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Liam M. Ashander, Rebecca A. Lindsay, Joao M. Furtado, Yuzhen Pan, Justine R. Smith; Infection of Human Retinal Pigment Epithelium with Natural Isolates of Toxoplasma gondii. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2963.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal destruction in ocular toxoplasmosis results from Toxoplasma gondii-induced cell lysis and host immune responses. The retinal pigment epithelium (RPE) participates in ocular immune privilege and might protect the eye in this disease. We studied the ability of natural T. gondii isolates to replicate within human RPE, comparing common haplogroup 1 strain (GT-1) with two ‘exotic’ haplogroup 6 strains (TgCatBR2 & GPHT), and measured expression of suppressor of cytokine signalling (SOCS) proteins after infection.
Human RPE was isolated from digested human retina-choroid by selective cloning based on morphology. Purity was assessed at 99% by immunostaining for RPE65. Cells (passage 8-9) were plated at confluence in DMEM + 10% heat-inactivated FBS + antibiotics, incubated for 72 hours at 37oC to promote formation of intercellular junctions, and subsequently infected at MOI of 5:1 with freshly egressed GT-1, TgCatBR2 or GPHT tachyzoites in serum-free DMEM for 4 hours. Parallel control cultures of RPE were maintained uninfected. Following washing, cultures were incubated for up to 48 hours at 37oC. At 4, 24 and 48 hours post-infection, RPE was fixed in formalin, immunostained with Alexafluor 594-tagged anti-human ZO-1, and imaged at 630x magnification by dark field and epifluoresence microscopy to identify tachyzoites and cell borders, respectively. Numbers of tachyzoites/infected cell were quantified from the images. At 4 hours post-infection, total RNA was extracted from RPE, and relative expression of SOCS1 and SOCS3, normalized to GAPDH, was determined by qRT-PCR using the Chromo4 Thermocycler.
Human RPE was relatively resistant to infection with T. gondii, with negligible cell lysis and modest tachyzoite replication over 48 hours. There was a significant difference in replication of different strains by 48 hours (mean tachyzoites/cell: GT-1, 31; GPHT, 57; TgCatBR2, 16; n=4 cultures; p = 0.05 by ANOVA). SOCS1, but not SOCS3, was up-regulated in RPE at 4 hours in infected versus uninfected cultures, and there was a difference between strains (mean fold-increase GT-1, 18.0; TgCatBR2, 4.6; and GPHT, 10.0; n=3 cultures; p<0.001 by ANOVA). Plaque assays indicated 13-24% tachyzoite viability at the time of infection, consistent with published studies using natural isolates.
Human RPE limits intracellular proliferation of T. gondii tachyzoites and produces immunomodulatory SOCS1 when infected, in a strain-dependent manner. Our results suggest that RPE may act to limit damage to retina and adjacent tissues in ocular toxoplasmosis.
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