Intestinal B cells license metabolic T-cell activation in NASH microbiota/antigen-independently and contribute to fibrosis by IgA-FcR signalling

BACKGROUND & AIMS: The progression of non-alcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC. METHODS: C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH-inducing diets or standard chow for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and μMT mice (containing B cells only in the gastrointestinal tract) were fed a choline-deficient high-fat diet, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with simple steatosis, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing analysis were performed in liver and gastrointestinal tissue to characterise immune cells in mice and humans. RESULTS: Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B cells; additionally, we observed a positive correlation between IgA levels and activated FcRg+ hepatic myeloid cells, as well the extent of liver fibrosis. CONCLUSIONS: Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for the treatment of NASH. IMPACT AND IMPLICATIONS: There is currently no effective treatment for non-alcoholic steatohepatitis (NASH), which is associated with a substantial healthcare burden and is a growing risk factor for hepatocellular carcinoma (HCC). We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we show that the absence of B cells prevented HCC development. B cell-intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets for combinatorial NASH therapies against inflammation and fibrosis

Intestinal B-cells license metabolic T-cell activation in NASH microbiota/antigen-independently and contribute to fibrosis by IgA-FcR signalling

BACKGROUND & AIMS The progression of nonalcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC. METHODS C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH diets (for example, choline-deficient high-fat diet, CD-HFD) or chow diet for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and μMT mice (containing B cells only in the gastrointestinal tract) were fed a CD-HFD, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with NAFL, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and scRNA-Seq analysis were performed in liver and gastrointestinal tissue for immune cells in mice and humans. RESULTS Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen-specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B-cells and showed a positive correlation between IgA levels and activated FcRγ+ hepatic myeloid cells as well extent of liver fibrosis. CONCLUSIONS Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for treating NASH. IMPACT AND IMPLICATIONS Nonalcoholic steatohepatitis (NASH) is a chronic inflammatory condition on the rise and can lead to hepatocellular carcinoma (HCC), the 3rd most common cause of cancer-related death worldwide. Currently, there is no effective treatment for this progressive disease that correlates with a marked risk of HCC mortality and carries a substantial healthcare burden. To date, among all the solid tumours, especially in HCC, the incidence and mortality rates are almost the same, making it crucial to find curative treatments for chronic diseases, such as NASH, which highly predispose to tumorigenesis. We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we could show that the absence of B cells prevented HCC development. B-cell intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets in combinatorial NASH therapies against inflammation and fibrosis

Platelet GPIbα is a mediator and potential interventional target for NASH and subsequent liver cancer

Non-alcoholic fatty liver disease ranges from steatosis to non-alcoholic steatohepatitis (NASH), potentially progressing to cirrhosis and hepatocellular carcinoma (HCC). Here, we show that platelet number, platelet activation and platelet aggregation are increased in NASH but not in steatosis or insulin resistance. Antiplatelet therapy (APT; aspirin/clopidogrel, ticagrelor) but not nonsteroidal anti-inflammatory drug (NSAID) treatment with sulindac prevented NASH and subsequent HCC development. Intravital microscopy showed that liver colonization by platelets depended primarily on Kupffer cells at early and late stages of NASH, involving hyaluronan-CD44 binding. APT reduced intrahepatic platelet accumulation and the frequency of platelet-immune cell interaction, thereby limiting hepatic immune cell trafficking. Consequently, intrahepatic cytokine and chemokine release, macrovesicular steatosis and liver damage were attenuated. Platelet cargo, platelet adhesion and platelet activation but not platelet aggregation were identified as pivotal for NASH and subsequent hepatocarcinogenesis. In particular, platelet-derived GPIbα proved critical for development of NASH and subsequent HCC, independent of its reported cognate ligands vWF, P-selectin or Mac-1, offering a potential target against NASH

Platelet GPIbα is a mediator and potential interventional target for NASH and subsequent liver cancer

Non-alcoholic fatty liver disease ranges from steatosis to non-alcoholic steatohepatitis (NASH), potentially progressing to cirrhosis and hepatocellular carcinoma (HCC). Here, we show that platelet number, platelet activation and platelet aggregation are increased in NASH but not in steatosis or insulin resistance. Antiplatelet therapy (APT; aspirin/clopidogrel, ticagrelor) but not nonsteroidal anti-inflammatory drug (NSAID) treatment with sulindac prevented NASH and subsequent HCC development. Intravital microscopy showed that liver colonization by platelets depended primarily on Kupffer cells at early and late stages of NASH, involving hyaluronan-CD44 binding. APT reduced intrahepatic platelet accumulation and the frequency of platelet-immune cell interaction, thereby limiting hepatic immune cell trafficking. Consequently, intrahepatic cytokine and chemokine release, macrovesicular steatosis and liver damage were attenuated. Platelet cargo, platelet adhesion and platelet activation but not platelet aggregation were identified as pivotal for NASH and subsequent hepatocarcinogenesis. In particular, platelet-derived GPIbα proved critical for development of NASH and subsequent HCC, independent of its reported cognate ligands vWF, P-selectin or Mac-1, offering a potential target against NASH.We thank D. Heide, J. Hetzer, R. Hillermann, C. Gropp, F. Muller, S. Prokosch, D. Kull, R. Dunkl, O. Seelbach, M. Bawohl, R. Maire, M. Bieri, C. Mittmann, H. HoncharovaBiletska, A. Fitsche, A. Adili, P. Munzer, T. Nussbaumer, F. Prutek, G. Dharmalingam and I. Singh for excellent technical assistance. We thank K. Nikolaou for the help with the human cohort recruitment and analysis. M. Malehmir was partially supported by grants from the University Zurich (Zurich Integrative Human Physiology (ZHIP) Sprint Fellowship) and from the Hartmann Muller Stiftung, Zurich. A.W. was supported by a grant from the Swiss National Science Foundation (320030_182764/1). M. Heikenwaelder was supported by an ERC Consolidator grant (HepatoMetaboPath), an EOS grant, SFBTR 209, SFBTR179, Research Foundation Flanders (FWO) under grant 30826052 (EOS Convention MODEL-IDI), Deutsche Krebshilfe projects 70113166 and 70113167, and the Helmholtz-Gemeinschaft, Zukunftsthema 'Immunology and Inflammation' (ZT-0027). This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement 667273 and the DFG (SFB/TR 240 (project 374031971) to B.N. and D. S.), ERC Consolidator grant 'CholangioConcept' (to L.Z.), and the German Research Foundation (DFG): grants FOR2314, SFB685 and the Gottfried Wilhelm Leibniz Program (to L.Z.). Further funding was provided by the German Ministry for Education and Research (BMBF) (eMed/Multiscale HCC), the German Universities Excellence Initiative (third funding line: 'future concept'), the German Center for Translational Cancer Research (DKTK) and the German-Israeli Cooperation in Cancer Research (DKFZ-MOST) (to L.Z. and M. Heikenwaelder). D. I. was supported by an EMBO Long-term Fellowship. J.M.L. is supported by Asociacion Espanola Contra el Cancer (Accelerator award: HUNTER), Spanish National Health Institute (SAF2013-41027), Generalitat de Catalunya (SGR 1162 and AGAUR, SGR-1358), the Samuel Waxman Cancer Research Foundation, the US Department of Defense (CA150272P3), the European Commission Horizon 2020 Program (HEPCAR, proposal number 667273-2), and the National Cancer Institute (P30 CA196521). D. A. M. is supported by CRUK grant C18342/A23390 and MRC grant MR/K001949/1. M. P. is supported by the German Research Foundation (DFG). M. G., T. G. and D. R. was supported by grants from the German Research Foundation (KFO274 and SFB/TR240 (project 374031971)). D. J. W. received a Wellcome Trust Strategic Award (098565/Z/12/Z) and funding from the Medical Research Council (MC-A654-5QB40). C.L.W. was funded by CRUK project Cancer Research UK Programme Grant C18342/A23390. H. G. A. has been supported by the Deutsche Forschungsgemeinschaft (SFB-TR209 'Liver Cancer').S