TP06: Role of platelets in control and clearance of hepatitis B virus infection via modulation of liver tissue environment and metabolism

The liver microenvironment containing different parenchymal and non-parenchymal liver cells and the hepatic metabolome play a pivotal role in the clearance of hepatic virus infections. Here we investigate the direct and indirect role of platelets in shaping the liver microenvironment, polarizing immune cell phenotypes and affecting liver metabolism in the context of viral infections. We analyse how platelets contribute to the reshaping of the liver immune niche, how they interact directly with hepatocytes in the context of antiviral therapies and how they affect clearance mechanisms. Our data might hint at novel targets used to support effective and long-lasting antiviral responses in the context of hepatic viral infections and reducing unwanted, immune-mediated side-effects.

Prof. Dr. Mathias Heikenwälder

Colasanti O, Burm R, Huang HE, Riedl T, Traut J, Gillich N, Li TF, Corneillie C, Faure-Dupuy S, Grünvogel O, Heide D, Lee JY, Tran CS, Merle U, Chironna M, Vondran FWF, Esser-Nobis K, Binder M, Bartenschlager R, Heikenwälder M, Meuleman P, Lohmann V. (2023) Comparison of HAV and HCV infections in vivo and in vitro reveals distinct patterns of innate immune evasion and activation. J Hepatol. 28;S0168-8278(23)00309-4. doi: 10.1016/j.jhep.2023.04.023.

Kotsiliti E, ..., Hofmann M, Rippe K, Zitzlesberger H, Weber A, Malek N, Lüdde T, Vucur M, Augustin HG, Flavell R, Parnas O, Rad R, Pabst O, Henderson NC, Huber S, Macpherson A, Knolle P, Claasen M, Geier A, Trautwein C, Unger K, Elinav E, Waisman A, Abdullah Z, Haller D, Tacke F, Anstee QM, Heikenwalder M. (2023) Intestinal B-cells license metabolic T-cell activation in NASH microbiota/antigen-independently and contribute to fibrosis by IgA-FcR signalling. J Hepatol. 2023 May 9:S0168-8278(23)00325-2. doi: 10.1016/j.jhep.2023.04.037.

Esser K, Cheng X, Wettengel JM, Lucifora J, Hansen-Palmus L, Austen K, Roca Suarez AA, Heintz S, Testoni B, Nebioglu F, Pham MT, Yang S, Zernecke A, Wohlleber D, Ringelhan M, Broxtermann M, Hartmann D, Hüser N, Mergner J, Pichlmair A, Thasler WE, Heikenwalder M, Gasteiger G, Blutke A, Walch A, Knolle PA, Bartenschlager R, Protzer U. (2023) Hepatitis B virus targets lipid transport pathways to infect hepatocytes,Cellular and Molecular Gastroenterology and Hepatology, 2023,ISSN 2352-345X,https://doi.org/10.1016/j.jcmgh.2023.03.011.

Delphin M, Desmares M, Schuehle S, Heikenwalder M, Durantel D, Faure-Dupuy S. (2021) How to get away with liver innate immunity? A viruses' tale. Liver Int. 41(11):2547-2559. doi: 10.1111/liv.15054.

Quitt O, Luo S, Meyer M, Xie Z, Golsaz-Shirazi F, Loffredo-Verde E, Festag J, Bockmann JH, Zhao L, Stadler D, Chou WM, Tedjokusumo R, Wettengel JM, Ko C, Noeßner E, Bulbuc N, Shokri F, Lüttgau S, Heikenwälder M, Bohne F, Moldenhauer G, Momburg F, Protzer U. (2021) T-cell engager antibodies enable T cells to control HBV infection and to target HBsAg-positive hepatoma in mice. J Hepatol. 75(5):1058-1071. doi: 10.1016/j.jhep.2021.06.022.

Faure-Dupuy S, Riedl T, Rolland M, Hizir Z, Reisinger F, Neuhaus K, Schuehle S, Remouchamps C, Gillet N, Schönung M, Stadler M, Wettengel J, Barnault R, Parent R, Schuster LC, Farhat R, Prokosch S, Leuchtenberger C, Öllinger R, Engleitner T, Rippe K, Rad R, Unger K, Tschaharganeh D, Lipka DB, Protzer U, Durantel D, Lucifora J, Dejardin E, Heikenwälder M. (2021) Control of APOBEC3B induction and cccDNA decay by NF-κB and miR-138-5p. JHEP Rep. 25;3(6):100354. doi: 10.1016/j.jhepr.2021.100354.

Stadler D, Kächele M, Jones AN, Hess J, Urban C, Schneider J, Xia Y, Oswald A, Nebioglu F, Bester R, Lasitschka F, Ringelhan M, Ko C, Chou WM, Geerlof A, van de Klundert MA, Wettengel JM, Schirmacher P, Heikenwälder M, Schreiner S, Bartenschlager R, Pichlmair A, Sattler M, Unger K, Protzer U. (2021) Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20. EMBO Rep. 4;22(6):e49568. doi: 10.15252/embr.201949568.

Rappez L, Stadler M, Triana S, Gathungu RM, Ovchinnikova K, Phapale P, Heikenwalder M, Alexandrov T. (2021) SpaceM reveals metabolic states of single cells. Nat Methods. 18(7):799-805. doi: 10.1038/s41592-021-01198-0.

Deczkowska A, David E, Ramadori P, Pfister D, Safran M, At The B, Giladi A, Jaitin DA, Barboy O, Cohen M, Yofe I, Gur C, Shlomi-Loubaton S, Henri S, Suhail Y, Qiu M, Kam S, Hermon H, Lahat E, Ben Yakov G, Cohen-Ezra O, Davidov Y, Likhter M, Goitein D, Roth S, Weber A, Malissen B, Weiner A, Ben-Ari Z, Heikenwälder M, Elinav E, Amit I. (2021) XCR1+ type 1 conventional dendritic cells drive liver pathology in non-alcoholic steatohepatitis. Nat Med. 27(6):1043-1054. doi: 10.1038/s41591-021-01344-3.

Riedl T, Faure-Dupuy S, Rolland M, Schuehle S, Hizir Z, Calderazzo S, Zhuang X, Wettengel J, Lopez MA, Barnault R, Mirakaj V, Prokosch S, Heide D, Leuchtenbergeg C, Schneider M, Heßling B, Stottmeier B, Wessbecher IM, Schirmacher P, McKeating JA, Protzer U, Durantel D, Lucifora J, Dejardin E, Heikenwalder M. (2021) Hepatology. 74(4):1766-1781. doi: 10.1002/hep.31902.

Dudek M, Pfister D, Donakonda S, ....Thimme R, Böttler T, Tacke F, Dufour J-F, Haller D, Murray PJ, Heeren R, Zehn D, Böttcher JP, Heikenwälder M, Knolle PA. (2021) Auto-aggressive CXCR6+ CD8 T cells cause liver immune pathology in NASH. Nature. 592, 444–449. doi: 10.1038/s41586-021-03568-2.

Pfister D, Núñez NG, Pinyol R, ... Knolle P, ... Heikenwalder M. (2021) NASH limits anti-tumour surveillance in immunotherapy-treated HCC. Nature. 592(7854):450-456. doi.org/10.1038/s41586-021-03362-0.

Conlon TM, John-Schuster G, Heide D, Pfister D, Lehmann M, Hu Y, Ertüz Z, Lopez MA, Ansari M, Strunz M, Mayr C, Angelidis I, Ciminieri C, Costa R, Kohlhepp MS, Guillot A, Günes G, Jeridi A, Funk MC, Beroshvili G, Prokosch S, Hetzer J, Verleden SE, Alsafadi H, Lindner M, Burgstaller G, Becker L, Irmler M, Dudek M, Janzen J, Goffin E, Gosens R, Knolle P, Pirotte B, Stoeger T, Beckers J, Wagner D, Singh I, Theis FJ, de Angelis MH, O'Connor T, Tacke F, Boutros M, Dejardin E, Eickelberg O, Schiller HB, Königshoff M, Heikenwalder M, Yildirim AÖ. (2021) Publisher Correction: Inhibition of LTβR signalling activates WNT-induced regeneration in lung. Nature. 589(7842):E6. doi: 10.1038/s41586-020-03087-6. Erratum for: Nature. 2020 Dec;588(7836):151-156.

Conlon TM, John-Schuster G, Heide D, Pfister D, Lehmann M, Hu Y, Ertüz Z, Lopez MA, Ansari M, Strunz M, Mayr C, Ciminieri C, Costa R, Kohlhepp MS, Guillot A, Günes G, Jeridi A, Funk MC, Beroshvili G, Prokosch S, Hetzer J, Verleden SE, Alsafadi H, Lindner M, Burgstaller G, Becker L, Irmler M, Dudek M, Janzen J, Goffin E, Gosens R, Knolle P, Pirotte B, Stoeger T, Beckers J, Wagner D, Singh I, Theis FJ, de Angelis MH, O'Connor T, Tacke F, Boutros M, Dejardin E, Eickelberg O, Schiller HB, Königshoff M, Heikenwalder M, Yildirim AÖ. Inhibition of LTβR signalling activates WNT-induced regeneration in lung. Nature. 2020 Nov 4. doi: 10.1038/s41586-020-2882-8. (TP05, TP06)

Lampl S, Janas MK, Donakonda S, Brugger M, Lohr K, Schneider A, Manske K, Sperl LE, Kläger S, Küster B, Wettmarshausen J, Müller C, Laschinger M, Hartmann D, Hüser N, Perocchi F, Schmitt-Kopplin P, Hagn F, Zender L, Hornung V, Borner C, Pichlmair A, Kashkar H, Klingenspor M, Prinz M, Schreiner S, Conrad M, Jost PJ, Zischka H, Steiger K, Krönke M, Zehn D, Protzer U, Heikenwälder M, Knolle PA, Wohlleber D. Reduced mitochondrial resilience enables non-canonical induction of apoptosis after TNF receptor signaling in virus-infected hepatocytes. J Hepatol. 2020 Jun 26:S0168-8278(20)30398-6. doi: 10.1016/j.jhep.2020.06.026. (TP05, TP06, TP10, TP11, TP13, TP16, TP18)

Baumann T, Dunkel A, Schmid C, Schmitt S, Hiltensperger M, Lohr K, Laketa V, Donakonda S, Ahting U, Lorenz-Depiereux B, Heil JE, Schredelseker J, Simeoni L, Fecher C, Körber N, Bauer T, Hüser N, Hartmann D, Laschinger M, Eyerich K, Eyerich S, Anton M, Streeter M, Wang T, Schraven B, Spiegel D, Assaad F, Misgeld T, Zischka H, Murray PJ, Heine A, Heikenwälder M, Korn T, Dawid C, Hofmann T, Knolle PA, Höchst B. 2020. Regulatory myeloid cells paralyze T cells through cell-cell transfer of the metabolite methylglyoxal. Nat Immunol. 21(5):555-566. (TP05, TP06)

Namineni S, O'Connor T, Faure-Dupuy S, Johansen P, Riedl T, Liu K, Xu H, Singh I, Shinde P, Li F, Pandyra A, Sharma P, Ringelhan M, Muschaweckh A, Borst K, Blank P, Lampl S, Durantel D, Farhat R, Weber A, Lenggenhager D, Kündig TM, Staeheli P, Protzer U, Wohlleber D, Holzmann B, Binder M, Breuhahn K, Assmus LM, Nattermann J, Abdullah Z, Rolland M, Dejardin E, Lang PA, Lang KS, Karin M, Lucifora J, Kalinke U, Knolle PA, Heikenwalder M. 2020. A dual role for hepatocyte-intrinsic canonical NF-κB signaling in virus control. J Hepatol. 72(5):960-975. (TP05, TP06, TP11, TP13, TP14)

Michler T, Kosinska AD, Festag J, Bunse T, Su J, Ringelhan M, Imhof H, Grimm D, Steiger K, Mogler C, Heikenwalder M, Michel ML, Guzman CA, Milstein S, Sepp-Lorenzino L, Knolle P, Protzer U. 2020. Knockdown of Virus Antigen Expression Increases Therapeutic Vaccine Efficacy in High-titer HBV Carrier Mice. Gastroenterology. 158(6):1762-1775. (TP05, TP06, TP18)

Ramadori P, Klag T, Malek NP, Heikenwalder M. 2019. Platelets in chronic liver disease, from bench to bedside. Journal Hepatol Reports. DOI: 10.1016/j.jhepr.2019.10.001.

Namineni S, O‘Connor T*, Faure-Dupuy S*, Johansen P, …, Protzer U, Wohlleber D, Holzmann B, Bin-der M, … Lucifora J, Kalinke U, Knolle PA, Heikenwalder M. 2019. A dual role for hepatocyte-intrinsic canonical NF-κB signaling in virus control. J Hepatol. 01/12 in press.

Faure-Dupuy S, Delphin M, Aillot L, Dimier L, Lebossé F, Fresquet J, Parent R, Matter MS, Rivoire M, Bendriss-Vermare N, Salvetti A, Heide D, Flores L, Klumpp K, Lam A, Zoulim F, Heikenwalder M, Du-rantel D, Lucifora J. 2019. Hepatitis B virus-induced modulation of liver macrophage function promotes hepatocyte infection. J Hepatol. 23(19) 30422-2.

Malehmir M, Pfister D, Gallage S, Szydlowska M, Inverso D, Kotsiliti E, Leone V, Peiseler M, Surewaard BGJ, Rath D, Ali A, Wolf MJ, Drescher H, Healy ME, Dauch D, Kroy D, Krenkel O, Kohl-hepp M, Engleitner T, Olkus A, Sijmonsma T, Volz J, Deppermann C, Stegner D, Helbling P, Nombela-Arrieta C, Rafiei A, Hinterleitner M, Rall M, Baku F, Borst O, Wilson CL, Leslie J, O'Connor T, Weston CJ, Adams DH, Sheriff L, Teijeiro A, Prinz M, Bogeska R, Anstee N, Bongers MN, Notohamiprodjo M, Geisler T, Withers DJ, Ware J, Mann DA, Augustin HG, Vegiopoulos A, Milsom MD, Rose AJ, Lalor PF, Llovet JM, Pinyol R, Tacke F, Rad R, Matter M, Djouder N, Kubes P, Knolle PA, Unger K, Zender L, Nieswandt B, Gawaz M, Weber A, Heikenwalder M. 2019. Platelet GPIbα is a mediator and potential in-terventional target for NASH and subsequent liver cancer. Nat Med. 25(4):641-655.

Schneider A, Kurz S, Manske K, Janas M, Heikenwalder M, Misgeld T, Aichler M, Weissmann SF, Zischka H, Knolle PA, Wohlleber D. 2019. Single organelle analysis to characterize mitochondrial func-tion and crosstalk during viral infection. Sci Rep. 9(1):8492.

Baazim H, Schweiger M, Moschinger M, Xu H, Scherer T, Popa A, Gallage S, Ali A, Khamina K, Kosack L, Vilagos B, Smyth M, Lercher A, Friske J, Merkler D, Aderem A, Helbich TH, Heikenwalder M, Lang PA, Zechner R, Bergthaler A. 2019. CD8+ T cells induce cachexia during chronic viral infection. Nat Im-munol. 20(6):701-710.

Ringelhan M, Pfister D, O'Connor T, Pikarsky E, Heikenwalder M. 2018. The immunology of hepatocel-lular carcinoma. Nat Immunol. 19(3):222-232.

Xia Y, Stadler D, Lucifora J, Reisinger F, Webb D, Hösel M, Michler T, Wisskirchen K, Cheng X, Zhang K, Chou WM, Wettengel JM, Malo A, Bohne F, Hoffmann D, Eyer F, Thimme R, Falk CS, Thasler WE, Heikenwalder M, Protzer U. 2016. Interferon-γ and Tumor Necrosis Factor-α Produced by T Cells Re-duce the HBV Persistence Form, cccDNA, Without Cytolysis. Gastroenterology. 150(1):194-205.

Schölzel K, Schildberg FA, Börner C, Geiger S, Kurts C, Heikenwalder M, Knolle PA, Wohlleber D. 2014. Cross-allocation of MHC-class-I molecules among liver sinusoidal cells facilitates hepatic immune surveillance. J Heaptol. 61(3):600-8.

Huang LR, Wohlleber D, Reisinger F, Jenne CN, Cheng RL, Abdullah Z, Schildberg FA, Odenthal M, Dienes HP, van Rooijen N, Schmitt E, Garbi N, Croft M, Kurts C, Kubes P, Protzer U, Heikenwalder M, Knolle PA. 2013. Intrahepatic myeloid-cell aggregates enable local proliferation of CD8+ T cells and successful immunotherapy against chronic viral liver infection. Nat Immunol. 14(6):574-83

Project in detail +

Hepatitis B virus (HBV) chronically infects around 250 million people worldwide (WHO data, 2016), drives chronic hepatitis and increases the risk for developing liver cirrhosis, liver dysfunction and liver cancer1. HBV has evolved several mechanisms to shape the hepatic inflammatory signature and to polarize innate and adaptive immune cells to achieve persistent infection. Notably, this HBV-dependent reprogramming of the hepatic micro-environment also affects the metabolism of liver cells - including hepatocytes and other parenchymal and non-parenchymal cells2,3. In the liver, platelets interplay with different resident and in-coming cellular populations to exert immune-regulatory functions in chronic liver diseases, such as chronic viral infections, fatty liver disease and liver cancer (Ramadori et al., 2019). This prompted us to consider selective modulation of platelet function as a promising approach to alter virus replica-tion/clearance as well as the metabolic and inflammatory hepatic environment. Recently, we identified a key role of platelets in shaping the microenvironment in murine and human liver tissue by applying pre-clinical mouse models of chronic liver damage (fatty liver disease) and by performing a clinical trial in-volving long term anti-platelet therapy (Mahlemir et al., 2019). Our data demonstrated that platelets control the influx of CD8+, CD4+ T-cells and other innate immune cells into the liver. Moreover, platelets - being the first cells to invade the liver upon damage - directly bind to hepatocyte- and Kupffer cell-derived hyaluronic acid through CD44 and affect immune cell activation status and hepatocyte metabolism subsequently (Mahlemir et al., 2019). In addition, platelets attract/retain specific adaptive and innate immune cells in the liver by secreting inflammatory mediators through α-granules. We selectively dissected the inflammatory role of platelets from their role in hemostasis, a process to prevent and stop bleeding. Our data indicate that platelets - besides their function in hemostasis - directly cross-talk to hepatocytes, liver sinusoidal epithelial cells (LSECs) and adaptive/innate immune cells, altering the metabolic, inflammatory and proliferative status, thus contributing to polarization and activation status of immune cells. In addition to these key results, we have demonstrated in the past funding period, that HBV modulates liver macro-phage function to favour the establishment and maintenance of infection (Faure-Dupuy et al., 2019). Moreover, in the past funding period we investigated whether T-cell derived Lymphotoxin (LT; LTαβ) plays a major role in shaping the liver microenvironment. We generated tools to abrogate or overexpress LT expres-sion specifically on T-cells in vivo (e.g. T-cell specific transgenic overexpression: lck-LTαβ tg mice; CD4-Cre LTα loxP/loxP). In the context of hepatic viral infection and antigen presentation (in vivo and in vitro), we have tested how intrahepatic T-cell migration, T-cell survival, T-cell mediated antigen recognition and viral clearance are affected by LTαβ overexpression or deletion. Our data indicate that LTαβ expression on T cells increases T cell homing into the liver and T-cell mediated antigen recognition. Since T-cell derived LTαβbut also virus infection itself, can induce NF-κB signalling in hepatocytes, we investigated how NF-κB signalling in hepatocytes affects the liver microenvironment and vial clearance (Namineni et al., 2019). In mice lacking canonical NF-κB signalling (IKKβΔHep) in hepatocytes and infected with LCMV, virus titers in the liver were elevated, concomitant with delayed and reduced expression of IFN-stimulated genes (ISGs). Importantly, IKKβΔHep livers displayed diminished intrahepatic influx of innate immune cells as well as reduced and delayed intrahepatic LCMV-specific CD8+ T-cell responses. Consequently, viral clearance and ISG-expression were also reduced in LCMV-infected, primary IKKβΔHep hepatocytes, demonstrating a hepatocyte-intrinsic effect. Similar to IKKβΔHep mice, enhanced hepatocytic LCMV accumulation was ob-served in livers lacking hepatocyte-specific IFN-α/-β signalling-(IFNARΔHep). In contrast, hepatocytes in mice lacking IFN-α/-β signalling in myeloid cells (IFNARΔMyel) controlled LCMV-infection, however, with delayed kinetics and efficacy, leaving Kupffer cells fully LCMV-infected. In summary, we show that hepatocyte-intrinsic NF-κB is a crucial amplifier of IFN-α/-β signalling pivotal for early, strong ISG responses, con-trolling hepatic influx of immune cells (e.g. T cells), and hepatic viral clearance (Namineni et al., 2019). Given the striking phenotype observed with platelets, we will focus on platelets and study platelet activa-tion, platelet-derived molecules, and platelet-triggered cross-talk with hepatocytes and immune cells (T cells and Kupffer cells) in the context of HBV-induced hepatitis and HBV replication in the next funding period. In close collaborations with partners of TRR179, we will focus on the identification of pathways that are direct-ly/indirectly activated or suppressed by platelets. We will study the role of these pathways for (1) HBV replication in hepatocytes, (2) hepatocyte metabolism, and (3) immune cells regarding their polariza-tion, immune status and anti-HBV responses (e.g. HBV clearance). Moreover, we will examine the role of platelets in developing and established chronic HBV infection. Using different knock-out mouse strains or specific, therapeutic anti-platelet regimens (e.g. Ticagrelor; anti-GPIbα antibodies) affecting plate-let biology (e.g. specifically blocking the inflammatory function of platelets), we will investigate chronic and acute HBV infection (e.g. AAV-based with Grimm/Protzer), as well as other surrogate models of acute and chronic virus infection (e.g. LCMV with Wohlleber, Bartenschlager and Knolle). A prime interest will be the role of platelets in contributing to infection-related changes of the liver tissue microenvironment/metabolism and in shaping anti-HBV responses (in collaboration with Z02, Wohlleber, Knolle, Protzer, and Thimme). In addition, we will collaborate with Bartenschlager, Binder/Pichlmair, Lohmann, Thimme and further apply new-ly established cell culture technologies and analysis tools that allow the correlation between HBV replication efficacy, transcriptomics and metabolic changes on single hepatocyte level in situ, and altered inflammatory signatures in the immediate environment of hepatocytes (with Knolle, Thimme, Bengsch). With the long-term goal of identifying platelet-derived targets that can be selectively investigated (e.g. knock-out/CRISP/Cas; pharmacologically) to modulate HBV infection and clearance, our results will contribute to a better under-standing of innate immune cell-derived mechanisms in the orchestration of inflammatory/metabolic path-ways in the context of hepatic viral infections.

Milestones of the first funding period:
- Identification and characterization of ex vivo isolated IHLs and antiviral T-cells expressing LT, LT and LIGHT and derived from livers of patients with chronic hepatitis B and C.
- Functional analysis of the role of T-cell derived LT, LT and LIGHT in antiviral activity in vitro.
- Mechanisms of T-cell derived LT and LIGHT expression in shaping T-cell migration, anigen/antigen receptor interaction and the hepatic environment in vivo