Inflammation of Dry Eye Syndrome: A Cellular Study of the Epithelial and Macrophagic Involvement of NFAT5 and RAGE.

Dry eye inflammation is a key step in a vicious circle and needs to be better understood in order to break it. The goals of this work were to, first, characterize alarmins and cytokines released by ocular surface cells in the hyperosmolar context and, second, study the role of NFAT5 in this process. Finally, we studied the potential action of these alarmins in ocular surface epithelial cells and macrophages via RAGE pathways. HCE and WKD cell lines were cultured in a NaCl-hyperosmolar medium and the expression of alarmins (S100A4, S100A8, S100A9, and HMGB1), cytokines (IL6, IL8, TNFα, and MCP1), and NFAT5 were assessed using RT-qPCR, ELISA and multiplex, Western blot, immunofluorescence, and luciferase assays. In selected experiments, an inhibitor of RAGE (RAP) or NFAT5 siRNAs were added before the hyperosmolar stimulations. HCE and WKD cells or macrophages were treated with recombinant proteins of alarmins (with or without RAP) and analyzed for cytokine expression and chemotaxis, respectively. Hyperosmolarity induced epithelial cell inflammation depending on cell type. NFAT5, but not RAGE or alarmins, participated in triggering epithelial inflammation. Furthermore, the release of alarmins induced macrophage migration through RAGE. These in vitro results suggest that NFAT5 and RAGE have a role in dry eye inflammation.

The receptor for advanced glycation end-products enhances lung epithelial wound repair: An in vitro study.

Re-epithelialization of the alveolar surface is a key process of lung alveolar epithelial barrier repair after acute lung injury. The receptor for advanced glycation end-products (RAGE) pathway plays key roles in lung homeostasis, and its involvement in wound repair has been already reported in human bronchial epithelial cells. However, its effects on lung alveolar epithelial repair after injury remain unknown. We investigated whether RAGE stimulation with its ligands high-mobility group box 1 protein (HMGB1) or advanced glycation end-products (AGEs), alone or associated with RAGE inhibition using RAGE antagonist peptide, affects in vitro wound healing in human alveolar epithelial A549 cells. We further asked whether these effects could be associated with changes in cell proliferation and migration. We found that treatment of A549 cells with HMGB1 or AGEs promotes RAGE-dependent wound healing after a scratch assay. In addition, both RAGE ligands increased cell proliferation in a RAGE-dependent manner. Treatment with HMGB1 increased migration of alveolar epithelial cells at 12 h, independently of RAGE, whereas AGEs stimulated migration as measured 48 h after injury in a RAGE-dependent manner. Taken together, these results suggest that RAGE pathway is involved in lung alveolar epithelial wound repair, possibly through enhanced cell migration and proliferation.

Oxidative and antioxidative stress markers in dry eye disease: A systematic review and meta-analysis.

PURPOSE: To conduct a systematic review and meta-analysis on the levels of oxidative stress markers and antioxidants in dry eye disease (DED) compared with healthy subject. METHOD: The PubMed, Cochrane Library, Embase, Science Direct and Google Scholar databases were searched on 10 January 2021 for studies reporting oxidative and antioxidative stress markers in DED and healthy controls. Main meta-analysis was stratified by type of biomarkers, type of samples (tears, conjunctival cells or biopsies), Sjögren's syndrome (SS) (patients with or without SS) and by geographical zones (Asia or Europe). RESULTS: We included nine articles, for a total of 333 patients (628 eye samples) with DED and 165 healthy controls (451 eye samples). There is an overall increase in oxidative stress markers in DED compared with healthy controls (standard mean deviation = 2.39, 95% confidence interval 1.85-2.94), with a significant increase in lipid peroxide (1.90, 0.69-3.11), myeloperoxidase (2.17, 1.06-3.28), nitric oxide synthase 3 (2.52, 0.95-4.08), xanthine oxidase/oxidoreductase (2.41, 1.40-5.43), 4-hydroxy-2-nonenal (4HNE) (4.75, 1.67-7.84), malondialdehyde (3.00, 2.55-3.45) and reactive oxygen species (1.31, 0.94-1.68). Oxidative stress markers were higher in tears, conjunctival cells and conjunctival biopsies of DED than controls. Even if small number of studies were included for antioxidants, catalase seemed to be decreased in DED compared with healthy controls (-2.17, -3.00 to -1.34), with an increase of antioxidants in tears of DED patients without SS (1.13, 0.76-1.49). CONCLUSION: Oxidative stress markers, and probably antioxidants, were dysregulated in DED, establishing a local oxidative environment in tears, conjunctival cells and tissues.

Dysregulation of the Amniotic PPARγ Pathway by Phthalates: Modulation of the Anti-Inflammatory Activity of PPARγ in Human Fetal Membranes.

Phthalates are reprotoxic pollutants that are omnipresent in the environment. Detectable in amniotic fluid, these compounds (with the most concentrated being mono-2-ethylhexyl phthalate (MEHP)) are in direct contact with fetal membranes (FMs). They can lead to the premature rupture of FMs by deregulating cellular and molecular pathways, such as, for example, the nuclear transcription factor peroxysome proliferator-activated receptor gamma (PPARγ) pathway. The objective was to study the impact of MEHP on the PPARγ pathway in FMs using amnion and choriodecidua across the three trimesters of pregnancy and the amniotic epithelial AV3 cell model by analyzing (i) PPARγ expression (mRNA and proteins) using RT-qPCR and Western blot assays; (ii) cytotoxicity and cell viability following MEHP treatment by lactate dehydrogenase (LDH) measurement and using Cell-counting Kit 8; and (iii) modulation by MEHP of PPARγ transcriptional activity (using a reporter gene assay) and PPARγ anti-inflammatory properties (by measuring IL6 and IL8 levels). PPARγ is expressed in the human amnion and choriodecidua during the three trimesters of pregnancy and in amniotic cells. In the AV3 cell line, MEHP is not cytotoxic and does not reduce cell viability, but it reduces PPARγ activity, here induced by a classical agonist without influencing its expression. MEHP also reduces PPARγ's anti-inflammatory properties. In conclusion, PPARγ signaling is dysregulated by MEHP; this paves the way for future explorations to highlight the hypothesis of phthalates as an amniotic PPARγ disruptor that can explain the premature rupture of FMs.

Dysregulation of Receptor for Advanced Glycation End Products (RAGE) Expression as a Biomarker of Keratoconus.

BACKGROUND: Because of the implications of Receptor for Advanced Glycation End Products (RAGE) in keratoconus (KC), we describe a differential expression of RAGE transcripts and proteins in corneal tissues and tears of KC and healthy patients. METHODS: Using a case-controlled study, corneal epitheliums and tears of KC and healthy subjects were obtained during corneal collagen cross-linking and photorefractive keratectomy (PKR) and during usual consultations. Quantitative reverse transcription (RT-qPCR) and Western-Blot were performed to analyze RAGE transcripts and proteins' expression in corneal tissues and tears. RESULTS: One hundred and six patients were included in this study. The characteristics of the patients were as follows: 56 KC (25 corneal epithelium and 31 tears) and 50 control subjects (25 corneal epithelium and 25 tears). Transcripts of RAGE, HMGB1, and S100 family ligands were quantified by RT-qPCR, identifying a significantly higher expression of RAGE and HMGB1 in the healthy group than in the KC group (p = 0.03 and 0.04, respectively). Western Blot showed a significantly higher fl-RAGE expression in KC corneal epithelium than control (p < 0.001) and lower s-RAGE expression in KC tears than control (p = 0.04). CONCLUSIONS: Linked with the inflammatory process occurring in KC pathophysiology, we propose for the first time that the RAGE expression (total and truncated forms of receptor and ligands) in KC corneal tissues and tear samples provides viable biomarkers.