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Cathepsin S (Ctss) is a protease that is proinflammatory on epithelial cells. The purpose of this study was to investigate the role of Ctss in age-related dry eye disease. Ctss -/ - mice [in a C57BL/6 (B6) background] of different ages were compared to B6 mice. Ctss activity in tears and lacrimal gland (LG) lysates was measured. The corneal barrier function was investigated in naïve mice or after topical administration of Ctss eye drops 5X/day for two days. Eyes were collected, and conjunctival goblet cell density was measured in PAS-stained sections. Immunoreactivity of the tight junction proteins, ZO-1 and occludin, was investigated in primary human cultured corneal epithelial cells (HCEC) without or with Ctss, with or without a Ctss inhibitor. A significant increase in Ctss activity was observed in the tears and LG lysates in aged B6 compared to young mice. This was accompanied by higher Ctss transcripts and protein expression in LG and spleen. Compared to B6, 12 and 24-month-old Ctss -/ - mice did not display age-related corneal barrier disruption and goblet cell loss. Treatment of HCEC with Ctss for 48 h disrupted occludin and ZO-1 immunoreactivity compared to control cells. This was prevented by the Ctss inhibitor LY3000328 or Ctss-heat inactivation. Topical reconstitution of Ctss in Ctss -/ - mice for two days disrupted corneal barrier function. Aging on the ocular surface is accompanied by increased expression and activity of the protease Ctss. Our results suggest that cathepsin S modulation might be a novel target for age-related dry eye disease., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The concept of innate immunity has been expanded to recognize environmental pathogens other than microbial components. However, whether and how the innate immunity is initiated by epithelium in response to environmental physical challenges such as low humidity and high osmolarity in an autoimmune disease, dry eye, is still largely unknown. Using two experimental dry eye models, primary human corneal epithelial cultures exposed to hyperosmolarity and mouse ocular surface facing desiccating stress, we uncovered novel innate immunity pathway by ocular surface epithelium, where oxidized mitochondrial DNA induces imbalanced activation of NLRP3/NLRP6 inflammasomes via stimulation of caspase-8 and BRCC36 in response to environmental stress. Activated NLRP3 with suppressed NLRP6 stimulates caspase-1 activation that leads to IL-1β and IL-18 maturation and secretion. NLRP3-independent caspase-8 noncanonically activates caspase-1 via reciprocal regulation of NLRP3/NLRP6-mediated inflammasomes. Reactive oxygen species-induced mitochondrial DNA oxidative damage and BRCC36 deubiquitinating activity provide a missing link and mechanism by which innate immunity responds to environmental stress via caspase-8-involved NLRP3/NLRP6 inflammasomes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The association and mechanism of bacteria linking to the allergic inflammation have not been well elucidated. This study was to explore a potential link between bacterial pathogens and allergic conjunctivitis by dendritic cells (DCs). Bone marrow-derived DCs from BALB/c and MyD88 knockout mice were treated with or without bacterial pathogens or thymic stromal lymphopoietin (TSLP). Two murine models of the topical challenge with LPS or flagellin and experimental allergic conjunctivitis (EAC) were used for in vivo study. The mRNA expression was determined by reverse transcription and real time PCR, and protein production was evaluated by ELISA, Western blotting, immunofluorescent staining and flow cytometry. TSLP mRNA and protein were found to be largely induced by DCs challenged with microbial pathogens, highly by lipopolysaccharide (LPS) and flagellin. The expression of MyD88, NFκB1, NFκB2 and RelA accompanied by NFκB p65 nuclear translocation and TSLP induction were significantly stimulated by flagellin, but blocked by TLR5 antibody or NFκB inhibitor in DCs from MyD88(+/+) but not MyD88(-/-) mice. TSLP promoted the expression of CD40, CD80, OX40 ligand (OX40L), IL-13 and CCL17 by DCs. TSLP-producing DCs were identified in vivo in ocular surface conjunctiva and draining cervical lymph nodes from two murine models of topical challenge with LPS or flagellin, and EAC in BALB/c mice. TSLP/TSLPR/OX40L signaling was observed in DCs of EAC mice. Our findings demonstrate that DCs not only respond to TSLP, but also produce TSLP via TLR/MyD88/NFκB pathways in response to bacterial pathogens, suggesting a potential link between bacteria and allergic disease., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Evidence suggests that the cytokine interferon (IFN)-γ released by natural killer and CD4(+) T cells contributes to the conjunctival goblet cell (GC) loss in dry eye. The purpose of this study was to investigate if topical neutralization of IFN-γ prevents or alleviates GC loss in an experimental desiccating stress (DS) model of dry eye. In this study, we found that topical IFN-γ neutralization significantly decreased DS-induced conjunctival GC loss. This was accompanied by decreased epithelial apoptosis, and increased IL-13 and decreased FoxA2 expression in the forniceal conjunctiva. To establish that IFN-γ produced by pathogenic CD4(+) T cells contributes to DS-induced GC loss, adoptive transfer of CD4(+) T cells isolated from DS exposed donors to naïve RAG-1(-/-) recipient mice was performed. Similar to the donor mice, topical IFN-γ neutralization decreased conjunctival GC loss, suppressed apoptosis and increased IL-13 expression in adoptive transfer recipients. In summary, this study demonstrated that topical neutralization of IFN-γ prevents GC loss via modulating apoptosis and maintaining IL-13 signaling., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Human peptidoglycan recognition proteins (PGLYRPs) are a novel family of pattern recognition receptors, and also act as anti-bacterial proteins. This study was to explore the toll-like receptor (TLR)-mediated regulation of PGLYRPs in human corneal epithelial cells (HCECs). Fresh human donor corneoscleral tissues were used to prepare cryosections. Primary HCECs, established from limbal explants, were treated with microbial ligands to TLRs 1-9 for 4-48 h, with or without pretreatment of TLR antibodies, NFkB inhibitor, or siRNA transfection. The mRNA of PGLYRPs was evaluated by RT and real-time PCR, and their proteins and NFkB activation were determined by immunostaining and Western blot. The nuclear IRF3 activity was quantified using an ELISA-based TransAM kit. PGLYRP-2, -3 and -4 were found to be expressed by human corneal epithelium while PGLYRP-1 was not detected. In primary HCEC cultures, PGLYRP-3 and -4 were constitutively expressed while PGLYRP-2 was largely inducible. PGLYRP-2 was induced by bacterial components, Pam3CSK4, PGN, flagellin and FSL-1, ligands for TLR2/1, 2, 5 and 2/6, respectively. Interestingly, PGLYRP-2 was strongest stimulated by polyI:C representing viral dsRNA. TLR3 antibody or NFkB inhibitor blocked IRF3 and NFkB p65 activation as well as polyI:C-stimulated PGLYRP-2. RNA interference indicates that the polyI:C-induced PGLYRP-2 was dramatically blocked in the cells transfected with siRNA-TRIF but neither siRNA-MyD88 nor the negative control siRNA-F. These findings suggest that human corneal epithelium may response to viral or bacterial infection by producing PGLYRPs through TLRs, and the induction of PGLYRP-2 by dsRNA was through TLR3-TRIF-IRF3-NFkB signaling pathways.

This paper evaluates the effects of adenoviral vector-mediated glial cell-derived neurotrophic factor (GDNF) gene delivery on survival of primary human corneal epithelial cells (PHCEC) established from limbal explants in vitro and the overexpression of GDNF gene in bioengineered human corneal constructs on substrate of corneal stromal discs followed by autograft ex vivo. In vitro, the overexpression of GDNF in the supernatant of PHCEC peaked at day 4, but lasted for at least 4 weeks after the transduction mediated by adenoviral vector. At day 10, the cell viability was 2-fold greater (P < 0.001), the number of terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL)-positive cells was more than 50% lower (P < 0.01) in the GDNF transduction group than the non-transduction group. 5 weeks after the transduction, the living cell population was greater in the GDNF transduction group than the non-transduction group (P < 0.01). In the ex vivo autograft of the bioengineered human corneal constructs, outgrowth of enhanced green fluorescent protein (eGFP) positive cells on the recipient corneoscleral tissue was observed. Overexpression of GDNF in the supernatant peaked at day 2, but was observed for at least 4 weeks after transplantation. At day 5, immunofluorescent staining showed expression of GDNF by all layers of epithelial cells on the graft. Our findings revealed that GDNF is a survival growth factor for cultured human corneal epithelium. The use of bioengineered human corneal constructs containing GDNF-transduced epithelial cells represents a novel method for delivering of this gene to promote survival of transplanted corneal epithelium to treat various corneal surface diseases.

The non-obese diabetic (NOD) mouse is prone to develop autoimmune disease, including Sjögren's syndrome. The purpose of this study was to determine if desiccating environmental stress exacerbates the development of Sjögren's syndrome-like lacrimal keratoconjunctivitis in the NOD.B10.H2(b) mouse. Four-week-old male mice were used as young controls. Sixteen-week-old male mice were untreated or subjected to desiccating stress with a fan alone or with a fan plus subcutaneous injections of the anticholinergic agent scopolamine for 5 or 10 days to inhibit tear production. Mice spontaneously developed Sjögren's syndrome-like lacrimal keratoconjunctivitis as they aged. Desiccating stress increased CD4+ and CCR5+ cells and decreased CD8+ cells in the conjunctival epithelium and lacrimal gland. Intraepithelial gammadelta T cells significantly decreased after 5 days and returned to baseline levels after 10 days in both groups exposed to desiccating stress. These immunopathological changes were accompanied by a decrease in conjunctival goblet cell density. Greater matrix metalloproteinase-9 production, gelatinase activity and loss of epithelial cell membrane CD25 immunoreactivity was noted in the ocular surface epithelia of stressed mice. These findings indicate that desiccating environmental stress aggravates Sjögren's syndrome-like lacrimal keratoconjunctivitis in the NOD mouse which has defective immunoregulation.

Nerve growth factor (NGF), a member of the neurotrophin family, has been identified as an essential growth factor supporting stem cell self-renewal outside the nervous system and was previously shown to stimulate corneal epithelial proliferation both in vivo and in vitro. In this study, we evaluated the expression of NGF and its corresponding receptors in the human corneal and limbal tissues, as well as in primary limbal epithelial cultures by immunofluorescent staining and relatively quantitative real-time polymerase chain reaction. We found that NGF was uniquely expressed in the human limbal basal epithelium, together with its two corresponding receptors: the high-affinity receptor TrkA and the low-affinity receptor p75NTR. TrkA was shown to preferentially localize to limbal basal epithelial cells. NGF and TrkA were also found co-localized with stem cell-associated molecular markers (drug-resistance transporter ABCG2 and p63), but not with the differentiation marker cytokeratin 3 in the human limbal basal epithelial layer. In cultured limbal epithelial cells, NGF and TrkA were found to be preferentially expressed by a small population of limbal epithelial cells. The NGF and TrkA immuno-positive subpopulations were enriched for certain properties (including ABCG2 and p63 expression) of putative limbal epithelial stem cells (P<0.01, compared with the entire cell population). Levels of NGF and TrkA transcripts were found to be much more abundant in limbal than in corneal tissues, and in young cultured cells in the proliferative stage than in airlifted stratified cultures containing differentiated cells. The co-expression of NGF with its two corresponding receptors in limbal basal epithelial cells, but not in the cornea, suggests that NGF may function as a critical autocrine or paracrine factor supporting stem cell self-renewal in the limbal stem cell niche. The spatial expression of NGF and TrkA by small clusters of basal cells interspersed between negative cell patches suggests that they are potential markers for human corneal epithelial progenitor cells.

The development of vectors and techniques to transfer therapeutic genes to corneal epithelium has broad clinical applications. To determine if adenoviral (Ad5) vectors could be tailored to increase transduction of corneal epithelial progenitor cells expressing epidermal growth factor receptor (EGFR), the feasibility of targeting gene therapy vectors to genetically modify primary cultured human corneal epithelial cells (PHCEC) was evaluated. PHCECs were cultured from human limbal explants and transduced with Ad5 vectors containing the enhanced green fluorescent protein (GFP) reporter cassette to mediate gene transfer. The efficiencies of transduction with different Ad5 dosages and different time periods of exposure were compared. Metabolically biotinylated Ad5 vectors were retargeted to PHCECs using biotinylated epidermal growth factor (EGF) as a cell-targeting ligand. Phenotypes and function assays of transduced cells were determined by real-time PCR and BrdU incorporation. Ad5 vectors transduced approximately 50-93% of PHCEC at 10-100 PFU/cell in a dose-dependent manner and the transgene persisted for more than 2 weeks in vitro. Retargeting of biotinylated Ad5 with EGF increased transduction of EGFR and ABCG2-expressing corneal epithelial progenitor cells up to nine-fold and reduced transduction of K12 and involucrin-expressing differentiated corneal epithelial cells and had higher BrdU incorporation indexes. These data provide proof of principle that ligand-bearing modified Ad5 vectors can target a population of corneal epithelial progenitor cells for corneal gene therapy.

We investigated the effects of corticosteroid and doxycycline on expression of matrix metalloproteinase (MMP)-9 and inflammatory cytokines and activation of mitogen-activated protein kinase (MAPK) signaling pathways, c-jun N-terminal kinases (JNK), extracellular-regulated kinases (ERK) and p38, in experimental murine dry eye. Experimental dry eye (EDE) was created in C57BL6 mice, with or without or topical treatment consisting of 1% methylprednisolone, 0.025% doxycycline or balanced salt solution four times per day. MMP-9 expression in the cornea epithelia was evaluated by laser scanning confocal microscopy. Gelatinase activity in the cornea was evaluated by in situ zymography and MMP-9 activity in tear washings was evaluated by gelatin zymography. Total and phosphorylated MAPKs (JNK1/2, ERK1/2, p38) were detected by Luminex immunobead assay. Levels of MMP-9, interleukin (IL)-1alpha, IL-1beta and tumor necrosis factor (TNF)-alpha RNA transcripts were evaluated by real-time PCR. MMP-9 immunoreactivity was localized to the apical corneal epithelial cell membranes in normal control eyes. Desiccating stress significantly increased production of MMP-9 by the corneal epithelium and increased its activity in the corneal epithelium and tear fluid. Dryness also increased expression of IL-1alpha, IL-1beta and TNF-alpha mRNA and stimulated phosphorylation of JNK1/2, ERK1/2 and p38 MAPKs in the corneal epithelium. Both methylprednisolone and doxycycline reduced expression and activity of MMP-9, decreased levels of inflammatory cytokines transcripts and reduced activation of MAPKs in the corneal epithelium in response to EDE. Desiccating stress stimulates expression of MMP-9, IL-1alpha, IL-1beta and TNF-alpha mRNA , as well as activates MAPK signaling pathways in the corneal epithelium. Both corticosteroid and doxycycline suppressed this molecular stress response.

Hyperosmolarity has been recognized to be a pro-inflammatory stress to the corneal epithelium. The cell signalling pathways linking hyperosmolar stress and inflammation have not been well elucidated. This study investigated whether exposure of human limbal epithelial cells to hyperosmotic stress activates the mitogen-activated protein kinase (MAPK) pathways and induces production of pro-inflammatory cytokines, interleukin (IL) -1beta, tumor necrosis factor (TNF) alpha, and the C-X-C chemokine IL-8. Primary human limbal epithelial cultures in normal osmolar media (312 mOsM) were exposed to media with higher osmolarity (400-500 mOsM) by adding 50-90 mM NaCl, with or without SB202190, an inhibitor of c-Jun N-terminal kinases (JNK) pathway, PD 98059, an inhibitor of extracellular-regulated kinase (ERK) pathway, dexamethasone or doxycycline for different lengths of time. The conditioned media were collected after 24 hr of treatment for ELISA. Total RNA was extracted from cultures treated for 6 hr for semi-quantitative RT-PCR. Cells treated for 15-60 min were lysed in RIPA buffer and subjected to Western blot with phospho (p)-specific antibodies against p-JNK and p-ERK. The concentrations of IL-1beta, TNF-alpha and IL-8 proteins in 24 hr conditioned media of limbal epithelial cells progressively increased as the media osmolarity increased from 312 to 500 mOsM. Active p-JNK-1/p-JNK-2 and p-ERK-1/p-ERK-2 were detected by Western blot and peaked at 60 min in cells exposed to hyperosmolar media. The levels of p-JNK-1/p-JNK-2 and p-ERK1/p-ERK2 were positively correlated with the medium osmolarity. SB202190, PD98059 and doxycycline markedly suppressed the levels of p-JNK-1/p-JNK-2 and/or p-ERK1/p-ERK2, as well as IL-1beta, TNF-alpha and IL-8 mRNAs and proteins stimulated by hyperosmolar media. These findings provide direct evidence that hyperosmolarity induces inflammation in human limbal epithelial cells by increasing expression and production of pro-inflammatory cytokines and chemokines, a process that appears to be mediated through activation of the JNK and ERK MAPK signalling pathways. The efficacy of doxycycline in treating ocular surface diseases may be due to its ability to suppress JNK and ERK signalling activation and inflammatory mediator production in the limbal epithelium.

The concept that corneal epithelium stem cells reside in limbus has been recognized for more than a decade, but isolation of these stem cells has not been accomplished. This study was an initial attempt to isolate a population of human limbal epithelial cells enriched for certain putative stem cell properties based on their phenotype. Epithelial cells harvested from fresh human limbal rings and their primary cultures were allowed to adhere to collagen IV-coated dishes for 20 min and 2 hr, sequentially. The rapidly adherent cells (RAC), slowly adherent cells and non-adherent cells were evaluated for certain stem cell properties: (a) BrdU-label retention, (b) expression of basal cell (integrin beta1, p63, ABCG2) and differentiation (involucrin, keratin 12) markers, and (c) colony forming efficiency (CFE) and growth capacity on a 3T3 fibroblast feeder layer. Among unfractionated cells and the three selected populations, the RAC, accounting for about 10% of whole population, were enriched 5-fold in BrdU label-retaining cells, displayed the highest number of integrin beta1 and p63 positive and involucrin negative cells, expressed high levels of DeltaNp63 and ABCG2 mRNA, and lacked involucrin and K12 expression, and possessed the greatest CFE and growth capacity. These findings demonstrated for the first time that human limbal epithelial cells with stem cell properties can be partially enriched by their adhesiveness to collagen IV. The RAC population enriched for certain putative stem cell properties may prove useful in the future for transplantation to diseased and damaged corneas with limbal stem cell deficiency.

Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of ocular surface diseases. This study investigated the regulated expression of gelatinases (MMP-2 and -9), collagenases (MMP-1 and -13) and stromelysins (MMP-3, -10, and -11) by TGF-beta1 in cultured human corneal epithelial cells. Primary human corneal epithelial cell cultures were grown to confluence and treated with different concentrations (0.1, 1.0, 10 ng ml(-1)) of TGF-beta1 in serum-free medium for 6-24 hr. Total RNA was isolated from cultured cells with or without TGF-beta1 treatment for 6 hr and subjected to semi-quantitative RT-PCR and Northern hybridization. Conditioned media were collected from cultures with or without TGF-beta1 treatment for 24 hr to evaluate the MMP production by ELISA and activity assays. Semi-quantitative RT-PCR revealed that the expressions of MMP-9, -1, -13, -3, -10 and -11 mRNA were up-regulated by TGF-beta1 in a concentration-dependent fashion, while MMP-2 and MMP-14 production did not change. Northern hybridization confirmed these findings. Gelatin zymography, MMP ELISA and activity assays showed concentration-dependent stimulated production and activity of MMP-9, -1, -13, -3 and -10 protein in the conditioned media of cultures treated for 24 hr with TGF-beta1. In conclusion, our results demonstrate that TGF-beta1 stimulates the expression and production of gelatinase (MMP-9), collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, -11) in human corneal epithelial cells. These findings suggest that TGF-beta1 may play a role in the pathogenesis of MMP mediated ocular surface diseases, such as sterile corneal ulceration.

Cultivated human corneal epithelial cells have been successfully used for corneal reconstruction. Explant and single cell systems are currently used for human corneal epithelial cultivation. This study was conducted to characterize the phenotypes of human corneal epithelial cells expanded ex vivo by these two culture systems with regard to their growth potential, morphology and antigen expression patterns. Human corneal epithelial cells were expanded by limbal explant culture or limbal single cell suspension culture on a mitomycin C treated 3T3 fibroblast feeder layer. The phenotypes of primary cultured cells were evaluated by morphology and immunohistochemical staining with antibodies for proposed keratinocyte stem cell markers (p63, EGFR, K19 and integrin beta1) and differentiation markers (K3, involucrin and gap junction protein connexin 43). BrdU labeling was performed to identify the label-retaining cells. Human corneal epithelial cells were grown from limbal tissues preserved as long as 16 days by both culture systems. The growth rate depended on the tissue freshness, the time from death to preservation and the time from death to culture, but not on the donor age. Cell growth was observed in 96.2% (n = 43) of single cell suspension cultures and in 90.8% (n = 213) of explant cultures. The cell expansion was confluent in 10-14 days in single cell suspension cultures and 14-21 days in explant cultures. The cell morphology in single cell suspension culture was smaller, more compact and uniform than that in explant culture. Immunostaining showed a greater number of the small cells expressing p63, EGFR, K19 and integrin beta1, while more larger cells stained positively for K3, involucrin and connexin 43 in both culture systems. BrdU-label retaining cells were identified in 2.3+/-0.7% of explant cultures and 3.73+/-1.5% of single cell cultures chased for 21 days. In conclusion, the limbal rims are a great treasure for ex vivo expansion of human corneal epithelial cells. The phenotypes of corneal epithelial cells, ranging from basal cells to superficial differentiated cells, are well maintained in both culture systems. Slow-cycling BrdU-label retaining cells, that are characteristic of stem cells, were identified in the cultures.

The matrix metalloproteinases, MMP-2 and MMP-9, are known to be critical extracellular-remodeling enzymes in wound healing and other diseases of the ocular surface. This study investigated the regulation of MMP-2 and MMP-9 in human corneal epithelial cells by growth factors and pro-inflammatory cytokines (IL-1beta and TNF-alpha) they are exposed to, and by doxycycline, a medication used to treat ocular surface disease. Primary human corneal epithelial cell cultures were treated with one of the following cytokines (IL-1alpha, IL-1beta, IL-6, IL-8, TNF-alpha) or growth factors (EGF, HGF, KGF, PDGF-BB, TGF-alpha, TGF-beta), with or without their corresponding inhibitors. The conditioned media were collected after 24 hr for gelatin zymography and MMP-9 activity assay. Total RNA was extracted from the cells treated for 6 hr and was subjected to RT-PCR and Northern hybridization. Between the two gelatinases, MMP-2 and MMP-9, detected by zymography, the 92 kDa MMP-9 in the conditioned medium was markedly up-regulated by the pro-inflammatory cytokines, IL-1beta and TNF-alpha. The MMP-9 protein and activity were dose-dependently stimulated by IL-1beta or TNF-alpha at 0.1, 1.0 and 10 ng ml(-1). This up-regulation was nearly abolished by neutralizing antibodies (IL-1beta and TNF-alpha) and by IL-1 receptor antagonist. Semi-quantitative RT-PCR and Northern hybridization disclosed that the MMP-9 transcript was also markedly up-regulated in a dose-dependent manner by IL-1beta and TNF-alpha. Doxycycline (10 microg ml(-1)) suppressed MMP-9 protein level and activity, but not its mRNA, that was stimulated by IL-1beta and TNF-alpha (1 ng ml(-1)). In contrast, the 72 kDa MMP-2 was not significantly modulated by any of these cytokines. In conclusion, production of MMP-9 is stimulated by the pro-inflammatory cytokines, IL-1beta and TNF-alpha. These factors may play a role in the pathogenesis of MMP-9 mediated corneal matrix degradation. The efficacy of doxycycline in treating ocular surface diseases may be related to its ability to suppress MMP-9 production in the corneal epithelium., (Copyright 2001 Academic Press.)