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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 pithelial 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 hours 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 CTSS modulation might be a novel target for age-related dry eye disease.

Human peptidoglycan recognition proteins (PGLYRPs) is a novel family of pattern recognition receptors, and also act as antibacterial 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 hours, with or without pretreatment of TLR antibodies, NFκB inhibitor, or siRNA transfection. The mRNA of PGLYRPs was evaluated by RT and real time PCR, and their proteins and NF-κB 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 NF-κB inhibitor blocked IRF3 and NF-κB 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-NFκB signaling pathways.

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.

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.

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.

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.

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.

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.