Abstract
Purpose :
To study the interaction between retinal pigment epithelial cells (RPE) and uveal choroidal melanocytes (UCM) in the presence and absence of pro-inflammatory cytokines. In order to understand the homeostasis in the posterior part of the eye.
Methods :
Human ARPE19 cells (passage 20-26) were cultured at transwell inserts for two weeks and tested in combination with five primary UCMs cell cultures (derived from three women and two males, donors, age between 15 to 62 years). The UCM cells were used at passage 4-7. ARPE19 and UCM cells were cultured alone or in co-cultured for 48 hours. Cells were left unstimulated or stimulated with recombinant cytokines IFNγ (100 ng/ml), TNFα (20 ng/ml), IL1β (20 ng/ml), IL6 (20 ng/ml). Individual set ups of all five UCM was alone or in co-culture with ARPE19 was analysed. For ARPE-19 TransEpithelial Electrical Resistance (TEER) was determined and metabolic activity, using MTT were measured. Gene expression analysis using Affymetrix Microarray was performed on ARPE19 and the individual UCMs. Data were analyzed using Transcriptome Analysis Console 4.0 (TAC) with defalult settings: Fold change <-2 or >2, p-value 0.05, FDR<0.05, Anova Method eBayers.
Results :
Increased TEER of ARPE19 was observed depending on days of culture, but no significant effect was seen after co-culture with UCMs. No significant changes in metabolic activity can observed in UCMs or ARPE19 in response to co-culture.
Only three genes where differential expressed in ARPE19 after co-culture with UCM, whereas 31 genes were upregulated in UCMs in response to co-culture with ARPE19. These include FLNB and APOLD1, both related to the vascular system. A significant number of genes were upregulated both in ARPE19 (#614) and UCMs (#275) in response to pro-inflammatory cytokines. Whereas an increased number of genes (#685) were, upregulated in UCMs after co-culture with ARPE19 and pro-inflammatory cytokines. An inverse correlation was found for ARPE19, when comparing number of up-regulated genes after stimulation with pro-inflammatory cytokines in monoculture to co-culture with UCMs in combination with pro-inflammatory cytokines, the numbers was 1042 and 794, respectively.
Conclusions :
Our results demonstrate that cross-talk between UCMs and ARPE19 in vitro leading to differential gene expression can occur depending on soluble factors, which can be influenced by the presence of pro-inflammatory cytokines.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.