NETosis as mediator of the metabolic effects of high-fat diet modified microbiota

Introduction. Emerging evidence suggest an essential role for neutrophils in the pathogenesis of metabolic diseases. Obesity and type 2 diabetes (T2D) are associated with an alteration in the composition of gut microbiota, termed dysbiosis, and with intestinal barrier dysfunction, which favors the translocation of bacterial lipopolysaccharide (LPS) into the bloodstream, contributing to systemic chronic inflammation and metabolic derangement. Microbiota also plays a crucial role in shaping host immunity, modulating the magnitude of inflammatory responses, and regulating neutrophil production and function. In diabetes, the response of neutrophils to pathogens is abnormal as they are primed to release NETs (neutrophil extracellular traps) composed by sticky chromatin filaments decorated with granular enzymes, and undergo NETosis Aims. We aimed to investigate whether NETosis abridges the effects of dysbiosis toward systemic dysmetabolism. Methods and results. Since the Padi4 (peptidyl arginine deiminase) activity is required for the release of NETs, we used a hematopoietic-restricted Padi4 knockout mouse (Padi4KO) to study the role of NETosis in the development of obesity and T2D. Two-photon confocal imaging, kinetic assays, and luminol-based bioluminescence showed that Padi4 deletion results in less release of NETs in response to PMA or calcium ionophore. Histone citrullination, the key hallmark of NET release, was not present in neutrophils from Padi4KO mice, as assessed by Western blot. After 12 weeks of HFD (60% of calories from fat, 21% carbohydrates and 19% from proteins) Padi4KO and control mice became equally obese, but Padi4KO mice were protected from the development of glucose intolerance and insulin resistance. Furthermore, Padi4KO mice displayed less accumulation of neutrophils and macrophages within the VAT and liver in comparison to their littermates after HFD, as shown by flow cytometry. In line with that, HFD-fed Padi4KO mice showed a seemingly “anergic” cytokine fingerprint and lower levels of plasma dsDNA, compared to control mice, indicating they were protected from the onset of inflammation that accompanies T2D. Through the FITC-dextran gavage assay, we showed that Padi4KO mice were protected from intestinal hyperpermeability induced by HFD and from the surge in plasma concentrations of LPS, a marker of metabolic endotoxemia. Shotgun sequencing of the microbiota showed similar HFD-induced dysbiosis in both control and Padi4KO mice. However, intestinal inflammation (fecal s100a8 levels) and gut neutrophils (CD45+CD115-Ly6G+SiglecF- cells) abundance were lower in Padi4KO mice. To test whether NETosis is a first-line response to dysbiosis and drives systemic dysmetabolism, we performed fecal microbiota transplantation. Control mice transplanted with gut flora from HFD mice became glucose intolerant despite being fed a chow diet. Remarkably, Padi4KO mice transplanted with gut flora from HFD mice were protected from the surge of LPS and displayed a less proinflammatory signature in the peripheral circulation. Finally, pharmacological inhibition of PAD4, like hematopoietic Padi4 deletion, prevented HFD-induced body weight gain, glucose intolerance, and intestinal hyperpermeability. Conclusions. We show for the first time that NETosis is involved in the pathogenesis of dysmetabolism during the onset of obesity. Our study contributed new insight into the role of neutrophils in translating the deleterious effects of a diet-modified microbiota. Therefore, inhibition of NETosis emerges as a target to counter metabolic diseases like T2D.