. We uncovered that isolated, washed human platelets exposed to hypoxic conditions show reduced thrombin receptor-activating peptide-6 (TRAP-6) and convulxin-induced aggregation. Of note, this hypoxia-triggered effect was not observed in platelet-rich plasma. Independent of the agonist used (TRAP-6, ADP), activation of the platelet fibrinogen receptor integrin αIIbß3 (GPIIbIIIa, CD41/CD61) was strongly reduced at 1% and 8% oxygen. The difference in agonist-induced integrin αIIbß3 activation was apparent within 5 minutes of stimulation. Following hypoxia, re-oxygenation resulted in the recovery of integrin αIIbß3 activation. Importantly, platelet secretion was not impaired by hypoxia. Static adhesion experiments revealed decreased platelet deposition to fibrinogen coatings, but not to collagen or vitronectin coatings, indicating that specifically the function of the integrin subunit αIIb is impaired by exposure of platelets to reduced oxygen levels. Our results reveal an unexpected effect of oxygen deprivation on platelet aggregation mediated by the fibrinogen receptor integrin αIIbß3.