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Catharina Busch, Balasubramaniam Annamalai, Maria Diedrichs-Möhring, Gerhild Wildner, Olaf Strauss, Baerbel Rohrer; Anaphylatoxins activate Akt/PI3-kinase and FOXO1/FoxP3-signaling pathways in the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2278.
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The retinal pigment epithelium (RPE) is a main target for complement activation in age-related macular degeneration. The anaphylatoxins C3a and C5a, have been thought to mostly play a role as chemoattractants for macrophages and immune cells; here we explore whether they trigger RPE alterations. Specifically, we investigated the RPE as a potential immunoregulatory gate, allowing for active changes in the RPE microenvironment in response to complement.
Gene expression of the parent proteins for anaphylatoxins and their receptors as well as protein secretion of different cytokines in ARPE-19 cells in response to anaphylatoxins were analyzed by qPCR and Bio-Plex bead analysis. ARPE-19 cells, human primary RPE cells and rat retina sections were investigated for the expression of Akt, FOXO1 and FoxP3 by means of PCR and immunohistochemistry. Phosphorylation of Akt, FOXO1 and FoxP3 in response to C3a and C5a was analyzed by Western blotting.
Combined application of C3a and C5a decreased C3 mRNA expression in ARPE-19 cells when compared to the unstimulated control, whereas mRNA levels of C3aR, C5 and C5aR were not affected by anaphylatoxin stimulation. Analysis of secreted cytokines revealed significantly elevated levels of IL-8 and VEGF-A in response to C3a+C5a. Secretion of IL-8, VEGF-A and MCP-1 was almost completely blocked by the application of PI3-kinase inhibitor LY294002. ARPE-19 cells were found to express Akt, FOXO1 and FoxP3 protein. In response to anaphylatoxins phosphorylation of Akt, FOXO1 and FoxP3 was significantly induced. Combination of C3a+C5a significantly increased phosphorylation of FOXO1 and FoxP3 compared to single C3a or C5a stimulation. However, phosphorylation of Akt was significantly reduced in response to C3a+C5a. FOXO1 and FoxP3 gene expression was confirmed in human native RPE cells (FOXO1) and retina sections of uveitis rats (FoxP3).
RPE cells show active changes in the activity of intracellular kinases, the transcription factors FOXO1 and FoxP3 as well as an altered protein secretion in response to anaphylatoxins. These data suggest that the RPE shares certain characteristics with immune cells (FoxP3 expression, cytokine secretion) confirming its role in the “immunoregulatory” gate; and indicates that the RPE thus actively contributes to the establishment of a proinflammatory environment in the presence of complement activation.
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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