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Objective: We evaluated the influence of sex on the pathophysiology of non-alcoholic fatty liver disease (NAFLD). We investigated diet-induced phenotypic responses to define sex-specific regulation between healthy liver and NAFLD to identify influential pathways in different preclinical murine models and their relevance in humans., Design: Different models of diet-induced NAFLD (high-fat diet, choline-deficient high-fat diet, Western diet or Western diet supplemented with fructose and glucose in drinking water) were compared with a control diet in male and female mice. We performed metabolic phenotyping, including plasma biochemistry and liver histology, untargeted large-scale approaches (liver metabolome, lipidome and transcriptome), gene expression profiling and network analysis to identify sex-specific pathways in the mouse liver., Results: The different diets induced sex-specific responses that illustrated an increased susceptibility to NAFLD in male mice. The most severe lipid accumulation and inflammation/fibrosis occurred in males receiving the high-fat diet and Western diet, respectively. Sex-biased hepatic gene signatures were identified for these different dietary challenges. The peroxisome proliferator-activated receptor α (PPARα) co-expression network was identified as sexually dimorphic, and in vivo experiments in mice demonstrated that hepatocyte PPARα determines a sex-specific response to fasting and treatment with pemafibrate, a selective PPARα agonist. Liver molecular signatures in humans also provided evidence of sexually dimorphic gene expression profiles and the sex-specific co-expression network for PPARα., Conclusions: These findings underscore the sex specificity of NAFLD pathophysiology in preclinical studies and identify PPARα as a pivotal, sexually dimorphic, pharmacological target., Trial Registration Number: NCT02390232., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

SNORD115 has been proposed to promote the activity of serotonin (HTR2C) receptor via its ability to base pair with its pre-mRNA and regulate alternative RNA splicing and/or A-to-I RNA editing. Because SNORD115 genes are deleted in most patients with the Prader-Willi syndrome (PWS), diminished HTR2C receptor activity could contribute to the impaired emotional response and/or compulsive overeating characteristic of this disease. In order to test this appealing but never demonstrated hypothesis in vivo, we created a CRISPR/Cas9-mediated Snord115 knockout mouse. Surprisingly, we uncovered only modest region-specific alterations in Htr2c RNA editing profiles, while Htr2c alternative RNA splicing was unchanged. These subtle changes, whose functional relevance remains uncertain, were not accompanied by any discernible defects in anxio-depressive-like phenotypes. Energy balance and eating behavior were also normal, even after exposure to high-fat diet. Our study raises questions concerning the physiological role of SNORD115 , notably its involvement in behavioural disturbance associated with PWS., Competing Interests: JH, VM, JP, PM, NK, HN, MA, HS, JP, BG, JC No competing interests declared, (© 2020, Hebras et al.)

Cell death is a key driver of disease progression and carcinogenesis in chronic liver disease (CLD), highlighted by the well-established clinical correlation between hepatocellular death and risk for the development of cirrhosis and hepatocellular carcinoma (HCC). Moreover, hepatocellular death is sufficient to trigger fibrosis and HCC in mice. However, the pathways through which cell death drives CLD progression remain elusive. Here, we tested the hypothesis that high-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) with key roles in acute liver injury, may link cell death to injury responses and hepatocarcinogenesis in CLD. While liver-specific HMGB1 deficiency did not significantly affect chronic injury responses such as fibrosis, regeneration, and inflammation, it inhibited ductular/progenitor cell expansion and hepatocyte metaplasia. HMGB1 promoted ductular expansion independently of active secretion in a nonautonomous fashion, consistent with its role as a DAMP. Liver-specific HMGB1 deficiency reduced HCC development in 3 mouse models of chronic injury but not in a model lacking chronic liver injury. As with CLD, HMGB1 ablation reduced the expression of progenitor and oncofetal markers, a key determinant of HCC aggressiveness, in tumors. In summary, HMGB1 links hepatocyte death to ductular reaction, progenitor signature, and hepatocarcinogenesis in CLD.

Based on data from publications and field observations, this study analyses the interactions between animal health, rural poverty and the performance and environmental impact of livestock farming in low-income countries and middle-income countries. There are strong statistical correlations between the quality of Veterinary Services, livestock productivity and poverty rates. In countries with effective Veterinary Services, livestock growth stems mainly from productivity gains and poverty rates are the lowest. Conversely, these analyses identify no statistical link between the quality of Veterinary Services and increased livestock production volumes. However, where animal diseases are poorly controlled, productivity is low and livestock growth is extensive, based mainly on a steady increase in animal numbers. Extensive growth is less effective than intensive growth in reducing poverty and aggravates the pressure of livestock production on natural resources and the climate.

Background & Aims: Transforming growth factor-β (TGFβ) exerts key functions in fibrogenic cells, promoting fibrosis development in the liver and other organs. In contrast, the functions of TGFβ in liver epithelial cells are not well understood, despite their high level of responsiveness to TGFβ. We sought to determine the contribution of epithelial TGFβ signaling to hepatic fibrogenesis and carcinogenesis., Methods: TGFβ signaling in liver epithelial cells was inhibited by albumin-Cre-, K19-CreERT-, Prom1-CreERT2-, or AAV8-TBG-Cre-mediated deletion of the floxed TGFβ 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 TGFβ 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., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

In contrast to microbially triggered inflammation, mechanisms promoting sterile inflammation remain poorly understood. Damage-associated molecular patterns (DAMPs) are considered key inducers of sterile inflammation following cell death, but the relative contribution of specific DAMPs, including high-mobility group box 1 (HMGB1), is ill defined. Due to the postnatal lethality of Hmgb1-knockout mice, the role of HMGB1 in sterile inflammation and disease processes in vivo remains controversial. Here, using conditional ablation strategies, we have demonstrated that epithelial, but not bone marrow-derived, HMGB1 is required for sterile inflammation following injury. Epithelial HMGB1, through its receptor RAGE, triggered recruitment of neutrophils, but not macrophages, toward necrosis. In clinically relevant models of necrosis, HMGB1/RAGE-induced neutrophil recruitment mediated subsequent amplification of injury, depending on the presence of neutrophil elastase. Notably, hepatocyte-specific HMGB1 ablation resulted in 100% survival following lethal acetaminophen intoxication. In contrast to necrosis, HMGB1 ablation did not alter inflammation or mortality in response to TNF- or FAS-mediated apoptosis. In LPS-induced shock, in which HMGB1 was considered a key mediator, HMGB1 ablation did not ameliorate inflammation or lethality, despite efficient reduction of HMGB1 serum levels. Our study establishes HMGB1 as a bona fide and targetable DAMP that selectively triggers a neutrophil-mediated injury amplification loop in the setting of necrosis.

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs)., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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 antitumor 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 toward antitumor 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 antitumor pathways, and discuss clinical applications of TLR-targeted therapies for tumor prevention and treatment.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 by the American Association for the Study of Liver Diseases.)

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.

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., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: Hepatic stellate cells (HSCs) contain a number of bioactive metabolites or their precursors including retinoids in their characteristic lipid droplets. The loss of lipid droplets and retinoids is a hallmark of HSC activation, but it remains unclear whether this loss promotes HSC activation, liver fibrogenesis or carcinogenesis., Design: Spontaneous and experimental fibrogenesis as well as a diethylnitrosamine-induced hepatocarcinogenesis were investigated in lecithin-retinol acyltransferase (LRAT)-deficient mice which lack retinoid-containing lipids droplets in their HSCs., Results: Following HSC activation, LRAT expression was rapidly lost, emphasising its importance in lipid droplet biology in HSCs. Surprisingly, there was no difference in fibrosis induced by bile duct ligation (BDL) or by eight injections of carbon tetrachloride (CCl4) between wild-type and LRAT-deficient mice. To exclude the possibility that the effects on fibrogenesis were missed due to the rapid downregulation of LRAT following HSC activation, acute as well as spontaneous liver fibrosis was investigated. However, there was no increased fibrosis in 3-, 8- and 12-month-old LRAT-deficient mice and in LRAT-deficient mice after a single injection of CCl4 compared with wild-type mice. To determine whether the absence of retinoids in HSCs affects hepatocarcinogenesis, wild-type and LRAT-deficient mice were injected with diethylnitrosamine. LRAT deficiency decreased diethylnitrosamine-induced injury and tumour load and increased the expression of the retinoic acid responsive genes Cyp26a1, RARb and p21, suggesting that the lower tumour load of LRAT-deficient mice was a result of increased retinoid signalling and subsequent p21-mediated inhibition of proliferation., Conclusions: The absence of retinoid-containing HSC lipid droplets does not promote HSC activation but reduces hepatocarcinogenesis.

Inflammation is strongly associated with chronic hepatic injury and the ensuing wound-healing process. Recent evidence from mouse models and human studies implicates Toll-like receptors (TLRs) as important regulators of the inflammatory response and a functional link between inflammation and fibrosis in the chronically injured liver. Here, we review mechanisms by which TLR4 and TLR4 ligands from the intestinal microbiota contribute to hepatic injury, inflammation, hepatic stellate cell activation, and fibrosis., (Copyright Thieme Medical Publishers.)

Background & Aims: c-Jun N-terminal kinase (JNK) is activated by multiple profibrogenic mediators; JNK activation occurs during toxic, metabolic, and autoimmune liver injury. However, its role in hepatic fibrogenesis is unknown., Methods: JNK phosphorylation was detected by immunoblot analysis and confocal immunofluorescent microscopy in fibrotic livers from mice after bile duct ligation (BDL) or CCl(4) administration and in liver samples from patients with chronic hepatitis C and non-alcoholic steatohepatitis. Fibrogenesis was investigated in mice given the JNK inhibitor SP600125 and in JNK1- and JNK2-deficient mice following BDL or CCl(4) administration. Hepatic stellate cell (HSC) activation was determined in primary mouse HSCs incubated with pan-JNK inhibitors SP600125 and VIII., Results: JNK phosphorylation was strongly increased in livers of mice following BDL or CCl(4) administration as well as in human fibrotic livers, occurring predominantly in myofibroblasts. In vitro, pan-JNK inhibitors prevented transforming growth factor (TGF) beta-, platelet-derived growth factor-, and angiotensin II-induced murine HSC activation and decreased platelet-derived growth factor and TGF-beta signaling in human HSCs. In vivo, pan-JNK inhibition did not affect liver injury but significantly reduced fibrosis after BDL or CCl(4). JNK1-deficient mice had decreased fibrosis after BDL or CCl(4), whereas JNK2-deficient mice displayed increased fibrosis after BDL but fibrosis was not changed after CCl(4). Moreover, patients with chronic hepatitis C who displayed decreased fibrosis in response to the angiotensin receptor type 1 blocker losartan showed decreased JNK phosphorylation., Conclusions: JNK is involved in HSC activation and fibrogenesis and represents a potential target for antifibrotic treatment approaches., (Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)