Background & Aims
Hepatocyte apoptosis and activation of hepatic stellate cells (HSC) are critical events in fibrogenesis. We previously demonstrated that phagocytosis of apoptotic hepatocytes by HSC is profibrogenic. Based on this, as well as the observation that reduced nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase induction is central to fibrogenesis, our aim was to study the phagocytic NADPH oxidase NOX2.

Methods
An in vivo phagocytosis model was developed by injecting wild type (wt) or NOX2-/- mice with lentiviral-green fluorescence protein (GFP) containing a hepatocyte-specific promoter, and adeno-tumor necrosis factor-related apoptosis-inducing ligand (ad-TRAIL). Fibrosis was evaluated in bile duct ligated (BDL) wt and NOX2-/- mice with or without gadolinium treatment. NOX2 expression was studied in human liver samples and in HSC isolated from fibrotic livers. The fibrogenic activity of NOX2 was assessed by collagen reporter assays.

Results
In the phagocytosis model, engulfment of GFP-labeled apoptotic bodies was seen, and the expression of a-smooth muscle actin (a-SMA) and collagen I increased significantly in the wt but not in the NOX2-/- mice. Inhibiting apoptosis decreased the profibrogenic response. NOX2-/- animals exhibited significantly less fibrosis following BDL. Inactivating macrophages in wt BDL mice did not lower collagen production to the level observed in NOX2-/- mice, suggesting that NOX2-expressing HSC are important in fibrogenesis. NOX2 was up-regulated in HSC from fibrotic livers, and phagocytosis-induced NOX2 expression and activity were demonstrated. Based on reporter assays, production of NOX2-mediated reactive oxygen species directly induced collagen promoter activity in HSC.

Conclusions
Apoptosis and phagocytosis of hepatocytes directly induce HSC activation and initiation of fibrosis. NOX2, the phagocytic NADPH oxidase, plays a key role in this process and in liver fibrogenesis in vivo.

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