Abstract
Purpose :
In the human choroid, melanocytes (HCMs) contribute functions including melanin-related light absorption and uptake of free radicals, with a potential for immunoregulation via local secretion of chemokines/cytokines and expression of Toll-like receptors (TLRs). The biological responses of HCMs to a proinflammatory stimulus (lipolysaccharide (LPS) - a TLR4 ligand), related to possible roles in the choroidal stromal microenvironment were studied using transcriptome and pathway analyses, and protein-based methods.
Methods :
Primary HCMs isolated from human donor eyes (n=4; <18 hours post-mortem delay; University of Sydney HREC) were established in melanocyte growth medium. RNA from HCMs (P3 to P5: control and LPS-stimulated (1μg/ml,18 hours) was used to establish cDNA libraries, that were sequenced (Illiumina HiSeq 2500) and analysed using bioinformatics (including gene ontology (GO) and gene set enrichment analysis. We confirmed several novel cell-cell adhesion and cell-extracellular matrix (ECM) genes using immunoblotting and immunohistochemistry.
Results :
100 differentially expressed genes were detected (98 genes upregulated, 2 genes downregulated; fold change >±2, p<0.01) were found for control versus LPS-stimulated HCMs. As expected, a number of most differentially expressed genes included CCL and CXCL cytokines (for example, CCL20, CCL2 and CXCL5; 43 to 316-fold, p<0.01). Genes that can mediate intercelleular adhesion and cell-ECM interactions also were highlighted (for example, ITGA1 and 11, CCN3, MMP14, COL7A1 and FN1; 1.4 to 23-fold; p<0.01). We also verified several LPS-induced genes, finding changes in HCM protein expresson for CCN3, MMP14, FN1 and ICAM1. We also confirmed in situ immunolabelling in donor human eye choroid sections with confocal microscopy.
Conclusions :
Our study confrims a proinflammatory shift in biological processes and genes for LPS-stimulated HCMs, Furthermore, we observed gene and protein expression patterns not previously highlghted, including significant GO terms related to "cellular adhesion" and "cell-matrix adhesion", "cell adhesion mediated by integrins" and "collagen binding involved in cell matrix adhesion". Taken together, these observations suggest HCMs can contribute more to the complex choroidal biology than light-absorption, with roles in maintaining and regulating the local choroidal microenvironment, normally and during inflammatory conditions.
This is a 2021 ARVO Annual Meeting abstract.