NETosis in the gut as mediator of the metabolic effects of high fat died-modified microbiota Ludovica Migliozzi1,2, Stefano Ciciliot2, Valentina Scattolini3, Serena Tedesco1,2, Roberta Cappellari1,2, Mattia Albiero1,2 and Gian Paolo Fadini1,2 1Veneto Institute of Molecular Medicine, Padova, Italy 2Department of Medicine (DIMED), University of Padova, Padova, Italy 3Department of Biology (DiBio), University of Padova, Padova, Italy Introduction. The discovery of metabolic inflammation has been a major achievement in the understanding of type 2 diabetes (T2D) development. Yet, T2D represents a major threat to human health. Metabolic endotoxemia, i.e. the elevation in circulating concentrations of lypopolysaccharide, is the result of gut dysbiosis and increased intestinal permeability. The role of some immune cells in this process has been investigated, but neutrophils are largely neglected. In response to bacterial challenge, neutrophils cast NETs (neutrophil extracellular traps), composed by sticky chromatin filaments decorated with granule enzymes, and eventually die by NETosis. Since the microbiota can prime neutrophils to undergo NETosis, we aimed to investigate whether NETosis could bridge the effects of a diabetogenic gut flora toward systemic metabolism Methods and results. To blunt NETs release in vivo, we generated a hematopoietic-restricted Padi4 Knock-out (Padi4KO) mouse by crossing Vav1-Cre mice with Padi4fl/fl mice. Padi4 is peptidyl arginine deiminase required for the release of NETs. After 12 weeks of HFD (60% of calories from fat, 21% carbohydrates and 19% from proteins) both Padi4KO and control mice became equally obese. Intraperitoneal Glucose Tolerant Test (ipGTT, 1g/kg) and intraperitoneal Insulin Tolerant Test (ipITT, 0.75 U/kg) showed that Padi4KO mice were protected from the development of glucose intolerance and insulin resistance after HFD. Furthermore, Padi4KO mice showed less recruitment of gut neutrophils, and were completely protected from the surge in circulating LPS induced by HFD and the increase in intestinal permeability, assessed by FITC-dextran permeability assay in vivo. The analysis of intestinal barrier integrity showed a deranged tight-junctions profile in HFD-fed control mice, but not in Padi4KO mice. To perform gut flora transplantation, we ablated the endogenous flora of control and Padi4KO mice with a cocktail of broad-spectrum of antibiotics. Control mice transplanted with a HFD flora became dysmetabolic, even on a chow diet. Interestingly, Padi4KO mice transplanted with an HFD flora were completely protected from the onset of dysmetabolism, as assessed by ipGTT. Conclusions. Our results showed for the first time that NETosis is involved in the pathogenesis of dysmetabolism during the onset of obesity. Mechanistically, we envisage that HFD-modified microbiota promotes the recruitment of neutrophils and triggers NETosis which affect intestinal barrier function. These new data will provide an unprecedented view of the role of neutrophils as mediators of metabolic dysfunction.