eritoran and Keratitis

Purpose. To investigate the corneal expression of toll-like receptor (TLR) 4 and determine its contribution to the immunopathogenesis of dry eye disease (DED). Methods. Seven to 8-week-old female C57BL/6 mice were housed in a controlled environment chamber and administered scopolamine to induce experimental DED. Mice received intravenous TLR4 inhibitor (Eritoran) to block systemic TLR4-mediated activity. The expression of TLR4 by the corneal epithelium and stroma was evaluated using real-time polymerase chain reaction and flow cytometry. Corneal fluorescein staining (CFS) was performed to evaluate clinical disease severity. The corneal expression of proinflammatory cytokines (IL-1β, IL-6, TNF, and CCL2), corneal infiltration of CD11b(+) antigen-presenting cells, and lymph node frequency of mature MHC-II(hi) CD11b(+) cells were assessed. Results. The epithelial cells of normal corneas expressed TLR4 intracellularly; however, DED significantly increased the cell surface expression of TLR4. Similarly, flow cytometric analysis of stromal cells revealed a significant increase in the expression of TLR4 proteins by DED-induced corneas as compared with normal corneas. DED increased the mRNA expression of TLR4 in corneal stromal cells, but not epithelial cells. TLR4 inhibition decreased the severity of CFS and significantly reduced the mRNA expression of IL-1β, IL-6, and TNF. Furthermore, TLR4 inhibition significantly reduced the corneal infiltration of CD11b(+) cells and the lymph node frequency of MHC-II(hi) CD11b(+) cells. Conclusions. These results suggest that DED increases the corneal expression of TLR4 and that TLR4 participates in the inflammatory response to ocular surface desiccating stress.

Topics: Animals; Cholinergic Antagonists; Corneal Stroma; Cytoplasm; Dendritic Cells; Disaccharides; Disease Models, Animal; Dry Eye Syndromes; Epithelium, Corneal; Female; Gene Expression; Interleukin-1beta; Interleukin-6; Keratitis; Mice; Mice, Inbred C57BL; Receptors, Cell Surface; RNA, Messenger; Scopolamine; Sugar Phosphates; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

To investigate the role of the TLR4/MD-2 antagonist eritoran tetrasodium in a murine model of contact lens-associated corneal infiltrates.. C57BL/6 mouse corneas were abraded and treated with eritoran tetrasodium or placebo, either before or after stimulation with either LPS, the TLR2 ligand Pam(3)Cys, or antibiotic-killed Pseudomonas aeruginosa. A 2-mm punch from a silicon hydrogel contact lens was used to cover the corneal surface throughout the inhibition and stimulation period. Corneal infiltrates were detected by in vivo confocal microscopy and by immunohistochemistry for neutrophils. The effect of eritoran tetrasodium on stimulated human corneal epithelial cells (HCECs), macrophages, and neutrophils was also assessed.. Eritoran tetrasodium significantly inhibited CXC chemokine production in the cornea and development of corneal infiltrates, specifically neutrophils, in response to stimulation with LPS (TLR4), but not Pam(3)Cys (TLR2). When the antagonist was applied after LPS stimulation, neutrophil infiltration was also inhibited, although a higher concentration was needed. Furthermore, IL-8 production by TLR4- but not TLR2-stimulated HCECs, macrophages and neutrophils was also significantly reduced. Corneal inflammation induced by P. aeruginosa in the presence of tobramycin was found to be dependent on expression of TLR4 and MD-2 and is inhibited by eritoran tetrasodium.. Eritoran tetrasodium is a highly effective antagonist of TLR4/MD-2-dependent corneal inflammation.

Topics: Animals; Apoptosis; Chemokine CXCL1; Cysteine; Disaccharides; Epithelium, Corneal; Eye Infections, Bacterial; Fluorescent Antibody Technique, Indirect; In Situ Nick-End Labeling; Keratitis; Lipopolysaccharides; Lipoproteins; Lymphocyte Antigen 96; Macrophages; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Sugar Phosphates; Toll-Like Receptors