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Elise Rochet, Yuefang Ma, Liam M. Ashander, Binoy Appukuttan, Justine R Smith; Understanding the Role of Retinal Müller Glial Cells in Ocular Toxoplasmosis. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4648.
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© ARVO (1962-2015); The Authors (2016-present)
Ocular toxoplasmosis is the most common infectious posterior uveitis globally, responsible for vision loss in up to one-quarter of affected eyes. Understanding the role of different retinal cells in this disease may suggest new options for treatment. We investigated the response of human retinal Müller glial cells to infection with the parasite, Toxoplasma gondii, which causes ocular toxoplasmosis.
Müller cells were isolated from human cadaveric donor eye-pairs by culture of Dispase II-digested and triturated retinal tissue in Dulbecco's modified Eagle medium with Non-Essential Amino Acid and GlutaMAX supplements (Thermo Fisher Scientific), 1 mM sodium pyruvate and 10% fetal bovine serum, with 50% of medium replaced biweekly. Confluent cells were infected with freshly egressed T. gondii tachyzoites (natural virulent strains, GT-1 [haplogroup 1] and GPHT [haplogroup 6], generously gifted by Drs. JP Dubey and LD Sibley) (multiplicity of infection=5:1) or mock-infected (n=6 isolates). Cell phenotype was assessed by immunolabeling for glutamine synthase, CRALBP, vimentin and GFAP. Parasite viability was assessed in a fibroblast plaque assay. Total RNA was isolated 24 hours post-infection. Real-time quantitative RT-PCR was used to examine expression of immunologic molecules. Transcript expression by infected and uninfected cells was compared by paired t-test (p<0.05 indicating difference).
Müller cell isolates were positively immunolabeled for glutamine synthase, CRALBP, vimentin and GFAP, confirming phenotype. Parasite viability was >35%, which is above expected for natural strains. In response to GT-1 and GPHT infections, cells increased expression of IL-6, CCL2, NFκB, and decreased expression of TGF-β2 and the lncRNA, FoxD3-AS1. CCL5, CCL7, CXCL8, CXCL10, and PD-L1 transcripts and mir-212 were upregulated, and IL-12A transcript was downregulated in GT-1-infected cells. mir-146b was downregulated in GPHT-infected cells. No change in the level of transcript was measured at 24 hours post-infection for TNF-α, IL-10 and iNOS, which are expressed by other cell types after T. gondii infection.
Our findings show that human retinal Müller glial cells may participate in the immune response against T. gondii, and this response may differ with parasite strain. Future molecular studies will involve RNA-sequencing, to further elucidate the potential role of Müller cells in ocular toxoplasmosis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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