Corneal Epithelial Cells Research Articles

Development of a Probability-Based In Vitro Eye Irritation Screening Platform.

Traditional eye irritation assessments, which rely on animal models or ex vivo tissues, face limitations due to ethical concerns, costs, and low throughput. Although numerous in vitro tests have been developed, none have successfully reconciled the need for high experimental throughput with the accurate prediction of irritation potential, attributable to the complexity of irritation mechanisms. Simple cell models, while suitable for high-throughput screening, offer limited mechanistic insights, contrasting with more physiologically relevant but less scalable complex organotypic corneal tissue constructs. This study presents a novel strategy to enhance the predictive accuracy of screening-compatible simple cell models in eye irritation testing. Our method combines the results of two in vitro assays-cell apoptosis and nociceptor (TRPV1) activation-using micropatterned chips to partition human corneal epithelial cells into numerous discrete small populations. Following exposure to test compounds, we measure apoptosis and nociceptor activation responses. The large datasets collected from the cell micropatterns facilitate binarization and statistical fitting to calculate a mathematical probability, which assesses the compound's potential to cause eye irritation. This method potentially enables the amalgamation of multiple mechanistic readouts into a singular index, providing a more accurate and reliable prediction of eye irritation potential in a format amenable to high-throughput screening.

OCTN2- and ATB0,+-targeted nanoemulsions for improving ocular drug delivery

Traditional eye drops are administered via topical instillation. However, frequent dosing is needed due to their relatively rapid precorneal removal and low ocular bioavailability. To address these issues, stearoyl L-carnitine-modified nanoemulsions (SC-NEs) were fabricated. The physicochemical properties of SC-NEs in terms of size, morphology, zeta potential, encapsulation efficiency, and in vitro drug release behavior were characterized. The cellular uptake and mechanisms of SC-NEs were comprehensively studied in human corneal epithelial cells and the stearoyl L-carnitine ratio in SC-NEs was optimized. The optimized SC-NEs could target the novel organic cation/carnitine transporter 2 (OCTN2) and amino acid transporter B (0 +) (ATB0,+) on the corneal epithelium, which led to superior corneal permeation, ocular surface retention ability, ocular bioavailability. Furthermore, SC-NEs showed excellent in vivo anti-inflammatory efficacy in a rabbit model of endotoxin-induced uveitis. The ocular safety test indicated that the SC-NEs were biocompatible. In general, the current study demonstrated that OCTN2 and ATB0,+-targeted nanoemulsions were promising ophthalmologic drug delivery systems that can improve ocular drug bioavailability and boost the therapeutic effects of drugs for eye diseases.Graphical

Staphylococcus pseudintermedius induces pyroptosis of canine corneal epithelial cells by activating the ROS–NLRP3 signaling pathway

ABSTRACT Staphylococcus pseudintermedius (S. pseudintermedius) is a common pathogen that causes canine corneal ulcers. However, the pathogenesis remained unclear. In this study, it has been demonstrated that S. pseudintermedius invaded canine corneal epithelial cells (CCECs) intracellularly, mediating oxidative damage and pyroptosis by promoting the accumulation of intracellular reactive oxygen species (ROS) and activating the NLRP3 inflammasome. The canine corneal stroma was infected with S. pseudintermedius to establish the canine corneal ulcer model in vivo. The intracellular infectious model in CCECs was established in vitro to explore the mechanism of the ROS – NLRP3 signalling pathway during the S. pseudintermedius infection by adding NAC or MCC950. Results showed that the expression of NLRP3 and gasdermin D (GSDMD) proteins increased significantly in the infected corneas (p < 0.01). The intracellular infection of S. pseudintermedius was confirmed with transmission electron microscopy and immunofluorescent 3D imaging. Flow cytometry analysis revealed that ROS and pyroptosis rates increased in the experimental group in contrast to the control group (p < 0.01). Furthermore, NAC or MCC950 inhibited activation of the ROS – NLRP3 signalling pathway and pyroptosis rate significantly, by suppressing pro-IL-1β, cleaved- IL-1β, pro-caspase-1, cleaved-caspase-1, NLRP3, GSDMD, GSDMD-N, and HMGB1 proteins. Thus, the research confirmed that oxidative damage and pyroptosis were involved in the process of CCECs infected with S. pseudintermedius intracellularly by the ROS – NLRP3 signalling pathway. The results enrich the understanding of the mechanisms of canine corneal ulcers and facilitate the development of new medicines and prevention measures.

Abstract 1338: Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells

Abstract Human leukocyte antigen-G (HLA-G) suppresses lymphoid and myeloid immune systems by binding ILT2 and ILT4 respectively and activating ITIM signaling. HLA-G is abundantly expressed in placental cytotrophoblasts to block fetal rejection. HLA-G is not expressed in most normal cells but is derepressed in in about 50% of malignancies. We engineered ILT2 and ILT4 to produce activating signals as Chimeric ILT Receptors or CIR. Preliminary results indicated robust killing of Acute Myeloid Leukemia (AML) cells by Natural Killer (NK) cells expressing either ILT2 or ILT4 CIRs. Here, we evaluated the effect of alteration of activating domains in the CIR constructs to produce signals that mimic the effect of IL-18 signaling in CIR-NK cells to enhance cytotoxicity, NK cell growth and persistence. Further, HLA-G expression and CIR-NK cell targeting of primary human cell types were rigorously screened. Activated NK cells derived from peripheral blood (2 to 5 donors) were transduced with γ-retroviruses directing expression of CIR proteins (CIR.X.Y or CIR.Y.X) where X stimulates cytotoxicity through CD3ζ, DAP10 or DAP12, and Y directs coactivation through 4-1BB, fusion of the MyD88 death domain or recruitment of endogenous MyD88 with receptor-derived TIR domains and soluble IL-15. CIR-NK cells were cocultured with GFPffluc-expressing AML and solid tumor target cell lines or normal primary human cells. NK cell proliferation and cytotoxicity of CIR-NK cells was monitored by Incucyte microscopy and supernatants were analyzed for cytokine release by ELISA. At low E:T (1:20 - 1:40) in 7-day cocultures, mock-transduced NK cells displayed innate killing activity against HLA-G+ Molm13 and Kasumi1 AML cells that was augmented by CIR constructs containing direct fusions of MyD88 and DAP10 or DAP12 (mock = 2.06E7 ± 4.53E6 vs. CIR-ILT4.MyD88.DAP10 = 1.03E7 ± 5.46E6) with 7-fold elevation of IFN-γ production. CIR constructs that recruited endogenous MyD88 signaling through the TLR2 TIR domain (4-1BB.DAP10.TLR2) also exhibited enhanced anti-HLA-G activity (9.13E6 ± 5.33E6). Against HLA-G+ HT-1376 bladder cancer cells (E:T = 1:10-1:20), CIR-NK cells also displayed enhanced tumor cell killing with enhanced NK cell growth. Conversely, CIR-enhanced killing was not observed against HLA-G− HCT-116 colorectal cancer cells. The persistence of CIR-NK cell potency after 4 weeks of expansion varied markedly with different activation domains employed. When screened against primary human cells (cardiomyocytes, hepatocytes, hepatic endothelial cells, corneal, colon and lung epithelial cells), no CIR-specific targeting was observed at high E:T ratios (1:1 and 1:5). In conclusion, CIR-NK cells exhibit potent and specific anti-tumor activity against HLA-G+ solid and leukemia cells. Alteration of CIR-NK cell intracellular signaling can optimize the potency and persistence of NK cell anti-tumor activity. Citation Format: MyLinh Duong, Jihyun Park, Raphel Ognar, Simon Wain-Hobson, Henri Bayle. Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1338.

Topical Insulin for Ocular Surface Disease.

Background: Insulin and insulin-like growth factor (IGF)-1 receptors are present in ocular tissues such as corneal epithelium, keratocytes, and conjunctival cells. Insulin plays a crucial role in the growth, differentiation, and proliferation of corneal epithelial cells, as well as in wound healing processes in various tissues. Purpose: This review explores the potential role of topical insulin in the treatment of ocular surface diseases. Specifically, it examines its impact on corneal nerve regeneration, sub-basal plexus corneal nerves, and its application in conditions like corneal epithelial defects, dry eye disease, and diabetic keratopathy. Methods: The review analyzes studies conducted over the past decade that have investigated the use of topical insulin in ocular surface diseases. It focuses on indications, drug preparation methods, side effects, efficacy outcomes, and variations in insulin concentrations and dosages used. Results: While off-label use of topical insulin has shown promising results in refractory corneal epithelial defects, its efficacy in dry eye disease is yet to be demonstrated. Variations in concentrations, dilutions, and dosing guidelines have been reported. However, limited data on ocular penetration, ocular toxicity, and systemic side effects pose challenges to its widespread utility. Conclusion: This review synthesizes findings from ocular investigations on topical insulin to assess its potential applicability in treating ocular surface and corneal diseases. By highlighting indications, preparation methods, side effects, and efficacy outcomes, it aims to provide insights into the current status and future prospects of using topical insulin in ophthalmic practice.

Development of an Eye Irritation Test Method Using an In-House Fabrication of a Reconstructed Human Cornea-like Epithelium Model for Eye Hazard Identification.

In a previous study, a novel human corneal-like epithelium model utilizing an immortalized human corneal epithelial cell line (iHCE-NY1) was developed as an alternative to animal models to identify chemicals not classified under the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS) and was evaluated following the criteria of Test Guideline 492 of the Organization for Economic Co-operation and Development (OECD). In the present study, our aim was to establish an eye irritation test protocol using the iHCE-NY1 model to classify liquid chemicals under the GHS ocular hazard categories: no effect, no classification (No Cat.), Category 2 (Cat. 2) reversible effects, and Category 1 (Cat. 1) irreversible eye damage. The protocol involved exposing the iHCE-NY1 model to 31 liquid test chemicals for 5 min, followed by observation at post-incubation periods (PIPs) to assess recovery. Classification was based on cell viability, and histopathological findings on PIP days 7, 14, and 21. The outcomes were compared with an established database of classifications. All Cat. 1 liquid chemicals, 62.5% of No Cat., and 63.2% of Cat. 2 were correctly categorized. This study demonstrates that the iHCE-NY1 model can not only distinguish No Cat. test liquid chemicals but also differentiate between Cat. 2 and Cat. 1 liquid chemicals.

Single nuclei transcriptomics of the in situ human limbal stem cell niche

The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent, quiescent limbal stem cells (LSCs) located at the limbus, where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood, which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study, we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover, TP63, KRT15, CXCL14, and ITGβ4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs), which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGβ4 could be used to enrich human corneal epithelial cell progenitors, which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1.

The potential application of formulation with pimelic acid as eye drops for corneal collagen cross-linking

Background and aimKeratoconus is a slowly progressive and non-inflammatory disease of eye of unknown etiology characterized by significant structural degeneration, thinning and protrusion of cornea. Collagen cross linking using Riboflavin/ultraviolet A, paraformaldehyde and glutaraldehyde has been widely used to treat keratoconus, but these techniques have several clinical complications. This study aimed to develop a rapid, safer, less toxic and more effective method to increase the corneal stiffness hence treating keratoconus by using EDC/NHS coupling with pimelic acid (PA) as cross-linkers. MethodsThe viability of corneal epithelial and endothelial cells was examined using Alamar blue. The corneal collagen and tissue integrity were evaluated with Masson and hematoxylin and eosin stains respectively. Uniaxial tensile testing was conducted to determine the tissue stiffness. We further assessed the effective penetration depth of cross-linking by fluorescence dye and confocal microscopy. TUNEL assay was performed to detect the damaged DNA fragments in the presence of PA. ResultsThe effect of PA solution on corneal cross-linking showed a significant increase in corneal rigidity without affecting the epithelium integrity under neutralized pH condition, while DEC/NHS alone did not induce the mechanical property. The penetration depth showed consistent results with increased stiffness, which could be affected by the parameters including pH condition, coupling with or without PA. In addition, PA significantly decreased the percentage of TUNEL-positive cells compared to that of EDC/NHS only condition. The cytotoxic effect to endothelium was considered minimal and protected in the presence of PA. ConclusionsWe demonstrated PA as a potential alternative for cross-linking in the model of whole eye with intact epithelium and its protective role on endothelium. The administration of this chemical cross-linker stands as an extremely promising technique for the treatment of corneal keratoconus.

Neuropathic Corneal Pain: Tear Proteomic and Neuromediator Profiles, Imaging Features, and Clinical Manifestations

To investigate the tear proteomic and neuromediator profiles, in vivo confocal microscopy (IVCM) imaging features, and clinical manifestations in neuropathic corneal pain (NCP) patients. Cross-sectional study. Twenty NCP patients and twenty age-matched controls were recruited. All subjects were evaluated by corneal sensitivity, Schirmer's test, tear break-up time, corneal and ocular surface staining, Ocular Surface Disease Index and Ocular Pain Assessment Survey questionnaires, as well as IVCM examinations for corneal nerves, microneruomas, epithelial and dendritic cells. Tears were collected for neuromediator and proteomic analysis using enzyme-linked immunosorbent assay and data-independent acquisition mass spectrometry. Burning and sensitivity to light were the two most common symptoms in NCP. A total of 188 significantly dysregulated proteins, such as elevated metallothionein-2, creatine kinases B-type, vesicle-associated membrane protein 2, neurofilament light polypeptide, and myelin basic protein, were identified in the NCP patients. The top 10 dysregulated biological pathways in NCP include neurotoxicity, axonal signaling, wound healing, neutrophil degradation, apoptosis, thrombin signaling mitochondrial dysfunction, RHOGDI and P70S6K signaling pathways. Compared to controls, the NCP cohort presented with significantly decreased corneal sensitivity (P<0.001), decreased corneal nerve fiber length (P=0.003), corneal nerve fiber density (P=0.006), nerve fiber fractal dimension (P=0.033), as well as increased in corneal nerve fiber width (P=0.002), increased length, total area and perimeter of microneuromas (P<0.001, P<0.001, P=0.019), smaller corneal epithelial size (P=0.017), and higher nerve growth factor level in tears (p=0.006). These clinical manifestations, imaging features, and molecular characterizations would contribute to the diagnostics and potential therapeutic targets for NCP.

Toxicological responses of A549 and HCE-T cells exposed to fine particulate matter at the air–liquid interface

Fine particulate matter (PM2.5) can enter the human body in various ways and have adverse effects on human health. Human lungs and eyes are exposed to the air for a long time and are the first to be exposed to PM2.5. The “liquid immersion exposure method” has some limitations that prevent it from fully reflecting the toxic effects of particulate matter on the human body. In this study, the collected PM2.5 samples were chemically analyzed. An air–liquid interface (ALI) model with a high correlation to the in vivo environment was established based on human lung epithelial cells (A549) and immortalized human corneal epithelial cells (HCE-T). The VITROCELL Cloud 12 system was used to distribute PM2.5 on the cells evenly. After exposure for 6 h and 24 h, cell viability, apoptosis rate, reactive oxygen species (ROS) level, expression of inflammatory factors, and deoxyribonucleic acid (DNA) damage were measured. The results demonstrated significant dose- and time-dependent effects of PM2.5 on cell viability, cell apoptosis, ROS generation, and DNA damage at the ALI, while the inflammatory factors showed dose-dependent effects only. It should be noted that even short exposure to low doses of PM2.5 can cause cell DNA double-strand breaks and increased expression of γ-H2AX, indicating significant genotoxicity of PM2.5. Increased abundance of ROS in cells plays a crucial role in the cytotoxicity induced by PM2.5 exposure These findings emphasize the significant cellular damage and genotoxicity that may result from short-term exposure to low levels of PM2.5.

Semaphorin 7A impairs barrier function in cultured human corneal epithelial cells in a manner dependent on nuclear factor-kappa B.

To evaluate the role of semaphorin 7A (Sema7A) and its associated regulatory mechanisms in modulating the barrier function of cultured human corneal epithelial cells (HCEs). Barrier models of HCEs were treated with recombinant human Sema7A at concentrations of 0, 125, 250, or 500 ng/mL for 24, 48, or 72h in vitro. Transepithelial electrical resistance (TEER) as well as Dextran-fluorescein isothiocyanate (FITC) permeability assays were conducted to assess barrier function. To quantify tight junctions (TJs) such as occludin and zonula occludens-1 (ZO-1) at the mRNA level, reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed. Immunoblotting was used to examine the activity of the nuclear factor-kappa B (NF-κB) signaling pathway and the production of TJs proteins. Immunofluorescence analyses were employed to localize the TJs. Enzyme-linked immunosorbent assay (ELISA) and RT-PCR were utilized to observe changes in interleukin (IL)-1β levels. To investigate the role of NF-κB signaling activation and IL-1β in Sema7A's anti-barrier mechanism, we employed 0.1 µmol/L IκB kinase 2 (IKK2) inhibitor IV or 500 ng/mL IL-1 receptor (IL-1R) antagonist. Treatment with Sema7A resulted in decreased TEER and increased permeability of Dextran-FITC in HCEs through down-regulating mRNA and protein levels of TJs in a time- and dose-dependent manner, as well as altering the localization of TJs. Furthermore, Sema7A stimulated the activation of inhibitor of kappa B alpha (IκBα) and expression of IL-1β. The anti-barrier function of Sema7A was significantly suppressed by treatment with IKK2 inhibitor IV or IL-1R antagonists. Sema7A disrupts barrier function through its influence on NF-κB-mediated expression of TJ proteins, as well as the expression of IL-1β. These findings suggest that Sema7A could be a potential therapeutic target for the diseases in corneal epithelium.

Carrageenan films as promising mucoadhesive ocular drug delivery systems

Polymer mucoadhesive films being developed for use in ophthalmology represent a new tool for drug delivery and are considered an alternative to traditional dosage forms. Due to their mucoadhesive properties, carrageenans (CRGs) are widely used in various forms for drug delivery. In this study, films based on CRGs of various structural types (κ/β, κ, x, and λ) for use in ophthalmology were studied. The films were loaded with the active substance echinochrome (ECH), a sea urchin pigment used in ophthalmology. Spectral data showed that ECH remained stable after its incorporation into the CRG films and did not oxidize for at least six months. Hydrophilic CRG films with a thickness of 10−12 µm were characterized in terms of their swelling and mucoadhesive properties. The rheological properties of solutions formed after film dissolution in artificial tears were also assessed. κ- and κ/β-CRG films with ECH exhibited pseudoplastic behavior after rehydrating films with an artificial tear solution. The CRG-loaded films had different swelling characteristics depending on the structure of the CRG used. The films based on highly sulfated CRGs dissolved in artificial tears, while the films of low-sulfated κ/β-CRG exhibited limited swelling. All studied ECH-loaded films exhibited mucoadhesive properties, which were evaluated by a texture analyzer using mucous tissue of the small intestine of the pig as a model. There was a slight prolongation of ECH release from CRG films in artificial tears. The effect of CRG/ECH on the epithelial cell lines of the outer shell of the human eye was investigated. At low concentrations, ECH in the composition of the CRG/ECH complex had no cytotoxic effect on corneal epithelial and conjunctival human cells. The use of ECH-containing films can prevent the drug from being immediately washed away by tears and help to retain it by increasing viscosity and having mucoadhesive properties.

Mesenchymal stem cell-based adjunctive therapy for Pseudomonas aeruginosa-induced keratitis: A proof-of-concept in-vitro study

PurposePseudomonas aeruginosa-induced keratitis is one of the most severe and challenging forms of corneal infection, owing to its associated intense inflammatory reactions leading to corneal necrosis and dense corneal scar with loss of vision. Since mesenchymal stem cells (MSCs) are reported to possess antimicrobial and immunomodulatory properties, they can be tested as an adjuvant treatment along with the antibiotics which are the current standard of care. This study aims to investigate the anti-bacterial and immunomodulatory roles of human bone marrow MSC-derived conditioned medium (MSC-CM) in P. aeruginosa-infected human corneal epithelial cells (HCECs) in vitro. MethodsThe effect of MSC-CM on the growth of clinical isolates of P. aeruginosa was evaluated by colony-forming unit assay. The expression of inflammatory cytokines (IL-6 and TNF-α) and an antimicrobial peptide (Lipocalin 2) in lipopolysaccharide-treated MSCs and HCECs was analyzed through ELISA. Corneal epithelial repair following infection with P. aeruginosa was studied through scratch assay. ResultsCompared to control (P. aeruginosa (5*105) incubated in DMEM (1 ml) at 37 °C for 16 h), MSC-CM significantly: i) inhibits the growth of P. aeruginosa (159*109 vs. 104*109 CFU/ml), ii) accelerates corneal epithelial repair following infection with P. aeruginosa (9% vs. 24% closure of the wounded area after 12 h of infection), and iii) downregulates the lipopolysaccharide-induced expression of IL-6, TNF-α and Lipocalin 2 in HCECs. A combination of MSC-CM with an antibiotic, Ciprofloxacin moderately regulated the expression of IL-6, TNF-α, and Lipocalin 2. ConclusionMSC-CM holds promise as an adjunctive therapeutic approach for P. aeruginosa-induced corneal epithelial damage.

Deoxynivalenol Damages Corneal Epithelial Cells and Exacerbates Inflammatory Response in Fungal Keratitis.

Fungal keratitis (FK) is a kind of infectious keratopathy with a high rate of blindness worldwide. Deoxynivalenol (DON) has been proven to have multiple toxic effects on humans and animals. The aim of this study was to explore a possible pathogenic role of DON in FK. We first made an animal model of FK in New Zealand white rabbits, and then attempted to detect DON in a culture medium in which Fusarium solani had been grown and also in the corneal tissue of the animal model of Fusarium solani keratitis. Next, a model of DON damage in human corneal epithelial cells (HCECs) was constructed to evaluate effects of DON on the activity, migration ability, cell cycle, and apoptosis in the HCECs. Then, putative the toxic damaging effects of DON on rabbit corneal epithelial cells and the impact of the repair cycle were studied. The expression levels of inflammatory factors in the corneas of the animal model and in the model of DON-damaged HCECs were measured. The Fusarium solani strain used in this study appeared to have the potential to produce DON, since DON was detected in the corneal tissue of rabbits which had been inoculated with this Fusarium solani strain. DON was found to alter the morphology of HCECs, to reduce the activity and to inhibit the proliferation and migration of HCECs. DON also induced the apoptosis and S-phase arrest of HCECs. In addition, DON was found to damage rabbit corneal epithelial cells, to prolong the corneal epithelial regeneration cycle, and to be associated with the upregulated expression of inflammatory factors in HCECs and rabbit corneas. DON appears to have a toxic damaging effect on HCECs in FK, and to induce the expression of inflammatory factors, leading to the exacerbation of keratitis and the formation of new blood vessels. Future studies will explore the possibility of developing a test to detect DON in ophthalmic settings to aid the rapid diagnosis of FK, and to develop DON neutralizers and adsorbents which have the potential to improve keratocyte status, inhibit apoptosis, and alleviate inflammation, therein providing new thinking for therapy of clinical FK.

Ectoine protects corneal epithelial survival and barrier from hyperosmotic stress by promoting anti-inflammatory cytokine IL-37

PurposeTo explore novel role and molecular mechanism of a natural osmoprotectant ectoine in protecting corneal epithelial cell survival and barrier from hyperosmotic stress. MethodsPrimary human corneal epithelial cells (HCECs) were established from donor limbus. The confluent cultures in isosmolar medium were switched to hyperosmotic media (400–500 mOsM), with or without ectoine or rhIL-37 for different time periods. Cell viability and proliferation were evaluated by MTT or WST assay. The integrity of barrier proteins and the expression of cytokines and cathepsin S were evaluated by RT-qPCR, ELISA, and immunostaining with confocal microscopy. ResultsHCECs survived well in 450mOsM but partially damaged in 500mOsM medium. Ectoine well protected HCEC survival and proliferation at 500mOsM. The integrity of epithelial barrier was significantly disrupted in HCECs exposed to 450mOsM, as shown by 2D and 3D confocal immunofluorescent images of tight junction proteins ZO-1 and occludin. Ectoine at 5–20 mM well protected these barrier proteins under hyperosmotic stress. The expression of TNF-α, IL-1β, IL-6 and IL-8 were dramatically stimulated by hyperosmolarity but significantly suppressed by Ectoine at 5–40 mM. Cathepsin S, which was stimulated by hyperosmolarity, directly disrupted epithelial barrier. Interestingly, anti-inflammatory cytokine IL-37 was suppressed by hyperosmolarity, but restored by ectoine at mRNA and protein levels. Furthermore, rhIL-37 suppressed cathepsin S and rescued cell survival and barrier in HCECs exposed to hyperosmolarity. ConclusionOur findings demonstrate that ectoine protects HCEC survival and barrier from hyperosmotic stress by promoting IL-37. This provides new insight into pathogenesis and therapeutic potential for dry eye disease.

Progranulin Facilitates Corneal Repair Through Dual Mechanisms of Inflammation Suppression and Regeneration Promotion.

The cornea serves as a vital protective barrier for the eye; however, it is prone to injury and damage that can disrupt corneal epithelium and nerves, triggering inflammation. Therefore, understanding the biological effects and molecular mechanisms involved in corneal wound healing and identifying drugs targeting these pathways is crucial for researchers in this field. This study aimed to investigate the therapeutic potential of progranulin (PGRN) in treating corneal injuries. Our findings demonstrated that PGRN significantly enhanced corneal wound repair by accelerating corneal re-epithelialization and re-innervation. In vitro experiments with cultured epithelial cells and trigeminal ganglion cells further revealed that PGRN stimulated corneal epithelial cell proliferation and promoted axon growth in trigeminal ganglion cells. Through RNA-sequencing (RNA-seq) analysis and other experimental techniques, we discovered that PGRN exerted its healing effects modulating Wnt signaling pathway, which played a critical role in repairing epithelial cells and promoting axon regeneration in trigeminal neurons. Importantly, our study highlighted the anti-inflammatory properties of PGRN by inhibiting the NF-κB signaling pathway, leading to decreased infiltration of macrophages. In conclusion, our findings underscored the potential of PGRN in facilitating corneal wound healing by promoting corneal epithelial cell proliferation, trigeminal ganglion cell axon regeneration, and suppressing ocular inflammation. These results suggest that PGRN could potentially expedite the healing process and improve visual outcomes in patients with corneal injuries.

BMSC Alleviates Dry Eye by Inhibiting the ROS-NLRP3-IL-1β Signaling Axis by Reducing Inflammation Levels

Purpose Bone marrow mesenchymal stem cells (BMSC) have multiple biological functions and are widely involved in regulating inflammatory diseases, tissue repair and regeneration. However, the mechanism of their action in dry eye disease (DED) is currently unclear. The purpose of this study was to investigate the effect of BMSCs in the treatment of dry eye mice and to explore its specific therapeutic mechanism. Methods Mouse corneal epithelial cells (MCECs) were treated with 500 mOsM sodium chloride hypertonic solution to induce a DED cell model. The dry eye animal model was constructed by adding 5 μL 0.2% benzalkonium chloride solution to mouse eyes. Western blotting was used to detect the expression of related proteins, and flow cytometry, enzyme-linked immunosorbent assay (ELISA), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, hematoxylin-eosin (HE) staining, and periodic acid schiff (PAS) staining were used to detect cell and eye tissue damage. Results The experimental results showed that BMSCs can reduce the levels of reactive oxygen species (ROS) and inflammatory factors in MCECs, promote cell proliferation, inhibit cell apoptosis, improve the integrity of the corneal epithelial layer in vivo, promote an increase in the number of goblet cells, and alleviate DED. Further exploration of the molecular mechanism of BMSCs treatment revealed that BMSCs alleviate the progression of DED by inhibiting the ROS-NLRP3-IL-1β signaling pathway. Conclusion BMSCs inhibit ROS-NLRP3-IL-1β signaling axis, reducing inflammation levels and alleviating dry eye symptoms. These findings provide new ideas and a basis for the treatment of DED and provide an experimental basis for further research on the application value of BMSCs in alleviating DED.

Analysis of the mucosal chemokines CCL28, CXCL14, and CXCL17 in dry eye disease: An in vitro and clinical investigation

Mucosal chemokines have antimicrobial properties and play an important role in mucosal immunity. However, little is known about their expression on the ocular surface. This study aimed to analyze the expression of the mucosal chemokines CCL28, CXCL14 and CXCL17 in corneal and conjunctival epithelial cells under in vitro dry eye (DE) conditions, and in conjunctival samples from healthy subjects and DE patients. Human corneal epithelial cells (HCE) and immortalized human conjunctival epithelial cells (IM-HConEpiC) were incubated under hyperosmolar (400–500 mOsM) or inflammatory (TNF-α 25 ng/mL) conditions for 6 h and 24 h to measure CCL28, CXCL14, and CXCL17 gene expression by RT-PCR and their secretion by immunobead-based analysis (CCL28, CXCL14) and ELISA (CXCL17). Additionally, twenty-seven DE patients and 13 healthy subjects were included in this study. DE-related questionnaires (OSDI, mSIDEQ and NRS) evaluated symptomatology. Ocular surface integrity was assessed using vital staining. Tactile sensitivity was measured with Cochet-Bonnet esthesiometer, and mechanic and thermal (heat and cold) sensitivity using Belmonte's non-contact esthesiometer. Subbasal nerve plexus and dendritic cell density were analyzed by in vivo confocal microscopy. Conjunctival cells from participants were collected by impression cytology to measure mucosal chemokines gene expression by RT-PCR. Our results showed that HCE and IM-HConEpiC cells increased CCL28, CXCL14, and CXCL17 secretion under hyperosmolar conditions. The gene expression of CCL28 was significantly upregulated in conjunctival samples from DE patients. CCL28 expression correlated positively with symptomatology, corneal staining, heat sensitivity threshold, and dendritic cell density. CXCL14 expression correlated positively with age, ocular pain, conjunctival staining, tactile sensitivity, and image reflectivity. CXCL17 expression correlated positively with corneal staining. These results suggest that corneal and conjunctival epithelial cells could be a source of CCL28, CXCL14, and CXCL17 on the ocular surface and that CCL28 might be involved in DE pathogenesis.

Examination of Subbasal Nerve Plexus and Central Corneal Stromal Microstructure in Subjects With Congenital Aniridia, Using in Vivo Confocal Laser Scanning Microscopy

Purpose During life up to 70% of aniridia subjects develop aniridia-associated keratopathy (AAK). AAK is characterized by limbal stem cell insufficiency, impaired corneal epithelial cell differentiation and abnormal cell adhesion, which leads to centripetal spreading vascularization, conjunctivalization, and thickening of the cornea. Our aim was to examine the subbasal nerve plexus and central corneal stromal microstructure in subjects with congenital aniridia, using in vivo confocal laser scanning microscopy CLSM. Methods 31 eyes of 18 patients (55.6% males, mean age: 25.22 ± 16.35 years) with congenital aniridia and 46 eyes of 29 healthy subjects (41.4% males, mean age 30 ± 14.82 years) were examined using the Rostock Cornea Module of Heidelberg Retina Tomograph-III. At the subbasal nerve plexus, corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL), corneal total branch density (CTBD), and corneal nerve fiber width (CNFW) were analyzed using ACCMetrics software. Keratocyte density in the anterior, middle and posterior stroma was assessed manually. Results The CNFD (2.02 ± 4.08 vs 13.99 ± 6.34/mm2), CNFL (5.78 ± 2.68 vs 10.56 ± 2.82 mm/mm2) and CTBD (15.08 ± 15.62 vs 27.44 ± 15.05/mm2) were significantly lower in congenital aniridia subjects than in controls (p < 0.001 for all). CNFW was significantly higher in aniridia subjects than in controls (0.03 ± 0.004 vs 0.02 ± 0.003 mm/mm2) (p = 0.003). Keratocyte density was significantly lower in all stromal layers of aniridia subjects than in controls (p < 0.001 for all). Stromal alterations included confluent keratocytes, keratocytes with long extensions and hyperreflective dots between keratocytes in aniridia. Conclusions Decrease in CNFD, CNFL, and CTBD, as well as increase in CNFW well refer to the congenital aniridia-associated neuropathy. The decreased keratocyte density and the stromal alterations may be related to an increased cell death in congenital aniridia, nevertheless, stromal changes in different stages of AAK have to be further analyzed in detail.

Chlorine-Induced Toxicity on Murine Cornea: Exploring the Potential Therapeutic Role of Antioxidants.

Chlorine (Cl2) exposure poses a significant risk to ocular health, with the cornea being particularly susceptible to its corrosive effects. Antioxidants, known for their ability to neutralize reactive oxygen species (ROS) and alleviate oxidative stress, were explored as potential therapeutic agents to counteract chlorine-induced damage. In vitro experiments using human corneal epithelial cells showed decreased cell viability by chlorine-induced ROS production, which was reversed by antioxidant incubation. The mitochondrial membrane potential decreased due to both low and high doses of Cl2 exposure; however, it was recovered through antioxidants. The wound scratch assay showed that antioxidants mitigated impaired wound healing after Cl2 exposure. In vivo and ex vivo, after Cl2 exposure, increased corneal fluorescein staining indicates damaged corneal epithelial and stromal layers of mice cornea. Likewise, Cl2 exposure in human ex vivo corneas led to corneal injury characterized by epithelial fluorescein staining and epithelial erosion. However, antioxidants protected Cl2-induced damage. These results highlight the effects of Cl2 on corneal cells using in vitro, ex vivo, and in vivo models while also underscoring the potential of antioxidants, such as vitamin A, vitamin C, resveratrol, and melatonin, as protective agents against acute chlorine toxicity-induced corneal injury. Further investigation is needed to confirm the antioxidants' capacity to alleviate oxidative stress and enhance the corneal healing process.