Human Conjunctival Epithelium Research Articles

Transmembrane Protein CMTM6 Alleviates Ocular Inflammatory Response and Improves Corneal Epithelial Barrier Function in Experimental Dry Eye.

To investigate the impact of transmembrane protein CMTM6 on the pathogenesis of dry eye disease (DED) and elucidate its potential mechanisms. CMTM6 expression was confirmed by database analysis, real-time polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry. Tear secretion was measured using the phenol red thread test. Immune cell infiltration was assessed through flow cytometry. Barrier function was evaluated by fluorescein sodium staining, immunofluorescence staining of zonula occludens 1 (ZO-1), and electric cell-substrate impedance sensing (ECIS) assessment. For silencing CMTM6 expression, siRNA and shRNA were employed, along with lentiviral vector-mediated overexpression of CMTM6. Proinflammatory cytokine levels were analyzed by RT-PCR and cytometric bead array (CBA) analysis. CMTM6 showed high expression in healthy human and mouse corneal and conjunctival epithelium but was notably reduced in DED. Notably, this downregulation was correlated with disease severity. Cmtm6-/- dry eye (DE) mice displayed reduced tear secretion, severe corneal epithelial defects, decreased conjunctival goblet cell density, and upregulated inflammatory response. Additionally, Cmtm6-/- DE mice and CMTM6 knockdown human corneal epithelial cell-transformed (HCE-T) cells showed more severe barrier disruption and reduced expression of ZO-1. Knockdown of CMTM6 in HCE-T cells increased inflammatory responses induced by hyperosmotic stress, which was significantly mitigated by CMTM6 overexpression. Moreover, the level of phospho-p65 in hyperosmolarity-stimulated HCE-T cells increased after silencing CMTM6. Nuclear factor kappa B (NF-κB) p65 inhibition (JSH-23) reversed the excessive inflammatory responses caused by hyperosmolarity in CMTM6 knockdown HCE-T cells. The reduction in CMTM6 expression on the ocular surface contributes to the pathogenesis of DED. The CMTM6-NF-κB p65 signaling pathway may serve as a promising therapeutic target for DED.

Epithelial Immune Cell Response to Initial Soft Contact Lens Wear in the Human Corneal and Conjunctival Epithelium.

The purpose of this study was to assess the immediate ocular immune response to soft contact lens (CL) wear by examining presumed epithelial immune cell (EIC) density and morphology at the central, peripheral, limbal cornea, and conjunctiva. Fifty-four participants naïve to CL wear (mean age = 24.8 ± 9.8years, 44% female participants), were examined using in vivo confocal microscopy at baseline and after 2hours of CL wear (1-Day ACUVUE MOIST). Images were captured at the central, temporal far peripheral and limbal cornea, and bulbar conjunctiva. EIC density was counted manually and morphology was graded. Differences in EIC parameters pre- and post-CL wear were examined using a generalized estimating equation model with appropriate post hoc analyses. After 2hours of soft CL wear, there was a significant increase in EIC density in all regions other than the central cornea (all P < 0.001). Cell body size was significantly larger, and a higher proportion of participants exhibited EIC with long dendrites after lens wear at the central and peripheral cornea (both P < 0.001). There was a significant increase in the number of participants displaying EIC with thick dendrites at the peripheral (P = 0.04) and limbal cornea (P < 0.001) after lens wear. EICs were primarily recruited to the peripheral regions, whereas the central cornea shows no significant recruitment after short-term CL wear. Both central and peripheral corneas exhibited an enhanced antigen capture capacity, whereas migratory capacity was increased in the peripheral corneal regions suggesting EIC activation following a short period of CL wear.

Immortalized Human Conjunctival Epithelial Cells Produce Functional Complement C3 and C4 Proteins.

The aim of this study was to assess whether complement proteins C3 and C4 are produced by immortalized human conjunctival epithelial (HCjE) cells. Supernatants and cell lysates from undifferentiated and differentiated HCjE cells were assayed for C3 and C4 by enzyme-linked immunosorbent assay. To measure complement protein function, supernatants and lysates were treated with heat-aggregated IgG, and soluble C5b-9 was measured. C3 was upregulated in supernatants from differentiated HCjE cells compared with undifferentiated HCjE cells (556.55 ± 91.75 vs. 56.95 ± 12.09 ng/mL, P <0.001). C4 was also increased in supernatants but to a much lesser extent (0.599 ± 0.476 vs. 0.172 ± 0.0133 ng/mL, P = 0.03). From HCjE cell lysates, total C3 production was 9.03 times higher in differentiated HCjE cells ( P <0.001), whereas total C4 remained relatively unchanged. After activation with heat-aggregated IgG, sC5b-9 could be detected from both undifferentiated and differentiated HCjE cell lysates, but not in the HCjE supernatants. HCjE cells produce C3 and C4 in sufficient quantities to support the formation of sC5b-9, confirming their biological activity and suggesting that HCjE cells likely produce all complement proteins C1 through C9.

Transmembrane Mucin Response in Conjunctival Epithelial Cells Exposed to Wall Shear Stresses

Human conjunctival epithelium cells (HCEC) line the inner surface of the eyelid and cover the sclera and are continuously subjected to wall shear stresses (WSS). The effects of external forces on the conjunctival epithelium are not fully known. The conjunctival epithelium contains stratified squamous cells that synthesize the membrane-spanning mucins MUC1 and MUC16, which play important roles in protecting the ocular surface. Alterations in both gel-forming and membrane-tethered mucins occur in drying ocular surface diseases. The aim of this study was to explore the mechanobiological characteristics of transmembrane mucin secretion and cellular alterations of primary HCEC exposed to airflow-induced WSS perturbations. We exposed the HCEC to a steady WSS of 0.5 dyne/cm2 for durations of 15 and 30 min. Cytoskeletal alterations and MUC1 secretions were studied using immunohistochemically fluorescent staining with specific antibodies. We investigated for the first time an in vitro model of membrane-tethered mucin secretion by HCEC in response to WSS. The exposure of HCEC to WSS increased the polymerization of F-actin, altered the cytoskeletal shape and reduced the secretion of membrane-tethered MUC1.

Effect of immobilized hyaluronidase and subtilisin enzymes on the ultrastructure of human conjunctival epithelial cells

The use of enzyme preparations is a traditional trend in various fields of medicine. The usage of immobilized enzymes hyaluronidase (IG) and subtilisin (IS) seems to be very promising for the treatment of damage to the ocular surface. The aim of the study was to establish the nature of changes in the ultrastructural organization of human conjunctival epithelium under the influence of immobilized hyaluronidase and subtilisin in vitro. Material and methods. In the experiment, a culture of normal cells of the human conjunctiva Chanqconjunctiva, clone 1–5 C-4, was seeded in 96well plates in the amount of 2 × 104 cells/well, after 24 h the medium was removed, Eagle’s medium MEM and fetal calf serum were added and cells were cultured for another 48 h. 5 experimental groups were formed: group 1 – without drugs (except for the composition of the incubation scheme), groups 2 and 3 – with IS at a concentration of 37 and 150 U/ml, respectively, groups 4 and 5 – with IG at a concentration of 37 and 150 U/ml, respectively. After preparation of cell preparations under a JEM 1400 electron microscope (Japan), ultrathin sections of 70–100 nm were studied at ×1000 magnification. Results. The article presents the results of electron microscopy of a culture of normal cells of the human conjunctiva in 5 experimental groups of cells with a description of changes in cytoarchitectonics under the influence of IG and IS. Conclusions. The introduction of immobilized enzymes into human conjunctival cell culture at a low dose (37 U/ml) affects the organization of cells without cytotoxic effects, while increasing the dose directly correlates with the occurrence of a cytotoxic effect.

Expression of GPR-68 in Human Corneal and Conjunctival Epithelium. Possible indicator and mediator of attrition associated inflammation at the ocular surface

Attrition and osmotic stress have been identified as major forces in the pathophysiology of dry eye. Impaired tolerance to mechano-transduction in the presence of insufficient lubrication has been associated with disturbances of ocular surface homeostasis and encouragement of inflammatory reactions, challenging the usual regulatory coping mechanisms. In spite of the probable link between enhanced attrition and secondary inflammation, the key mediators driving the vicious cycle of severe dry eye disease have not yet been identified. The goal of this study was therefore to investigate human corneal and conjunctival epithelium for the presence of the G protein-coupled receptor GPR-68. This protein had most recently been shown to be not only chemically activated but also mechanically, possibly through attrition. De-identified sections of human cornea and conjunctiva were stained for the presence of G protein-coupled receptor 68 with specific antibodies using immunohistochemical methods. Results Specific staining for G-protein-coupledreceptor 68 (GPR68) was observed in all samples of the cornea throughout the epithelial layers of the corneal epithelium, most prominently in the area of the wing cells and the basement membrane. Even in the conjunctiva, specific staining for GPR-68 was found. The detection of G protein-coupled receptor GPR-68 in human corneal and conjunctival epithelium raises the question of its function and purpose. The mechanical activation of GPR68 in situations with enhanced friction and attrition could modify various cellular functions and possibly jeopardize normal inflammatory homeostasis at the ocular surface. Accordingly, decreased lubrication in dry eye disease could result in activation of GPR-68. This could lead to secondary inflammation, initially in the epithelium and surrounding stroma. Continuous mechanical stress could result in chronic inflammation, also reaching deeper structures of the cornea, possibly making GPR-68 an important actor in the vicious cycle of dry eye disease. G protein-coupled receptor GPR-68, sensitive to flow and mechanic stimulation, is present in the human corneal epithelium and conjunctiva. Decreased lubrication and increased attrition, accompanied by sensations typical for dry eye, might lead to local inflammation. It is possible that subtle signs of conjunctival, and later corneal, surface damage in the context of these sensations could be a better indicator of the need for and success of therapy than the clinical signs of dry eye disease alone, at least in the early stages of the disease. Inhibition of G protein-coupled receptor GPR-68 could represent a new strategy in the treatment of dry eye disease.

The effect of amniotic membrane proteins released by ocular inserts containing self‐healing hydrogels

AbstractPurpose: Growth factors found in the amniotic membrane (AM) help accelerate epithelium regeneration in the damaged cornea or conjunctiva. This study compares the effects of hydrogels releasing the AM proteins on the proliferation, migration, and time‐to‐closure (healing) of the conjunctival epithelium.Methods: Proteins extracted from the AM using sonication were incorporated into two hydrogels used as ocular inserts. The tested inserts materials were the self‐healing, dynamic hyaluronic‐acid hydrogel based on gold‐thiolate/disulfide exchange (Hydrogel A) and a physically cross‐linked hydrogel based on a combination between Eudragit S100 and hyaluronic acid with a reinforced matrix by polyvinyl alcohol, polyvinyl pyrrolidone and polyethylene glycol (Hydrogel B). In an in vitro experiment, a scratch was made on the human conjunctival epithelium cells (Chang Conjunctiva, Clone 1‐5c‐4) to mimic an incision wound. The cells were cultured with hydrogels for 48 hours to study the effect of these materials on epithelium repair. The samples were examined using a Zeiss Observer Z1 inverted microscope, and the results were evaluated employing the Image‐J program to obtain the cell migration rate (μm/h).Results: Hydrogel A increased the rate of closure at 22 h (121 ± 19 μm) in comparison with Hydrogel B (394 ± 78 μm). After 46 h, the scratches in Hydrogel A and control cultures were completely closed, while the Hydrogel B samples had an unclosed area of 54 ± 4 μm.Conclusions: Both hydrogels promoted wound healing in cultured conjunctival epithelium cells. However, a lower rate of wound closure was observed for Hydrogel B than for Hydrogel A and the control, probably because the speed of cell proliferation was slower in Hydrogel B. The wound‐healing assay showed that the Hydrogel A and B increased cell viability and significantly stimulated cell migration.

Cytotoxicity, Mitochondrial Functionality, and Redox Status of Human Conjunctival Cells after Short and Chronic Exposure to Preservative-Free Bimatoprost 0.03% and 0.01%: An In Vitro Comparative Study.

Prostaglandin analogues (PGAs), including bimatoprost (BIM), are generally the first-line therapy for glaucoma due to their greater efficacy, safety, and convenience of use. Commercial solutions of preservative-free BIM (BIM 0.03% and 0.01%) are already available, although their topical application may result in ocular discomfort. This study aimed to evaluate the in vitro effects of preservative-free BIM 0.03% vs. 0.01% in the human conjunctival epithelial (HCE) cell line. Our results showed that long-term exposure to BIM 0.03% ensues a significant decrease in cell proliferation and viability. Furthermore, these events were associated with cell cycle arrest, apoptosis, and alterations of ΔΨm. BIM 0.01% does not exhibit cytotoxicity, and no negative influence on conjunctival cell growth and viability or mitochondrial activity has been observed. Short-time exposure also demonstrates the ability of BIM 0.03% to trigger reactive oxygen species (ROS) production and mitochondrial hyperpolarisation. An in silico drug network interaction was also performed to explore known and predicted interactions of BIM with proteins potentially involved in mitochondrial membrane potential dissipation. Our findings overall strongly reveal better cellular tolerability of BIM 0.01% vs. BIM 0.03% in HCE cells.

Novel CFTR Activator Cact-3 Ameliorates Ocular Surface Dysfunctions in Scopolamine-Induced Dry Eye Mice.

Cystic fibrosis transmembrane conductance regulator (CFTR) is highly expressed on the ocular epithelium and plays a pivotal role in the fluid secretion driven by chloride transport. Dry eye disease is one of the most common diseases with limited therapeutic options. In this study, a high-throughput screening was performed to identify novel CFTR activators capable of inducing chloride secretion on the ocular surface. The screening of 50,000 small molecules revealed three novel CFTR activators. Among them, the most potent CFTR activator, Cact-3 (7-(3,4-dimethoxyphenyl)-N-(4-ethoxyphenyl)pyrazolo [1,5-α]pyrimidine-2-carboxamide), produced large and sustained Cl− currents in WT-CFTR-expressing FRT cells with no alterations of ANO1 and hERG channel activity. The application of Cact-3 strongly activated CFTR in the ocular epithelia of mice and it also significantly increased CFTR-mediated Cl− transport in a primary cultured human conjunctival epithelium. Cact-3 strongly stimulated tear secretion in normal mice. In addition, Cact-3 significantly reduced ocular surface damage and the expression of proinflammatory factors, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in an experimental mouse model of dry eye disease. These results suggest that Cact-3, a novel CFTR activator, may be a potential development candidate for the treatment of dry eye disease.

SARS-CoV-2 infects human adult donor eyes and hESC-derived ocular epithelium.

The SARS-CoV-2 pandemic has caused unparalleled disruption of global behavior and significant loss of life. To minimize SARS-CoV-2 spread, understanding the mechanisms of infection from all possible routes of entry is essential. While aerosol transmission is thought to be the primary route of spread, viral particles have been detected in ocular fluid, suggesting that the eye may be a vulnerable point of viral entry. To this end, we confirmed SARS-CoV-2 entry factor and antigen expression in post-mortem COVID-19 patient ocular surface tissue and observed productive viral replication in cadaver samples and eye organoid cultures, most notably in limbal regions. Transcriptional analysis of ex vivo infected ocular surface cells and hESC-derived eye cultures revealed robust induction of NF-κB in infected cells as well as diminished type I/III interferon signaling. Together these data suggest that the eye can be directly infected by SARS-CoV-2 and implicate limbus as a portal for viral entry.

Lactobacillus sakei Pro-Bio65 Reduces TNF-α Expression and Upregulates GSH Content and Antioxidant Enzymatic Activities in Human Conjunctival Cells.

PurposeThe study investigates the regulatory effects exhibited by lysate of Lactobacillus sakei pro-Bio65 (4%; L.SK) on the human conjunctival epithelial (HCE) cell line.MethodsTrypan blue and methylthiazol tetrazolium (MTT) methods were used to assess cell growth and viability. Mitochondrial membrane potential was assessed by JC-1 staining and cytofluorimetric detection methods. The antioxidant pattern and the intracellular reactive oxygen species (ROS) levels were analyzed by spectrophotometric and spectrofluorimetric methods. NF-κB luciferase activity was quantified by luminometric detection. NF-κB nuclear translocation, as well as mitochondrial morphology, were investigated by immunofluorescence using confocal microscopy. Cytokines and COX2 expression levels were determined by Western blot analyses.ResultsThis study demonstrates that L.SK exposure does not influence HCE cell proliferation and viability in vitro. L.SK paraprobiotic induces mild-low levels of intracellular ROS. It is coupled to changes in the mitochondrial membrane potential (ΔΨm), in a context of a regular mitochondrial-network organization. The negative modulation of tumor necrosis factor alpha (TNF-α) expression levels and rising antioxidant defense efficiency, mediated by the upregulation of glutathione (GSH) and increased antioxidant enzymatic activities, were observed.ConclusionsThis study demonstrates that L.SK empowers the antioxidant endogenous efficiency of HCE cells, by the upregulation of the GSH content and the enzymatic antioxidant pattern, and concurrently reduces TNF-α protein expression.Translational RelevanceAlthough the obtained in vitro results should be confirmed by in vivo investigations, our data suggest the possibility of L.SK paraprobiotic application for promoting eye health, exploring its use as an endogen antioxidant system inducer in preventing and treating different oxidative stress-based, inflammatory, and age-related conditions.

In the eye of the storm: SARS-CoV-2 infection and replication at the ocular surface?

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) first emerged in December 2019 and spread quickly causing the coronavirus disease 2019 (COVID‐19) pandemic. Recent single cell RNA‐Seq analyses have shown the presence of SARS‐CoV‐2 entry factors in the human corneal, limbal, and conjunctival superficial epithelium, leading to suggestions that the human ocular surface may serve as an additional entry gateway and infection hub for SARS‐CoV‐2. In this article, we review the ocular clinical presentations of COVID‐19 and the features of the ocular surface that may underline the overall low ocular SARS‐CoV‐2 infection. We critically evaluate the studies performed in nonhuman primates, ex vivo organ culture ocular models, stem cell derived eye organoids and the differences in infection efficiency observed in different parts of human ocular surface epithelium. Finally, we highlight the additional work that needs to be carried out to understand the immune response of the ocular surface to SARS‐CoV‐2 infection, which can be translated into prophylactic treatments that may be applied to other organ systems.

Tryptophan as a Safe Compound in Topical Ophthalmic Medications: In Vitro and In Vivo Studies

ABSTRACT Background To evaluate the effects of tryptophan (TRP) on normal human corneal and conjunctival epithelium in vitro and the re-epithelization of corneal erosion in rabbits. Materials and methods Corneal epithelial cell (10.014 pRSV-T) and conjunctival epithelial cell (HC0597) cultures were used. The cellular metabolism, viability, secretion of IL-1β, IL-6, IL-10, cytoskeleton organization, transwell migration were determined. Cells were incubated in the presence of TRP at 1–100 μM. After corneal de-epithelization rabbits received TRP drops (100 μM), 5 times a day. Results TRP increased conjunctival epithelium metabolism at 50 μM and increased the viability of corneal epithelium at 100 μM. TRP (10 μM) enhanced the production of IL-6 by the corneal epithelium and had no effect on IL-1β and IL-10. Conclusions TRP had no influence on the cellular cytoskeleton but induced a significant pseudopodia projection in both epithelia. TRP did not influence corneal re-epithelization in vivo. TRP was not toxic for corneal and conjunctival epithelia.

Generation of functional conjunctival epithelium, including goblet cells, from human iPSCs

The conjunctival epithelium, which covers the sclera (the white of the eye) and lines the inside of the eyelids, is essential for mucin secretion and the establishment of a healthy tear film. Here, we describe human conjunctival development in a self-formed ectodermal autonomous multi-zone (SEAM) of cells that were derived from human-induced pluripotent stem cells (hiPSCs) and mimic whole-eye development. Our data indicate that epidermal growth factor (EGF) drives the generation of cells with a conjunctival epithelial lineage. We also show that individual conjunctival cells can be sorted and reconstituted by cultivation into a functional conjunctival epithelium that includes mucin-producing goblet cells. Keratinocyte growth factor (KGF), moreover, is necessary for the maturation of hiPSC-derived conjunctival epithelium-particularly the goblet cells-indicating key complementary roles of EGF and KGF in directing the differentiation and maturation, respectively, of the human conjunctival epithelium.

Cross-Reactive Effects of Vaccines: Heterologous Immunity between Tetanus and Chlamydia.

Vaccines can have heterologous effects on the immune system, i.e., effects other than triggering an immune response against the disease targeted by the vaccine. We investigated whether monoclonal antibodies (mAbs) specific for tetanus could cross-react with Chlamydia and confer heterologous protection against chlamydial infection. The capability of two tetanus-specific mAbs, namely mAb26 and mAb51, to prevent chlamydial infection has been assessed: (i) in vitro, by performing a neutralization assay using human conjunctival epithelial (HCjE) cells infected with Chlamydia trachomatis serovar B, and (ii) in vivo, by using a guinea pig model of Chlamydia caviae-induced inclusion conjunctivitis. The mAb26 has been superior in comparison with mAb51 in the prevention of chlamydial infection in HCjE cells. The mAb26 has conferred ≈40% inhibition of the infection, compared to less than 5% inhibition in the presence of the mAb51. In vivo, mAb26 significantly diminished ocular pathology intensity in guinea pigs infected with C. caviae compared to either the mAb51-treated or sham-treated guinea pigs. Our data provide insights that tetanus immunization generates antibodies which induce heterologous chlamydial immunity and promote protection beyond the intended target pathogen.

Regulation of gene expression by miRNA-455-3p, upregulated in the conjunctival epithelium of patients with Stevens\u2013Johnson syndrome in the chronic stage

To investigate the role of miRNA in the pathogenesis underlying ocular surface complications in patients with Stevens–Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) in the chronic stage. Using oligonucleotide microarrays, we performed comprehensive miRNA analysis of the conjunctival epithelium of SJS/TEN patients with severe ocular complications (SOC) in the chronic stage (n = 3). Conjunctival epithelium of patients with conjunctival chalasis (n = 3) served as the control. We confirmed the down- and up-regulation of miRNA of interest by quantitative real-time polymerase chain reaction (RT-PCR) assays using the conjunctival epithelium from 6 SJS/TEN with SOC patients and 7 controls. We focused on miRNA-455-3p, which is significantly upregulated in the conjunctival epithelium of the SJS/TEN patients, and investigated its function by inhibiting miR-455-3p in primary human conjunctival epithelial cells (PHCjEs). Comprehensive miRNA expression analysis showed that the expression of 5 kinds of miRNA was up-regulated more than fivefold, and that the expression of another 5 kinds of miRNA was down-regulated by less than one-fifth. There was a significant difference between the SJS/TEN patients and the controls [analysis of variance (ANOVA) p < 0.05]. Quantitative miRNA PCR assay showed that hsa-miR-31* and hsa-miR-455-3p were significantly up-regulated in the conjunctival epithelium of the SJS/TEN patients. Comprehensive gene expression analysis of PHCjEs transfected with the hsa-miR-455-3p inhibitor and quantitative RT PCR assay showed that ANKRD1, CXCL8, CXCL2, GEM, PTGS2, RNASE8, IL6, and CXCL1 were down-regulated by the hsa-miR-455-3p inhibitor. Quantitative RT-PCR, focused on the genes that tended to be up-regulated in SJS/TEN with SOC, revealed that the expression of IL1A, KPRP, IL36G, PPP1R3C, and ADM was significantly down-regulated in PHCjEs transfected with the hsa-miR-455-3p inhibitor. Our results suggest that miRNA-455-3p could regulate many genes including innate immune related genes in human conjunctival epithelium, and that its up-regulation contributes to the pathogenesis on the ocular surface in SJS/TEN patients with the SOC in the chronic stage. Our findings may lead to the development of new treatments using the miRNA-455-3p inhibitor.

Advance in research of beta coronavirus receptors on ocular surface

2019-Novel coronavirus (2019-nCoV) caused an outbreak of corona virus disease 2019 (COVID-19) from December 2019 in China.2019-nCoV which was identified as a kind of beta coronavirus belongs to one of four coronavirus genera.Except 2019-nCoV, two other beta coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are also quite harmful to human beings.2019-nCoV uses the same cell entry receptor, angiotensin-converting enzyme 2 (ACE2), as SARS-CoV.And dipeptidyl peptidase 4 (DPP4) or CD26 is the cell receptor for MERS-CoV.The expression of ACE2 was found to have obvious positive expression in human corneal and conjunctival epithelium, and corneal endothelium.DPP4 activity was presented in normal animal conjunctival epithelium and fibroblasts of the subjacent connective tissue.It was also presented in the whole corneal epithelium and tear fluid of animal with severe injured corneas.The two receptors, ACE2 and DPP4, are involved in many cellular signaling pathways and pathophysiological processes.Their expression in the cells of ocular surface may be an access route of corona virus in eye, which provides clues to elucidating the pathogenesis of corona virus in the eyeballs. Key words: Coronavirus; Receptor; Angiotensin-converting enzyme 2; Dipeptidyl peptidase 4; Ocular surface

Conjunctival reconstruction via enrichment of human conjunctival epithelial stem cells by p75 through the NGF-p75-SALL2 signaling axis.

Severe conjunctival diseases can cause significant conjunctival scarring, which seriously limits eye movement and affects patients' vision. Conjunctival reconstruction remains challenging due to the lack of efficient methods for stem cells enrichment. This study indicated that p75 positive conjunctival epithelial cells (CjECs) were mainly located in the basal layer of human conjunctival epithelium and showed an immature differentiation state in vivo. The p75 strongly positive (p75++) CjECs enriched by immuno‐magnetic beads exhibited high expression of stem cell markers and low expression of differentiated keratins. During continuous cell passage cultivation, p75++ CjECs showed the strongest proliferation potential and were able to reconstruct the conjunctiva in vivo with the most complete structure and function. Exogenous addition of NGF promoted the differentiation of CjECs by increasing nuclear localization of SALL2 in p75++ CjECs while proNGF played an opposite role. Altogether, p75++ CjECs present stem cell characteristics and exhibit the strongest proliferation potential so can be used as seed cells for conjunctival reconstruction, and NGF‐p75‐SALL2 signaling pathway was involved in regulating the differentiation of CjECs.

Co-expression of SARS-CoV-2 entry genes in the superficial adult human conjunctival, limbal and corneal epithelium suggests an additional route of entry via the ocular surface

PurposeThe high infection rate of SARS-CoV-2 necessitates the need for multiple studies identifying the molecular mechanisms that facilitate the viral entry and propagation. Currently the potential extra-respiratory transmission routes of SARS-CoV-2 remain unclear. MethodsUsing single-cell RNA Seq and ATAC-Seq datasets and immunohistochemical analysis, we investigated SARS-CoV-2 tropism in the embryonic, fetal and adult human ocular surface. ResultsThe co-expression of ACE2 receptor and entry protease TMPRSS2 was detected in the human adult conjunctival, limbal and corneal epithelium, but not in the embryonic and fetal ocular surface up to 21 post conception weeks. These expression patterns were corroborated by the single cell ATAC-Seq data, which revealed a permissive chromatin in ACE2 and TMPRSS2 loci in the adult conjunctival, limbal and corneal epithelium. Co-expression of ACE2 and TMPRSS2 was strongly detected in the superficial limbal, corneal and conjunctival epithelium, implicating these as target entry cells for SARS-CoV-2 in the ocular surface. Strikingly, we also identified the key pro-inflammatory signals TNF, NFKβ and IFNG as upstream regulators of the transcriptional profile of ACE2+TMPRSS2+ cells in the superficial conjunctival epithelium, suggesting that SARS-CoV-2 may utilise inflammatory driven upregulation of ACE2 and TMPRSS2 expression to enhance infection in ocular surface. ConclusionsTogether our data indicate that the human ocular surface epithelium provides an additional entry portal for SARS-CoV-2, which may exploit inflammatory driven upregulation of ACE2 and TMPRSS2 entry factors to enhance infection.

DNA methylation of miR-200 clusters promotes epithelial to mesenchymal transition in human conjunctival epithelial cells

Epithelial to mesenchymal transition (EMT) contributes to fibrosis associated pathologies including scarring of different ocular tissues. Recently targeting EMT is seen as an appropriate therapeutic approach for different fibrosis related eye diseases such as macular degeneration or glaucoma surgery related fibrosis. Nevertheless, for ocular surface diseases, target genes specific for particular cell type or condition are still undefined. This study aimed to expose the complex regulatory mechanisms that trigger EMT in human conjunctival epithelial (HCjE) cells. EMT was induced by prolonged treatment with two TGF-β isoforms, TGF-β1 and TGF-β2, and their combination. TGF-β1 showed the strongest potential for initiating EMT in HCjE cells, reflected on morphological changes, cell migration and the levels of mRNA expression of different epithelial (CDH1, OCLN, DSP) and mesenchymal (CDH2, FN1, VIM, SNAI1, ZEB2, TWIST1) marker genes. Co-treatment with the DNA demethylating agent 5-Azacytidine (5-AzaC) was capable of stopping the transition of HCjE cells towards a mesenchymal phenotype, based on morphological features, reduced cell mobility and mRNA and protein expression levels of epithelial and mesenchymal marker genes. An EMT qRT-PCR-based array revealed that EMT induced considerable alterations in gene expression, with downregulation of the majority of epithelial marker genes and upregulation of genes specific for the mesenchymal state. The major effect of 5-AzaC treatment was observed as a suppression of mesenchymal marker genes, suggesting the involvement of upstream negative regulator(s) whose promoter demethylation and subsequent expression will in turn promote EMT switch off. The expression level of miRNAs potentially important for EMT induction was determined using qRT-PCR-based array which pointed at members of miR-200 family as main regulators of EMT process in HCjE cells. 5-AzaC treatment induced increased expression of miR-200a, −200b, −200c and miR-141 towards the control level, indicating important role of DNA methylation in their regulation. The DNA methylation status of both miR-200 family clusters, analyzed with high-resolution melting (HRM) and bisulfite sequencing (Bis-Seq), revealed that TGF-β1-induced EMT was accompanied by increase in promoter CpG methylation of both miR-200 loci, which was reverted after 5-AzaC treatment. In conclusion, our results indicate that DNA demethylation of promoters of miR-200 loci is critically important for stopping and reverting the EMT in human conjunctival epithelial cells, suggesting the potential for the development of novel epigenetic-based therapeutic strategies for treating conjunctival conditions associated with EMT.