FoxP3 expression by retinal pigment epithelial cells: transcription factor with potential relevance for the pathology of age-related macular degeneration

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-1 beta 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-1 beta 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

Anaphylatoxins Activate Ca2+, Akt/PI3-Kinase, and FOXO1/FoxP3 in the Retinal Pigment Epithelium

Purpose: The retinal pigment epithelium (RPE) is a main target for complement activation in age-related macular degeneration (AMD). 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. Design: In vitro and in vivo analysis of signaling pathways. Methods: Individual activities of and interaction between the two anaphylatoxin receptors were tested in cultured RPE cells by fluorescence microscopy, western blot, and immunohistochemistry. Main outcome measures: Intracellular free calcium, protein phosphorylation, immunostaining of tissues/cells, and multiplex secretion assay. Results: Similar to immune cells, anaphylatoxin exposure resulted in increases in free cytosolic Ca2+, PI3-kinase/Akt activation, FoxP3 and FOXO1 phosphorylation, and cytokine/chemokine secretion. Differential responses were elicited depending on whether C3a and C5a were co-administered or applied consecutively, and response amplitudes in co-administration experiments ranged from additive to driven by C5a (C3a + C5a = C5a) or being smaller than those elicited by C3a alone (C3a + C5a < C3a). Conclusion: We suggest that this combination of integrative signaling between C3aR and C5aR helps the RPE to precisely adopt its immune regulatory function. These data further contribute to our understanding of AMD pathophysiology

Dynamics of Intraocular IFN-γ, IL-17 and IL-10-Producing Cell Populations during Relapsing and Monophasic Rat Experimental Autoimmune Uveitis

<div><p>A major limitation of most animal models of autoimmune diseases is that they do not reproduce the chronic or relapsing-remitting pattern characteristic of many human autoimmune diseases. This problem has been overcome in our rat models of experimentally induced monophasic or relapsing-remitting autoimmune uveitis (EAU), which depend on the inducing antigen peptides from retinal S-Antigen (monophasic EAU) or interphotoreceptor retinoid-binding protein (relapsing EAU). These models enable us to compare autoreactive and regulatory T cell populations. Intraocular, but not peripheral T cells differ in their cytokine profiles (IFN-γ, IL-17 and IL-10) at distinct time points during monophasic or relapsing EAU. Only intraocular T cells concomitantly produced IFN-γ, IL-17 and/or IL-10. Monophasic EAU presented rising numbers of cells expressing IFN-γ and IL-17 (Th1/Th17) and cells expressing IL-10 or Foxp3. During relapsing uveitis an increase of intraocular IFN-γ+ cells and a concomitant decrease of IL-17+ cells was detected, while IL-10+ populations remained stable. Foxp3+ cells and cells expressing IL-10, even in combination with IFN-γ or IL-17, increased during the resolution of monophasic EAU, suggesting a regulatory role for these T cells. In general, cells producing multiple cytokines increased in monophasic and decreased in relapsing EAU. The distinct appearance of certain intraocular populations with characteristics of regulatory cells points to a differential influence of the ocular environment on T cells that induce acute and monophasic or relapsing disease. Here we provide evidence that different autoantigens can elicit distinct and differently regulated immune responses. IFN-γ, but not IL-17 seems to be the key player in relapsing-remitting uveitis, as shown by increased, synchronized relapses after intraocular application of IFN-γ. We demonstrated dynamic changes of the cytokine pattern during monophasic and relapsing-remitting disease with strongly increasing IL-10 expression in intraocular T cells during monophasic uveitis.</p> </div

Expression IL-10, IFN-γ, IL-17 and Foxp3 in the late phase of remission of EAU.

<p>(A) Time course showing mean daily clinical uveitis scores. Eyes were collected after complete resolution of clinical uveitis (“late remission”) 10 days after resolution of EAU induced as indicated. (B) Coexpression of IFN-γ (left panel) and IL-17 (right panel) with IL-10 in quiescent eyes of late remission. Cells were gated on lymphocytes based on FSC and SSC. (C) Expression of Foxp3 in intraocular TCR-αβ+ cells from quiescent eyes of late remission. Data show means of at least 3 independent experiments with n = 6–10 eyes per group.</p

MHC-class II restriction of PDSAg- and R14-specific T cells.

<p>Proliferation of a PDSAg- and a R14-specific T cell line in response to the respective antigen peptide and mouse mastocytoma cells P80, expressing rat CD80 and rat MHC-class II antigens RT1.B or RT1.D. T cells were stimulated for 3 days and T cell responses are shown as stimulation index (SI)+SD.</p

In vivo administration of IFN-γ.

<p>Rats immunized with R14-CFA were injected intraocularly (i.o.) into the anterior chamber or intraperitoneally (i.p.) with IFN-γ or saline (as indicated) at day 19, when the clinical signs of EAU were ≤0.5 in all eyes. The time course shows the mean daily clinical scores of all eyes per group ± SE. The arrow marks the time point of IFN-γ or saline application. Significant difference between the group receiving intraocular IFN-γ and the other treatment groups is indicated (*, p≤0.05).</p

Expression of TCR-αβ, IFN-γ and IL-17 by intraocular cells during EAU.

<p>(A, B) Representative clinical courses of monophasic EAU induced with PDSAg (A) or relapsing EAU induced with R14 (B). Black and white symbols represent clinical scores of right and left eyes, respectively. (C, D) Corresponding mean clinical uveitis scores of n = 18–27 eyes from at least 3 independent experiments after immunization with PDSAg-CFA (C) or R14-CFA (D). (E–H) Intraocular cells were isolated from rat eyes at indicated time points after immunization with PDSAg (E, G) or R14 (F, H). Cells from 2–6 eyes were pooled for each experiment and analyzed by flow cytometry. Lymphocytes were gated based on FSC and SSC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049008#pone.0049008.s001" target="_blank">Fig. S1</a>). (E, F) TCR-αβ+ intraocular cells during PDSAg-CFA- (E) and R14-CFA-induced EAU (F). (G, H) Expression of IFN-γ, IL-17 or both together. Data show means from at least 3 independent experiments+SD. Significant differences between the time points of cell collection (*, p<0.05) and PDSAg and R14 (°, p<0.05) are indicated.</p

Expression of IFN-γ and IL-17 by intraocular TCR-αβ or TCR-γδ cells.

<p>(A, B) Representative dot plots from the FACS analysis of double staining for TCR-αβ and IFN-γ or IL-17 from PDSAg- (A) and R14-induced (B) uveitis. (C, D) Dot plots showing double staining of TCR-γδ and IL-17 or IFN-γ at different time points during PDSAg- (C) and R14-induced EAU (D). Lymphocytes were gated based on FSC and SSC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049008#pone.0049008.s001" target="_blank">Fig. S1</a>). For corresponding EAU scores see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049008#pone-0049008-g001" target="_blank">Fig. 1C/D</a>.</p

Expression of Foxp3 and IL-10 by intraocular cells during EAU.

<p>Cells were isolated from eyes at indicated time points during EAU. Corresponding clinical EAU scores are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049008#pone-0049008-g001" target="_blank">Fig. 1C and D</a>. Lymphocytes were gated based on FSC and SSC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049008#pone.0049008.s001" target="_blank">Fig. S1</a>). (A) Ex vivo staining for Foxp3. Mean % of cells+SD is shown for at least 3 independent experiments with n = 2–6 eyes per group. (B, C) Ex vivo staining for intracellular IL-10 and IFN-γ expression (left panels) as well as for expression of IL-10 and IL-17 (right panels). Data are shown as mean+SD for at least 3 independent experiments with n = 2–6 eyes per group. Significant differences between the time points of cell collection (*, p<0.05) and PDSAg and R14 are indicated (°, p<0.05). (D) Quadruple staining of intraocular cells for expression of TCR-αβ, IFN-γ, IL-17 and IL-10. Dot plots show IFN-γ and IL-17 staining of TCR-αβ+/IL-10+ gated cells.</p