Cytokine gene expression and protein production in West Nile virus‐infected retinal pigment epithelium

Abstract

AbstractPurpose To investigate if West Nile virus (WNV) infection of human retinal pigment epithelium (RPE) cells induces differential expression of mRNA and proteins for cytokines important for leucocyte recruitment (e.g. CCL5) and integrity of the outer blood retinal barrier (BRB) (e.g. TNF).Methods Primary human RPE cells (P3‐5) were cultured until a complete monolayer was observed, and then infected with 1, 5 or 10 plaque‐forming units (p.f.u.) of WNV (Sarafend) for 24, 48 and 72hrs. Supernatants were collected for Luminex‐based cytokine assay. RNA was prepared from cells and processed for real‐time (RT)‐PCR. Pro‐inflammatory, leucocyte recruitment and viral defense cytokines and chemokines were studied.Results CCL5, IL8, IL6 (pro‐inflammatory) and IFNB1 (anti‐viral) showed increases in mRNA expression from 24 h post‐infection (p.i.) and protein levels from 48h p.i. However in WNV‐infected RPE, protein levels of VEGF and TNF were similar to mock‐infected cells, despite significant increases in mRNA (TNF 24 h p.i., p <0.05; VEGF 48 h, p<0.01). Interestingly, CCL2 mRNA and protein showed no change in expression post‐WNV infection at 24, 48 and 72hConclusion We previously observed that WNV induced transepithelial electrical resistance (TEER) changes in human RPE (outer BRB) after 48 hours, although the mechanisms(s) remain unclear. Studies in mice show that both TNF and CCL2 are important for blood‐brain barrier integrity and leucocyte recruitment following WNV infection. Surprisingly we find little or no effect on TNF or CCL2 expression in WNV‐infected RPE, suggesting alternate mechanisms operate in RPE post‐viral infection. Funding‐ NFMRI and NHMRC