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Sara Touhami, Fanny Beguier, Sébastien Augustin, Sacha Reichman, Olivier Goureau, Emeline F Nandrot, Xavier Guillonneau, Bahram Bodaghi, Florian Sennlaub; Chronic exposure to TNFα impairs RPE barrier and immunosuppressive functions.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5743. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal pigment epithelium (RPE) is a monolayer of pigmented cells with important functions in the outer blood-retinal barrier and subretinal immune suppression. Failure of RPE functions and chronic inflammation have been both hypothesized to play a role in the pathophysiology of age related macular degeneration (AMD). We have previously shown that acute stimulation of RPE cells by TNFα down-regulates gene expression of OTX2 (Orthodenticle homeobox 2) and that of major visual cycle-related genes . We here investigated the long-term effects of TNFα on RPE morphology and function in vitro.
Primary porcine RPE cells were cultivated until confluence, then recombinant TNFα was added daily in the culture medium (at 0.8, 4, 20 or 100ng/ml=C1,C2,C3 and C4) for 10 days. RPE cell morphology and gene expression, barrier, phagocytosis and immunosuppressive functions were assessed.
Cell morphology and gene expression: 10 day stimulation by TNFα (i) decreased RPE cell numbers (3653.6, 3428, 3227, 2791 and 2020 cells/mm2 respectively for control, C1,C2,C3 and C4, all p<0.01); (ii) increased cell size (+5.3, +12.6, +13.9 and +9.5% for C1,C2,C3 and C4 as compared to control, all p<0.05); (iii) increased the number of multinucleated cells (5.7, 7.7, 9.4, 9.9, 15.9% of multinucleated cells for control, C1,C2,C3 and C4, all p<0.05); (iv) and decreased OTX2 expression (-11.1, -19.7, -52 and -82.9% for C1,C2,C3 and C4 as compared to control, all p<0.05). The number of apoptotic TUNEL+ cells was increased upon TNFα adjunction (112,3 , 158.7 and 189.9% of control for C2,C3 and C4, all p<0.05). Barrier function: 10 day stimulation by TNFα (i) disturbed Zonula Occludens 1 cellular distribution and cytoskeletal architecture (actin F distribution) and (ii) significantly decreased RPE transepithelial resistance in a dose-dependent manner ( -70, -88.5 and -90.8% of control for C2,C3 and C4, p<0.05). Immunossupressive function: 10 day pre-stimulation with -TNFα significantly decreased RPE capacity to induce monocyte death after 24h of co-culture (p<0.05).
Chronic exposure to TNFα deteriorates major RPE functions that are essential to visual function and might play a key role in the pathophysiology of AMD.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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