Publications by authors named "Mathias Heikenwalder"

259 Publications

Therapeutic Effects of Inhibition of Sphingosine-1-Phosphate Signaling in HIF-2α Inhibitor-Resistant Clear Cell Renal Cell Carcinoma.

Cancers (Basel) 2021 Sep 25;13(19). Epub 2021 Sep 25.

Department of Medicine I, Medical Center, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany.

Specific inhibitors of HIF-2α have recently been approved for the treatment of ccRCC in VHL disease patients and have shown encouraging results in clinical trials for metastatic sporadic ccRCC. However, not all patients respond to therapy and pre-clinical and clinical studies indicate that intrinsic as well as acquired resistance mechanisms to HIF-2α inhibitors are likely to represent upcoming clinical challenges. It would be desirable to have additional therapeutic options for the treatment of HIF-2α inhibitor resistant ccRCCs. Here we investigated the effects on tumor growth and on the tumor microenvironment of three different direct and indirect HIF-α inhibitors, namely the HIF-2α-specific inhibitor PT2399, the dual HIF-1α/HIF-2α inhibitor Acriflavine, and the S1P signaling pathway inhibitor FTY720, in the autochthonous mutant ccRCC mouse model and validated these findings in human ccRCC cell culture models. We show that FTY720 and Acriflavine exhibit therapeutic activity in several different settings of HIF-2α inhibitor resistance. We also identify that HIF-2α inhibition strongly suppresses T cell activation in ccRCC. These findings suggest prioritization of sphingosine pathway inhibitors for clinical testing in ccRCC patients and also suggest that HIF-2α inhibitors may inhibit anti-tumor immunity and might therefore be contraindicated for combination therapies with immune checkpoint inhibitors.
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http://dx.doi.org/10.3390/cancers13194801DOI Listing
September 2021

A human liver cell-based system modeling a clinical prognostic liver signature for therapeutic discovery.

Nat Commun 2021 09 17;12(1):5525. Epub 2021 Sep 17.

Liver Tumor Translational Research Program, Simmons Comprehensive Cancer Center, Division of Digestive and Liver Diseases, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Chronic liver disease and hepatocellular carcinoma (HCC) are life-threatening diseases with limited treatment options. The lack of clinically relevant/tractable experimental models hampers therapeutic discovery. Here, we develop a simple and robust human liver cell-based system modeling a clinical prognostic liver signature (PLS) predicting long-term liver disease progression toward HCC. Using the PLS as a readout, followed by validation in nonalcoholic steatohepatitis/fibrosis/HCC animal models and patient-derived liver spheroids, we identify nizatidine, a histamine receptor H2 (HRH2) blocker, for treatment of advanced liver disease and HCC chemoprevention. Moreover, perturbation studies combined with single cell RNA-Seq analyses of patient liver tissues uncover hepatocytes and HRH2, CLEC5A, MARCO liver macrophages as potential nizatidine targets. The PLS model combined with single cell RNA-Seq of patient tissues enables discovery of urgently needed targets and therapeutics for treatment of advanced liver disease and cancer prevention.
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http://dx.doi.org/10.1038/s41467-021-25468-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8448834PMC
September 2021

Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF.

J Exp Med 2021 Nov 16;218(11). Epub 2021 Sep 16.

Institute of Microbiology, Department of Biology, Eidgenössische Technische Hochschule Zurich, Zurich, Switzerland.

Intestinal epithelial cell (IEC) NF-κB signaling regulates the balance between mucosal homeostasis and inflammation. It is not fully understood which signals tune this balance and how bacterial exposure elicits the process. Pure LPS induces epithelial NF-κB activation in vivo. However, we found that in mice, IECs do not respond directly to LPS. Instead, tissue-resident lamina propria intercrypt macrophages sense LPS via TLR4 and rapidly secrete TNF to elicit epithelial NF-κB signaling in their immediate neighborhood. This response pattern is relevant also during oral enteropathogen infection. The macrophage-TNF-IEC axis avoids responses to luminal microbiota LPS but enables crypt- or tissue-scale epithelial NF-κB responses in proportion to the microbial threat. Thereby, intercrypt macrophages fulfill important sentinel functions as first responders to Gram-negative microbes breaching the epithelial barrier. The tunability of this crypt response allows the induction of defense mechanisms at an appropriate scale according to the localization and intensity of microbial triggers.
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http://dx.doi.org/10.1084/jem.20210862DOI Listing
November 2021

How to get away with liver innate immunity? A viruses' tale.

Liver Int 2021 Sep 14. Epub 2021 Sep 14.

Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany.

In their never-ending quest towards persistence within their host, hepatitis viruses have developed numerous ways to counteract the liver innate immunity. This review highlights the different and common mechanisms employed by these viruses to (i) establish in the liver (passive entry or active evasion from immune recognition) and (ii) actively inhibit the innate immune response (ie modulation of pattern recognition receptor expression and/or signalling pathways, modulation of interferon response and modulation of immune cells count or phenotype).
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http://dx.doi.org/10.1111/liv.15054DOI Listing
September 2021

Histone H3K27 demethylase KDM6A is an epigenetic gatekeeper of mTORC1 signalling in cancer.

Gut 2021 Sep 11. Epub 2021 Sep 11.

Helmholtz-University Group "Cell Plasticity and Epigenetic Remodeling", German Cancer Research Center (DKFZ) & Institute of Pathology, University Hospital, Heidelberg, Germany

Objective: Large-scale genome sequencing efforts of human tumours identified epigenetic modifiers as one of the most frequently mutated gene class in human cancer. However, how these mutations drive tumour development and tumour progression are largely unknown. Here, we investigated the function of the histone demethylase KDM6A in gastrointestinal cancers, such as liver cancer and pancreatic cancer.

Design: Genetic alterations as well as expression analyses of KDM6A were performed in patients with liver cancer. Genetic mouse models of liver and pancreatic cancer coupled with Kdm6a-deficiency were investigated, transcriptomic and epigenetic profiling was performed, and in vivo and in vitro drug treatments were conducted.

Results: KDM6A expression was lost in 30% of patients with liver cancer. Kdm6a deletion significantly accelerated tumour development in murine liver and pancreatic cancer models. Kdm6a-deficient tumours showed hyperactivation of mTORC1 signalling, whereas endogenous Kdm6a re-expression by inducible RNA-interference in established Kdm6a-deficient tumours diminished mTORC1 activity resulting in attenuated tumour progression. Genome-wide transcriptional and epigenetic profiling revealed direct binding of Kdm6a to crucial negative regulators of mTORC1, such as Deptor, and subsequent transcriptional activation by epigenetic remodelling. Moreover, in vitro and in vivo genetic epistasis experiments illustrated a crucial function of Deptor and mTORC1 in Kdm6a-dependent tumour suppression. Importantly, KDM6A expression in human tumours correlates with mTORC1 activity and KDM6A-deficient tumours exhibit increased sensitivity to mTORC1 inhibition.

Conclusion: KDM6A is an important tumour suppressor in gastrointestinal cancers and acts as an epigenetic toggle for mTORC1 signalling. Patients with KDM6A-deficient tumours could benefit of targeted therapy focusing on mTORC1 inhibition.
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http://dx.doi.org/10.1136/gutjnl-2021-325405DOI Listing
September 2021

The immunological and metabolic landscape in primary and metastatic liver cancer.

Nat Rev Cancer 2021 09 29;21(9):541-557. Epub 2021 Jul 29.

Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany.

The liver is the sixth most common site of primary cancer in humans, and generally arises in a background of cirrhosis and inflammation. Moreover, the liver is frequently colonized by metastases from cancers of other organs (particularly the colon) because of its anatomical location and organization, as well as its unique metabolic and immunosuppressive environment. In this Review, we discuss how the hepatic microenvironment adapts to pathologies characterized by chronic inflammation and metabolic alterations. We illustrate how these immunological or metabolic changes alter immunosurveillance and thus hinder or promote the development of primary liver cancer. In addition, we describe how inflammatory and metabolic niches affect the spreading of cancer metastases into or within the liver. Finally, we review the current therapeutic options in this context and the resulting challenges that must be surmounted.
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http://dx.doi.org/10.1038/s41568-021-00383-9DOI Listing
September 2021

CCL2 in the Tumor Microenvironment.

Adv Exp Med Biol 2021 ;1302:1-14

Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany.

The C-C motif chemokine ligand 2 (CCL2) is a crucial mediator of immune cell recruitment during microbial infections and tissue damage. CCL2 is also frequently overexpressed in cancer cells and other cells in the tumor microenvironment, and a large body of evidence indicates that high CCL2 levels are associated with more aggressive malignancies, a higher probability of metastasis, and poorer outcomes in a wide range of cancers. CCL2 plays a role in recruiting tumor-associated macrophages (TAMs), which adopt a pro-tumorigenic phenotype and support cancer cell survival, facilitate tumor cell invasion, and promote angiogenesis. CCL2 also has direct, TAM-independent effects on tumor cells and the tumor microenvironment, including recruitment of other myeloid subsets and non-myeloid cells, maintaining an immunosuppressive environment, stimulating tumor cell growth and motility, and promoting angiogenesis. CCL2 also plays important roles in the metastatic cascade, such as creating a pre-metastatic niche in distant organs and promoting tumor cell extravasation across endothelia. Due to its many roles in tumorigenesis and metastatic processes, the CCL2-CCR2 signaling axis is currently being pursued as a potential therapeutic target for cancer.
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http://dx.doi.org/10.1007/978-3-030-62658-7_1DOI Listing
July 2021

Corrigendum to "Dietary protein dilution limits dyslipidemia in obesity through FGF21-driven fatty acid clearance" [The Journal of Nutritional Biochemistry 57 (2018) 189-196].

J Nutr Biochem 2021 Nov 9;97:108807. Epub 2021 Jul 9.

Joint Research Division Molecular Metabolic Control, German Cancer Research Center, Center for Molecular Biology, Heidelberg University and Heidelberg University Hospital, 69120 Heidelberg, Germany; Nutrient Metabolism and Signalling Lab, Dept. of Biochemistry and Molecular Biology, School of Biomedical Sciences, and Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Clayton, 3800, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.jnutbio.2021.108807DOI Listing
November 2021

SpaceM reveals metabolic states of single cells.

Nat Methods 2021 07 5;18(7):799-805. Epub 2021 Jul 5.

Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.

A growing appreciation of the importance of cellular metabolism and revelations concerning the extent of cell-cell heterogeneity demand metabolic characterization of individual cells. We present SpaceM, an open-source method for in situ single-cell metabolomics that detects >100 metabolites from >1,000 individual cells per hour, together with a fluorescence-based readout and retention of morpho-spatial features. We validated SpaceM by predicting the cell types of cocultured human epithelial cells and mouse fibroblasts. We used SpaceM to show that stimulating human hepatocytes with fatty acids leads to the emergence of two coexisting subpopulations outlined by distinct cellular metabolic states. Inducing inflammation with the cytokine interleukin-17A perturbs the balance of these states in a process dependent on NF-κB signaling. The metabolic state markers were reproduced in a murine model of nonalcoholic steatohepatitis. We anticipate SpaceM to be broadly applicable for investigations of diverse cellular models and to democratize single-cell metabolomics.
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http://dx.doi.org/10.1038/s41592-021-01198-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611214PMC
July 2021

T cell engager antibodies enable T cells to control HBV infection and to target HBsAg-positive hepatoma in mice.

J Hepatol 2021 Jun 23. Epub 2021 Jun 23.

Institute of Virology, School of Medicine, Technical University of Munich, Helmholtz Zentrum München, Munich, Germany; German Center for Infection Research (DZIF), Munich and Hamburg Partner sites, Germany. Electronic address:

Background & Aims: Current antiviral therapies control but rarely eliminate HBV, leaving chronic HBV carriers at risk of developing hepatocellular carcinoma (HCC). Lacking or dysfunctional virus-specific adaptive immunity prevents control of HBV and allows the virus to persist. Restoring antiviral T cell immunity could lead to HBV elimination and cure of chronically infected patients.

Methods: We constructed bispecific T cell engager antibodies that are designed to induce antiviral immunity through simultaneous binding of HBV envelope proteins (HBVenv) on infected hepatocytes and CD3 or CD28 on T cells. T cell engager antibodies were employed in co-cultures with healthy donor lymphocytes and HBV-infected target cells. Activation of the T cell response was determined by detection of pro-inflammatory cytokines, effector function (by cytotoxicity) and antiviral effects. To study in vivo efficacy, immune-deficient mice were transplanted with HBVenv-positive and -negative hepatoma cells.

Results: The 2 T cell engager antibodies synergistically activated T cells to become polyfunctional effectors that in turn elicited potent antiviral effects by killing infected cells and in addition controlled HBV via non-cytolytic, cytokine-mediated antiviral mechanisms. In vivo in mice, the antibodies attracted T cells specifically to the tumors expressing HBVenv resulting in T cell activation, tumor infiltration and reduction of tumor burden.

Conclusion: This study demonstrates that the administration of HBVenv-targeting T cell engager antibodies facilitates a robust T cell redirection towards HBV-positive target cells and provides a feasible and promising approach for the treatment of chronic viral hepatitis and HBV-associated HCC.

Lay Summary: T cell engager antibodies are an interesting, novel therapeutic tool to restore immunity in patients with chronic hepatitis B. As bispecific antibodies, they bind envelope proteins on the surface of the hepatitis B virus (HBV) and CD3 or CD28 on T cells. This way, they induce a potent antiviral and cytotoxic T cell response that leads to the elimination of HBV-positive cells. These bispecific T cell engager antibodies are exciting therapeutic candidates for chronic hepatitis B and HBV-associated hepatocellular carcinoma.
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http://dx.doi.org/10.1016/j.jhep.2021.06.022DOI Listing
June 2021

A spatial vascular transcriptomic, proteomic, and phosphoproteomic atlas unveils an angiocrine Tie-Wnt signaling axis in the liver.

Dev Cell 2021 06 25;56(11):1677-1693.e10. Epub 2021 May 25.

Division of Vascular Oncology and Metastasis Research, German Cancer Research Center (DKFZ), Heidelberg, Germany; European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany; German Cancer Consortium, Heidelberg, Germany. Electronic address:

Single-cell transcriptomics (scRNA-seq) has revolutionized the understanding of the spatial architecture of tissue structure and function. Advancing the "transcript-centric" view of scRNA-seq analyses is presently restricted by the limited resolution of proteomics and genome-wide techniques to analyze post-translational modifications. Here, by combining spatial cell sorting with transcriptomics and quantitative proteomics/phosphoproteomics, we established the spatially resolved proteome landscape of the liver endothelium, yielding deep mechanistic insight into zonated vascular signaling mechanisms. Phosphorylation of receptor tyrosine kinases was detected preferentially in the central vein area, resulting in an atypical enrichment of tyrosine phosphorylation. Prototypic biological validation identified Tie receptor signaling as a selective and specific regulator of vascular Wnt activity orchestrating angiocrine signaling, thereby controlling hepatocyte function during liver regeneration. Taken together, the study has yielded fundamental insight into the spatial organization of liver endothelial cell signaling. Spatial sorting may be employed as a universally adaptable strategy for multiomic analyses of scRNA-seq-defined cellular (sub)-populations.
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http://dx.doi.org/10.1016/j.devcel.2021.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8191494PMC
June 2021

XCR1 type 1 conventional dendritic cells drive liver pathology in non-alcoholic steatohepatitis.

Nat Med 2021 06 20;27(6):1043-1054. Epub 2021 May 20.

Division of Chronic Inflammation and Cancer, German Cancer Research Center Heidelberg (DKFZ), Heidelberg, Germany.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are prevalent liver conditions that underlie the development of life-threatening cirrhosis, liver failure and liver cancer. Chronic necro-inflammation is a critical factor in development of NASH, yet the cellular and molecular mechanisms of immune dysregulation in this disease are poorly understood. Here, using single-cell transcriptomic analysis, we comprehensively profiled the immune composition of the mouse liver during NASH. We identified a significant pathology-associated increase in hepatic conventional dendritic cells (cDCs) and further defined their source as NASH-induced boost in cycling of cDC progenitors in the bone marrow. Analysis of blood and liver from patients on the NAFLD/NASH spectrum showed that type 1 cDCs (cDC1) were more abundant and activated in disease. Sequencing of physically interacting cDC-T cell pairs from liver-draining lymph nodes revealed that cDCs in NASH promote inflammatory T cell reprogramming, previously associated with NASH worsening. Finally, depletion of cDC1 in XCR1 mice or using anti-XCL1-blocking antibody attenuated liver pathology in NASH mouse models. Overall, our study provides a comprehensive characterization of cDC biology in NASH and identifies XCR1 cDC1 as an important driver of liver pathology.
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http://dx.doi.org/10.1038/s41591-021-01344-3DOI Listing
June 2021

Molecular characterisation of hepatocellular carcinoma in patients with non-alcoholic steatohepatitis.

J Hepatol 2021 Oct 13;75(4):865-878. Epub 2021 May 13.

Sema4, Stamford, Connecticut, USA; Department of Genetics and Genomic Sciences and Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, USA.

Background And Aims: Non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is increasing globally, but its molecular features are not well defined. We aimed to identify unique molecular traits characterising NASH-HCC compared to other HCC aetiologies.

Methods: We collected 80 NASH-HCC and 125 NASH samples from 5 institutions. Expression array (n = 53 NASH-HCC; n = 74 NASH) and whole exome sequencing (n = 52 NASH-HCC) data were compared to HCCs of other aetiologies (n = 184). Three NASH-HCC mouse models were analysed by RNA-seq/expression-array (n = 20). Activin A receptor type 2A (ACVR2A) was silenced in HCC cells and proliferation assessed by colorimetric and colony formation assays.

Results: Mutational profiling of NASH-HCC tumours revealed TERT promoter (56%), CTNNB1 (28%), TP53 (18%) and ACVR2A (10%) as the most frequently mutated genes. ACVR2A mutation rates were higher in NASH-HCC than in other HCC aetiologies (10% vs. 3%, p <0.05). In vitro, ACVR2A silencing prompted a significant increase in cell proliferation in HCC cells. We identified a novel mutational signature (MutSig-NASH-HCC) significantly associated with NASH-HCC (16% vs. 2% in viral/alcohol-HCC, p = 0.03). Tumour mutational burden was higher in non-cirrhotic than in cirrhotic NASH-HCCs (1.45 vs. 0.94 mutations/megabase; p <0.0017). Compared to other aetiologies of HCC, NASH-HCCs were enriched in bile and fatty acid signalling, oxidative stress and inflammation, and presented a higher fraction of Wnt/TGF-β proliferation subclass tumours (42% vs. 26%, p = 0.01) and a lower prevalence of the CTNNB1 subclass. Compared to other aetiologies, NASH-HCC showed a significantly higher prevalence of an immunosuppressive cancer field. In 3 murine models of NASH-HCC, key features of human NASH-HCC were preserved.

Conclusions: NASH-HCCs display unique molecular features including higher rates of ACVR2A mutations and the presence of a newly identified mutational signature.

Lay Summary: The prevalence of hepatocellular carcinoma (HCC) associated with non-alcoholic steatohepatitis (NASH) is increasing globally, but its molecular traits are not well characterised. In this study, we uncovered higher rates of ACVR2A mutations (10%) - a potential tumour suppressor - and the presence of a novel mutational signature that characterises NASH-related HCC.
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http://dx.doi.org/10.1016/j.jhep.2021.04.049DOI Listing
October 2021

Hypoxia-Inducible Factor 1 Alpha-Mediated RelB/APOBEC3B Down-regulation Allows Hepatitis B Virus Persistence.

Hepatology 2021 Oct 15;74(4):1766-1781. Epub 2021 Aug 15.

Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Background And Aims: Therapeutic strategies against HBV focus, among others, on the activation of the immune system to enable the infected host to eliminate HBV. Hypoxia-inducible factor 1 alpha (HIF1α) stabilization has been associated with impaired immune responses. HBV pathogenesis triggers chronic hepatitis-related scaring, leading inter alia to modulation of liver oxygenation and transient immune activation, both factors playing a role in HIF1α stabilization.

Approach And Results: We addressed whether HIF1α interferes with immune-mediated induction of the cytidine deaminase, apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B; A3B), and subsequent covalently closed circular DNA (cccDNA) decay. Liver biopsies of chronic HBV (CHB) patients were analyzed by immunohistochemistry and in situ hybridization. The effect of HIF1α induction/stabilization on differentiated HepaRG or mice ± HBV ± LTβR-agonist (BS1) was assessed in vitro and in vivo. Induction of A3B and subsequent effects were analyzed by RT-qPCR, immunoblotting, chromatin immunoprecipitation, immunocytochemistry, and mass spectrometry. Analyzing CHB highlighted that areas with high HIF1α levels and low A3B expression correlated with high HBcAg, potentially representing a reservoir for HBV survival in immune-active patients. In vitro, HIF1α stabilization strongly impaired A3B expression and anti-HBV effect. Interestingly, HIF1α knockdown was sufficient to rescue the inhibition of A3B up-regulation and -mediated antiviral effects, whereas HIF2α knockdown had no effect. HIF1α stabilization decreased the level of v-rel reticuloendotheliosis viral oncogene homolog B protein, but not its mRNA, which was confirmed in vivo. Noteworthy, this function of HIF1α was independent of its partner, aryl hydrocarbon receptor nuclear translocator.

Conclusions: In conclusion, inhibiting HIF1α expression or stabilization represents an anti-HBV strategy in the context of immune-mediated A3B induction. High HIF1α, mediated by hypoxia or inflammation, offers a reservoir for HBV survival in vivo and should be considered as a restricting factor in the development of immune therapies.
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http://dx.doi.org/10.1002/hep.31902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611739PMC
October 2021

Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20.

EMBO Rep 2021 06 9;22(6):e49568. Epub 2021 May 9.

Institute of Virology, School of Medicine, Technical University of Munich / Helmholtz Zentrum München, Munich, Germany.

Hepatitis B virus (HBV) persists by depositing a covalently closed circular DNA (cccDNA) in the nucleus of infected cells that cannot be targeted by available antivirals. Interferons can diminish HBV cccDNA via APOBEC3-mediated deamination. Here, we show that overexpression of APOBEC3A alone is not sufficient to reduce HBV cccDNA that requires additional treatment of cells with interferon indicating involvement of an interferon-stimulated gene (ISG) in cccDNA degradation. Transcriptome analyses identify ISG20 as the only type I and II interferon-induced, nuclear protein with annotated nuclease activity. ISG20 localizes to nucleoli of interferon-stimulated hepatocytes and is enriched on deoxyuridine-containing single-stranded DNA that mimics transcriptionally active, APOBEC3A-deaminated HBV DNA. ISG20 expression is detected in human livers in acute, self-limiting but not in chronic hepatitis B. ISG20 depletion mitigates the interferon-induced loss of cccDNA, and co-expression with APOBEC3A is sufficient to diminish cccDNA. In conclusion, non-cytolytic HBV cccDNA decline requires the concerted action of a deaminase and a nuclease. Our findings highlight that ISGs may cooperate in their antiviral activity that may be explored for therapeutic targeting.
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http://dx.doi.org/10.15252/embr.201949568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8183418PMC
June 2021

Dietary Essential Amino Acid Restriction Promotes Hyperdipsia via Hepatic FGF21.

Nutrients 2021 Apr 26;13(5). Epub 2021 Apr 26.

Nutrient Metabolism & Signalling Laboratory, Department of Biochemistry and Molecular Biology, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, 3800 Clayton, Australia.

Prior studies have reported that dietary protein dilution (DPD) or amino acid dilution promotes heightened water intake (i.e., hyperdipsia) however, the exact dietary requirements and the mechanism responsible for this effect are still unknown. Here, we show that dietary amino acid (AA) restriction is sufficient and required to drive hyperdipsia during DPD. Our studies demonstrate that particularly dietary essential AA (EAA) restriction, but not non-EAA, is responsible for the hyperdipsic effect of total dietary AA restriction (DAR). Additionally, by using diets with varying amounts of individual EAA under constant total AA supply, we demonstrate that restriction of threonine (Thr) or tryptophan (Trp) is mandatory and sufficient for the effects of DAR on hyperdipsia and that liver-derived fibroblast growth factor 21 (FGF21) is required for this hyperdipsic effect. Strikingly, artificially introducing Thr de novo biosynthesis in hepatocytes reversed hyperdipsia during DAR. In summary, our results show that the DPD effects on hyperdipsia are induced by the deprivation of Thr and Trp, and in turn, via liver/hepatocyte-derived FGF21.
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http://dx.doi.org/10.3390/nu13051469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8144947PMC
April 2021

Downregulation of TGR5 (GPBAR1) in biliary epithelial cells contributes to the pathogenesis of sclerosing cholangitis.

J Hepatol 2021 Sep 17;75(3):634-646. Epub 2021 Apr 17.

Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital Düsseldorf, Medical Faculty of Heinrich Heine University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany.

Background & Aims: Primary sclerosing cholangitis (PSC) is characterized by chronic inflammation and progressive fibrosis of the biliary tree. The bile acid receptor TGR5 (GPBAR1) is found on biliary epithelial cells (BECs), where it promotes secretion, proliferation and tight junction integrity. Thus, we speculated that changes in TGR5-expression in BECs may contribute to PSC pathogenesis.

Methods: TGR5-expression and -localization were analyzed in PSC livers and liver tissue, isolated bile ducts and BECs from Abcb4, Abcb4/Tgr5 and ursodeoxycholic acid (UDCA)- or 24-norursodeoxycholic acid (norUDCA)-fed Abcb4 mice. The effects of IL8/IL8 homologues on TGR5 mRNA and protein levels were studied. BEC gene expression was analyzed by single-cell transcriptomics (scRNA-seq) from distinct mouse models.

Results: TGR5 mRNA expression and immunofluorescence staining intensity were reduced in BECs of PSC and Abcb4 livers, in Abcb4 extrahepatic bile ducts, but not in intrahepatic macrophages. No changes in TGR5 BEC fluorescence intensity were detected in liver tissue of other liver diseases, including primary biliary cholangitis. Incubation of BECs with IL8/IL8 homologues, but not with other cytokines, reduced TGR5 mRNA and protein levels. BECs from Abcb4 mice had lower levels of phosphorylated Erk and higher expression levels of Icam1, Vcam1 and Tgfβ2. Overexpression of Tgr5 abolished the activated inflammatory phenotype characteristic of Abcb4 BECs. NorUDCA-feeding restored TGR5-expression levels in BECs in Abcb4 livers.

Conclusions: Reduced TGR5 levels in BECs from patients with PSC and Abcb4 mice promote development of a reactive BEC phenotype, aggravate biliary injury and thus contribute to the pathogenesis of sclerosing cholangitis. Restoration of biliary TGR5-expression levels represents a previously unknown mechanism of action of norUDCA.

Lay Summary: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease-associated with progressive inflammation of the bile duct, leading to fibrosis and end-stage liver disease. Bile acid (BA) toxicity may contribute to the development and disease progression of PSC. TGR5 is a membrane-bound receptor for BAs, which is found on bile ducts and protects bile ducts from BA toxicity. In this study, we show that TGR5 levels were reduced in bile ducts from PSC livers and in bile ducts from a genetic mouse model of PSC. Our investigations indicate that lower levels of TGR5 in bile ducts may contribute to PSC development and progression. Furthermore, treatment with norUDCA, a drug currently being tested in a phase III trial for PSC, restored TGR5 levels in biliary epithelial cells.
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http://dx.doi.org/10.1016/j.jhep.2021.03.029DOI Listing
September 2021

JNK signaling prevents biliary cyst formation through a CASPASE-8-dependent function of RIPK1 during aging.

Proc Natl Acad Sci U S A 2021 03;118(12)

Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital Duesseldorf, Medical Faculty, Heinrich Heine University, 40225 Duesseldorf, Germany.

The c-Jun N-terminal kinase (JNK) signaling pathway mediates adaptation to stress signals and has been associated with cell death, cell proliferation, and malignant transformation in the liver. However, up to now, its function was experimentally studied mainly in young mice. By generating mice with combined conditional ablation of and in liver parenchymal cells (LPCs) (JNK1/2 mice; KO, knockout), we unraveled a function of the JNK pathway in the regulation of liver homeostasis during aging. Aging JNK1/2 mice spontaneously developed large biliary cysts that originated from the biliary cell compartment. Mechanistically, we could show that cyst formation in livers of JNK1/2 mice was dependent on receptor-interacting protein kinase 1 (RIPK1), a known regulator of cell survival, apoptosis, and necroptosis. In line with this, we showed that RIPK1 was overexpressed in the human cyst epithelium of a subset of patients with polycystic liver disease. Collectively, these data reveal a functional interaction between JNK signaling and RIPK1 in age-related progressive cyst development. Thus, they provide a functional linkage between stress adaptation and programmed cell death (PCD) in the maintenance of liver homeostasis during aging.
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http://dx.doi.org/10.1073/pnas.2007194118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000530PMC
March 2021

Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner.

EMBO J 2021 May 25;40(9):e106048. Epub 2021 Mar 25.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA.

Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease.
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http://dx.doi.org/10.15252/embj.2020106048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8090854PMC
May 2021

Auto-aggressive CXCR6 CD8 T cells cause liver immune pathology in NASH.

Nature 2021 Apr 24;592(7854):444-449. Epub 2021 Mar 24.

Department of Internal Medicine I, Gastroenterology, Hepatology, Endocrinology and Metabolism, Medical University Innsbruck, Innsbruck, Austria.

Nonalcoholic steatohepatitis (NASH) is a manifestation of systemic metabolic disease related to obesity, and causes liver disease and cancer. The accumulation of metabolites leads to cell stress and inflammation in the liver, but mechanistic understandings of liver damage in NASH are incomplete. Here, using a preclinical mouse model that displays key features of human NASH (hereafter, NASH mice), we found an indispensable role for T cells in liver immunopathology. We detected the hepatic accumulation of CD8 T cells with phenotypes that combined tissue residency (CXCR6) with effector (granzyme) and exhaustion (PD1) characteristics. Liver CXCR6 CD8 T cells were characterized by low activity of the FOXO1 transcription factor, and were abundant in NASH mice and in patients with NASH. Mechanistically, IL-15 induced FOXO1 downregulation and CXCR6 upregulation, which together rendered liver-resident CXCR6 CD8 T cells susceptible to metabolic stimuli (including acetate and extracellular ATP) and collectively triggered auto-aggression. CXCR6 CD8 T cells from the livers of NASH mice or of patients with NASH had similar transcriptional signatures, and showed auto-aggressive killing of cells in an MHC-class-I-independent fashion after signalling through P2X7 purinergic receptors. This killing by auto-aggressive CD8 T cells fundamentally differed from that by antigen-specific cells, which mechanistically distinguishes auto-aggressive and protective T cell immunity.
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http://dx.doi.org/10.1038/s41586-021-03233-8DOI Listing
April 2021

NASH limits anti-tumour surveillance in immunotherapy-treated HCC.

Nature 2021 Apr 24;592(7854):450-456. Epub 2021 Mar 24.

Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle, UK.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need. Here we report the progressive accumulation of exhausted, unconventionally activated CD8PD1 T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8PD1 T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8PD1CXCR6, TOX, and TNF T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8 T cells or TNF neutralization, suggesting that CD8 T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8PD1 T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.
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http://dx.doi.org/10.1038/s41586-021-03362-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046670PMC
April 2021

Liver alanine catabolism promotes skeletal muscle atrophy and hyperglycaemia in type 2 diabetes.

Nat Metab 2021 03 18;3(3):394-409. Epub 2021 Mar 18.

Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia.

Both obesity and sarcopenia are frequently associated in ageing, and together may promote the progression of related conditions such as diabetes and frailty. However, little is known about the pathophysiological mechanisms underpinning this association. Here we show that systemic alanine metabolism is linked to glycaemic control. We find that expression of alanine aminotransferases is increased in the liver in mice with obesity and diabetes, as well as in humans with type 2 diabetes. Hepatocyte-selective silencing of both alanine aminotransferase enzymes in mice with obesity and diabetes retards hyperglycaemia and reverses skeletal muscle atrophy through restoration of skeletal muscle protein synthesis. Mechanistically, liver alanine catabolism driven by chronic glucocorticoid and glucagon signalling promotes hyperglycaemia and skeletal muscle wasting. We further provide evidence for amino acid-induced metabolic cross-talk between the liver and skeletal muscle in ex vivo experiments. Taken together, we reveal a metabolic inter-tissue cross-talk that links skeletal muscle atrophy and hyperglycaemia in type 2 diabetes.
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http://dx.doi.org/10.1038/s42255-021-00369-9DOI Listing
March 2021

Analysis of chronic inflammatory lesions of the colon for BMMF Rep antigen expression and CD68 macrophage interactions.

Proc Natl Acad Sci U S A 2021 03;118(12)

Division of Episomal-Persistent DNA in Cancer and Chronic Diseases, German Cancer Research Center, 69120 Heidelberg, Germany;

Consumption of Eurasian bovine meat and milk has been associated with cancer development, in particular with colorectal cancer (CRC). In addition, zoonotic infectious agents from bovine products were proposed to cause colon cancer (zur Hausen et al., 2009). Bovine meat and milk factors (BMMF) are small episomal DNA molecules frequently isolated from bovine sera and milk products, and recently, also from colon cancer (de Villiers et al., 2019). BMMF are bioactive in human cells and were proposed to induce chronic inflammation in precancerous tissue leading to increased radical formation: for example, reactive oxygen and reactive nitrogen species and elevated levels of DNA mutations in replicating cells, such as cancer progenitor cells (zur Hausen et al., 2018). Mouse monoclonal antibodies against the replication (Rep) protein of H1MSB.1 (BMMF1) were used to analyze BMMF presence in different cohorts of CRC peritumor and tumor tissues and cancer-free individuals by immunohistochemistry and Western blot. BMMF DNA was isolated by laser microdissection from immunohistochemistry-positive tissue regions. We found BMMF Rep protein present specifically in close vicinity of CD68 macrophages in the interstitial lamina propria adjacent to CRC tissues, suggesting the presence of local chronic inflammation. BMMF1 (modified H1MSB.1) DNA was isolated from the same tissue regions. Rep and CD68 detection increased significantly in peritumor cancer tissues when compared to tissues of cancer-free individuals. This strengthens previous postulations that BMMF function as indirect carcinogens by inducing chronic inflammation and DNA damage in replicating cells, which represent progress to progenitor cells for adenoma (polyps) formation and cancer.
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http://dx.doi.org/10.1073/pnas.2025830118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000208PMC
March 2021

Liver Inflammation and Hepatobiliary Cancers.

Trends Cancer 2021 07 2;7(7):606-623. Epub 2021 Mar 2.

Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany. Electronic address:

Immune regulation has an important role in cancer development, particularly in organs with continuous exposure to environmental pathogens, such as the liver and gastrointestinal tract. Chronic liver inflammation can lead to the development of hepatobiliary cancers, namely hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (iCCA), or combined HCC (cHCC)-CCA. In this review, we discuss the link between oxidative stress and the hepatic immune compartments, as well as how these factors trigger hepatocyte damage, proliferation, and eventually cancer initiation and its sustainment. We further give an overview of new anticancer therapies based on immunomodulation.
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http://dx.doi.org/10.1016/j.trecan.2021.01.012DOI Listing
July 2021

Hypoxia inducible factors regulate hepatitis B virus replication by activating the basal core promoter.

J Hepatol 2021 07 29;75(1):64-73. Epub 2021 Jan 29.

Nuffield Department of Medicine, University of Oxford, Oxford, UK; Chinese Academy of Medical Sciences (CAMS) Oxford Institute (COI), University of Oxford, Oxford, UK. Electronic address:

Background & Aims: Hypoxia inducible factors (HIFs) are a hallmark of inflammation and are key regulators of hepatic immunity and metabolism, yet their role in HBV replication is poorly defined. HBV replicates in hepatocytes within the liver, a naturally hypoxic organ, however most studies of viral replication are performed under conditions of atmospheric oxygen, where HIFs are inactive. We therefore investigated the role of HIFs in regulating HBV replication.

Methods: Using cell culture, animal models, human tissue and pharmacological agents inhibiting the HIF-prolyl hydroxylases, we investigated the impact of hypoxia on the HBV life cycle.

Results: Culturing liver cell-based model systems under low oxygen uncovered a new role for HIFs in binding HBV DNA and activating the basal core promoter, leading to increased pre-genomic RNA and de novo HBV particle secretion. The presence of hypoxia responsive elements among all primate members of the hepadnaviridae highlights an evolutionary conserved role for HIFs in regulating this virus family.

Conclusions: Identifying a role for this conserved oxygen sensor in regulating HBV transcription suggests that this virus has evolved to exploit the HIF signaling pathway to persist in the low oxygen environment of the liver. Our studies show the importance of considering oxygen availability when studying HBV-host interactions and provide innovative routes to better understand and target chronic HBV infection.

Lay Summary: Viral replication in host cells is defined by the cellular microenvironment and one key factor is local oxygen tension. Hepatitis B virus (HBV) replicates in the liver, a naturally hypoxic organ. Hypoxia inducible factors (HIFs) are the major sensors of low oxygen; herein, we identify a new role for these factors in regulating HBV replication, revealing new therapeutic targets.
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http://dx.doi.org/10.1016/j.jhep.2020.12.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214165PMC
July 2021

Niche derived netrin-1 regulates hematopoietic stem cell dormancy via its receptor neogenin-1.

Nat Commun 2021 01 27;12(1):608. Epub 2021 Jan 27.

Laboratory of Ageing Biology and Stem Cells, European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential associated to dormancy. Here we identify the cell surface receptor neogenin-1 as specifically expressed in dormant HSCs. Loss of neogenin-1 initially leads to increased HSC expansion but subsequently to loss of self-renewal and premature exhaustion in vivo. Its ligand netrin-1 induces Egr1 expression and maintains quiescence and function of cultured HSCs in a Neo1 dependent manner. Produced by arteriolar endothelial and periarteriolar stromal cells, conditional netrin-1 deletion in the bone marrow niche reduces HSC numbers, quiescence and self-renewal, while overexpression increases quiescence in vivo. Ageing associated bone marrow remodelling leads to the decline of netrin-1 expression in niches and a compensatory but reversible upregulation of neogenin-1 on HSCs. Our study suggests that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1 function. Decline of netrin-1 production during ageing leads to the gradual decrease of Neo1 mediated HSC self-renewal.
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http://dx.doi.org/10.1038/s41467-020-20801-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840807PMC
January 2021
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