Critical Role of Tnf-receptor 1 but Not 2 in Hepatic Stellate Cell-induced Extracellular Matrix Remodeling and in Vivo Liver Fibrosis

Background & Aims: TNF has been implicated in the progression of many chronic liver diseases leading to fibrosis; however, the specific contribution of TNF or its receptors, TNF-R1 and TNF-R2, to HSC activation remains to be established. Methods: We have isolated hepatic stellate cells (HSC) from wild-type, and TNF-Receptors 1 (TNFR1), 2 (TNFR2), and double (TNFR-DKO) knockout mice, and cultured on plastic. Proliferation was assessed by [H]-Thymidine incorporation to DNA. Expression of α-SMA was determined by western-blot. TGF-β, TIMP-1, pro-Colα1(I), MMP-2, and MMP-9 mRNA expression was determined by quantitative real-time PCR. Bile duct ligation was performed in wild-type and TNFR-DKO mice to analyze liver damage and fibrogenesis. Results: Loss of both TNF receptors reduced pro-Colα1(I) expression, slowed down HSC proliferation, and are required for PDGF-induced pro-mitogenic signaling in HSC. Antagonism of TNFR1 by specific neutralizing antibodies reproduced the behavior of TNFR-DKO HSC accompanied with decreased pAKT and proliferation in response to PDGF, due to the absence of TNF-R1. In addition, MMP-9 expression was dependent on TNF binding to TNF-R1 in primary mouse HSC. These results were validated in the human HSC cell line LX2 using neutralizing antibodies against TNF-R1 and TNF-R2. Moreover, in vivo liver damage and fibrosis were reduced after cholestasis induction in TNFR-DKO mice compared to wild-type mice. Conclusions: TNF receptors regulate HSC-induced extracellular matrix remodeling in vitro and in early liver fibrosis. Tarrats et al. 3 -