Double-stranded RNA mediates interferon regulatory factor 3 activation and interleukin-6 production by engaging Toll-like receptor 3 in human brain astrocytes Introduction Toll-like receptors (TLR) play a key role in the innate immune response by recognizing pathogens. They regulate the production of inflammatory mediators and cytokines. 1 SummaryToll-like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. In this study, human brain astrocytes were found to constitutively express TLR3, and this expression was increased by interferon-c (IFN-c) or double-stranded RNA (dsRNA). Treatment employing dsRNA in astrocytes induced IFN regulatory factor 3 (IRF3) phosphorylation, dimer formation and nuclear translocation followed by STAT1 activation. This treatment also activated nuclear factor-jB, p38 and c-Jun N-terminal kinase significantly, while activating extracellular signal-regulated kinase to a lesser extent. Treatment with anti-TLR3 antibody inhibited dsRNA-mediated interleukin-6 (IL-6) production. In the presence of mitogen-activated protein kinase inhibitors, astrocytes failed to secrete IL-6 in response to dsRNA treatment. Therefore, dsRNAinduced IL-6 production is dependent on mitogen-activated protein kinases and type I IFN production is dependent on IRF3 in brain astrocytes. These results suggest that brain inflammation, which produces inflammatory cytokines and type I IFNs, may enhance TLR3 expression in astrocytes. Additionally, upregulated TLR3 might modulate inflammatory processes by producing proinflammatory cytokines.Keywords: astrocytes; cytokine; interferon-regulatory factor 3; mitogenactivated protein kinase; Toll-like receptor 3Abbreviations: CNS, central nervous system; dsRNA, double-stranded RNA; DMEM, Dulbecco's modified Eagle's minimum essential medium; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; IFN, interferon; IgG, immunoglobulin G; IL-6, interleukin-6; IRF3, interferon regulatory factor 3; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAL, MyD88-adaptor-like; MAPK, mitogen-activated protein kinase; NF-jB, nuclear factor-jB; PBS, phosphate-buffered saline; PBST, PBS-Tween-20; RT-PCR, reverse transcription-polymerase chain reaction; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; STAT1, signal transducer and activator of transcription 1; TICAM-1, Toll/interleukin-1 receptor-containing adaptor molecule-1; TIRAP, Toll/interleukin-1 receptor domain-containing adaptor protein; TLR, Toll-like receptor; TRIF, Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-b.