Cells In Hepatitis C Virus Infection Research Articles

Peripheral blood iNKT cell activation correlates with liver damage during acute hepatitis C.

Invariant NK T (iNKT) cells are implicated in viral clearance; however, their role in hepatitis C virus (HCV) infection remains controversial. Here, iNKT cells were studied during different stages of HCV infection. iNKT cells from patients with acute HCV infection and people who inject drugs (PWID) with chronic or spontaneously resolved HCV infection were characterized by flow cytometry. In a longitudinal analysis during acute HCV infection, frequencies of activated CD38+ iNKT cells reproducibly declined in spontaneously resolving patients, whereas they were persistently elevated in patients progressing to chronic infection. During the first year of infection, the frequency of activated CD38+ or CD69+ iNKT cells strongly correlated with alanine transaminase levels with particularly pronounced correlations in spontaneously resolving patients. Increased frequencies of activated iNKT cells in chronic HCV infection were confirmed in cross-sectional analyses of PWID with chronic or spontaneously resolved HCV infection; however, no apparent functional differences were observed with various stimulation protocols. Our data suggest that iNKT cells are activated during acute hepatitis C and that activation is sustained in chronic infection. The correlation between the frequency of activated iNKT cells and alanine transaminase may point toward a role of iNKT cells in liver damage.

Diverse Functions of γδ T Cells in the Progression of Hepatitis B Virus and Hepatitis C Virus Infection.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are primary risk factors for a wide spectrum of liver diseases that severely affect human health. The liver is an immunological organ that has an abundance of immune cells. Thus, various innate or adaptive immune cells are involved in the progression of HBV or HCV infection. Among those cells, a unique kind of immune cell, the γδ T cell, contributes to promoting or inhibiting the progression of liver diseases. To reveal the diverse roles of γδ T cells in HBV or HCV infection, the properties and functions of these cells in human and mouse models are analyzed. Here, we briefly describe the characteristics and functions of γδ T cells subsets in liver diseases. Then, we fully discuss the diverse roles of γδ T cells in the progression of HBV or HCV infection, including stages of acute infection, chronic infection, liver cirrhosis, and hepatocellular carcinoma. Finally, the functions and existing problems of γδ T cells in HBV or HCV infection are summarized. A better understanding of the function of γδ T cells during the progression of HBV and HCV infection will be helpful for the treatment of virus infection.

Immunomodulation of the Natural Killer Cell Phenotype and Response during HCV Infection.

Hepatitis C virus (HCV) infection develops into chronic hepatitis in over two-thirds of acute infections. While current treatments with direct-acting antivirals (DAAs) achieve HCV eradication in >95% of cases, no vaccine is available and re-infection can readily occur. Natural killer (NK) cells represent a key cellular component of the innate immune system, participating in early defence against infectious diseases, viruses, and cancers. When acute infection becomes chronic, however, NK cell function is altered. This has been well studied in the context of HCV, where changes in frequency and distribution of NK cell populations have been reported. While activating receptors are downregulated on NK cells in both acute and chronic infection, NK cell inhibiting receptors are upregulated in chronic HCV infection, leading to altered NK cell responsiveness. Furthermore, chronic activation of NK cells following HCV infection contributes to liver inflammation and disease progression through enhanced cytotoxicity. Consequently, the NK immune response is a double-edged sword that is a significant component of the innate immune antiviral response, but persistent activation can drive tissue damage during chronic infection. This review will summarise the role of NK cells in HCV infection, and the changes that occur during HCV therapy.

A juggernaut of innate & adaptive immune cells in chronic hepatitis C.

Hepatitis C virus (HCV) is a small positive-sense, single-stranded RNA virus, the causal organism for chronic hepatitis. Chronic hepatitis leads to inflammation of liver, causing cirrhosis, fibrosis and steatosis, which may ultimately lead to liver cancer in a few cases. Innate and adaptive immune responses play an important role in the pathogenesis of HCV infection, thus acting as an important component in deciding the fate of the disease. Numerous studies have indicated that the derangement of these immune responses results in the persistence of infection leading to chronic state of the disease. Interactions between virus and host immune system generally result in the elimination of virus, but as the virus evolves with different evading mechanisms, it makes environment favourable for its survival and replication. It has been reported that HCV impairs the immune system by functional modulation of the cells of innate as well as adaptive immune responses, resulting in chronic state of the disease, influencing the response to antiviral therapy in these patients. These defects in the immune system lead to suboptimal immune responses and therefore, impaired effector functions. This review highlights the involvement or association of different immune cells such as natural killer cells, B cells, dendritic cells and T cells in HCV infection and how the virus plays a role in manipulating certain regulatory mechanisms to make these cells dysfunctional for its own persistence and survival.

Increased killer B cells in chronic HCV infection may lead to autoimmunity and increased viral load

Regulatory B (Breg ) cells are characterized by various membrane markers and the secretion of different inhibitory cytokines. A new subset of Breg cells was identified as CD5hi Fas-ligand (FasL)hi . Their main reported role is to suppress anti-viral and anti-tumour immune responses, and, hence they have been dubbed 'killer' B cells. In this study, we aim to assess the role of these cells in chronic hepatitis C virus (HCV) infection, and determine if they contribute to the increased viral load and persistence of HCV and its related autoimmunity. (i) FasL expression on CD5hi B cells is increased significantly in HCV-infected patients compared to healthy individuals [28·06±6·71 mean fluorescence intensity (MFI)±standard error of the mean (s.e.m.), median = 27·9 versus 10·87±3·97 MFI±s.e.m., median = 10·3, respectively, P<0·0001]. (ii) Killer B cells from HCV patients increased autologous CD4+ T cell apoptosis compared to the apoptosis in healthy individuals [39·17%±7·18% mean±standard deviation (s.d.), median = 39·6 versus 25·92±8·65%, mean±s.d., median = 24·1%, P<0·0001, respectively]. A similar increase was observed in CD8+ T cell apoptosis (54·67±15·49% mean±s.d., median=57·3 versus 21·07%±7·4%, mean±s.d., median=20%, P=0·0006, respectively). (iii) By neutralizing FasL with monoclonal anti-FasL antibodies, we have shown that the induction of apoptosis by killer B cells is FasL-dependent. (iv) Increased expression of FasL on CD5hi B cells is correlated positively with an increased viral load and the presence of anti-nuclear antibodies and rheumatoid factor in HCV. This is the first study in which killer B cells have been suggested to play a pathogenic role in HCV. They seem to be involved in HCV's ability to escape efficient immune responses.

Insufficiency of DNA repair enzyme ATM promotes naive CD4 T-cell loss in chronic hepatitis C virus infection

T cells have a crucial role in viral clearance and vaccine response; however, the mechanisms regulating their responses to viral infections or vaccinations remain elusive. In this study, we investigated T-cell homeostasis, apoptosis, DNA damage, and repair machineries in a large cohort of subjects with hepatitis C virus (HCV) infection. We found that naive CD4 T cells in chronically HCV-infected individuals (HCV T cells) were significantly reduced compared with age-matched healthy subjects. In addition, HCV T cells were prone to apoptosis and DNA damage, as evidenced by increased 8-oxoguanine expression and γH2AX/53BP1-formed DNA damage foci—hallmarks of DNA damage responses. Mechanistically, the activation of DNA repair enzyme ataxia telangiectasia mutated (ATM) was dampened in HCV T cells. ATM activation was also diminished in healthy T cells exposed to ATM inhibitor or to HCV (core protein) that inhibits the phosphoinositide 3 kinase pathway, mimicking the biological effects in HCV T cells. Importantly, ectopic expression of ATM was sufficient to repair the DNA damage, survival deficit, and cell dysfunctions in HCV T cells. Our results demonstrate that insufficient DNA repair enzyme ATM leads to increased DNA damage and renders HCV T cells prone to apoptotic death, which contribute to the loss of naive T cells in HCV infection. Our study reveals a novel mechanism for T-cell dysregulation and viral persistence, providing a new strategy to improve immunotherapy and vaccine responses against human viral diseases.

Notch Signaling Regulates Circulating T Helper 22 Cells in Patients with Chronic Hepatitis C.

Notch signaling enhanced the response of interleukin (IL)-22-producing CD4+ T cells that were defined as T helper 22 (Th22) cells, and Notch-aryl hydrocarbon receptor (AhR)-IL-22 axis fine-tuned inflammatory response. Previous studies have demonstrated that both Notch signaling and Th22 cells took part in the pathogenesis of chronic hepatitis C virus (HCV) infection. Thus, in this study, we aimed at examining the regulatory role of Notch signaling in Th22 cells in HCV infection. A total of 59 patients with chronic hepatitis C and 22 normal controls (NCs) were enrolled in this study. The percentage of Th22 cells and mRNA expression of related transcriptional factors and cytokines were analyzed in response to γ-secretase inhibitor. Th22 cell frequency was significantly elevated in chronic hepatitis C in comparison with that in NCs. Inhibition of Notch signaling downregulated HCV-specific Th22 cells and IL-22 production, which was accompanied by the reduction of AhR and modulatory cytokines (IL-6 and tumor necrosis factor-α). Moreover, the suppression of Notch signaling also decreased the IL-22-mediated antimicrobial response in both normal and HCV-infected HepG2 cells/Huh7.5 cells. This process was also accompanied by the depression of signal transducers and activators of transcription 3 signaling. In conclusion, the current results suggested that Notch signaling acted as a critical pathway in determining the response to IL-22 in chronic hepatitis C. Thus, Notch-Th22 axis might be considered a new therapeutic target for HCV-infected patients.

Natural killer cells in hepatitis C: Current progress.

Patients infected with the hepatitis C virus (HCV) are characterized by a high incidence of chronic infection, which results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The functional impairment of HCV-specific T cells is associated with the evolution of an acute infection to chronic hepatitis. While T cells are the important effector cells in adaptive immunity, natural killer (NK) cells are the critical effector cells in innate immunity to virus infections. The findings of recent studies on NK cells in hepatitis C suggest that NK cell responses are indeed important in each phase of HCV infection. In the early phase, NK cells are involved in protective immunity to HCV. The immune evasion strategies used by HCV may target NK cells and might contribute to the progression to chronic hepatitis C. NK cells may control HCV replication and modulate hepatic fibrosis in the chronic phase. Further investigations are, however, needed, because a considerable number of studies observed functional impairment of NK cells in chronic HCV infection. Interestingly, the enhanced NK cell responses during interferon-α-based therapy of chronic hepatitis C indicate successful treatment. In spite of the advances in research on NK cells in hepatitis C, establishment of more physiological HCV infection model systems is needed to settle unsolved controversies over the role and functional status of NK cells in HCV infection.

NK cell function and receptor diversity in the context of HCV infection.

Hepatitis C virus (HCV) infects over 170 million people in the world. While a minority of individuals are able to naturally clear this hepatotropic virus using their immune system, most people go on to develop a lifetime chronic infection that can result in severe liver pathology, potentially leading to liver cirrhosis and hepatic cellular carcinoma. Investigations into acute immune responses and spontaneous clearance of the virus are severely hampered by difficulties in identification of relevant patient cohorts. While the role for the adaptive immune response in viral clearance is well established, it is becoming clear that the innate immune system also impacts on HCV outcome. The innate immune response to infection is likely to influence the type of adaptive immune response that develops and will ultimately influence if the virus is cleared or develops into a chronic infection. Natural Killer (NK) cells are lymphocytes that have important anti-viral functions including direct cytotoxicity of infected cells and the production of inflammatory cytokines, e.g., IFN-γ. They are generally considered to be cells of the innate immune system, although there is increasing evidence that NK cells adapt and persist in response to particular viral infections. NK cells are altered in patients with acute and chronic HCV infection. There is increasing evidence from both cellular and genetic studies that NK cells modulate HCV outcome. This review will describe and discuss the current experimental and clinical evidence of a role for NK cells in HCV infection and describe recent discoveries that are likely to play a role in future research.

Editorial: Recent Advances in HBV and HCV Immunology.

Viral hepatitis is a major public health problem. While a prophylactic vaccine is now available for hepatitis B virus (HBV), an estimated 240–350 million people worldwide are persistently infected with HBV (1–3). There is not yet an approved vaccine to prevent hepatitis C virus (HCV) infection, and between 130 and 200 million people are believed to be chronically infected worldwide (4–6). Untreated, HBV and HCV can each cause liver inflammation, fibrosis, and cirrhosis, and predispose patients to liver failure and hepatocellular carcinoma (2, 5). These infections are difficult and expensive to treat. Therapy for HBV infection may be lifelong, and a true cure with loss of HBV cccDNA from the liver is rarely achieved (2). IFNα-based therapy was the only option for chronic HCV infection until recently; these regimens were often poorly tolerated and frequently ineffective. An array of direct-acting antiviral drugs has greatly improved the outlook for HCV therapy since 2011 (7), but the expense of the new drugs may limit the number of people who benefit. We still do not understand the immunologic mechanisms leading to chronic HBV and HCV infection, how the immune system controls these viruses, and how the immune system contributes to disease pathogenesis. These topics must be understood in order to develop improved therapies and, at least in the case of HCV, an effective vaccine. Our goal in this Research Topic is to cover advancements toward this understanding. The immune response in the liver – where HBV and HCV each flourish – is incompletely understood. Many of our insights into the immune responses to HBV and HCV have come from studies of human peripheral blood, with limited comparisons to the cells in the liver. The liver is believed to constitute a tolerogenic environment as it receives portal venous blood rich in microbial products from the gut (8, 9). During persistent infection with either HCV or HBV, virus-specific T cells may decrease in number. The virus-specific T cells that can be found tend to have an exhausted phenotype including expression of inhibitory co-receptors, such as PD-1, CTLA-4, or Tim3 (10). Age at the time of exposure is an important predictor of the outcome of HBV infection: those infected in the perinatal period or early in childhood are likely to experience chronic infection, while those infected as adults often mount a successful immune response, with elimination of the template cccDNA from the liver (2). Antiviral immune responses wax and wane during chronic HBV infection. In this Topic, Schuch and colleagues review the roles of CD8+ T cells and natural killer cells in HBV infection (11). Sharma and colleagues present a study of Treg cells in HBV-associated hepatocellular carcinoma (12). Rajoriya and colleagues review the roles of γδ T cells in HBV or HCV infection as well as other conditions (13). HCV causes persistent infection in an estimated 50–85% of those exposed (14). Innate and adaptive immune responses can drive spontaneous clearance of HCV infection. In this Topic, Abdel-Hakeem and Shoukry review the role of the immune response in resolution of HCV infection (15). Sung and colleagues focus on the roles played by CD8+ T cells during acute HCV (16). Genome-wide association studies have highlighted a role for IFNλ in determining the outcome of HCV infection and IFNα-based antiviral therapy; this issue is reviewed by Laidlaw and Dustin (17). Cashman and colleagues discuss the roles of humoral immunity in acute and persistent HCV infection (18). Chronic HCV infection is also associated with extrahepatic disease; Sidharthan and colleagues present gene expression study showing a role for monocytes in HCV-associated mixed cryoglobulinemic vasculitis (19). Because of shared modes of transmission, up to 30% of individuals with HIV infection may also be infected with HCV; co-infected individuals have increased inflammation and accelerated liver damage (14). Cho and colleagues present a study of T cell phenotype, activation, and function in HCV–HIV co-infected patients, highlighting distinct changes in T cell function during co-infection – even when HIV is well controlled with highly active antiretroviral therapy (20).

Dendritic cells in hepatitis C virus infection: key players in the IFNL3-genotype response.

Recently, single nucleotide polymorphisms, in the vicinity of the interferon lambda 3 (IFNL3) gene have been identified as the strongest predictor of spontaneous and treatment induced clearance of hepatitis C virus (HCV) infection. Since then, increasing evidence has implicated the innate immune response in mediating the IFNL3 genotype effect. Dendritic cells (DCs) are key to the host immune response in HCV infection and their vital role in the IFNL3 genotype effect is emerging. Reports have identified subclasses of DCs, particularly myeloid DC2s and potentially plasmacytoid DCs as the major producers of IFNL3 in the setting of HCV infection. Given the complexities of dendritic cell biology and the conflicting current available data, this review aims to summarize what is currently known regarding the role of dendritic cells in HCV infection and to place it into context of what is know about lambda interferons and dendritic cells in general.

Innate immune responses in hepatitis C virus-exposed healthcare workers who do not develop acute infection

Hepatitis C virus (HCV) infection typically results in chronic disease with HCV outpacing antiviral immune responses. Here we asked whether innate immune responses are induced in healthcare workers who are exposed to small amounts of HCV, but do not develop systemic infection and acute liver disease. Twelve healthcare workers with accidental percutaneous exposure to HCV-infected blood were prospectively studied for up to 6 months for phenotype and function of natural killer T (NKT) and NK cells, kinetics of serum chemokines, and vigor and specificity of HCV-specific T-cell responses. Eleven healthcare workers tested negative for HCV RNA and HCV antibodies. All but one of these aviremic cases displayed NKT cell activation, increased serum chemokines levels, and NK cell responses with increased CD122, NKp44, NKp46, and NKG2A expression, cytotoxicity (as determined by TRAIL and CD107a expression), and interferon-gamma (IFN-γ) production. This multifunctional NK cell response appeared a month earlier than in the one healthcare worker who developed high-level viremia, and it differed from the impaired IFN-γ production, which is typical for NK cells in chronic HCV infection. The magnitude of NKT cell activation and NK cell cytotoxicity correlated with the magnitude of the subsequent HCV-specific T-cell response. T-cell responses targeted nonstructural HCV sequences that require translation of viral RNA, which suggests that transient or locally contained HCV replication occurred without detectable systemic viremia. Exposure to small amounts of HCV induces innate immune responses, which correlate with the subsequent HCV-specific T-cell response and may contribute to antiviral immunity.

Functional Attributes of Responding T Cells in HCV Infection: The Recent Advances in Engineering Functional Antiviral T Cells

Hepatitis C virus (HCV) is one of the major causes of hepatocellular carcinoma (HCC) around the world. HCV promotes characteristics of cancer stem cells and the infected cells are insensitive to apoptotic signals, which lead to persistent antigen stimulation and T cell exhaustion in the host. In spite of new effective antiviral drugs, new challenges are around the corner as drug-resistant viral strains and drug-drug interactions have already been reported. Considering that there are few effective treatments available for HCC, novel immunotherapies to prevent HCC and late stage HCV-related liver diseases should be considered. Given that adoptive immunotherapy with antigen-specific T lymphocytes has emerged as an effective therapeutic strategy for combating cancer, there is, therefore, reason to examine the possibility of using highly functional HCV-reactive T cells in immunotherapy. This review aims to provide the current understanding of natural HCV responding T cells in HCV infection and to give an update on the novel approaches that have the capacity to ex vivo generate functional T cells for potential adoptive cell therapy. Approaches based on the pMHC tetramer-associated magnetic enrichment, exogenous HCV T cell receptor transfer, and induced pluripotent stem cell technologies are described herein. Their potentials as immunotherapeutic against HCV-related diseases are discussed.

Intrahepatic natural killer cell NKp46 expression drives HCV-associated liver inflammation and viral resistance to treatment with interferon alpha

BackgroundThere is evidence that natural killer (NK) cells help control persistent viral infections including hepatitis C virus (HCV). HCV infection is treated with interferon (IFN) alpha, which stimulates the immune system, and is successful in 40–80% of patients. Detailed comparison of the phenotype and function of blood and intrahepatic NK cells in chronic HCV infection and in response to treatment with IFN alpha has not been elucidated. MethodsWe performed a comparison of NK cells derived from blood and intrahepatic compartments in multiple paired samples from 24 HCV infected patients pretreatment and 22 patients with non-viral chronic liver disease (CLD). NK phenotype (CD16, NKp30, NKp46, NKG2D, and NKG2A) and functional profile (CD107a, IFNγ, and granzyme B) were assessed with flow cytometry. In a separate cohort of 17 patients with HCV, who had completed treatment, rate of viral clearance was calculated and pretreatment peripheral blood NK phenotype and CD107a expression (degranulation) in response to increasing stimulation was measured. FindingsNK cells in the liver demonstrate a distinct phenotype compared with blood, manifested as downregulation of the NK cell activation receptors CD16, NKG2D, and NKp30. By contrast, NKp46 expression was not downregulated. Intrahepatic NK cells appeared to be more activated with increased spontaneous degranulation (reduced granzyme B, p<0·001 and increased CD107a, p=0·006) and production of IFNγ (p=0·05). NKp46 expression correlated with NK-cell activation, and correlated closely with the severity of liver inflammation (p=0·035). The rate of viral clearance during treatment with IFN alpha inversely correlated with NKp46 expression at baseline (p=0·01). However, the ability to increase cytotoxic NK function in response to increasing stimulation ex vivo correlated with viral clearance (p=0·029) and inversely with NKp46 (p=0·005). InterpretationThese findings indicate that NKp46 marks out pathologically activated NK cells in HCV, which are unlikely to be involved in viral control in IFN alpha-treated individuals. FundingWelsh Assembly.

Viral Proteins Mediate Upregulation of Negative Regulatory Factors Causing Down-Modulated Dendritic Cell Functions In Chronic Hepatitis C Virus Infection

Stunted cellular immune response against a narrow range of epitopes is the hallmark of chronic hepatitis C infection, but the underneath molecular mechanisms have not been well elucidated. Suboptimal antigen presentation through defective antigen presenting cells, have been suggested. The myeloid dendritic cells as professional antigen presenting cells have been found to be phenotypically and functionally defective in chronic hepatitis C-infected patients in our recently published study. In order to find out if the maturation defects in dendritic cells (DC) are induced by the persistence of virus, we tried to differentiate CD14+ monocytes isolated from the peripheral blood of a healthy volunteer in dendritic cell culture medium containing GM-CSF and IL-4 supplemented with hepatitis C virus (HCV) proteins, core and NS5. The results indicated that a lesser number of monocytes differentiated to DC in presence of HCV proteins. Moreover, the differentiated cells depicted immature phenotype, which will not respond to the TLR-4 mediated stimulation ex vivo with significantly lesser upregulation of activation markers, HLA-DR, CD83, CD80 and CD86 as compared to cells differentiated in the absence of HCV proteins. Besides, these immature cells showed characteristics of defective antigen presentation, with significantly lower allostimulatory capacity towards lymphocytes from a healthy donor. Semi-quantitative reverse-transcription polymerase chain reaction (RT- PCR) showed upregulated expression of negative regulatory genes SOCS3, PDL1 and IDO in cells grown in presence of HCV proteins, suggesting the role of HCV and associated antigens in functional down- modulation of dendritic cells. This may correlate with the antigen persistence and maturation-defective status of dendritic cells in chronic HCV infection.

Dendritic Cells in HIV-1 and HCV Infection: Can They Help Win the Battle?

Persistent infections with human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) are a major cause of morbidity and mortality worldwide. As sentinels of our immune system, dendritic cells (DCs) play a central role in initiating and regulating a potent antiviral immune response. Recent advances in our understanding of the role of DCs during HIV-1 and HCV infection have provided crucial insights into the mechanisms employed by these viruses to impair DC functions in order to evade an effective immune response against them. Modulation of the immunological synapse between DC and T-cell, as well as dysregulation of the crosstalk between DCs and natural killer (NK) cells, are emerging as two crucial mechanisms. This review focuses on understanding the interaction of HIV-1 and HCV with DCs not only to understand the immunopathogenesis of chronic HIV-1 and HCV infection, but also to explore the possibilities of DC-based immunotherapeutic approaches against them. Host genetic makeup is known to play major roles in infection outcome and rate of disease progression, as well as response to anti-viral therapy in both HIV-1 and HCV-infected individuals. Therefore, we highlight the genetic variations that can potentially affect DC functions, especially in the setting of chronic viral infection. Altogether, we address if DCs’ potential as critical effectors of antiviral immune response could indeed be utilized to combat chronic infection with HIV-1 and HCV.

The long way toward understanding host and viral determinants of therapeutic success in HCV infection

The T helper type 1 (Th1)/Th2 hypothesis of immune regulation arose in the late 1980s, stemming from observations in mice [1]. Since then, the Th1/Th2 cytokine balance has been extensively described in humans as a pivotal component of antiviral immunity and pathogenesis in infections. In a simplistic view of the literature, Th1 cells are commonly associated with interferon-gamma (IFN-c), interleukin (IL)-2 and IL-12 secretion, and these cells are hypothesized to mediate the response against intracellular pathogens such as viruses. Inversely, Th2 cells, which are most heavily reliant on IL-4, IL-5, IL-10 and IL-13, are supposed to emphasize protection against extracellular pathogens. In its modern incarnation, the role played by the Th1/Th2 balance in the regulation of cellular immunity against infections seems vaster and influenced by a large number of natural variables [2, 3]. In addition, since 1986 and the emergence of the Th1/Th2 hypothesis, other T-cell subpopulations, such as Th17 cells, have been described as functionally integrated actors of the antiinfectious immunity and crucial component of an effective response against pathogens [4]. Despite increasing evidence demonstrating the importance of these subpopulations in the effectiveness of the immune response against pathogens, the Th1/Th2 balance remains a critical ratio to clear invaders. For hepatitis C virus (HCV), a robust Th1 cell response and a mild Th2 activity, with a vigorous multi-epitopespecific CD4 and CD8 T-cell responses, are associated with HCV clearance in chronic infection [5]. IFN-c, a cytokine strongly associated with the Th1 response, exerts direct and durable antiviral effects inhibiting the replication of subgenomic and genomic HCV RNAs by stimulating the cytotoxic T-lymphocyte response. In patients with chronic hepatitis C, the cytotoxic CD8 T-cell activity may also be inversely related to the viral load. Viral sequence mutations are an important mechanism to escape the adaptative cellular immune response. A high level of replication, high mutation rate (estimated to be 1 in 1000 bases per year) due to the lack of proof-reading capacity of its polymerase, and pre-existing quasi-species contribute to the rapid diversification of the viral population and limits the efficiency of an adaptive immune response [6]. In addition, infiltrated cells in the liver belong mostly to the Th1 cells, unable to secrete IL-4 and IL-5 [7]. In this context, the overproduction of the initially protective immune response and soluble factors at the site of infection leads to adverse effects resulting in hepatocellular damage. Th1 cells release TNF-a, IFN-c, and IL-2 causing selfinflicted inflammation and necrosis. To limit these events and the persistence of HCV, the Th1 immune response must be kept under control. Th2 cells such as IL-4and IL-10-secreting cells have a vital role in protecting the host from the potentially damaging consequences of Th1 cells in chronic HCV infection by suppressing the Th1 response and counteracting the fibrogenic effects of TNF-a, IFN-c, and IL-2. On the other hand, the Th2 response is also more frequently associated with viral persistence and resistance to treatment in contrast to the viral clearance associated with Th1 [8]. Hence, intrahepatic IL-10-producing T cells are more frequently detected in patients with chronic hepatitis. An increased Th1 and Th2 response has been shown during chronic HCV infection with a greater elevated level of Th2 than Th1 cytokines [9]. This suggests that an enhanced Th2 response is at least partly responsible P.-A. Rubbo (&) P. Van de Perre E. Tuaillon INSERM U1058, CHU Montpellier and Montpellier 1 University, Montpellier, France e-mail: pierrealainrubbo@gmail.com

Immune-Related Disorders and Extrahepatic Diseases in Chronic HCV Infection

Hepatitis C virus (HCV) represents one of the most important causes of chronic active liver disease worldwide, potentially resulting in cirrhosis, and hepatocellular carcinoma. HCV is mainly characterized by two major immunologic fingerprints, namely, escape of immune response in more than 80% of infected patients and production of autoantibodies in almost half of them. Within the infected host, HCV exists as a quasispecies or a flock of related viral sequences. HCV can evolve rapidly as its replicative cycle yields 1012 HCV particles per day in the liver, whereas in the serum the newly produced viral particles have an estimated halflife of about 3 hours. This high rate of virus production coupled with the lack of proofreading capacity of the HCV's NS5B RNA-dependent RNA polymerase lead to its rapid evolution and escape from immune recognition in each host. In addition, HCV profoundly deranges the functions of immune system. It deregulates both innate and adaptive antiviral response. It has been shown that HCV-encoded proteins subvert type I interferon (IFN) receptor signal transduction and the function of downstream IFN effector pathway. The virus decreases the ability of NK cells to promote dendritic cell maturation modifying antigen presentation and production of soluble mediators including IL-10 and IL-12. Resolution of HCV infection is mainly dependent on adaptive immunity and is associated with a robust and sustained specific T-cell response targeting multiple epitopes and intrahepatic IFN-γ production. Defective CD4+ T cells in both acute and chronic HCV infection lead to CD8+ T-cell exhaustion. While HCV evolves under immunological pressure, the cellular immune response remains focused on viral sequences encountered early in the course of the infection. Indeed, humoral immune response seems more flexible in that the continued emergence of new antibody specificities sharply contrasts with the static T-cell response. Indubitable, interaction of HCV with B cells may promote favourable conditions for lymphocyte proliferation. Viral replicative intermediary was found in the B cell from patients with mixed cryoglobulinemia (MC), whereas no traces of HCV productive particles were demonstrable in HCV-infected individuals without cryoglobulin production, supporting the notion that HCV is not a genuine lymphotropic virus, but its entry and replication are largely dependent on host selective interactions. HCV binding to BCR triggers both the initiation of signaling cascade and internalization of the BCR and bound antigen into the cell. These interactions result in in vivo polyclonal activation and expansions of CD19+ CD5+ cells. Extrahepatic disease manifestations, which include autoimmune phenomena and frank autoimmune and/or rheumatic diseases, may complicate the clinical features of chronic HCV infection and sometimes dominate its course in almost half of chronic HCV carriers. Possibly, progression to frank B-cell lymphoid malignancy may also be superposed as additional stochastic oncogenic event. However, there are many dark areas in the comprehension of several aspects of their pathogenetic mechanisms. The nature of the process during which B cells expand with preferential involvement of rheumatoid factor- (RF-) producing B cells is undefined. Whether B-cell clonal expansion of a particular specificity occurs as a result of distinct selection is not clear. The process of B-cell clonal expansion occurring in an environment favourable to the immortalization of one specific clone must be clarified. Predisposing factors for transforming events must be identified. Against this background, it was felt that times were ripe to produce a state-of-the-art survey of the multifaceted pictures of HCV-related immune disorders. This special issue is therefore devoted to expand our knowledge on the features and mechanisms underlying the relationship between B-cell immune response and extrahepatic manifestations of chronic HCV infection. It comprises 16 review articles and 1 clinical study. The paper by T. Himoto and T. Masaki is a general overview on HCV-related extrahepatic manifestations including cryoglobulinemia, Sjogren's syndrome, autoimmune thyroid disease, lichen planus, CREST syndrome, IFN-induced autoimmunity, and autoimmune cytopenia. Few data are available on this topic in children. The paper by G. Indolfi et al. deals with the clinical significance of non-organ specific autoantibodies in the course of paediatric chronic hepatitis C. Subclinical hypothyroidism and membranoproliferative glomerulonephritis have been described. Dysregulation of the cytokine/chemokine network, involving proinflammatory and Th1 chemokines, is addressed by P. Fallahi et al. Both the humoral and viral counterparts at the basis of cryoglobulins production in HCV-induced type II MC, with particular attention to the most frequently involved single IgV subfamilies have been analyzed by G. Sautto et al. Neurological complications that occur in a large number of patients and range from peripheral neuropathy to cognitive impairment are discussed by S. Monaco et al. Autoimmune thrombocytopenia is a frequent complication of HCV chronic infection. D. Dimitroulis et al. estimated the epidemiological characteristics of the disease and discussed the potential treatment strategies. Extrahepatic manifestations of HCV infection include a multitude of disease processes affecting the small vessels, skin, kidneys, salivary gland, eyes, thyroid, and immunologic system. The majority of these conditions are thought to be immune-mediated. H. M. Ko et al. highlighted the histomorphologic features of these pathologic conditions. G. Lauletta et al. described epidemiological, clinical, and pathogenetic mechanisms underlying the cryoglobulinemic vasculitis. In MC, consumption of complement component 4 may be due to activation of complement cascade. Potential activators include monoclonal IgM-RF, IgG antibodies, and the complexing of the two in the cold, perhaps modulated by the rheology and stoichiometry of cryocomplexes in specific microcirculations. P. D. Gorevic elucidated the role of the RF and the complement in the vessel damage. The association between HCV infection and B-cell non-Hodgkin's lymphomas has led to search for molecular signatures capable of predicting patients' characteristics. V. De Re et al. underscored that HCV-related lymphomas are subject to specific deregulation induced by the virus. Epidemiology and mechanisms of HCV-induced lymphoproliferation have been elucidated by F. Forghieri et al. Different biological mechanisms, namely, chronic antigen stimulation, high-affinity interaction between HCV-E2 protein and its cellular receptors, direct HCV infection of B cells, and “hit and run” transforming events, may cooperate in a multifactorial model of HCV-associated lymphomagenesis. A comprehensive review of molecular mechanisms involved in HCV-related lymphomagenesis has been resumed by A. L. Zignego et al. It is concluded that HCV lymphomagenesis is a complex, multistep, multifactorial process. L. Arcaini et al. illustrated the relationship between HCV infection and different subtypes of indolent B-cell lymphomas. They summarized the data from the therapeutic studies reporting the use of antiviral treatment as hematological therapy. L. Chiche et al. provided an updated overview on the place of immunotherapy, especially biologics, in the management of HCV-induced cryoglobulinaemic vasculitis. Rituximab has been used to treat oncohaematological diseases, B-cell-related autoimmune diseases, rheumatoid arthritis, and, more recently, HCV-associated mixed cryoglobulinaemic vasculitis. E. Sagnelli et al. discussed rituximab-based treatment and conclude that this drug is capable of enhancing HCV replication with potential liver failure. Y. Kishida et al. explored the hypothesis that an induction approach with nIFN-beta followed by PEG-IFN-alpha plus ribavirin would increase the initial virologic response in chronic hepatitis C. They concluded that this combined therapeutic scheme results in high rate of sustained virologic response and improvement of innate and adaptive immunity. F. Bellanti et al. accomplished an overview about the biological activity and clinical applications of interferon lambda 3, summarizing the available data on its impact on HCV infection. The potential usefulness of this type of interferon in the treatment of HCV infection has been also discussed. Domenico Sansonno

Rituximab Treatment in Hepatitis C Infection: An In Vitro Model to Study the Impact of B Cell Depletion on Virus Infectivity

Hepatitis C virus (HCV) infected patients with vasculitis are often treated with the B-cell-depleting anti-CD20 antibody rituximab. Treatment reduces the cryoglobulins that cause vasculitis, yet it also leads to a transient increase in liver enzymes and HCV genomic RNA in the periphery. The mechanism underlying the increased viral load is unclear and both direct and indirect roles have been proposed for B cells in HCV infection. We previously reported that HCV can associate with B cells and can trans-infect hepatocytes. We established an in vitro assay to study the effect(s) of rituximab on B cell-associated HCV infectivity. Rituximab-mediated lysis of B cells in vitro increases the level of infectious HCV released from B cells. Our results, using a model where virus does not replicate in B cells, recapitulate observations seen in patients and may explain in part the rapid increase in blood HCV RNA observed after rituximab treatment.

Peripheral Blood B Cell Subset Skewing Is Associated with Altered Cell Cycling and Intrinsic Resistance to Apoptosis and Reflects a State of Immune Activation in Chronic Hepatitis C Virus Infection

Chronic hepatitis C virus (HCV) infection is associated with B cell activation, although underlying mechanisms are unclear. To investigate B cell regulation during HCV infection, we measured bulk B cell CpG and Staphylococcus aureus Cowan-induced IgG Ab-secreting cell (ASC) frequency, HCV and tetanus-specific ASC frequency, BCR- and CD40L-dependent CD80/CD86 expression, and activation of memory CD4 cells. Immature transitional, naive, resting memory, mature activated, tissue-like memory, and plasma B cell subset frequencies, cell cycling, and intrinsic apoptosis were quantified. We observed intact or enhanced tetanus-specific and total IgG ASC frequency, serum IgG, BCR- and CD40L-dependent CD80/CD86 expression, and CD40L-dependent bulk B cell activation of memory CD4 cells in HCV infection. HCV-specific ASCs were observed in HCV-infected but not control subjects, although frequencies were lower compared with tetanus-specific cells. Immature transitional and mature activated B cell subset frequencies were increased in HCV-infected subjects, with immature transitional frequency associated with liver inflammation and serum B cell-activating factor. Mature activated B cells less commonly expressed Ki67, more commonly expressed Bcl2, and were more intrinsically resistant to apoptosis, whereas immature transitional B cells more commonly expressed Ki67, the latter associated with plasma HCV level. Taken together, these results indicate that in the setting of chronic HCV infection, a state of activation results in B cell subset skewing that is likely the result of alterations in homeostasis, cell cycling, and intrinsic resistance to apoptosis and that results in an overall intact or enhanced B cell response to BCR and CD40L.