Hepatitis C Virus NS3 Mediated Microglial Inflammation via TLR2/TLR6 MyD88/NF-κB Pathway and Toll Like Receptor Ligand Treatment Furnished Immune Tolerance.

BACKGROUND: Recent evidence suggests the neurotrophic potential of hepatitis C virus (HCV). HCV NS3 protein is one of the potent antigens of this virus mediating inflammatory response in different cell types. Microglia being the immune surveillance cells in the central nervous system (CNS), the inflammatory potential of NS3 on microglia was studied. Role of toll like receptor (TLR) ligands Pam2CSK3 and Pam3CSK4 in controlling the NS3 mediated microglial inflammation was studied using microglial cell line CHME3. METHODS: IL (Interleukin)-8, IL-6, TNF-α (Tumor nicrosis factor alpha) and IL-1ß gene expressions were measured by semi quantitative RT-PCR (reverse transcription-PCR). ELISA was performed to detect IL-8, IL-6, TNF-α, IL-1ß and IL-10 secretion. FACS (Flourescent activated cell sorting) was performed to quantify TLR1, TLR2, TLR6, MyD88 (Myeloid differntiation factor 88), IkB-α (I kappaB alpha) and pNF-κB (phosphorylated nuclear factor kappaB) expression. Immunofluorescence staining was performed for MyD88, TLR6 and NF-κB (Nuclear factor kappaB). Student's t-test or One way analysis of variance with Bonferoni post hoc test was performed and p < 0.05 was considered significant. RESULTS: Microglia responded to NS3 by secreting IL-8, IL-6, TNF-α and IL-1ß via TLR2 or TLR6 mediated MyD88/NF-κB pathway. Transcription factor NF-κB was involved in activating the cytokine gene expression and the resultant inflammatory response was controlled by NF-κB inhibitor, Ro106-9920, which is known to down regulate pro-inflammatory cytokine secretion. Activation of the microglia by TLR agonists Pam3CSK4 and Pam2CSK4 induced immune tolerance against NS3. TLR ligand treatment significantly down regulated pro-inflammatory cytokine secretion in the microglia. IL-10 secretion was suggested as the possible mechanism by which TLR agonists induced immune tolerance. NS3 as such was not capable of self-inducing immune tolerance in microglia. CONCLUSION: In conclusion, NS3 protein was capable of activating microglia and the inflammatory response could be controlled via blocking the transcription factor NF-κB, or by treating the microglia with TLR ligands which likely function via secreting anti-inflammatory cytokines such as IL-10. This can have therapeutic potential in controlling HCV mediated neuroinflammation.

Patients with dry eye without hepatitis C virus infection possess the viral RNA in their tears.

PURPOSE: Dry eye is one of the suggested extrahepatic complications associated with hepatitis C virus (HCV) infection. HCV RNA has been detected from the tear fluid of patients with chronic HCV. There has been no literature evidence on the presence of HCV RNA in the tear fluid of patients with dry eye without HCV infection. In this study, tear fluid of patients with dry eye with no HCV infection was screened for the presence of HCV RNA. METHODS: Tear fluid was collected from patients with dry eye (n = 36) and healthy controls (n = 20). Real-time polymerase chain reaction was performed to detect HCV RNA in the tear fluid. Anti-HCV enzyme-linked immunosorbent assay, alkaline phosphatase, and alanine aminotransferase tests were performed in the serum samples collected from 15 patients with dry eye. RESULTS: Viral RNA was detected in 58.3% of the patients. Serum samples collected from 15 patients with dry eye were negative for anti-HCV. Alkaline phosphatase levels were elevated in 12 of 15 patients. Alanine aminotransferase levels were normal in all 15 patients. The odds ratio for the presence of HCV RNA in patients with dry eye was 22.4. CONCLUSIONS: These results indicate a direct correlation between dry eye and HCV in non-HCV patients.

Hepatitis C virus core and NS3 antigens induced conjunctival inflammation via toll-like receptor-mediated signaling.

PURPOSE: Dry eye condition is an extrahepatic manifestation associated with chronic hepatitis C virus (HCV) infection. Since conjunctival inflammation can contribute to the dry eye condition, in the present study we analyzed the conjunctival inflammatory response to HCV core and NS3 proteins. METHODS: We used primary human conjunctival fibroblasts for our study. Cytokines were measured with enzyme-linked immunosorbent assay (ELISA). Toll-like receptor (TLR) and cell adhesion molecule gene expression patterns were analyzed with semiquantitative reverse transcription (RT)-PCR. Immunofluorescence staining was performed for the MyD88, nuclear factor-kappa B (NF-kB), and inducible nitric oxide synthase (iNOS) proteins. Nitric oxide (NO) was measured with the Griess assay; terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed for apoptosis and cell viability, respectively. RESULTS: When exposed to the HCV core and NS3 proteins, the conjunctival fibroblasts secreted interleukin-8 (IL-8), IL-6, tumor necrosis factor-alpha (TNF-α), and IL-10 in a dose-dependent manner. Various TLRs were involved in the innate immune response via MyD88 signaling without NF-kB involvement. The gene expression of cell adhesion molecules such as CD44 and ICAM-1 was upregulated, and the cells secreted NO via iNOS. As the sum of these stress responses, the cells underwent apoptosis, which eventually lead to cell death. CONCLUSIONS: HCV core and NS3 proteins induced conjunctival inflammation that may form the pathogenesis of dry eye condition.

HCV core and NS3 proteins mediate toll like receptor induced innate immune response in corneal epithelium.

Direct association of dry eye syndrome and hepatitis C virus (HCV) infection is a well established fact. In this context, the current study examines the in vitro corneal inflammatory response with respect to HCV core and NS3 antigens. Toll like receptors (TLRs) are pattern recognition receptors which can mediate innate immune response. In the present study, corneal epithelial cells responded to HCV core and NS3 proteins by secreting pro-inflammatory cytokines IL-8, IL-6 and TNF-α via TLR1, TLR2 and TLR6 mediated innate immune response. MyD88/NF-kB signalling was involved in pro-inflammatory cytokine production. Corneal epithelium synthesised nitric oxide (NO) via iNOS during HCV core and NS3 exposure. On later stages of inflammation, cells underwent apoptosis which lead to cell death. SiRNA mediated silencing of TLR1, TLR2 and TLR6 resulted in a significant down regulation of IL-8 and NO. In conclusion, this study indicates that HCV core and NS3 proteins are capable of inducing immune response in corneal epithelium which can potentiate the pathology of HCV associated dry eye condition. Blocking specific TLR response can have therapeutic application in controlling the inflammatory response associated with this dry eye condition.