SUMMARY Increased translocation of intestinal bacteria is a hallmark of chronic liver disease and contributes to hepatic inflammation and fibrosis. Here we tested the hypothesis that the intestinal microbiota and Toll-like receptors (TLRs) promote hepatocellular carcinoma (HCC), a long-term consequence of chronic liver injury, inflammation and fibrosis. Hepatocarcinogenesis in chronically injured livers depended on the intestinal microbiota, and TLR4 activation in non-bone marrow-derived resident liver cells. TLR4 and the intestinal microbiota were not required for HCC initiation but for HCC promotion, mediating increased proliferation, expression of the hepatomitogen epiregulin, and prevention of apoptosis. Gut sterilization restricted to late stages of hepatocarcinogenesis reduced HCC suggesting that the intestinal microbiota and TLR4 represent therapeutic targets for HCC prevention in advanced liver disease.

Although organ fibrosis causes significant morbidity and mortality in chronic diseases, the lack of detailed knowledge about specific cellular contributors mediating fibrogenesis hampers the design of effective anti-fibrotic therapies. Different cellular sources including tissue-resident and bone marrow-derived fibroblasts, pericytes and epithelial cells have been suggested to give rise to myofibroblasts, but their relative contributions remain controversial, with profound differences between organs and different diseases. Here we employ a novel Cre-transgenic mouse that marks 99% of hepatic stellate cells (HSCs), a liver-specific pericyte population, to demonstrate that HSCs give rise to 82-96% of myofibroblasts in models of toxic, cholestatic and fatty liver disease. Moreover, we exclude that HSCs function as facultative epithelial progenitor cells in the injured liver. On the basis of these findings, HSCs should be considered the primary cellular target for anti-fibrotic therapies across all types of liver disease.

Although it is well established that hepatic macrophages play a crucial role in the development of liver fibrosis, the underlying mechanisms remain largely elusive. Moreover, it is not known whether other mononuclear phagocytes such as dendritic cells contribute to hepatic stellate cell (HSC) activation and liver fibrosis. Here we show for the first time that hepatic macrophages enhance myofibroblast survival in an NF-κB-dependent manner, and thereby promote liver fibrosis. Microarray and pathway analysis revealed no induction of HSC activation pathways by hepatic macrophages but a profound activation of the nuclear factor-kappa B (NF-κB) pathway in HSCs. Conversely, depletion of mononuclear phagocytes during fibrogenesis in vivo resulted in suppressed NF-κB activation in HSCs. Macrophage-induced activation of NF-κB in HSC in vitro and in vivo was mediated by IL-1 and TNF. Notably, IL-1 and TNF did not promote HSC activation but promoted survival of activated HSC in vitro and in vivo and thereby increased liver fibrosis, as demonstrated by neutralization in co-culture experiments, and genetic ablation of IL-1 and TNF receptor in vivo. Co-culture and in vivo ablation experiments revealed only a minor contribution to NF-κB activation in HSCs by dendritic cells, and no contribution of dendritic cells to liver fibrosis development, respectively. Conclusion Promotion of NF-κB-dependent myofibroblast survival by macrophages but not dendritic cells provides a novel link between inflammation and fibrosis.

Background & Aims Activated hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver, undergo apoptosis after cessation of liver injury, thereby contributing to the resolution of liver fibrosis. In this study, we investigated whether HSC deactivation constitutes an additional mechanism of liver fibrosis resolution. Methods HSC activation and deactivation were investigated by single cell PCR and genetic tracking in transgenic mice expressing tamoxifen-inducible CreER under control of the endogenous vimentin promoter (VimCreER). Results Single cell quantitative PCR demonstrated activation of virtually the entire HSC population in fibrotic livers, and a gradual decrease of HSC activation during fibrosis resolution, indicating deactivation of HSCs. VimCreER marked activated HSCs, demonstrated by a 6- to 16-fold induction of a membrane-bound green fluorescent protein (mGFP) Cre-reporter following injection of carbontetrachloride (CCl4) in both liver and isolated HSCs, and a shift in localization of mGFP-marked HSCs from perisinusoidal to fibrotic septa. Tracking of mGFP-positive HSCs revealed the persistence of 40–45% of mGFP expression in livers and isolated HSCs 30–45 days after cessation of CCl4, despite normalization of fibrogenesis parameters, thereby confirming reversal of HSC activation. After fibrosis resolution, mGFP expression was observed again in desmin-positive perisinusoidal HSCs; no mGFP expression was detected in hepatocytes or cholangiocytes, thereby excluding mesenchymal-epithelial transition. Notably, reverted HSCs remained in a primed state, with higher responsiveness to profibrogenic stimuli. Conclusion In mice, reversal of HSC activation contributes to the termination of fibrogenesis during fibrosis resolution but results in higher responsiveness of reverted HSCs to recurring fibrogenic stimulation.

Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has subsequently been adapted to mice, allowing to study fibrogenesis by genetic approaches in transgenic mice. However, mouse HSC isolation is commonly hampered by low yield and purity. Here we present an easy-to-perform protocol for high-purity and high-yield isolation of quiescent and activated HSCs in mice, based on retrograde pronase-collagenase perfusion of the liver and subsequent density-gradient centrifugation. We describe an optional add-on protocol for ultrapure HSC isolation from normal and fibrotic livers via subsequent flow-cytometric sorting, thus providing a validated method to determine gene expression changes during HSC activation devoid of cell culture artefacts or contamination with other cells. The described isolation procedure takes approximately four hours to complete.