Ruzhi Deng

Oxidative stress markers induced by hyperosmolarity in primary human corneal epithelial cells.

Oxidative stress has been known to be involved in pathogenesis of dry eye disease. However, few studies have comprehensively investigated the relationship between hyperosmolarity and oxidative damage in human ocular surface. This study was to explore whether and how hyperosmolarity induces oxidative stress markers in primary human corneal epithelial cells (HCECs). Primary HCECs were established from donor limbal explants. The hyperosmolarity model was made in HCECs cultured in isosmolar (312 mOsM) or hyperosmotic (350, 400, 450 mOsM) media. Production of reactive oxygen species (ROS), oxidative damage markers, oxygenases and anti-oxidative enzymes were analyzed by DCFDA kit, RT-qPCR, immunofluorescent and immunohistochemical staining and Western blotting. Compared to isosmolar medium, ROS production significantly increased at time- and osmolarity-dependent manner in HCECs exposed to media with increasing osmolarities (350–450 mOsM). Hyperosmolarity significantly induced oxidative damage markers in cell membrane with increased toxic products of lipid peroxidation, 4–hydroxynonenal (4-HNE) and malondialdehyde (MDA), and in nuclear and mitochondria DNA with increased aconitase-2 and 8-OHdG. Hyperosmotic stress also increased the mRNA expression and protein production of heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2), but reduced the levels of antioxidant enzymes, superoxide dismutase-1 (SOD1), and glutathione peroxidase-1 (GPX1). In conclusion, our comprehensive findings demonstrate that hyperosmolarity induces oxidative stress in HCECs by stimulating ROS production and disrupting the balance of oxygenases and antioxidant enzymes, which in turn cause cell damage with increased oxidative markers in membrane lipid peroxidation and mitochondrial DNA damage.

Blueberry Component Pterostilbene Protects Corneal Epithelial Cells from Inflammation via Anti-oxidative Pathway

Blueberries have been recognized to possess protective properties from inflammation and various diseases, but not for eye and ocular disorders. This study explores potential benefits of pterostilbene (PS), a natural component of blueberries, in preventing ocular surface inflammation using an in vitro culture model of human corneal epithelial cells (HCECs) exposed to hyperosmotic medium at 450 mOsM. Gene expression was detected by RT-qPCR, and protein production or activity was determined by ELISA, zymography, Western blotting and immunofluorescent staining. Reactive oxygen species (ROS) production was measured using DCFDA kit. The addition of PS significantly reduced the expression of pro-inflammatory mediators, TNF-α, IL-1 β, IL-6, MMP-2 and MMP-9 in HCECs exposed to hyperosmotic medium. Pre-treatment with PS (5 to 20 μM) suppressed ROS overproduction in a dose-dependent manner. Additionally, PS significantly decreased the levels of oxidative damage biomarkers, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), aconitase-2 and 8-hydroxydeoxyguanosine (8-OHdG). Importantly, PS was found to rebalance homeostasis between oxygenases and anti-oxidative enzymes by decreasing cyclooxygenase 2 (COX2) expression and restoring the activity of antioxidant enzymes, superoxide dismutase 1 (SOD1) and peroxiredoxin-4 (PRDX4) during hyperosmotic stress. Our findings demonstrate that PS protects human cornea from hyperosmolarity-induced inflammation and oxidative stress, suggesting protective effects of PS on dry eye.

Effects of l-Carnitine, Erythritol and Betaine on Pro-inflammatory Markers in Primary Human Corneal Epithelial Cells Exposed to Hyperosmotic Stress

Abstract Purpose: To explore the effects of osmoprotectants on pro-inflammatory mediator production in primary human corneal epithelial cells (HCECs) exposed to hyperosmotic stress. Methods: HCECs cultured in iso-osmolar medium (312 mOsM) were switched to hyperosmotic media with or without prior incubation with 2–20 mM of l-carnitine, erythritol or betaine for different time periods. The mRNA expression and protein production of pro-inflammatory markers in HCECs were evaluated by RT-qPCR and ELISA. Results: Hyperosmolar media significantly stimulated the mRNA and protein expression of pro-inflammatory cytokines, TNF-α, IL-1β and IL-6, and chemokines, IL-8, CCL2 and CCL20 in HCECs in an osmolarity dependent manner. The stimulated expression of these pro-inflammatory mediators was significantly but differentially suppressed by l-carnitine, erythritol or betaine. l-Carnitine displayed the greatest inhibitory effects and down-regulated 54–77% of the stimulated mRNA levels of TNF-α (down from 12.3–5.7 fold), IL-1β (2.2–0.9 fold), IL-6 (7.3–2.9 fold), IL-8 (4.6–2.0 fold), CCL2 (15.3–3.5 fold) and CCL20 (4.1–1.5 fold) in HCECs exposed to 450 mOsM. The stimulated protein production of TNF-α, IL-1β, IL-6 and IL-8 was also significantly suppressed by l-carnitine, erythritol and betaine. l-carnitine suppressed 49–79% of the stimulated protein levels of TNF-α (down from 81.3 to 17.4 pg/ml), IL-1β (56.9–29.2 pg/ml), IL-6 (12.8–4.6 ng/ml) and IL-8 (21.2–10.9 ng/ml) by HCECs exposed to 450 mOsM. Interestingly, hyperosmolarity stimulated increase in mRNA and protein levels of TNF-α, IL-1β and IL-6 were significantly suppressed by a transient receptor potential vanilloid channel type 1 (TRPV1) activation inhibitor capsazepine. Conclusions: l-carnitine, erythritol and betaine function as osmoprotectants to suppress inflammatory responses via TRPV1 pathway in HCECs exposed to hyperosmotic stress. Osmoprotectants may have efficacy in reducing innate inflammation in dry eye disease.

A Novel Interleukin 33/ST2 Signaling Regulates Inflammatory Response in Human Corneal Epithelium

Interleukin (IL) 33, a member of IL-1 cytokine family, is well known to promote Th2 type immune responses by signaling through its receptor ST2. However, it is not clear whether ST2 is expressed by mucosal epithelium, and how it responds to IL-33 to induce inflammatory mediators. This study was to identify the presence and function of ST2 and explore the role of IL-33/ST2 signaling in regulating the inflammatory cytokine production in corneal epithelial cells. Human corneal tissues and cultured primary human corneal epithelial cells (HCECs) were treated with IL-33 in different concentrations without or with different inhibitors to evaluate the expression, location and signaling pathways of ST2 in regulating production of inflammatory cytokine and chemokine. The mRNA expression was determined by reverse transcription and real time PCR, and protein production was measured by enzyme-linked immunosorbent assay (ELISA), immunohistochemical and immunofluorescent staining. ST2 mRNA and protein were detected in donor corneal epithelium and cultured HCECs, and ST2 signal was enhanced by exposure to IL-33. IL-33 significantly stimulated the production of inflammatory cytokines (TNF-α, IL-1β and IL-6) and chemokine IL-8 by HCECs at both mRNA and protein levels. The stimulated production of inflammatory mediators by IL-33 was blocked by ST2 antibody or soluble ST2 protein. Interestingly, the IκB-α inhibitor BAY11-7082 or NF-κB activation inhibitor quinazoline blocked NF-κB p65 protein phosphorylation and nuclear translocation, and also suppressed the production of these inflammatory cytokines and chemokine induced by IL-33. These findings demonstrate that ST2 is present in human corneal epithelial cells, and IL-33/ST2 signaling plays an important role in regulating IL-33 induced inflammatory responses in ocular surface.

Unique Expression Pattern and Functional Role of Periostin in Human Limbal Stem Cells

Periostin is a non-structural matricellular protein. Little is known about periostin in human limbal stem cells (LSCs). This study was to explore the unique expression pattern and functional role of periostin in maintaining the properties of human LSCs. Fresh donor corneal tissues were used to make cryosections for evaluation of periostin expression on ex vivo tissues. Primary human limbal epithelial cells (HLECs) were generated from limbal explant culture. In vitro culture models for proliferation and epithelial regeneration were performed to explore functional role of periostin in LSCs. The mRNA expression was determined by reverse transcription and quantitative real-time PCR (RT-qPCR), and the protein production and localization were detected by immunofluorescent staining and Western blot analysis. Periostin protein was found to be exclusively immunolocalized in the basal layer of human limbal epithelium. Periostin localization was well matched with nuclear factor p63, but not with corneal epithelial differentiation marker Keratin 3. Periostin transcripts was also highly expressed in limbal than corneal epithelium. In primary HLECs, periostin expression at mRNA and protein levels was significantly higher in 50% and 70% confluent cultures at exponential growth stage than in 100% confluent cultures at slow growth or quiescent condition. This expression pattern was similar to other stem/progenitor cell markers (p63, integrin β1 and TCF4). Periostin expression at transcripts, protein and immunoreactivity levels increased significantly during epithelial regeneration in wound healing process, especially in 16-24 hours at wound edge, which was accompanied by similar upregulation and activation of p63, integrin β1 and TCF4. Our findings demonstrated that periostin is exclusively produced by limbal basal epithelium and co-localized with p63, where limbal stem cells reside. Periostin promotes HLEC proliferation and regeneration with accompanied activation of stem/progenitor cell markers p63, integrin β1 and TCF4, suggesting its novel role in maintaining the phenotype and functional properties of LSC.

Identification for Differential Localization of Putative Corneal Epithelial Stem Cells in Mouse and Human

Human Corneal epithelial stem cells (CESCs) have been identified to reside in limbus for more than 2 decades. However, the precise location of CESCs in other mammalian remains elusive. This study was to identify differential localization of putative CESCs in mice. Through a series of murine corneal cross-sections from different directions, we identified that anatomically and morphologically the murine limbus is composed of the thinnest epithelium and the thinnest stroma without any palisades of Vogt-like niche structure. The cells expressing five of stem/progenitor cell markers are localized in basal layer of entire murine corneal epithelium. BrdU label-retaining cells, a key characteristic of epithelial stem cells, are detected in both limbal and central cornea of mouse eye. Functionally, corneal epithelium can be regenerated in cultures from central and limbal explants of murine cornea. Such a distribution of mouse CESCs is different from human cornea, where limbal stem cell concept has been well established and accepted. We are aware that some new evidence supports limbal stem cell concept in mouse recently. However, it is important to know that central cornea may provide an alternative source of stem cells when one utilizes mice as animal model for corneal research.

Pollen/TLR4 Innate Immunity Signaling Initiates IL-33/ST2/Th2 Pathways in Allergic Inflammation

Innate immunity has been extended to respond environmental pathogen other than microbial components. Here we explore a novel pollen/TLR4 innate immunity in allergic inflammation. In experimental allergic conjunctivitis induced by short ragweed (SRW) pollen, typical allergic signs, stimulated IL-33/ST2 signaling and overproduced Th2 cytokine were observed in ocular surface, cervical lymph nodes and isolated CD4+ T cells of BALB/c mice. These clinical, cellular and molecular changes were significantly reduced/eliminated in TLR4 deficient (Tlr4-d) or MyD88 knockout (MyD88−/−) mice. Aqueous SRW extract (SRWe) directly stimulated IL-33 mRNA and protein expression by corneal epithelium and conjunctiva in wild type, but not in Tlr4-d or MyD88−/− mice with topical challenge. Furthermore, SRWe-stimulated IL-33 production was blocked by TLR4 antibody and NF-kB inhibitor in mouse and human corneal epithelial cells. These findings for the first time uncovered a novel mechanism by which SRW pollen initiates TLR4-dependent IL-33/ST2 signaling that triggers Th2-dominant allergic inflammation.

Mitochondrial DNA oxidation induces imbalanced activity of NLRP3/NLRP6 inflammasomes by activation of caspase-8 and BRCC36 in dry eye

The concept of innate immunity has been expanded to recognize environmental pathogens other than microbial components. However, whether and how the innate immunity is initiated by epithelium in response to environmental physical challenges such as low humidity and high osmolarity in an autoimmune disease, dry eye, is still largely unknown. Using two experimental dry eye models, primary human corneal epithelial cultures exposed to hyperosmolarity and mouse ocular surface facing desiccating stress, we uncovered novel innate immunity pathway by ocular surface epithelium, where oxidized mitochondrial DNA induces imbalanced activation of NLRP3/NLRP6 inflammasomes via stimulation of caspase-8 and BRCC36 in response to environmental stress. Activated NLRP3 with suppressed NLRP6 stimulates caspase-1 activation that leads to IL-1β and IL-18 maturation and secretion. NLRP3-independent caspase-8 noncanonically activates caspase-1 via reciprocal regulation of NLRP3/NLRP6-mediated inflammasomes. Reactive oxygen species-induced mitochondrial DNA oxidative damage and BRCC36 deubiquitinating activity provide a missing link and mechanism by which innate immunity responds to environmental stress via caspase-8-involved NLRP3/NLRP6 inflammasomes.