Kathie Béland

Chronic hepatitis E infection in children with liver transplantation

Objective Chronic hepatitis E virus (HEV) infection has been described in immunosuppressed adult patients. A study was undertaken to establish the presence of HEV infection in children after orthotopic liver transplantation (OLT). Methods Children undergoing liver transplantation between 1992 and 2010 with available serum were classified into two groups: group 1 (control group, n=66) with normal serum aminotransferases and group 2 (n=14) with persistently increased serum aminotransferases and histological features of chronic hepatitis. Patients were tested for HEV RNA by reverse transcription-polymerase chain reaction (RT-PCR). HEV amplicons were sequenced and compared with published sequences. Antibody titres (IgG and IgM) to 12 HEV immunodominant regions were measured by enzyme-linked immunosorbent assays. Results In group 1 (control group), 15% of children were anti-HEV IgG-positive during follow-up. No anti-HEV IgM antibodies were detected in any of these children. After OLT, 86% of patients in group 2 had anti-HEV IgG compared with 36% pre-OLT. Thus, two-thirds of children acquired anti-HEV IgG after OLT. Seven anti-HEV IgG-positive patients (58%) were also anti-HEV IgM-positive more than once during follow-up after OLT. Eight years post-OLT, one girl presented with anti-HEV IgG and IgM that remained positive afterwards. In this patient, HEV RNA was found in five different annual samples from 10 years post-OLT, concomitantly with increased serum aminotransferases and cirrhosis development during that period. Phylogenetic analysis revealed two different HEV strains (detected 3 years apart) that were highly similar to swine genotype 3, suggesting a possible case of zoonotic re-infection. Conclusion The diagnosis of HEV infection is technically challenging and should be made simultaneously with RT-PCR methods, viral load quantification and serological markers. In immunosuppressed children who develop chronic hepatitis, the prevalence of HEV is high and could explain the chronic liver inflammation potentially leading to cirrhosis. Re-infection by different HEV strains from zoonotic transmission can result in progressive liver disease in immunocompromised children.

Adoptive transfer of ex vivo expanded regulatory T cells in an autoimmune hepatitis murine model restores peripheral tolerance.

Autoimmune hepatitis (AIH) is characterized by a loss of immunological tolerance to hepatocytes. Patients respond well to immunosuppression but progression to endstage liver disease occurs in 10%‐20% of cases, leading to liver transplantation. Using a murine model of type 2 AIH, we identified susceptibility factors for autoimmune hepatitis and attempted to restore immunological tolerance to liver autoantigens. An increased ectopic expression of a liver autoantigen (FTCD) in the thymus leading to reduced numbers of circulating autoreactive T cells was sufficient to prevent development of AIH in mice. However, in the presence of a reduced central tolerance to FTCD, a strong regulatory T‐cell response was able to inhibit proliferation of liver‐specific autoreactive T cells and prevent AIH. Development of a severe AIH stemmed from reduced numbers of functional regulatory T cell (Tregs) leading to an increased proliferation of FTCD‐specific autoreactive T and B cells. Adoptive transfer of ex vivo expanded CXCR3+ Tregs in mice with AIH efficiently targeted the inflamed liver, restored peripheral tolerance to FTCD, and induced remission of AIH. Conclusion: Peripheral tolerance to liver autoantigens in AIH is paramount. Autologous infusion of ex vivo expanded CXCR3+ Tregs in AIH patients could be an effective therapeutic approach to restore peripheral tolerance and induce remission of AIH. (HEPATOLOGY 2013)

LKM1 autoantibodies in chronic hepatitis C infection: A case of molecular mimicry?

Anti‐liver‐kidney microsome type 1 (LKM1) autoantibodies directed against the cytochrome P450 2D6 (CYP2D6) are considered specific markers of type 2 autoimmune hepatitis, but are also found in 5% of sera from patients chronically infected by hepatitis C virus (HCV). Molecular mimicry between HCV proteins and CYP2D6 has been proposed to explain the emergence of these autoantibodies. Anti‐LKM1 autoantibodies from hepatitis C–infected patients were affinity‐purified against immobilized CYP2D6 protein and used to screen a phage display library. CYP2D6 conformational epitopes were identified using phage display analysis and the identification of statistically significant pairs (SSPs). Cross‐reactivity between CYP2D6 and HCV protein candidates was tested by immunoprecipitation. Nineteen different clones were isolated, and their sequencing resulted in the mapping of a conformational epitope to the region of amino acids 254‐288 of CYP2D6. Candidate HCV proteins for molecular mimicry included: core, E2, NS3 and NS5a. Affinity‐purified autoantibodies from HCV+/LKM1+ patients immunoprecipitated either NS3, NS5a, or both, and these reactivities were specifically inhibited by immobilized CYP2D6. In conclusion, HCV+/LKM1+ sera recognize a specific conformational epitope on CYP2D6 between amino acids 254 to 288, the region that contains the major linear epitope in type 2 autoimmune hepatitis patients. Cross‐reactivity due to molecular mimicry at the B‐cell level was shown between the CYP2D6 and the HCV NS3 and NS5a proteins and could explain the presence of anti‐LKM1 in patients chronically infected with HCV. Further investigation of the role played by this molecular mimicry in HCV‐infected patients may lead to more specific strategies for diagnosis and treatment. (HEPATOLOGY 2005.)

Anti-soluble liver antigen (SLA) antibodies in chronic HCV infection.

Hepatitis C infection is associated with autoimmune disorders, such as the production of autoantibodies. Anti-LKM1 and anti-LC1, immunomarkers of type 2 autoimmune hepatitis, have been previously associated with a HCV infection. Anti-Soluble-Liver-Antigen autoantibodies (SLA) are specifically associated with type 1 and type 2 autoimmune hepatitis and more closely related to patients who relapse after steroid therapy. The recent molecular cloning of the soluble liver antigen provides the opportunity to develop more specific tests for the detection of antibodies against it. The aim of this work is to characterize anti-soluble-liver autoantibodies in sera from patients chronically infected by HCV. A recombinant cDNA from activated Jurkat cells coding for the full length tRNP(Ser)Sec/SLA antigen was obtained. ELISA, Western Blot and immunoprecipitation tests were developed and used to search for linear and conformational epitopes recognized by anti-SLA antibodies in sera from patients chronically infected by HCV. Anti-soluble liver antigen antibodies were found in sera from 10.4% of HCV-infected patients. The prevalence was significantly increased to 27% when anti-LKM1 was also present. Most anti-SLA reactivity was directed against conformational epitopes on the antigen. The means titers by ELISA were lower than those obtained in type 2 AIH. The result of autoantibody isotyping showed a subclass restriction to IgG1 and also IgG4. This study shows the presence of anti-SLA antibodies in approximately 10% of HCV infected patients. The prevalence of SLA autoantibodies in HCV infected patients increases when LKM1 autoantibodies are also present. The relationship between the prevalence of this characteristic autoimmune hepatitis autoantibody and the implication of an autoimmune phenomenon in the liver injury of patients chronically infected by HCV needs further investigation.

Forkhead box p3+ regulatory T cell underlies male resistance to experimental type 2 autoimmune hepatitis†

Autoimmune hepatitis (AIH), like many autoimmune diseases, is most prevalent in young women. The immunological basis of this age and sex susceptibility bias was investigated in a murine model of AIH. Xenoimmunization of 7‐week‐old female C57BL/6 mice resulted in more severe AIH with higher levels of liver inflammation, serum alanine aminotransferase, specific T‐cell cytotoxicity, and autoantibody than younger and older females. Vaccinated males developed minimal liver inflammation and higher percentages of CD4+CD25+FoxP3+ regulatory T cell in peripheral blood mononuclear cells, spleen, and liver than females. Regulatory T cells (Tregs) were virtually absent in liver‐lymphocytes infiltrates of females. Castration of C57BL/6 mice, with or without 17β‐estradiol supplementation, did not modify susceptibility in males, nor Treg numbers, suggesting minimal contribution of testosterone and estradiol to autoimmune hepatitis (AIH) susceptibility. Xenoimmunized Aire(+/0) mouse displayed similar AIH susceptibility, sex bias, and Tregs numbers as C57BL/6 mice, suggesting that susceptibility in females is not the result of less stringent thymic central tolerance. Autoreactive B cell response against formiminotransferase‐cyclodeaminase correlated with disease activity, possibly linking B‐cell autoreactivity and AIH pathogenesis. Conclusion: Peripheral tolerance and development of regulatory T cells after self‐mimicking antigen exposure, and not sexual hormone nor central tolerance, are the main factors for susceptibility to AIH in females. HEPATOLOGY 2010

Torque Teno Virus in Children Who Underwent Orthotopic Liver Transplantation: New Insights About a Common Pathogen

BACKGROUND  Torque Teno virus (TTV) is a ubiquitous infectious agent. Transplant recipients are at risk of hepatitis E virus (HEV) infection and could be vulnerable to TTV-associated adverse effects. The aim of this study was to evaluate the influence of immunosuppression and HEV infection on TTV replication and liver injury in pediatric patients after orthotopic liver transplantation (OLT). METHODS  Pediatric recipients of liver transplants were classified into the following 2 groups: (1) those with normal serum aminotransferases levels and (2) those with persistently increased serum aminotransferases levels and histological features of chronic hepatitis of unknown etiology. The TTV load was assessed in 342 serum samples by use of TaqMan real-time polymerase chain reaction, along with TTV genogroups and coinfection with HEV. RESULTS  TTV DNA was detected in 96% of tested serum samples. Viral load was significantly lower in patients with features of chronic hepatitis, of whom 78% had liver fibrosis scores of ≥2. Viral load decreased during posttransplantation follow-up. Viral load and genogroups were influenced by immunosuppression. Lower viral load was observed in patients coinfected with HEV. CONCLUSIONS  TTV infection is widespread, and its replication is closely related to immune status and viral coinfection. High TTV viremia is not associated with hepatitis after OLT, but, conversely, liver inflammatory activity impairs TTV replication.

A novel “humanized mouse” model for autoimmune hepatitis and the association of gut microbiota with liver inflammation

Autoimmune hepatitis (AIH) in humans is a severe inflammatory liver disease characterized by interface hepatitis, the presence of circulating autoantibodies, and hyper‐gammaglobulinemia. There are two types of AIH, type 1 (AIH‐1) and type 2 (AIH‐2), characterized by distinct autoimmune serology. Patients with AIH‐1 are positive for anti–smooth muscle and/or antinuclear autoantibodies, whereas patients with AIH‐2 have anti–liver kidney microsomal type 1 and/or anti–liver cytosol type 1 autoantibodies. Cytochrome P4502D6 is the antigenic target of anti–liver kidney microsomal type 1, and formiminotransferase cyclodeaminase is the antigenic target of anti–liver cytosol type 1. It is known that AIH, both types 1 and 2, is strongly linked to the human leukocyte antigen (HLA) alleles ‐DR3, ‐DR4, and ‐DR7. However, direct evidence of the association of HLA with AIH is lacking. We developed a novel mouse model of AIH using the HLA‐DR3 transgenic mouse on the nonobese‐diabetic background by immunization of HLA‐DR3– and HLA‐DR3+ nonobese‐diabetic mice with a DNA plasmid, coding for human cytochrome P4502D6/formiminotransferase cyclodeaminase fusion protein. Immunization with cytochrome P4502D6/formiminotransferase cyclodeaminase leads to a sustained elevation of alanine aminotransferase, development of antinuclear autoantibodies and anti–liver kidney microsomal type 1/anti–liver cytosol type 1 autoantibodies, chronic immune cell infiltration, and parenchymal fibrosis on liver histology in HLA‐DR3+ mice. Immunized mice also showed an enhanced T helper 1 immune response and paucity of the frequency of regulatory T cells in the liver. Moreover, HLA‐DR3+ mice with exacerbated AIH showed reduced diversity and total load of gut bacteria. Conclusion: Our humanized animal model has provided a novel experimental tool to further elucidate the pathogenesis of AIH and to evaluate the efficacy and safety of immunoregulatory therapeutic interventions in vivo. (Hepatology 2015;62:1536–1550)

Depletion of B cells induces remission of autoimmune hepatitis in mice through reduced antigen presentation and help to T cells

Autoimmune hepatitis (AIH) is known as a T cell–mediated disease. However, AIH patients refractory to conventional treatment have been successfully treated with anti‐CD20‐mediated B‐cell depletion. The aim of this project was to understand the immunological changes underlying the AIH remission caused by B‐cell depletion in an experimental model of AIH. C57BL/6 AIH mice, xenoimmunized with DNA coding for human liver antigens, were treated with a single dose of depleting mouse anti‐CD20 antibody at the peak of liver inflammation. Liver inflammation, alanine aminotransferase levels, chemokine (C‐X‐C) ligand 10 expression, and circulating B‐cell, autoantibody, and total immunoglobulin G levels were monitored following depletion. T‐cell and B‐cell phenotype and function were characterized. Administration of a single dose of anti‐CD20 resulted in a drastic reduction of liver inflammation accompanied by a significant reduction of alanine aminotransferase levels and of proinflammatory chemokine (C‐X‐C) ligand 10 expression. The treatment did not result in significant changes in total immunoglobulin G levels or autoantibodies. There were significantly more naive and less antigen‐experienced CD4+ and CD8+ T cells, and T‐cell proliferation was significantly reduced following anti‐CD20 treatment. B cells served as antigen‐presenting cells to CD4+ T cells. Anti‐CD20 treatment also led to a profound reduction of T follicular helper cells. Conclusion: B cells play an active role in the pathogenesis of AIH in antigen presentation processes and the modulation of T‐cell functions and influence the T follicular helper–cell population; this active role of B cells could explain the success of B‐cell depletion for remission of AIH despite its classification as a T cell–mediated autoimmune liver disease. (Hepatology 2015;62:1511–1523)

Liver Restores Immune Homeostasis after Local Inflammation despite the Presence of Autoreactive T Cells

The liver must keep equilibrium between immune tolerance and immunity in order to protect itself from pathogens while maintaining tolerance to food antigens. An imbalance between these two states could result in an inflammatory liver disease. The aims of this study were to identify factors responsible for a break of tolerance and characterize the subsequent restoration of liver immune homeostasis. A pro-inflammatory environment was created in the liver by the co-administration of TLR ligands CpG and Poly(I:C) in presence or absence of activated liver-specific autoreactive CD8+ T cells. Regardless of autoreactive CD8+ T cells, mice injected with CpG and Poly(I:C) showed elevated serum ALT levels and a transient liver inflammation. Both CpG/Poly(I:C) and autoreactive CD8+T cells induced expression of TLR9 and INF-γ by the liver, and an up-regulation of homing and adhesion molecules CXCL9, CXCL10, CXCL16, ICAM-1 and VCAM-1. Transferred CFSE-labeled autoreactive CD8+ T cells, in presence of TLR3 and 9 ligands, were recruited by the liver and spleen and proliferated. This population then contracted by apoptosis through intrinsic and extrinsic pathways. Up-regulation of FasL and PD-L1 in the liver was observed. In conclusion, TLR-mediated activation of the innate immune system results in a pro-inflammatory environment that promotes the recruitment of lymphocytes resulting in bystander hepatitis. Despite this pro-inflammatory environment, the presence of autoreactive CD8+ T cells is not sufficient to sustain an autoimmune response against the liver and immune homeostasis is rapidly restored through the apoptosis of T cells.

Different sites of xenoantigen delivery lead to a virally induced late-onset hepatitis in mice through molecular mimicry

Epidemiological and laboratory evidences led to the hypothesis that molecular mimicry between viruses and self‐proteins could be linked to the onset of autoimmune hepatitis (AIH). Hepatotropic viruses could be good candidates, as a pro‐inflammatory environment may facilitate the development of AIH.