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

Shervi Lie, Elise Rochet, Erik Segerdell, Yuefang Ma, Liam M. Ashander, Audra M.A. Shadforth, Timothy A. Blenkinsop, Michael Z. Michael, Binoy Appukuttan, Beth Wilmot, Justine R. Smith

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Number of pages1
JournalFrontiers in immunology
Volume10
DOIs
StatePublished - Jan 1 2019

Fingerprint

Retinal Pigments
Toxoplasmosis
Toxoplasma
Ocular Toxoplasmosis
Epithelial Cells
Transcriptome
RNA
Retinal Pigment Epithelium
MicroRNAs
Software
RNA Sequence Analysis
Gene Ontology
Health Services Needs and Demand
Infection
Genes
Retina
Parasites
Inflammation
Cell Line

Keywords

  • gene ontology
  • human
  • infection
  • network
  • pathway
  • retinal pigment epithelium
  • RNA-sequencing
  • Toxoplasma gondii

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

Immunological Molecular Responses of Human Retinal Pigment Epithelial Cells to Infection With Toxoplasma gondii. / Lie, Shervi; Rochet, Elise; Segerdell, Erik; Ma, Yuefang; Ashander, Liam M.; Shadforth, Audra M.A.; Blenkinsop, Timothy A.; Michael, Michael Z.; Appukuttan, Binoy; Wilmot, Beth; Smith, Justine R.

In: Frontiers in immunology, Vol. 10, 01.01.2019.

Research output: Contribution to journalArticle

Lie, S, Rochet, E, Segerdell, E, Ma, Y, Ashander, LM, Shadforth, AMA, Blenkinsop, TA, Michael, MZ, Appukuttan, B, Wilmot, B & Smith, JR 2019, 'Immunological Molecular Responses of Human Retinal Pigment Epithelial Cells to Infection With Toxoplasma gondii', Frontiers in immunology, vol. 10. https://doi.org/10.3389/fimmu.2019.00708
Lie, Shervi ; Rochet, Elise ; Segerdell, Erik ; Ma, Yuefang ; Ashander, Liam M. ; Shadforth, Audra M.A. ; Blenkinsop, Timothy A. ; Michael, Michael Z. ; Appukuttan, Binoy ; Wilmot, Beth ; Smith, Justine R. / Immunological Molecular Responses of Human Retinal Pigment Epithelial Cells to Infection With Toxoplasma gondii. In: Frontiers in immunology. 2019 ; Vol. 10.
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