Expression of Toll-Like Receptor 4 Contributes to Corneal Inflammation in Experimental Dry Eye Disease

Abstract

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-1b, IL-6, TNF, and CCL2), corneal infiltration of CD11b 3 Recent studies have demonstrated that corneal epithelial cells respond to hyperosmolar stress by producing proinflammatory cytokines, chemokines, and matrix metalloproteinases (MMPs).4,5 Furthermore, hyperosmolar stress and proinflammatory cytokines such as interferon (IFN)-c promote epithelial cell apoptosis. [5][6][7][8] Toll-like receptors (TLRs) are pattern recognition receptors of the innate immune system that recognize highly conserved microbial structures and products.9,10 To date, 12 murine TLRs have been identified, and TLRs are expressed by a variety of cell types, including epithelial cells, dendritic cells, macrophages, and lymphocytes.10-13 TLR stimulation leads to the activation of nuclear factor-kappaB (NF-jB) that upregulates the production of proinflammatory cytokines and antimicrobial proteins.10,14 The NF-jB signaling pathway is important for the induction of innate and adaptive immune responses. 10,14 TLR4 recognizes the Gram-negative bacterial cell wall component lipopolysaccharide (LPS) in association with cofactors such as CD14, LPS-binding protein (LBP), and myeloid differentiation factor-2 (MD-2). 15,16 It has also been suggested that TLR4 is a receptor for endogenous ligands associated with noninfectious diseases such as myocardial ischemia-reperfusion injury and central nervous system autoimmune disease. 17,18 We hypothesized that DED-induced corneal inflammation and injury may lead to the production of endogenous TLR4 ligands that activate the immune system. Therefore, we investigated the corneal expression of TLR4 and sought to determine the expression pattern of TLR4 in DED. MATERIALS AND METHODS AnimalsSeven to 8-week-old female C57BL/6 mice (Charles River Laboratories, Wilmington, MA) were used for these experiments. The experimental protocol was approved by the Institutional Animal Care and Use Committee, and all animals were managed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Dry Eye ModelDED was induced by placing mice in a controlled environment chamber (CEC) and administering scopolamine (Sigma-Aldrich, St. Louis, MO) to maximize ocular surface dryness, as previously described. 19,20 Mice placed in the CEC were exposed to a relative humidity < 25%, temperature of 20 to 228C, and airflow of 15 L/min, 2...

Dry Eye Disease and Microbial Keratitis: Is There a Connection?

“…138 In the same mouse model, we have observed upregulation of TLR2-4 and 9 in conjunctiva, cornea, and/or lacrimal gland. 137 These investigations show that TLR expression is modulated at the ocular surface in dry eye conditions.…”

Dry eye modifies the thermal and menthol responses in rat corneal primary afferent cool cells

“…Induction of dry eye in rodents, produced by either environmentally controlled arid conditions or the injection of choleratoxin B into the lacrimal gland, increased the corneal expression of inflammatory cytokines (Dursun et al 2002;Lee et al 2012;Okanobo et al 2012;Shim et al 2012;Stevenson et al 2012;Zhu et al 2009Zhu et al , 2012. However, just as nerve injury reduced cold-and menthol-evoked activity, inflammation also appears to inhibit TRPM8-mediated responses.…”

The CCR6/CCL20 Axis Mediates Th17 Cell Migration to the Ocular Surface in Dry Eye Disease

“…6,20 Mice were housed in a low-humidity (relative humidity: <20%) controlled environment chamber (CEC) with constant air flow (15 L/min) for 12 days and received 5 mg/mL subcutaneous scopolamine hydrobromide (Sigma-Aldrich, St. Louis, MO) injections three times per day. Topical atropine sulfate (1%; Bausch & Lomb, Rochester, NY) was administered twice per day on days 1 and 2 to enhance anticholinergic effects.…”

“…To validate our hypothesis, we systematically explored a well-characterized murine model of dry eye. 6,20 We evaluated the generation of Th17 cells and their expression of the chemokine receptor CCR6, as well as the presence of CCL20 at the ocular surface. Furthermore, we used local, in vivo neutralization of CCL20 to demonstrate the functional releCopyright 2013 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783 vance of the CCR6/CCL20 axis in mediating Th17 cell migration and DED pathogenesis.…”