Contribution of IPS-1 to polyI:C-induced cytokine production in conjunctival epithelial cells.

We previously demonstrated that ocular surface epithelium expressed TLR3 and that its ligand, polyI:C, stimulation induced the secretion of inflammatory cytokines and type I IFN. It was recently reported that RIG-I and MDA5 also recognize viral dsRNA mimicking polyI:C. In this study, we investigated whether RIG-I and/or MDA5 contribute to polyI:C-inducible responses in conjunctival epithelium. The expression of RIG-I, MDA5, and TLR3 in human conjunctival epithelium was examined by RT-PCR and their up-regulation after polyI:C stimulation by quantitative RT-PCR and immunoblot analysis. Human conjunctival epithelial cells also expressed RIG-I, MDA5 and TLR3 mRNA and protein. The expression of RIG-I and MDA5, but not of TLR3, was markedly up-regulated upon polyI:C stimulation. We also examined the function of IPS-1 (an adaptor molecule common to RIG-I and/or MDA5) and TLR3 in conjunctival epithelium using IPS-1 KO and TLR3 KO mice. To analyze in vivo murine conjunctival epithelial cells, 10 μl of a 100 μg/ml polyI:C solution were delivered subconjunctivally and as eye drops, then conjunctival epithelial cells were subjected to gene expression analysis. We focused on 10 transcripts up-regulated in murine conjunctival epithelium upon polyI:C stimulation. Cxcl10, Mx1, Ifi44, Ifi203, Iigp2 and Rtp4 were dominantly regulated by IPS-1, Ccl5 by TLR3, and Rsad2, Mx2 and Cmpk2 were regulated by TLR3 and IPS-1. Our results showed that conjunctival epithelial cells express RIG-I and MDA5, and IPS-1, an adaptor molecule common to RIG-I and MDA5, contributes to polyI:C-inducible cytokine production in conjunctival epithelial cells.