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 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.

Non-canonical proteolytic activation of human prothrombin by subtilisin from Bacillus subtilis may shift the procoagulant-anticoagulant equilibrium toward thrombosis.

Blood coagulation is a finely regulated physiological process culminating with the factor Xa (FXa)-mediated conversion of the prothrombin (ProT) zymogen to active α-thrombin (αT). In the prothrombinase complex on the platelet surface, FXa cleaves ProT at Arg-271, generating the inactive precursor prethrombin-2 (Pre2), which is further attacked at Arg-320-Ile-321 to yield mature αT. Whereas the mechanism of physiological ProT activation has been elucidated in great detail, little is known about the role of bacterial proteases, possibly released in the bloodstream during infection, in inducing blood coagulation by direct proteolytic ProT activation. This knowledge gap is particularly concerning, as bacterial infections are frequently complicated by severe coagulopathies. Here, we show that addition of subtilisin (50 nm to 2 µm), a serine protease secreted by the non-pathogenic bacterium Bacillus subtilis, induces plasma clotting by proteolytically converting ProT into active σPre2, a nicked Pre2 derivative with a single cleaved Ala-470-Asn-471 bond. Notably, we found that this non-canonical cleavage at Ala-470-Asn-471 is instrumental for the onset of catalysis in σPre2, which was, however, reduced about 100-200-fold compared with αT. Of note, σPre2 could generate fibrin clots from fibrinogen, either in solution or in blood plasma, and could aggregate human platelets, either isolated or in whole blood. Our findings demonstrate that alternative cleavage of ProT by proteases, even by those secreted by non-virulent bacteria such as B. subtilis, can shift the delicate procoagulant-anticoagulant equilibrium toward thrombosis.

Noncoded amino acids in protein engineering: Structure-activity relationship studies of hirudin-thrombin interaction.

The advent of recombinant DNA technology allowed to site-specifically insert, delete, or mutate almost any amino acid in a given protein, significantly improving our knowledge of protein structure, stability, and function. Nevertheless, a quantitative description of the physical and chemical basis that makes a polypeptide chain to efficiently fold into a stable and functionally active conformation is still elusive. This mainly originates from the fact that nature combined, in a yet unknown manner, different properties (i.e., hydrophobicity, conformational propensity, polarizability, and hydrogen bonding capability) into the 20 standard natural amino acids, thus making difficult, if not impossible, to univocally relate the change in protein stability or function to the alteration of physicochemical properties caused by amino acid exchange(s). In this view, incorporation of noncoded amino acids with tailored side chains, allowing to finely tune the structure at a protein site, would facilitate to dissect the effects of a given mutation in terms of one or a few physicochemical properties, thus much expanding the scope of physical organic chemistry in the study of proteins. In this review, relevant applications from our laboratory will be presented on the use of noncoded amino acids in structure-activity relationships studies of hirudin binding to thrombin.

Gut Microbiota and the Microvasculature.

The gut microbiota is increasingly recognized as an actuating variable shaping vascular development and endothelial cell function in the intestinal mucosa but also affecting the microvasculature of remote organs. In the small intestine, colonization with gut microbiota and subsequent activation of innate immune pathways promotes the development of intricate capillary networks and lacteals, influencing the integrity of the gut-vascular barrier as well as nutrient uptake. Since the liver yields most of its blood supply via the portal circulation, the hepatic microcirculation steadily encounters microbiota-derived patterns and active signaling metabolites that induce changes in the organization of the liver sinusoidal endothelium, influencing immune zonation of sinusoids and impacting on metabolic processes. In addition, microbiota-derived signals may affect the vasculature of distant organ systems such as the brain and the eye microvasculature. In recent years, this gut-resident microbial ecosystem was revealed to contribute to the development of several vascular disease phenotypes.

Sterility testing of germ-free mouse colonies.

In biomedical research, germ-free and gnotobiotic mouse models enable the mechanistic investigation of microbiota-host interactions and their role on (patho)physiology. Throughout any gnotobiotic experiment, standardized and periodic microbiological testing of defined gnotobiotic housing conditions is a key requirement. Here, we review basic principles of germ-free isolator technology, the suitability of various sterilization methods, and the use of sterility testing methods to monitor germ-free mouse colonies. We also discuss their effectiveness and limitations, and share the experience with protocols used in our facility. In addition, possible sources of isolator contamination are discussed and an overview of reported contaminants is provided.

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.

Exogenous human α-Synuclein acts in vitro as a mild platelet antiaggregant inhibiting α-thrombin-induced platelet activation.

α-Synuclein (αSyn) is a small disordered protein, highly conserved in vertebrates and involved in the pathogenesis of Parkinson's disease (PD). Indeed, αSyn amyloid aggregates are present in the brain of patients with PD. Although the pathogenic role of αSyn is widely accepted, the physiological function of this protein remains elusive. Beyond the central nervous system, αSyn is expressed in hematopoietic tissue and blood, where platelets are a major cellular host of αSyn. Platelets play a key role in hemostasis and are potently activated by thrombin (αT) through the cleavage of protease-activated receptors. Furthermore, both αT and αSyn could be found in the same spatial environment, i.e. the platelet membrane, as αT binds to and activates platelets that can release αSyn from α-granules and microvesicles. Here, we investigated the possibility that exogenous αSyn could interfere with platelet activation induced by different agonists in vitro. Data obtained from distinct experimental techniques (i.e. multiple electrode aggregometry, rotational thromboelastometry, immunofluorescence microscopy, surface plasmon resonance, and steady-state fluorescence spectroscopy) on whole blood and platelet-rich plasma indicate that exogenous αSyn has mild platelet antiaggregating properties in vitro, acting as a negative regulator of αT-mediated platelet activation by preferentially inhibiting P-selectin expression on platelet surface. We have also shown that both exogenous and endogenous (i.e. cytoplasmic) αSyn preferentially bind to the outer surface of activated platelets. Starting from these findings, a coherent model of the antiplatelet function of αSyn is proposed.

NOG-Derived Peptides Can Restore Neuritogenesis on a CRASH Syndrome Cell Model.

Homo- and heterophilic binding mediated by the immunoglobulin (Ig)-like repeats of cell adhesion molecules play a pivotal role in cell-cell and cell-extracellular matrix interactions. L1CAM is crucial to neuronal differentiation, in both mature and developing nervous systems, and several studies suggest that its functional interactions are mainly mediated by Ig2-Ig2 binding. X-linked mutations in the human L1CAM gene are summarized as L1 diseases, including the most diagnosed CRASH neurodevelopmental syndrome. In silico simulations provided a molecular rationale for CRASH phenotypes resulting from mutations I179S and R184Q in the homophilic binding region of Ig2. A synthetic peptide reproducing such region could both mimic the neuritogenic capacity of L1CAM and rescue neuritogenesis in a cellular model of the CRASH syndrome, where the full L1CAM ectodomain proved ineffective. Presented functional evidence opens the route to the use of L1CAM-derived peptides as biotechnological and therapeutic tools.

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.

Interaction of Lactoferrin with Unsaturated Fatty Acids: In Vitro and In Vivo Study of Human Lactoferrin/Oleic Acid Complex Cytotoxicity.

As shown recently, oleic acid (OA) in complex with lactoferrin (LF) causes the death of cancer cells, but no mechanism(s) of that toxicity have been disclosed. In this study, constitutive parameters of the antitumor effect of LF/OA complex were explored. Complex LF/OA was prepared by titrating recombinant human LF with OA. Spectral analysis was used to assess possible structural changes of LF within its complex with OA. Structural features of apo-LF did not change within the complex LF:OA = 1:8, which was toxic for hepatoma 22a cells. Cytotoxicity of the complex LF:OA = 1:8 was tested in cultured hepatoma 22a cells and in fresh erythrocytes. Its anticancer activity was tested in mice carrying hepatoma 22a. In mice injected daily with LF-8OA, the same tumor grew significantly slower. In 20% of animals, the tumors completely resolved. LF alone was less efficient, i.e., the tumor growth index was 0.14 for LF-8OA and 0.63 for LF as compared with 1.0 in the control animals. The results of testing from 48 days after the tumor inoculation showed that the survival rate among LF-8OA-treated animals was 70%, contrary to 0% rate in the control group and among the LF-treated mice. Our data allow us to regard the complex of LF and OA as a promising tool for cancer treatment.

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.

A holobiont view on thrombosis: unravelling the microbiota's influence on arterial thrombus growth.

The commensal microbiota has co-evolved with its host, colonizing all body surfaces. Therefore, this microbial ecosystem is intertwined with host physiology at multiple levels. While it is evident that microbes that reach the blood stream can trigger thrombus formation, it remains poorly explored if the wealth of microbes that colonize the body surfaces of the mammalian host can be regarded as a modifier of cardiovascular disease (CVD) development. To experimentally address the microbiota's role in the development of atherosclerotic lesions and arterial thrombosis, we generated a germ-free (GF) low-density lipoprotein receptor-deficient (Ldlr-/- ) atherosclerosis mouse model (Kiouptsi et al., mBio, 2019) and explored the role of nutritional composition on arterial thrombogenesis.

Protease-activated receptor signaling in intestinal permeability regulation.

Protease-activated receptors (PARs) are a unique class of G-protein-coupled transmembrane receptors, which revolutionized the perception of proteases from degradative enzymes to context-specific signaling factors. Although PARs are traditionally known to affect several vascular responses, recent investigations have started to pinpoint the functional role of PAR signaling in the gastrointestinal (GI) tract. This organ is exposed to the highest number of proteases, either from the gut lumen or from the mucosa. Luminal proteases include the host's digestive enzymes and the proteases released by the commensal microbiota, while mucosal proteases entail extravascular clotting factors and the enzymes released from resident and infiltrating immune cells. Active proteases and, in case of a disrupted gut barrier, even entire microorganisms are capable to translocate the intestinal epithelium, particularly under inflammatory conditions. Especially PAR-1 and PAR-2, expressed throughout the GI tract, impact gut permeability regulation, a major factor affecting intestinal physiology and metabolic inflammation. In addition, PARs are critically involved in the onset of inflammatory bowel diseases, irritable bowel syndrome, and tumor progression. Due to the number of proteases involved and the multiple cell types affected, selective regulation of intestinal PARs represents an interesting therapeutic strategy. The analysis of tissue/cell-specific knockout animal models will be of crucial importance to unravel the intrinsic complexity of this signaling network. Here, we provide an overview on the implication of PARs in intestinal permeability regulation under physiologic and disease conditions.

A serine protease secreted from Bacillus subtilis cleaves human plasma transthyretin to generate an amyloidogenic fragment.

Aggregation of human wild-type transthyretin (hTTR), a homo-tetrameric plasma protein, leads to acquired senile systemic amyloidosis (SSA), recently recognised as a major cause of cardiomyopathies in 1-3% older adults. Fragmented hTTR is the standard composition of amyloid deposits in SSA, but the protease(s) responsible for amyloidogenic fragments generation in vivo is(are) still elusive. Here, we show that subtilisin secreted from Bacillus subtilis, a gut microbiota commensal bacterium, translocates across a simulated intestinal epithelium and cleaves hTTR both in solution and human plasma, generating the amyloidogenic fragment hTTR(59-127), which is also found in SSA amyloids in vivo. To the best of our knowledge, these findings highlight a novel pathogenic mechanism for SSA whereby increased permeability of the gut mucosa, as often occurs in elderly people, allows subtilisin (and perhaps other yet unidentified bacterial proteases) to reach the bloodstream and trigger generation of hTTR fragments, acting as seeding nuclei for preferential amyloid fibrils deposition in the heart.

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.

Germ-free housing conditions do not affect aortic root and aortic arch lesion size of late atherosclerotic low-density lipoprotein receptor-deficient mice.

The microbiota has been linked to the development of atherosclerosis, but the functional impact of these resident bacteria on the lesion size and cellular composition of atherosclerotic plaques in the aorta has never been experimentally addressed with the germ-free low-density lipoprotein receptor-deficient (Ldlr-/- ) mouse atherosclerosis model. Here, we report that 16 weeks of high-fat diet (HFD) feeding of hypercholesterolemic Ldlr-/- mice at germ-free (GF) housing conditions did not impact relative aortic root plaque size, macrophage content, and necrotic core area. Likewise, we did not find changes in the relative aortic arch lesion size. However, late atherosclerotic GF Ldlr-/- mice had altered inflammatory plasma protein markers and reduced smooth muscle cell content in their atherosclerotic root plaques relative to CONV-R Ldlr-/- mice. Neither absolute nor relative aortic root or aortic arch plaque size correlated with age. Our analyses on GF Ldlr-/- mice did not reveal a significant contribution of the microbiota in late aortic atherosclerosis.

Comment on "Endothelial Protein C Receptor (EPCR), Protease Activated Receptor-1 (PAR-1) and Their Interplay in Cancer Growth and Metastatic Dissemination" Cancers 2019, 11, 51.

Although the interplay between tumor progression and blood coagulation has been recognized [...].