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Ken Simmons, Stephen C Pflugfelder, Cintia S De Paiva; Toll-like receptor 4 activation on the ocular surface in experimental dry eye. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4811.
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TLR4 alerts cells to the presence of bacteria by initiating an inflammatory response. However, it is unknown if TLR4 can generate a strong immune response on the ocular surface. We hypothesize that TLR4 activation on the ocular surface leads to production/release of inflammatory mediators. In addition, we hypothesize that TLR4 activation in a dry eye leads to greater expression of inflammatory mediators than in a normal eye.
A single dose of increasing concentrations (10ng/µl, 100ng/µl, 1µg/µl, or 10µg/µl) of lipopolysaccharide (LPS) from salmonella minnesota was dissolved in endotoxin-free water and applied to the cornea of non-stressed (NS) female C57BL/6J mice (5µl/eye). Endotoxin-free water eyedrops were used as a control. After 4 hours, the mice were euthanized and the cornea and conjunctiva was extracted to analyze expression of inflammatory mediators via RT-PCR. To analyze expression of inflammatory mediators after TLR4 activation in dry eye, mice were subjected to desiccating stress (DS) and received either a single dose of LPS (1µg/µl) or vehicle at day 5.
LPS administration increased expression of inflammatory mediators in a dose dependent manner. In general, 10µg/µl had the highest effect. 1µg/µl LPS drops increased expression of inflammatory mediators IL-1β (21±10 fold), IL-6 (20±10 fold), CXCL10 (185±41 fold), IL-12a (4±0.27 fold), and IFN-γ (13±4 fold) in conjunctiva and IL-1β (2±0.31 fold) and CXCL10 (37±8 fold) in cornea compared to untreated mice. DS5 mice treated with LPS exhibited significantly increased expression of IL-1β (12 vs. 0.18 fold), IL-6 (48 vs. 0.65 fold), CXCL10 (132 vs. 0.42 fold), IL-12a (8 vs. 2 fold) and IFN-γ (8 vs.0.4 fold) in conjunctiva and IL-1β (7 vs. 0.73 fold) and CXCL10 (125 vs. 0.71 fold) in cornea compared to DS5 mice treated with water. LPS drops in DS mice further increased expression of IL-1β (3 fold) and CXCL10 (3 fold) in cornea and IL-12a (2 fold) in conjunctiva compared to LPS treated control mice.
LPS administration leads to increased expression of inflammatory cytokines on the ocular surface in a dose-dependent manner. This expression appears to increase in a dry eye, which may indicate that epithelial barrier disruption enhances exposure of LPS to TLR4+ cells and that the inflammatory response to commensal or pathogenic bacteria may be more severe during dry eye disease.
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