Differential Effects of Dexamethasone and Doxycycline on Inflammation and MMP Production in Murine Alkali-Burned Corneas Associated with Dry Eye.

Alkali burns to the cornea are among the most devastating injuries to the eye. The purpose of this study was to evaluate the effects of dexamethasone (Dex) or doxycycline (Doxy) on protease activity and corneal complications in a combined model (CM) of alkali burn and dry eye. C57BL/6 mice were subjected to the CM for 2 or 5 days (D). Mice were topically treated either with Dex (0.1%), Dox (0.025%) or vehicle QID and observed daily for appearance of corneal perforation. Quantitative real time PCR was performed to measure expression of inflammation cytokines and matrix metalloproteinases (MMPs) in whole cornea lysates. No perforations were observed in the Dex-treated corneas. All wounds in Doxy-treated corneas were closed 2D post-injury, and they had significantly lower corneal opacity scores at days 4 and 5 post-injury compared to BSS treatment. Dex-treated corneas had the lowest corneal opacity scores. Dex treatment significantly decreased expression of IL-1ß, IL-6, MMPs -1, -9, -13, and TIMP-1 after 2 days but increased levels of MMP-8, while Doxy treatment significantly decreased IL-1ß, IL-6, MMP-8, and -9, compared to vehicle. Decreased MMP-1, -9 and -13 immunoreactivity and gelatinolytic activity were seen in corneas treated with Doxy and Dex compared to vehicle. Increased neutrophil infiltration and myeloperoxidase activity was noted in the vehicle group compared to Dex 2 days post-injury. These findings demonstrate that early initiation of anti-inflammatory therapy is very efficacious in preserving corneal clarity and facilitating wound healing, while modulating MMP production and suppressing neutrophil infiltration.

Improvement of Outcome Measures of Dry Eye by a Novel Integrin Antagonist in the Murine Desiccating Stress Model.

PURPOSE: We investigated the effects of GW559090, a novel, competitive, and high-affinity α4 integrin antagonist, in a murine model of dry eye. Through interaction with vascular cell adhesion molecule 1 (VCAM-1) and fibronectin α4ß1 integrin is involved in leukocyte trafficking and activation. METHODS: Female C57BL/6 mice, aged 6 to 8 weeks, were subjected to desiccating stress (DS). Bilateral topical twice daily treatment with GW559090 was compared to vehicle-treated controls. Treatment was initiated at the time of DS induction. Treatment effects were assessed on corneal staining with Oregon Green Dextran (OGD) and expression of inflammatory markers in ocular surface tissues by real time PCR. Dendritic cell activation was measured in draining cervical lymph nodes (CLN) by flow cytometry. Separate groups of mice received GW559090 subcutaneously to evaluate the effects of systemic administration on corneal staining and cells in CLN. RESULTS: Topical GW559090 significantly reduced corneal uptake of OGD compared to vehicle-treated disease controls in a dose-dependent manner (1, 3, 10, and 30 mg/mL) with 30 mg/mL showing the greatest reduction in OGD staining. When administered topically, corneal expression of IL-1α, matrix metalloproteinase (MMP)-9, chemokine ligand 9 (CXCL9), and TGF-ß1 was reduced in GW559090-treated eyes. Topical treatment with GW559090 decreased dendritic cell activation in lymph nodes. The effects on corneal staining and cellular composition in CLN were not reproduced by systemic administration of GW559090, suggestive of a local role for integrin antagonism in the treatment of dry eye. CONCLUSION: The novel α4 integrin antagonist, GW559090, improved outcome measures of corneal staining and ocular surface inflammation in this murine model of dry eye. These results indicate the potential of this novel agent for the treatment of dry eye disease.

Desiccating Stress-Induced MMP Production and Activity Worsens Wound Healing in Alkali-Burned Corneas.

PURPOSE: To evaluate the effects of dry eye on ocular surface protease activity and sight threatening corneal complications following ocular surface chemical injury. METHODS: C57BL/6 mice were subjected to unilateral alkali burn (AB) with or without concomitant dry eye for 2 or 5 days. Mice were observed daily for appearance of corneal perforation. Whole corneas were harvested and lysed for RNA extraction. Quantitative real-time PCR was performed to measure expression of inflammation cytokines, matrix metalloproteinases (MMP). Matrix metalloproteinase-9 activity, gelatinase activity, and myeloperoxidase (MPO) activity were evaluated in corneal lysates. Presence of infiltrating neutrophils was evaluated by immunohistochemistry and flow cytometry. RESULTS: Eyes subjected to the combined model of AB and dry eye (CM) had 20% sterile corneal perforation rate as soon as 1 day after the initial injury, which increased to 35% by 5 days, delayed wound closure and increased corneal opacity. Increased levels of IL-1ß, -6, and MMPs-1, -3, -8, -9, and -13, and chemokine (C-X-C motif) ligand 1 (CSCL1) transcripts were found after 2 days in CM compared with AB corneas. Increased MMP-1, -3, -9, and -13 immunoreactivity and gelatinolytic activity were seen in CM corneas compared with AB. Increased neutrophil infiltration and MPO activity was noted in the CM group compared with AB 2 days post injury. CONCLUSIONS: Desiccating stress worsens outcome of ocular AB, creating a cytokine and protease storm with greater neutrophil infiltration, increasing the risk of corneal perforation.

Altered balance of interleukin-13/interferon-gamma contributes to lacrimal gland destruction and secretory dysfunction in CD25 knockout model of Sjögren's syndrome.

INTRODUCTION: The lacrimal gland (LG) of the CD25-/- model of Sjögren's syndrome (SS) has high interleukin (IL)-17, IL-13 and interferon-gamma (IFN-γ) cytokines. The specific contribution of these cytokines to the onset and severity of dacryoadenitis in the CD25-/- mice has not been evaluated. METHODS: CD25-/-IL-17A-/-, CD25-/-IL-17-/-IFN-γ-/- and CD25-/-IFN-γ-/- were used at 4, 8, 12, 16 weeks (W). Total lymphocytic infiltration was evaluated by histology and characterized by flow cytometry. Epidermal growth factor (EGF) concentration was measured in tears. Immunofluorescent staining evaluated expression of IFN-γ receptor (IFN-γR) and apoptosis. Real-time PCR evaluated inflammatory and T cell-related cytokines expression in LG. Caspase-3, -8, -9 activities was assayed in LG lysates. T helper cytokines were measured in serum by Luminex assay. RESULTS: The greatest total LG infiltration at 8 W was seen in CD25-/-IL-17A-/- (95%), followed by CD25-/- (71%) and IL-17-/- (12%). Tear EGF concentration was in normal range in CD25-/- at 4 W and in very low levels in both CD25-/- and CD25-/-IL-17A-/-. CD25-/- had high levels of inflammatory cytokines transcripts in LG compared to IL-17-/- mice; however, CD25-/-IL-17A-/- had even higher IL-1ß, IFN-γR, caspase-3, -8, -9 mRNA levels, greater immunoreactivity to IFN-γR in LG acini, greater number of apoptotic+ cells and greater caspases activities in the LG at 8 W. CD25-/-IL-17A-/- had lower IL-13 concentration and lower IL-13/IFN-γ ratio compared to CD25-/- in serum. CD25-/-IFN-γ-/- had lower number of apoptotic+ cells and decreased caspase-3 expression in LG. CD25-/-IL-17-/-IFN-γ-/- had lower total lymphocytic cell infiltration at 8 W (48%), CD4+T cell infiltration and expression of IFN-γR and apoptotic+ cells in the LG and increased tear EGF concentration in tears. CONCLUSIONS: IFN-γ is critical for LG destruction and secretory dysfunction in the CD25-/- model of SS. Altered balance between IFN-γ and IL-13 in the CD25-/-IL-17A-/- mice accelerates LG destruction by increasing glandular apoptosis and facilitating apoptosis through increased expression of IFN-γR by glandular epithelium and activation of caspases. Targeting both IFN-γ and IL-17 may be beneficial for treating the LG inflammation in SS.

Dendritic cell-derived thrombospondin-1 is critical for the generation of the ocular surface Th17 response to desiccating stress.

TSP-1 is a physiologic activator of TGF-ß, a critical induction factor for Th17-mediated immunity. The purpose of this study was to investigate the role of TSP-1 in the induction of the Th17 ocular surface response to DS. TSP-1KO and WT mice were subjected to DS5 and DS10), and parameters of ocular surface disease, including corneal barrier function, conjunctival CD4(+) T cell infiltration, and GC density, were evaluated. TSP-1KO mice subjected to DS had less corneal barrier disruption, reduced loss of PAS+ GC, and decreased CD4(+) T cell infiltration in the conjunctiva. In contrast to WT, TSP-1KO mice failed to up-regulate MMP-3 and MMP-9 mRNA transcripts in the cornea and IL-17A mRNA transcripts in the conjunctiva. RAG-1KO recipients of adoptively transferred CD4(+) T cells isolated from TSP-1KO mice subjected to DS5 showed milder dry-eye phenotype and less conjunctival inflammation than recipients of CD4(+) T cells from DS5 WT control. Reconstitution of TSP-1KO mice with WT DCs prior to DS reversed the resistance of the TSP-1KO to DS-induced immunopathology. In conclusion, DC-derived TSP-1 is critical for generating the Th17 ocular surface response to DS.

Deletion of interferon-γ delays onset and severity of dacryoadenitis in CD25KO mice.

INTRODUCTION: To investigate the role of interferon-gamma (IFN-γ) in the onset and severity of dacryoadenitis in the CD25 knockout (KO) mouse model of Sjögren Syndrome. METHODS: CD25/IFN-γ double KO (γDKO) mice were created by crossbreeding CD25KO and IFN-γKO mice. Mice were used at 8, 12, and 16 weeks. Lacrimal gland (LG) infiltrating lymphocytes were characterized with flow cytometry. Tear epidermal growth factor (EGF) concentration was measured with enzyme-linked immunosorbent assay (ELISA). Quantitative polymerase chain reaction (PCR) evaluated T-cell-related cytokines in LGs. Serum autoantibodies against M3R in LG lysates were detected with Western blot. RESULTS: γDKO LG showed lower lymphocytic infiltration at 8 weeks than in the CD25KO parental strain (˜20% versus ˜60%, respectively), which increased to CD25KO levels at 16 weeks. Flow-cytometry analysis showed an increase in CD4+ and CD8+ T cells with aging in γDKO LG, similar to that in CD25KO. γDKO had lower levels of interleukin (IL)-17A, transforming growth-factor (TGF)-ß1, IL-21, and CCL20, and higher IL-1ß and IL-13 mRNA transcripts in the LG than in the parental CD25KO strain. Autoantibodies to M3R were observed in both strains and significantly increased with aging in both strains. CD25KO mice had very low tear EGF concentrations at all ages, whereas the ear EGF concentration in γDKO mice significantly decreased with aging and inversely correlated with the presence of M3R autoantibodies and the degree of LG CD4 and CD8+ T-cell infiltration. CONCLUSIONS: The deletion of IFN-γ in the CD25KO mice strain delays glandular destruction and preserves glandular function. M3R autoantibodies increased with aging in both the γDKO and the CD25KO strains. The decrease in LG function in γDKO correlated with the degree of T-cell infiltration and the presence of M3R autoantibodies.

Pharmacological cholinergic blockade stimulates inflammatory cytokine production and lymphocytic infiltration in the mouse lacrimal gland.

PURPOSE: To investigate the effects of cholinergic blockade on inflammatory cell infiltration and cytokine production in the mouse lacrimal gland (LG). METHODS: C57BL/6 mice were untreated (UT) or received subcutaneous injections of either scopolamine hydrobromide (SCOP; 0.5 mg/0.2 mL) or saline (SAL) four times daily for 2 or 5 days (2D, 5D). This was followed by a 7-day rest period in separate groups. Tear volume (cotton thread) and tear epidermal growth factor (EGF, by ELISA) concentrations were measured. Extraorbital LGs were surgically excised and sectioned or lysed for gene expression analysis. Immunohistochemistry evaluated immunophenotype of infiltrating cells. Expression of EGF and T helper (Th)-1, -2, and -17-associated cytokines in LGs was evaluated by real-time PCR. Goblet cell density was evaluated in periodic acid Schiff-stained conjunctival sections. RESULTS: Tear volume and EGF protein levels were significantly reduced in SCOP5D mice compared with controls, indicating that cholinergic blockade decreased LG secretory function. LGs of SCOP2D and SCOP5D mice showed an increased density of CD4(+), CD11c+, CD11b+, and myeloperoxidase+ cells compared with UT controls. At day 5, these cells were significantly elevated compared with SAL-treated counterparts. Elevated levels of IL-17A, IL-17R, IFN-γ, IL-12Rß1, IL-2, IL-13, IL-6, IL-1ß, and TNF-α transcripts were noted in SCOP2D mice and IFN-γ, TGF-ß1, and IL-18R transcripts in SCOP5D mice. CONCLUSIONS: Pharmacological blockade of lacrimal secretion induced a significant CD4(+) infiltration in the LG, mimicking Sjögren's syndrome. The mRNA expression profile revealed elevations of a mix of inflammatory cytokines and Th-1-associated factors.