June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Expression of complement and other inflammatory pathway genes is co-ordinated in the human RPE cell transcriptome.
Author Affiliations & Notes
  • Paul N Bishop
    Faculty of Biology, Medicine & Health, University of Manchester, Manchester, United Kingdom
    Manchester Royal Eye Hospital, CMFT, Manchester Academic Health Sciences Centre, Manchester, United Kingdom
  • Selina McHarg
    Faculty of Biology, Medicine & Health, University of Manchester, Manchester, United Kingdom
  • Nicole Brace
    Faculty of Biology, Medicine & Health, University of Manchester, Manchester, United Kingdom
  • Alexander W.W. Langford-Smith
    Healthcare Science Research Institute, Manchester Metropolitan University, Manchester, United Kingdom
  • Richard Unwin
    Faculty of Biology, Medicine & Health, University of Manchester, Manchester, United Kingdom
  • Rahat Perveen
    Faculty of Biology, Medicine & Health, University of Manchester, Manchester, United Kingdom
    St. Mary's Hospital, CMFT, Manchester Academic Health Sciences Centre, Manchester, United Kingdom
  • Graeme C. Black
    Faculty of Biology, Medicine & Health, University of Manchester, Manchester, United Kingdom
    St. Mary's Hospital, CMFT, Manchester Academic Health Sciences Centre, Manchester, United Kingdom
  • Anthony Day
    Faculty of Biology, Medicine & Health, University of Manchester, Manchester, United Kingdom
  • Simon John Clark
    Faculty of Biology, Medicine & Health, University of Manchester, Manchester, United Kingdom
  • Footnotes
    Commercial Relationships   Paul Bishop, None; Selina McHarg, None; Nicole Brace, None; Alexander Langford-Smith, None; Richard Unwin, None; Rahat Perveen, None; Graeme Black, None; Anthony Day, None; Simon Clark, None
  • Footnotes
    Support  Supported by Fight for Sight (1517/1518) and The Macular Society (12928)
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3378. doi:
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      Paul N Bishop, Selina McHarg, Nicole Brace, Alexander W.W. Langford-Smith, Richard Unwin, Rahat Perveen, Graeme C. Black, Anthony Day, Simon John Clark; Expression of complement and other inflammatory pathway genes is co-ordinated in the human RPE cell transcriptome.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3378.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : To investigate the expression of complement genes in primary cultures of human RPE cells.

Methods : RPE cells isolated from adult human donor eye tissue were cultured for 7-10 days prior to RNA extraction. Quantitative polymerase chain reaction (qPCR) was used to determine the expression of a range of complement genes including complement factor H (CFH) and complement component C3 (C3). Illumina Hi-Seq RNA expression analysis was performed on RNA from 12 x RPE cultures; 6 x expressing high levels of CFH and C3 and 6 x expressing low levels.

Results : qPCR analysis of 108 primary human RPE cell cultures from different donors demonstrated that the expression levels of CFH and C3 correlate significantly (R2 = 0.521, p < 0.0001). This observation was investigated further by RNA-seq transcriptome analysis. RNA-seq detected, in total, RNA from 14,187 genes (13,141 of which were protein-coding) expressed in RPE cell cultures. Ingenuity Pathways Analysis identified 1289 genes which were differentially expressed (fold change > 50%, p < 0.05) in RPE cells which had high CFH and C3 expression, versus those with low expression. The complement pathway was shown to be the most upregulated canonical pathway, with 76% of complement genes detected being significantly increased in expression in high expressers of CFH and C3. Several canonical pathways relating to inflammation and immunology were upregulated in donors with elevated CFH/C3 expression, with the highest fold gene changes in cytokines, transcription factors, lipid metabolism and adhesion molecules. Top upstream regulators of altered canonical pathways in the high CFH/C3 group included TNF, IL1B, IFNG and TGFB. Spearman’s rank analysis against CFH and C3 expression similarly identified members of the TNF superfamily, vasoregulatory and retinoic acid metabolism genes as significantly correlating with altered expression (p < 0.001).

Conclusions : Expression of the majority of complement genes detected in primary cultures of RPE cells is co-ordinated. This co-ordination of expression extends to genes involved in other pathways including cytokines, transcription factors, lipid metabolism and adhesion molecules.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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