Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a metabolic liver disease that progresses from simple steatosis to the disease state of inflammation and fibrosis. Previous studies suggest that apoptosis and necroptosis may contribute to the pathogenesis of NASH, based on several murine models. However, the mechanisms underlying the transition of simple steatosis to steatohepatitis remain unclear, because it is difficult to identify when and where such cell deaths begin to occur in the pathophysiological process of NASH. In the present study, our aim is to investigate which type of cell death plays a role as the trigger for initiating inflammation in fatty liver. By establishing a simple method of discriminating between apoptosis and necrosis in the liver, we found that necrosis occurred prior to apoptosis at the onset of steatohepatitis in the choline-deficient, ethionine-supplemented (CDE) diet model. To further investigate what type of necrosis is involved in the initial necrotic cell death, we examined the effect of necroptosis and ferroptosis inhibition by administering inhibitors to wild-type mice in the CDE diet model. In addition, necroptosis was evaluated using mixed lineage kinase domain-like protein (MLKL) knockout mice, which is lacking in a terminal executor of necroptosis. Consequently, necroptosis inhibition failed to block the onset of necrotic cell death, while ferroptosis inhibition protected hepatocytes from necrotic death almost completely, and suppressed the subsequent infiltration of immune cells and inflammatory reaction. Furthermore, the amount of oxidized phosphatidylethanolamine, which is involved in ferroptosis pathway, was increased in the liver sample of the CDE diet-fed mice. These findings suggest that hepatic ferroptosis plays an important role as the trigger for initiating inflammation in steatohepatitis and may be a therapeutic target for preventing the onset of steatohepatitis.

Neutrophils alleviate fibrosis in the CCl4-induced mouse chronic liver injury model.

Tribbles pseudokinase 1 (Trib1) is a negative regulator of CCAAT/enhancer binding protein α (C/EBPα) and is known to induce granulopoiesis while suppressing monocyte differentiation. Loss of Trib1 was previously shown to increase the neutrophil population in the spleen but lead to M2-like macrophage reduction. Because M2 macrophages are anti-inflammatory and promote tissue repair by producing fibrogenic factors, we investigated liver fibrosis in Trib1-deficient mice. Interestingly, loss of Trib1 suppressed fibrosis in the CCl4-induced chronic liver injury model. Trib1 knockout increased neutrophils but had a minimal effect on the macrophage population in the liver. Hepatic expressions of neutrophil matrix metalloproteinases (Mmp)8 and Mmp9 were increased, but the production of fibrogenic factors, including transforming growth factor ß1, was not affected by loss of Trib1. These results suggest that neutrophils are responsible for the suppression of fibrosis in Trib1-deficient liver. Consistently, transplantation of Trib1-deficient bone marrow cells into wild-type mice alleviated CCl4-induced fibrosis. Furthermore, expression of chemokine (C-X-C motif) ligand 1 (Cxcl1) by adeno-associated viral vector in the normal liver recruited neutrophils and suppressed CCl4-induced fibrosis; infusion of wild-type neutrophils in CCl4-treated mice also ameliorated fibrosis. Using recombinant adeno-associated virus-mediated expression of Mmp8 and Mmp9 alleviated liver fibrosis. Finally, neutrophil depletion by infusion of Ly6G antibody significantly enhanced CCl4-induced fibrosis. Conclusion: While neutrophils are well known to exacerbate acute liver injury, our results demonstrate a beneficial role of neutrophils in chronic liver injury by promoting fibrolysis. (Hepatology Communications 2018;2:703-717).