Hyperosmolar conditions, from 450 or 500 mOsM, induced cell death in an osmo-dependent manner, CCL2 secretion and gene expression, and NFAT5 gene expression and translocation in HeLa-modified conjunctiva-derived cells.

<p>Cells exposed to supplemented medium (340 mOsM) or different hyperosmolar conditions from 400 to 600 mOsM for 24 h were tested for cellular viability (A) with CellTiterBlue^® assay and CCL2 secretion (B) by ELISA, and cells treated for 4 h were lysed for mRNA expression by RT-qPCR to determine the CCL2 and NFAT5 gene expression (C, D). Data show mean ± SEM, <i>n</i> = 3 or more. Statistical analysis: one-way ANOVA followed by Dunnett's multiple comparison test. * <i>p</i> ≤ 0.05; ** <i>p</i> ≤ 0.01; *** <i>p</i> ≤ 0.001; **** <i>p</i> ≤ 0.0001, compared to 340 mOsM. Cells exposed to supplemented medium (340 mOsM) or hyperosmolar medium (500 mOsM) for 24 h were analyzed for NFAT5 translocation with NFAT5 immunofluorescent staining (E) in green. White scale bar: 20μm.</p

Effects of a p38 inhibitor (SB203580), a JNK inhibitor (SP600125), a MEK/ERK inhibitor (U0126) and a NFκB inhibitor (PDTC) on HO-induced CCL2 secretion and CCL2 and NFAT5 gene expression in HeLa-modified conjunctiva-derived cells.

<p>Cells were treated for 1 h with DMSO 0.1%, SB203580 10 μM, SP600125 10 μM, U0126 10 μM or PDTC 50 μM before adding supplemented medium 340 mOsM or hyperosmolar medium to reach 500 mOsM. Cells exposed to medium or HO for 24 h were tested for CCL2 secretion (A) by ELISA and cells exposed for 4 h were lysed for mRNA expression using RT-qPCR to determine the CCL2 and NFAT5 gene expression (B, C). Data showing mean ± SEM, <i>n</i> = 3 or more. Statistical analysis: two-way ANOVA followed by Tukey’s multiple comparison test. * <i>p</i> ≤ 0.05; ** <i>p</i> ≤ 0.01; *** <i>p</i> ≤ 0.001; **** <i>p</i> ≤ 0.0001, compared to respective DMSO 0.1%.</p

Effects of CsA, Dex and Dox on HO-induced CCL2 secretion and CCL2 and NFAT5 gene expression in HeLa-modified conjunctiva-derived cells.

<p>Cells were treated for 1 h with DMSO 0.1%, CsA (0.1, 1 and 10 μg/mL), Dex (10⁻¹⁰, 10⁻⁸ and 10^-6M), Dox 10 μg/mL or a mix of CsA 10 μg/mL and Dex 10^-8M before adding supplemented medium 340 mOsM or HO to reach 500 mOsM. Cells exposed to medium or HO for 24 h were tested for CCL2 secretion (A) by ELISA and cells exposed for 4 h were lysed for mRNA expression using RT-qPCR to determine the CCL2 and NFAT5 gene expression (B, C). Data showing mean ± SEM, <i>n</i> = 3 or more. Statistical analysis: two-way ANOVA followed by Tukey’s multiple comparison test. * <i>p</i> ≤ 0.05; ** <i>p</i> ≤ 0.01; *** <i>p</i> ≤ 0.001; **** <i>p</i> ≤ 0.0001, compared to respective DMSO 0.1%.</p

Schematic figure summarizing the hypotheses of the signaling mechanisms investigated in HeLa-modified conjunctiva-derived cells exposed to hyperosmolar conditions.

<p>1) Is HO able to stimulate CCL2 or NFAT5? 2) Is NFAT5 involved in the CCL2 stimulation? 3) Are MAPKs or NFĸB implicated in the CCL2 and NFAT5 stimulations? 4) What are the effects of CsA, Dex or Dox on these stimulations?</p

NFAT5 siRNA increased HO-induced cell death and inhibited HO-induced CCL2 secretion and CCL2 and NFAT5 gene expression in HeLa-modified conjunctiva-derived cells.

<p>Cells treated for 24 h with negative control siRNA or NFAT5 siRNA were then exposed to supplemented medium (340 mOsM) or hyperosmolar condition (500 mOsM). Cells exposed for 24 h were tested for cellular viability (A) with CellTiterBlue^® assay, CCL2 secretion (B) by ELISA and cells exposed for 4 h were lysed for mRNA expression using RT-qPCR to determine the CCL2 and NFAT5 gene expression (C, D). Data showing mean ± SEM, <i>n</i> = 3 or more. Statistical analysis: two-way ANOVA followed by Tukey’s multiple comparison test. § <i>p</i> ≤ 0.05; §§ <i>p</i> ≤ 0.01; §§§ <i>p</i> ≤ 0.001; §§§ <i>p</i> ≤ 0.0001, compared to respective 340 mOsM. * <i>p</i> ≤ 0.05; ** <i>p</i> ≤ 0.01; *** <i>p</i> ≤ 0.001; **** <i>p</i> ≤ 0.0001, compared to respective control siRNA.</p

Hyperosmolar condition of 500 mOsM induced cell death, secretion and gene expression of CCL2 and NFAT5 gene expression in HeLa-modified conjunctiva-derived cells in a time-dependent manner.

<p>Cells exposed to supplemented medium (340 mOsM) or the hyperosmolar condition (500 mOsM) for different times from 30 min to 24 h were tested for cellular viability (A) with CellTiterBlue^® assay, CCL2 secretion (B) with ELISA and were lysed for mRNA expression using RT-qPCR to determine CCL2 and NFAT5 gene expression (C, D). Data showing mean ± SEM, <i>n</i> = 3 or more. Statistical analysis: two-way ANOVA followed by Sidak’s multiple comparison test. * <i>p</i> ≤ 0.05; ** <i>p</i> ≤ 0.01; *** <i>p</i> ≤ 0.001; **** <i>p</i> ≤ 0.0001, compared to 340 mOsM at the same time.</p

The morphological correlation between IVCM analysis (A, C, E) and immunohistology (B, whole-mount conjunctiva; D, impression cytology; F, cryosections) for observing the microstructure of superior conjunctiva in normal rabbits.

<p>Bar: 50 µm. IVCM observation for meibomian acinar glands (Fig. A, circle) is similar to that of whole-mount conjunctiva with partial eyelids (Fig. B, circle). Goblet cells presented large hyperreflective, round or oval aspects (arrows in C for IVCM and D for impression cytology). Several white hyperreflective inflammatory cells were found (triangles). The CALT structure under the IVCM presented a round/oval aspect (Fig. E) with the goblet cells surrounding them. Immunohistology of MUC-5AC in cryosections confirmed this distribution of goblet cells (F).</p

Conjunctiva-Associated Lymphoid Tissue (CALT) Reactions to Antiglaucoma Prostaglandins with or without BAK-Preservative in Rabbit Acute Toxicity Study

<div><p>Conjunctiva-associated lymphoid tissue (CALT) is closely associated with ocular surface immunity. This study investigated the effects of antiglaucoma prostaglandin analogs with or without benzalkonium chloride (BAK) preservative on organized CALT using an acute toxic model. A total of 48 albino rabbits were used and seven groups of treatments were constituted. Solutions (50 µl) of PBS, 0.02%BAK, ^0.02%BAK+latanoprost, ^0.015%BAK+travoprost, ^0.005%BAK+bimatoprost, ^BAK-freetravoprost preserved with the SofZia® system or ^BAK-freetafluprost were instilled 15 times at 5-min intervals in both eyes. CALT changes were analyzed using <em>in vivo</em> confocal microscopy (IVCM), immunohistology in cryosections for detecting MUC-5AC+ mucocytes and CD45+ hematopoietic cells. Antiglaucoma eye drops stimulated inflammatory cell infiltration in the CALT, and seemed to be primarily related to the concentration of their BAK content. The CALT reaction after instillation of BAK-containing eye drops was characterized by inflammatory cell infiltration in the dome and intrafollicular layers and by cell circulation inside the lymph vessels. CD45 was strongly expressed in the CALT after instillation of all BAK-containing solutions at 4 h and decreased at 24 h. The number of MUC-5AC+ mucocytes around the CALT structure decreased dramatically after instillation of BAK-containing solutions. This study showed for the first time the <em>in vivo</em> aspect of rabbit CALT after toxic stimuli, confirming the concentration-dependent toxic effects of BAK. IVCM-CALT analysis could be a pertinent tool in the future for understanding the immunotoxicologic challenges in the ocular surface and would provide useful criteria for evaluating newly developed eye drops.</p> </div

Immunofluorescence staining of MUC-5AC in the cryosections at 4 h: normal goblet cell patterns after PBS applications (A).

<p>The follicle size increased, and no or rare goblet cells were observed after the instillations of 0.02%BAK (B) or ^0.02%BAK+latanoprost (C) with unstructured green paths. BAK-free travoprost Z (D) and BAK-free tafluprost (E) presented almost normal MUC-5AC staining. (Images ×200).</p

IVCM analysis of rabbit CALT, 75 min (A, B), 2 h (C, D) and 4 h (E, F) after the first instillation of ^0.02%BAK+latanoprost in dome layers (A, C, E: 0–15 µm) and in intrafollicular layers (B, D, F: 15–30 µm).

<p>All images: 400 µm×400 µm. Note the disappearance of goblet cells around the CALT structure with the increase of inflammatory cell infiltration in dome layers and the activation of lymphatic vessels with the increase of inflammatory cell infiltration in the intrafollicular layers.</p