Neutrophil Extracellular Traps Promote Inflammation And Development Of Hepatocellular Carcinoma In Nonalcoholic Steatohepatitis

Background: Nonalcoholic steatohepatitis(NASH)is a progressive,inflammatory form of nonalcoholic fatty liver disease(NAFLD)characterized by steatosis,hepatocellular injury and chronic inflammation,which affects 3 to 5% of the population and approximately 10% NASH patients will progress to cirrhosis.NASH with or without cirrhosis is itself a significant risk factor for hepatocellular carcinoma(HCC).The inflammatory signals promoting the progression of NASH to HCC remain largely unknown.Neutrophils are recognized as key regulators of the pro-tumorigenic inflammatory environment which can expel their chromatin structures along with proinflammatory proteins into the extracellular environment leading to the formation of neutrophil extracellular traps(NETs).Neutrophils form neutrophil extracellular traps(NETs),which are large,extracellular,weblike structures composed of cytosolic and granule proteins that are assembled on a scaffold of decondensed chromatin,which has been shown to be important in chronic inflammatory conditions and in cancer progression.In this study,we asked whether NETs formation occurs in NASH and contributes to the progression of HCC.Objective 1.Determine the role of NETs formation mediating NAFLD development?2.Clarify the mechanism of NETs formation mediated-inflammatory and immune responses foster NASH development and NASH-HCC tumor growth.3.Investigate the potential for NETs-related molecules as biomarkers and as targets for therapeutic intervention in NASH-HCC.Methods: Serum samples were collected preoperatively from patients scheduled to undergo partial hepatectomy at the University of Pittsburgh(Pittsburgh,PA,US)between 2010 and 2017 for MPO-DNA complexes.NASH model was created by exposing C57Bl/6 mice to streptozotocin(200ug I.P.<5d from birth)and high fat diet food.The development of NASH was evaluated over time by serum and tissue levels of inflammatory cytokines,flow cytometry of liver non-parenchymal cells,and histology.NETs blockade was achieved with injections of DNase1(50mg I.P.daily),or by using peptidylarginine deiminase 4 knockout(PAD4 KO)mice(that are genetically unable to form NETs).Overexpress NETs in vivo by injecting exogenous NETs.Stimulating neutrophils isolated from mouse bone marrow with the three Free Fatty Acids,palmitic(C16:0),linoleic(C18:2),and oleic(C18:1),NETs formation was measured by MPO-DNA complexes in culture supernatants.Results: 1.Elevated levels of a NETs marker in serum of patients with NASH.2.In livers from STAM mice,early neutrophil infiltration and NETs formation were seen,followed by an influx of monocyte-derived macrophages,production of inflammatory cytokines,and progression of HCC.3.Blocking NETs did not affect steatosis and free fatty acids accumulation in the liver,but prevented macrophage infiltration and changed the inflammatory environment to one less favorable to HCC growth.4.Overexpress NETs in vivo increased the macrophage infiltration,production of inflammatory cytokines,and progression of HCC.5.Mechanistically,commonly elevated free fatty acids stimulate NETs formation in vitro.Conclusion: NETs blockade reduces macrophage infiltration and significantly alters the chronic inflammation,thereby reduces the risks of developing HCC.The elevated levels of a NETs marker in NASH patients indicating the clinical relevance of our findings.