Pigment Epithelium-Derived Factor Enhances the Suppressive Phenotype of Regulatory T Cells in a Murine Model of Dry Eye Disease

Abstract

Pigment epithelium-derived factor (PEDF) is a widely expressed 50-kDa glycoprotein belonging to the serine protease inhibitor family, with well-established anti-inflammatory functions. Recently, we demonstrated the immunoregulatory role played by PEDF in dry eye disease (DED) by suppressing the maturation of antigen-presenting cells at the ocular surface following exposure to the desiccating stress. In this study, we evaluated the effect of PEDF on the immunosuppressive characteristics of regulatory T cells (Tregs), which are functionally impaired in DED. In the presence of PEDF, the in vitro cultures prevented proinflammatory cytokine (associated with type 17 helper T cells)-induced loss of frequency and suppressive phenotype of Tregs derived from normal mice. Similarly, PEDF maintained the in vitro frequency and enhanced the suppressive phenotype of Tregs derived from DED mice. On systemically treating DED mice with PEDF, moderately higher frequencies and significantly enhanced suppressive function of Tregs were observed in the draining lymphoid tissues, leading to the efficacious amelioration of the disease. Our results demonstrate that PEDF promotes the suppressive capability of Tregs and attenuates their type 17 helper T-cell-mediated dysfunction in DED, thereby playing a role in the suppression of DED.

Figure 1

Pigment epithelium–derived factor (PEDF) enhances the suppressive phenotype of regulatory T cells in a murine model of dry eye disease (DED). Dendritic cells (DCs) are activated in response to the proinflammatory cytokines, released by corneal epithelium on exposure to desiccating stress, and migrate from the cornea to the draining lymph nodes (DLNs) to form the afferent arm of immunopathogenesis in DED. In the DLNs, DCs prime naïve T cells to generate proinflammatory cytokines secreting type 17 helper T (Th17) cells. The regulatory T cells (Tregs) are known to limit the interaction between DCs and naïve T cells, thereby preventing the generation of Th17 cells. However, in DED, Tregs are rendered dysfunctional, leading to loss of their immunosuppressive functions. This study shows a protective effect of recombinant PEDF (rPEDF) on Tregs in DED and subsequent amelioration of DED. CD86, cluster of differentiation 86; CTLA-4, cytotoxic T-lymphocyte–associated protein 4; FOXP3, forkhead box P3; GITR, glucocorticoid-induced TNFR-related protein; IFN-γ, interferon-γ; MHC, major histocompatibility complex; Th1, T helper cells type 1; TNF-α, tumor necrosis factor-α.

Figure 2

Pigment epithelium-derived factor (PEDF) prevents proinflammatory cytokine–induced loss of regulatory T-cell (Treg) frequency and suppressive phenotype. Cervical and submandibular draining lymph nodes were harvested from normal mice, and CD4⁺CD25⁺ cells were isolated by magnetic sorting. Sorted regulatory T cells (Tregs) were cultured with dysfunction, inducing proinflammatory cytokines IL-17A (100 ng/mL) and interferon (IFN)-γ (10 ng/mL) for 72 hours. The culture medium was supplemented with IL-2 (100 ng/mL) for Treg maintenance in vitro. Representative flow cytometry plots (A) and bar charts (B), showing frequencies of CD4⁺CD25⁺ FOXP3⁺ Tregs cultured with (100 ng/mL) or without recombinant pigment epithelium–derived factor (rPEDF). Bar charts showing forkhead box P3 (FOXP3) (C), cytotoxic T-lymphocyte–associated protein 4 (CTLA-4; D), and glucocorticoid-induced TNFR-related protein (GITR) (E) expression levels [mean fluorescence intensity (MFI)] in Tregs cultured with or without rPEDF. Real-time analysis showing expression levels of IL-10 (F) and transforming growth factor (TGF)-β (G) by Tregs cultured with (100 ng/mL) or without rPEDF. Data are presented as a representative experiment of the four performed, each consisting of four mice per group. Data are presented as means ± SEM (B–G). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Figure 3

Pigment epithelium–derived factor (PEDF) maintains the frequency and enhances the suppressive phenotype of regulatory T cells (Tregs) derived from dry eye disease (DED) mice. Mice were placed in the controlled environment chamber for 14 days to induce DED. The cervical and submandibular draining lymph nodes were harvested from mice, and CD4⁺CD25⁺ cells were isolated by magnetic sorting. Representative flow cytometry plots (A) and bar charts (B), showing frequencies of CD4⁺CD25⁺ FOXP3⁺ Tregs cultured with or without recombinant PEDF (rPEDF). Bar charts showing forkhead box P3 (FOXP3) (C), cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) (D), and glucocorticoid-induced TNFR-related protein (GITR) (E) expression levels [mean fluorescence intensity (MFI)] in Tregs cultured with or without rPEDF. Real-time analysis, showing the expression levels of IL-10 (F) and transforming growth factor-β (TGF-β) (G) by Tregs cultured with or without rPEDF. Data are presented as a representative experiment of the four performed, each consisting of four mice per group. Data are presented as means ± SEM (B–G). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 4

Systemic treatment with recombinant pigment epithelium–derived factor (rPEDF) maintains regulatory T-cell (Treg) phenotype in dry eye disease (DED). To delineate the effect of PEDF on dysfunctional Tregs in DED, mice were treated with i.p. injection of rPEDF (1 μg/mL) or murine serum albumin (MSA; control) once daily for 7 days. A: Schematic diagram showing the time points of DED induction, i.p. rPEDF or MSA injections, and tissue harvesting. B: Representative flow cytometry plots showing the frequency of CD4⁺CD25⁺FOXP3⁺ Tregs. C: Bar charts showing the frequency of forkhead box P3 (FOXP3), in mice treated with rPEDF or MSA. D: Bar charts showing the expression levels of FOXP3 in mice treated with rPEDF or MSA. Data are presented as a representative experiment of the four performed, each consisting of three mice per group. Data are presented as means ± SEM (C and D). ∗∗P < 0.01. DLN, draining lymph node; MFI, mean fluorescence intensity; qd, once a day.

Figure 5

In vitro regulatory T-cell (Treg) suppression assay. Naïve or primed T cells isolated from the draining lymph nodes (DLNs) of normal and dry eye disease (DED) mice, respectively, were stimulated with CD3 antibody for 3 days in the presence of Tregs isolated from the DLNs of normal or DED [pigment epithelium–derived factor (PEDF)–treated and untreated] mice. The activity of Tregs is measured at Treg/Teff (effector T cell) ratio of 1:2, as standardized previously. Proliferation was measured using the bromodeoxyuridine incorporation assay, compared with the proliferative responses of respective CD3-stimulated T cells in the absence of Tregs, and the percentage suppression calculated. Data are presented as a representative experiment of the four performed, each consisting of three mice per group. Data are presented as means ± SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. mAb, monoclonal antibody; rPEDF, recombinant PEDF.

Figure 6

Treatment with recombinant pigment epithelium–derived factor (rPEDF) reduces dry eye disease (DED) severity. Efficacy of systemic rPEDF treatment initiated after DED induction for 7 days was assessed. A: Clinical severity of DED on treatment with murine serum albumin (MSA; control) or rPEDF was evaluated using corneal fluorescein staining on days 7, 9, 12, and 14. B: Representative corneal fluorescein staining images captured on days 7, 9, 12, and 14. Data are presented as a representative experiment of the four performed, each consisting of four mice per group. Data are presented as means ± SEM (A). ∗P < 0.05, ∗∗P < 0.01.