Differential cellular release of alarmins (DAMPs) by conjunctival and corneal epithelial cells in hyperosmolar context leads to an inflammatory response of macrophage: test of specific membranous receptors implication

Abstract

Purpose : Alarmins (or DAMP: Damage Associated Molecular Pattern) are well-known cellular released actors which were able to trigger sterile inflammatory as encountered in the dry eye pathology. The goal of this work is firstly to identify alarmins released by ocular surface cells, secondly to study their ability to activate inflammatory response of macrophages and finally to test the implication of two membranous receptors (RAGE and TLR4) in such phenomenon.

Methods : HCE (Human Corneal Epithelial) and WKD (Wong-Kilbourne derivative of Chang conjunctival cell) were cultured in normal medium or hyperosmolar medium (addition of 70 mM or 90 mM NaCl). Western-Blot and Elisa tests were performed to analyze alarmin releases in culture media of corneal and conjunctival cells exposed to normal or hyperosmolar conditions for 48h (n=4 independent experiments). Conditioned media coming from HCE or WKD, used for alarmins quantification, were applied on THP1-differentiated macrophages for 24h in the presence or not of RAGE or TLR4 inhibitors (RAP and TAK respectively) (n=4 independent experiments). Then, intracellular cytokines synthesis analysis was performed by Multiplex assay. Statistical analyses for quantitative experiments were conducted using non-parametric test Kruskal-Wallis and Mann-Whitney U-test.

Results : Ocular surface epithelial cells leads to the production and release of alarmins such as HMGB1, S100A4 for both cell types and S100A8 and S100A9 specifically for WKD cells. Both cell-specific conditioned media exposition of macrophagic cells significantly induces various pro-inflammatory cytokines production (IL6 and IL8) which seems to be independent, in our conditions, of both membranous receptors tested.

Conclusions : Our results underlined a cellular specificity of danger message releases by both epithelial surface ocular cells in hyperosmolar condition. Furthermore, conditioned media demonstrate that WDK or HCE cells are independently able to trigger an inflammatory reaction of macrophage without passing by RAGE or TLR4 activation cascades.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.