The role of melanocytes in the human choroidal microenvironment and inflammation: Insights from the transcriptome.

The choroid within the human eye contains a rich milieu of cells including melanocytes. Human choroidal melanocytes (HCMs) absorb light, regulate free radical production, and were recently shown to modulate inflammation. This study aimed to identify key genes and pathways involved in the inflammatory response of HCMs through the use of RNA-seq. Primary HCMs were cultured from donor choroids, RNA was extracted from control and lipopolysaccharide (LPS)-treated HCMs, and mRNA was sequenced. Functional annotation and pathway analysis were performed using gene ontology and gene set enrichment analyses. Representative RNA-seq results were verified with RT-qPCR and protein measurements. We detected 100 differentially expressed genes including an array of CCL and CXCL cytokines and mediators of cell-cell and cell-matrix adhesion, such as ICAM1, CLDN1, CCN3, ITGA1 and ITGA11. Functional annotation showed that these gene sets control inflammatory pathways, immune cell trafficking, cell-cell adhesion, interactions with the extracellular matrix and blood vessels, angiogenesis and epithelial-to-mesenchymal transitions. Our study provides insights into the transcriptional regulation of primary HCMs in response to inflammatory stimuli and identifies novel melanocyte-driven mechanisms potentially involved in choroidal homeostasis and inflammation.

Extracellular matrix and oxidative stress regulate human retinal pigment epithelium growth.

Age-related macular degeneration (AMD), the most common cause of vision loss with ageing, is characterised by degeneration of the photoreceptors and retinal pigment epithelium (RPE) and changes in the extracellular matrix (ECM) underlying the RPE. The pathogenesis of AMD is still not fully understood. In this study we investigated the in vitro growth and function of primary human RPE cells in response to different ECM substrates, including nitrite-modified ECM. We initially confirmed the presence of disorganised retinal glial and photoreceptor cells, marked retinal cytoplasmic and Bruch's membrane expression of nitro-tyrosine (an oxidative stress marker) and increased numbers of Iba1+ macrophages/microglia in human donor eye sections (aged and AMD) using multi-marker immunohistochemistry (n = 3). Concurrently, we utilised two-photon microscopy to reveal topographical changes in flatmounts of RPE-associated ECM and in the underlying choroid of aged and AMD donor eyes (n = 3). To recapitulate these observations in vitro, we then used primary human RPE cells to investigate how different ECM proteins, including nitrite cross-linked RPE-secreted ECM, modified RPE cell growth and function. Collagen I or IV increased RPE attachment and spreading two-to three-fold, associated with significantly increased cell migration and proliferation, consistent with a preferential interaction with these matrix substrates. Primary human RPE cells grown on collagen I and IV also showed increased secretion of pro-inflammatory cytokines, MCP-1 and IL-8. Nitrite-modification of RPE-secreted ECM (simulating ageing of Bruch's membrane) significantly reduced in vitro RPE attachment to the ECM and this was mitigated with collagen IV coating of the modified ECM. Taken together, our observations confirm the importance of RPE-ECM interactions for normal RPE growth and function, and for inducing RPE secretion of pro-inflammatory cytokines. Furthermore, the findings are consistent with ageing and/or oxidative stress-induced disruption of RPE-ECM interactions contributing to the pathogenesis of AMD.