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X. Zheng, D.-Q. Li, C. S. De Paiva, W. J. Farley, S. C. Pflugfelder; Ocular Surface Epithelia-Derived Cytokines Induce Th17 Differentiation Through Dendritic Cell-Mediated Pathway in a Mouse Dry Eye Model. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3657.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous study revealed increased expression of Th17 associated cytokines in experimental murine dry eye. This study tested our hypothesis that ocular surface epithelia-derived cytokines induce Th17 differentiation through a dendritic cell(DC)-mediated pathway in a mouse dry eye model.
Experimental dry eye was created by subjecting C57BL/6 mice to desiccating environmental stress for 10 days. Corneal and conjunctival explants from dry eye or control mice were co-cultured with dendritic cells (DCs) isolated from bone marrow of C57BL/6 mice for 24 hours, prior to the addition of CD4+ T cells isolated from spleens and cervical lymph nodes for an additional 4-7 days. Expression of Th-17 associated genes in the cornea, conjunctiva, DCs and CD4+ T cells was evaluated by real-time PCR. Cytokine protein concentrations in co-culture supernatants were measured by Luminex immunobead assay. The number of IL-17 producing T cells was evaluated by ELISPOT bioassay. RORγt production in CD4+ T cells was detected by Western-blot.
Increased mRNA expression of TGF-β1, -β2, IL-6 (inducers for Th17 initiation), IL-23 and IL-1β (inducers for Th17 survival and expansion), as well as the Th17 cytokine IL-17A, was observed in corneal and conjunctival epithelia of the dry eye mice. Higher levels of TGF-β1&2, IL-6 and IL-1β were detected in the supernatants of 2 day corneal and conjunctival explants from dry eye than normal mice. DCs co-cultured with the explants from dry eye mice for 2 days produced higher levels of TGF-β1, IL-6, IL-23 and IL-1β mRNA transcripts and protein. Interestingly, when co-cultured with the DCs and epithelial explants from dry eye mice, CD4+ T cells expressed increased mRNA levels of the Th17 cytokines (IL-17A, IL-17F, IL-22 and CCL-20) and the Th17 cell transcription factor (RORγt). Increased IL-17A in the supernatant, and increased numbers of IL-17-producing T cells (Th17 cells) evaluated by ELISPOT bioassay, were also observed from these CD4+ T cells. Increased level of RORγt protein were detected in co-cultured CD4+ T cells by Western-blot.
These findings demonstrate that desiccating stress stimulates the expression and production of Th-17 inducing cytokines by corneal and conjunctival epithelia, and ocular surface epithelia-derived cytokines induce Th17 differentiation through a dendritic cell-mediated pathway in a mouse dry eye model.
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