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Platelet CXCL4 mediates neutrophil extracellular traps formation in ANCA-associated vasculitis.

ABSTRACT: Neutrophils form neutrophil extracellular traps (NETs), which are involved in the pathogenesis of ANCA-associated vasculitis (AAV). Recent reports suggest that platelets stimulated via toll-like receptor (TLR) pathways can induce NETs formation. However, the mechanism underlying the involvement of platelets in NETs formation in AAV is unknown. We investigated the role of platelets in the pathogenesis of AAV. Platelets from AAV patients and healthy controls (HCs) were co-cultured with peripheral neutrophils, and NETs formation was visualized and quantified. The expression levels of TLRs on platelets were examined by flow cytometry. Platelets were treated with a TLR agonist, platelet-derived humoral factor, CXCL4 (platelet factor 4: PF4), and/or anti-CXCL4 antibody to investigate the effects of TLR-CXCL4 signaling on NETs formation. Platelets from AAV significantly upregulated NETs formation in vitro. Flow cytometric analysis revealed that the proportion of TLR9 positive platelets was significantly higher in AAV than HCs. CXCL4 released from TLR9 agonist-stimulated platelets was significantly enhanced in AAV, which subsequently increased NETs formation. Further, neutralizing anti-CXCL4 antibody significantly inhibited NETs formation enhanced by platelets from AAV. TLR9 signaling and CXCL4 release underlie the key role that platelets play in NETs formation in the pathogenesis of AAV.

SUBMITTER: Matsumoto K

PROVIDER: S-EPMC7794527 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Platelet CXCL4 mediates neutrophil extracellular traps formation in ANCA-associated vasculitis.

Matsumoto Kotaro K Yasuoka Hidekata H Yoshimoto Keiko K Suzuki Katsuya K Takeuchi Tsutomu T

Scientific reports 20210108 1

Neutrophils form neutrophil extracellular traps (NETs), which are involved in the pathogenesis of ANCA-associated vasculitis (AAV). Recent reports suggest that platelets stimulated via toll-like receptor (TLR) pathways can induce NETs formation. However, the mechanism underlying the involvement of platelets in NETs formation in AAV is unknown. We investigated the role of platelets in the pathogenesis of AAV. Platelets from AAV patients and healthy controls (HCs) were co-cultured with peripheral ...[more]

PMID: 33420306

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Project description:Our understanding of immune recognition and response to infection and non-infectious forms of cell damage and death is rapidly increasing. The major focus is on host immunity and microbiological invasion. However, it is also clear that these same pathways are important in the initiation and maintenance of autoimmunity and the damage caused to targeted organs. Understanding the involvement of cell death in autoimmune disease is likely to help define critical pathways in the immunopathogenesis of autoimmune disease and new therapeutic targets. An important immune responder cell population in host defense and autoimmunity is the neutrophil. One autoimmune disease where neutrophils play important roles is MPO-ANCA Microscopic Vasculitis. This a severe disease that results from inflammation to small blood vessels in the kidney, the glomeruli (high blood flow and pressure filters). One of the best studied ways in which neutrophils participate in this disease is by cell death through NETosis resulting in the discharge of proinflammatory enzymes and nuclear fragments. In host defense against infection this process helps neutralize pathogens however in auto immunity NETosis results in injury and death to the surrounding healthy tissues. The major autoimmune target in this disease is myeloperoxidase (MPO) which is found uniquely in the cytoplasm of neutrophils. Although the kidney is the major organ targeted in this disease MPO is not expressed in the kidney. Autoantibodies target surface MPO on activated circulating neutrophils resulting in their lodgment in glomerular capillaries where they NETose releasing extracellularly MPO and nuclear fragments initiating injury and planting the key autoantigen MPO. It is the cell death of neutrophils that changes the kidney from innocent bystander to major autoimmune target. Defining the immunopathogenesis of this autoimmune disease and recognizing critical injurious pathways will allow therapeutic intervention to block these pathways and attenuate autoimmune injury. The insights (regarding mechanisms of injury and potential therapeutic targets) are likely to be highly relevant to many other autoimmune diseases.

Project description:Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections often characterized by robust neutrophilic infiltration. Neutrophils provide the first line of defense against P. aeruginosa. Aside from their defense conferred by phagocytic activity, neutrophils also release neutrophil extracellular traps (NETs) to immobilize bacteria. Although NET formation is an important antimicrobial process, the details of its mechanism are largely unknown. The identity of the main components of P. aeruginosa responsible for triggering NET formation is unclear. In this study, our focus was to identify the main bacterial factors mediating NET formation and to gain insight into the underlying mechanism. We found that P. aeruginosa in its exponential growth phase promoted strong NET formation in human neutrophils while its NET-inducing ability dramatically decreased at later stages of bacterial growth. We identified the flagellum as the primary component of P. aeruginosa responsible for inducing NET extrusion as flagellum-deficient bacteria remained seriously impaired in triggering NET formation. Purified P. aeruginosa flagellin, the monomeric component of the flagellum, does not stimulate NET formation in human neutrophils. P. aeruginosa-induced NET formation is independent of the flagellum-sensing receptors TLR5 and NLRC4 in both human and mouse neutrophils. Interestingly, we found that flagellar motility, not flagellum binding to neutrophils per se, mediates NET release induced by flagellated bacteria. Immotile, flagellar motor-deficient bacterial strains producing paralyzed flagella did not induce NET formation. Forced contact between immotile P. aeruginosa and neutrophils restored their NET-inducing ability. Both the motAB and motCD genetic loci encoding flagellar motor genes contribute to maximal NET release; however the motCD genes play a more important role. Phagocytosis of P. aeruginosa and superoxide production by neutrophils were also largely dependent upon a functional flagellum. Taken together, the flagellum is herein presented for the first time as the main organelle of planktonic bacteria responsible for mediating NET release. Furthermore, flagellar motility, rather than binding of the flagellum to flagellum-sensing receptors on host cells, is required for P. aeruginosa to induce NET release.

Project description:The inflammatory microenvironment is commonly characterized by extracellular acidosis (pH < 7.35). Sensitivity to pH, CO2 or bicarbonate concentrations allows neutrophils to react to changes in their environment and to detect inflamed areas in the tissue. One important antimicrobial effector mechanism is the production of neutrophil extracellular traps (NETs), which are released during a programmed reactive oxygen species (ROS)-dependent cell death, the so-called NETosis. Although several functions of neutrophils have been analyzed under acidic conditions, the effect of extracellular acidosis on NETosis remains mainly unexplored and the available experimental results are contradictory. We performed a comprehensive study with the aim to elucidate the effect of extracellular acidosis on ROS-dependent NETosis of primary human neutrophils and to identify the underlying mechanisms. The study was performed in parallel in a CO2-bicabonate-buffered culture medium, which mimics in vivo conditions, and under HEPES-buffered conditions to verify the effect of pH independent of CO2 or bicarbonate. We could clearly show that extracellular acidosis (pH 6.5, 6.0, and 5.5) and intracellular acidification inhibit the release of ROS-dependent NETs upon stimulation of neutrophils with phorbol myristate acetate and immobilized immune complexes. Moreover, our findings suggest that the diminished NET release is a consequence of reduced ROS production and diminished glycolysis of neutrophils under acidic conditions. It was suggested previously that neutrophils can sense the border of inflamed tissue by the pH gradient and that a drop in pH serves as an indicator for the progress of inflammation. Following this hypothesis, our data indicate that an acidic inflammatory environment results in inhibition of extracellular operating effector mechanisms of neutrophils such as release of ROS and NETs. This way the release of toxic components and tissue damage can be avoided. However, we observed that major antimicrobial effector mechanisms such as phagocytosis and the killing of pathogens by neutrophils remain functional under acidic conditions.

Dataset Information

Platelet CXCL4 mediates neutrophil extracellular traps formation in ANCA-associated vasculitis.

Publications

Platelet CXCL4 mediates neutrophil extracellular traps formation in ANCA-associated vasculitis.

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