Data_Sheet_1_Immunological Molecular Responses of Human Retinal Pigment Epithelial Cells to Infection With Toxoplasma gondii.PDF

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.

眼弓形虫病是专性胞内寄生虫弓形虫（Toxoplasma gondii）感染最常见的临床表现。活动性眼弓形虫病以弓形虫速殖子（tachyzoites）在视网膜内复制并引发反应性炎症为特征。具有多种功能的视网膜色素上皮细胞（retinal pigment epithelium）是弓形虫的关键靶细胞群。由于全基因表达谱有助于理解视网膜色素上皮细胞在眼弓形虫病中的分子作用机制，我们对感染弓形虫速殖子的人类细胞开展了RNA测序（RNA-Sequencing, RNA-Seq）。我们将来自3名遗体捐赠者眼部的原代细胞分离株，以及ARPE-19人视网膜色素上皮细胞系，以感染复数（multiplicity of infection, MOI）=5的弓形虫GT-1株速殖子感染24小时，同时设置未感染的对照组细胞进行孵育。从细胞中提取总RNA和小RNA后，在Illumina NextSeq 500平台上进行测序，并将测序结果比对至人类hg19参考基因组序列。多维尺度分析显示，感染组与未感染组的原代细胞分离株转录组之间存在良好的分离效果，我们通过edgeR软件对二者进行了差异表达分析。此次差异表达分析显示，总RNA转录组存在显著的应答反应：共筛选得到7234个显著差异表达基因，占已注释转录本的28.9%；而小RNA转录组的变化则极为有限：仅30个转录本发生显著变化，占已注释转录本的0.35%，其中包含8个微RNA（microRNA）。我们通过CAMERA软件对差异表达的总RNA进行基因本体论（Gene Ontology, GO）和通路富集分析，发现了显著的免疫相关转录组特征。为验证感染细胞的免疫活性，我们对另外3名不同遗体捐赠者来源的上皮细胞分离株（采用不同的分离方法制备，但同样感染了弓形虫）中的26个免疫相关蛋白编码转录本和长链非编码RNA转录本进行了实时定量聚合酶链反应（RT-qPCR）验证。针对微RNA的RT-qPCR检测显示，在这3株独立的细胞分离株中，分别有2株和3株检测到miR-146b与miR-212的表达上调。我们在InnateDB平台上开展了生物学网络分析，纳入735个已注释的差异表达基因以及2046个一级互作蛋白，最终在细胞针对弓形虫的转录组免疫应答中鉴定出10个核心枢纽和5个子网。本研究结果为后续探究弓形虫与人视网膜色素上皮细胞之间的分子与细胞互作、阐明眼弓形虫病的发病机制提供了坚实的研究基础。