Bile acid-induced IRF3 phosphorylation mediates cell death, inflammatory responses, and fibrosis in cholestasis-induced liver and kidney injury via regulation of ZBP1.

BACKGROUND AND AIMS: Cell death and inflammation play critical roles in chronic tissue damage caused by cholestatic liver injury leading to fibrosis and cirrhosis. Liver cirrhosis is often associated with kidney damage, which is a severe complication with poor prognosis. Interferon regulatory factor 3 (IRF3) is known to regulate apoptosis and inflammation, but its role in cholestasis remains obscure. In this study. APPROACH AND RESULTS: We discovered increased IRF3 phosphorylation in the liver of patients with primary biliary cholangitis and primary sclerosing cholangitis. In the bile duct ligation model of obstructive cholestasis in mice, we found that tissue damage was associated with increased phosphorylated IRF3 (p-IRF3) in the liver and kidney. IRF3 knockout ( Irf3-/- ) mice showed significantly attenuated liver and kidney damage and fibrosis compared to wide-type mice after bile duct ligation. Cell-death pathways, including apoptosis, necroptosis, and pyroptosis, inflammasome activation, and inflammatory responses were significantly attenuated in Irf3-/- mice. Mechanistically, we show that bile acids induced p-IRF3 in vitro in hepatocytes. In vivo , activated IRF3 positively correlated with increased expression of its target gene, Z-DNA-Binding Protein-1 (ZBP1), in the liver and kidney. Importantly, we also found increased ZBP1 in the liver of patients with primary biliary cholangitis and primary sclerosing cholangitis. We discovered that ZBP1 interacted with receptor interacting protein 1 (RIP1), RIP3, and NLRP3, thereby revealing its potential role in the regulation of cell-death and inflammation pathways. In conclusion. CONCLUSIONS: Our data indicate that bile acid-induced p-IRF3 and the IRF3-ZBP1 axis play a central role in the pathogenesis of cholestatic liver and kidney injury.

Protective role of cGAS in NASH is related to the maintenance of intestinal homeostasis.

BACKGROUND & AIMS: Various intracellular pathways regulate inflammation in NASH. Cyclic GMP-AMP synthase (cGAS) is a DNA sensor that activates STING and plays a role in inflammatory diseases. Here, we explored the role of cGAS in hepatic damage, steatosis, inflammation, and liver fibrosis in mouse models of NASH. METHODS: cGAS deficient (cGAS-KO) and STING deficient (STING-KO) mice received high fat-high cholesterol-high sugar diet (HF-HC-HSD) or relevant control diets. Livers were evaluated after 16 or 30 weeks. RESULTS: HF-HC-HSD diet, both at 16 and 30 weeks, resulted in increased cGAS protein expression as well as in increased ALT, IL-1ß, TNF-α and MCP-1 in wild-type (WT) mice compared to controls. Surprisingly, liver injury, triglyceride accumulation, and inflammasome activation were greater in HF-HC-HSD cGAS-KO compared to WT mice at 16 and to a lesser extent at 30 weeks. STING, a downstream target of cGAS was significantly increased in WT mice after HF-HC-HSD. In STING-KO mice after HF-HC-HSD feeding, we found increased ALT and attenuated MCP1 and IL-1ß expression compared to WT mice. Markers of liver fibrosis were increased in cGAS- and STING-KO mice compared to WT on HF-HC-HSD. We discovered that cGAS-KO mice had a significant increase in circulating endotoxin levels on HF-HC-HSD that correlated with changes in intestinal morphology which was exacerbated by HF-HC-HSD compared to WT mice. CONCLUSION: Our findings indicate that cGAS or STING deficiency exacerbate liver damage, steatosis, and inflammation in HF-HC-HSD diet-induced NASH, which might be linked to the disruption of the gut barrier.

Pharmacological Inhibition of CCR2/5 Signaling Prevents and Reverses Alcohol-Induced Liver Damage, Steatosis, and Inflammation in Mice.

Kupffer cell and macrophage (MØ) activation contributes to steatosis, inflammation, and fibrosis in alcoholic liver disease (ALD). We found increased frequency of MØ, T cells, and expression of C-C chemokine receptor type 2 (Ccr2) and C-C chemokine receptor type 5 (Ccr5) in the livers of patients with ALD, and increased circulating chemokines, C-C chemokine ligand types 2 (CCL2), and C-C chemokine ligand types 5 (CCL5) in patients with alcoholic hepatitis. We hypothesized that inhibition of CCL2 signaling with the dual CCR2/5 inhibitor, cenicriviroc (CVC), would attenuate ALD. In a mouse model of ALD, liver injury (alanine aminotransferase [ALT]) and steatosis were prevented by CVC whether administered as "prevention" throughout the alcohol feeding or as "treatment" started after the development of ALD. Alcohol-induced increases in early liver fibrosis markers (sirius red, hydroxyproline, and collagen-1) were normalized by both modes of CVC administration. We found that prevention and treatment with CVC reversed alcohol-related increases in liver mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and CCL2. CVC administration regimens prevented the increase in infiltrating MØ (F4/80lo CD11bhi ) and reduced proinflammatory Ly6Chi MØ in livers of alcohol-fed mice. CVC increased liver T-cell numbers and attenuated Il-2 expression without an effect on CD69+ or CD25+ T-cell expression. In vitro, CVC inhibited CCL2-induced increases in hepatocyte fatty acid synthase (Fasn) and adipose differentiation-related protein (Adrp), whereas it augmented acyl-coenzyme A oxidase 1 (Acox-1), proliferator-activated receptor gamma co-activator alpha (Pgc1α) and uncoupling protein 2 expression, suggesting mechanisms for attenuated hepatocyte steatosis. We found that CCL2 and CCL5 sensitized hepatocytes to lipopolysaccharide-induced liver injury (TNF-α, ALT, and lactate dehydrogenase release). Alcohol feeding induced apoptosis (poly ADP-ribose polymerase [PARP] and caspase-3 [CASP-3] cleavage) and pyroptosis (gasdermin D [GSDMD] cleavage) in livers, and CVC prevented both of these forms of cell death. Conclusion: Together, our data demonstrate preclinical evidence for CCR2/CCR5 inhibition with CVC as a potent intervention to ameliorate alcohol-induced steatohepatitis and liver damage.