CXC chemokine receptor 3 promotes steatohepatitis in mice through mediating inflammatory cytokines, macrophages and autophagy.

CXC chemokine receptor 3 (CXCR3) is involved in virus-related chronic liver inflammation. However, the role of CXCR3 in non-alcoholic steatohepatitis (NASH) remains unclear. We aimed to investigate the role of CXCR3 in NASH. Human liver tissues were obtained from 24 non-alcoholic fatty liver disease (NAFLD) patients and 20 control subjects. CXCR3 knockout (CXCR3(-/-)), obese db/db mice and their wild-type (WT) littermates were used in both methionine-and-choline-deficient (MCD) diet and high-fat high-carbohydrate high-cholesterol (HFHC) diet-induced NASH models. In addition, MCD-fed WT mice were administrated with CXCR3 specific antagonists. CXCR3 was significantly upregulated in liver tissues of patients with NAFLD and in dietary-induced NASH animal models. Compared with WT littermates, CXCR3(-/-) mice were more resistant to both MCD and HFHC diet-induced steatohepatitis. Induction of CXCR3 in dietary-induced steatohepatitis was associated with the increased expression of hepatic pro-inflammatory cytokines, activation of NF-κB, macrophage infiltration and T lymphocytes accumulation (Th1 and Th17 immune response). CXCR3 was also linked to steatosis through inducing hepatic lipogenic genes. Moreover, CXCR3 is associated with autophagosome-lysosome impairment and endoplasmic reticulum (ER) stress in steatohepatitis as evidenced by LC3-II and p62/SQSTM1 accumulation and the induction of GRP78, phospho-PERK and phospho-eIF2α. Inhibition of CXCR3 using CXCR3 antagonist significantly suppressed MCD-induced steatosis and hepatocytes injury in AML-12 hepatocytes. Blockade of CXCR3 using CXCR3 antagonists in mice reversed the established steatohepatitis. CXCR3 plays a pivotal role in NASH development by inducing production of cytokines, macrophage infiltration, fatty acid synthesis and causing autophagy deficiency and ER stress.

Zhang X ，Han J ，Man K ，Li X ，Du J ，Chu ES ，Go MY ，Sung JJ ，Yu J ... -  《-》

Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice.

Gasdermin D (GSDMD)-executed programmed necrosis is involved in inflammation and controls interleukin (IL)-1β release. However, the role of GSDMD in non-alcoholic steatohepatitis (NASH) remains unclear. We investigated the role of GSDMD in the pathogenesis of steatohepatitis. Human liver tissues from patients with non-alcoholic fatty liver disease (NAFLD) and control individuals were obtained to evaluate GSDMD expression. Gsdmd knockout (Gsdmd-/-) mice, obese db/db mice and their wild-type (WT) littermates were fed with methionine-choline deficient (MCD) or control diet to induce steatohepatitis. The Gsdmd-/- and WT mice were also used in a high-fat diet (HFD)-induced NAFLD model. In addition, Alb-Cre mice were administered an adeno-associated virus (AAV) vector that expressed the gasdermin-N domain (AAV9-FLEX-GSDMD-N) and were fed with either MCD or control diet for 10 days. GSDMD and its pyroptosis-inducing fragment GSDMD-N were upregulated in liver tissues of human NAFLD/NASH. Importantly, hepatic GSDMD-N protein levels were significantly higher in human NASH and correlated with the NAFLD activity score and fibrosis. GSDMD-N remained a potential biomarker for the diagnosis of NASH. MCD-fed Gsdmd-/- mice exhibit decreased severity of steatosis and inflammation compared with WT littermates. GSDMD was associated with the secretion of pro-inflammatory cytokines (IL-1β, TNF-α, and MCP-1 [CCL2]) and persistent activation of the NF-ĸB signaling pathway. Gsdmd-/- mice showed lower steatosis, mainly because of reduced expression of the lipogenic gene Srebp1c (Srebf1) and upregulated expression of lipolytic genes, including Pparα, Aco [Klk15], Lcad [Acadl], Cyp4a10 and Cyp4a14. Alb-Cre mice administered with AAV9-FLEX-GSDMD-N showed significantly aggravated steatohepatitis when fed with MCD diet. As an executor of pyroptosis, GSDMD plays a key role in the pathogenesis of steatohepatitis, by controlling cytokine secretion, NF-ĸB activation, and lipogenesis. Non-alcoholic fatty liver disease has become one of the most feared chronic liver diseases, because it is the most rapidly growing indication for adult liver transplantation and a major cause of hepatocellular carcinoma. However, the mechanisms involved in the transformation of simple steatosis to steatohepatitis remain unclear. Herein, we show that gasdermin D driven pyroptosis is prominent in patients with non-alcoholic steatohepatitis (NASH), and gasdermin-N domain remains a potential biomarker for the diagnosis of NASH. Gasdermin D plays a key role in the pathogenesis of NASH by regulating lipogenesis, the inflammatory response, and the NF-ĸB signaling pathway, revealing potential treatment targets for NASH in humans.

Xu B ，Jiang M ，Chu Y ，Wang W ，Chen D ，Li X ，Zhang Z ，Zhang D ，Fan D ，Nie Y ，Shao F ，Wu K ，Liang J ... -  《-》

Macrophage p38α promotes nutritional steatohepatitis through M1 polarization.

p38 mitogen-activated protein kinases are important inflammatory factors. p38α alteration has been implicated in both human and mouse inflammatory disease models. Therefore, we aimed to characterize the cell type-specific role of p38α in non-alcoholic steatohepatitis (NASH). Human liver tissues were obtained from 27 patients with non-alcoholic fatty liver disease (NAFLD) and 20 control individuals. NASH was established and compared between hepatocyte-specific p38α knockout (p38αΔHep), macrophage-specific p38α knockout (p38αΔMΦ) and wild-type (p38αfl/fl) mice fed with high-fat diet (HFD), high-fat/high-cholesterol diet (HFHC), or methionine-and choline-deficient diet (MCD). p38 inhibitors were administered to HFHC-fed wild-type mice for disease treatment. p38α was significantly upregulated in the liver tissues of patients with NAFLD. Compared to p38αfl/fl littermates, p38αΔHep mice developed significant nutritional steatohepatitis induced by HFD, HFHC or MCD. Meanwhile, p38αΔMΦ mice exhibited less severe steatohepatitis and insulin resistance than p38αfl/fl mice in response to a HFHC or MCD. The effect of macrophage p38α in promoting steatohepatitis was mediated by the induction of pro-inflammatory factors (CXCL2, IL-1β, CXCL10 and IL-6) secreted by M1 macrophages and associated signaling pathways. p38αΔMΦ mice exhibited M2 anti-inflammatory polarization as demonstrated by increased CD45+F4/80+CD11b+CD206+ M2 macrophages and enhanced arginase activity in liver tissues. Primary hepatocytes from p38αΔMΦ mice showed decreased steatosis and inflammatory damage. In a co-culture system, p38α deleted macrophages attenuated steatohepatitic changes in hepatocytes through decreased secretion of pro-inflammatory cytokines (TNF-α, CXCL10 and IL-6), which mediate M1 macrophage polarization in p38αΔMΦ mice. Restoration of TNF-α, CXCL10 or IL-6 induced lipid accumulation and inflammatory responses in p38αfl/fl hepatocytes co-cultured with p38αΔMΦ macrophages. Moreover, pharmacological p38 inhibitors suppressed HFHC-induced steatohepatitis. Macrophage p38α promotes the progression of steatohepatitis by inducing pro-inflammatory cytokine secretion and M1 polarization. p38 inhibition protects against steatohepatitis. LAY SUMMARY: p38 mitogen-activated protein kinases are important inflammatory factors. In the present study, we demonstrated that p38α is upregulated in liver tissues of patients with non-alcoholic fatty liver diseases. Genetic deletion of p38α in macrophages led to ameliorated nutritional steatohepatitis in mice through decreased pro-inflammatory cytokine secretion and increased M2 macrophage polarization.

Zhang X ，Fan L ，Wu J ，Xu H ，Leung WY ，Fu K ，Wu J ，Liu K ，Man K ，Yang X ，Han J ，Ren J ，Yu J ... -  《-》

Protective role of autophagy in methionine-choline deficient diet-induced advanced nonalcoholic steatohepatitis in mice.

The methionine choline-deficient (MCD) diet leads to severe liver injury similar to human nonalcoholic steatohepatitis (NASH). Autophagy has emerged as a critical lysosomal pathway that maintains cell function and survival through the degradation of cellular components such as organelles and proteins. The goal of this study was to elucidate the role of autophagy in MCD-induced steatosis, fibrosis, inflammation, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress in mice. Mice were fed with MCD diet and treated with rapamycin (an autophagy enhancer) or chloroquine (an autophagy inhibitor) for 10 weeks. Liver injury was evaluated biochemically and histologically together with hepatic gene expression analysis. Autophagic flux was impaired in livers of mice fed with MCD diet, evidenced by reduced ratio of LC3-II/LC3-I and increased protein expression of p62. It was found that autophagy activation by rapamycin attenuated MCD-induced steatosis, fibrosis, inflammation, mitochondrial dysfunction, and ER stress. By contrast, MCD mice treated with chloroquine developed more liver injury. In conclusions, the autophagic pathway plays an important protective role in MCD-induced advanced NASH. Thus, pharmacological promotion of autophagy may provide a novel therapeutic strategy for treatment of NASH.

Chen R ，Wang Q ，Song S ，Liu F ，He B ，Gao X ... -  《-》

Lipocalin-2 mediates non-alcoholic steatohepatitis by promoting neutrophil-macrophage crosstalk via the induction of CXCR2.

Inflammatory cell infiltration in the liver is a hallmark of non-alcoholic steatohepatitis (NASH). However, the pathological events which trigger the infiltration of inflammatory cells to mediate NASH pathogenesis remains poorly understood. This study aims to investigate the role of neutrophil-derived lipocalin 2 (LCN2) in mediating the transition from simple steatosis to NASH. Animal models of NASH were induced by high fat high cholesterol (HFHC) diet and methionine- and choline-deficient (MCD) diet in LCN2 knockout mice and wild-type controls. Circulating levels of LCN2 and its hepatic expression were markedly increased in both murine models and human subjects with NASH, and these changes were associated with increased infiltration of neutrophils. In diet-induced NASH models, hepatic injury, necroinflammation and infiltration of neutrophils and macrophages were substantially attenuated by genetic depletion of LCN2. In contrast, chronic infusion of recombinant LCN2 exacerbated diet-induced liver injury, inflammation and macrophage accumulation in a neutrophil-dependent manner. Primary mouse neutrophils lacking LCN2 exhibited a defective migration capacity, which can be reversed by replenishment with recombinant LCN2. Mechanistically, LCN2 induced the expression of the chemokine (C-X-C motif) receptor 2 (CXCR2), thereby leading to activation of ERK1/2 and production of proinflammatory chemokines. LCN2-induced inflammation, infiltration of macrophages and liver injury was abrogated in CXCR2-deficient mice. These findings demonstrated that LCN2 acts as a central mediator to facilitate the crosstalk between neutrophils and hepatic macrophages via induction of the chemokine receptor CXCR2, thereby exacerbating steatohepatitis. Lipocalin-2 levels in blood and the liver were markedly increased in both mouse models and human subjects with NASH, and these changes were associated with increased infiltration of neutrophils in the liver. In diet-induced NASH models, hepatic injury, necroinflammation and infiltration of neutrophils and macrophages were substantially attenuated by genetic depletion of lipocalin-2, but was augmented by chronic infusion of recombinant lipocalin-2. Lipocalin-2 induced the expression of the chemokine receptor CXCR2, thereby leading to activation of the mitogen-activated protein (MAP) kinase ERK1/2 and production of proinflammatory chemokines. Lipocalin-2-induced inflammation, infiltration of macrophages and liver injury was abrogated in CXCR2-deficient mice.

Ye D ，Yang K ，Zang S ，Lin Z ，Chau HT ，Wang Y ，Zhang J ，Shi J ，Xu A ，Lin S ，Wang Y ... -  《-》