Monoacylglycerol lipase reprograms hepatocytes and macrophages to promote liver regeneration

Background & Aims: Liver regeneration is a repair process in which metabolic reprogramming of parenchymal and inflammatory cells plays a major role. Monoacylglycerol lipase (MAGL) is an ubiquitous enzyme at the crossroad between lipid metabolism and inflammation. It converts monoacylglycerols into free fatty acids and metabolises 2-arachidonoylglycerol into arachidonic acid, being thus the major source of pro-inflammatory prostaglandins in the liver. In this study, we investigated the role of MAGL in liver regeneration. Methods: Hepatocyte proliferation was studied in vitro in hepatoma cell lines and ex vivo in precision-cut human liver slices. Liver regeneration was investigated in mice treated with a pharmacological MAGL inhibitor, MJN110, as well as in animals globally invalidated for MAGL (MAGL-/-) and specifically invalidated in hepatocytes (MAGLHep-/-) or myeloid cells (MAGLMye-/-). Two models of liver regeneration were used: acute toxic carbon tetrachloride injection and two-thirds partial hepatectomy. MAGLMye-/- liver macrophages profiling was analysed by RNA sequencing. A rescue experiment was performed by in vivo administration of interferon receptor antibody in MAGLMye-/- mice. Results: Precision-cut human liver slices from patients with chronic liver disease and human hepatocyte cell lines exposed to MJN110 showed reduced hepatocyte proliferation. Mice with global invalidation or mice treated with MJN110 showed blunted liver regeneration. Moreover, mice with specific deletion of MAGL in either hepatocytes or myeloid cells displayed delayed liver regeneration. Mechanistically, MAGLHep-/- mice showed reduced liver eicosanoid production, in particular prostaglandin E2 that negatively impacts on hepatocyte proliferation. MAGL inhibition in macrophages resulted in the induction of the type I interferon pathway. Importantly, neutralising the type I interferon pathway restored liver regeneration of MAGLMye-/- mice. Conclusions: Our data demonstrate that MAGL promotes liver regeneration by hepatocyte and macrophage reprogramming. Impact and Implications: By using human liver samples and mouse models of global or specific cell type invalidation, we show that the monoacylglycerol pathway plays an essential role in liver regeneration. We unveil the mechanisms by which MAGL expressed in both hepatocytes and macrophages impacts the liver regeneration process, via eicosanoid production by hepatocytes and the modulation of the macrophage interferon pathway profile that restrains hepatocyte proliferation.The authors thank V. Fauveau, Institut Cochin, for help in surgery experiments; Olivier Thibaudeau of the Plateau de Morphologie Facility (INSERM UMR 1152, France) and Nicolas Sorhaindo of the Plateforme de Biochimie (CRI, INSERM UMR1149) for their help in the histology and liver function tests; and K. Bailly from the cytometry platform of Cochin Institute and H. Fohrer-Ting from the Centre de Recherche des Cordeliers, Paris University, for cell sorting analyses.Scopu

Primary cilia sensitize endothelial cells to BMP and prevent excessive vascular regression

Blood flow shapes vascular networks by orchestrating endothelial cell behavior and function. How endothelial cells read and interpret flow-derived signals is poorly understood. Here, we show that endothelial cells in the developing mouse retina form and use luminal primary cilia to stabilize vessel connections selectively in parts of the remodeling vascular plexus experiencing low and intermediate shear stress. Inducible genetic deletion of the essential cilia component intraflagellar transport protein 88 (IFT88) in endothelial cells caused premature and random vessel regression without affecting proliferation, cell cycle progression, or apoptosis. IFT88 mutant cells lacking primary cilia displayed reduced polarization against blood flow, selectively at low and intermediate flow levels, and have a stronger migratory behavior. Molecularly, we identify that primary cilia endow endothelial cells with strongly enhanced sensitivity to bone morphogenic protein 9 (BMP9), selectively under low flow. We propose that BMP9 signaling cooperates with the primary cilia at low flow to keep immature vessels open before high shear stress-mediated remodeling

Role of autophagy in liver sinusoidal endothelial cells on nonalcoholic steatohepatitis

Contexte et hypothèse : La stéatohépatite non alcoolique (NASH) est définie par une accumulation excessive de lipides dans le foie (stéatose), une atteinte hépatocytaire et une inflammation hépatique avec ou sans fibrose. La NASH peut évoluer vers la cirrhose et le carcinome hépatocellulaire. De récentes études suggèrent que des altérations microvasculaires et une dysfonction endothéliale sinusoïdale précèdent l'atteinte fibreuse et inflammatoire de la NASH. L'autophagie est un processus cellulaire par lequel du matériel cytoplasmique rejoint les lysosomes pour dégradation. L'autophagie a été étudiée dans les différents types cellulaires du foie, mais le rôle de l'autophagie dans les cellules endothéliales sinusoïdales du foie (LSECs) n'a à ce jour jamais été évalué dans la NASH. Le but de mon projet de thèse a été d'évaluer le rôle de l'autophagie dans les LSECs dans la NASH. Matériel et méthodes : (a) échantillons humains : j'ai utilisé des biopsies hépatiques humaines de patients avec un foie histologiquement normal, de patients atteints de stéatose ou de patients atteints de NASH, afin d'évaluer par microscopie électronique l'autophagie dans les LSECs. (b) LSECs en culture : j'ai testé l'effet du TNFa et de l'IL6, aux concentrations rapportées dans le sang portal d'individus atteints de syndrome métabolique, sur l'autophagie dans une lignée de LSECs soumises à des contraintes de cisaillement. Enfin, j'ai caractérisé l'effet du défaut d'autophagie sur le phénotype endothélial en utilisant une lignée de LSECs que j'ai transduites avec un shARN ciblant ATG5 pour les rendre déficientes en autophagie. (c) souris transgéniques : J'ai évalué l'effet du défaut d'autophagie endothéliale sur les stades précoces de la NASH, en utilisant une ligné de souris déficientes en autophagie spécifiquement dans l'endothélium (Atg5lox/lox VE-cadhérine-Cre) soumises à un régime riche en matières grasses pendant 16 semaines, et sur les stades avancés de la fibrose hépatique en traitant les souris Atg5lox/lox VE-cadhérine-Cre au tétrachlorure de carbone (CCl4). Résultats : (a) échantillons humains : Le pourcentage de LSECs contenant des vacuoles d'autophagie était 2 fois moins important dans le foie des patients atteints de NASH comparativement aux témoins et aux patients atteints de stéatose. (b) LSECs en culture : La combinaison de TNFa et d'IL6 réduisait significativement l'autophagie dans les LSECs. Cette réduction passait par l'inhibition de l'AMPKa. Les LSECs déficientes en autophagie surexprimaient les gènes inflammatoires Mcp1 et Rantes et la protéine VCAM1. Le défaut d'autophagie dans les LSECs induisait une surexpression des marqueurs de transition endothélio-mésenchymateuse a-SMA, Collagène1a1, Collagène1a2 et Tgf-b1. (c) souris transgéniques : Comparativement aux souris contrôles, les souris Atg5lox/lox VE-cadhérine-Cre avaient une inflammation hépatique augmentée (surexpression hépatique des gènes inflammatoires Mcp1 et Rantes et de la protéine VCAM1). La déficience en autophagie endothéliale augmentait la fibrose (surexpression hépatique des gènes fibrogéniques Collagène1a2 et Tgf-b1, de la protéine a-SMA et de la surface de collagène). Les souris Atg5lox/lox VE-cadhérine-Cre traitées au CCl4 présentaient plus de fibrose que les souris contrôles soumises au même traitement (surexpression hépatique des gènes fibrogéniques a-SMA, Collagène 1a1, Collagène 1a2 et Tgf-b1, augmentation de la surface de collagène). Conclusion : Un défaut d'autophagie dans les LSECs se produit chez les patients atteints de NASH. Le TNFa et l'IL6 présents dans le sang portal de ces patients pourraient être responsables de ce défaut en altérant l'activité de l'AMPKa. Le défaut d'autophagie dans les LSECs favorise le développement de l'inflammation et de la fibrose hépatique aux stades précoces et avancées de la maladie. Cibler l'autophagie dans les LSECs constitue une stratégie thérapeutique intéressante pour le traitement de la NASH.Background and Aims: Non alcoholic steatohepatitis (NASH) is defined as the excessive lipids accumulation in the liver, hepatocellular injury and inflammation with or without fibrosis. NASH has the potential for cirrhosis and hepatocellular carcinoma. Recent studies suggest that microvascular alterations and sinusoidal endothelial dysfunction precede inflammation and fibrosis in NASH. Autophagy is a cellular process by which the dysfunctional cytoplasmic material joins lysosomes for degradation. The role of autophagy in hepatocytes, in hepatic stellate cells and in liver monocyte-macrophages has been studied but nothing is known about the role of autophagy in liver sinusoidal endothelial cells (LSECs) in NASH. The aim of my thesis work was to investigate the potential implication of autophagy in LSECs in NASH and liver fibrosis. Method: (a) Human samples: I used liver biopsies from patient without liver histological abnormalities, with simple steatosis or with NASH to analyze autophagy in LSECs by electron microscopy. (b) Cultured LSECs: I tested the effect of TNFa and IL6 (at concentrations present in the portal venous blood of patients with metabolic syndrome) on autophagy in LSECs exposed to shear stress. I characterized the effect of autophagy deficiency on the phenotype of LSECs by transducing transformed LSECs with a shRNA targeting ATG5. (c) Transgenic mice: I analyzed the effect of a defect in endothelial autophagy on early stages of NASH, by using mice deficient in autophagy specifically in endothelial cells (Atg5lox/lox-VE-CadherinCre), fed a high fat diet (HFD) for 16 weeks, and on advanced stages of liver fibrosis by treating Atg5lox/lox-VE-CadherinCre mice with carbon tetrachloride (CCl4). Results: (a) Human samples: Patients with NASH had twice less LSECs containing autophagic vacuoles than patients without liver histological abnormalities or patients with simple steatosis. (b) Cultured LSECs: The combination of TNFa and IL6 decreased autophagy level in LSECs. This reduction of autophagy involved the inhibition of AMPKa. LSECs deficient in autophagy overexpressed Mcp1 and Rantes genes and VCAM1 protein expression. Deficiency in autophagy in LSECs induced the expression of the endothelial to mesenchymal transition markers a-SMA, Collagen1a1, Collagen1a2 and Tgf-b1. (c) Transgenic mice: As compared to littermate controls, mice deficient in endothelial ATG5 fed a HFD had a more frequent nodular liver surface, a higher liver inflammation (increased liver gene expression of Mcp-1 and Rantes and VCAM1 protein) and more liver fibrosis (increased liver gene expression of Collagen1a2 and Tgf-b1, higher expression a-SMA protein and more collagen deposition). Mice deficient in ATG5 in endothelial cells treated with CCl4 had more liver fibrosis (increased liver gene expression of a-SMA, Collagen1a1, Collagen 1a2 and Tgf-b1 and more collagen deposition). Conclusion: Autophagy is defective in LSECs of patients with NASH. TNFa and IL6 at concentrations present in the portal blood of patients with NASH could be responsible for this defect through the impairment of AMPKa activity. Autophagy defect in LSECs contributes to the development of liver inflammation and fibrosis at early and advanced stages of the disease. Stimulating endothelial autophagy could be an attractive strategy for NASH treatment

Rôle de l’autophagie des cellules endothéliales sinusoïdales du foie dans la stéatohépatite non alcoolique

International audienc

MAIT cell inhibition promotes liver fibrosis regression via macrophage phenotype reprogramming

Liver cirrhosis is characterised by extensive fibrosis of the liver, and understanding the underpinning immunological processes is important in designing intervention. Here authors show that Mucosal-Associated Invariant T cells are instrumental to controlling the balance between profibrogenic and restorative macrophages and inhibiting their activation might reverse liver fibrosis

Monoacylglycerol lipase reprograms hepatocytes and macrophages to promote liver regeneration

Background & Aims: Liver regeneration is a repair process in which metabolic reprogramming of parenchymal and inflammatory cells plays a major role. Monoacylglycerol lipase (MAGL) is an ubiquitous enzyme at the crossroad between lipid metabolism and inflammation. It converts monoacylglycerols into free fatty acids and metabolises 2-arachidonoylglycerol into arachidonic acid, being thus the major source of pro-inflammatory prostaglandins in the liver. In this study, we investigated the role of MAGL in liver regeneration. Methods: Hepatocyte proliferation was studied in vitro in hepatoma cell lines and ex vivo in precision-cut human liver slices. Liver regeneration was investigated in mice treated with a pharmacological MAGL inhibitor, MJN110, as well as in animals globally invalidated for MAGL (MAGL-/-) and specifically invalidated in hepatocytes (MAGLHep-/-) or myeloid cells (MAGLMye-/-). Two models of liver regeneration were used: acute toxic carbon tetrachloride injection and two-thirds partial hepatectomy. MAGLMye-/- liver macrophages profiling was analysed by RNA sequencing. A rescue experiment was performed by in vivo administration of interferon receptor antibody in MAGLMye-/- mice. Results: Precision-cut human liver slices from patients with chronic liver disease and human hepatocyte cell lines exposed to MJN110 showed reduced hepatocyte proliferation. Mice with global invalidation or mice treated with MJN110 showed blunted liver regeneration. Moreover, mice with specific deletion of MAGL in either hepatocytes or myeloid cells displayed delayed liver regeneration. Mechanistically, MAGLHep-/- mice showed reduced liver eicosanoid production, in particular prostaglandin E2 that negatively impacts on hepatocyte proliferation. MAGL inhibition in macrophages resulted in the induction of the type I interferon pathway. Importantly, neutralising the type I interferon pathway restored liver regeneration of MAGLMye-/- mice. Conclusions: Our data demonstrate that MAGL promotes liver regeneration by hepatocyte and macrophage reprogramming. Impact and Implications: By using human liver samples and mouse models of global or specific cell type invalidation, we show that the monoacylglycerol pathway plays an essential role in liver regeneration. We unveil the mechanisms by which MAGL expressed in both hepatocytes and macrophages impacts the liver regeneration process, via eicosanoid production by hepatocytes and the modulation of the macrophage interferon pathway profile that restrains hepatocyte proliferation

Endothelial autophagic flux hampers atherosclerotic lesion development

International audienceBlood flowing in arteries generates shear forces at the surface of the vascular endothelium that control its anti-atherogenic properties. However, due to the architecture of the vascular tree, these shear forces are heterogeneous and atherosclerotic plaques develop preferentially in areas where shear is low or disturbed. Here we review our recent study showing that elevated shear forces stimulate endothelial autophagic flux and that inactivating the endothelial macroautophagy/autophagy pathway promotes a proinflammatory, prosenescent and proapoptotic cell phenotype despite the presence of atheroprotective shear forces. Specific deficiency in endothelial autophagy in a murine model of atherosclerosis stimulates the development of atherosclerotic lesions exclusively in areas of the vasculature that are normally resistant to atherosclerosis. Our findings demonstrate that adequate endothelial autophagic flux limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence and inflammation

Autophagy is required for endothelial cell alignment and atheroprotection under physiological blood flow

International audienceIt has been known for some time that atherosclerotic lesions preferentially develop in areas exposed to low SS and are characterized by a proinflammatory, apoptotic, and senescent endothelial phenotype. Conversely, areas exposed to high SS are protected from plaque development, but the mechanisms have remained elusive. Autophagy is a protective mechanism that allows recycling of defective organelles and proteins to maintain cellular homeostasis. We aimed to understand the role of endothelial autophagy in the atheroprotective effect of high SS. Atheroprotective high SS stimulated endothelial autophagic flux in human and murine arteries. On the contrary, endothelial cells exposed to atheroprone low SS were characterized by inefficient autophagy as a result of mammalian target of rapamycin (mTOR) activation, AMPKα inhibition, and blockade of the autophagic flux. In hypercholesterolemic mice, deficiency in endothelial autophagy increased plaque burden only in the atheroresistant areas exposed to high SS; plaque size was unchanged in atheroprone areas, in which endothelial autophagy flux is already blocked. In cultured cells and in transgenic mice, deficiency in endothelial autophagy was characterized by defects in endothelial alignment with flow direction, a hallmark of endothelial cell health. This effect was associated with an increase in endothelial apoptosis and senescence in high-SS regions. Deficiency in endothelial autophagy also increased TNF-α-induced inflammation under high-SS conditions and decreased expression of the antiinflammatory factor KLF-2. Altogether, these results show that adequate endothelial autophagic flux under high SS limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence, and inflammation

Characterization and Pharmacological Validation of a Preclinical Model of NASH in Göttingen Minipigs

International audienceBackground: Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease, which is associated with features of metabolic syndrome. NAFLD may progress in a subset of patients into nonalcoholic steatohepatitis (NASH) with liver injury resulting ultimately in cirrhosis and potentially hepatocellular carcinoma. Today, there is no approved treatment for NASH due to, at least in part, the lack of preclinical models recapitulating features of human disease. Here, we report the development of a dietary model of NASH in the Göttingen minipig.Methods: First, we performed a longitudinal characterization of diet-induced NASH and fibrosis using biochemical, histological, and transcriptional analyses. We then evaluated the pharmacological response to Obeticholic acid (OCA) treatment for 8 weeks at 2.5mg/kg/d, a dose matching its active clinical exposure.Results: Serial histological examinations revealed a rapid installation of NASH driven by massive steatosis and inflammation, including evidence of ballooning. Furthermore, we found the progressive development of both perisinusoidal and portal fibrosis reaching fibrotic septa after 6 months of diet. Histological changes were mechanistically supported by well-defined gene signatures identified by RNA Seq analysis. While treatment with OCA was well tolerated throughout the study, it did not improve liver dysfunction nor NASH progression. By contrast, OCA treatment resulted in a significant reduction in diet-induced fibrosis in this model.Conclusions: These results, taken together, indicate that the diet-induced NASH in the Göttingen minipig recapitulates most of the features of human NASH and may be a model with improved translational value to prioritize drug candidates toward clinical development

Paradoxical Suppression of Atherosclerosis in the Absence of microRNA-146a

Rationale: Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identified as a critical brake on proinflammatory nuclear factor kappa light chain enhancer of activated B cells signaling in several cell types, including endothelial cells and bone marrow (BM)-derived cells. Importantly, miR-146a expression is elevated in human atherosclerotic plaques, and polymorphisms in the miR-146a precursor have been associated with risk of coronary artery disease. Objective: To define the role of endogenous miR-146a during atherogenesis. Methods and Results: Paradoxically, Ldlr(-/-) (low-density lipoprotein receptor null) mice deficient in miR-146a develop less atherosclerosis, despite having highly elevated levels of circulating proinflammatory cytokines. In contrast, cytokine levels are normalized in Ldlr(-/-);miR-146a(-/-) mice receiving wild-type BM transplantation, and these mice have enhanced endothelial cell activation and elevated atherosclerotic plaque burden compared with Ldlr(-/-) mice receiving wild-type BM, demonstrating the atheroprotective role of miR-146a in the endothelium. We find that deficiency of miR-146a in BM-derived cells precipitates defects in hematopoietic stem cell function, contributing to extramedullary hematopoiesis, splenomegaly, BM failure, and decreased levels of circulating proatherogenic cells in mice fed an atherogenic diet. These hematopoietic phenotypes seem to be driven by unrestrained inflammatory signaling that leads to the expansion and eventual exhaustion of hematopoietic cells, and this occurs in the face of lower levels of circulating low-density lipoprotein cholesterol in mice lacking miR-146a in BM-derived cells. Furthermore, we identify sortilin-1 (Sort1), a known regulator of circulating low-density lipoprotein levels in humans, as a novel target of miR-146a. Conclusions: Our study reveals that miR-146a regulates cholesterol metabolism and tempers chronic inflammatory responses to atherogenic diet by restraining proinflammatory signaling in endothelial cells and BM-derived cells