D. J. Van Der Windt1, V. Sud1, P. Varley1, J. Goswami1, H. Zhang1, H. Yazdani1, P. Loughran2, M. I. Minervini4, H. Huang1, R. L. Simmons1, A. Tsung1 1University Of Pittsburgh,Surgical Oncology,Pittsburgh, PENNSYLVANIA, USA 2University Of Pittsburgh,Center For Biologic Imaging,Pittsburgh, PENNSYLVANIA, USA 3University Of Pittsburgh,Department Of Medicine,Pittsburgh, PENNSYLVANIA, USA 4University Of Pittsburgh,Department Of Pathology,Pittsburgh, PENNSYLVANIA, USA
Introduction:
Nonalcoholic steatohepatitis (NASH) is a rapidly increasing precursor of hepatocellular carcinoma (HCC) whether or not cirrhosis is evident, underlining the importance of inflammation as a hallmark of cancer development. Neutrophils are increasingly recognized as regulators of the protumorigenic inflammatory environment. Neutrophils can expel their chromatin structures along with pro-inflammatory proteins into the extracelluar environment leading to the formation of neutrophil extracellular traps (NETs). We have previously reported that blocking NETs can reduce the development of HCC in an experimental model of NASH. Here we sought to further characterize the role of NETs in the inflammatory environment in NASH that can give rise to HCC.
Methods:
NASH mice were created by exposing C57Bl/6 mice to streptozotocin (200ug I.P. <5d from birth) and high fat diet. 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. NET blockade was achieved with injections of DNase1 (100U I.P. 3x/wk), or by using peptidylarginine deiminase 4 knockout (PAD4 KO) mice (that are genetically unable to form NETs)
Results:
Neutrophil infiltration was seen in NASH mice as early as 5 weeks by flow cytometry (6.6±0.8% of CD45+ non-parenchymal cells in NASH vs. 3.6±0.5% in control mice, p<0.05). NASH mouse livers exhibited NET formation by immunofluorescence and western blot for citrullinated histone, a specific NET marker. In vitro, the common free fatty acids, palmitic and linoleic acid (but not oleic acid), were able to stimulate neutrophils to form NETs, suggesting a mechanism for NASH to stimulate NETs. In NASH mice in vivo, neutrophil infiltration was followed by an influx of infiltrating macrophages at 8 weeks with increased levels of IL-6 and TNFa. NET blockade with DNase1 reduced macrophage infiltration from 6.2±1.3% to 3.2±0.7% (p<0.05), and reduced inflammation in the liver (IL-6 was reduced from 22.2±6.7 to 5.8±1.9 pg/mL, TNFa expression was reduces 4-fold, all p<0.05), resulting in a decrease in NASH activity scores (p<0.05). In the long term, the alterations in the liver inflammatory environment resulted in significantly fewer tumors in DNase1 treated NASH mice and PAD4KO NASH mice, compared to wild type NASH mice (1.3±1.1 and 2.2±1.8, vs. 8.7±3.4, p<0.05).
Conclusion:
In steatohepatitis, neutrophil extracellular traps (NETs) attract macrophages, which are a known effector cell in NASH. NET blockade reduces macrophage infiltration and significantly alters the chronic inflammation, thereby reduces the risks of developing HCC.