Platelet-derived CXCL7 induces neutrophil extracellular traps via CXCL7/CXCR2 axis, exacerbating the pathogenesis of diabetic retinopathy

BackgroundProlonged hyperglycemia in diabetes activates platelets and immune cells, forming platelet-immune complexes that damage blood vessels in the retina. However, the role of platelet-neutrophil interactions and neutrophil extracellular traps(NETs) in the development of diabetic retinopathy (DR) was not well studied. In this study, we investigated the mechanisms underlying platelet-mediated NET formation in DR. MethodologyPlatelet activation markers, platelet-neutrophil aggregates (PNA), and NETs markers were assessed by flowcytometry, and the circulatory level of inflammatory markers was measured by Luminex assays in healthy control(HC), type 2 diabetes mellitus(T2DM), non-proliferative DR(NPDR) and proliferative DR(PDR) subjects. In vitro studies investigated platelet-neutrophil interaction in NETs formation using an immunofluorescence assay. Proteomics analysis identified the mechanistic regulators of platelet-induced NETs in DR. Platelet pellet and plasma CXCL7 were quantified using western blot and ELISA, respectively. The role of the CXCL7/CXCR2 axis in inducing NETs formation was examined using CXCL7 recombinant protein, anti-CXCL7 antibody and CXCR2 antagonist (SB225002). ResultsPlatelet activation markers (p-selectin & PF4), PNA, and NETs markers (%NETs, proteinase-3 (PR3), neutrophil elastase (NE)) were significantly increased in the DR group. In vitro studies confirmed that DR-platelets aggregate with healthy neutrophils and form NETs compared to T2DM and HC-platelets. Furthermore, platelet activation and NETs markers were positively correlated with pro-angiogenic (ANGPT2, VEGFA) and inflammatory markers (IL18, ICAM1). In vitro studies reveal that NETs induce inflammation, endothelial dysfunction, disrupt the endothelial monolayer and exacerbate angiogenesis in RF/6A endothelial cell spheroids. Proteomics analysis of platelet-induced NETs in DR revealed dysregulation of proteins involved in platelet activation and NET formation, including CXCL7. Furthermore, increased CXCL-7 levels were observed in platelet pellet and plasma samples from the DR group. Additionally, CXCL7-treated neutrophils formed NETs via the CXCR2 receptor, and inhibition of NETosis was observed in neutrophils exposed to an anti-CXCL-7 antibody and a CXCR2 antagonist. ConclusionOur findings revealed that platelets released CXCL-7 induce NETs formation via the CXCL7/CXCR2 axis and blockade of CXCL7/CXCR2 axis inhibits the NETosis in DR, thereby inhibiting the pathogenesis of DR. Circulating CXCL7 serves as a potential prognostic marker, and the CXCL7/CXCR2 axis may be a therapeutic target for the treatment of DR. What Are the Clinical Implications?Platelets have emerged as immune cells, and platelet-neutrophil interactions are reported to play a significant role in the pathogenesis of various metabolic diseases. The role of platelet-neutrophil interactions and neutrophil extracellular traps (NETs) in the development of diabetic retinopathy (DR) remains poorly understood. Investigating the mechanistic regulators of platelet-mediated NETs in DR is crucial for identifying new therapeutic approaches. Our study observed increased platelet activation, platelet-neutrophil aggregates and NETs among DR subjects. Further, DR-platelet induces NET in healthy neutrophils, and NETs induce inflammation, angiogenesis, and disrupt endothelial barrier function in RF/6A cells in vitro. These findings strengthen the evidence that platelet-neutrophil interactions play a major role in DR pathogenesis. Proteomic analysis identified CXCL7 as a mechanistic regulator of platelet-induced NETs formation in DR. Inhibitors that target the platelet-derived CXCL7/CXCR2 axis for NETosis can be used for the prevention of retinal injury in DR. Overall, our work emphasises the mechanistic understanding of platelet-neutrophil interactions and CXCL7/CXCR2 axis as a therapeutic target for inhibiting the pathogenesis of DR. Graphical abstractScheme for the platelet-derived CXCL7 regulation of NETs in DR. Activated platelets release CXCL7 and platelet aggregates with neutrophils to form NETs via the CXCL7:CXCR2 axis. Blockade of the CXCL7:CXCR2 axis by anti-CXCL7 antibody and CXCR2 inhibitor prevents NETs formation in DR.