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PURPOSE: To explore novel role and molecular mechanism of a natural anti-inflammatory cytokine interleukin (IL) 37 in preventing corneal epithelial barrier disruption from hyperosmolar stress as can occur in dry eye disease. METHODS: Primary human corneal epithelial cells (HCECs) were cultured from fresh donor limbal explants. An in vitro dry eye model with hyperosmolar stress was established by switching HCECs from isosmolar (312mOsM) to hyperosmolar medium (350–500 mOsM), and some cells were treated with rhIL-37 or rhTNF-α, for different periods (2–48 h). The expression of cytokines and cathepsin S, and barrier protein integrity were evaluated by RT-qPCR, ELISA, and immunofluorescent staining with confocal microscopy. RESULTS: The integrity of epithelial barrier was significantly disrupted in HCECs exposed to hyperosmolar medium, as shown by immunofluorescent images of tight junction (TJ, ZO-1, occludin and claudin-1) and adheren junction (E-cadherin) proteins. TNF-α accentuated hyperosmolar-induced disruption of TJ barrier functional integrity whereas exposure to IL-37 blunted or even reversed these changes. Cathepsin S, encoded by CTSS gene, was found to directly disrupt epithelial barrier integrity. Interestingly, CTSS expression was significantly induced by TNF-α and hyperosmolarity, while exogenous rhIL-37 inhibited TNF-α and CTSS expression at mRNA and protein levels following hyperosmolar stress. Furthermore, rhIL-37 restored barrier protein integrity, observed in 2D and 3D confocal immunofluorescent images, in HCECs under hyperosmolar stress. CONCLUSION: Our findings demonstrate a novel signaling pathway by which anti-inflammatory cytokine IL-37 prevents corneal epithelial barrier disruption under hyperosmotic stress via suppressing TNF-α and CTSS expression. This study provides new insight into mechanisms protecting corneal barrier in dry eye disease.

s Purpose To explore the distinct expression and diverse roles of IL-36 cytokines in dry eye disease using an in vitro hyperosmolarity model of human corneal epithelial cells (HCECs). Methods Primary HCECs were cultured from fresh donor limbal explants. Hyperosmolarity model was established by switching HCECs from isosmotic (312 mOsM) to hyperosmotic medium (350–500 mOsM) alone or with addition of rhIL-36RA or rhIL-38 for 2–48 h. Some cultures were treated with IL-36α (1–10 ng/ml) with or without rhIL-36RA or rhIL-38. Gene expression was detected by RT-qPCR; and protein production and barrier disruption were evaluated by ELISA and/or immunofluorescent staining. Results IL-36 cytokines were differential expressed in primary HCECs. Among 3 pro-inflammatory agonists, IL-36α, but not IL-36β and IL-36γ, was distinctly induced at osmolarity-dependent manner while two antagonist IL-36RA and IL-38 were significantly suppressed in HCECs exposed to hyperosmotic stress. IL-36α increased to 4.4-fold in mRNA and 6.9-fold at protein levels (116.0 ± 36.33 pg/ml vs 16.79 ± 6.51 pg/ml in controls) by 450 mOsM, but dramatically inhibited by addition of rhIL-36RA or rhIL-38. Exogenous rhIL-36α stimulated expression of TNF-α and IL-1β at mRNA and protein levels and disrupted tight junction proteins ZO-1 and occludin. However, rhIL-36RA or rhIL-38 suppressed TNF-α and IL-1β production and protected HCECs from barrier disruption in response to IL-36α or hyperosmolarity. Conclusions Our findings demonstrate that the stimulated pro-inflammatory IL-36α with the suppressed antagonists IL-36RA and IL-38 is a novel mechanism by which hyperosmolarity induces inflammation in dry eye. IL-36RA and IL-38 may have a therapeutic potential in dry eye.

Purpose This study aimed to uncover novel cell types in heterogenous basal limbus of human cornea for identifying LSC at single cell resolution. Methods Single cells of human limbal basal epithelium were isolated from young donor corneas. Single-cell RNA-Sequencing was performed using 10x Genomics platform, followed by clustering cell types through the graph-based visualization method UMAP and unbiased computational informatic analysis. Tissue RNA in situ hybridization with RNAscope, immunofluorescent staining and multiple functional assays were performed using human corneas and limbal epithelial culture models. Results Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters belonging to three lineages. A smallest cluster (0.4% of total cells) was identified as LSCs based on their quiescent and undifferentiated states with enriched marker genes for putative epithelial stem cells. TSPAN7 and SOX17 are discovered and validated as new LSC markers based on their exclusive expression pattern and spatial localization in limbal basal epithelium by RNAscope and immunostaining, and functional role in cell growth and tissue regeneration models with RNA interference in cultures. Interestingly, five cell types/states mapping a developmental trajectory of LSC from quiescence to proliferation and differentiation are uncovered by Monocle3 and CytoTRACE pseudotime analysis. The transcription factor networks linking novel signaling pathways are revealed to maintain LSC stemness. Conclusions This human corneal scRNA-Seq identifies the LSC population and uncovers novel cell types mapping the differentiation trajectory in heterogenous limbal basal epithelium. The findings provide insight into LSC concept and lay the foundation for understanding the corneal homeostasis and diseases.

Alkali burns to the cornea are among the most devastating injuries to the eye. The purpose of this study was to evaluate the effects of dexamethasone (Dex) or doxycycline (Doxy) on protease activity and corneal complications in a combined model (CM) of alkali burn and dry eye. C57BL/6 mice were subjected to the CM for 2 or 5 days (D). Mice were topically treated either with Dex (0.1%), Dox (0.025%) or vehicle QID and observed daily for appearance of corneal perforation. Quantitative real time PCR was performed to measure expression of inflammation cytokines and matrix metalloproteinases (MMPs) in whole cornea lysates. No perforations were observed in the Dex-treated corneas. All wounds in Doxy-treated corneas were closed 2D post-injury, and they had significantly lower corneal opacity scores at days 4 and 5 post-injury compared to BSS treatment. Dex-treated corneas had the lowest corneal opacity scores. Dex treatment significantly decreased expression of IL-1β, IL-6, MMPs -1, -9, -13, and TIMP-1 after 2 days but increased levels of MMP-8, while Doxy treatment significantly decreased IL-1β, IL-6, MMP-8, and -9, compared to vehicle. Decreased MMP-1, -9 and -13 immunoreactivity and gelatinolytic activity were seen in corneas treated with Doxy and Dex compared to vehicle. Increased neutrophil infiltration and myeloperoxidase activity was noted in the vehicle group compared to Dex 2 days post-injury. These findings demonstrate that early initiation of anti-inflammatory therapy is very efficacious in preserving corneal clarity and facilitating wound healing, while modulating MMP production and suppressing neutrophil infiltration.

Inflammation is recognized as a key component in the pathogenesis of dry eye and a variety of ocular surface diseases. Stress-activated protein kinases have been identified as pathways signaling ocular surface stresses, such as increased tear film osmolarity and ultraviolet light exposure. Activation of these stress pathways results in transcription of stress related genes, including inflammatory cytokines (e.g. interleukin-1 and tumor necrosis factor (TNF)-alpha and matrix metalloproteinases (MMPs), such as MMP-9. Treatment of osmo-stressed cultured corneal epithelia with inhibitors of the stress associated kinase c-jun N-terminal kinases was found to decrease production of MMPs by these cells. These findings suggest that SAPKs may be key therapeutic targets for dry eye and ocular surface diseases.

Matrix metalloproteinases (MMPs) are a family of calcium-dependent, zinc-containing endoproteinases capable of degrading the extracellular matrix and basement membrane. Many inflammatory stimuli, such as wound healing, ultraviolet, hyperosmolarity, and stress, can stimulate production of inflammatory cytokines and a variety of MMPs, including gelatinases, collagenases, and stromelysins. Pro-inflammatory cytokines and growth factors can further promote the production of MMPs. Thus, MMPs participate in and promote the inflammation process. MMP-9 may be a most important amplifying factor for corneal inflammation. Mitogen activated protein kinases are involved in MMPs amplified inflammation. These pathway studies provide new therapeutic potential to block MMP mediated inflammation.

Avascularity of normal cornea is a result of homeostasis between anti-angiogenic and pro-angiogenic stimuli. Disruption of this delicate balance during corneal wound healing can lead to pathological corneal vascularization. Several unique characteristics in the ocular surface epithelia modulate corneal avascularity. Normal cornea contains heparan sulfate to prevent the release of potent angiogenic cytokines, such as basic fibroblast growth factor (bFGF) from the Bowman's layer. Potent angiostatic factors, such as endostatin and angiostatin, can be produced from degradation of corneal extracelluar matrix. In contrast, conjunctiva contains angiogenic cytokines, such as bFGF and vascular endothelial growth factor. In addition to regulating release of angiogenic and angiostatic cytokines, matrix metalloproteinases (MMPs) and other proteolytic enzymes can modulate corneal vascularization via matrix degradation to allow endothelial migration and tissue remodeling. When the cornea becomes vascularized, inflammatory cells and mediators gain uninhibited access to the cornea, thus rendering immune sensitization and increased risk of corneal graft rejection. Novel therapies targeting angiogenic cytokines or MMPs have been shown to suppress corneal vascularization effectively in animal models, and may have therapeutic potential for clinical use.