Expression of complement and other inflammatory pathway genes is co-ordinated in the human RPE cell transcriptome.

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.