Both MC5r and A2Ar Are Required for Protective Regulatory Immunity in the Spleen of Post–Experimental Autoimmune Uveitis in Mice

Darren J. Lee and Andrew W. Taylor
J Immunol October 15, 2013, 191 (8) 4103-4111; DOI: https://doi.org/10.4049/jimmunol.1300182

Abstract

The ocular microenvironment uses a poorly defined melanocortin 5 receptor (MC5r)-dependent pathway to recover immune tolerance following intraocular inflammation. This dependency is seen in experimental autoimmune uveoretinitis (EAU), a mouse model of endogenous human autoimmune uveitis, with the emergence of autoantigen-specific regulatory immunity in the spleen that protects the mice from recurrence of EAU. In this study, we found that the MC5r-dependent regulatory immunity increased CD11b+F4/80+Ly-6ClowLy-6G+CD39+CD73+ APCs in the spleen of post-EAU mice. These MC5r-dependent APCs require adenosine 2A receptor expression on T cells to activate EAU-suppressing CD25+CD4+Foxp3+ regulatory T cells. Therefore, in the recovery from autoimmune disease, the ocular microenvironment induces tolerance through a melanocortin-mediated expansion of Ly-6G+ regulatory APCs in the spleen that use the adenosinergic pathway to promote activation of autoantigen-specific regulatory T cells.

Introduction

Experimental autoimmune uveitis (EAU) is a mouse model of endogenous uveitis in humans (1). EAU has been studied to better understand the mechanisms contributing to the relapsing and remitting nature of chronic autoimmune uveitis. C57BL/6J mice immunized with human interphotoreceptor retinoid binding protein (IRBP) peptide aa 1–20 display uveoretinitis within 2–3 wk of immunization. This inflammation resolves on its own 70–90 d after immunization. The resolution of EAU establishes regulatory immunity specific to IRBP in the spleen (2). This post-EAU regulatory immunity suppresses memory/recall immune responses to IRBP, as well as suppresses induction and reactivation of EAU. The post-EAU protective regulatory immunity involves an undefined interaction between regulatory APCs and their Ag-stimulated IRBP-specific CD25+CD4+ regulatory T cells (Tregs) (2). The regulatory activity is not seen with IRBP immunization only; it requires a uveitic response. This suggested that, during uveitis, the ocular mechanisms of immune privilege induce distinctive regulatory APCs to activate IRBP-specific Tregs in the spleen.

The immunomodulating neuropeptide α-melanocyte stimulating hormone (α-MSH) is a central mediator of immunosuppression within the ocular microenvironment (3). Therapeutic injections of α-MSH are effective in suppressing EAU (4, 5), experimental autoimmune encephalomyelitis (6), and corneal allograft rejection (7). The melanocortin 5 receptor (MC5r) is one of four melanocortin receptors for which α-MSH is the ligand (8, 9). The presence of post-EAU regulatory APCs requires expression of MC5r (10). When reimmunized after recovery from the initial episode of EAU, MC5r knockout [MC5r(−/−)] mice have a second episode of EAU with a rapid onset and enhanced severity that is characteristic of a memory/recall response (2). This is in contrast to reimmunized wild-type (WT) mice, which display an immunologically naive-like response for a second episode of EAU. The severity of the second episode in reimmunized MC5r(−/−) mice is suppressed by the adoptive transfer (AT) of spleen cells from post-EAU WT mice. This failure of MC5r(−/−) mice to generate post-EAU regulatory immunity is not due to an inability of MC5r(−/−) mice to generate Tregs but is a failure of MC5r(−/−) mice to generate post-EAU regulatory APCs (10). Therefore, the dependence on MC5r expression is associated with the induction of regulatory APCs.

Myeloid-derived suppressor cells (MDSCs) are APCs capable of suppressing inflammation and promoting regulatory immunity (11–13). MDSCs are identified by CD11b and can be divided into Ly-6ClowLy-6G+ and Ly-6ChighLy-6G− cell populations (14, 15). They can emerge as inflammatory diseases progress to resolution (16), and it was suggested that they are more characteristic of alternatively activated macrophages (12, 14). The melanocortin pathways that include the melanocortin receptors and their ligand α-MSH were shown to induce alternative activation of macrophages and dendritic cells, and to promote activation of regulatory immunity (17, 18). Recently, it was found that α-MSH with Neuropeptide Y mediates the induction of myeloid suppressor cell–like activity in resting macrophages and contributes to the regulation of similar activity in retinal microglial cells (19). The dependence on MC5r expression for the induction of post-EAU regulatory APCs suggests that the post-EAU APCs may be similar to MDSCs.

In this study, we identified the MC5r-dependent post-EAU APCs as CD11b+F4/80+Ly-6ClowLy-6G+ cells with adenosine-generating ectonucleotidases CD39 and CD73 that mediate activation of Tregs through adenosine 2A receptor (A2Ar) expressed on T cells. These findings demonstrate a link between two highly conserved pathways of immune regulation through MC5r expression on APCs and A2Ar on T cells in establishing protective post-EAU regulatory immunity to ocular autoantigen.

Materials and Methods

Mice

The Boston University Institutional Animal Care and Use Committee approved all procedures used on mice in this study. C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). MC5r(−/−) mice on a C57BL/6J background were obtained from Roger D. Cone (Oregon Health Sciences, Portland, OR), and A2Ar(−/−) mice, also on a C57BL/6J background, were obtained from Dr. Jiang-Fen Chen (Boston University School of Medicine). Both strains were housed and bred in the Boston University Laboratory Animal Science Center.

Induction of EAU

EAU was induced by immunizing the mice with an emulsion of CFA with 5 mg/ml desiccated Mycobacterium tuberculosis (Difco Laboratories, Detroit, MI) and 2 mg/ml IRBP peptide aa 1–20 (GenScript, Piscataway, NJ). Mice received a s.c. injection of the emulsion (100 μl) into two sites on the lower back, followed by an i.p. injection of 0.3 μg pertussis toxin. Every 3–4 d, the course of EAU was evaluated by fundus examination. The ocular fundus was examined using a slit lamp microscope. The corneas were flattened with a glass coverslip, the cornea was numbed with 0.5% proparacaine, and the iris was dilated with 1% tropicamide. The clinical signs of observable infiltration and vasculitis in the retina were scored on a 5-point scale, as previously described (20). The maximum score from either eye/mouse was recorded, and the mean score for each group/d was calculated. The mean maximum score was calculated by averaging the maximum scores of each mouse in each group over the entire course of disease.

Flow cytometry analysis

The spleens from post-EAU mice (days 70–80 after immunization for EAU) were collected and placed in RPMI 1640 supplemented with 5% FBS, 10 μg/ml gentamicin (Sigma, St. Louis, MO), 10 mM HEPES (BioWhittaker, Walkersville, MD), 1 mM sodium pyruvate (BioWhittaker), and nonessential amino acids (BioWhittaker). Cells were made into a single-cell suspension, depleted of RBCs using RBC Lysis Buffer (Sigma), washed, and resuspended in serum-free medium (SFM). SFM consisted of RPMI 1640 supplemented with 10 μg/ml gentamicin (Sigma), 10 mM HEPES, 1 mM sodium pyruvate (BioWhittaker), nonessential amino acids 0.2% (BioWhittaker), 1% ITS+1 solution (Sigma), and 0.1% BSA (Sigma). Splenocytes were incubated in SFM at 37°C and 5% CO2 for 90 min in tissue culture plates and washed twice, and adherent cells were scraped off the plastic in ice-cold SFM. The cells were washed with staining buffer, PBS with 1% BSA, blocked with mouse IgG in staining buffer, and stained with the conjugated Abs. Abs used in this study were CD11b-FITC (clone M1/70; BioLegend, San Diego, CA), Ly-6C (clone HK1.4; BioLegend), Ly-6G (clone 1A8: BioLegend), CD73 (clone 496406: R&D Systems, Minneapolis, MN), CD39 (clone 495826: R&D Systems), and F4/80 (clone BM8, eBioscience, San Diego, CA). Stained cells were analyzed in the Boston University Flow Cytometry Core Facility on a BD LSR II (BD Biosciences), and data were analyzed using FlowJo software (TreeStar, Ashland, OR).

Cell sorting and AT

Adherent spleen cells were collected as described above and pooled from post-EAU mice (days 70–80 after immunization for EAU). Staining was performed as described above. Stained cells were sorted in the Boston University Flow Cytometry Core Facility on a MoFlo Cell Sorter (Beckman Coulter, Brea, CA) or a FACSAria III (BD Biosciences). Cells were sorted into tubes containing 10% FBS; immediately after sorting an aliquot of cells was analyzed on the FACSAria III and found to be ≥96% pure (data not shown). Sorted cells were washed with SFM and plated at 5 × 104 cells/well. CD3-enriched T cells were obtained from post-EAU spleens using a CD3 enrichment column (R&D Systems), added to the sorted APCs at 8 × 105 cells/well with 50 μg IRBP peptide, and cultured at 37°C in 5% CO2 for 48 h. Subsequently, T cells and APCs were collected and washed in PBS. Mice were injected in the tail vein with 1 × 106 activated post-EAU cells in PBS. Following the AT, mice were immunized for EAU as described above. Each group of AT experiments was repeated at least two times, and the EAU scores shown were pooled from all repeated experiments.

Cytokine analysis

Cytokine production was measured in the supernatant of cells cultured for 48 h. After incubation, the supernatants were assayed for IFN-γ and TGF-β. The concentration of IFN-γ was measured using a sandwich ELISA (IFN-γ detection and biotinylated IFN-γ Abs from BD Pharmingen). The concentration of TGF-β was measured using the standard Mv1Lu bioassay (21) or TGF-β ELISA (R&D Systems). The concentration of IL-17 in the culture media was measured with an IL-17 ELISA kit (R&D Systems).

α-MSH plasmid treatment

Treatment of mice immunized for EAU with α-MSH expression plasmid was performed as previously described (4). Briefly, the expression vector used was pCMV-Script with the portion of the POMC gene that encodes ACTH residues 1–17, which results in production of a properly structured α−MSH peptide (22). Mice received 6-μl subconjunctival injections of 1 mg/ml in PBS on days 17 and 19 after EAU immunization. Subsequently, mice were evaluated for EAU, as described above.

CGS21680 treatment

CGS21680 (CGS) (Tocris, Bristol, U.K.) was reconstituted in DMSO at 27.7 mg/ml and then diluted in PBS to 0.05 mg/ml. Mice were immunized for EAU, as described above, and followed until the maximum EAU score was obtained (day 23 after immunization). Subsequently, mice were given an i.p. injection of CGS, 0.5 mg/kg once a day for three consecutive days, and evaluated for EAU.

In vitro α-MSH treatment and RT-PCR analysis

Spleen APCs were collected, as described above, from naive mice. APCs were plated at 6 × 106 cells/well in a 24-well plate in SFM, with or without 1 ng/ml α-MSH (19). Cells were incubated at 37°C in 5% CO2 for 48 h and then collected, sorted, and processed for RT-PCR. The RNA was extracted from the sorted APC subsets with a RNeasy kit (QIAGEN, Germantown, MD). Reverse transcription of extracted RNA into cDNA was done with the QuantiTect Reverse Transcription kit (QIAGEN), and PCR reactions were run with the QuantiTect Probe PCR kit (QIAGEN). GAPDH, ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1, CD39), and 5-nucleotidase (NT5E, CD73) primers and probes were purchased from Integrated DNA Technologies (Skokie, IL). PCR reactions were run in the Boston University School of Medicine RT-PCR Core Facility on an Applied Biosystems 7300 (Applied Biosystems, Foster City, CA). Data were analyzed by the 2−ΔΔCT method (23).

Statistical analysis

The experimental results for EAU scoring used the nonparametric Mann–Whitney U test to assess statistical differences between groups of mice. In addition, changes in the tempo of disease between the groups of treated EAU mice were analyzed by two-way ANOVA. Comparisons of flow cytometry results and cytokine concentrations were analyzed statistically using one-way ANOVA and a Bonferroni posttest. Statistical significance was determined when p ≤ 0.05.

Results

There is an MC5r-dependent increase in post-EAU CD11b+F4/80+Ly-6ClowLy-6G+ spleen cells

It was shown previously that MC5r is required for the emergence of post-EAU protective regulatory immunity in the spleen and that the APCs must express MC5r to have post-EAU regulatory immunity (10). The post-EAU APCs from WT mice were compared with post-EAU APCs from MC5r(−/−) mice for their expression of CD11b, F4/80, Gr-1 (Ly-6G), and Ly-6C. Flow cytometry analysis of stained cells showed a significant expansion of CD11b+F4/80+ post-EAU APCs compared with APCs found in the spleen of unimmunized mice (Fig. 1A). In contrast, post-EAU MC5r(−/−) APCs showed no significant change in CD11b or F4/80 expression compared with unimmunized MC5r(−/−) mice. Also, post-EAU WT spleen cells showed a significant increase in CD11b+F4/80+ cells compared with post-EAU MC5r(−/−) spleen cells (Fig. 1B). Post-EAU WT and MC5r(−/−) expression of Ly-6G and Ly-6C was observed for cells gated on CD11b+F4/80+ APCs (Fig. 1C). Cells from WT post-EAU spleen, but not from post-EAU MC5r(−/−) mice, had a significant increase in the proportion of cells expressing CD11b+F4/80+Ly-6ClowLy-6G+ compared with spleen cells from unimmunized mice (Fig. 1D). In contrast, there was only a moderate increase in CD11b+F4/80+Ly-6ChighLy-6G− cells in both post-EAU WT and MC5r(−/−) CD11b+F4/80+ spleen cells. Overall, there was a significant increase in both populations in post-EAU spleens of WT mice compared with post-EAU MC5r(−/−) mice (Fig. 1D). Hereafter, the CD11b+F4/80+Ly-6ClowLy-6G+ population of APCs is labeled Ly-6ClowLy-6G+, and the CD11b+F4/80+Ly-6ChighLy-6G− APCs are labeled Ly-6ChighLy-6G−. These results show that, in the post-EAU spleen, there was an MC5r expression–dependent expansion of Ly-6ClowLy-6G+ cells.

FIGURE 1.
FIGURE 1.

MC5r expression is required for expansion of CD11b+F4/80+Ly-6ClowLy-6G+ APCs. WT and MC5r(−/−) mice were immunized for EAU, and APCs were collected from spleens when the mice recovered from EAU (day 70–80). (A) The spleen post-EAU APCs were stained for CD11b and F4/80 and analyzed by flow cytometry. (B) Bar graph shows the quantified CD11b+F4/80+ APCs from all WT and MC5r(−/−) mice (mean percentage ± SEM). *p = 0.007. (C) Expression of Ly-6G and Ly-6C on gated CD11b+F4/80+ APCs from post-EAU spleens was assayed by flow cytometry. (D) Bar graphs show quantified Ly-6ClowLy-6G+ and Ly-6Chigh Ly-6G– APC populations from all mice (mean percentage ± SEM). The bar graphs compare post-EAU WT and unimmunized WT mice (left panel, *p = 0.0037), post-EAU MC5r(−/−) and unimmunized MC5r(−/−) mice (middle panel), and post-EAU WT and post-EAU MC5r(−/−) mice (right panel, *p ≤ 0.05). Data in (A–D) are representative of five mice assayed individually. n.s., Not significant.

Post-EAU Ly-6ClowLy-6G+ APCs mediate regulatory immunity

The regulatory activity of the post-EAU Ly-6ClowLy-6G+ and Ly-6ChighLy-6G− cells was determined by pulsing sorted Ly-6ClowLy-6G+ and Ly-6ChighLy-6G− cells with IRBP and using these cells as APCs to activate post-EAU CD3+ T cells in culture for 48 h. The T cells and APCs were adoptively transferred into WT mice immunized for EAU. Mice that received the post-EAU T cells activated with Ly-6ChighLy-6G− APCs showed no significant change in the tempo and severity of EAU compared with mice that were not injected with cells (Fig. 2A). In contrast, mice that received post-EAU T cells activated by the post-EAU Ly-6ClowLy-6G+ APCs showed a significant change in the tempo of EAU, with an early resolution (Fig. 2A). This demonstrates that the post-EAU regulatory immunity was mediated by Ly-6ClowLy-6G+ APCs.

FIGURE 2.
FIGURE 2.

Ly-6ClowLy-6G+ post-EAU APCs require MC5r to activate post-EAU Tregs. APCs and T cells were collected from the spleens of EAU (day 70–80) WT and MC5r(−/−) mice. CD11b+F4/80+ APCs were sorted based on Ly-6C and Ly-6G expression, and the sorted APCs were used to activate T cells. (A–C) EAU scores (mean ± SEM) for each group of mice that received AT of both APCs and T cells. Mice that received no cells are shown as a control in each graph (n = 11). *p ≤ 0.05. (A) Cells transferred were T cells activated with Ly-6ChighLy-6G− APCs (n = 7 recipient mice) or Ly-6ClowLy-6G+ APCs (n = 6 recipient mice), p ≤ 0.0001. (B) Mice received T cells activated by MC5r(−/−) Ly-6ChighLy-6G− APCs (n = 9 recipient mice) or MC5r(−/−) Ly-6ClowLy-6G+ APCs (n = 13 recipient mice). (C) Transferred cells were MC5r(−/−) T cells activated by Ly-6ChighLy-6G− APCs (n = 4 recipient mice) or Ly-6ClowLy-6G+ APCs (n = 10 recipient mice). *p = 0.02. (D) EAU scores (mean ± SEM) of mice treated with α-MSH expression plasmid (n = 10, α-MSH Tx) on days 17 and 19 (arrows) or untreated EAU mice (n = 11, No Tx). p = 0.002. (E) IFN-γ, IL-17, and TGF-β were measured from spleen APCs and T cells from treated or untreated mice on day 42. Data are mean ± SEM (ng/ml) for each combination of APCs and T cells for five individual mice. *p ≤ 0.05. (F) Expression of Ly-6C and Ly-6G on CD11b+F4/80+ APCs from spleens of treated (α-MSH Tx) or untreated (No Tx) EAU mice on day 42. Scatter plot is representative of seven mice. n.s., Not significant.

As with the post-EAU WT spleen cells, cells from post-EAU MC5r(−/−) mice spleens were sorted and used to activate post-EAU WT T cells to determine whether these cells can suppress EAU. Neither the post-EAU Ly-6ChighLy-6G− APCs nor the post-EAU Ly-6ClowLy-6G+ APCs from MC5r(−/−) mice activated post-EAU T cells to suppress EAU (Fig. 2B). Therefore, although there are potential Ly-6ClowLy-6G+ APCs present in the post-EAU spleen of MC5r(−/−) mice, they do not have the ability to activate regulatory immunity to suppress EAU. This corresponds with previously published observations on post-EAU MC5r(−/−) spleen cells (10). However, it was reported that APCs from post-EAU WT spleens stimulate regulatory activity in T cells from post-EAU spleens of MC5r(−/−) mice. To determine whether the post-EAU Ly-6ChighLy-6G− or Ly-6ClowLy-6G+ APCs of WT mice stimulate regulatory activity in post-EAU MC5r(−/−) spleen T cells, MC5r(−/−) post-EAU spleen T cells were Ag stimulated with sorted Ly-6ChighLy-6G− or Ly-6ClowLy-6G+ APCs from post-EAU WT mice. The post-EAU Ly-6ClowLy-6G+ APCs, but not the Ly-6ChighLy-6G− APCs, with MC5r(−/−) post-EAU T cells significantly changed the tempo and severity of EAU in the recipient mice (Fig. 2C). These results demonstrate that the spleen Ly-6ClowLy-6G+ APCs mediate the post-EAU regulatory immunity.

α-MSH treatment of EAU significantly increases regulatory APCs in the spleen

Because the results show a dependency on MC5r expression for the emergence of post-EAU regulatory APCs in the spleen of mice (10), this suggests that if MC5r is stimulated with its ligand, α-MSH, it should mediate expansion of the regulatory APCs in the spleens of EAU mice. It is known that local application of α-MSH expression plasmid at the onset of EAU is effective in promoting an early resolution of EAU and preserving retinal structure (4). EAU mice treated with the α-MSH plasmid showed suppressed EAU, as seen previously (Fig. 2D). Analysis of culture supernatants of spleen cells stimulated for 48 h with IRBP from α-MSH plasmid–treated and untreated mice showed no change in the low levels of IL-17, with a decrease in IFN-γ production; however, there was a significant increase in TGF-β production (Fig. 2E). This shows that, in the spleens of α-MSH–treated EAU mice, the Ag-specific response is tilted toward Treg activation. To determine whether this is because α-MSH treatment induced regulatory APCs in the spleen, APCs from treated EAU mice were used to activate spleen T cells from untreated EAU mice. Significantly higher levels of TGF-β with significantly suppressed production of IFN-γ and IL-17 were seen when T cells were stimulated by APCs from the α-MSH–treated EAU mice (Fig. 2E). To determine whether α-MSH treatment promoted expansion of post-EAU Ly-6ClowLy-6G+ APCs in the spleen, the spleen cells were collected on day 42 from treated and untreated EAU mice and stained for CD11b, F4/80, Ly-6G, and Ly-6C. There was an overall expansion of CD11b+F4/80+ cells in the spleens of α-MSH–treated EAU mice (data not shown), with a 2-fold expansion of both Ly-6ChighLy-6G− and Ly-6ClowLy-6G+ cells (Fig. 2F). Therefore, α-MSH plasmid treatment promotes the expansion of Ly-6ClowLy-6G+ cells and regulatory activity in post-EAU mice.

Post-EAU protective regulatory immunity also requires A2Ar expression

Because the adenosinergic pathway has the potential to govern the intensity of an immune response (24, 25), and there is some hint in the literature of a potential link between the melanocortin and adenosinergic pathways (26), a possible role for A2Ar in post-EAU protective regulatory immunity and a link with MC5r were investigated. Mice with A2Ar knocked out were immunized to induce EAU and clinically scored for uveoretinitis. The course and severity of EAU in WT and A2Ar(−/−) mice were nearly the same and not significantly different (Fig. 3A). The post-EAU spleen cells were collected and restimulated in vitro with IRBP to assay their regulatory potential, as done previously with MC5r(−/−) post-EAU spleen cells (2, 10). The post-EAU spleen cells from A2Ar(−/−) mice were unable to suppress EAU, in contrast to the AT of post-EAU spleen cells from WT mice (Fig. 3B–D). Therefore, like MC5r(−/−) mice, A2Ar(−/−) mice do not develop post-EAU regulatory immunity in the spleen.

FIGURE 3.
FIGURE 3.

A2Ar(−/−)mice do not develop protective post-EAU regulatory immunity. WT and A2Ar(−/−) mice were immunized for EAU, and the fundus was examined until resolution. (A) EAU scores for WT (n = 10) and A2Ar(−/−) (n = 15) mice (left panel) and maximum EAU score for each mouse over the entire course of disease (right panel). (B–D) Spleen cells from post-EAU mice (day 70–80) were collected and stimulated with IRBP for 48 h and then transferred to mice immunized for EAU. Each graph shows the EAU score (mean ± SEM) for each group over time. Cells transferred to recipient mice were post-EAU spleen cells from WT mice (n = 14), p = 0.0001 (B) or post-EAU spleen cells from A2Ar(−/−) mice (n = 8) (C). (D) Maximum EAU score for each mouse over the course of disease. *p ≤ 0.05.

Induction of post-EAU regulatory immunity requires A2Ar expression on T cells

Because A2Ar expression was reported on both APCs and T cells (27), post-EAU A2Ar(−/−) APCs were used to activate Tregs from post-EAU WT spleen with IRBP. Both APCs and T cells were transferred into mice immunized for EAU. Mice that received post-EAU Tregs activated by post-EAU A2Ar(−/−) APCs showed significantly suppressed clinical scoring of EAU, as well as a significantly reduced severity of EAU (Fig. 4A). In contrast, mice that received T cells isolated from spleen of post-EAU A2Ar(−/−) mice restimulated by APCs from post-EAU WT mice showed no significant change in the course of EAU or severity of disease (Fig. 4B). This suggests that the lack of post-EAU regulatory immunity in spleens of A2Ar(−/−) mice is because the T cells are required to express A2Ar. A2Ar(−/−) post-EAU T cells were stained for Foxp3 and the surface markers CD4 and CD25. Post-EAU spleen T cells from A2Ar(−/−) mice showed a significantly lower proportion of Foxp3+CD25+CD4+ T cells compared with post-EAU T cells from spleens of WT mice (Fig. 4C). Therefore, the expression of A2Ar is necessary for there to be Tregs associated with the regulatory immunity in spleens of post-EAU mice.

FIGURE 4.
FIGURE 4.

A2Ar on the post-EAU Treg is necessary for activation by post-EAU regulatory APCs. (A and B) EAU scores (mean ± SEM) over the course of disease (left panels) and maximum scores (right panels) for mice immunized for EAU that received AT of both post-EAU (day 70–80) APCs and T cells. The cells transferred to recipient mice were WT post-EAU T cells activated by post-EAU A2Ar(−/−) APCs (n = 10) (A) or post-EAU A2Ar(−/−) T cells activated by WT post-EAU APCs (n = 7) (B). (C) Foxp3 and CD25 expression of IRBP-restimulated post-EAU CD4+ T cells from WT or A2Ar(−/−) mice. Scatter plot is representative of three mice. Cells were gated on CD4+ T cells and stained for Foxp3 and CD25. The scatter plot labeled Foxp3 control is the CD25 isotype control of cells stained for CD4 and Foxp3. The scatter plot labeled CD25 control is the Foxp3 isotype control of cells stained for CD4 and CD25. Bar graph (right panel) is the percentage (mean ± SEM) of CD4+CD25+Foxp3+ T cells from three post-EAU mice. (D) EAU scores of mice that received CGS21680 (n = 9) on days 23–25 (arrows) or PBS injections (n = 9). p = 0.0017. Arrowhead indicates day 38, when the spleen cells were collected, IRBP restimulated, and assayed for IFN-γ, IL-17, and TGF-β production. (E) Bar graph shows mean (± SEM) concentration of IFN-γ, IL-17, and TGF-β in the cultures of spleen cells from EAU mice (day 70–80) of CGS-treated or untreated mice (n = 4). p = 0.007. (F) EAU scores over time (left panel) and maximum EAU scores (right panel) of A2Ar(−/−) mice treated with α-MSH expression plasmid on days 17 and 19 (arrows) (n = 7). (G) Concentration of IFN-γ, IL-17, and TGF-β production (mean ± SEM) by IRBP-restimulated spleen cells from treated (Tx, n = 6) or untreated A2Ar(−/−) mice (n = 14) on day 71. Spleen cells incubated without IRBP was used as a no-Ag control. n.s., no significant differences were seen between groups.

If the stimulation of A2Ar is sufficient to induce post-EAU Tregs, then stimulation of A2Ar should establish regulatory immunity in mice with EAU. Mice with EAU were injected systemically with a selective A2Ar agonist, CGS, on days 2–25, when they first reach the height of retinal inflammation. On day 35, treated mice began to show resolution of ocular inflammation and, by day 60, all mice had a score ≤ 1 (Fig. 4D). In contrast, PBS-injected mice maintained EAU through day 60 (Fig. 4D). To determine whether there was an early emergence of regulatory immunity in the spleen, spleen cells were collected on day 38 and assayed for IRBP-stimulated expression of IFN-γ, IL-17, and TGF-β. Spleen cells from CGS-treated mice had significantly lower IFN−γ production and no change in TGF-β production compared with T cells from untreated mice (Fig. 4E). The levels of IL-17 production were very low in both untreated and CGS-treated mice. These findings showed that CGS treatment suppressed Th1 cell activity while promoting or maintaining Treg activity. In addition, the results showed that stimulating A2Ar during EAU promotes early resolution of uveitis.

To determine whether α-MSH plasmid treatment of EAU can induce regulatory immunity and early resolution of EAU in A2Ar(−/−) mice, A2Ar(−/−) EAU mice were treated with α−MSH expression plasmid on days 17 and 19 after immunization to induce EAU. These treated mice showed a normal course of EAU, with no early EAU resolution and no change in disease severity (Fig. 4F). At the resolution of EAU, spleen cells were restimulated, and cytokine production was measured. Untreated A2Ar(−/−) mice and α−MSH expression plasmid–treated A2Ar(−/−) mice showed no significant differences in IL-17, IFN−γ, and TGF−β production (Fig. 4G). Therefore, the melanocortin-driven induction of regulatory immunity also required the expression of A2Ar on T cells.

Emergence of post-EAU regulatory immunity is linked through MC5r and A2Ar

If adenosine is a mediator in the induction of post-EAU Tregs, then post-EAU APCs should express the ectonucleotidases CD39 and CD73 that convert ATP, ADP, and AMP into adenosine (28). CD39 and CD73 mRNA in the sorted Ly-6ClowLy-6G+ and Ly-6ChighLy-6G− subsets from unimmunized and post-EAU mice was measured by real-time PCR (Fig. 5A). Both CD39 and CD73 mRNA expression was significantly greater in the Ly-6ClowLy-6G+ subset compared with the Ly-6ChighLy-6G− subset (Fig. 5A). Moreover, the Ly-6ClowLy-6G+ subset from post-EAU mice had significantly higher levels of CD39 and CD73 expression compared with cells from unimmunized mice (Fig. 5A). In contrast, CD39 and CD73 mRNA expression was suppressed in the Ly-6ChighLy-6G− subset from post-EAU mice (Fig. 5A). This showed that the regulatory Ly-6ClowLy-6G+ APC subset in post-EAU mice spleens highly expresses CD39 and CD73 mRNA.

FIGURE 5.
FIGURE 5.

Expression of CD39 and CD73 by Ly-6ChighLy-6G− and Ly-6ClowLy-6G+ APCs. (A) CD39 and CD73 mRNA in Ly-6ClowLy-6G+ APCs and Ly-6ChighLy-6G− APCs sorted from the spleen of unimmunized or post-EAU mice were assayed by RT-PCR. The 2−ΔΔCT for CD39 and CD73 mRNA was normalized to GAPDH, and their relative expression is presented. Data are mean ± SEM; two mice were pooled per experiment, and the experiment was done twice. (B) CD39 and CD73 mRNA was measured in Ly-6ClowLy-6G+ APCs and Ly-6ChighLy-6G− APCs that were sorted from α-MSH–treated and untreated APCs from unimmunized mice spleens. Data are relative expression (mean ± SEM) of CD39 and CD73 mRNA normalized to GAPDH mRNA expression for five mice pooled per experiment; the experiment was done twice. *p ≤ 0.05.

To determine whether the increased expression of CD39 and CD73 mRNA was dependent on MC5r, APCs were collected from spleens of unimmunized mice, treated with α-MSH, sorted into Ly-6ClowLy-6G+ and Ly-6ChighLy-6G− subsets, and analyzed for CD39 and CD73 expression by RT-PCR. Ly-6ClowLy-6G+ APCs showed no change in CD39 and CD73 expression with α-MSH treatment (Fig. 5B). Ly-6ChighLy-6G− APCs showed suppression of CD39 and increased CD73 expression with α-MSH treatment (Fig. 5B). This demonstrated that the MC5r-dependent induction of post-EAU regulatory immunity was not associated with the upregulation of CD39 and CD73 in regulatory APCs. Therefore, the induction of post-EAU protective immunity in the spleen is an MC5r-driven expansion of ocular autoantigen–presenting Ly-6ClowLy-6G+ APCs in the spleen that activate Tregs through the adenosinergic pathway (Fig. 6).

FIGURE 6.
FIGURE 6.

Induction of post-EAU protective regulatory immunity. At resolution of EAU, through MC5r, presumably stimulated by the neuropeptide α-MSH, regulatory Ly-6ClowLy-6G+ APCs expressing CD39 (ectonucleoside triphosphate diphosphohydrolase) and CD73 (ecto-5′-nucleotidase) localize or accumulate in the spleen. Ly-6ClowLy-6G+ APCs present ocular autoantigens and, through their expression of CD39 and CD73, generate adenosine from ATP to induce, in an A2Ar-dependent manner, autoantigen-specific Tregs, which mediate a post-EAU protective regulatory immune response to ocular autoantigens.

Discussion

Our results demonstrated that the emergence of post-EAU protective regulatory immunity in the spleen is dependent on a sequential activity of the melanocortin and adenosinergic pathways of regulatory APC and T cells. At resolution of EAU, there was a melanocortin-driven expansion of CD11b+F4/80+Ly-6ClowLy-6G+ APCs that mediate the induction of regulatory T cells in the spleen. These APCs and the induction of regulatory immunity were not seen in spleens of post-EAU MC5r(−/−) mice, but the APCs were seen in spleens of post-EAU A2Ar(−/−) mice. This difference was caused by the different roles that the melanocortin and adenosinergic pathways have in the induction of post-EAU regulatory immunity. The melanocortin pathway through MC5r on APCs was associated with the induction and expansion of regulatory APCs, and the adenosinergic pathway through A2Ar on T cells was associated with APC induction and activation of Tregs.

How the expression of MC5r is associated with the expansion or localization of suppressor cells in the spleen is unknown. This is a new finding. There are at least two other melanocortin receptors on macrophages and dendritic cells: MC1r and MC3r. α-MSH suppression of macrophage and dendritic cell production and response to proinflammatory cytokines occurs through MC1r and MC3r (29–31). Through these receptors, α-MSH blocks TLR-signaling pathways and inhibits activation of NF-κB and p38 MAPK (31–33). The macrophages treated with α-MSH produce TGF-β and IL-10, which also is seen with α-MSH–treated Langerhans cells that prevent contact hypersensitivity (17). Although these studies suggest that it is a cytokine-mediated suppression of immunity caused by α-MSH treatment, our results demonstrated that mediation of melanocortin-induced regulatory activity in the post-EAU mouse is through APCs with CD39 and CD73 expression that have localized to the spleen and subsequently activate Tregs through the immunoregulating adenosinergic pathway.

Ly-6ClowLy-6G+ suppressor cells have been found in the retina of mice recovering from EAU and are considered important in mediating local resolution of inflammation (34). Recently, it was found that microglia in healthy retina express characteristics of myeloid suppressor cells that are mediated by the presence of α-MSH (19). Because we showed that this regulatory immunity requires an intact eye, as well as recovery from uveitis, it is possible that the source of the splenic post-EAU regulatory APC is the eye itself. It is known that foreign Ag placed into the anterior chamber of the healthy eye ends up being presented in the spleen by regulatory/tolerogenic F4/80+ APCs that activate CD8+ Tregs as part of the anterior chamber–associated immune deviation phenomenon (35). This suggests that a healthy or inflamed ocular microenvironment is uniquely capable of driving APCs toward suppressive functions where induction of immune tolerance is found in the spleen. The post-EAU–induced tolerance is dependent on a functional MC5r melanocortin pathway and a significantly different mechanism of tolerance induction than is the anterior chamber–associated immune deviation phenomenon.

Through MC5r, α-MSH induces regulatory activity in effector T cells (21, 36, 37), and, from our results, regulatory APCs. Although most of the intracellular signaling pathways associated with the melanocortin receptors are unclear, α-MSH stimulation of MC5r activates the JAK2 and STAT1 pathway (38). The activation of STAT1 is necessary in the activity of tumor-derived myeloid suppressor cells, suggesting that α-MSH uses STAT1 to induce regulatory activity in APCs (13, 39, 40). However, there are other receptors on APCs through which α-MSH suppresses inflammatory cytokine production and certain innate immune responses, and it is not possible to rule out their role in α-MSH–mediated induction of regulatory APCs. Recently, it was found that α-MSH, together with another constitutive neuropeptide in the eye, Neuropeptide Y, promotes myeloid suppressor cell characteristics in macrophages (19). It is not known which melanocortin receptor is involved in this interaction; however, the findings suggest that it most likely is MC5r. Although MC5r is required for the expansion or localization of Ly-6ClowLy-6G+ APCs seen in the post-EAU spleen, the effects of α-MSH cannot account for the expression of the ectonucleotidases, nor is it understood how MC5r regulates cell migration to the spleen. It is known that α-MSH regulates the migratory activity of neutrophils, as well as melanocyte adhesion and migration, but this occurs through MC1r (41, 42). Therefore, although the presence of regulatory Ly-6ClowLy-6G+ APCs in the spleen is dependent on MC5r expression, the induction of the regulatory activity must involve more than the melanocrotin pathway.

Adenosine stimulation of A2Ar on T cells was demonstrated to be effective and possibly required for Treg induction (24, 43, 44). A2Ar stimulation protects against inflammatory bowel disease (45), neuroinflammatory injury (46), reperfusion injury in the lung and liver (47, 48), and allograft rejection (49). Stimulating another adenosine receptor, A3Ar, suppresses EAU by suppressing the activation of effector T cells and upregulating IL-10 production in cultured splenocytes from treated mice (50). Our results demonstrated that post-EAU protective immunity is mediated by a peripheral mechanism of Treg activation through A2Ar.

Our results demonstrated that there is a pathway for inducing regulatory immunity that is through MC5r-dependent expansion in the spleen of ectonucleotidase-expressing CD11b+F4/80+Ly-6ClowLy-6G+ regulatory APCs, which, through A2Ar on T cells, activates Foxp3+CD4+ Tregs (Fig. 6). The result of this pathway is the activation of Ag-specific inducible CD4+ Tregs that, when specific to ocular autoantigens, protect mice from the recrudescence of uveitis. Therefore, the highly conserved melanocortin- and adenosinergic-signaling pathways of immune regulation and homeostasis function together to induce a protective regulatory immune response that can be exploited to (re)establish tolerance to autoantigens.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank David Yee for technical assistance and the Boston University Flow Cytometry facility for technical assistance in the sorting experiments.

Footnotes

  • This work was supported in part by the Massachusetts Lions Eye Research Foundation and National Institutes of Health/National Eye Institute Public Health Service Grant EY010752.

  • Abbreviations used in this article:

    A2Ar
    adenosine 2A receptor
    AT
    adoptive transfer
    CGS
    CGS21680
    EAU
    experimental autoimmune uveoretinitis
    IRBP
    interphotoreceptor retinoid binding protein
    MC5r
    melanocortin 5 receptor
    MDSC
    myeloid-derived suppressor cell
    α-MSH
    α-melanocyte stimulating hormone
    SFM
    serum-free medium
    Treg
    regulatory T cell
    WT
    wild-type.

  • Received January 17, 2013.
  • Accepted August 15, 2013.

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