In Vitro Inhibition of NFAT5-Mediated Induction of CCL2 in Hyperosmotic Conditions by Cyclosporine and Dexamethasone on Human HeLa-Modified Conjunctiva-Derived Cells

Abstract

PurposeTo investigate the pro-inflammatory intracellular mechanisms induced by an in vitro model of dry eye disease (DED) on a Hela-modified conjunctiva-derived cells in hyperosmolarity (HO) stress conditions. This study focused on CCL2 induction and explored the implications of the nuclear factor of activated T-cells 5 (NFAT5) as well as mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NFĸB). This work was completed by an analysis of the effects of cyclosporine A (CsA), dexamethasone (Dex) and doxycycline (Dox) on HO-induced CCL2 and NFAT5 induction.MethodsA human HeLa-modified conjunctiva-derived cell line was cultured in NaCl-hyperosmolar medium for various exposure times. Cellular viability, CCL2 secretion, NFAT5 and CCL2 gene expression, and intracytoplasmic NFAT5 were assessed using the Cell Titer Blue® assay, enzyme-linked immunosorbent assay (ELISA), RT-qPCR and immunostaining, respectively. In selected experiments, inhibitors of MAPKs or NFκB, therapeutic agents or NFAT5 siRNAs were added before the hyperosmolar stimulations.ResultsHO induced CCL2 secretion and expression as well as NFAT5 gene expression and translocation. Adding NFAT5-siRNA before hyperosmolar stimulation led to a complete inhibition of CCL2 induction and to a decrease in cellular viability. p38 MAPK (p38), c-Jun NH2-terminal kinase (JNK) and NFĸB inhibitors, CsA and Dex induced a partial inhibition of HO-induced CCL2, while Dox and extracellular signal-regulated kinase (ERK) inhibitor did not. Dex also induced a partial inhibition of HO-induced NFAT5 gene expression but not CsA or Dox.ConclusionsThese in vitro results suggest a potential role of CCL2 in DED and highlight the crucial role of NFAT5 in the pro-inflammatory effect of HO on HeLa-modified conjunctiva-derived cells, a rarely studied cellular type. This inflammatory pathway involving NFAT5 and CCL2 could offer a promising target for developing new therapies to treat DED, warranting further investigations to fully grasp the complete intracellular mechanisms.

Blue light phototoxicity toward human corneal and conjunctival epithelial cells in basal and hyperosmolar conditions

“…However, additional HO stress probably broke down the negative loop regulation leading to a significant increase in their expression. HO also slightly increased the expression NFκB, a protein complex that controls transcription of DNA, cytokine production, and cell survival, in accordance with previous studies where NFκB was reported to have a role in HO-induced cellular signaling [38,75]. In HCE, the observed mRNA up-regulation of SOD1, one of the main antioxidant enzymes [57,76], meaning a setting-up of cellular defense system, even better adjusted under HO hormetic effect.…”

“…The measured osmolarity values were within the range of the hyperosmolarity commonly used in experimental settings (e.g. [38,45],). For both cell types, we decided to keep the same composition of culture media, in order not to induce an additional stress and to be able to assess the specific phototoxic effect.…”

“…Moreover, it was reported that IOBA line had a highly similar profile of biomarkers concerning cellular defense, communication and development when compared to primary culture of human conjunctival epithelial cells [42]. In order to mimic in vitro dry eye conditions, HO stress was applied to these cells [38]. To make HO media, we added 69 mM of NaCl since greater values were reported to induce an important cell death [38,44] and would probably bias the impact of light phototoxicity.…”

“…In order to mimic in vitro dry eye conditions, HO stress was applied to these cells [38]. To make HO media, we added 69 mM of NaCl since greater values were reported to induce an important cell death [38,44] and would probably bias the impact of light phototoxicity. The measured osmolarity values were within the range of the hyperosmolarity commonly used in experimental settings (e.g.…”

“…Given our specialized practice in the clinical management of DED and previous basic studies on ocular surface inflammation and toxicity [34][35][36][37], we hypothesized about a potential harmful influence of blue light on the triggering and evolution of DED. Thus, the aim of this in vitro study was to investigate the impact of blue-light exposure on human epithelial cells of ocular surface, cultured either in basal conditions or additionally stressed by hyperosmolar conditions (HO) frequently used as an in vitro model of dry eye [38]. In particular, we studied the impact of various blue wavebands on cellular viability and health, oxidative stress, mitochondrial function and inflammatory cytokines expression.…”