title: Hepatitis C Virus NS3 Mediated Microglial Inflammation via TLR2/TLR6 MyD88/NF-κB Pathway and Toll Like Receptor Ligand Treatment Furnished Immune Tolerance.
abstract: 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-1β gene expressions were measured by semi quantitative RT-PCR (reverse transcription-PCR). ELISA was performed to detect IL-8, IL-6, TNF-α, IL-1β 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-1β 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: L & T Microbiology Research Centre, Vision Research Foundation, Chennai, India; Centre for Nanotechnology and Advanced Biomaterials, SASTRA University, Thanjavur, India.
authors.forename: Ayilam Ramachandran
authors.initials: AR
authors.lastname: Rajalakshmy
authors.affiliation: L & T Microbiology Research Centre, Vision Research Foundation, Chennai, India.
authors.forename: Jambulingam
authors.initials: J
authors.lastname: Malathi
authors.affiliation: L & T Microbiology Research Centre, Vision Research Foundation, Chennai, India.
authors.forename: Hajib Naraharirao
authors.initials: HN
authors.lastname: Madhavan
mesh_terms: D002460:Cell Line; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D016174:Hepacivirus / Q000276:immunology*; D006801:Humans; D007249:Inflammation / Q000276:immunology* / Q000821:virology; D017628:Microglia / Q000276:immunology / Q000821:virology*; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051193:Toll-Like Receptors / Q000819:agonists; D017361:Viral Nonstructural Proteins / Q000276:immunology*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D016207:Cytokines; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D051193:Toll-Like Receptors; D017361:Viral Nonstructural Proteins; C507779:MYD88 protein, human; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases