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

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          Abstract

          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 NH 2-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.

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          Mechanisms of action of cyclosporine.

          Satoshi Matsuda, Shigeo Koyasu (2000)
          Cyclosporine (cyclosporin A, CsA) has potent immunosuppressive properties, reflecting its ability to block the transcription of cytokine genes in activated T cells. It is well established that CsA through formation of a complex with cyclophilin inhibits the phosphatase activity of calcineurin, which regulates nuclear translocation and subsequent activation of NFAT transcription factors. In addition to the calcineurin/NFAT pathway, recent studies indicate that CsA also blocks the activation of JNK and p38 signaling pathways triggered by antigen recognition, making CsA a highly specific inhibitor of T cell activation. Here we discuss the action of CsA on JNK and p38 activation pathways. We also argue the potential of CsA and its natural counterparts as pharmacological probes.
          Article 0 comments Cited 184 times – based on 0 reviews     Review now
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            An objective approach to dry eye disease severity.

            Allison Porreco, Diane Whitmer, Alan Tomlinson … (2010)
            A prospective, multisite clinical study (10 sites in the European Union and the United States) evaluated the clinical utility of commonly used tests and tear osmolarity for assessing dry eye disease severity. Three hundred fourteen consecutive subjects between the ages of 18 and 82 years were recruited from the general patient population, 299 of which qualified with complete datasets. Osmolarity testing, Schirmer test without anesthesia, tear film breakup time (TBUT), corneal staining, meibomian dysfunction assessment, and conjunctival staining were performed bilaterally. A symptom questionnaire, the Ocular Surface Disease Index (OSDI), was also administered to each patient. Distributions of clinical signs and symptoms against a continuous composite severity index were evaluated. Osmolarity was found to have the highest correlation coefficient to disease severity (r(2) = 0.55), followed by conjunctival staining (r(2) = 0.47), corneal staining (r(2) = 0.43), OSDI (r(2) = 0.41), meibomian score (r(2) = 0.37), TBUT (r(2) = 0.30), and Schirmer result (r(2) = 0.17). A comparison of standard threshold-based classification with the composite severity index revealed significant overlap between the disease severities of prospectively defined normal and dry eye groups. Fully 63% of the subjects were found to be poorly classified by combinations of clinical thresholds. Tear film osmolarity was found to be the single best marker of disease severity across normal, mild/moderate, and severe categories. Other tests were found to be informative in the more severe forms of disease; thus, clinical judgment remains an important element in the clinical assessment of dry eye severity. The results also indicate that the initiation and progression of dry eye is multifactorial and supports the rationale for redefining severity on the basis of a continuum of clinical signs. (ClinicalTrials.gov number, NCT00848198.).
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              Analysis of inflammatory cytokines in the tears of dry eye patients.

              Xiaohong Li, Penny Asbell, Dongmei Chen … (2009)
              To determine the levels of 8 important cytokines and 1 chemokine in tears of patients with dry eye disease. Tear samples were collected from 7 patients with dry eye disease and 7 healthy volunteers, and impression cytology samples were collected from 3 of the dry eye patients and 3 of the normal controls. Tears were analyzed for the presence of 8 cytokines [interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, IL-1beta] and 1 chemokine (IL-8). The cytokines and chemokine in each tear sample were measured using Invitrogen's Multiplex Bead Immunoassays. The impression cytology samples were analyzed for IL-1beta, IL-6, IL-8, and TNF-alpha mRNA expression using real-time reverse transcriptase polymerase chain reaction anlaysis. All cytokines and the chemokine measured were significantly increased in the tears of dry eye patients as compared to normal controls. mRNA of all four markers was increased, and the fold increase correlated well with the fold increase of the cytokine concentration found in the tear samples. Tears from dry eye patients contain significantly increased concentrations of cytokines that show correlation to severity of the disease. The upregulation of their respective genes in the conjunctiva suggests that the concentration increase is not the result of evaporative effects, but of overproduction. These findings suggest that cytokines may play an important role in dry eye disease and topical cytokine modulators may be explored as a therapeutic approach to dry eye disease.
              Article 0 comments Cited 133 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Michele Madigan: Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, CA USA )
                ISSN (Electronic): 1932-6203
                Publication date (Electronic): 3 August 2016
                Publication date Collection: 2016
                Volume: 11
                Issue: 8
                Electronic Location Identifier: e0159983
                Affiliations
                [1 ]Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, Paris, France
                [2 ]CHNO des Quinze-Vingts, Service Pharmacie, Paris, France
                [3 ]CHNO des Quinze-Vingts, Service III, Paris, France
                [4 ]Horus Pharma, Saint-Laurent du Var, France
                [5 ]Faculté de Pharmacie de Paris, Univ Paris Descartes, Sorbonne Paris Cité, Paris, France
                Save Sight Institute, AUSTRALIA
                Author notes

                Competing Interests: This work was funded by an unrestricted grant from Horus Pharma. Carole Gard is employed by Horus Pharma. Christophe Baudouin: Financial support and consultant (Alcon; Allergan; Santen; Laboratoires Théa). There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

                • Conceptualization: EW CB CG FB.

                • Formal analysis: EW FB.

                • Funding acquisition: CB FB.

                • Investigation: EW.

                • Methodology: EW CB FB.

                • Project administration: CB FB.

                • Resources: CG.

                • Supervision: FB CB.

                • Validation: EW FB.

                • Visualization: EW FB.

                • Writing - original draft: EW FB.

                • Writing - review & editing: EW CB CG FB.

                Author information
                http://orcid.org/0000-0002-5521-4650
                Article
                Publisher ID: PONE-D-15-51951
                DOI: 10.1371/journal.pone.0159983
                PMC ID: 4972436
                PubMed ID: 27486749
                SO-VID: bf720ab2-6f47-42fc-b7a3-a1a58df557ed
                Copyright © © 2016 Warcoin et al
                License:

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Date received : 29 November 2015
                Date accepted : 12 July 2016
                Page count
                Figures: 6, Tables: 0, Pages: 19
                Funding
                Funded by: HORUS PHARMA
                Carole Gard is employed by Horus Pharma. Concerning her participation, as a co-author, we had regular scientific meetings with her and she participated in the design of the methods, as well as in the manuscript review. She also contributed to reagents, material and analysis tolls. Horus Pharma provided support in the form of salary for author CG, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the ‘author contributions’ section.
                Categories
                Subject: Research Article
                Subject: Biology and Life Sciences
                Subject: Genetics
                Subject: Gene Expression
                Subject: Biology and Life Sciences
                Subject: Physiology
                Subject: Physiological Processes
                Subject: Secretion
                Subject: Medicine and Health Sciences
                Subject: Physiology
                Subject: Physiological Processes
                Subject: Secretion
                Subject: Biology and life sciences
                Subject: Genetics
                Subject: Gene expression
                Subject: Gene regulation
                Subject: Small interfering RNAs
                Subject: Biology and life sciences
                Subject: Biochemistry
                Subject: Nucleic acids
                Subject: RNA
                Subject: Non-coding RNA
                Subject: Small interfering RNAs
                Subject: Research and Analysis Methods
                Subject: Immunologic Techniques
                Subject: Immunoassays
                Subject: Enzyme-Linked Immunoassays
                Subject: Biology and Life Sciences
                Subject: Immunology
                Subject: Immune Response
                Subject: Inflammation
                Subject: Medicine and Health Sciences
                Subject: Immunology
                Subject: Immune Response
                Subject: Inflammation
                Subject: Medicine and Health Sciences
                Subject: Diagnostic Medicine
                Subject: Signs and Symptoms
                Subject: Inflammation
                Subject: Medicine and Health Sciences
                Subject: Pathology and Laboratory Medicine
                Subject: Signs and Symptoms
                Subject: Inflammation
                Subject: Research and Analysis Methods
                Subject: Bioassays and Physiological Analysis
                Subject: Cell Analysis
                Subject: Cell Viability Testing
                Subject: Biology and Life Sciences
                Subject: Anatomy
                Subject: Head
                Subject: Eyes
                Subject: Medicine and Health Sciences
                Subject: Anatomy
                Subject: Head
                Subject: Eyes
                Subject: Biology and Life Sciences
                Subject: Anatomy
                Subject: Ocular System
                Subject: Eyes
                Subject: Medicine and Health Sciences
                Subject: Anatomy
                Subject: Ocular System
                Subject: Eyes
                Subject: Biology and Life Sciences
                Subject: Cell Biology
                Subject: Cell Processes
                Subject: Cell Death
                Custom metadata
                Data Availability All relevant data are within the paper and its Supporting Information files.

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