Nadine Reichhart1, Gerhild Wildner2, Olaf Strauß3, Ahmad Samir Alfaar1,4, Lucas Stürzbecher1, Maria Diedrichs-Möhring5, Marion Lam6, Christophe Roubeix6, Julia Ritter7, Kathrin Schumann7, Balasubramaniam Annamalai8, Inga-Marie Pompös1, Bärbel Rohrer8, Florian Sennlaub6. 1. Experimental Ophthalmology, Department of Ophthalmology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität, Berlin Institute of Health, Humboldt-University, 10117, Berlin, Germany. 2. Section of Immunobiology, Department of Ophthalmology, University Hospital, LMU Munich, 80336, Munich, Germany. Gerhild.Wildner@med.uni-muenchen.de. 3. Experimental Ophthalmology, Department of Ophthalmology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität, Berlin Institute of Health, Humboldt-University, 10117, Berlin, Germany. Olaf.strauss@charite.de. 4. Department of Ophthalmology, University Hospital of Ulm, 89075, Ulm, Germany. 5. Section of Immunobiology, Department of Ophthalmology, University Hospital, LMU Munich, 80336, Munich, Germany. 6. Institut de La Vision, Sorbonne Université, INSERM, CNRS, 75012, Paris, France. 7. Institut Für Med. Mikrobiologie, Immunologie Und Hygiene, TU München, 81675, Munich, Germany. 8. Department of Ophthalmology, College of Medicine, Medical University South Carolina, Charleston, SC, 29425, USA.
Abstract
BACKGROUND: Forkhead-Box-Protein P3 (FoxP3) is a transcription factor and marker of regulatory T cells, converting naive T cells into Tregs that can downregulate the effector function of other T cells. We previously detected the expression of FoxP3 in retinal pigment epithelial (RPE) cells, forming the outer blood-retina barrier of the immune privileged eye. METHODS: We investigated the expression, subcellular localization, and phosphorylation of FoxP3 in RPE cells in vivo and in vitro after treatment with various stressors including age, retinal laser burn, autoimmune inflammation, exposure to cigarette smoke, in addition of IL-1β and mechanical cell monolayer destruction. Eye tissue from humans, mouse models of retinal degeneration and rats, and ARPE-19, a human RPE cell line for in vitro experiments, underwent immunohistochemical, immunofluorescence staining, and PCR or immunoblot analysis to determine the intracellular localization and phosphorylation of FoxP3. Cytokine expression of stressed cultured RPE cells was investigated by multiplex bead analysis. Depletion of the FoxP3 gene was performed with CRISPR/Cas9 editing. RESULTS: RPE in vivo displayed increased nuclear FoxP3-expression with increases in age and inflammation, long-term exposure of mice to cigarette smoke, or after laser burn injury. The human RPE cell line ARPE-19 constitutively expressed nuclear FoxP3 under non-confluent culture conditions, representing a regulatory phenotype under chronic stress. Confluently grown cells expressed cytosolic FoxP3 that was translocated to the nucleus after treatment with IL-1β to imitate activated macrophages or after mechanical destruction of the monolayer. Moreover, with depletion of FoxP3, but not of a control gene, by CRISPR/Cas9 gene editing decreased stress resistance of RPE cells. CONCLUSION: Our data suggest that FoxP3 is upregulated by age and under cellular stress and might be important for RPE function.
BACKGROUND: Forkhead-Box-Protein P3 (FoxP3) is a transcription factor and marker of regulatory T cells, converting naive T cells into Tregs that can downregulate the effector function of other T cells. We previously detected the expression of FoxP3 in retinal pigment epithelial (RPE) cells, forming the outer blood-retina barrier of the immune privileged eye. METHODS: We investigated the expression, subcellular localization, and phosphorylation of FoxP3 in RPE cells in vivo and in vitro after treatment with various stressors including age, retinal laser burn, autoimmune inflammation, exposure to cigarette smoke, in addition of IL-1β and mechanical cell monolayer destruction. Eye tissue from humans, mouse models of retinal degeneration and rats, and ARPE-19, a human RPE cell line for in vitro experiments, underwent immunohistochemical, immunofluorescence staining, and PCR or immunoblot analysis to determine the intracellular localization and phosphorylation of FoxP3. Cytokine expression of stressed cultured RPE cells was investigated by multiplex bead analysis. Depletion of the FoxP3 gene was performed with CRISPR/Cas9 editing. RESULTS: RPE in vivo displayed increased nuclear FoxP3-expression with increases in age and inflammation, long-term exposure of mice to cigarette smoke, or after laser burn injury. The human RPE cell line ARPE-19 constitutively expressed nuclear FoxP3 under non-confluent culture conditions, representing a regulatory phenotype under chronic stress. Confluently grown cells expressed cytosolic FoxP3 that was translocated to the nucleus after treatment with IL-1β to imitate activated macrophages or after mechanical destruction of the monolayer. Moreover, with depletion of FoxP3, but not of a control gene, by CRISPR/Cas9 gene editing decreased stress resistance of RPE cells. CONCLUSION: Our data suggest that FoxP3 is upregulated by age and under cellular stress and might be important for RPE function.