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.

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.

Recognition of non-self molecular patterns by pattern recognition receptors is a cornerstone of innate immunity. Toll-like receptors (TLRs) exert a key role in recognizing pathogen-associated molecular patterns (PAMPs) but have also been implicated in the recognition of damage-associated molecular patterns (DAMPs). As such, TLRs regulate a wide range of biological responses including inflammatory and immune responses during carcinogenesis. The high expression of TLRs by antigen-presenting cells, including dendritic cells, and their ability to induce anti-tumor mediators such as type I interferon has led to efforts to utilize TLR agonists in tumor therapy in order to convert the often tolerant immune response towards anti-tumor responses. However, TLRs are also increasingly recognized as regulators of tumor-promoting inflammation and promoters of tumor survival signals. Here, we will review in detail the dichotomous role of TLRs in tumor biology, focusing on relevant TLR-dependent pro- and anti-tumor pathways, and discuss clinical applications of TLR-targeted therapies for tumor prevention and treatment.

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