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

NKG2D and DNAM-1 immunosurveillance mechanisms in hepatocellular carcinoma

Le carcinome hépatocellulaire (CHC) est la 4ème cause de mortalité par cancer dans le monde sans traitement efficace. Deux principales tumeurs se distinguent : 1) agressives de mauvais pronostic, proliférantes, inflammatoires fréquemment mutées dans le gène TP53, 2) de meilleur pronostic bien différenciées, stable dans leur génome, peu inflammatoires et associées à des mutations activatrices dans le gène CTNNB1 codant la beta-caténine. De nombreux travaux ayant montré le rôle essentiel du microenvironnement immunitaire dans le cancer, l'immunothérapie représente une perspective très intéressante pour le traitement du CHC. Dans ce contexte, mes travaux de thèse visent à étudier les mécanismes d'immunosurveillance appelés NKG2D et DNAM-1 dont le rôle n'a pas encore été clairement établi dans le CHC. Ces systèmes sont basés sur la reconnaissance de ligands (NKG2DL et DNAM-1L) exprimés lors d'un stress oncogénique à la surface des cellules tumorales et qui sont reconnus par le récepteur spécifique (NKG2D et DNAM-1) sur les cellules immunitaires permettant leur activation et la lyse des cellules tumorales. Tout d'abord, nous avons analysé l'expression des ligands de NKG2D (MICA/B et ULBP1/2) dans une cohorte de patients atteints de CHC et dans nos modèles murins récapitulant les deux types de tumorigenèse humaine. De façon intéressante, nous montrons que l'expression des ligands de NKG2D est associée aux CHC de mauvais pronostic. Plusieurs études ont d'ailleurs montré que ce mécanisme était associé à une inflammation du tissu hépatique (hépatite) et à une tumorigenèse aggravée chez la souris. De façon intéressante, nous avons observé que l'expression des ligands ULBP1/2 est drastiquement diminuée dans les CHC mutés pour la beta-caténine (non inflammatoire) et corrèle négativement avec les cibles de la signalisation beta-caténine. Nous avons analysé plus précisément ces résultats dans nos modèles murins et démontrons que l'expression des ligands de NKG2D, Rae-1, orthologues des ULBP1/2 chez l'Homme, sont directement inhibés par la signalisation beta-caténine. Ainsi, nous démontrons que les ligands de NKG2D sont associés à une tumorigenèse aggravée et inflammatoire et que l'inhibition d'une partie de ces ligands par la signalisation beta-caténine expliquerait le phénotype non inflammatoire des tumeurs CTNNB1 mutées. Pour le système DNAM-1, nous montrons que l'expression des ligands CD155 et CD112 est augmentée dans le CHC, sans distinction entre les différents types de CHC. L'analyse de l'expression du récepteur DNAM-1 à la surface des cellules immunitaires à fort potentiel anti-tumoral (NK, NKT et lymphocytes T CD8+) a permis de montrer que ces populations expriment différemment le récepteur DNAM-1 en terme de proportion et de quantité relative (MFI) et que seules les cellules NKT exprimaient toutes le récepteur DNAM-1 (100%) dans un foie normal. De façon intéressante, cette proportion est diminuée spécifiquement dans les CHC mutés pour la beta-caténine laissant alors apparaitre une population de NKT DNAM-1 négatives. L'analyse fonctionnelle par cytométrie en flux et RNAseq de des populations de NKT DNAM-1 positives et négatives extraites des tumeurs démontre que les NKT DNAM-1 négatives arborent un phénotype de cellules épuisées (diminution de leur production d'IFNg et de l'expression des molécules cytotoxiques granzyme B et perforine). Ces résultats montrent que la perte du récepteur DNAM-1 à la surface des cellules NKT signe la perte de leur capacité anti-tumoral. Mes travaux de thèse ont permis de démontrer le rôle délétère du mécanisme NKG2D dans la tumorigenèse hépatique et dévoilent la présence d'une population épuisée de NKT DNAM-1 négatives dans les CHC mutés pour la beta-caténine. L'ensemble de ces résultats montre que les mécanismes NKG2D et DNAM-1 sont moins sollicités dans les CHC mutés CTNNB1 ce qui expliquerait possiblement leur statut peu inflammatoire.Hepatocellular carcinoma (HCC) is the 4th leading cause of cancer-related worldwide without effective treatment. Two main types of tumors can be distinguished: 1) aggressive with poor prognosis, proliferative, inflammatory, harbouring chromosomal instability and frequently mutated in the TP53 gene 2) better prognosis, well differentiated, stable in their genome, low inflammatory status and associated with activating mutations in the CTNNB1 gene encoding for beta-catenin. As many studies have shown the essential role of the immune microenvironment in cancer, immunotherapy represents a very interesting perspective for the treatment of HCC. Interestingly, my thesis work aims to study immunosurveillance mechanisms called NKG2D and DNAM-1 whose role has not yet been clearly established in HCC. These systems are based on the recognition of ligands (NKG2DL and DNAM-1L) expressed after oncogenic stress at the surface of tumor cells and which are recognized by the specific receptor (NKG2D and DNAM-1) on immune cells allowing their activation. We have studied these mechanisms in hepatic tumorigenesis in both humans and mice. First, we analysed the expression of NKG2D ligands (MICA/B and ULBP1/2) in human and in our mouse models recapitulating the two types of human tumorigenesis. Several studies have shown that NKG2D ligands expression is associated with inflammation of liver tissue (hepatitis) and aggravated tumorigenesis in mice. In this context, we observed that the expression of ULBP1/2 ligands is drastically decreased in beta-catenin-mutated HCC (non-inflammatory) and negatively correlates with the targets of beta-catenin signalling. We analysed these results more precisely in our mouse models and demonstrate that the expression of NKG2D ligands, Rae-1, ULBP1/2 orthologous in human, was directly inhibited by beta-catenin signalling. Thus, we demonstrated that NKG2D ligands are associated with an aggravated and inflammatory tumorigenesis and that the inhibition of part of these ligands by beta-catenin signalling could explain the non-inflammatory phenotype of CTNNB1-mutated HCC. For the DNAM-1 system, we show that the expression of its ligands (CD155 and CD112) is increased in HCC, without distinction between the different type of HCCs. Analysis of DNAM-1 receptor expression on immune cells with high anti-tumor potential (NK, NKT and CD8+ T cells) showed that these populations express DNAM-1 receptor differently in terms of proportion and relative quantity (MFI) and that all NKT cells expressed the DNAM-1 receptor (100%) in a normal liver. Interestingly, this proportion is specifically reduced in beta-catenin mutated HCCs resulting in the presence of a DNAM-1 negative NKT population. Functional analysis by cytometry and RNAseq of DNAM-1 negative versus DNAM-1 positive populations extracted from tumors shows that DNAM-1 negative NKT exhibits an exhausted phenotype (decrease in their IFNg production and granzyme B, perforin cytotoxic molecules expression). These results convincely show that the loss of DNAM-1 receptor on NKT cells signifies the loss of their anti-tumor capacities. My thesis work evidenced the deleterious role of NKG2D mechanism in hepatic tumorigenesis and revealed the presence of an exhausted DNAM-1 negative NKT population in beta-catenin mutated HCC. Collectively, these results show that both NKG2D and DNAM-1 mechanisms are less solicited in beta-catenin-mutated HCC, confering possibly to the tumors their the low inflammatory status

Hepatospecific ablation of p38α MAPK governs liver regeneration through modulation of inflammatory response to CCl4-induced acute injury

International audienceMammalian p38α MAPK (Mitogen-Activated Protein Kinase) transduces a variety of extracellular signals that regulate cellular processes, such as inflammation, differentiation, proliferation or apoptosis. In the liver, depending of the physiopathological context, p38α acts as a negative regulator of hepatocyte proliferation as well as a promotor of inflammatory processes. However, its function during an acute injury, in adult liver, remains uncharacterized. In this study, using mice that are deficient in p38α specifically in mature hepatocytes, we unexpectedly found that lack of p38α protected against acute injury induced by CCl4 compound. We demonstrated that the hepatoprotective effect alleviated ROS accumulation and shaped the inflammatory response to promote efficient tissue repair. Mechanistically, we provided strong evidence that Ccl2/Ccl5 chemokines were crucial for a proper hepatoprotective response observed secondary to p38α ablation. Indeed, antibody blockade of Ccl2/Ccl5 was sufficient to abrogate hepatoprotection through a concomitant decrease of both inflammatory cells recruitment and antioxidative response that result ultimately in higher liver damages. Our findings suggest that targeting p38α expression and consequently orientating immune response may represent an attractive approach to favor tissue recovery after acute liver injury

Polyploidy spectrum: a new marker in HCC classification

International audienceOBJECTIVES: Polyploidy is a fascinating characteristic of liver parenchyma. Hepatocyte polyploidy depends on the DNA content of each nucleus (nuclear ploidy) and the number of nuclei per cell (cellular ploidy). Which role can be assigned to polyploidy during human hepatocellular carcinoma (HCC) development is still an open question. Here, we investigated whether a specific ploidy spectrum is associated with clinical and molecular features of HCC.DESIGN: Ploidy spectra were determined on surgically resected tissues from patients with HCC as well as healthy control tissues. To define ploidy profiles, a quantitative and qualitative in situ imaging approach was used on paraffin tissue liver sections.RESULTS: We first demonstrated that polyploid hepatocytes are the major components of human liver parenchyma, polyploidy being mainly cellular (binuclear hepatocytes). Across liver lobules, polyploid hepatocytes do not exhibit a specific zonation pattern. During liver tumorigenesis, cellular ploidy is drastically reduced; binuclear polyploid hepatocytes are barely present in HCC tumours. Remarkably, nuclear ploidy is specifically amplified in HCC tumours. In fact, nuclear ploidy is amplified in HCCs harbouring a low degree of differentiation and TP53 mutations. Finally, our results demonstrated that highly polyploid tumours are associated with a poor prognosis.CONCLUSIONS: Our results underline the importance of quantification of cellular and nuclear ploidy spectra during HCC tumorigenesis

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

Lomustine is beneficial to older AML with ELN2017 adverse risk profile and intermediate karyotype: a FILO study

International audienc