Luca Guidotti
Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy, Department of Immunology & Microbial Sciences, The Scripps Research Institute, La Jolla, CA 92037, USA

Q. WHAT IS THE EVIDENCE THAT IN HEPATITIS B PLATELETS PLAY A ROLE IN THE PERPETUATION OF LIVER DAMAGE?

The first evidence involving platelets in the pathogenesis of hepatitis B emerged from our laboratory over 10 years ago 1. Using mouse models of acute viral hepatitis (e.g. HBV replication-competent transgenic mice transferred with HBV-specific effector CD8+ T cells or wild-type mice infected with hepatotropic adenoviruses), we showed that platelets are detectable at sites of organ damage and their depletion reduces the number of intrahepatic HBV-specific CD8+ T cells – and the number of antigen-nonspecific inflammatory cells that these CD8+ T cells secondarily recruit into the liver – ameliorating disease severity 1. This outcome was reversed by reconstituting thrombocytopenic animals with normal but not dysfunctional platelets 1. In an ongoing effort to explain mechanistically how platelets support the intrahepatic accumulation of HBV-specific CD8+ T cells, we found that this function was unaffected by anticoagulant treatments preventing intrahepatic fibrin deposition without reducing platelet counts 1. Platelet-mediated induction of intrahepatic CD8+ T homing was instead hindered by two specific inhibitors of platelet activation pathways, aspirin that blocks thromboxane (TX) A2 production and clopidogrel that blocks the P2Y12 ADP receptor. Indeed, treating mice with aspirin, clopidogrel, or a combination of the two attenuated the acute accumulation of effector CD8+ T cells in the liver and the associated organ damage 2.

As oral administration of aspirin and clopidogrel is often used for long-term treatment of patients at risk of thrombosis, we then used a mouse model of immune-mediated chronic hepatitis B (CHB) to evaluate how the sustained inhibition of platelet activation may impact the severity of persistent liver injury and its complications. Treating these animals with clinically relevant doses of aspirin and clopidogrel – beginning after the induction of hepatitis and continuing indefinitely – limited the intrahepatic accumulation of HBV-specific CD8+ T cells and the consequent hepatocellular injury, reduced the compensatory hepatocellular proliferation, diminished the severity of liver fibrosis/cirrhosis, prevented the development of HCC and, more importantly, improved the overall survival without causing bleeding side effects 3. These results indicate that platelets influence the chronic stages of a pathogenic mechanism triggered by HBV-specific CD8+ T cells and they also imply that immune-mediated inflammatory reactions are the predominant cause of HCC transformation during persistent HBV infection 4.

Q. WHAT ARE THE LABORATORY SYSTEMS THAT YOU DEVELOPED TO STUDY THE INTERACTION BETWEEN HBV AND PLATELETS?

A definitive comprehension of the platelet-dependent mechanisms implicated in HBV pathogenesis required the development of advanced imaging systems allowing the dynamic visualization of intrahepatic platelet-CD8+ T cell interactions in vivo. This concept prompted us to put into operation both epifluorescence and multiphoton intravital microscopy in dedicated mouse models of HBV infection. Thanks to these efforts, we could demonstrate that platelets and CD8+ T cells do physically interact within the liver microcirculation 5. Indeed, we showed that the initial sinusoidal arrest of circulating effector CD8+ T cells depends on their capacity to dock onto platelets that have previously adhered to sinusoidal hyaluronan via CD44 5. Conversely, we showed that selectins, β2- and α4-integrins, Platelet Endothelial Cell Adhesion Molecule (PECAM)-1, Vascular Adhesion Protein (VAP)-1, Gαi-coupled chemokine receptors, endothelial activation and antigen recognition (molecules and processes previously thought to be variably relevant for leukocyte trafficking in other organs) are not required for the hepatic homing of effector CD8+ T cells 5.

Q. WILL SUCH AN INTERACTION OPEN NEW THERAPEUTIC OPTIONS FOR HEPATITIS B?

The notion that antiplatelet drugs such as aspirin and clopidogrel safely and effectively prevent HCC and improve overall survival in a highly relevant mouse model of immune-mediated chronic hepatitis B suggests that drugs targeting platelet function may represent new therapeutic options in CHB patients. Notably, recent retrospective analyses performed on aspirin- and/or clopidogrel-treated CHB patients under antiviral therapy or after HCC-related liver resections showed reduced cancer risk and better recurrence-free survival or overall survival 6,7. An obvious concern about the use of aspirin- and/or clopidogrel in CHB patients is the risk of bleeding and, indeed, the two analyses above-mentioned suggested that the overall bleeding risk is higher in patients under antiplatelet therapy 6,7. It should be noted, however, that the excessive bleeding that can occur in advanced liver cirrhosis may be a lesser risk than thrombosis in some patients with chronic liver disease 8. Thus, further investigation aimed at identifying predictors of bleeding in CHB patients will be highly relevant for the design of including/excluding criteria fostering the future launch of dedicated clinical trials with existing antiplatelet drugs.

Q. WHAT ARE THE NEXT STEPS OF YOUR RESEARCH PROGRAM IN VIRAL HEPATITIS?”

We are continuously developing new mouse models and new technological advances in the field of static and dynamic imaging to tackle yet unresolved issues in HBV pathogenesis. Such issues include the definition of the mechanisms whereby innate and adaptive immune cells traffic and function within the normal, cirrhotic or cancerous liver. Pertinent to platelets, we are actively working on the identification of yet unknown platelet surface molecules that promote CD8+ T cell interaction without affecting hemostasis. Identification of such molecules will help creating novel and potentially safer platelet inhibitors for the treatment of CHB and its life-threatening complications.

References

  1. Iannacone M, Sitia G, Isogawa M, Marchese P, Castro MG, Lowenstein PR, Chisari FV, et al. Platelets mediate cytotoxic T lymphocyte-induced liver damage. Nat Med 2005;11:1167-1169.
  2. Iannacone M, Sitia G, Narvaiza I, Ruggeri ZM, Guidotti LG. Antiplatelet drug therapy moderates immune-mediated liver disease and inhibits viral clearance in mice infected with a replication-deficient adenovirus. Clin Vaccine Immunol 2007;14:1532-1535.
  3. Sitia G, Aiolfi R, Di Lucia P, Mainetti M, Fiocchi A, Mingozzi F, Esposito A, et al. Antiplatelet therapy prevents hepatocellular carcinoma and improves survival in a mouse model of chronic hepatitis B. Proc Natl Acad Sci U S A 2012;109:E2165-2172.
  4. Sitia G, Iannacone M, Guidotti LG. Anti-platelet therapy in the prevention of hepatitis B virus-associated hepatocellular carcinoma. J Hepatol 2013;59:1135-1138.
  5. Guidotti LG, Inverso D, Sironi L, Di Lucia P, Fioravanti J, Ganzer L, Fiocchi A, et al. Immunosurveillance of the liver by intravascular effector CD8(+) T cells. Cell 2015;161:486-500.
  6. Lee M, Chung GE, Lee JH, Oh S, Nam JY, Chang Y, Cho H, et al. Antiplatelet therapy and the risk of hepatocellular carcinoma in chronic hepatitis B patients on antiviral treatment. Hepatology 2017.
  7. Lee PC, Yeh CM, Hu YW, Chen CC, Liu CJ, Su CW, Huo TI, et al. Antiplatelet Therapy is Associated with a Better Prognosis for Patients with Hepatitis B Virus-Related Hepatocellular Carcinoma after Liver Resection. Ann Surg Oncol 2016;23:874-883.
  8. Tripodi A, Mannucci PM. The coagulopathy of chronic liver disease. The New England journal of medicine 2011;365:147-156.