D. J. Van Der Windt1, V. Sud1, P. Varley1, J. Goswami1, H. Yazdani1, P. Loughran3, M. I. Minervini4, H. Huang1,2, R. L. Simmons1, A. Tsung1 1University Of Pittsburgh Medical Center,Surgery,Pittsburgh, PA, USA 2Huazhong University Of Science And Technology,Surgery,Wuhan, HUBEI, China 3Center For Biologic Imaging,Cell Biology,Pittsburgh, PA, USA 4University Of Pittsburgh Medical Center,Pathology,Pittsburgh, PA, USA
Introduction: Non-alcoholic steatohepatitis (NASH) is a progressive, inflammatory form of non-alcoholic fatty liver disease. Its prevalence is rapidly increasing, resulting in more patients with NASH-related hepatocellular carcinoma (HCC). Surgical resection and liver transplantation are the only curative options for HCC. As a consequence, there is an increased number of patients with NASH undergoing liver resection, and those patients have higher risks of post-operative complications. The inflammatory pathogenesis of NASH progression to HCC remains incompletely understood. Neutrophils are increasingly recognized as cells that regulate the inflammatory environment and can affect the progression of inflammatory cancers. The formation of neutrophil extracellular traps (NETs) has been discovered as a novel neutrophil function, during which chromatin structures with pro-inflammatory proteins are exposed to the extracellular environment. Here we sought to evaluate the role of NETs in the inflammatory development of NASH and the progression to HCC.
Methods: Wild type (WT) and peptidylarginine deiminase 4 knockout (PAD4 KO) mice (that are unable to form NETs) were exposed to a model of NASH-related HCC, and compared to healthy control (HC) mice. NASH was established by injection of streptozotocin to induce diabetes (200ug I.P., 1 dose within 5d from birth), and feeding of a high fat diet starting at age 3 weeks. WT and PAD4KO mice were sacrificed at various time points for analysis of liver inflammation and the formation of HCC. To test the efficacy of a clinically available treatment strategy for eliminating NETs, WT mice were treated with DNase1 (100U I.P. 3x/wk).
Results: WT male mice developed NASH as evidenced by progressive increase in NASH activity score (NAS), and developed multiple liver tumors by age 20 weeks. Neutrophil infiltration was seen as early as age 6 weeks as evidenced by flow cytometry (12.6±0.5% in WT vs. 4.0±0.2% of CD45+ liver non-parenchymal cells in HC, p<0.001). NETs were observed on liver immunofluorescence. At age 8 weeks, WT mice had elevated serum ALT and IL-6 levels, indicating liver injury and inflammation. ALT and IL-6 levels were reduced to baseline in PAD4 KO mice and by DNase1 treatment, respectively (ALT [U/L]: WT 166±35, PAD4 KO 102±4, DNase1 73±16, p<0.05. IL-6 [pg/mL]: WT 23±7.9, PAD4 KO 3.9±0.7, DNase1 5.8±1.2, p<0.05). Most importantly, PAD4KO mice developed significantly fewer tumors at age 20wks than WT mice (1.7±0.6 vs. 6.3±1.8, p<0.05).
Conclusion: Elimination of NETs reduces inflammation and prevents progression to HCC in murine NASH. Clinically, inhibition of NETs in patients with NASH may reduce their risk of developing HCC.