Table_13_Immunological Molecular Responses of Human Retinal Pigment Epithelial Cells to Infection With Toxoplasma gondii.XLSX

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刚地弓形虫（Toxoplasma gondii）感染最常见的临床表现。活动性眼弓形虫病以刚地弓形虫速殖子在视网膜内复制并伴随反应性炎症为特征。多功能视网膜色素上皮（retinal pigment epithelium, RPE）是刚地弓形虫的关键靶细胞群。由于全局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，我们对刚地弓形虫速殖子感染后的人源细胞开展了RNA测序（RNA-Sequencing, RNA-Seq）。本研究纳入3具尸体供者眼部来源的原代细胞分离株，以及ARPE-19人视网膜色素上皮细胞系；将上述样本以感染复数为5的GT-1株刚地弓形虫速殖子感染24小时，同时设置未感染的空白对照组。从细胞中提取总RNA与小RNA，随后在Illumina NextSeq 500测序平台进行测序，测序结果比对至人类hg19参考序列。多维尺度分析结果显示，感染组与未感染组原代细胞分离株的转录组分离效果良好，随后通过edgeR软件对两组转录组进行差异表达分析。本次差异表达分析结果显示，总RNA转录组存在显著的转录应答：共计7234个基因呈现显著差异表达（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个转录本发生表达改变（占已注释转录本的0.35%），其中包含8个微小RNA（microRNA）。通过CAMERA软件对差异表达的总RNA进行基因本体论（GO）富集分析与通路富集分析，鉴定出显著的免疫相关转录组特征。为验证感染细胞的免疫活性，我们针对另外3名尸体供者来源的上皮细胞分离株（采用不同的细胞分离方案制备，但同样以刚地弓形虫感染），开展了针对26个免疫应答相关蛋白编码转录本与长链非编码转录本的实时定量PCR（RT-qPCR）验证。针对微小RNA的检测结果显示，在这3株独立细胞分离株中，有2株检测到miR-146b表达上调，3株均检测到miR-212表达上调。借助InnateDB平台开展生物学网络分析，纳入735个已注释的差异表达基因与2046个一阶互作蛋白，最终在细胞针对刚地弓形虫的转录组免疫应答中鉴定出10个核心枢纽与5个子网。本研究结果为后续探究刚地弓形虫与人视网膜色素上皮之间的分子与细胞相互作用、阐明眼弓形虫病的发病机制提供了坚实的研究基础。