Dehnad A1,2, Sasaki Y1,2, Jiang JX1, Sato A1, Chao TI1, Tian J1 ,Török NJ1,2
1Division of Gastroenterology and Hepatology, University of California Davis, Sacramento, CA, USA
2VA Medical Center, VA Northern California Health Care System, Mather, CA, USA
Inflammatory cell recruitment is a major feature of alcoholic liver injury however; the signals and cellular sources regulating the migration of these cells to the liver are still not well defined. C-C chemokine receptor type 2 (CCR2) is mainly expressed by active hepatic stellate cells (HSC) and is a key recruitment signal. Active HSC are also important sources of hydrogen peroxide resulting from the activation of NADPH oxidase 4 (NOX4). As the role of this NOX in early alcoholic liver injury has not been well studied, we hypothesized that NOX4 via hydrogen peroxide modulates the mRNA stability of CCR2 thereby inducing recruitment of inflammatory cells. Methods: NOX4 was studied in healthy human livers and in patients with alcoholic liver disease. Fl/fl and HSC specific [GFAP-cre NOX4 knockout mice (KO)] were pair-fed with the Lieber deCarli or control isocaloric diets, and CCR2, MCP-1, TNFα, IL-1β, IL-6, and Ly6C expression in the livers were assessed by real time qPCR. In vitro, NOX4 expression was studied in primary HSC exposed to acetaldehyde (Ac). CCR2 mRNA stability was assessed 1) in primary wt or NOX4KO HSC, and 2) in LX2 human HSC line transfected by Ad-NOX4 or control vector. Immunohistochemistry and western blots were done to detect the mRNA binding protein HuR subcellular localization and phosphorylation. Results: NOX4 was co-localized with αSMA-expressing activated HSC in liver biopsies of patients with alcoholic hepatitis. NOX4 mRNA was significantly induced in patients, as well as in mice on the Lieber deCarli diet. In the fl/fl mice on Lieber deCarli diet, TG content, lipid peroxidation and the expression of CCR2, TNFα, IL-6, MCP-1, Ly6C were significantly increased compared to the HSCNOX4Komice (p <0.05). NOX4 was induced in primary HSC by Ac treatment (p<0.05). NOX4 has significantly increased transcript stability of CCR2 (p<0.05) coinciding with cytoplasmic shuttling and phosphorylation of HuR. In conclusion: NOX4 is induced in early alcoholic liver injury in HSC and regulates CCR2 mRNA stability thereby promotes recruitment of inflammatory cells and production of proinflammatory cytokines.