Corneal Epithelial Cell Line Research Articles

Host cell-type and pathogen-specific immunomodulatory functions of macrophage migration inhibitory factor (MIF) in infectious keratitis

Therapeutic management of inflammation in infectious keratitis (IK) requires new strategy and targets for selective immunomodulation. Targeting host cell-type specific inflammatory responses might be a viable strategy to curtail unnecessary inflammation and reduce tissue damage without affecting pathogen clearance. This study explores the possibility of pathogen and host cell-type dependent differences in the inflammatory pathways relevant in the pathogenesis of IK. Human corneal epithelial cell line (HCEC) and phorbol 12-myristate-13 acetate (PMA) differentiated THP-1 macrophage line were infected with either Aspergillus flavus conidia or Acanthamoeba castellanii trophozoites and the elicited inflammatory responses were studied in terms of gene expression and secretion of proinflammatory factors interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) and an upstream inflammatory regulator and mediator protein-the Macrophage Migration Inhibitory Factor (MIF). Given the pleotropic mode of MIF function in diverse cell types relevant in many human diseases, we tested if MIF driven responses to infection is different in HCECs and THP-1 macrophages by studying its expression, secretion and involvement in inflammation by siRNA mediated knockdown. We also examined IK patient tear samples for MIF levels. Infection with A. flavus or A. castellanii induced IL-8 and TNF-α responses in HCECs and THP-1 macrophages but to different levels. Our preliminary human data showed that the level of secreted MIF protein was elevated in IK patient tear, however, MIF secretion by the two cell types were strikingly different in-vitro, under both normal and infected conditions. We found that HCECs released MIF constitutively, which was significantly inhibited with infection, whereas THP-1 macrophages were stimulated to release MIF during infection. MIF gene expression remained largely unaffected by infection in both the cell lines. Although MIF in HCECs appeared to be intracellularly captured during infection, MIF knockdown in HCECs associated with a partial reduction of the IL-8 and TNF-α expression produced by either of the pathogens, suggesting a pro-inflammatory role for MIF in HCECs, independent of its canonical cytokine like function. In contrast, MIF knockdown in THP-1 macrophages accompanied a dramatic increase in IL-8 and TNF-α expression during A. castellanii infection, while the responses to A. flavus infection remained unchanged. These data imply a host cell-type and pathogen specific distinction in the MIF- related inflammatory signaling and MIF as a potential selective immunomodulatory target in infectious keratitis.

L-Carnitine Suppresses Transient Receptor Potential Vanilloid Type 1 Activation in Human Corneal Epithelial Cells.

Tear film hyperosmolarity induces dry eye syndrome (DES) through transient receptor potential vanilloid type 1 (TRPV1) activation. L-carnitine is a viable therapeutic agent since it protects against this hypertonicity-induced response. Here, we investigated whether L-carnitine inhibits TRPV1 activation by blocking heat- or capsaicin-induced increases in Ca2+ influx or hyperosmotic stress-induced cell volume shrinkage in a human corneal epithelial cell line (HCE-T). Single-cell fluorescence imaging of calcein/AM-loaded cells or fura-2/AM-labeled cells was used to evaluate cell volume changes and intracellular calcium levels, respectively. Planar patch-clamp technique was used to measure whole-cell currents. TRPV1 activation via either capsaicin (20 µmol/L), hyperosmolarity (≈450 mosmol/L) or an increase in ambient bath temperature to 43 °C induced intracellular calcium transients and augmented whole-cell currents, whereas hypertonicity induced cell volume shrinkage. In contrast, either capsazepine (10 µmol/L) or L-carnitine (1-3 mmol/L) reduced all these responses. Taken together, L-carnitine and capsazepine suppress hypertonicity-induced TRPV1 activation by blocking cell volume shrinkage.

Formulation and In-Vitro/Ex-Vivo Characterization of Pregelled Hybrid Alginate-Chitosan Microparticles for Ocular Delivery of Ketorolac Tromethamine.

Innovative hybrid chitosan-sodium alginate (Ch-Ag) microparticles (MPs) were fabricated using both the ionic gelation method as well as the pre-gelation technique. The hybrid Ch-Ag MPs were studied for size, zeta potential, morphology, mucoadhesion, in-vitro release, corneal permeation, and ocular irritation using lens and corneal epithelial cell lines. The average particle size ranged from 1322 nm to 396 nm. The zeta potential for the prepared formulations showed an increase with increasing Ch concentrations up to a value of >35 mV; the polydispersity index (PDI) of some optimized MPs was around 0.1. Compared to drug-free MPs, ketorolac-loaded Ch-Ag MPs demonstrated a drug proportion-dependent increase in their size. SEM, as well as TEM of KT-loaded MPs, confirmed that the formed particles were quasi-spherical to elliptical in shape. The KT release from the MPs demonstrated a prolonged release profile in comparison to the control KT solution. Further, mucoadhesion studies with porcine mucin revealed that the KT-loaded MPs had effective mucoadhesive properties, and polymeric particles were stable in the presence of mucin. Corneal permeation was studied on bovine eyes, and the results revealed that Ch-based MPs were capable of showing more sustained KT release across the cornea compared with that for the control drug solution. Conclusively, the cytotoxicity assay confirmed that the investigated MPs were non-irritant and could confer protection from direct drug irritation of KT on the ocular surface. The MTT cytotoxicity assay confirmed that KT-loaded MPs showed acceptable and reasonable tolerability with both human lens and corneal epithelial cell lines compared to the control samples.

Long Noncoding RNA MIATNB Regulates Hyperosmotic Stress-induced Corneal Epithelial Cell Injury by Inhibiting Autophagy in Dry Eye Disease

Purpose Dry eye disease (DED) has a complex etiology and the roles of long noncoding RNAs (lncRNAs) in its pathophysiology are not completely understood. Autophagy is a self-eating process important for cell survival and homeostasis. The present study explored the role of myocardial infarction-associated transcript neighbor (MIATNB) long non-coding RNA in hyperosmolarity-induced autophagy and apoptosis in human corneal epithelial cell (HCEC)-based model of dry eye disease. Methods In vitro assays were performed with a human SV40 immortalized corneal epithelial cell line. Different concentrations of NaCl were used to create hyperosmolarity. HCECs were cultured in presence of 70–120 mM NaCl for 24 h to create an in vitro model of dry eye. RT-qPCR was performed to assess the expression of dry eye related LC3B, ATG16L, BECN1, ATG1, ATG7, ATG13, ATG5, ATG10, and ATG101 mRNAs and western blot analysis of LC3B and P62 and RFP -GFP-tagged LC3. Flow cytometry and western blot analysis of caspase 3, BCL2 and BAX were performed to detect apoptosis. Chloroquine (CQ) was used to inhibit autophagy pharmacologically. Results Autophagy flux was activated in HCECs subjected to hyperosmotic stress. Hyperosmolarity activated apoptosis and inhibited HCEC migration and autophagy. Hyperosmolarity upregulated MIATNB expression, while MIATNB knockdown inhibited autophagosome degradation and promoted HCEC apoptosis. Under hyperosmolar conditions, MIATNB knockdown also inhibited the degradation of autophagolysosomes and stimulated HCEC apoptosis. Conclusion MIATNB plays a vital role in dry eye pathogenesis and serves as a bridge between autophagy and apoptosis. Targeting MIATNB for DED treatment should be further evaluated.

Binary Polymeric Surfactant Mixtures for the Development of Novel Loteprednol Etabonate Nanomicellar Eyedrops.

The treatment of several ocular inflammatory conditions affecting different areas of the ocular globe involves the administration of topical ophthalmic formulations containing corticosteroids. This research was aimed at evaluating the solubilising efficacy of 5.0% w/w of different binary mixtures of commercial amphiphilic polymeric surfactants with the purpose of obtaining nanomicellar solutions containing a high amount of loteprednol etabonate (LE). The selected LE-TPGS/HS nanomicelles, containing 0.253 mg/mL of the drug, had a small size (=13.57 nm) and uniform distribution (Polydispersity Index = 0.271), appeared completely transparent and perfectly filterable through 0.2 μm membrane filter, and remained stable up to 30 days at 4 °C. The critical micellar concentration (CMCTPGS/HS) was 0.0983 mM and the negative value of the interaction parameter between the polymeric-surfactant-building unit (βTPGS/HS = -0.1322) confirmed the ability of the polymeric surfactants to interact, favouring the dissolution of LE into nanomicelles. The disappearance of the endothermic peak of LE in the DSC analysis confirmed the interactions of LE with the polymeric surfactants. LE-TPGS/HS produced in vitro LE which sustained diffusion for 44 h (more than 40% of encapsulated LE). Furthermore, the lack of a significant cytotoxic effect on a sensitive corneal epithelial cell line makes it a candidate for further biological studies.

C1q/TNF-Related Proteins 1, 6 and 8 Are Involved in Corneal Epithelial Wound Closure by Targeting Relaxin Receptor RXFP1 In Vitro.

Inadequate wound healing of ocular surface injuries can lead to permanent visual impairment. The relaxin ligand-receptor system has been demonstrated to promote corneal wound healing through increased cell migration and modulation of extracellular matrix formation. Recently, C1q/tumor necrosis factor-related protein (CTRP) 8 was identified as a novel interaction partner of relaxin receptor RXFP1. Additional data also suggest a role for CTRP1 and CTRP6 in RXFP1-mediated cAMP signaling. However, the role of CTRP1, CTRP6 and CTRP8 at the ocular surface remains unclear. In this study, we investigated the effects of CTRP1, CTRP6, and CTRP8 on epithelial ocular surface wound closure and their dependence on the RXFP1 receptor pathway. CTRP1, CTRP6, and CTRP8 expression was analyzed by RT-PCR and immunohistochemistry in human tissues and cell lines derived from the ocular surface and lacrimal apparatus. In vitro ocular surface wound modeling was performed using scratch assays. We analyzed the effects of recombinant CTRP1, CTRP6, and CTRP8 on cell proliferation and migration in human corneal and conjunctival epithelial cell lines. Dependence on RXFP1 signaling was established by inhibiting ligand binding to RXFP1 using a specific anti-RXFP1 antibody. We detected the expression of CTRP1, CTRP6, and CTRP8 in human tissue samples of the cornea, conjunctiva, meibomian gland, efferent tear ducts, and lacrimal gland, as well as in human corneal, conjunctival, and meibomian gland epithelial cell lines. Scratch assays revealed a dose-dependent increase in the closure rate of surface defects in human corneal epithelial cells after treatment with CTRP1, CTRP6, and CTRP8, but not in conjunctival epithelial cells. Inhibition of RXFP1 fully attenuated the effect of CTRP8 on the closure rate of surface defects in human corneal epithelial cells, whereas the CTRP1 and CTRP6 effects were not completely suppressed. Conclusions: Our findings demonstrate a novel role for CTRP1, CTRP6, and CTRP8 in corneal epithelial wound closure and suggest an involvement of the relaxin receptor RXFP1 signaling pathway. This could be a first step toward new approaches for pharmacological and therapeutic intervention.

IFN-γ Facilitates Corneal Epithelial Cell Pyroptosis Through the JAK2/STAT1 Pathway in Dry Eye.

To investigate the effect of gamma interferon (IFN-γ) on corneal epithelial pyroptosis in an experimental dry eye (DE) model and explore the underlying molecular mechanisms. Experimental DE was established in adult wild-type (WT) C57BL/6 mice and Ifng-knockout mice on a C57BL/6 background by subcutaneous injection of scopolamine (1.5 mg/0.3 mL, three times per day) and exposure to desiccating stress. An immortalized human corneal epithelial cell line (HCE-T) was treated with IFN-γ under hyperosmolar conditions. Corneal epithelial defects, tear production, and conjunctival goblet cells were detected by fluorescein sodium staining, the phenol red cotton test, and periodic acid-Schiff staining. The mRNA expression was measured by quantitative real-time PCR. Changes in protein expression were analyzed by Western blotting and immunofluorescence staining. Cell Counting Kit-8 and lactate dehydrogenase assays and in situ TUNEL staining were used to assess cell death. The expression of IFNG and its related genes was increased in the corneas of DE mice, whereas genetic deletion of Ifng ameliorated desiccating stress-induced dry eye symptoms. We further found that IFN-γ activated the JAK2/STAT1 signaling pathway inducing corneal epithelial pyroptosis. Topical application of a STAT1 inhibitor in vivo or siRNA targeting STAT1 in vitro suppressed pyroptosis of corneal epithelial cells. In addition, the production of reactive oxygen species (ROS) was elevated in DE, and a reduction in excessive ROS release prevented pyroptosis. The increase in IFN-γ participates in the pathogenesis of dry eye and promotes corneal epithelial pyroptosis by activating the JAK2/STAT1 signaling pathway. Oxidative stress might be in downstream of JAK2/STAT1, thereby contributing to pyroptosis.

Ethosomal Nanoformulations for Combinational Photothermal Therapy of Fungal Keratitis

AbstractFungal keratitis/mycotic keratitis or keratomycosis, is characterized by the inflammation of underlying stroma of corneal epithelium due to fungal infection. Multi‐drug resistance and subsequent recurrence of infection limits the clinical outcomes of anti‐fungal agents such as Amphotericin B and Natamycin; and therefore, demands research on newer anti‐microbial strategies. In this study, the authors have synthesized and characterized ethosomes (IAEs) encapsulating a NIR dye, Indocyanine green (ICG), and an anti‐fungal drug, Amphotericin B, for a combinational photothermal therapy of fungal keratitis. The in vitro anti‐fungal efficacy of the IAEs is validated against Candida albicans by colony formation assay, calcofluor staining, and SEM analysis. The results indicate that the IAEs, upon NIR irradiation, displays a synergistic anti‐fungal efficacy. The IAEs are highly biocompatible when tested using a human corneal epithelial (HCE) cell line model (in vitro) and a Draize eye test model in rabbits (in vivo). Further, it is observed that the IAEs,upon NIR laser exposure significantly eliminate the fungal infection in rat model of occular fungal keratitis. The research indicates that the IAEs nano‐formulation exhibits a synergistic and sustained anti‐fungal effect paving its way for further translation from bench to bedside for the efficient management of fungal keratitis.

Establishing an accurate and sensitive in vitro drug screening system for human adenovirus infection with human corneal cells

Epidemic keratoconjunctivitis (EKC) is a hazardous and highly contagious disease, with the potential to cause epidemic outbreaks in hospitals and other community settings. There are currently no approved drugs for human adenovirus (HAdV), the causative agent of EKC. To establish a novel drug screening system for ocular HAdV infections, we employed CRL11516, a non-cancerous but immortalized human corneal epithelial cell line. Brincidoforvir and 3′-deoxy-3′-fluorothymidine inhibit replication of HAdV species C type 1 (C1), C2, E4, and C6 to the same extent. This alternative assay system may allow for the evaluation of anti-HAdV activity and cell cytotoxicity of compounds within 2 days and without the need of the rabbit eye infection model.

Considerations for the optimization of in vitro models of chloropicrin toxicity.

Chloropicrin (CP) is a common agricultural fumigant historically used as a chemical warfare agent and is a concern for potential use in warfare and terrorist applications. Our inability to effectively treat CP-induced injuries makes it essential to better understand CP toxicity. We set out to elucidate variables that must be understood to achieve optimal exposure conditions for in vitro investigations given that such models are important for the study of CP injury and potential therapeutics. To this end, we evaluated the effects of volatility, cell seeding density, and serum concentration of cell culture medium on CP toxicity in an immortalized human corneal epithelial cell line. We found that even with very dilute solutions, CP remained highly volatile, so much so that a 0.0019% CP solution resulted in 90% cell death at time 0, but was nearly nontoxic 45min later. Not surprisingly, the CP-induced IL-8 response was shown to vary with cell viability in this experiment. After exposure with 0.00115% CP, cells that were 12% confluent experienced over 40% more cell death than cells exposed at 87% confluency. Exposure with the same CP dose in medium containing concentrations of fetal bovine serum (FBS) ranging from 0.1% to 15% exhibited a 17% difference in cell viability. Given that chemical toxicity can be significantly influenced by volatility, cell density, and serum content of cell culture medium, these phenomena should be explored during the development and optimization of toxicant exposure models.

Crocetin protects against mitochondrial damage induced by UV-A irradiation in corneal epithelial cell line HCE-T cells

The corneal epithelium is located at the outermost layer of the ocular surface and continuously exposed to environmental factors, such as ultraviolet (UV) radiation from sunlight. UV irradiation causes excessive production of reactive oxygen species (ROS) in cells, which results in oxidative damage to membrane-bound organelles such as mitochondria, eventually leading to cell death. Crocetin, a natural carotenoid found in plants, has various biological properties including antioxidant activity. In this study, we investigated the effects of crocetin on UV-A-induced cell injury in the corneal epithelium. Using an in vitro system with the human corneal epithelial cell-transformed (HCE-T) cell line, pretreatment with 10 μM crocetin suppressed the reduction of cell viability induced by UV-A exposure. Crocetin ameliorated the decrease in oxygen consumption rates and the mitochondrial fragmentation that occurred following UV-A irradiation. Crocetin inhibited both ROS production and the activation of the apoptosis pathway; it also preserved the defects of epithelial cell polarity and barrier function in UV-A-irradiated HCE-T cells. The reduction in apical Mucin-16 expression was partially recovered in the presence of crocetin. Taking these findings together, we conclude that crocetin has a protective effect against UV-A irradiation-induced mitochondrial injury in corneal epithelial cells.

Cytotoxic, Apoptotic, and Oxidative Effects of Preserved and Preservative-Free Brimonidine in a Corneal Epithelial Cell Line.

Purpose: This study aims to compare the cytotoxic, apoptotic, and oxidative effects of preserved and preservative-free forms of brimonidine 0.15% on the human corneal epithelial cell (HCEC) line. Methods: Time-dependent cytotoxicity studies were performed with the Alamar Blue method. For apoptotic studies, PE Annexin V and 7-amino-actinomycin (7-AAD) staining and flow cytometry were performed. Messenger RNA (mRNA) expressions of Bax, Bcl-2, and caspase-3, -9, -12, and protein expressions of Bax and Bcl-2 were evaluated by quantitative real-time polymerase chain reaction and Western blot method, respectively. Results: Cell viability was 76.4% with the preserved solution and 36.05% with the preservative-free solution at the fifth minute. No significant difference was observed with either solution at the 15-min mark, whereas cell viability did not change significantly after 1 h. In the apoptosis evaluation, it was observed that the preservative-free solution increased the early apoptotic activity to a greater degree (P < 0.05). Preservative-free solution also induced gene expression of proapoptotic Bax, caspase-9 and -12, and protein expression of Bax while reducing the protein expression of anti-apoptotic Bcl-2 (P < 0.0001). Preserved solution induced only the gene expression of caspase-12, and reduced the protein expression of Bcl-2 (P < 0.0001). No significant difference was observed in the reactive oxygen species (ROS) levels of either solution compared with the control group (P > 0.05). Conclusion: It was demonstrated that the preserved solution is less cytotoxic to the HCEC line in the early period, has less early apoptotic activity, and does not significantly increase ROS levels.

A novel biodegradable polymer scaffold for in vitro growth of corneal epithelial cells.

The shortage of donor corneal tissue worldwide has led to extensive research for alternate corneal equivalents utilizing tissue engineering methods. We conducted experiments using Poly D, L lactic acid polymer along with a copolymer (Eudragit) in varying concentrations to create a biodegradable scaffold suitable for in vitro growth of corneal epithelial stem cells. It was found that stable, spherical, and porous microparticles can be prepared by combining PDLLA and Eudragit RL100 polymers in the ratio of 90:10 and 70:30. The microparticles can then be fused to form scaffold membranes with porous architecture and good water retention capacity at room temperature using methanol, which can withstand handling during transplantation procedures. The scaffolds made using a 70:30 ratio were found to be suitable for the promotion of growth of laboratory corneal epithelial stem cell lines (SIRC cell lines). This innovation can pave way for further developments in corneal stem cell research and growth, thus providing for viable laboratory-derived corneal substitutes.

AKR1C1 Protects Corneal Epithelial Cells Against Oxidative Stress-Mediated Ferroptosis in Dry Eye.

PurposeTo evaluate the precise mode of cell death and to investigate the molecular mechanism underlying the initiation of inflammation in dry eye disease (DED).MethodsC57BL/6 mice were injected with scopolamine subcutaneously and exposed to desiccating stress to establish a DED mouse model. An immortalized human corneal epithelial cell line (HCEC) was cultured under hyperosmolarity (500 mOsM). Protein expressions were measured using western blot assay and immunofluorescence staining. mRNA expression was analyzed by RNA-sequencing and quantitative RT-PCR. Transmission electron microscopy was used to observe the intracellular ultrastructure. Intracellular Fe2+ was detected by a FerroOrange fluorescent probe. Flow cytometry was used to evaluate the cellular reactive oxygen species and lipid peroxidation.ResultsMarked changes in ferroptosis-related markers expression, intracellular iron accumulation, and lipid peroxidation were observed in corneal epithelial cells of DED models. When excessive oxidative stress was suppressed, ferroptosis induced by hyperosmolarity in HCECs was restrained, as indicated by decreased iron content and lipid peroxidation levels. Moreover, AKR1C1 was upregulated by the activation of NRF2 in HCECs under hyperosmolarity. When AKR1C1 was knocked down, cell viability was decreased, accompanied by increased lipid peroxidation, whereas overexpression of AKR1C1 produced the opposite results. It was observed consistently that corneal defects and the inflammatory response were promoted after inhibition of AKR1C1 in vivo.ConclusionsExcessive oxidative stress-induced ferroptosis participates in DED pathogenesis. The expression of AKR1C1 is triggered by NRF2 to decrease ferroptosis-induced cell damage and inflammation in HCECs. These findings may provide potential makers targeting ferroptosis and AKR1C1 for DED therapy.

Hyaluronan supports the limbal stem cell phenotype during ex vivo culture

BackgroundHyaluronan (HA) has previously been identified as an integral component of the limbal stem cell niche in vivo. In this study, we investigated whether a similar HA matrix is also expressed in vitro providing a niche supporting limbal epithelial stem cells (LESCs) during ex vivo expansion. We also investigated whether providing exogenous HA in vitro is beneficial to LESCs during ex vivo expansion.MethodHuman LESCs (hLESCs) were isolated from donor corneas and a mouse corneal epithelial progenitor cell line (TKE2) was obtained. The HA matrix was identified surrounding LESCs in vitro using immunocytochemistry, flow cytometry and red blood exclusion assay. Thereafter, LESCs were maintained on HA coated dishes or in the presence of HA supplemented in the media, and viability, proliferation, cell size, colony formation capabilities and expression of putative stem cell markers were compared with cells maintained on commonly used coated dishes.ResultshLESCs and TKE2 cells express an HA-rich matrix in vitro, and this matrix is essential for maintaining LESCs. Further supplying exogenous HA, as a substrate and supplemented to the media, increases LESC proliferation, colony formation capabilities and the expression levels of putative limbal stem cell markers.ConclusionOur data show that both exogenous and endogenous HA help to maintain the LESC phenotype. Exogenous HA provides improved culture conditions for LESC during ex vivo expansion. Thus, HA forms a favorable microenvironment for LESCs during ex vivo expansion and, therefore, could be considered as an easy and cost-effective substrate and/or supplement for culturing LESCs in the clinic.

SiRNA high throughput screening identifies regulators of chloropicrin and hydrogen fluoride injury in human corneal epithelial cell models

Corneal injuries induced by various toxicants result in similar clinical presentations such as corneal opacity and neovascularization. Many studies suggest that several weeks post-exposure a convergence of the molecular mechanisms drives these progressive pathologies. However, chemical agents vary in toxicological properties, and early molecular responses are anticipated to be somewhat dissimilar for different toxicants. We chose 3120 targets from the Dharmacon Human Druggable genome to screen for chloropicrin (CP) and hydrogen fluoride (HF) corneal injury as we hypothesized that targets identified in vitro may be effective as therapeutic targets in future studies. Human immortalized corneal epithelial cells (SV40-HCEC) were used for screening. Cell viability and IL-8 were analyzed to down-select hits into validation studies, where multiplex cytokine analysis and high content analysis were performed to understand toxicant effect and target function. Some endpoints were also evaluated in a second human immortalized corneal epithelial cell line, TCEpi. Over 20 targets entered validation studies for CP and HF; of these, only three targets were shared: NR3C1, RELA, and KMT5A. These findings suggest that early molecular responses to different toxicants may be somewhat distinctive and present dissimilar targets for possible early intervention.

Integrated Stress Response Regulation of Corneal Epithelial Cell Motility and Cytokine Production.

PurposeTo investigate the effect of an active integrated stress response (ISR) on human corneal epithelial cell motility and cytokine production.MethodsISR agonists tunicamycin (TUN) and SAL003 (SAL) were used to stimulate the ISR in immortalized corneal epithelial cell lines, primary human limbal epithelial stem cells, and ex vivo human corneas. Reporter lines for ISR-associated transcription factors activating transcription factor 4 (ATF4) and XBP1 activity were generated to visualize pathway activity in response to kinase-specific agonists. Scratch assays and multiplex magnetic bead arrays were used to investigate the effects of an active ISR on scratch wounds and cytokine production. A C/EBP homologous protein (CHOP) knockout cell line was generated to investigate the effects of ISR ablation. Finally, an ISR antagonist was assayed for its ability to rescue negative phenotypic changes associated with an active ISR.ResultsISR stimulation, mediated through CHOP, inhibited cell motility in both immortalized and primary human limbal epithelial cells. Scratch wounding of ex vivo corneas elicited an increase in the ISR mediators phosphorylated-eIF2α and ATF4. ISR stimulation also increased the production of vascular endothelial growth factor (VEGF) and proinflammatory cytokines. ISR ablation, through CHOP knockout or inhibition with integrated stress response inhibitor (ISRIB) rescued epithelia migration ability and reduced VEGF secretion.ConclusionsWe demonstrate that the ISR has dramatic effects on the ability of corneal epithelial cells to respond to wounding models and increases the production of proinflammatory and angiogenic factors. Inhibition of the ISR may provide a new therapeutic option for corneal diseases in which the ISR is implicated.

Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells

BackgroundPrimary canine corneal epithelial cells (CCECs) easily become senescent, and cell proliferation is limited. Therefore, sampling for experimentation requires a large number of animals, which is problematic in terms of animal welfare and fails to maintain the stability of the cells for in vitro analyses.ResultsIn this study, CCECs were separated and purified by trypsin and dispase II enzymatic analysis. Next, the cells were immortalized by transfection with a lentiviral vector expressing Simian vacuolating virus 40 large T (SV40T). The immortalized canine corneal epithelial cell line (CCEC-SV40T) was established by serial passages and monoclonal selection. The biological characteristics of CCEC-SV40T cells were evaluated based on the cell proliferation rate, cell cycle pattern, serum dependence, karyotype, and cytokeratin 12 immunofluorescence detection. In addition, we infected CCEC-SV40T cells with Staphylococcus pseudintermedius (S. pseudintermedius) and detected the inflammatory response of the cells. After the CCEC-SV40T cells were passaged continuously for 40 generations, the cells grew in a cobblestone pattern, which was similar to CCECs. The SV40T gene and cytokeratin 12 can be detected in each generation. CCEC-SV40T cells were observed to have a stronger proliferation capacity than CCECs. CCEC-SV40T cells maintained the same diploid karyotype and serum-dependent ability as CCECs. After CCEC-SV40T cells were infected with S. pseudintermedius, the mRNA expression levels of NLRP3, Caspase-1 and proinflammatory cytokines, including IL-1β, IL-6, IL-8 and TNF-α, were upregulated, and the protein levels of MyD88, NLRP3 and the phosphorylation of Iκbα and p65 were upregulated.ConclusionsIn conclusion, the CCEC-SV40T line was successfully established and can be used for in vitro studies, such as research on corneal diseases or drug screening.

An efficient simplified method for the generation of corneal epithelial cells from human pluripotent stem cells.

Corneal epithelial cells derived from human pluripotent stem cells (hPSCs) are an important cell source for preclinical models to test ophthalmic drugs. However, current differentiation protocols lack instructions regarding optimal culturing conditions, which hinders the quality of cells and limits scale-up. Here, we introduce a simplified small molecule-based corneal induction method (SSM-CI) to generate corneal epithelial cells from hPSCs. SSM-CI provides the advantage of minimizing cell-culturing time using two defined culturing media containing TGF-β, and Wnt/β-catenin pathway inhibitors, and bFGF growth factor over 25days. Compared to the conventional human corneal epithelial cell line (HCE-T) and human primary corneal epithelial cells (hPCEpCs), corneal epithelial cells generated by SSM-CI are well differentiated and express relevant maturation markers, including PAX6 and CK12. RNA-seq analysis indicated the faithful differentiation of hPSCs into corneal epithelia, with significant upregulation of corneal progenitor and adult corneal epithelial phenotypes. Furthermore, despite the initial inhibition of TGF-β and Wnt/β-catenin, upregulation of these pathway-related transcripts was observed in the later stages, indicating their necessity in the generation of mature corneal epithelial cells. Moreover, we observed a shift in gene signatures associated with the metabolic characteristics of mature corneal epithelial cells, involving a decrease in glycolysis and an increase in fatty acid oxidation. This was also attributed to the overexpression of metabolic enzymes and transporter-related transcripts responsible for fatty acid metabolism. Thus, SSM-CI provides a comprehensive method for the generation of functional corneal epithelial cells for use in preclinical models.

Early Interventions for Toxicant Ocular Injury May Need to be Toxicant‐Specific

Corneal injuries induced by various toxicants share similar pathologies such as corneal opacity and neovascularization. Several studies suggest that the molecular mechanisms that drive these delayed and progressive pathologies may converge at later time points. However, chemicals with different toxicological properties are likely to elicit different molecular responses early after exposure. These early responses may present opportunities for therapeutic intervention. Our objective was to study the molecular mechanisms of toxicant‐induced corneal injury, utilizing siRNA high throughput screening (HTS). Our hypothesis was that early molecular responses to each toxicant would be dissimilar and targets validated in vitro may be effective for treating toxicant injury. We selected ~3000 targets from preconfigured Dharmacon Human Druggable libraries for three toxicants: chloropicrin (CP), hydrogen fluoride (HF), and sulfur mustard (HD). Microarray data from injured mouse corneas were used to focus library selection. Screening was conducted in the human immortalized corneal epithelial cell line SV40‐HCEC. Endpoints included IL‐8 levels in cell culture supernatants and cell viability. Target validation studies utilized phenotype restoration in SV40‐HCEC and siRNA function in the human immortalized corneal epithelial cell line, TCEpi. Validation endpoints included multiplex cytokine analysis and high content analysis to understand toxicant effect and target function. Approximately 20 target genes entered in vitro validation studies for each toxicant, but only one gene was in common, NR3C1, a glucocorticoid receptor. For each toxicant, 5‐10 target genes entered testing in mice. These results demonstrated that the drug chlorpheniramine (target HRH1) was effective in preventing central progression of corneal neovascularization in CP‐induced corneal injury during 8 weeks of study even after treatment was withdrawn 2 weeks post‐exposure. In conclusion, these findings show that there is little similarity in the early molecular responses to these three toxicants and suggest that some early interventions for toxicant ocular injury may need to be toxicant‐specific.