HCV Infection and Innate Immunity

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HCV is a major public health problem, infecting more than 170 million people worldwide. Most cases of HCV infection become persistent and may eventually lead to chronic liver disease, cirrhosis, and hepatocellular carcinoma. Although hepatocytes are the major site of viral replication, a broad clinical spectrum of extrahepatic complications and diseases are associated with chronic HCV infection, including mixed cryoglobulinemia, non-Hodgkins lymphoma, cutaneous vasculitis, glomerulonephritis, neuropathy, and lymphoproliferative disorders. The existence of extrahepatic reservoirs of HCV replication, particularly in PBMCs, remains highly controversial. It is unclear how B cells become dysregulated during the course of chronic HCV infection. 1) Our group has demonstrated that free HCV is opsonized by complement and binds to CR1 on erythrocytes (ECR1); the presence of HCVspecific antibody significantly increased this binding. Whether binding of free HCV to erythrocytes is related to HCV clearance or pathogenesis has not been investigated. Mixed cryoglobulinemia (type II) is among the most common IC diseases associated with chronic HCV infection. Whereas nearly half of patients with HCV have detectable cryoglobulins, less than 10% develop clinically apparent disease. We have demonstrated that HCVIC binds to erythrocytes, and it is therefore plausible that factors affecting HCVIC/erythrocyte interaction could influence the expression of clinically evident ICrelated disease. (Hepatology 2018). 2)To further explore the pathogenesis of HCV-related immune complex disease, we tested the levels of complement-activated immune complexes (IC), rheumatoid factors, and several chemokines and cytokines in plasma samples from chronic HCV patients and matched healthy control in order to elucidate the possible biological responses and consequences of HCV-IC/erythrocyte interaction. We also investigate the genetic polymorphism of complement receptor 1 (CR1/CD35) gene associated with CR1 expression on erythrocytes from both groups of individuals, and how this CR1 expression level correlates with IC level. We have found that: a) several chemokine levels including CXCL10, CXCL12, and BAFF in blood from patients with chronic HCV infection are significantly elevated when compared to those from healthy controls; b) Similar elevated levels of circulating immune complexes (CIC) and rheumatoid factors are also found in patients with chronic HCV infection; c) levels of CIC in blood from chronic HCV patients with HH genotype in CR1 gene are significantly lower than those from chronic HCV patients with HL genotype. (Manuscript in preparation). 3)We observed that the association of HCV with CD19+ B cells is mediated by the complement system. In addition, using antibodies against cell surface markers, we showed that the binding complex mainly involved CD21 (complement receptor 2), CD19, CD20, and CD81. In human B cells, CD21 is known to form a costimulatory complex with CD19 and CD81. Co-ligation of the B cell antigen receptor (BCR) with this costimulatory complex can lower the threshold required for BCR-mediated B cell activation and proliferation. Epidemiological studies have demonstrated an increased risk of developing B-cell non-Hodgkin lymphoma in patients with chronic HCV infection. Both the regression of HCV-associated lymphoma with antiviral treatment and the beneficial effects of antiviral treatment on overall survival of patients with HCV-related lymphoma have strengthened the aetiological link between HCV infection and NHL presumed from epidemiological, clinical and pathophysiological studies (Hepatology 2016). 4) Since B-cells proliferation, in response to antigenic stimulation or polyclonal activation, may predispose to genetic aberrations (mutation, gene translocation, gene fusion, chromosomal amplification or deletion) we are using RNA-sequencing, to obtain both mutational and gene expression data from B-cells obtained from patients who are HCV+, HBV+, HDV+ and B-NHL+, and compare to patients who are HCV-, HBV-, HDV-, NHL+; HCV+, HBV+, HDV+, NHL-, and healthy controls. We will validate/ confirm the results on paired Paraffin Embedded lymphatic tissues then. The adoption of RNA-sequencing has been proven useful in clinical oncology as most of the clinically relevant somatic mutations are also expressed on the RNA level. We are recipient of an NIAID-NCI grant: "Investigation of HCV and HBV-Mediated B-Cell Malignant Transformation and Prioritization of Treatment for HCV-Related NHL in Mongolia. We have received more than 200 samples from Mongolia, including PBMC, plasma, and matched paraffin embedded lymphatic tissues, and have performed RNA-sequencing on 75 peripheral B cell samples isolated from PBMC. 5)Peripheral B cells from HCV-only patients demonstrated a gene signature consistent with chronic viral infection that indicated elevated Type I interferon signaling and general immunosuppression. This pattern was not observed in B cells from patients with HCV-associated BNHL. In these patients, we observed enrichment of an anergic-like gene signature mirroring that observed in autoimmune disorders and other HCV-associated lymphoproliferative disorders. Additionally, we observed significant clonal enrichment in HCV-associated BNHL samples of immunoglobulin genes known to be associated with autoimmunity and lymphoproliferative disorders. Clonal enrichment was correlated with expression of multiple epigenetic regulatory genes, which were found to be overexpressed in BNHL patients with and without concurrent HCV infection. Our data support viral-mediated clonal expansion of anergic-like B cells as a potential contributing factor to HCV-associated BNHL development, and suggest epigenetic dysregulation may be a common pathway in viral- and non-viral-associated lympho-progression. In HCV-associated BNHL, epigenetic mechanisms may also be involved in regulation of B cell anergy and represent an attractive target for clinical interventions (Table 1).