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

• Published in: PloS one 2016-08, Vol.11 (8), p.e0159983-e0159983
• Main Authors: Warcoin, Elise, Baudouin, Christophe, Gard, Carole, Brignole-Baudouin, Françoise
• Format: Article
• Language: English
• Subjects: Actors; Analysis; Apoptosis; Aquaporins; Biology and Life Sciences; Cell death; Cell Line; Central nervous system depressants; Chemical compounds; Chemokine CCL2 - genetics; Chemokine CCL2 - metabolism; Conjunctiva; Conjunctiva - cytology; Conjunctiva - drug effects; Conjunctiva - metabolism; Cornea; Cyclosporine - pharmacology; Cyclosporins; Cytokines; Dexamethasone; Dexamethasone - pharmacology; Down-Regulation - drug effects; Down-Regulation - genetics; Doxycycline; Enzyme-linked immunosorbent assay; Enzymes; Experiments; Extracellular signal-regulated kinase; Eye diseases; Gene expression; Gene Expression Regulation - drug effects; Growth factors; HeLa Cells; Human health and pathology; Humans; Inflammation; Inhibition; Inhibitors; Intracellular; JNK protein; Kinases; Life Sciences; MAP kinase; Medicine and Health Sciences; Mitogens; Monocyte chemoattractant protein 1; NF-κB protein; Osmotic pressure; Osmotic Pressure - physiology; Pathology; Pharmaceutical sciences; Pharmacology; Protein kinases; Protein Transport - drug effects; Protein Transport - genetics; Proteins; Research and Analysis Methods; Sensory Organs; siRNA; Sodium chloride; Steroids; Steroids (Organic compounds); T cells; Transcription factors; Transcription Factors - physiology; Translocation
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0159983

To 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. A 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. HO 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. These 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.