Immunological Molecular Responses of Human Retinal Pigment Epithelial Cells to Infection With Toxoplasma gondii

Abstract

Ocular toxoplasmosis is the commonest clinical manifestation of infection with obligate intracellular parasite, Toxoplasma gondii . Active ocular toxoplasmosis is characterized by replication of T. gondii tachyzoites in the retina, with reactive inflammation. The multifunctional retinal pigment epithelium is a key target cell population for T. gondii . Since the global gene expression profile is germane to understanding molecular involvements of retinal pigment epithelial cells in ocular toxoplasmosis, we performed RNA-Sequencing (RNA-Seq) of human cells following infection with T. gondii tachyzoites. Primary cell isolates from eyes of cadaveric donors ( n = 3), and the ARPE-19 human retinal pigment epithelial cell line, were infected for 24 h with GT-1 strain T. gondii tachyzoites (multiplicity of infection = 5) or incubated uninfected as control. Total and small RNA were extracted from cells and sequenced on the Illumina NextSeq 500 platform; results were aligned to the human hg19 reference sequence. Multidimensional scaling showed good separation between transcriptomes of infected and uninfected primary cell isolates, which were compared in edgeR software. This differential expression analysis revealed a sizeable response in the total RNA transcriptome—with significantly differentially expressed genes totaling 7,234 (28.9% of assigned transcripts)—but very limited changes in the small RNA transcriptome—totaling 30 (0.35% of assigned transcripts) and including 8 microRNA. Gene ontology and pathway enrichment analyses of differentially expressed total RNA in CAMERA software, identified a strong immunologic transcriptomic signature. We conducted RT-qPCR for 26 immune response-related protein-coding and long non-coding transcripts in epithelial cell isolates from different cadaveric donors ( n = 3), extracted by a different isolation protocol but similarly infected with T. gondii , to confirm immunological activity of infected cells. For microRNA, increases in miR-146b and miR-212 were detected by RT-qPCR in 2 and 3 of these independent cell isolates. Biological network analysis in the InnateDB platform, including 735 annotated differentially expressed genes plus 2,046 first-order interactors, identified 10 contextural hubs and 5 subnetworks in the transcriptomic immune response of cells to T. gondii . Our observations provide a solid base for future studies of molecular and cellular interactions between T. gondii and the human retinal pigment epithelium to illuminate mechanisms of ocular toxoplasmosis.

Systems Biology Approaches to Understanding the Human Immune System

“…Indeed, IRF5 −/− and IL-10RA −/− mutant iPSDM cells were both shown to have increased susceptibility to C. trachomatis infection. These results, along with numerous other published studies [e.g., (73)(74)(75)(76)(77)], demonstrate that multi-omics integration using PPI networks can reveal novel insight into the factors that play a significant role in the host immune response to infections.…”

Neutrophil Activities in Human Ocular Toxoplasmosis: An In Vitro Study With Human Cells

“…Instead, this work indicates that neutrophils are likely to participate actively in an inflammatory response to T. gondii, which often is aggressive and may contribute to retinal damage, 3 through their interaction with the retinal pigment epithelium, which is an intraocular target for the parasite. 14 Monocytes and dendritic cells have been identified as leukocytes with capacity to carry T. gondii tachyzoites from the gut via the circulation into target tissues. 23 Using a transwell system, we have shown that dendritic cells infected with tachyzoites use Ig superfamily adhesion molecules to move the parasite across human retinal endothelial cell monolayers, simulating movement from the bloodstream into the retina.…”

“…Human retinal pigment epithelial cells were isolated from human cadaver donor eyes. The method, which we have reported previously, 14 yields cells that express cytokeratin-8, retinal pigment epithelium-specific protein 65, cellular retinaldehyde-binding protein, and zonula occludens 1, but not asmooth muscle actin, which is indicative of mesenchymal differentiation. In brief, the retinal pigment epithelium-choroid was digested with 0.5 mg/mL collagenase IA and 0.5 mg/mL collagenase IV, and epithelial sheets were isolated by scraping free from Bruch's membrane followed by sucrose density gradient centrifugation.…”

“…The retinal pigment epithelium is a principal target for T. gondii tachyzoites in ocular toxoplasmosis. 14 We examined the activities of neutrophils in response to T. gondii in a coculture system with retinal pigment epithelial cells that had been infected with GT-1 strain T. gondii tachyzoites 24 hours previously. In the first series of experiments, the ARPE-19 human retinal pigment epithelial cell line was used ( Figs.…”

“…During a microbial infection, neutrophils swarm to the area, where they release granules containing antimicrobial molecules and other Copyright 2019 The Authors iovs.arvojournals.org j ISSN: 1552-5783 proteins that promote an immune response, including reactive oxygen species (ROS) and inflammatory cytokines. 11 In the posterior eye, the retinal pigment epithelium is a key target cell for T. gondii, 5,13,14 and has capacity to promote or suppress neutrophil activity, by synthesis of diverse inflammatory mediators or immuno-modulatory products, respectively. [15][16][17] We explored the potential activities of neutrophils in human ocular toxoplasmosis, using neutrophils isolated from human peripheral blood in Bowden transwell migration assays, and in activation assays that involved coculture with T. gondiiinfected human retinal pigment epithelial cells.…”