Sub theme 4.2
Liverspecific immunopathology; mechanism and therapeutic modulation

Goals of research: general outline
Scientific achievements
Future plans: special goals and approach
Running projects
Associated staff

Workgroup leaders   Department
Dr.  P.A.  Boonstra   Gastro-enterology and Hepatology
Dr.  J.  Kwekkeboom   Gastro-enterology and Hepatology
dr.  L.J.W.  van der  Laan   Surgery
Dr.  A.M.  Woltman   Gastro-enterology and Hepatology

Goals of research: general outline

Liver Immunology research within the Erasmus MC has a strongly translational character with basic scientists and clinicians working closely together. The basic research is performed by a rapidly growing group of scientist from the Department of Gastroenterology and Hepatology and the Department of Surgery. In order to form a coherent group, all investigators are situated in the Laboratory of Gastroenterology and Hepatology. Basic and clinical research is tightly integrated with basic scientists and clinical researchers from both departments having research meetings together. Liver immunology is focussed on three research lines, all using similar techniques and collaborating closely together:

Viral Hepatitis

The aim of this research line is to investigate which virus- and host related conditions are required for immunological control of hepatitis B (HBV) and hepatitis C (HCV) viruses, and, in parallel, for sustained response to antiviral therapy. With the foundation of our laboratory in 2000, our major effort was to conduct basic experimental studies on HBV- and HCV-immune reactivity. These fundamental studies are focussed on the innate immune response and the T-cell response both in the peripheral and intrahepatic compartment of patients. Our main objective is to delineate the mechanisms of host-pathogen interaction and the lack of immune control over the pathogen. Important deficiencies in dendritic cells, natural killer cells and the T cell compartment were observed in patients with chronic viral hepatitis and downstream mechanisms are currently unravelled. Besides these fundamental studies, observations are validated in experimental animal models and viral hepatitis patients to improve therapeutic strategies and disease outcome.

Liver transplantation

Liver transplantation is on the short-term a successful treatment for end-stage liver diseases, but long-term survival and quality of life are severely impaired by complications caused by continuous immunosuppressive treatment and HCV re-infection. One part of our studies concentrates on defining parameters that identify on the one hand patients which are at risk of rejection and on the other hand patients that are tolerant to their liver graft. Prediction of the risk of immune reactivity to the graft will enable individualization, and a subgroup of patients probably reduction, of immunosuppressive therapy. For this purpose we investigate both genetic and immune parameters. Immune parameters are studied preferentially in the graft itself. For this purpose we have introduced a liver Fine-Needle-Aspiration (FNAB) technique. Secondly, we aim to promote immunological conditions that are required for the development of tolerance against liver grafts. For this, we focus on modulation of donor- and recipient DC-migration and function and on the induction of donor-specific Treg. Thirdly, we are developing novel strategies to prevent HCV-recurrence. For this part we focus on anti-viral effects of combinations of immunosuppressive drugs and on the development of a ex vivo gene therapy approach based on RNA interference in which the liver transplant is transfected with anti-HCV iRNA before transplantation.

Portal Hypertension

The pathogenesis of hepatic and portal vein thrombosis is largely unclear. We investigate on an international level the etiology and prognostic factors of these rare but life-threatening liver disorders.

Scientific achievements

2.1 Innate immunity in chronic hepatitis B infection

Dendritic cells and NK cells derived from chronic hepatitis B patients are functionally impaired as compared to healthy volunteers. Antiviral therapy aimed to reduce viral load improved both dendritic cell and NK cell function in these patients. We recently demonstrated that the virus itself directly interferes with dendritic cell function in vitro. Of importance, in vivo host-pathogen interaction seemed to be most prevalent in the liver rather than in circulation. The molecular mechanisms underlying dendritic cell and NK cell functional impairment as well as the function of these cells during antiviral therapy are currently under investigation. 

2.2 Regulatory T cells in chronic hepatitis C infection

We showed that chronic HBV and HCV patients exhibit increased percentages of Treg in the liver. In addition, multiple mechanism of immune suppression to explain the weak immune responses to HCV were evaluated, including the presence and consequence of IL-10 and TGF-beta. Further studies will include the mechanism of suppression as well as changes in frequency and functionality of Tregs under antiviral therapy.

2.3 Immune responses in antiviral therapy of chronic hepatitis B and C infection

We used the FNAB-technique to analyse phenotype, functional status and specificity of subsets of intrahepatic immune cells in HBV- and HCV-infection. This allowed us to make a detailed evaluation of the immune status of patients with chronic viral hepatitis before and during anti-viral therapy.

2.4 Parameters identifying tolerant liver transplant recipients

We identified several genetic polymorphisms in cytokine- and co-stimulatory genes that are associated with the risk of rejection after liver transplantation. In addition we found, using the FNAB-technique, that intragraft immune parameters like enhanced granzyme- and reduced foxp3-expression are strongly associated with rejection or immunological quiescence. However, durable tolerance to liver grafts is probably dependent on active inhibition of the immune response of the recipient to donor antigen. We found that after liver transplantation, levels of reguatory T-cells in peripheral blood dropped significantly, possible as a consequence of immunosuppression.

2.5 Modulation of immunological conditions to induce tolerance to liver grafts

We found that considerable numbers of DC detach from the graft during the transplantation procedure, and demonstrated that these donor-derived DC migrate via the blood circulation into the recipient. We hypothesized that these professional antigen-presenting cells initiate the rejection process by priming allogeneic recipients T-cells. However, investigating in vivo matured liver DC (which we isolated for hepatic lymph nodes), we found that liver DC are relatively weak allo-stimulators and produce high amounts of the immunoregulatory cytokine IL-10. Moreover, we demonstrated that treatment of liver transplant recipients with Intravenous Immunoglobulins (IVIg) prevented acute rejection, and found that the IVIg reduce the immunogenicity of DC. Presently, it is investigated whether IVIg can induce tolerance to liver grafts, and thereby may open a new treatment option to reduce treatment with immunosuppressive drugs

2.6 New therapies for Hepatitis C virus (HCV) recurrence after liver transplantation

The only current treatment for end-stage liver disease due to chronic HCV infection is liver transplantation. However, re-infection of the graft by persisting HCV causes progressive recurrence of disease and has a profound impact on survival of the patient and graft. We have developed a retrovirus-based gene therapy that blocks HCV-infection of cultured hepatocytes by RNA interference. The aim is to effectively deliver this therapy during isolated perfusion of the liver graft before transplantation. Alternatively, we found that specific combinations of immunosuppressive drugs have unexpected inhibitory effects on HCV-replication.

2.7 Portal Hypertension

With support of the European Community (5th framework) we set up a European network to construct an on-line common database and to federate sample banks. We showed the importance of several genetic defects in the etiology of vascular liver disease and constructed prognostic models for intervention. Currently we investigate the role of fibrin homeostasis in the pathogenesis and site-specificity of hepatic vein thrombosis.

Future plans: special goals and approach

Innate immune response to HBV and HCV

Dendritic cells (DC) and their interaction with natural killer (NK) cells are crucial in the initiation and regulation of (anti-viral) T cell immunity (Boonstra, 2001, 2003; Woltman, 2003, 2006). We showed that the activity of DC as well as NK cells is suppressed in patients with chronic HBV infections (van der Molen, 2004; 2x Op den Brouw 2009) and that the virus directly interferes with the function of DC. Currently, we are studying the mechanisms behind this observation in HBV and HCV infections, as well as the interaction of DC and NK cells during infection and anti-viral treatment.

Regulation of the immune response to HBV and HCV

Our group was one of the first to show that CD4+CD25+ regulatory T cells are increased in number in the periphery of chronic HBV patients as compared to healthy controls. More importantly, we showed that these regulatory T cells suppressed HBV-specific T cell responses (Stoop, 2005). Studies are ongoing to determine the underlying mechanisms of action of regulatory T cells during infection with both HBV and HCV.

The effect of antiviral therapy on the immunity to HBV and HCV

The close interaction of the laboratory and the clinical branch of the Rotterdam Liver Unit enables us to perform studies examining the effect of antiviral therapy on the immune status of patients. This has resulted in a number of publications describing at a mechanistic level how anti-viral therapy leads to viral eradication (van der Molen, 2006; Stoop, 2007). These studies are crucial for the development of novel treatment strategies. 

The intrahepatic immune response to HBV and HCV

The local immune response to HBV and HCV in the liver is studied using a unique method to collect lymphocytes directly from the liver using Fine Needle Aspiration Biopsies. Using this technique, intrahepatic lymphocytes can be examined by flow-cytometry. Phenotypic studies using tetramer technology, as well as functional assays have already provided valuable information on the local immune response during viral hepatitis, and for evaluation of the effect of anti-viral therapy (Sprengers, 2005; Vrolijk, 2004).

Tolerance induction in liver transplantation

In the field of liver transplantation, we will try to establish which conditions are favorable for the induction of donor antigen-specific Treg. In particular we will investigate whether transplant tolerance can be induced by ex-vivo manipulation of DC in the graft. In addition, the suitability of IVIg to induce donor-specific hyporesponsiveness will be explored in experimental animal studies and clinical research. To improve detection of a tolerant state, we will develop a sensitive technique to quantify the recipient T-cell response against indirectly presented donor MHC. For this purpose we will use a completely new approach, namely donor antigen presentation by recipient DC genetically transduced with donor MHC-genes.

Gene therapy to prevent HCV recurrence

Finally, we aim to develop a gene therapy approach to prevent HCV infection of the liver graft using interfering-RNA (iRNA) to inhibit HCV replication. Conditions to achieve effective lentiviral transduction during the ex vivo perfusion of the liver graft will be determined.

Most recent publications
  1. Woltman AM, Boonstra A, Janssen HLA. Dendritic Cells in Chronic Viral Hepatitis B and C: Victims or Guardian Angels? Gut in press (IF 9.7).
  2. Woltman AM, van der Kooij SW, Coffer PJ, Offringa R, Daha MR, van Kooten C (2003) Rapamycin specifically interferes with GM-CSF signaling in human dendritic cells, leading to apoptosis via increased p27KIP1 expression. Blood 101, 1439-1445. (IF 10.4)
  3. Boonstra A, Asselin-Paturel C, Gilliet M, Crain C, Trinchieri G, Liu Y-J, O'Garra A. Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential Toll-like receptor ligation. J Exp Med. 2003. 197: 101-109. (IF 15.2)
  4. Boonstra A, van der Laan LJW, Vanwolleghem T, Janssen HLA. Experimental models for hepatitis C viral infection. Accepted for publication. Hepatology. 2009. June 24. ahead of print. (IF 11.3)
  5. Janssen HLA, Zonneveld van M, Schalm SW. Hepatitis B virus infection natural history and clinical consequences. New Engl J Med 2004;350:2719-2720 (IF 52.5).
  6. Janssen HLA, Zonneveld van M, Senturk H, Zeuzem S, Akarca U, Cakaloglu Y, Simon K, So Man Kit T, Gerken G, Man de RA, Niesters HG, Zondervan P, Schalm SW. Pegylated interferon a-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial. Lancet 2005;365:123-129 (IF 28.5).
  7. Veldt BJ, Heathcote EJ, Wedemeyer H, Reichen J, Hofmann WP, Zeuzem S, Manns M, Hansen BE, Schalm SW, Janssen HLA. Sustained virological response leads to an improved clinical outcome in patients with hepatitis C and advanced fibrosis. Annals of Internal Medicine 2007;147:677-84 (IF 17.4).
  8. Buster EH, Flink HJ, Cadaloglu Y, Simon K, Trojan J, Tabak F, So TM, Feinman SV, Mach T, Akarca US, Schutten M, Tielemans W, van Vuuren AJ, Hansen BE, Janssen HLA. Sustained HBeAg and HBsAg Loss After Long-term Follow-up of HBeAg-Positive Patients Treated With Peginterferon alpha-2b. Gastroenterology 2008;135:459-67 (IF 12.6).
  9. Sprengers D, Sille FC, Besra G, Janssen HLA, Schott E, Boes M. Critical role for CD1d-restricted V-alpha-14iNKT cells in stimulating intrahepatic CD8 T cell responses to liver-expressed antigen. Gastroenterology 2008;134:2132-43(IF 12.6).
  10. Sarwa Darwish Murad S, Plessier A, Hernandez-Guerra M, Fabris F, Eapen CE, Bahr MJ, Trebicka J, Morard I, Lasser L, Heller J, Hadengue A, Langlet P, Miranda H, Primignani M, Elias E, Leebeek FW, Rosendaal FR, Garcia-Pagan JC, Valla DC, Janssen HLA for the European Network for Vascular Disorders of the Liver (EN-Vie). Risk Factors, Management and Outcome of Budd-Chiari Syndrome. Annals of Internal Medicine, 2009;151:167-75 (IF 17.4)