Role of NK and NKT cells in the immunopathogenesis of HCV-induced hepatitis

Natural killer (NK) cells constitute the first line of host defense against invading pathogens. They usually become activated in an early phase of a viral infection. Liver is particularly enriched in NK cells, which are activated by hepatotropic viruses such as hepatitis C virus (HCV). The activated NK cells play an essential role in recruiting virus-specific T cells and in inducing antiviral immunity in liver. They also eliminate virus-infected hepatocytes directly by cytolytic mechanisms and indirectly by secreting cytokines, which induce an antiviral state in host cells. Therefore, optimally activated NK cells are important in limiting viral replication in this organ. This notion is supported by the observations that interferon treatment is effective in HCV-infected persons in whom it increases NK cell activity. Not surprisingly, HCV has evolved multiple strategies to counter host's NK cell response. Compromised NK cell functions have been reported in chronic HCV-infected individuals. It is ironic that activated NK cells may also contribute toward liver injury. Further studies are needed to understand the role of these cells in host defense and in liver pathology in HCV infections. Recent advances in understanding NK cell biology have opened new avenues for boosting innate and adaptive antiviral immune responses in HCV-infected individuals.     

Autoantigen-selected B cells are bystanders in spontaneous T cell-driven experimental autoimmune hepatitis

Autoreactive B cells are considered pathogenic drivers in many autoimmune diseases; however, it is not clear whether autoimmune B cells are invariably pathogenic or whether they can also arise as bystanders of T cell-driven autoimmune pathology. Here, we studied the B cell response in an autoantigen- and CD4⁺ T cell-driven model of autoimmune hepatitis (AIH), the Alb-iGP_Smarta mouse in which expression of a viral model antigen (GP) in hepatocytes and its recognition by GP-specific CD4⁺ T cells causes spontaneous AIH-like disease. T cell-driven AIH in Alb-iGP_Smarta mice was marked by autoantibodies and hepatic infiltration of plasma cells and B cells, particularly of isotype-switched memory B cells, indicating antigen-driven selection and activation. Immunosequencing of B cell receptor repertoires confirmed B cell expansion selectively in the liver, which was most likely driven by the hepatic GP model antigen, as indicated by branched networks of connected sequences and elevated levels of IgG antibodies to GP. However, intrahepatic B cells did not produce increased levels of cytokines and their depletion with anti-CD20 antibody did not alter the CD4⁺ T cell response in Alb-iGP_Smarta mice. Moreover, B cell depletion did not prevent spontaneous liver inflammation and AIH-like disease in Alb-iGP_Smarta mice. In conclusion, selection and isotype-switch of liver-infiltrating B cells was dependent on the presence of CD4⁺ T cells recognizing liver antigen. However, recognition of hepatic antigen by CD4⁺ T cells and CD4⁺ T cell-mediated hepatitis was not dependent on B cells. Thus, autoreactive B cells can be bystanders and need not be drivers of liver inflammation in AIH.     

Autoimmunity and hepatitis C

Hepatitis C is a widespread chronic liver disease leading to cirrhosis and to the complications of portal hypertension. Based on biochemical and clinical features, it is almost indistinguishable from autoimmune hepatitis, which is characterized by the absence of viral infection, and other causes of chronic liver diseases, and represents a classical autoimmune disease with loss of immunological tolerance of liver tissue. Although the differentiation between both diseases is not difficult due the availability of diagnostic viral markers, it is well recognized that not only are autoantibodies present in autoimmune hepatitis frequently detected in hepatitis C, but also that an array of immune-mediated symptoms and diseases occur in patients with chronic hepatitis C. This has prompted research aimed at identifying a link between hepatitis C and autoimmunity, and autoimmune hepatitis in particular. This review focuses on the general immunological mechanisms linking viral infections with autoimmunity and includes the specific features of hepatitis C- and D-associated autoimmunity. Virus infection remains at the center of molecular and cellular research aimed at identifying the forces driving human autoimmunity and autoimmune diseases.     

B cells and autoimmune liver diseases

Autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are the three major autoimmune diseases affecting the liver. They are all characterized by the presence of a variety of autoantibodies, some of which are found in all three diseases, whereas others are restricted to one or two of them or are even specific for the particular disease. In this review we will first provide details of the serological features of these three autoimmune diseases that target the liver. In addition, we will highlight the possible pathogenic roles of autoreactive B cells, focusing on their immunological functions as autoantibody producing cells and as antigen-presenting cells for T cell priming. As well, we will describe the contribution of toll-like receptor (TLR) signaling to the activation of autoimmune B cells and the putative role of defects in regulatory T cell function in the development of autoimmune liver diseases.     

Mouse hepatitis viral infection induces an extrathymic differentiation of the specific intrahepatic alpha beta-TCRintermediate LFA-1high T-cell population.

Mouse hepatitis virus type 3 (MHV3), a coronavirus, is an excellent model for the study of thymic and extrathymic T-cell subpopulation disorders induced during viral hepatitis. It was recently reported that, in addition to the intrathymic T-cell differentiation pathway, an extrathymic differentiation pathway of alpha beta-T-cell receptor (TCR) T lymphocytes exists in the liver, and becomes important under pathological situations such as autoimmune diseases, malignancies or hepatic bacterial infections. In the present study, we compared the phenotypes of resident hepatic, splenic or thymic T-cell subpopulations during the acute viral hepatitis induced by HMV3 in susceptible C57BL/6 mice. The number of liver-resident mononuclear cells (MNC) increased during the viral infection, while cellularity decreased. Single positive (SP) CD4+ cells strongly increased in both the liver and thymus, while double positive (DP) (CD4+ CD8+) cells, present in the liver and thymus of mock-infected mice, decreased in C57BL/6 mice during the viral infection. A shift of alpha beta-TCRintermediate T cells toward alpha beta-TCRhigh was evidenced in the liver and thymus of infected mice, but not in the spleen. The few alpha beta-TCRint double negative (DN) (CD4-CD8-) cells also decreased following viral infection. alpha beta-TCRint or high lymphocytes expressing high levels of leucocyte function antigen-1 (LFA-1) increased in the liver of MHV3-infected mice. In addition, liver-resident T cells expressed strongly the CD44 (Pgp-1) activation marker, suggesting that they were either activated or antigen experienced during the viral infection. No significant change in T-cell subpopulations was detected in the spleen, suggesting that MHV3 infection could induce an early in situ differentiation of resident hepatic T cells rather than a recruitment of lymphocytes from peripheral lymphoid organs.     

Pathogenesis of autoimmune hepatitis: from break of tolerance to immune-mediated hepatocyte apoptosis

Understanding the pathogenesis and progression of autoimmune hepatitis (AIH) at the molecular level could prove essential in developing new preventive and therapeutic strategies. Recently developed murine models have enabled the identification of various mechanisms involved in the development and perpetuation of this autoimmune disorder. Studies on these models have shown that a peripheral break of tolerance against liver-expressed antigens is sufficient to induce an autoimmune liver disease, which can occur without prior liver damage. Recent data have also shown that the liver selectively recruits and induces the apoptosis of activated CD8+ T cells after an immune response. This process of T-cell trapping involves the expression of specific chemokines and adhesion molecules, and these molecules are believed to play an important role in the initiation and perpetuation of autoimmune hepatitis. Hepatocyte apoptosis, induced by autoreactive T cells, follows specific pathways that could be targeted by new therapeutic agents. Basic research on the break of immune tolerance against liver antigens would be beneficial for patients with autoimmune hepatitis, as well as those suffering from other chronic inflammatory liver diseases, such as primary biliary cirrhosis and graft-versus-host diseases.     

Role of hepatitis A and E viruses in the development of autoimmune diseases

The mechanisms of development of autoimmune diseases may be associated with a complex of genetic, immune, hormonal, and infectious factors. Autoimmune diseases include a wide range of systemic and organ-specific diseases, including autoimmune hepatitis (AIH). It is currently assumed that the pathogenesis of AIH is due to compromised immune regulation in the presence of an exogenous triggering factor. Exogenous factors, such as viruses, may be triggers of AIH. There may be different ways of initiating an autoimmune response by viruses, which includes nonspecific T-lymphocyte activation and molecular mimicry. There is much evidence supporting the initiating role of hepatitis viruses in the development of AIH and other autoimmune diseases. The development of AIH symptoms during hepatitis A and E virus infections has been described elsewhere. The creation of animal models of viral hepatitis is required to confirm the hypothesis that the viruses trigger the development of AIH and other autoimmune manifestations.     

Liver-infiltrating T helper cells in autoimmune chronic active hepatitis stimulate the production of autoantibodies against the human asialoglycoprotein receptor in vitro.

Autoantibodies against the human asialoglycoprotein receptor (ASGPR) occur in the sera of patients with autoimmune liver disorders. Liver-infiltrating T cell clones that specifically recognize the ASGPR have been described in patients with autoimmune chronic active hepatitis (AI-CAH) and primary biliary cirrhosis (PBC). Recently, we have shown that peripheral blood mononuclear cells (PBMC) from patients with AI-CAH or PBC but not chronic viral hepatitis secreted anti-ASGPR antibodies in vitro. In this study we characterized the influence of liver-infiltrating T cells on the secretion of ASGPR-specific autoantibodies by autologous B cells in cell culture supernatants. T cell clones from liver biopsies of three patients with chronic autoimmune liver disorders (one with AI-CAH, two with PBC) were isolated and investigated for their proliferative response to soluble ASGPR and their helper function provided to autoantibody-secreting B lymphocytes. PBMC from these patients secreted autoantibodies spontaneously in their cell culture supernatants and showed a proliferative response to ASGPR. T cell-depleted PBMC, however, lacked spontaneous antibody secretion. Four CD4+CD8- liver-infiltrating T cell clones showed a proliferative response to ASGPR and also induced spontaneous anti-ASGPR antibody production in cell culture supernatants when added to autologous T cell depleted PBMC. Activated supernatants of these T cell clones failed to induce antibody production. None of seven CD4+CD8- and two CD4-CD8+ T cell clones non-responding to ASGPR provided this help for antibody secretion. Anti-ASGPR secretion in vitro could not be inhibited by the addition of MoAbs raised against monomorphic determinants on HLA class II molecules. The addition of purified ASGPR or polyclonal-activating pokeweed mitogen showed no influence on the production of autoantibodies in these cultures. These data show that B lymphocytes require T cell help for the production of ASGPR-specific antibodies. This help can be provided by ASGPR-responsive T helper cells via cellular interactions.     

Acute and chronic hepatitis

Most liver biopsies performed today are for grading and staging of chronic viral hepatitis and steatohepatitis; there are uncommon indications for liver biopsy in the setting of acute hepatitis. Pathologists must have a broad knowledge of many forms of acute and chronic hepatitis, as well as their variations; these include viral hepatitis, autoimmune hepatitis, drug-induced hepatitis, metabolic diseases, and reactive hepatitis secondary to systemic disease processes. In this article, the authors review the pathological features of acute and chronic hepatitis.     

Murine viral hepatitis involves NK cell depletion associated with virus-induced apoptosis

Mouse hepatitis virus type 3 (MHV3), a coronavirus, is an excellent animal model for the study of immunological disorders related to acute and chronic hepatitis. In this study, we have verified if the fulminant hepatitis induced by MHV3 could be related to an impairment of innate immunity. Groups of three C57BL/6 mice were infected with the pathogenic L2-MHV3 or attenuated YAC-MHV3 viruses, and the natural killer (NK) cell populations from liver, spleen and bone marrow were analysed. The percentage of intrahepatic NK1.1(+)T cell receptor (TCR)- cells did not increase while NK1.1(+)TCR(inter) cells decreased in both L2-MHV3- and YAC-MHV3-infected mice. Concurrently, splenic and myeloid NK1.1+ cells decreased in L2-MHV3-infected mice. However, the cytotoxic activity of NK cells increased in liver and decreased in bone marrow from pathogenic L2-MHV3-infected mice while no modification was detected in YAC-MHV3-infected mice. Flow cytometric analysis revealed that both normal and larger splenic or myeloid NK cells decreased more in pathogenic L2-MHV3-infected mice than in attenuated YAC-MHV3-infected mice. In vitro viral infections of interleukin (IL)-15-stimulated lymphoid cells from liver and bone marrow revealed that L2-MHV3 induced higher decreases in cell viability of NK1.1+ cells than the YAC-MHV3 variant. The NK cell decreases were due to the viral permissivity leading to cytopathic effects characterized by cell rounding, syncytia formation and apoptosis. Larger NK+ syncytia were observed in L2-MHV3-infected cells than in YAC-MHV3-infected cells. These results suggest that NK cell production is impaired by viral infection favouring fulminant hepatitis.     

Circulating immune complexes and cell-mediated immunity in patients with hepatitis B virus associated liver diseases

The prevalence of circulating immune complexes (CIC), their role and their relationship to cell-mediated immunity in patients with hepatitis B virus associated liver disease are still controversial. This study was designed to investigate the prevalence of CIC and their relationship to viral markers, to subsets of peripheral blood T lymphocytes and to suppressor cell activity in patients with hepatitis B virus associated liver diseases. CIC were positive in 29.3% of 41 healthy HBsAg carriers, 37.8% of 88 patients with hepatitis B virus associated liver diseases, and 15% of 41 healthy subjects by the platelet aggregation test (PAT). The prevalence of CIC in patients with acute hepatitis (40.0%) and in those with cirrhosis (61.5%) was significantly higher than in normal controls (p less than 0.05, p less than 0.005 respectively). There was no correlation between the titer of CIC and serum HBsAg titer or the status of HBeAg, and no significant decrease in the peripheral blood lymphocyte CD4/CD8 ratio in healthy HBsAg carriers (1.39 +/- 0.31) and in patients with liver diseases (1.40 +/- 0.54) compared to the normal controls (1.48 +/- 0.31). Concanavalin A induced suppressor cell activity on IgG producing cells was impaired in healthy HBsAg carriers (34.9%) (p less than 0.005) and in patients with liver diseases (25.3%) (p less than 0.0001), and this change was prominent in patients with chronic active hepatitis and cirrhosis (p less than 0.0001). And there was a significant reverse correlation between concanavalin A induced suppressor cell activity on IgG-producing cells and the titer of CIC in PAT positive patients with hepatitis B virus associated liver diseases. In conclusion, it was suggested that defective suppressor cell function may lead to an increased B cell activation and such activity may account for the presence of CIC.     

The role of virus-induced regulatory T cells in immunopathology

In recent years, regulatory T cells have received increased attention for their role in immune responses to microbial infections. The list of microbial pathogens associated with regulatory T cell responses is growing rapidly and includes bacteria, viruses, parasites, and fungi. As the biology of regulatory T cells is revealed, we are discovering that their induction during infection is a normal aspect of immunity, necessary to limit collateral damage from inflammatory responses and aggressive immunological effectors. Thus, these cells play a critical role in maintaining the delicate balance between preventing immunopathology and allowing the immune response to clear infections. While generally successful, there are notable exceptions where regulatory T cell-mediated suppression appears to be responsible for allowing certain viruses to establish and maintain a persistent state. In this review, we will discuss our current understanding of what virus-induced regulatory T cells are, how they are induced, and what mechanisms they use to suppress immunity. The complex role of Tregs in regulating immunity to viral infections, and the consequences their activity has on disease is illustrated by a review of specific viral infections including hepatitis C virus and human immunodeficiency virus.     

Human asialoglycoprotein receptor as an autoantigen in chronic hepatitis

ASGPR may play a role in the pathogenesis of autoimmune chronic liver diseases. Several lines of evidence support this hypothesis. Antibodies to rabbit ASGPR could be found in various inflammatory liver diseases. In contrast, ASGPR preparations derived from human liver (h-ASGPR) were recognized predominantly by sera from patients with ai-CAH. Moreover, anti-h-ASGPR showed a close correlation to the inflammatory activity of ai-CAH both in terms of prevalence (88% in histologically proven active diseases), immunoglobulin class (IgM antibodies restricted to active inflammation) and behavior during treatment. Anti-h-ASGPR secretion could also be found in vitro, when PBL of patients were stimulated in cell culture. Anti-h-ASGPR antibodies did not correlate with a subgroup of ai-CAH; they also occurred in nearly 15% of patients with PBC. Other inflammatory liver diseases, as well as nonhepatic autoimmune disorders, seldom exhibited anti-h-ASGPR (less than 5%). Additionally, PBL and T-cell clones obtained from liver biopsies in patients with ai-CAH and PBC responded to h-ASGPR. Liver-infiltrating T cells were predominantly of CD4 phenotype and HLA class II restricted. Thus, the human ASGPR has been shown to represent a common target for humoral and cellular autoimmune response in chronic hepatitis probably contributing to disease induction or perpetuation.     

Detection of a liver-membrane autoantibody in HBsAg-negative chronic active hepatitis.

Investigation of humoral immunity against hepatocellular membrane antigens in patients with chronic active hepatitis and other liver diseases showed two different immunofluorescence patterns of IgG on hepatocyte membranes. A linear pattern was seen in HBsAg-negative hepatitis, but HBsAg-positive cases and some of protracted, acute hepatitis B had a granular pattern. In patients with IgG bound to hepatocytes, continuing necrosis of parenchymal liver cells was seen. Conversely, hepatocytes without bound IgG were found in cases of chronic active hepatitis in remission, acute viral hepatitis without HBsAg and chronic persistent hepatitis, in "healthy" HBsAg-carriers and in patients with fatty liver or alcoholic cirrhosis. A liver-membrane autoantibody in serum, proved by fixation on membranes of isolated rabbit hepatocytes, could be demonstrated only in HBsAg-negative chronic active hepatitis with elevated IgG-concentrations. The results support the existence of different pathogenetic types of chronic active hepatitis, a so-called autoimmune type and a hepatitis virus-B-induced type.     

Interleukin-17 producing mucosal associated invariant T cells - emerging players in chronic inflammatory diseases?

Mucosal associated invariant T (MAIT) cells are a population of evolutionarily conserved T cells, which express an invariant T cell receptor (TCR) and represent a significant subset of innate-like T cells in humans, yet their role in immunity is still emerging. Unlike conventional αβ T cells, MAIT cells are not restricted by MHC molecules, but instead uniquely recognize microbially derived vitamin metabolites presented by the MHC-I like molecule MR1. MAIT cells are enriched in mucosal sites and tissues including liver and adipose tissue where they are thought to play an important role in immunosurveillance and immunity against microbial infection. In addition to their putative role in antimicrobial immunity, recent research on MAIT cells, in particular IL-17 producing MAIT cells, has demonstrated their involvement in numerous chronic inflammatory conditions. In this review, we give an overview of the work to date on the function and subsets of MAIT cells. We also examine the role of IL-17 producing MAIT cells in chronic inflammatory diseases ranging from autoimmune conditions, metabolic diseases to cancer. Furthermore, we discuss the most recent findings from the clinic that might help deepen our understanding about the biology of MAIT cells.     

Electron microscopic examination of congenital cytomegalovirus hepatitis

Summary The electron microscopic features of cytomegalovirus hepatitis in the liver biopsy of a three-week-old infant were studied.The liver cells did not contain virus, but severe alterations similar to virus hepatitis were observed. In the bile duct cells, nuclear and cytoplasmic virus inclusions were demonstrated. In the nuclear inclusions virus particles of various degrees of maturity were embedded in dense granular material. The cytoplasm of the infected cells contained vacuoles with mature viruses. The Golgi zone seemed to play an important role in vacuole formation. In another type of infected cell, viruses were lying free in the cytoplasm and passed into the lumen of the bile ducts. It is concluded that viruses are eliminimated by the bile. Based on this electron microscopical observations, the examination of duodenal fluid is recommended as a new diagnostic procedure for demonstrating viruses.     

The Incidence and Significance of Pattern-Recognition Receptors in Chronic Viral Hepatitis Types B and C in Man

Chronic viral hepatitis B and C are among the most common and devastating liver diseases worldwide. Immune response plays a crucial role in the course of both diseases. In spite of the importance of the adaptive arm of the immune response, there is a growing role of innate immunity, the earliest confronted with viral attack. Pattern-recognition receptors (PRRs) and, in particular, Toll-like receptors (TLRs) are molecules which are able not only to recognize foreign invaders, but also quickly mount an antiviral defense. Activation of PRRs has been demonstrated in both hepatitis types, i.e. in situ in the liver and on while blood cells. Both viruses, HCV and HBV, are able to subvert the PRR-mediated antiviral response by means of various proteins and enzymes. HCV acts via the non-structural proteins NS2 and NS3/4A, while HBV HBeAg is inversely correlated with TLR activity. Viral counterattack is particularly directed toward dendritic cells, those creating the link with the adaptive immune response. Apart from TLRs, other PRRs such as RIG-1 and MDA-5 are also able to recognize viral infection and participate in the activation of type I interferon synthesis. TLRs manifest gene polymorphism, which was shown to affect several consequences associated with chronic viral hepatitis such as liver cirrhosis and the outcome of liver allotransplantation. There have been numerous attempts to take advantage of the existence and activity of PRRs for the patients’ benefit. Several authors examined the role of TLR synthetic agonists as inducers of TLR activation. In hepatitis C the most promising agonists appear to be TLR3, 7, and 9 for potential antiviral therapy. PRRs may also act as potent adjuvants in HBV vaccines. Their baseline mRNA levels may have predictive value in the course of antiviral therapy.     

Critical roles of conventional dendritic cells in promoting T cell‐dependent hepatitis through regulating natural killer T cells

Dendritic cells (DCs) play critical roles in initiating and regulating innate immunity as well as adaptive immune responses. However, the role of conventional dendritic cells (cDCs) in concanavalin A (ConA)‐induced fulminant hepatitis is unknown. In this study, we demonstrated that depletion of cDCs using either CD11c‐diphtheria toxin receptor transgenic mice (DTR Tg) mice or anti‐CD11c antibody reduced the severity of liver injury significantly, indicating a detrimental role of cDCs in ConA‐induced hepatitis. We elucidated further the pathological role of cDCs as being the critical source of interleukin (IL)‐12, which induced the secretion of interferon (IFN)‐γ by natural killer (NK) T cells. Reconstitution of cDCs‐depleted mice with IL‐12 restored ConA‐induced hepatitis significantly. Furthermore, we determined that NK T cells were the target of DC‐derived IL‐12, and NK T cells contributed to liver inflammation and injury through production of IFN‐γ. In summary, our study demonstrated a novel function of cDCs in mediating ConA‐induced hepatitis through regulating IFN‐γ secretion of NK T cells in an IL‐12‐dependent fashion. Targeting cDCs might provide potentially therapeutic applications in treating autoimmune related liver diseases.     

CCR5 deficiency drives enhanced natural killer cell trafficking to and activation within the liver in murine T cell-mediated hepatitis

Natural killer (NK) cells are innate immune cells that are enriched in the liver, but the processes underlying NK cell trafficking to the liver and cellular activation within the liver of patients with T cell-mediated liver diseases remain poorly defined. Concanavalin A (Con A) hepatitis is a murine model mimicking many aspects of human T cell-mediated liver diseases. Here we demonstrate that severe hepatitis in CCR5-deficient (KO) mice is associated with increased hepatic NK cell recruitment driven by enhanced hepatic production of CCL5 acting via CCR1 and by enhanced hepatic NK cell activation relative to that observed in wild-type mice after Con A administration. Furthermore, NK cell depletion ameliorated severe hepatitis in CCR5 KO mice but did not alter hepatitis in wild-type mice after Con A treatment. We propose that in the setting of CCR5 deficiency NK cells assume a profound effector role in Con A hepatitis via enhanced CCL5-CCR1 driven hepatic recruitment in addition to augmented cytokine-driven NK cell activation to produce interferon-gamma. These results highlight the potential profound impact of altered chemokine receptor expression on the innate immune response in the setting of T cell-mediated hepatitis.