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Elise Warcoin, Christophe Baudouin, Françoise Baudouin; Cyclosporine inhibits hyperosmolarity-induced CCL2 secretion by conjunctival cells in an in vitro model of dry eye. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):332.
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© ARVO (1962-2015); The Authors (2016-present)
Dry eye disease is a multifactorial pathology characterized by elevated tear film osmolarity and inflammation on the ocular surface (OS). Cyclosporine (CsA) is actually the principal molecule prescribed to treat this disease. However, little is known about its mechanisms to reduce OS inflammation. This study investigated CCL2 secretion by conjunctival cells under hyperosmotic stress and analyzed the anti-inflammatory effects of CsA using dexamethasone (Dex), a corticoid used to treat ocular inflammation, as an anti-inflammatory control.
Human conjunctival cells were cultured in normo-osmolar (340mOsM) or in NaCl-hyperosmolar (360-600mOsM) media for various exposure times with or without Csa (0.1µg/mL to 100µg/mL) or Dex (10-8 to 10-5M) incubation. CCL2 was investigated using RT-qPCR, immunostaining and ELISA according to cellular viability (microtitration assays).
After 24h of stress, CCL2 secretion started increasing from 420mOsM to a peak at 550mOsM (300pg/mL). This CCL2 increase was confirmed by an intense immunostaining. Hyperosmolarity started to alter cell viability (-10% cell vs control) at 500mOsM, osmolarity considered for the following results. CCL2 secretion was not detectable after 2, 4 and 6 hours of stress; on the contrary, CCL2 gene expression increased as soon as 2 hours and continued to increase with time, reaching a 130-fold vs control cells after 24 hours. This expression was found to be osmodependent. CsA from 0.1µg/mL (0.00001%) to 100µg/mL (0.01%) decreased hyperosmolarity-induced CCL2 secretion (230pg/mL at 500mOsM) in a concentration-dependent manner to 170 and 90pg/mL respectively. As expected, Dex reduced CCL2 secretion to 180 and 165pg/mL for 10-8 and 10-5M respectively.
This study showed that hyperosmolar conditions stimulate both CCL2 secretion and gene expression in an osmolarity-dependent manner in conjunctival cells, an understudied cell type. CsA reduced this secretion even at very low concentration as 0.1µg/mL and in a concentration-dependent manner. These results offer insights into the anti-inflammatory properties of CsA on CCL2 secretion induced by hyperosmolarity, but the precise mechanisms remain to be further investigated.
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