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Insulin resistance is a key event in type 2 diabetes onset and a major comorbidity of obesity. It results from a combination of fat excess-triggered defects, including lipotoxicity and metaflammation, but the causal mechanisms remain difficult to identify. Here, we report that hyperactivation of the tyrosine phosphatase SHP2 found in Noonan syndrome (NS) led to an unsuspected insulin resistance profile uncoupled from altered lipid management (for example, obesity or ectopic lipid deposits) in both patients and mice. Functional exploration of an NS mouse model revealed this insulin resistance phenotype correlated with constitutive inflammation of tissues involved in the regulation of glucose metabolism. Bone marrow transplantation and macrophage depletion improved glucose homeostasis and decreased metaflammation in the mice, highlighting a key role of macrophages. In-depth analysis of bone marrow-derived macrophages in vitro and liver macrophages showed that hyperactive SHP2 promoted a proinflammatory phenotype, modified resident macrophage homeostasis, and triggered monocyte infiltration. Consistent with a role of SHP2 in promoting inflammation-driven insulin resistance, pharmaceutical SHP2 inhibition in obese diabetic mice improved insulin sensitivity even better than conventional antidiabetic molecules by specifically reducing metaflammation and alleviating macrophage activation. Together, these results reveal that SHP2 hyperactivation leads to inflammation-triggered metabolic impairments and highlight the therapeutical potential of SHP2 inhibition to ameliorate insulin resistance.

Dysregulations of lipid metabolism in the liver may trigger steatosis progression leading to potentially severe clinical consequences such as non-alcoholic fatty liver diseases (NAFLD). Molecular mechanisms underlying liver lipogenesis are very complex and fine-tuned by chromatin dynamics and the activity of multiple key transcription factors. Here, we demonstrate that the nuclear factor HMGB1 acts as a strong repressor of liver lipogenesis during metabolic stress in NAFLD. Mice with liver-specific Hmgb1-deficiency display exacerbated liver steatosis and hepatic insulin resistance when subjected to a high-fat diet or after fasting/refeeding. Global transcriptome and functional analysis revealed that the deletion of Hmgb1 gene enhances LXRα activity resulting in increased lipogenesis. HMGB1 repression is not mediated through nucleosome landscape re-organization but rather via a preferential DNA occupation in region carrying genes regulated by LXRα. Together these findings suggest that hepatocellular HMGB1 protects from liver steatosis development. HMGB1 may constitute a new attractive option to therapeutically target LXRα axis during NAFLD.

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.

Apelin treatment has been shown to improve insulin sensitivity in insulin resistant mice by acting in skeletal muscles. However, the effects of systemic apelin on the hepatic energy metabolism have not been addressed. We thus aimed to determine the effect of chronic apelin treatment on the hepatic lipid metabolism in insulin resistant mice. The apelin receptor (APJ) expression was also studied in this context since its regulation has only been reported in severe liver pathologies. Mice were fed a high-fat diet (HFD) in order to become obese and insulin resistant compared to chow fed mice (CD). HFD mice then received a daily intraperitoneal injection of apelin (0.1 µmol/kg) or PBS during 28 days. Triglycerides content and the expression of different lipogenesis-related genes were significantly decreased in the liver of HFD apelin-treated compared to PBS-treated mice. Moreover, at this stage of insulin resistance, the beta-oxidation was increased in liver homogenates of HFD PBS-treated mice compared to CD mice and reduced in HFD apelin-treated mice. Finally, APJ expression was not up-regulated in the liver of insulin resistant mice. In isolated hepatocytes from chow and HFD fed mice, apelin did not induce significant effect. Altogether, these results suggest that systemic apelin treatment decreases steatosis in insulin resistant mice without directly targeting hepatocytes.

Sarcopenia, the degenerative loss of skeletal muscle mass, quality and strength, lacks early diagnostic tools and new therapeutic strategies to prevent the frailty-to-disability transition often responsible for the medical institutionalization of elderly individuals. Herein we report that production of the endogenous peptide apelin, induced by muscle contraction, is reduced in an age-dependent manner in humans and rodents and is positively associated with the beneficial effects of exercise in older persons. Mice deficient in either apelin or its receptor (APLNR) presented dramatic alterations in muscle function with increasing age. Various strategies that restored apelin signaling during aging further demonstrated that this peptide considerably enhanced muscle function by triggering mitochondriogenesis, autophagy and anti-inflammatory pathways in myofibers as well as enhancing the regenerative capacity by targeting muscle stem cells. Taken together, these findings revealed positive regulatory feedback between physical activity, apelin and muscle function and identified apelin both as a tool for diagnosis of early sarcopenia and as the target of an innovative pharmacological strategy to prevent age-associated muscle weakness and restore physical autonomy.

Summary 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.

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.

The development of liver fibrosis constitutes one of the major complications of chronic liver disease with many clinical consequences such as the development of esophageal varices and ascites being directly related to the presence of liver fibrosis (1). The hepatic wound healing response is a concerted action of multiple resident and non-resident cell types that not only provides a scaffold for structural stability but also involves the removal of cellular debris by infiltrating hepatic macrophages (HM) and the regeneration of functional parenchyma (2,3). Hepatic stellate cells (HSCs) are considered the main fibrogenic cell type in the liver, and are responsible for the production of various types of extracellular matrix (ECM) (2,3). HSCs undergo a well-characterized activation process during which they lose their characteristic vitamin A and lipid stores, and obtain a myofibroblastic phenotype (2,3). The activation of HSCs is controlled by multiple soluble mediators including TGFβ and PDGF, and is part of a complex cellular network that controls the hepatic wound healing response. Previous studies have demonstrated that multiple cell populations including HM, myeloid-derived suppressor cell, B cells, T cells and natural killer cells influence the development of liver fibrosis (4-12). Among those, HM exert a profound effect on HSCs and hepatic fibrosis as shown by genetic or pharmacologic models of macrophage depletion (6,7,13). At the same time, HM also contribute to fibrosis resolution through MMP13 and matrix remodeling (6,14,15). However, the mechanisms by which HM promote liver fibrosis remain largely elusive. Dendritic cells (DC) are developmentally closely related to macrophages, and exert a profound effect on liver fibrosis regression (16) and the cytokine microenvironment during fibrogenesis (12), but their contribution to liver fibrosis development remains unknown. In the present study, we uncovered the promotion of HSC/myofibroblast survival as novel and to-date unknown mechanism through which macrophages promote fibrosis. Moreover, we demonstrate for the first time that neither classical DC (cDC) and plasmacytoid DC (pDC) contribute to fibrogenesis.

Nuclear factor κB (NF-κB) is a master regulator of inflammation and cell death. Whereas most of the activity of NF-κB is regulated through the inhibitor of κB (IκB) kinase (IKK)-dependent degradation of IκB, IKK also phosphorylates subunits of NF-κB. We investigated the contribution of the phosphorylation of the NF-κB subunit p65 at the IKK phosphorylation site serine 536 (Ser(536)) in humans, which is thought to be required for the activation and nuclear translocation of NF-κB. Through experiments with knock-in mice (S534A mice) expressing a mutant p65 with an alanine-to-serine substitution at position 534 (the murine homolog of human Ser(536)), we observed increased expression of NF-κB-dependent genes after injection of mice with the inflammatory stimulus lipopolysaccharide (LPS) or exposure to gamma irradiation, and the enhanced gene expression was most pronounced at late time points. Compared to wild-type mice, S534A mice displayed increased mortality after injection with LPS. Increased NF-κB signaling in the S534A mice was at least in part explained by the increased stability of the S534A p65 protein compared to that of the Ser(534)-phosphorylated wild-type protein. Together, our results suggest that Ser(534) phosphorylation of p65 in mice (and, by extension, Ser(536) phosphorylation of human p65) is not required for its nuclear translocation, but instead inhibits NF-κB signaling to prevent deleterious inflammation.

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.

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.

International audience; In mammals, birth entails complex metabolic adjustments essential for neonatal survival. Using a mouse knockout model, we identify crucial biological roles for the miR-379/miR-410 cluster within the imprinted Dlk1-Dio3 region during this metabolic transition. The miR-379/miR-410 locus, also named C14MC in humans, is the largest known placental mammal-specific miRNA cluster, whose 39 miRNA genes are expressed only from the maternal allele. We found that heterozygote pups with a maternal--but not paternal--deletion of the miRNA cluster display partially penetrant neonatal lethality with defects in the maintenance of energy homeostasis. This maladaptive metabolic response is caused, at least in part, by profound changes in the activation of the neonatal hepatic gene expression program, pointing to as yet unidentified regulatory pathways that govern this crucial metabolic transition in the newborn's liver. Not only does our study highlight the physiological importance of miRNA genes that recently evolved in placental mammal lineages but it also unveils additional layers of RNA-mediated gene regulation at the Dlk1-Dio3 domain that impose parent-of-origin effects on metabolic control at birth and have likely contributed to mammal evolution.

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.

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.

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.Promotion of NF-κB-dependent myofibroblast survival by macrophages but not DCs provides a novel link between inflammation and fibrosis.

Introduction L'apeline est une adipokine dont les effets benefiques sur le metabolisme energetique ont ete decrits notamment chez les souris obeses et resistantes a l'insuline. Elle permet, entre autre, d'augmenter l'utilisation des lipides et la biogenese mitochondriale dans le muscle. Par contre, les effets de l'apeline sur le metabolisme hepatique ne sont pas connus. Il est vrai que le foie exprime tres faiblement le recepteur APJ en situation physiologique. Le but de cette etude a donc ete de determiner quels sont les effets d'un traitement a l'apeline chez des souris obeses et insulino-resistantes sur le metabolisme lipidique hepatique. Materiels et Methodes Des souris mâles adultes ont ete nourries soit avec un regime standard soit hyperlipidique (HFD) pendant 10 semaines puis traitees pendant 28 jours avec une injection ip d'apeline (0,1 μmol/kg/j) ou de PBS. L'etat d'insulino-resistance a ete evalue. Au sacrifice, le foie a ete preleve afin de mesurer differentes fonctions metaboliques. En parallele, des hepatocytes isoles de souris standard ont ete traites au palmitate (250 μm) et a l'apeline (10 – 7M) a court (20 min) et moyen terme (7 h). Resultats Le traitement apeline chez les souris HFD reduit le gain de masse grasse et l'hyperinsulinemie comme precedemment montre. Dans le foie, le contenu en TG est diminue ainsi que l'expression de genes impliques dans la lipogenese de novo (SREBP-1c, FAS…). L'augmentation de la β-oxydation observee chez les souris HFD est reduite apres le traitement apeline ainsi que la secretion de VLDL. Sur les hepatocytes isoles traites au palmitate, l'apeline n'a pas d'effet sur l'expression des genes de la lipogenese de novo. En aigu l'apeline n'a pas d'effet sur la phosphorylation d'Akt decrite auparavant dans d'autres tissus. Conclusions La steatose hepatique est attenuee chez les souris insulino-resistantes traitees a l'apeline. Cependant il semblerait que les effets de l'apeline sur le metabolisme hepatique soient indirects et une consequence de l'amelioration globale de la sensibilite a l'insuline.

International audience; INTRODUCTION: The presence of fibrosis is associated with alterations in organ architecture and is responsible for the morbidity of diseases including pneumopathies, systemic sclerosis, liver cirrhosis, chronic cardiovascular diseases, progressive kidney diseases and diabetes. Although a growing number of pro-fibrotic molecules, mediators and other pathways have been reported, there are currently very few antifibrotic molecules being evaluated in clinical trials. AREAS COVERED: Current knowledge about the contribution of lysophosphatidic acid (LPA), a bioactive mediator acting via specific G-protein coupled receptors (LPAR), in the etiology of fibrosis. In a number of organs, fibrosis is associated with increased LPA production as well as with increased expression of some LPAR subtypes (mainly LPA1R). LPAR(-/-) knockout mice and treatment of animal models with specific antagonists clearly demonstrate the contribution of LPA1R subtype to the development of kidney, lung, vascular and dermal fibrosis. The involvement of LPA in liver fibrosis is also strongly suspected but still unproven. EXPERT OPINION: Experiments in animal models clearly demonstrate that LPA1R antagonists have interesting anti-fibrotic potencies. This reveals promising perspectives for the design of new therapeutic approaches to prevent fibrosis-associated diseases. Nevertheless, the number of efficient LPA1R antagonists currently available is still low, and none of them has been used in clinical trials so far.

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 (CCl 4 ) 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 CCl 4 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.

The release of lysophosphatidic acid (LPA) by adipocytes has previously been proposed to play a role in obesity and associated pathologies such as insulin resistance and diabetes. In the present work, the sensitivity to diet-induced obesity was studied in mice lacking one of the LPA receptor subtype (LPA1R). Conversely to what was observed in wild type (WT) mice, LPA1R-KO-mice fed a high fat diet (HFD) showed no significant increase in body weight or fat mass when compared to low fat diet (LFD). In addition, in contrast to what was observed in WT mice, LPA1R-KO mice did not exhibit over-consumption of food associated with HFD. Surprisingly, when fed a LFD, LPA1R-KO mice exhibited significant higher plasma leptin concentration and higher level of adipocyte leptin mRNA than WT mice. In conclusion, LPA1R-KO mice were found to be resistant to diet-induced obesity consecutive to a resistance to fat-induced over-consumption of food that may result at least in part from alterations in leptin expression and production.

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) β-, platelet-derived growth factor-, and angiotensin II-induced murine HSC activation and decreased platelet-derived growth factor and TGF-β 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.

International audience; The development of fibrosis involves a multitude of events and molecules. Until now the majority of these molecules were found to be proteins or peptides. But recent data show significant involvement of the phospholipid lysophosphatidic acid (LPA) in the development of pulmonary, liver and renal fibrosis. The latest data on the role of LPA and the G-protein-coupled LPA1 receptor in the development of renal fibrosis will be discussed. LPA1-receptor activation was found to be associated with increased vascular leakage and increased fibroblast recruitment in pulmonary fibrosis. Furthermore, in renal fibrosis LPA1-receptor activation stimulates macrophage recruitment and connective tissue growth factor expression. The observations make this receptor an interesting alternative and new therapeutic target in fibrotic diseases.

International audience; Tubulointerstitial fibrosis in chronic renal disease is strongly associated with progressive loss of renal function. We studied the potential involvement of lysophosphatidic acid (LPA), a growth factor-like phospholipid, and its receptors LPA(1-4) in the development of tubulointerstitial fibrosis (TIF). Renal fibrosis was induced in mice by unilateral ureteral obstruction (UUO) for up to 8 d, and kidney explants were prepared from the distal poles to measure LPA release into conditioned media. After obstruction, the extracellular release of LPA increased approximately 3-fold. Real-time reverse transcription PCR (RT-PCR) analysis demonstrated significant upregulation in the expression of the LPA(1) receptor subtype, downregulation of LPA3, and no change of LPA2 or LPA4. TIF was significantly attenuated in LPA1 (-/-) mice compared to wild-type littermates, as measured by expression of collagen III, alpha-smooth muscle actin (alpha-SMA), and F4/80. Furthermore, treatment of wild-type mice with the LPA1 antagonist Ki16425 similarly reduced fibrosis and significantly attenuated renal expression of the profibrotic cytokines connective tissue growth factor (CTGF) and transforming growth factor beta (TGFbeta). In vitro, LPA induced a rapid, dose-dependent increase in CTGF expression that was inhibited by Ki16425. In conclusion, LPA, likely acting through LPA1, is involved in obstruction-induced TIF. Therefore, the LPA1 receptor might be a pharmaceutical target to treat renal fibrosis.

Introduction: The cellular origin and contribution of mesenchymal stem cells (MSCs) in the gastrointestinal tract, bone marrow and tumor microenvironment are unknown. More specific markers of MSCs are needed. We hypothesized that Grem1 expression would be a specific marker of MSCs. Methods: We developed Grem1-BAC-CreERT, αSMA-BAC-CreERT, Grem1-BAC-EGFP-peptide 2A-DTR-peptide 2A-CreERT and Vimentin-BAC-CreERT transgenic lines to characterize the origin, function and kinetics of gastrointestinal, bone marrow and tumor-associated mesenchyme. These mice were crossed to reporters, Grem1fl/fl and diphtheria toxin associated lines. The cellular fate and function of the Grem1, αSMA and Vimentin expressing cells, was analyzed by direct fluorescence microscopy and immunostaining at several time points (up to 12 months) and in several cancer models. Results: In the bone marrow, Grem1 recombination was identified in a very rare population of CD45/Ter119/CD31 triple negative cells (0.004% of all cells from compact bone extraction). In the bone marrow, Grem1-EGFP identified a discrete population of perisinusoidal cells. Grem1-recombined cells dramatically expand within MSC cultures forming large colony forming units, with a CFU-F efficiency of 3%. Grem1-recombined cells could be differentiated in vitro into adipocytes, osteoblasts, chondrocytes and αSMA(+) myofibroblasts. Furthermore, these cells are long-lived, recoverable from the bone at least 9 months after tamoxifen induction and can be transplanted. In the small intestine, 24 hours after tamoxifen induction, there were single Grem1 recombined cells found immediately subjacent to the basement membrane. These initially rare αSMA(-) recombined cells expanded over the next 12 months to completely trace the periepithelial fibroblast sheath. In both the gut and bone marrow, perinatal tamoxifen induction led to dramatically accelerated lineage tracing. Syngeneic tumorigenicity and carcinogenesis studies (including hepatic metastatic model (splenic injection of MC38, C57Bl/6 colorectal cancer cell line), MNU/Hfelis and AOM/DSS gastrointestinal cancer models) in our Grem1-BAC-specific lines revealed that Grem1 marked a cellular origin of cancer-associated mesenchyme, although many cancer-associated fibroblasts also arise from a specific αSMA+ stromal population. The exact lineage relationship between these Grem1 and αSMA+ cells is being tested. Conclusions: Grem1-expression labels a specific mesenchymal progenitor in the bone and gut that can give rise to multiple mesenchymal lineages during development and in the peritumoral stroma. We are currently examining the functional relevance of these cells and Grem1 production in supporting the normal and pathological stem cell niche in the bone marrow, gut and tumor microenvironment. Citation Format: Daniel L. Worthley, Yiling Si, Samuel Asfaha, Benedikt Westphalen, Yagnesh Tailor, Michael Churchill, Jean-Philippe Pradere, Robert Schwabe, Siddhartha Mukherjee, Timothy Wang. Gremlin 1 labels a mesenchymal progenitor in the gastrointestinal tract, bone and tumor microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2299. doi:10.1158/1538-7445.AM2013-2299

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.

The gastrointestinal tract is supported by myofibroblasts (MF) that regulate epithelial homeostasis. In cancer the physiological stroma is replaced by activated cells including bone marrow (BM)-derived cancer-associated fibroblasts (CAFs) that drive carcinogenesis. MFs and a significant proportion of CAFs express αSMA. The origin of these cells and their relation to the stem cell niche in cancer, however, is unclear. We examined gene expression to determine markers of the MSC niche, from which MFs and CAFs develop, and then generated three transgenic mouse models to trace and examine the kinetics of the physiological and cancer stroma. MSC cultures from whole BM of αSMA-RFP transgenic reporter mice consist of a heterogeneous population of αSMA-RFP (+) and (-) cells (FACS). The αSMA-RFP (-) population contained the true MSC (CFU-F and differentiation). The BMP-antagonist, Grem1, was specifically upregulated in the combined RFP+/− population but not RFP+ cells suggesting that Grem1 may mark the MSC within its niche. Additionally, we confirmed the expansion of BM-derived Grem1 cells in the gastric peritumoral stroma supporting our hypothesis that the mesenchymal niche is recruited to the cancer microenvironment. In order to confirm that Grem1 labels MSCs, we developed a Grem1-BAC-CreER transgenic mouse to lineage trace this cell. 24 hours after tamoxifen induction single recombined cells were evident throughout the gastrointestinal stroma adjacent to the epithelial stem cell zone. By 3 months post-induction the recombined cells had expanded giving rise to αSMA+ MFs. Importantly, Grem1(+) cells were identified within BM-derived MSC cultures, where they also gave rise to αSMA(+) MFs and adipocytes, and are currently being differentiated into osteocytes and chondrocytes. Furthermore, in a syngeneic tumor model, subcutaneous injection of MC38 cells into Grem1-BAC-CreER mice, yielded infiltrating CAFs derived from Grem1 cells, suggesting that Grem1 marks the cellular origin of both physiological and pathological mesenchyme. Gastrointestinal carcinogenesis models (AOM/DSS, MNU/H. felis), metastasis models (splenic injection MC38) and longer term lineage tracing are proceeding. We also developed αSMA-BAC-CreER and Vimentin-BAC-CreER transgenic mice to characterize the cellular kinetics of the stroma and further refine markers co-expressed by MSCs. These studies have confirmed the turnover of intestinal MFs. In summary, Grem1 expressing stromal cells co-localize with the epithelial stem cell niche within the gastrointestinal tract and specifically label a mesenchymal cell that gives rise to αSMA+ MFs. Furthermore, syngeneic tumor models suggest that Grem1 cells also give rise to peritumoral mesenchyme. Grem1 expression identifies a likely progenitor to gastrointestinal MFs/CAFs in health and cancer. Grem1 is a promising marker of the MSC in health in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2969. doi:1538-7445.AM2012-2969

SummaryIncreased 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.