Proinflammatory Markers, Chemokines, and Enkephalin in Patients Suffering from Dry Eye Disease.

Dry eye symptoms are among the leading complaints in ophthalmology. Dry eye disease (DED) is associated with significant pain affecting quality of life. Cellular and molecular mechanisms underlying ocular pain associated with DED are not fully understood. In this study, we investigated the ocular surface of patients with DED using in vivo confocal microscopy (IVCM) to quantify corneal nerve density and its relation with corneal inflammation. Gene expression of the proinflammatory markers HLA-DR, IL-6, CXCL12, and CCL2 and the receptors CXCR4 and CCR2, as well as PENK (enkephalin precursor), was therefore quantified in conjunctival impression cytology specimens. Thirty-two patients with DED and 15 age-matched controls were included. Subbasal nerve density was significantly lower in DED patients compared to controls. IVCM analysis revealed that DED patients had a significantly higher corneal dendritic cell density compared to controls. Conjunctival impression cytology analysis revealed that HLA-DR, IL-6, CXCR4, and CCL2/CCR2 mRNA levels were significantly increased in DED patients compared to controls, whereas PENK mRNA levels were significantly decreased. Similar results were obtained in vitro on immortalized human conjunctiva-derived epithelial cells challenged with osmotic stress that mimics the DED condition. These results demonstrate that proinflammatory molecules and endogenous enkephalin have opposite gene regulation during DED.

Hyperosmolarity and Benzalkonium Chloride Differently Stimulate Inflammatory Markers in Conjunctiva-Derived Epithelial Cells in vitro.

Tear hyperosmolarity is known to cause ocular surface inflammation in dry eye syndrome. Benzalkonium chloride (BAK), an eyedrop preservative, is known to induce dry eye in long-term-treated patients. Analyzing the modulation of the proinflammatory potential of hyperosmolarity in the presence of BAK on the conjunctiva could give new insights into the effect of this preservative on the disease. In a hyperosmolar model on a conjunctiva-derived cell line, and in the presence of BAK, we evaluated key inflammatory markers [CCL2, IL-8, IL-6, macrophage migration inhibitory factor (MIF) and intercellular adhesion molecule (ICAM)-1] as well as the osmoprotectant element nuclear factor of activated T cells (NFAT)5 using ELISA, RT-qPCR or immunofluorescence staining. Hyperosmolarity highly stimulated CCL2 and NFAT5 in these cells. BAK alone only increased IL-6 expression. The stress-combined condition stimulated CCL2, NFAT5, MIF and IL-8 secretion. ICAM-1 was not modulated by any of the conditions tested. In this model, hyperosmolarity and BAK induced the release of different proinflammatory mediators, and, when combined, they lead to the release of additional inflammatory cytokines. This in vitro study highlights the importance of avoiding long-term ophthalmic treatments containing BAK, as tear film hyperosmolarity can be a result of its detergent action.

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

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.