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Sarah B Dale, Tomas Blanco, Rose Mathew, Priyatham S Mettu, Scott W Cousins, Virginia L Calder, Daniel Saban; CD11b+ Classical Dendritic Cells (cDC) Directly Effect Fibrosis in Ocular Surface Inflammation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5874.
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The pathobiology underlying conjunctival fibrosis, such as evidenced in vision-threatening forms of severe ocular allergy (e.g. vernal/atopic keratoconjunctivitis) is poorly understood. Our lab has established that CD11b+ cDCs are immunopathogenic in the mouse model of severe allergic eye disease (AED), and further demonstrated distinct appearance of conjunctival fibrosis in these mice. This led us to examine herein whether the production by CD11b+ cDCs of retinoic acid (RA), a paracrine pro-fibrotic factor, contributes to fibrosis development in AED.
AED is induced by i.p. injection of OVA (10µg), aluminium hydroxide (4mg), and pertussis toxin (300ng) mixture, followed 2wks later by once/day OVA instillations for 1wk. Flow cytometry defined CD11b+ cDCs, as CD45+ Siglec F- CD11b+ Ly6C- Ly6G- F4/80+ CD64- CD11c+ I-A/E+ CD103-. ALDEFLOUR was used to measure levels of ALDH (enzyme responsible for RA synthesis). Primary fibroblasts were used in vitro for gel contractility and proliferation assays. For RXR agonist experiments in vivo, bexarotene (0.2µg/µl) was administered subconjunctivally; for ALDH inhibition experiments in vivo, DEAB was applied topically (300µM). Fibrosis was evaluated clinically and histologically (trichrome staining).
Only CD11b+ cDCs in the conjunctiva possessed significantly increased ALDH activity (p<0.001) in the AED, suggesting augmented RA production by these cells. In vitro, the application of CD11b+ cDC conditioned media, containing RA, led to impaired fibroblast functionality (p<0.001). This impairment was prevented with blockade of the retinoic acid receptor RXR but not RAR (p<0.05), suggesting a role for 9-cis RA produced by CD11b+ cDCs in fibrosis. Furthermore, subconjonjunctival administration of RXR agonist led to the presence of conjunctival fibrosis, verifying in vivo, existence of an RA/RXR axis in fibrosis. Finally, inhibition of CD11b+ cDC derived ALDH activity, achieved with topical instillation of DEAB and verified via flow cytometry, led to the protection from conjunctival fibrosis in AED.
The data reveals a direct and novel role for CD11b+ cDCs in fibrosis. This mechanism involves production by these cells of RA, which in turn leads to fibroblast dysfunction via RXR activation. Additionally, our data suggests that targeting cDCs protects mice from development of conjunctival fibrosis in severe ocular allergy.
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