Hepatic Fibrosis is Independent of the Effects of Endotoxin (Lipopolysaccharide) on Hepatic Stellate Cells

Abstract

BackgroundBinding of Gram‐negative bacterial endotoxin lipopolysaccharide (LPS) to CD14 is critical for its cellular effects via TLR4. Activation of hepatic stellate cells (HSCs) from its quiescent phenotype to α‐smooth muscle actin (α‐sma)‐expressing fibrogenic and proliferative myofibroblast‐like phenotype is the major mechanism of liver fibrosis of various etiologies. LPS/TLR4 interactions in HSCs were reported to play an essential role in hepatic fibrosis in experimental murine models. However, LPS was found to induce synthesis of nitric oxide and inflammatory cytokines in HSCs without the aid of CD14 or TLR4, suggesting that CD14/TLR4‐independent pathways may play an important role in hepatic pathophysiology. To address this hypothesis, we investigated effects of LPS on HSCs from CD14‐knock‐out (KO) mice and carbon tetrachloride (CCl4)‐induced fibrosis in them.MethodsHSCs derived from wild type (WT) or CD14‐KO mice were stimulated with LPS (100 ng/ml) following their culture‐induced activation. Parameters of fibrogenic activity (expression collagen, α‐sma and fibrogenic cytokine TGFβ1and its receptor) and inflammatory cytokines/chemokines were measured. In vivo, mice were subjected to CCl4 treatment for 4 weeks, and then administered PBS or LPS (5 mg/kg; ip). Liver injury, inflammation and fibrosis were determined.ResultsLPS‐stimulated TNFα expression was greater, IL6 expression similar and IL10 expression lower in CD14‐KO HSCs compared to WT HSCs. Expression of the chemokines Ccl2, Cxcl1 and Cxcl9 also increased similarly in LPS‐stimulated CD14‐KO and WT HSCs. LPS down‐regulated Acta2 (encodes α‐sma), TGFbr1 and collagen 1 expression in both WT and CD14‐KO HSCs, and the inhibitory effects of LPS on α‐sma and collagen 1 in HSCs were not reversed by exogenous TGFβ1. In vivo, CCl4 treatment induced similar liver injury (histology and serum ALT) and fibrosis (Sirius red staining and hydroxyproline content) in WT and CD14‐KO mice. However, administration of LPS following CCl4 treatment increased liver injury and inflammation in WT mice but not in CD14‐KO mice. Essentially similar results were obtained when TLR4‐KO mice were treated with CCl4 and HSCs from TLR4‐KO mice were challenged with LPS.ConclusionThese data suggest that the direct effects of LPS on HSCs are not essential for hepatic fibrosis. In contrast, LPS may play an important role in limiting fibrosis by down‐modulating the critically responsible components in HSCs.Support or Funding InformationThis work was supported by a VA Merit Review Award 1IO1BX001174.