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Elise Rochet, Shervi Lie, Yuefang Ma, Liam M. Ashander, Audra M. A. Shadforth, Timothy A. Blenkinsop, Binoy Appukuttan, Beth Wilmot, Justine R. Smith; Gene Expression Changes in Human Retinal Pigment Epithelial (hRPE) Cells Following Infection with Toxoplasma gondii. Invest. Ophthalmol. Vis. Sci. 2018;59(9):499.
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© ARVO (1962-2015); The Authors (2016-present)
Ocular toxoplasmosis – caused by retinal infection with T. gondii – is a major cause of vision impairment worldwide in both immuno- compromised and competent individuals. Underlying pathophysiological mechanisms in the human are poorly understood, and available drug treatments do not prevent recurrent attacks of retinal inflammation. We investigated differential gene expression by hRPE cells infected with T. gondii to elucidate parasite-host cell interactions in the immune privileged retina.
One group of hRPE cells, harvested from 3 cadaveric donor eye-pairs, was infected with T. gondii natural virulent strain GT-1 (MOI = 5:1, parasite viability > 20%) or mock-infected (singlet samples). Total RNA was isolated 24 hours post-infection. RNA sequencing (RNA-seq) was performed on the Illumina NextSeq platform: samples were aligned to human hg19 and mirbase, and T. gondii reference sequences, and data were normalized in EdgeR. False Discovery Rate (FDR) < 0.05 defined significantly different expression between infected and mock-infected samples. To validate RNA-seq results by RT-qPCR, infections were repeated with hRPE cells isolated from 3 additional donor eye-pairs (quadruplicate samples). Protein coding and non-coding RNA were selected for validation based on documented involvement in the immune response, significant expression difference, and > 2 or < (-2) fold-change (FC).
A total of 7234 genes were significantly differentially expressed between T. gondii- and mock- infected hRPE cells: 1968 had FC > 2 and 1739 had FC < (-2), indicating 53% of differentially expressed genes were upregulated in infected cells. Thirty transcripts were small RNA, including 8 microRNA: 26 had FC > 2 and 4 had FC < (-2). Fold-changes of 15 protein-coding RNA (TGFb2, TSP1, IL6, ANGTPL7, IL1RN, PTGS2, ICAM1, CXCL3, CCL2, NFKB1, BIRC3, TSLP, IL17RB, LRP8 and IL11) and 4 long non-coding RNA (MIR155HG, LINC01105, LINC00968 and MALAT1) involved in the immune response were confirmed by RT-qPCR.
Our findings highlight the large number of genes that are modulated during T. gondii infection of human retinal pigment epithelium. Future studies will focus on gene ontology, pathway and network analyses to decipher inflammatory mechanisms involved in the pathogenesis of ocular toxoplasmosis. These results could provide clues to more effective treatments for infection with T. gondii.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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