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L. E. Munoz-Erazo, R. Natoli, A. W. S. Yeung, J. M. Provis, M. C. Madigan, S. R. Thomas, N. J. C. King; Microarray Analysis of Human Retinal Pigmented Epithelium Following Infection With West Nile Virus. Invest. Ophthalmol. Vis. Sci. 2010;51(13):411.
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Several studies have investigated the role of viral infection of the retinal pigment epithelium (RPE) in the pathogenesis of macular degeneration (AMD). We used a gene array approach to identify inflammatory genes expressed by RPE cells following West Nile virus (WNV) infection.
Monolayers of primary human RPE (n=4) and ARPE19 cells were infected with WNV at a multiplicity of infection (MOI) of 1 for 24 hrs. mRNA was extracted from uninfected and infected cells and reverse transcribed for microarray processing. Following subsequent analysis of the microarray data, 20 genes of interest were selected from the differentially regulated genes, along with 7 related genes, and qRT-PCR was performed to confirm the microarray data. For one of the differentially regulated genes, indolamine 2,3 dioxygenase (IDO), HPLC was used to determine levels of tryptophan and kynurenine in supernatant from infected primary RPE cells at 24, 48 and 72 hours.
A number of viral response genes were upregulated, including IDO and TLR3. Microarray and qRT-PCR data showed an increase in IDO mRNA at 24hrs post infection, however IDO activity was not detected until 48hrs post infection. TLR3 activation by double stranded RNA (such as WNV) can induce RPE apoptosis. Other gene clusters of interest include oxidative stress genes, immune privilege genes and genes involved in the TGF-beta pathway. Innate immunity complement proteins identified in AMD such as CFB, CFH and C3 were also upregulated. In addition, chemokines that are chemotactic for a variety of leukocytes, including CCL5, CCL2 and IL8 were upregulated.
Viral infection of the RPE induces expression of a number of response genes, including several implicated in the pathogenesis of AMD. These findings support the suggestion that viral infection plats a role in the pathogenesis and/or progression of AMD.
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