Notch-mediated hepatocyte MCP-1 secretion causes liver fibrosis.

Patients with nonalcoholic steatohepatitis (NASH) have increased expression of liver monocyte chemoattractant protein-1 (MCP-1), but its cellular source and contribution to various aspects of NASH pathophysiology remain debated. We demonstrated increased liver CCL2 (which encodes MCP-1) expression in patients with NASH, and commensurately, a 100-fold increase in hepatocyte Ccl2 expression in a mouse model of NASH, accompanied by increased liver monocyte-derived macrophage (MoMF) infiltrate and liver fibrosis. To test repercussions of increased hepatocyte-derived MCP-1, we generated hepatocyte-specific Ccl2-knockout mice, which showed reduced liver MoMF infiltrate as well as decreased liver fibrosis. Forced hepatocyte MCP-1 expression provoked the opposite phenotype in chow-fed wild-type mice. Consistent with increased hepatocyte Notch signaling in NASH, we observed a close correlation between markers of Notch activation and CCL2 expression in patients with NASH. We found that an evolutionarily conserved Notch/recombination signal binding protein for immunoglobulin kappa J region binding site in the Ccl2 promoter mediated transactivation of the Ccl2 promoter in NASH diet-fed mice. Increased liver MoMF infiltrate and liver fibrosis seen in opposite gain-of-function mice was ameliorated with concomitant hepatocyte Ccl2 knockout or CCR2 inhibitor treatment. Hepatocyte Notch activation prompts MCP-1-dependent increase in liver MoMF infiltration and fibrosis.

Opposing roles of hepatic stellate cell subpopulations in hepatocarcinogenesis.

Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality worldwide, develops almost exclusively in patients with chronic liver disease and advanced fibrosis1,2. Here we interrogated functions of hepatic stellate cells (HSCs), the main source of liver fibroblasts3, during hepatocarcinogenesis. Genetic depletion, activation or inhibition of HSCs in mouse models of HCC revealed their overall tumour-promoting role. HSCs were enriched in the preneoplastic environment, where they closely interacted with hepatocytes and modulated hepatocarcinogenesis by regulating hepatocyte proliferation and death. Analyses of mouse and human HSC subpopulations by single-cell RNA sequencing together with genetic ablation of subpopulation-enriched mediators revealed dual functions of HSCs in hepatocarcinogenesis. Hepatocyte growth factor, enriched in quiescent and cytokine-producing HSCs, protected against hepatocyte death and HCC development. By contrast, type I collagen, enriched in activated myofibroblastic HSCs, promoted proliferation and tumour development through increased stiffness and TAZ activation in pretumoural hepatocytes and through activation of discoidin domain receptor 1 in established tumours. An increased HSC imbalance between cytokine-producing HSCs and myofibroblastic HSCs during liver disease progression was associated with increased HCC risk in patients. In summary, the dynamic shift in HSC subpopulations and their mediators during chronic liver disease is associated with a switch from HCC protection to HCC promotion.

Quantification of adipocyte numbers following adipose tissue remodeling.

To analyze the capacity of white and brown adipose tissue remodeling, we developed two mouse lines to label, quantitatively trace, and ablate white, brown, and brite/beige adipocytes at different ambient temperatures. We show here that the brown adipocytes are recruited first and reach a peak after 1 week of cold stimulation followed by a decline during prolonged cold exposure. On the contrary, brite/beige cell numbers plateau after 3 weeks of cold exposure. At thermoneutrality, brown adipose tissue, in spite of being masked by a white-like morphology, retains its brown-like physiology, as Ucp1+ cells can be recovered immediately upon beta3-adrenergic stimulation. We further demonstrate that the recruitment of Ucp1+ cells in response to cold is driven by existing adipocytes. In contrast, the regeneration of the interscapular brown adipose tissue following ablation of Ucp1+ cells is driven by de novo differentiation.

Notch activity characterizes a common hepatocellular carcinoma subtype with unique molecular and clinicopathologic features.

BACKGROUND & AIMS: The hepatocyte Notch pathway is a pathogenic factor in non-alcoholic steatohepatitis (NASH)-associated fibrosis, but its role in hepatocellular carcinoma (HCC) is less well defined. Herein, we aimed to characterize the molecular and clinical features of Notch-active human HCC, and to investigate the mechanisms by which Notch affects NASH-driven HCC. METHODS: Using a 14-gene Notch score, we stratified human HCCs from multiple comprehensively profiled datasets. We performed gene set enrichment analyses to compare Notch-active HCCs with published HCC subtype signatures. Next, we sorted Notch-active hepatocytes from Notch reporter mice for RNA sequencing and characterized Notch-active tumors in an HCC model combining a carcinogen and a NASH-inducing diet. We used genetic mouse models to manipulate hepatocyte Notch to investigate the sufficiency and necessity of Notch in NASH-driven tumorigenesis. RESULTS: Notch-active signatures were found in ~30% of human HCCs that transcriptionally resemble cholangiocarcinoma-like HCC, exhibiting a lack of activating CTNNB1 (ß-catenin) mutations and a generally poor prognosis. Endogenous Notch activation in hepatocytes is associated with repressed ß-catenin signaling and hepatic metabolic functions, in lieu of increased interactions with the extracellular matrix in NASH. Constitutive hepatocyte Notch activation is sufficient to induce ß-catenin-inactive HCC in mice with NASH. Notch and ß-catenin show a pattern of mutual exclusivity in carcinogen-induced HCC; in this mouse model, chronic blockade of Notch led to ß-catenin-dependent tumor development. CONCLUSIONS: Notch activity characterizes a distinct HCC molecular subtype with unique histology and prognosis. Sustained Notch signaling in chronic liver diseases can drive tumor formation without acquiring specific genomic driver mutations. LAY SUMMARY: The Notch signaling pathway is known to be involved in the pathogenesis of liver fibrosis. However, its role in liver cancer has not been well defined. Herein, we show that Notch activity is increased in a subset of liver cancers and is associated with poor outcomes. We also used a mouse model to show that aberrant Notch activity can drive cancer progression in obese mice.

ESRRG and PERM1 Govern Mitochondrial Conversion in Brite/Beige Adipocyte Formation.

When exposed to cold temperatures, mice increase their thermogenic capacity by an expansion of brown adipose tissue mass and the formation of brite/beige adipocytes in white adipose tissue depots. However, the process of the transcriptional changes underlying the conversion of a phenotypic white to brite/beige adipocytes is only poorly understood. By analyzing transcriptome profiles of inguinal adipocytes during cold exposure and in mouse models with a different propensity to form brite/beige adipocytes, we identified ESRRG and PERM1 as modulators of this process. The production of heat by mitochondrial uncoupled respiration is a key feature of brite/beige compared to white adipocytes and we show here that both candidates are involved in PGC1α transcriptional network to positively regulate mitochondrial capacity. Moreover, we show that an increased expression of ESRRG or PERM1 supports the formation of brown or brite/beige adipocytes in vitro and in vivo. These results reveal that ESRRG and PERM1 are early induced in and important regulators of brite/beige adipocyte formation.

A Genetic Model to Study the Contribution of Brown and Brite Adipocytes to Metabolism.

UCP1-dependent thermogenesis is studied to define new strategies to ameliorate obesity and type 2 diabetes; however, animal models are mostly limited to germline mutations of UCP1, which can effect adaptive changes in UCP1-independent pathways. We develop an inducible mouse model for the sequential ablation of UCP1+ brown and brite/beige adipocytes in adult mice. We demonstrate that activated brown adipocytes can increase systemic energy expenditure (EE) by 30%, while the contribution of brite/beige UCP1+ cells is <5%. Notably, UCP1+ adipocytes do not contribute to circulating FGF21 levels, either at room temperature or after cold exposure. We demonstrate that the FGF21-mediated effects on EE and glucose homeostasis are partially dependent on the presence of UCP1+ cells, while the effect on weight loss is not. In conclusion, acute UCP1+ cell deletion may be a useful model to study the impact of brown and brite/beige adipocytes on metabolism.

Author Correction: Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

In the version of this article originally published, the months on the axis labeled projected month of conception in Fig. 1a were out of order. April and March should have been the first and last months listed, respectively. The error has been corrected in the print, PDF and HTML versions of this article.

Publisher Correction: Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

In the version of this article originally published, the bars in the mean temperature graph in Fig. 1a were incorrectly aligned. The left-most bar should have been aligned with the Apr label on the projected month of conception axis. The error has been corrected in the print, PDF and HTML versions of this article.

Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

Recent research has focused on environmental effects that control tissue functionality and systemic metabolism. However, whether such stimuli affect human thermogenesis and body mass index (BMI) has not been explored. Here we show retrospectively that the presence of brown adipose tissue (BAT) and the season of conception are linked to BMI in humans. In mice, we demonstrate that cold exposure (CE) of males, but not females, before mating results in improved systemic metabolism and protection from diet-induced obesity of the male offspring. Integrated analyses of the DNA methylome and RNA sequencing of the sperm from male mice revealed several clusters of co-regulated differentially methylated regions (DMRs) and differentially expressed genes (DEGs), suggesting that the improved metabolic health of the offspring was due to enhanced BAT formation and increased neurogenesis. The conclusions are supported by cell-autonomous studies in the offspring that demonstrate an enhanced capacity to form mature active brown adipocytes, improved neuronal density and more norepinephrine release in BAT in response to cold stimulation. Taken together, our results indicate that in humans and in mice, seasonal or experimental CE induces an epigenetic programming of the sperm such that the offspring harbor hyperactive BAT and an improved adaptation to overnutrition and hypothermia.

Interleukin-33-Activated Islet-Resident Innate Lymphoid Cells Promote Insulin Secretion through Myeloid Cell Retinoic Acid Production.

Pancreatic-islet inflammation contributes to the failure of ß cell insulin secretion during obesity and type 2 diabetes. However, little is known about the nature and function of resident immune cells in this context or in homeostasis. Here we show that interleukin (IL)-33 was produced by islet mesenchymal cells and enhanced by a diabetes milieu (glucose, IL-1ß, and palmitate). IL-33 promoted ß cell function through islet-resident group 2 innate lymphoid cells (ILC2s) that elicited retinoic acid (RA)-producing capacities in macrophages and dendritic cells via the secretion of IL-13 and colony-stimulating factor 2. In turn, local RA signaled to the ß cells to increase insulin secretion. This IL-33-ILC2 axis was activated after acute ß cell stress but was defective during chronic obesity. Accordingly, IL-33 injections rescued islet function in obese mice. Our findings provide evidence that an immunometabolic crosstalk between islet-derived IL-33, ILC2s, and myeloid cells fosters insulin secretion.

TLR4 Deficiency Protects against Hepatic Fibrosis and Diethylnitrosamine-Induced Pre-Carcinogenic Liver Injury in Fibrotic Liver.

BACKGROUND: The development of hepatocellular carcinoma (HCC) is a common consequence of advanced liver fibrosis but the interactions between fibrogenesis and carcinogenesis are still poorly understood. Recently it has been shown that HCC promotion depends on Toll-like receptor (TLR) 4. Pre-cancerogenous events can be modelled in mice by the administration of a single dose of diethylnitrosamine (DEN), with HCC formation depending amongst others on interleukin (IL) 6 production. Mice lacking the hepatocanalicular phosphatidylcholine transporter ABCB4 develop liver fibrosis spontaneously, resemble patients with sclerosing cholangitis due to mutations of the orthologous human gene, and represent a valid model to study tumour formation in pre-injured cholestatic liver. The aim of this study was to investigate DEN-induced liver injury in TLR4-deficient mice with biliary fibrosis. METHODS: ABCB4-deficient mice on the FVB/NJ genetic background were crossed to two distinct genetic backgrounds (TLR4-sufficient C3H/HeN and TLR4-deficient C3H/HeJ) for more than 10 generations. The two congenic knockout and the two corresponding wild-type mouse lines were treated with a single dose of DEN for 48 hours. Phenotypic differences were assessed by measuring hepatic collagen contents, inflammatory markers (ALT, CRP, IL6) as well as hepatic apoptosis (TUNEL) and proliferation (Ki67) rates. RESULTS: Hepatic collagen accumulation is significantly reduced in ABCB4-/-:TLR4-/-double-deficient mice. After DEN challenge, apoptosis, proliferation and inflammatory markers are decreased in TLR4-deficient in comparison to TLR4-sufficient mice. When combining ABCB4 and TLR4 deficiency with DEN treatment, hepatic IL6 expression and proliferation rates are lowest in fibrotic livers from the double-deficient line. Consistent with these effects, selective digestive tract decontamination in ABCB4-/- mice also led to reduced tumor size and number after DEN. CONCLUSION: This study demonstrates that liver injury upon DEN challenge depends on pre-existing fibrosis and genetic background. The generation of ABCB4-/: TLR4-/- double-deficient mice illustrates that TLR4-deficiency protects against hepatic injury in a preclinical mouse model of chronic liver disease.

Epithelial Transforming Growth Factor-ß Signaling Does Not Contribute to Liver Fibrosis but Protects Mice From Cholangiocarcinoma.

BACKGROUND & AIMS: Transforming growth factor-ß (TGFß) exerts key functions in fibrogenic cells, promoting fibrosis development in the liver and other organs. In contrast, the functions of TGFß in liver epithelial cells are not well understood, despite their high level of responsiveness to TGFß. We sought to determine the contribution of epithelial TGFß signaling to hepatic fibrogenesis and carcinogenesis. METHODS: TGFß signaling in liver epithelial cells was inhibited by albumin-Cre-, K19-CreERT-, Prom1-CreERT2-, or AAV8-TBG-Cre-mediated deletion of the floxed TGFß receptor II gene (Tgfbr2). Liver fibrosis was induced by carbon tetrachloride, bile duct ligation, or disruption of the multidrug-resistance transporter 2 gene (Mdr2). Hepatocarcinogenesis was induced by diethylnitrosamine or hepatic deletion of PTEN. RESULTS: Deletion of Tgfbr2 from liver epithelial cells did not alter liver injury, toxin-induced or biliary fibrosis, or diethylnitrosamine-induced hepatocarcinogenesis. In contrast, epithelial deletion of Tgfbr2 promoted tumorigenesis and reduced survival of mice with concomitant hepatic deletion of Pten, accompanied by an increase in tumor number and a shift from hepatocellular carcinoma to cholangiocarcinoma. Surprisingly, both hepatocyte- and cholangiocyte-specific deletion of Pten and Tgfbr2 promoted the development of cholangiocarcinoma, but with different latencies. The prolonged latency and the presence of hepatocyte-derived cholangiocytes after AAV8-TBG-Cre-mediated deletion of Tgfbr2 and Pten indicated that cholangiocarcinoma might arise from hepatocyte-derived cholangiocytes in this model. Pten deletion resulted in up-regulation of Tgfbr2, and deletion of Tgfbr2 increased cholangiocyte but not hepatocyte proliferation, indicating that the main function of epithelial TGFBR2 is to restrict cholangiocyte proliferation. CONCLUSIONS: Epithelial TGFß signaling does not contribute to the development of liver fibrosis or formation of hepatocellular carcinomas in mice, but restricts cholangiocyte proliferation to prevent cholangiocarcinoma development, regardless of its cellular origin.

High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers.

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, thus allowing the study of 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 artifacts or contamination with other cells. The described isolation procedure takes ∼4 h to complete.

Sequestration of fatty acids in triglycerides prevents endoplasmic reticulum stress in an in vitro model of cardiomyocyte lipotoxicity.

We used human cardiomyocyte-derived cells to create an in vitro model to study lipid metabolism and explored the effects of PPARγ; ACSL1 and ATGL on fatty acid-induced ER stress. Compared to oleate, palmitate treatment resulted in less intracellular accumulation of lipid droplets and more ER stress, as measured by upregulation of CHOP, ATF6 and GRP78 gene expression and phosphorylation of eukaryotic initiation factor 2a (EIF2a). Both ACSL1 and PPARγ adenovirus-mediated expression augmented neutral lipid accumulation and reduced palmitate-induced upregulation of ER stress markers to levels similar to those in the oleate and control treatment groups. This suggests that increased channeling of non-esterified free fatty acids (NEFA) towards storage in the form of neutral lipids in lipid droplets protects against palmitate-induced ER stress. Overexpression of ATGL in cells incubated with oleate-containing medium increased NEFA release and stimulated expression of ER stress markers. Thus, inefficient creation of lipid droplets as well greater release of stored lipids induces ER stress.

High-mobility group box 1 is dispensable for autophagy, mitochondrial quality control, and organ function in vivo.

In vitro studies have demonstrated a critical role for high-mobility group box 1 (HMGB1) in autophagy and the autophagic clearance of dysfunctional mitochondria, resulting in severe mitochondrial fragmentation and profound disturbances of mitochondrial respiration in HMGB1-deficient cells. Here, we investigated the effects of HMGB1 deficiency on autophagy and mitochondrial function in vivo, using conditional Hmgb1 ablation in the liver and heart. Unexpectedly, deletion of Hmgb1 in hepatocytes or cardiomyocytes, two cell types with abundant mitochondria, did not alter mitochondrial structure or function, organ function, or long-term survival. Moreover, hepatic autophagy and mitophagy occurred normally in the absence of Hmgb1, and absence of Hmgb1 did not significantly affect baseline and glucocorticoid-induced hepatic gene expression. Collectively, our findings suggest that HMGB1 is dispensable for autophagy, mitochondrial quality control, the regulation of gene expression, and organ function in the adult organism.

CCL20 mediates lipopolysaccharide induced liver injury and is a potential driver of inflammation and fibrosis in alcoholic hepatitis.

OBJECTIVE: Chemokines are known to play an important role in the pathophysiology of alcoholic hepatitis (AH), a form of acute-on-chronic liver injury frequently mediated by gut derived lipopolysaccharide (LPS). In our study, we hypothesise that chemokine CCL20, one of the most upregulated chemokines in patients with AH, is implicated in the pathogenesis of AH by mediating LPS induced liver injury. DESIGN: CCL20 gene expression and serum levels and their correlation with disease severity were assessed in patients with AH. Cellular sources of CCL20 and its biological effects were evaluated in vitro and in vivo in chronic, acute and acute-on-chronic experimental models of carbon tetrachloride and LPS induced liver injury. RNA interference technology was used to knockdown CCL20 in vivo. RESULTS: CCL20 hepatic and serum levels were increased in patients with AH and correlated with the degree of fibrosis, portal hypertension, endotoxaemia, disease severity scores and short term mortality. Moreover, CCL20 expression was increased in animal models of liver injury and particularly under acute-on-chronic conditions. Macrophages and hepatic stellate cells (HSCs) were identified as the main CCL20 producing cell types. Silencing CCL20 in vivo reduced LPS induced aspartate aminotransferase and lactate dehydrogenase serum levels and hepatic proinflammatory and profibrogenic genes. CCL20 induced proinflammatory and profibrogenic effects in cultured primary HSCs. CONCLUSIONS: Our results suggest that CCL20 upregulation is strongly associated with LPS and may not only represent a new potential biomarker to predict outcome in patients with AH but also an important mediator linking hepatic inflammation, injury and fibrosis in AH.

Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology.

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 antifibrotic 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 these findings, HSCs should be considered the primary cellular target for antifibrotic therapies across all types of liver disease.

Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice.

UNLABELLED: 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 (DCs) contribute to hepatic stellate cell (HSC) activation and liver fibrosis. We show for the first time that hepatic macrophages enhance myofibroblast survival in a nuclear factor kappa B (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 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 HSCs in vitro and in vivo was mediated by interleukin (IL)-1 and tumor necrosis factor (TNF). Notably, IL-1 and TNF did not promote HSC activation but promoted survival of activated HSCs in vitro and in vivo and thereby increased liver fibrosis, as demonstrated by neutralization in coculture experiments and genetic ablation of IL-1 and TNF receptor in vivo. Coculture and in vivo ablation experiments revealed only a minor contribution to NF-κB activation in HSCs by DCs, and no contribution of DCs to liver fibrosis development, respectively. CONCLUSION: Promotion of NF-κB-dependent myofibroblast survival by macrophages but not DCs provides a novel link between inflammation and fibrosis.

Deactivation of hepatic stellate cells during liver fibrosis resolution in mice.

BACKGROUND & AIMS: Activated hepatic stellate cells (HSCs), the main fibrogenic cell type in the liver, undergo apoptosis after cessation of liver injury, which contributes to resolution of 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 that expressed a tamoxifen-inducible CreER under control of the endogenous vimentin promoter (Vimentin-CreER). RESULTS: Single-cell quantitative polymerase chain reaction demonstrated activation of almost the entire HSC population in fibrotic livers, and a gradual decrease of HSC activation during fibrosis resolution, indicating deactivation of HSCs. Vimentin-CreER marked activated HSCs, demonstrated by a 6- to 16-fold induction of a membrane-bound green fluorescent protein (mGFP) Cre-reporter after injection of carbon tetrachloride, in liver and isolated HSCs, and a shift in localization of mGFP-marked HSCs from peri-sinusoidal 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 carbon tetrachloride was no longer administered, despite normalization of fibrogenesis parameters; these findings confirm reversal of HSC activation. After fibrosis resolution, mGFP expression was observed again in desmin-positive peri-sinusoidal HSCs; no mGFP expression was detected in hepatocytes or cholangiocytes, excluding mesenchymal-epithelial transition. Notably, reverted HSCs remained in a primed state, with higher levels of responsiveness to fibrogenic stimuli. CONCLUSIONS: In mice, reversal of HSC activation contributes to termination of fibrogenesis during fibrosis resolution, but results in higher responsiveness of reverted HSCs to recurring fibrogenic stimulation.

Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4.

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.