Innate immunity plays a crucial role in the response to sterile inflammation such as liver ischemia/reperfusion (I/R) injury. of ischemic liver lobes NETs increase hepatocyte death and induce Kupffer cells to release proinflammatory cytokines. DAMPs such as HMGB1 and histones released by hurt hepatocytes stimulate NET formation through Toll-like receptor (TLR4)- and TLR9-MyD88 signaling pathways. After neutrophil depletion in mice the adoptive transfer of TLR4 knockout (KO) or TLR9 KO neutrophils confers significant protection from liver I/R injury with significant decrease in NET formation. In addition we found inhibition of CW069 NET formation by PAD4 inhibitor or DNase I reduces HMGB1 and histone-mediated liver I/R injury. Conclusion DAMPs released during liver I/R promotes NET formation through TLRs signaling pathway. Development of NETs subsequently exacerbates organ damage and initiates inflammatory responses during liver I/R. and (9); however NETs have recently been implicated as harmful contributors in various sterile inflammatory conditions including atherosclerosis venous thrombosis lung injury and tumor metastasis among others (10 11 The role of DAMPs released following ischemic liver injury in activating neutrophils to form NETs and the role of NETs themselves in liver I/R remain unknown. Elucidating the mechanisms of NET formation in liver I/R will increase our understanding of the molecular pathophysiology of liver ischemic injury and provide significant insight into the mechanisms by which ischemic tissues notify the immune system of impending cell damage. We found in this study that neutrophils form NETs in the setting of liver I/R. NET formation is dependent on DAMPs such as HMGB1 and histones released from stressed hepatocytes and mediate NET CW069 formation through TLR4 and TLR9 signaling. Targeting NETs using DNase I or specific PAD4 inhibitors ameliorated the hepatic I/R-induced injury in mice. As liver resection or transplantation represent potential cures for patients with malignancies or end stage liver disease liver protective therapeutic strategies using DNase I or PAD4 inhibitors could minimize liver I/R injury and improve clinical outcomes. Materials and Methods Animals Male wild-type (WT C57BL/6) mice (8-12weeks aged) were purchased from Jackson ImmunoResearch Laboratories. TLR4 knockout (KO) and WT TLR9CpG/CpG mutant and WT TLR4/TLR9 double KO and WT MyD88?/? and MyD88+/+ mice were provided by Dr. Timothy Billiar (University or college of Pittsburgh Medical Center Pittsburgh PA). LysMeGFP knockin mice were provided by Dr. Thomas Graf. Animal protocols were approved by the Animal Care and Use Committee of the University or college of Pittsburgh and the experiments were performed in adherence to National Institutes of Health guidelines for the use of laboratory animals. Liver ischemia/reperfusion A nonlethal model of segmental (70%) hepatic warm ischemia and reperfusion was used as previously explained (12). Mice received intraperitoneal injections of histones (25mg/kg Sigma-Aldrich) recombined HMGB1 (rHMGB1 10 μg per mouse) DNase I (2.5 mg or 5 mg/kg Roche) or PAD4 inhibitor YW3-56 (10 mg/kg) or YW4-03 (10 mg/kg) (13) immediately after ischemia CW069 or PBS 1h prior to ischemia. Sham animals underwent anesthesia laparotomy and exposure of the portal triad without hepatic ischemia. Neutrophil depletion isolation and adoptive transfer Mouse neutrophils were isolated from bone marrow of tibias and femurs as explained previously (10). Neutrophils were sorted CW069 on a BD Aria Plus high-speed sorter after incubation with APC-conjugated anti-mouse Ly6G antibody and APC-Cy7 CD11b (BD Biosciences) (purity >96%) (Supplementary Fig. 1). Neutrophil depletion Rabbit Polyclonal to ELOA3. was performed CW069 as explained previously (14) with an intra-peritoneal injection of 500 μg anti-Ly6G antibody (1A8) (BioXCell) 24 and 2 hours before I/R. TLR9KO TLR4 KO or WT freshly isolated neutrophils were injected into the spleens of WT mice just before I/R. Quantification of NETs To quantify NETs in cell culture supernatant and in mouse serum a capture ELISA myeloperoxidase (MPO) associated with DNA was performed as explained previously (15). For the capture antibody Mouse MPO ELISA kit (Hycult biotech HK210-01) was used according to the manufacturer’s directions. A.