Conjunctival infiltrates and cytokines in an experimental immune-mediated blepharoconjunctivitis rat model.

Purpose: To characterize the histopathological and immunological findings of a rat model of allergic blepharoconjunctivitis (BC) and demonstrate its potential utility for the assessment of BC therapies. Methods: Sprague-Dawley (SD) rats were immunized with ovalbumin (OVA) and topically challenged with OVA (BC group) or PBS (control group), while a corticosteroid group was pre-treated with triamcinolone acetate 24 h before the challenge. Morphological features were evaluated and tissues were harvested for histological, flow cytometry and cytokine analysis. Results: The BC group rats developed eyelid excoriations, redness, and conjunctival edema 24 h after the OVA challenge, while corticosteroid pre-treated and PBS-challenged rats were unaffected. The BC features were reduced despite repeated challenges for 5 days. Massive immune cell infiltration was observed in conjunctivae of BC rats, while no significant infiltration was seen in the other groups. Populations of T cells, mono-macrophages, neutrophils, and NK cells made up more than 77% of CD45+7AAD- cells in the conjunctival tissues. T cell proportions were increased at 96 h compared to 24 h post-challenge, while macrophages decreased during the same time period. Eosinophils and intraepithelial neutrophils were detected in the BC rats, but not in the PBS and corticosteroid groups. BC eyes had significantly higher levels of IFN-γ and IL-2, while IL-4 and IL-6 levels were similar to controls. Conclusion: A robust BC response was detected in this rat model which was suppressed by corticosteroid pre-treatment. Immune cell composition and cytokine profiles changed over time.

Toll-like receptor 2-mediated NF-kappa B pathway activation in ocular surface epithelial cells.

BACKGROUND: Gram-positive bacteria stimulate Toll-like receptor (TLR) 2 and then activate the pro-inflammatory nuclear factor-kappa B (NF-κB) pathway. As the human ocular surface is heavily colonised by gram-positive cocci bacteria, a balance of activation/repression of NF-κB target genes is essential to avoid uncontrolled infection or autoimmune-related inflammation. It is advantageous to test NF-κB targeting molecules in an ocular surface culture system that allows assessment of temporal NF-κB activation in a longitudinal fashion without destruction of cells. Such initial testing under standardised conditions should reduce the number of molecules that progress to further evaluation in animal models. This study aims to establish an in-vitro cell culture system to assess NF-κB activation in the context of ocular surface cells. METHODS: NF-κB activity was evaluated through a secretory alkaline phosphatase reporter assay (SEAP). Immunoblots and immunofluorescence were used to examine IκBα phosphorylation and p65/p50 nuclear localization. Monocyte chemoattractant protein-1 (MCP-1) transcripts were evaluated by real time PCR and protein levels were measured by ELISA. RESULTS: NF-κB activity in HCE-T cells treated with TLR2 activator Pam3CSK4 was higher than control cells at both 6 and 24 h. Pam3CSK4-stimulated NF-κB activation was inhibited by IκK inhibitors, Wedelolactone and BMS-345541. In Pam3CSK4 treated cells, active NF-κB subunits p50 and p65 increased in cell nuclear fractions as early as 1.5 h. Although the level of total IκB-α remained constant, phospho-IκB-α increased with treatment over time. In the culture media of Pam3CSK4-stimulated cells, MCP-1 protein level was increased, which was suppressed in the presence of IκK inhibitors. CONCLUSION: NF-κB pathway can be activated by the TLR2 ligand and inhibited by IκK inhibitors in the ocular surface cell culture system. This cell culture system may be used to evaluate TLR-related innate defences in ocular surface diseases.

In vitro secretomics study of pterygium-derived fibroblasts by iTRAQ-based quantitative proteomics strategy.

Pterygium is a triangular shaped ocular fibrous surface lesion growing from conjunctiva towards central cornea, causing ocular irritation, astigmatism, and visual disturbance. The condition is characterized by epithelial proliferation, fibrovascular growth, chronic inflammation, and prominent extracellular matrix remodeling. Studies have suggested that aberrant extracellular proteins secreted by fibroblasts lead to abnormal matrix production and tissue invasion contributing to the development of the disease. In this study, secreted proteins collected from paired pterygium and conjunctival fibroblasts in vitro were identified and quantified by LC-MS iTRAQ-based analysis, in which 433 proteins common to all samples were identified. Among these proteins, 48.0% (208) were classified as classically secreted proteins, 17.1% (74) were exported out of the cells via non-classical secretion pathways, and 31.2% (135) were exosome proteins. A minority (3.7%) was not previously known to be secreted, or might be contaminants. 31 and 27 proteins were found up- or down-regulated in the conditioned media of pterygium fibroblasts relative to the media of control cells, respectively. Molecular function analysis showed that these proteins either belonged to catalytic proteins, structural molecules or were involved with receptor activities and protein binding. Further pathway analysis revealed that these proteins were involved in ECM-receptor interaction, focal adhesion, cancer-related, p53 signaling, complement and coagulation, and TGF-beta signaling pathways. These molecules identified may serve as extracellular ligands to activate intracellular pathways, possibly serving as potential therapeutic targets.

Evaluation of global differential gene and protein expression in primary Pterygium: S100A8 and S100A9 as possible drivers of a signaling network.

PURPOSE: Pterygium is a wing shaped fibrovascular growth on the ocular surface, characterized by fibrosis, angiogenesis, extracellular matrix remodeling, and inflammatory infiltrates. Epidemiologic studies have linked pterygium formation to various chronic inflammatory conditions, such as ultraviolet radiation, sawdust exposure, and dry eye disease. The purpose of this study is to identify proteins that are differentially expressed in primary pterygium by using a combination of gene microarray and proteomic platforms. METHODS: Paired pterygium and uninvolved conjunctiva tissues of four patients were evaluated for differences in global gene transcript levels using a genechip microarray. Proteins extracted from another four pairs of tissues were quantified by iTRAQ approach. Western blot and immunofluorescent staining on additional patients were used to validate dysregulated protein expression obtained from microarray and proteomics data. In addition, primary conjunctival fibroblasts were treated with recombinant S100A8, S100A9 or both. Transcript level changes of a panel of potential target genes were evaluated by real time-PCR. RESULTS: The following were up-regulated at both protein and transcript levels S100 A8 and A9, aldehyde dehydrogenase 3 family, member1 (ALDH3A1) and vimentin (VIM). Conversely, serpin peptidase inhibitor clade A member 1 (SERPINA1) and transferrin (TF) were down-regulated. Upon adding S100A8, S100A9 or both, the inflammatory chemokine CXCL1, matrix proteins vimentin, biglycan, and gelsolin, as well as annexin-A2, thymosin-ß4, chymase (CMA1), member of Ras oncogene family RAB10 and SERPINA1 were found to be up-regulated. CONCLUSIONS: We identified 3 up-regulated and 2 down-regulated proteins by using a stringent approach comparing microarray and proteomic data. On stimulating cells with S100A8/9, a repertoire of key genes found to be up-regulated in pterygium tissue, were induced in these cells. S100A8/9 may be an upstream trigger for inflammation and other disease pathways in pterygium.