Corneal Epithelial Barrier Function Research Articles

Bone morphogenetic protein 4 inhibits corneal neovascularization by blocking NETs-induced disruption to corneal epithelial barrier

Corneal neovascularization (CoNV) is the second leading cause of visual impairment worldwide, and current drugs have certain limitations. Inflammatory response is the core pathological process of CoNV. Neutrophil extracellular traps (NETs) are generated after neutrophil activation, which promotes neovascularization. Prior studies demonstrated that bone morphogenetic protein 4 (BMP4) could significantly reduce inflammation and CoNV formation, its exact molecular mechanism remains unclear. Therefore, we stimulated human peripheral blood neutrophils with phorbol myristate acetate (PMA) or deoxyribonuclease I (DNase I) to induce or inhibit NETs formation. By using corneal sutures and subconjunctival injections of NETs or DNase I, rat CoNV models were established. Compared with the suture group, NETs formation and inflammatory cell infiltration in the corneal stroma were significantly increased, corneal edema was aggravated, and the length, area and diameter of CoNV were significantly enhanced in the NETs group. Furthermore, by curetting the corneal epithelial apical junctional complexes (AJCs), a crucial component in preserving the function of the corneal epithelial barrier, we discovered that the damage of AJCs had a significant role in inducing CoNV formation. NETs could induce CoNV formation by injuring corneal epithelial AJCs. Finally, by comparing the aforementioned indicators after the intervention of BMP4, BMP4 inhibitor Noggin and NADPH oxidase (NOX) inhibitor, we finally demonstrated that BMP4 could inhibit NETs-induced inflammation and corneal epithelial AJC injury, repair corneal epithelial barrier function and eventually inhibit CoNV formation by blocking NOX-2-dependent NETs formation.

Therapeutic effects of low-color-temperature light-emitting diodes on dry eye.

As a new technology for treating dry eye diseases, phototherapy has attracted great attention, but the research on its safety and effectiveness is limited. In this study, the therapeutic effects of low-color-temperature light-emitting diodes on dry eye in humans, rabbits, and rats were investigated. In clinical experiments, subjects in both groups read the same paper for 3 h under light sources of two color temperatures: 1900 K (low-color-temperature light-emitting diodes) or 4000 K (artificial fluorescent white light-emitting diodes). The differences in the non-invasive tear film breakup time, tear meniscus height, and conjunctival congestion scores before and after the experiment were compared between the two groups. In animal experiments, corneal epithelial barrier function and tear production of Sprague-Dawley rats and New Zealand white rabbits with dry eye were compared before and after low-color-temperature light-emitting diodes treatment. TUNEL staining and Western blotting were used to detect the apoptosis of corneal and conjunctival cells and the expression of inflammatory factor IL-1β. Low-color-temperature light-emitting diodes prolonged tear film breakup time in patients with dry eye. Moreover, it increased tear secretion, decreased fluorescein sodium staining scores, corneal and conjunctival cell apoptosis, and inflammatory factor expression in rabbits and rats with dry eye. Low-color-temperature light-emitting diodes phototherapy can be used as an effective treatment for dry eye, reducing its symptoms and related ocular surface damage in humans, rabbits, and rats.

Conditional deletion of CD25 in the corneal epithelium reveals sex differences in barrier disruption

PurposeIL-2 promotes activation, clonal expansion, and deletion of T cells. IL-2 signals through its heterotrimeric receptor (IL-2R) consisting of the CD25, CD122 and CD132 chains. CD25 knockout (KO) mice develop Sjögren Syndrome-like disease. This study investigates whether corneal CD25/IL-2 signaling is critical for ocular health. MethodsEyes from C57BL/6 mice were collected and prepared for immunostaining or in-situ hybridization. Bulk RNA sequencing was performed on the corneal epithelium from wild-type and CD25KO mice. We generated a conditional corneal-specific deletion of CD25 in the corneal epithelium (CD25Δ/ΔCEpi). Corneal barrier function was evaluated based on the uptake of a fluorescent dye. Mice were subjected to unilateral corneal debridement, followed by epithelial closure over time. ResultsIn C57BL/6 mice, CD25 mRNA was expressed in ocular tissues. Protein expression of CD25, CD122, and CD132 was confirmed in the corneal epithelium. Delayed corneal re-epithelization was seen in female but not male CD25KO mice. There were 771 differentially expressed genes in the corneal epithelium of CD25KO compared to wild-type mice. While barrier function is disrupted in CD25Δ/ΔCEpi mice, re-epithelialization rates are not delayed. ConclusionsAll three chains of the IL-2R are expressed in the corneal epithelium. Our results indicate for the first time, deleting CD25 systemically in all tissues in the mouse and deleting CD25 locally in just the corneal epithelium compromises corneal epithelial barrier function, leading to dry eye disease in female mice. Future studies are needed to delineate the pathways used by IL-2 signaling to influence cornea homeostasis.

Protection of Barrier Function in Cultured Human Corneal Epithelial Cells by Semaphorin 4D

Purpose Corneal epithelial barrier function is important to maintain corneal homeostasis and is impaired by inflammation. We aimed to investigate the localization of semaphorin 4D (Sema4D) in the cornea, and its effects on the barrier function of cultured corneal epithelial cells. Methods The expressions of semaphorin4 D and its receptor in the murine cornea were examined by immunoblot, immunofluorescent staining and confocal microscopy observations. Human corneal epithelial (HCE) cells stimulated by TNF-α or IL-1β were cultured with or without Sema4D. Cell viability was examined by CCK8, cell migration was evaluated by scratch wound assay, and barrier function was assessed by transepithelial electrical resistance (TEER) and Dextran-FITC permeability assay. The expression of tight junction proteins in HCE cells was examined by immunoblot, immunofluorescent staining and qRT-PCR. Results We demonstrated that the protein of Sema4D and its receptor, plexin-B1, was expressed in murine cornea. Sema4D induced an increase in the TEER and a decrease in the permeability of HCE cells. It also induced the expression of tight junction protein ZO-1, occludin and claudin-1 in HCE cells. Furthermore, under stimulation of TNF-α or IL-1β, Sema4D treatment could inhibit the decreased TEER and the elevated permeability of HCE cells. Conclusions Sema4D is located distinctly in corneal epithelial cells and promoted their barrier function by increasing the expression of tight junction proteins. Sema4D may act as a preventive for maintaining corneal epithelial barrier function during ocular inflammation.

Transglutaminase-2 is critical for corneal epithelial barrier function via positive regulation of Claudin-1

PurposeTransglutaminase (TG)-2 is a ubiquitous multi-functional protein expressed in all living cells. The purpose of the current study was to investigate the role of TG-2 in corneal barrier function and its potential regulation of epithelial junctional proteins and transcription factors. MethodsCorneal barrier function to ions in TG-2−/− and TG-2+/+ mice was assessed by Ussing chamber assay. Hypo-osmolar water or FITC-dextran was applied on top of mouse eyes to evaluate the corneal barrier function to water and macromolecules. Western blots, qPCR and immunofluorescent staining were used to investigate the expression of tight junction proteins in TG-2−/− and TG-2+/+ mouse corneas, and also in TG-2 knockdown human corneal epithelial cells. ResultsCorneal explants from TG-2−/− mice had a lower trans-epithelial electrical resistance compared to TG-2+/+ mice. When challenged by hypo-osmolar water, the central corneal thickness of TG-2−/− mice increased faster, and these mice had a faster rise of fluorescence in the anterior chamber after ocular exposure to FITC-dextran, compared to TG-2+/+. Claudin-1 protein and transcript levels were reduced in the cornea of TG-2−/− mice and in TG-2 knockdown human corneal epithelial cells. Slug which previously reported suppressing Claudin-1 transcription, was increased at both protein and transcript level in TG-2 knockdown cells. TG-2 and Claudin-1 protein levels were unchanged in shRNA and shTG cells after MG132 treatment, while Slug accumulated in treated cells. ConclusionTG-2 may positively regulate Claudin-1 through repressing Slug at transcript level, and thus it is critical for normal corneal barrier function.

Involvement of aberrant acinar cell proliferation in scopolamine-induced dry eye mice

Dry eye is a multifactorial disease that causes dryness, inflammation and damage of ocular surface. Subcutaneous injection of the muscarinic cholinergic antagonist scopolamine under desiccating stress reduces tear production and induces dry eye symptoms in mice. However, the expression profile and pathogenic changes of the lacrimal gland remain incompletely understood. In the present study, we performed comparative transcriptomic analysis of lacrimal glands from the control and scopolamine-treated mice. Primary analysis identified 677 upregulated genes and 269 downregulated genes in the lacrimal gland of mice with scopolamine treatment. Unexpectedly, KEGG pathway and hierarchical clustering analysis showed the enrichment of “DNA replication” and “cell cycle” categories in the upregulated genes. Subsequently, we confirmed that the acinar cells were the major proliferating cells of lacrimal gland, which exhibited significant increasing of the proliferating cell nuclear antigen (PCNA) expression after scopolamine treatment, accompanied with the upregulation of DNA damage marker γ-H2AX. More importantly, both prophylactic and therapeutic administration of the cyclin-dependent kinase (CDK) inhibitor AT7519 rescued the tear reduction and alleviated dry eye severity in the scopolamine-treated mice, including corneal epithelial barrier function, lacrimal and corneal inflammation, and conjunctival goblet cell density. Therefore, we conclude that aberrant acinar cell proliferation is involved in the scopolamine-induced tear reduction and dry eye onset, which can be improved by AT7519 treatment.

Determining the effect of ocular chemical injuries on topical drug delivery

Ocular chemical injuries (OCIs) commonly cause ocular damage and visual loss and treatment uses topical therapies to facilitate healing and limit complications. However, the impact of chemical injury on corneal barrier function and treatment penetration is unknown. Therefore, the aim of this study was to determine the effect of OCI on drug penetration and absorption. Porcine corneal explants were used to assess histological damage, electrical resistance, and the trans-corneal penetration/corneal adsorption of reference compounds (sodium fluorescein and rhodamine B) and dexamethasone. Corneal explants were injured with either 1 M sulfuric acid, or 1 M sodium hydroxide. Dexamethasone penetration was measured using high-performance liquid chromatography (HPLC) and that of fluorescein and rhodamine using fluorescence. Dexamethasone corneal adsorption was measured using enzyme-linked immunoabsorbant assay (ELISA). Both acid and alkaline injuries reduced trans-corneal electrical resistance. NaOH injury increased hydrophilic fluorescein penetration (NaOH 8.59 ± 1.50E–05 cm.min−1 vs. Hanks' Balanced Salt Solution (HBSS) 1.64 ± 1.01E–06 cm.min−1) with little impact on hydrophobic rhodamine B (1 M NaOH 6.55 ± 2.45E–04 cm.min−1 vs. HBSS 4.60 ± 0.972E–04 cm.min−1) and dexamethasone penetration (1 M NaOH 3.00 ± 0.853E–04 cm.min−1 vs. HBSS 2.69 ± 0.439E–04 cm.min−1). By contrast, H2SO4 decreased trans-corneal penetration of hydrophilic fluorescein (H2SO4 1.16 ± 14.2E–07 cm.min−1) and of hydrophobic dexamethasone (H2SO4 1.88 ± 0.646E–04 cm.min−1) and rhodamine B (H2SO4 4.60 ± 1.42E–05 cm.min−1). Acid and alkaline OCI differentially disrupted the corneal epithelial barrier function. Acid injury reduced penetration of hydrophobic dexamethasone and rhodamine B as well as hydrophilic fluorescein, which may translate clinically into reduced drug penetration after OCI, while alkaline injury increased fluorescein penetration, with minimal effect on dexamethasone and rhodamine B penetration.

Differential effects of Hsp90 inhibition on corneal cells in vitro and in vivo

The transformation of quiescent keratocytes to activated fibroblasts and myofibroblasts (KFM transformation) largely depends on transforming growth factor beta (TGFβ) signaling. Initiation of the TGFβ signaling cascade results from binding of TGFβ to the labile type I TGFβ receptor (TGFβRI), which is stabilized by the 90 kDa heat shock protein (Hsp90). Since myofibroblast persistence within the corneal stroma can result in stromal haze and corneal fibrosis in patients undergoing keratorefractive therapy, modulation of TGFβ signaling through Hsp90 inhibition would represent a novel approach to prevent myofibroblast persistence.In vitro, rabbit corneal fibroblasts (RCFs) or stratified immortalized human corneal epithelial cells (hTCEpi) were treated with a Hsp90 inhibitor (17AAG) in the presence/absence of TGFβ1. RCFs were cultured either on tissue culture plastic, anisotropically patterned substrates, and hydrogels of varying stiffness. Cellular responses to both cytoactive and variable substrates were assessed by morphologic changes to the cells, and alterations in expression patterns of key keratocyte and myofibroblast proteins using PCR, Western blotting and immunocytochemistry. Transepithelial electrical resistance (TEER) measurements were performed to establish epithelial barrier integrity. In vivo, the corneas of New Zealand White rabbits were wounded by phototherapeutic keratectomy (PTK) and treated with 17AAG (3× or 6× daily) either immediately or 7 days after wounding for 28 days. Rabbits underwent clinical ophthalmic examinations, SPOTS scoring and advanced imaging on days 0, 1, 3, 7, 10, 14, 21 and 28. On day 28, rabbits were euthanized and histopathology/immunohistochemistry was performed.In vitro data demonstrated that 17AAG inhibited KFM transformation with the de-differentiation of spindle shaped myofibroblasts to dendritic keratocyte-like cells accompanied by significant upregulation of corneal crystallins and suppression of myofibroblast markers regardless of TGFβ1 treatment. RCFs cultured on soft hydrogels or patterned substrates exhibited elevated expression of α-smooth muscle actin (αSMA) in the presence of 17AAG. Treatment of hTCEpi cells disrupted zonula occludens 1 (ZO-1) adherens junction formation. In vivo, there were no differences detected in nearly all clinical parameters assessed between treatment groups. However, rabbits treated with 17AAG developed greater stromal haze formation compared with controls, irrespective of frequency of administration. Lastly, there was increased αSMA positive myofibroblasts in the stroma of 17AAG treated animals when compared with controls.Hsp90 inhibition promoted reversion of the myofibroblast to keratocyte phenotype, although this only occurred on rigid substrates. By contrast, in vivo Hsp90 inhibition was detrimental to corneal wound healing likely due to impairment in corneal epithelial closure and barrier function restoration. Collectively, our data demonstrated a strong interplay in vitro between biophysical cues and soluble signaling molecules in determining corneal stromal cell phenotype.

Effect of High Glucose on Ocular Surface Epithelial Cell Barrier and Tight Junction Proteins.

PurposePatients with diabetes mellitus are reported to have ocular surface defects, impaired ocular surface barrier function, and a higher incidence of corneal and conjunctival infections. Tight junctions are critical for ocular surface barrier function. The present study was designed to investigate the effect of high glucose exposure on human corneal and conjunctival epithelial cell barrier function and tight junction proteins.MethodsHuman corneal and conjunctival epithelial cells were exposed to 15 mM and 30 mM glucose for 24 and 72 hours. The barrier function was measured using transepithelial electrical resistance (TEER). The cell migration was quantified using scratch assay. The cells were harvested for protein extraction and mRNA isolation. Gene and protein expression of claudins, zonula occludens (ZOs), and occludin was quantified using real-time PCR and Western blot.ResultsGlucose caused a significant decrease in TEER after 72 hours of exposure in both corneal and conjunctival epithelial cells. Glucose did not cause any notable change in migration of either corneal or conjunctival epithelial cells. Glucose exposure did not cause any notable change in protein expression of claudin-1, ZO-1, ZO-2, ZO-3, or occludin. On the other hand, 15 mM glucose caused an increase in gene expression of claudin-1, claudin-3, ZO-2, ZO-3, and occludin, a likely response to osmotic stress since 15 mM mannitol also caused consistently similar increase in gene expression of these proteins.ConclusionsHigh glucose exposure causes impairment of corneal and conjunctival epithelial cell barrier function, but this detrimental effect is not caused by a decrease in expression of tight junction proteins: claudin-1, ZO-1, ZO-2, ZO-3, and occludin.

Corneal Health during Three Months of Scleral Lens Wear.

This study evaluated the effects scleral lens wear has on corneal health using fluorometry and in vivo confocal microscopy. No subclinical changes on healthy corneas of young subjects were observed during 3 months of scleral lens wear. This study aimed to evaluate the effects 3 months of scleral lens wear has on the corneal epithelial barrier function, dendritic cell density, and nerve fiber morphology. Twenty-seven neophytes (mean [standard deviation] age, 21.4 [3.9] years) wore scleral lenses of a fluorosilicone acrylate material bilaterally (97 Dk, 15.6 to 16.0-mm diameter) for 3 months without overnight wear. Subjects were randomized to use either Addipak (n = 12) or PuriLens Plus (n = 15) during lens insertion. Measurements of corneal epithelial permeability to fluorescein were performed with automated scanning fluorophotometer (Fluorotron Master; Ocumetrics, Mountain View, CA) on the central cornea of the right eye and the temporal corneal periphery of the left eye. Images of the distributions of corneal nerve fibers and dendritic cells and nerve fibers were captured in vivo with a confocal laser scanning microscope (Heidelberg Retina Tomograph, Rostock Cornea Module; Heidelberg Engineering, Heidelberg, Germany) on the central and inferior peripheral cornea of the left eye. Corneal measurements and imaging were performed at baseline and after 1 and 3 months of lens wear. The corneal permeability values in natural log, dendritic cell densities, and nerve fiber morphology did not significantly change from baseline to 1 and 3 months of lens wear, for both central and peripheral corneal regions (P > .05). Dendritic cell density at the inferior cornea was higher than the central cornea throughout the study (P < .001). No relationships were observed between each outcome measurements and the saline solution groups (P > .05). Scleral lens wear for 3 months on healthy cornea of young subjects did not affect corneal epithelial barrier function, nerve fiber, and dendritic cell densities. Buffered and nonbuffered saline solutions impacted the corneal health in similar ways.

High-fat diet induces dry eye-like ocular surface damages in murine

PurposeA high-fat diet leads to dysfunction in multiple systems of the body. Herein we investigate the effects of a high-fat diet on the ocular surface using a murine model. MethodsFour-week-old male C57BL/6 mice were fed with a standard-fat diet (10 kcal% fat, SFD) or a high-fat diet (60 kcal% fat, HFD) for 1 or 3 months. Phenol red thread test was used to detect tear production, oregon green dextran (OGD) staining was performed to assess corneal epithelial permeability, and PAS staining was conducted to ascertain the presence of conjunctival goblet cells. Squamous metaplasia in the ocular surface and corneal epithelial barrier function were detected by immunofluorescent staining, zymography and Western blot analysis. Oxidative stress related protein expression was evaluated by immunostaining and Western blot analysis. Corneal and conjunctival cell apoptosis was determined by TUNEL assay and caspase-3 expression. ResultsA HFD induced obvious ocular surface damages, including decreased tear production, notable OGD staining and distinct goblet cell loss. It also resulted in corneal epithelial barrier dysfunction and significant squamous metaplasia of the corneal and conjunctival epithelia. The HFD also upregulated key factors that regulate oxidative stress in the ocular surface, and upregulated cell apoptosis in ocular surface epithelial cells. ConclusionsA HFD induces dry eye-like ocular surface damages in mice via the activation of oxidative stress and an induction of apoptosis in the cells of the ocular surface.

Therapeutic Effects of STAT3 Inhibition on Experimental Murine Dry Eye

To investigate the therapeutic effects of targeting signal transducer and activator of transcription-3 (STAT3) activation on the ocular surface damage of dry eye in mice. Adult Balb/C and C57BL/6 mice with benzalkonium chloride (BAC) treatment, lacrimal gland excision, and meibomian gland dysfunction were used as dry eye models. The levels of phosphorylated STAT3 (p-STAT3) were detected with immunofluorescence staining and Western blotting. STAT3 inhibition was performed by topical application of STAT3 inhibitor S3I-201. Corneal epithelial barrier function, tear production, and conjunctival goblet cell density were quantified with fluorescein sodium staining, phenol red cotton test, and histochemical staining. The expressions of matrix metalloproteinase (MMP)-3/9, TUNEL, and inflammation cytokines were assessed with immunofluorescence staining, qPCR, and ELISA assays. The therapeutic effect of S3I-201 was further compared with the Janus kinase inhibitor tofacitinib and ruxolitinib. Elevated levels of nuclear p-STAT3 were detected in the corneal and conjunctival epithelium of three dry eye models. Topical application of S3I-201 improved corneal epithelial barrier function, increased tear production and conjunctival goblet cell density in BAC-induced dry eye mice. Moreover, S3I-201 decreased the expression of MMP-3/9, suppressed the apoptosis of corneal and conjunctival epithelial cells, and reduced the levels of IL-1β, IL-6, IL-17A, and IFN-γ. Compared with tofacitinib and ruxolitinib, the STAT3 inhibitor S3I-201 showed superior improvement of tear production and inflammatory cytokine expression in lacrimal gland. Elevated STAT3 activation is involved in the pathogenesis of dry eye, while targeting STAT3 effectively alleviates BAC-induced ocular surface damage.

Selective Serotonin Reuptake Inhibitors Aggravate Depression-Associated Dry Eye Via Activating the NF-κB Pathway.

Our study aimed to evaluate the side effects of selective serotonin reuptake inhibitors (SSRIs) on the ocular surface. Twenty patients with depression and dry eye disease (DED) were randomly picked to receive SSRI treatment, whereas another 20 patients received placebo treatment. The serotonin, inflammatory cytokine, and proapoptotic protein levels were determined by using protein chip, qRT-PCR, and ELISA analyses. A rat depression model was established, and SSRIs were applied for 3 or 6 weeks. Tear production and corneal epithelial barrier function were evaluated. The serotonin and inflammatory cytokine levels were analyzed by qRT-PCR, immunohistochemical staining, and ELISA. Human corneal epithelial cells were subjected to serotonin, a HTR antagonist, and/or an NF-κB signaling inhibitor. The inflammatory cytokine and proapoptotic protein levels were determined by qRT-PCR, Western blot analysis, and ELISA. The cell apoptosis rate was assessed by using flow cytometry. The SSRI group had higher tear serotonin levels and more serious inflammation and cell apoptosis on the ocular surface. In the rat depression model, depression decreased tear secretion and increased IL-1β and TNF-α production, whereas the serotonin, TLR2, and TLR4 levels were not increased. SSRI aggravated DED, disrupted the corneal epithelial barrier, and promoted an inflammatory response on the ocular surface by increasing the tear serotonin levels. In addition, serotonin induced an inflammatory response and cell apoptosis in corneal epithelial cells by activating NF-κB signaling. SSRIs aggravate depression-associated DED via activating the NF-κB pathway. The antagonist of HTRs or the inhibitor of NF-κB signaling presents a potential therapeutic strategy for depression-associated DED. (Trial registration number, ChiCTR1800015592).

Water extract of indoor dust induces tight junction disruption in normal human corneal epithelial cells

In corneal epithelium, tight junctions play a vital role in its barrier function. Human cornea is highly susceptible to damage by dust. Continued daily exposure to dust has been associated with increased risks of corneal injury. Studies demonstrated that water extract of dust induced cytotoxicity in human corneal epithelial cells (HCECs); however, its effects on corneal epithelial barrier function are unknown. In this study, we determined the concentrations of heavy metals in water extracts of dust, with office dust having higher concentrations of heavy metals than housedust, and Cu and Zn being highest among metals for both dust. Changes in barrier function and its associated mechanism after exposing HCECs to water extracts of dust at 48 μg/100 μ L for 7 d were evaluated. Water extracts of both dust caused decrease of TEER value (39–73%), down-regulation of gene expression related to tight junction and mucin (0.2–0.8 fold), and loss of ZO-1 immunoreactivity from cellular borders, with office dust having greater potential than housedust to disrupt corneal epithelial barrier function. Our data implied the importance to reduce heavy metals in dust to reduce their adverse impacts on human eyes.

0.005% Preservative-Free Latanoprost Induces Dry Eye-Like Ocular Surface Damage via Promotion of Inflammation in Mice.

To investigate the side effects of preservative-free 0.005% latanoprost on the murine ocular surface. We applied 0.005% latanoprost or vehicle in mice in two patterns for 14 to 28 days. Tear production was measured by phenol red cotton test, and corneal epithelial barrier function was assessed by Oregon-green-dextran (OGD) staining. Periodic acid-Schiff (PAS) staining was used to quantify conjunctival goblet cells (GCs). The expression of matrix metalloproteinase (MMP)-3 and -9, occludin-1 and zonula occludens (ZO)-1 in corneal epithelium was assessed by immunofluorescent staining and/or quantitative real-time PCR (qRT-PCR). Inflammation in conjunctiva was assessed by activation of P38 and NF-κB, infiltration of CD4+ T cells, and production inflammatory cytokines including TNF-α, IL-1β, IFN-γ, IL-17A, and IL-13. Apoptosis in ocular surface was assessed by TUNEL and immunofluorescent staining for activated caspase-3 and -8. Cell viability assay was performed in human corneal epithelial cells. Topical latanoprost treatment decreased tear production, induced conjunctival GC loss, disrupted the corneal epithelial barrier, and promoted cell apoptosis in the ocular surface. Topical latanoprost treatment increased the expression of MMP-3 and -9, and decreased the expression of ZO-1 and occludin-1 in the corneal epithelium. Topical application of latanoprost promoted activation of P38-NF-κB signaling and production of TNF-α and IL-1β in conjunctiva. Topical application of latanoprost increased CD4+ T cells infiltration, with increased production of IFN-γ and IL-17A and decreased production of IL-13 in conjunctiva. 0.005% latanoprost induced dry eye-like ocular surface damage via promotion of inflammation in mice.

A Mouse Model of Schnyder Corneal Dystrophy with the N100S Point Mutation

Schnyder corneal dystrophy (SCD) is a rare autosomal dominant disease in humans, characterized by abnormal deposition of cholesterol and phospholipids in cornea caused by mutations in the UbiA prenyltransferase domain containing 1 (UBIAD1) gene. In this study, we generated a mouse line carrying Ubiad1 N100S point mutation using the CRISPR/Cas9 technique to investigate the pathogenesis of SCD. In vivo confocal microscopy revealed hyper-reflective dot-like deposits in the anterior cornea in heterozygotes and homozygotes. No significant change was found in corneal epithelial barrier function or wound healing. Electron microscopy revealed abnormal mitochondrial morphology in corneal epithelial, stromal, and endothelial cells. Mitochondrial DNA copy number assay showed 1.27 ± 0.07 fold change in homozygotes versus 0.98 ± 0.05 variation in wild type mice (P < 0.05). Lipidomic analysis indicated abnormal metabolism of glycerophosphoglycerols, a lipid class found in mitochondria. Four (34:1, 34:2, 36:2, and 44:8) of the 11 glycerophosphoglycerols species identified by mass spectrometry showed a significant increase in homozygous corneas compared with heterozygous and wild-type mouse corneas. Unexpectedly, we did not find a difference in the corneal cholesterol level between different genotypes by filipin staining or lipidomic analysis. The Ubiad1N100S mouse provides a promising animal model of SCD revealing that mitochondrial dysfunction is a prominent component of the disease. The different phenotype in human and mouse may due to difference in cholesterol metabolism between species.

Topical Application of Mizoribine Suppresses CD4+ T-cell-Mediated Pathogenesis in Murine Dry Eye.

We investigate the effect of topical application of mizoribine (MZR) eye drops on CD4+ T-cell-mediated immunity and epithelial damage in ocular surface of dry eye disease (DED). Topical application of MZR or vehicle eye drops was performed in mice subjected to desiccating stress (DS). The phenol red cotton test was used to measure tear production, and Oregon-green-dextran (OGD) staining was performed to assess corneal epithelial barrier function. PAS staining was used to quantify conjunctival goblet cells. Immunofluorescent staining and quantitative (q) RT-PCR were used to assess the expression of matrix metalloproteinase (MMP)-9 in corneal epithelium. qRT-PCR and ELISA were used to assess the production of TNF-α and IL-1β in conjunctiva. Apoptosis in ocular surface was assessed by TUNEL and activation of caspase-8. CD4+ T-cell-mediated immunity was evaluated by CD4 immunohistochemistry and production of T helper cytokines, including IFN-γ, IL-13, and IL-17A in conjunctiva. Compared to vehicle control mice, topical MZR-treated mice showed increased tear production, decreased goblet cell loss, and improved corneal barrier function. Topical application of MZR suppressed the expression of MMP-9 in corneal epithelium and apoptosis in ocular surface, while it had no obvious effect on production of TNF-α and IL-1β in conjunctiva. Topical application of MZR decreased CD4+ T cells infiltration, with decreased production of IFN-γ and IL-17A, and increased production of IL-13 in conjunctiva. Topical application of MZR could alleviate epithelial damage and CD4+ T-cell-mediated immunity in ocular surface of DED.

Contributions of MyD88-dependent receptors and CD11c-positive cells to corneal epithelial barrier function against Pseudomonas aeruginosa

Previously we reported that corneal epithelial barrier function against Pseudomonas aeruginosa was MyD88-dependent. Here, we explored contributions of MyD88-dependent receptors using vital mouse eyes and confocal imaging. Uninjured IL-1R (−/−) or TLR4 (−/−) corneas, but not TLR2 (−/−), TLR5 (−/−), TLR7 (−/−), or TLR9 (−/−), were more susceptible to P. aeruginosa adhesion than wild-type (3.8-fold, 3.6-fold respectively). Bacteria adherent to the corneas of IL-1R (−/−) or TLR5 (−/−) mice penetrated beyond the epithelial surface only if the cornea was superficially-injured. Bone marrow chimeras showed that bone marrow-derived cells contributed to IL-1R-dependent barrier function. In vivo, but not ex vivo, stromal CD11c+ cells responded to bacterial challenge even when corneas were uninjured. These cells extended processes toward the epithelial surface, and co-localized with adherent bacteria in superficially-injured corneas. While CD11c+ cell depletion reduced IL-6, IL-1β, CXCL1, CXCL2 and CXCL10 transcriptional responses to bacteria, and increased susceptibility to bacterial adhesion (>3-fold), the epithelium remained resistant to bacterial penetration. IL-1R (−/−) corneas also showed down-regulation of IL-6 and CXCL1 genes with and without bacterial challenge. These data show complex roles for TLR4, TLR5, IL-1R and CD11c+ cells in constitutive epithelial barrier function against P. aeruginosa, with details dependent upon in vivo conditions.

Therapeutic Effect of MK2 Inhibitor on Experimental Murine Dry Eye.

To investigate the role of mitogen-activated protein kinase-activated protein kinase-2 (MK2) in ocular surface damage of dry eye. MK2 inhibition was performed in mice subjected to desiccating stress (DS) by topical application of MK2 inhibitor (MK2i) or vehicle eye drops. The total and phosphorylated MK2 in conjunctiva were detected by Western blot. The phenol red cotton test was used to measure tear production, and Oregon green dextran staining was performed to assess corneal epithelial barrier function. PAS staining was used to quantify conjunctival goblet cells. Immunofluorescent staining and quantitative RT-PCR were used to assess the expression of matrix metalloproteinase (MMP)-3 and -9 in corneal epithelium. Apoptosis in ocular surface was assessed by TUNEL and immunofluorescent staining for activated caspase-3 and -8. Inflammation was evaluated by CD4+ T-cell infiltration and production of T helper (Th) cytokines, including IFN-γ, IL-13, and IL-17A in conjunctiva. DS promoted MK2 activation in conjunctiva. Compared with vehicle control mice, MK2i-treated mice showed increased tear production, decreased goblet cell loss, and improved corneal barrier function. Topical MK2 inhibition decreased the expression of MMP-3 and -9 in corneal epithelium, and suppressed cell apoptosis in ocular surface under DS. Topical MK2 inhibition decreased CD4+ T-cell infiltration, with decreased production of IFN-γ and IL-17A and increased production of IL-13 in conjunctiva. Topical MK2 inhibition effectively alleviated ocular surface damage via suppressing cell apoptosis and CD4+ T-cell-mediated inflammation in ocular surface of dry eye.

Changes in Ocular Surface Characteristics after Switching from Benzalkonium Chloride-Preserved Latanoprost to Preservative-Free Tafluprost or Benzalkonium Chloride-Preserved Tafluprost.

Purpose The aim of the present study was to examine the effects of switching from Latanoprost ophthalmic solution containing a preservative to preservative-free Tafluprost ophthalmic solution or Tafluprost containing a preservative on ocular surfaces. Materials and Methods Forty patients (40 eyes) with glaucoma (mean age: 62.0 ± 10.9 years) using Latanoprost with preservative for six months or longer were assigned either to a Tafluprost-containing-preservative group (20 eyes) or preservative-free-Tafluprost group (20 eyes). The intraocular pressure, corneal epithelial barrier function (fluorescein uptake concentration with fluorophotometer FL-500), superficial punctate keratopathy (AD classification), and tear film breakup time (TBUT) were assessed before switching and at 12 weeks after switching. Results No significant differences in intraocular pressure were noted after switching in either group. Corneal epithelial barrier function was improved significantly after switching in both the Tafluprost-containing-preservative and the preservative-free-Tafluprost groups. There were no significant differences in AD scores after switching in the Tafluprost-containing-preservative group, but significant improvements were noted in the preservative-free-Tafluprost group. No significant differences in TBUT were noted in the Tafluprost-containing-preservative or preservative-free-Tafluprost groups after switching. Conclusion After switching from preservative Latanoprost to Tafluprost containing-preservative or preservative-free Tafluprost, corneal epithelial barrier function was improved while the intraocular pressure reduction was retained.