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An A2B Adenosine Receptor Agonist Promotes Th17 Autoimmune Responses in Experimental Autoimmune Uveitis (EAU) via Dendritic Cell Activation.

Chen M, Liang D, Zuo A, Shao H, Kaplan HJ, Sun D - PLoS ONE (2015)

Bottom Line: We have recently reported that, although adenosine receptor (AR) agonists have a suppressive effect on Th1 autoreactive T cells, their effect on Th17 autoreactive T cells and γδ T cells is stimulatory and this effect is mainly mediated via A2A adenosine receptors (A2ARs).In the present study, we showed that exposure of DCs to A2BR agonist facilitated γδ T cell activation, leading to augmented Th17 responses and progressive EAU development.Our results further support our previous finding that AR agonists have distinct effects on Th1 and Th17 autoimmune responses.

View Article: PubMed Central - PubMed

Affiliation: Doheny Eye Institute and Department of Ophthalmology, University of California, Los Angeles, CA90033, United States of America.

ABSTRACT
We have recently reported that, although adenosine receptor (AR) agonists have a suppressive effect on Th1 autoreactive T cells, their effect on Th17 autoreactive T cells and γδ T cells is stimulatory and this effect is mainly mediated via A2A adenosine receptors (A2ARs). In this study, we further demonstrate that treatment of C57BL/6 (B6) mice with a selective A2B adenosine receptor (A2BR) agonist greatly enhanced the development of experimental autoimmune uveitis (EAU), whereas treatment with an A2BR antagonist significantly ameliorated severity of EAU. The A2BR agonist-treated mice showed augmented Th17, but not Th1, responses. Mechanistic studies showed that the A2BR agonist-induced enhancement of the Th17 response was significantly lower when TCR-δ-/- mice received the same treatment and that transfer of γδ T cells into TCR-δ-/- mice partially restored this effect. We also showed that dendritic cells (DCs) from A2BR agonist-treated mice showed a significantly increased ability to activate γδ T cells and Th17 autoreactive T cells. Thus, our previous studies have shown that, in EAU, activated γδ T cells possess greatly increased ability to enhance Th17 autoimmune responses. In the present study, we showed that exposure of DCs to A2BR agonist facilitated γδ T cell activation, leading to augmented Th17 responses and progressive EAU development. Our results further support our previous finding that AR agonists have distinct effects on Th1 and Th17 autoimmune responses.

No MeSH data available.


Related in: MedlinePlus

Administration of A2BR agonist to EAU-prone B6 mice enhanced EAU induction and activation of Th17 autoreactive T cells.(A&B). Two groups of B6 mice (n = 6) were immunized with IRBP1-20/CFA and injected i.p. with an A2BR agonist (A2BR-A, BAY 60–6583, 1 mg/kg) or vehicle (DMSO) on days 1, 4, 7, and 10 post-immunization. EAU was clinically scored by fundoscopy (A). On day 25 post-immunization, when the mice were sacrificed and the eye were taken and subjected to pathological examination (B). The H&E staining of the eye sections from naive, immunized but not BAY 60-6583-treated, and BAY60-6583-treated mice was shown. (C). Serum cytokine (IFN-γ and IL-17) levels measured by ELISA on day 13 post immunization. (D). Cytoplasmic staining assessing the percentage of IL-17+ cells among the in vitro antigen-stimulated IRBP-specific T cells. After 5 days’ of in vitro stimulation, the activated T cells were treated with PMA, ionomycin, and brefeldin, then were intracellularly stained with FITC-conjugated anti-TCR antibodies and PE-conjugated anti-IL-17 (upper panels)or anti-IFN-γ (lower panels) antibodies, followed by FACS analysis. (E). Determination of serum cytokine. 48-hour culture supernatants were assessed for IL-17 and IFN-γ by ELISA. Data are from one single experiment, which are representative of three independent experiments. **p< 0.01.
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pone.0132348.g001: Administration of A2BR agonist to EAU-prone B6 mice enhanced EAU induction and activation of Th17 autoreactive T cells.(A&B). Two groups of B6 mice (n = 6) were immunized with IRBP1-20/CFA and injected i.p. with an A2BR agonist (A2BR-A, BAY 60–6583, 1 mg/kg) or vehicle (DMSO) on days 1, 4, 7, and 10 post-immunization. EAU was clinically scored by fundoscopy (A). On day 25 post-immunization, when the mice were sacrificed and the eye were taken and subjected to pathological examination (B). The H&E staining of the eye sections from naive, immunized but not BAY 60-6583-treated, and BAY60-6583-treated mice was shown. (C). Serum cytokine (IFN-γ and IL-17) levels measured by ELISA on day 13 post immunization. (D). Cytoplasmic staining assessing the percentage of IL-17+ cells among the in vitro antigen-stimulated IRBP-specific T cells. After 5 days’ of in vitro stimulation, the activated T cells were treated with PMA, ionomycin, and brefeldin, then were intracellularly stained with FITC-conjugated anti-TCR antibodies and PE-conjugated anti-IL-17 (upper panels)or anti-IFN-γ (lower panels) antibodies, followed by FACS analysis. (E). Determination of serum cytokine. 48-hour culture supernatants were assessed for IL-17 and IFN-γ by ELISA. Data are from one single experiment, which are representative of three independent experiments. **p< 0.01.

Mentions: To determine the in vivo effect of an A2BR agonist on EAU development, randomly grouped B6 mice were immunized with a pathogenic dose of IRBP1-20, then received i.p. injections of the A2BR agonist BAY 60–6583 (1.0 mg/kg) or DMSO (vehicle) on days 1, 4, 7, and 10 post-immunization. EAU was monitored by fundoscopic examination for 15 days starting 10 days post-immunization and the mice were sacrificed at day 25 post-immunization and the eyes subjected to pathological examination. As shown in Fig 1A and 1B, the EAU peptide immunized mice treated with BAY 60–6583 showed significantly increased severity of ocular inflammation than those mice treated with vehicle. Fig 1C shows that serum IL-17 levels at day 13 post-immunization were markedly increased in immunized mice compared to naïve mice, and levels were twice as high in the BAY 60-6583-treated mice than the vehicle-treated mice. In contrast, serum IFN-γ levels were increased by immunization, but BAY 60–6583 had no effect (Fig 1C). To determine the effect of treatment on Th1 and Th17 autoimmune responses, CD3+ T cells from BAY 60-6583- or vehicle-treated immunized mice on day 13 post-immunization were prepared using a magnetic sorter and stimulated in vitro for 5 days with the immunizing peptide and APCs in the presence of IL-12 (Th1 polarizing conditions) or IL-23 (Th17 polarizing conditions), then the number of responding T cells expressing IL-17 or IFN-γ was assessed. As shown in Fig 1D, the percentage of IL-17+ T cells among T cells activated under Th17-polarizing conditions was significantly increased in BAY 60-6583-treated mice compared to control mice, whereas the percentage of IFN-γ+ T cells in the Th1 polarized T cells did not differ significantly between the two groups. Measurement of IL-17 and IFN-γ levels in the supernatants of T cells stimulated in vitro for 48 h with the immunizing peptide showed that T cells from BAY 60-6583-treated mice produced significantly higher levels of IL-17 than vehicle-treated mice, whereas similar IFN-γ production was seen in the two sets of cultures.


An A2B Adenosine Receptor Agonist Promotes Th17 Autoimmune Responses in Experimental Autoimmune Uveitis (EAU) via Dendritic Cell Activation.

Chen M, Liang D, Zuo A, Shao H, Kaplan HJ, Sun D - PLoS ONE (2015)

Administration of A2BR agonist to EAU-prone B6 mice enhanced EAU induction and activation of Th17 autoreactive T cells.(A&B). Two groups of B6 mice (n = 6) were immunized with IRBP1-20/CFA and injected i.p. with an A2BR agonist (A2BR-A, BAY 60–6583, 1 mg/kg) or vehicle (DMSO) on days 1, 4, 7, and 10 post-immunization. EAU was clinically scored by fundoscopy (A). On day 25 post-immunization, when the mice were sacrificed and the eye were taken and subjected to pathological examination (B). The H&E staining of the eye sections from naive, immunized but not BAY 60-6583-treated, and BAY60-6583-treated mice was shown. (C). Serum cytokine (IFN-γ and IL-17) levels measured by ELISA on day 13 post immunization. (D). Cytoplasmic staining assessing the percentage of IL-17+ cells among the in vitro antigen-stimulated IRBP-specific T cells. After 5 days’ of in vitro stimulation, the activated T cells were treated with PMA, ionomycin, and brefeldin, then were intracellularly stained with FITC-conjugated anti-TCR antibodies and PE-conjugated anti-IL-17 (upper panels)or anti-IFN-γ (lower panels) antibodies, followed by FACS analysis. (E). Determination of serum cytokine. 48-hour culture supernatants were assessed for IL-17 and IFN-γ by ELISA. Data are from one single experiment, which are representative of three independent experiments. **p< 0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492970&req=5

pone.0132348.g001: Administration of A2BR agonist to EAU-prone B6 mice enhanced EAU induction and activation of Th17 autoreactive T cells.(A&B). Two groups of B6 mice (n = 6) were immunized with IRBP1-20/CFA and injected i.p. with an A2BR agonist (A2BR-A, BAY 60–6583, 1 mg/kg) or vehicle (DMSO) on days 1, 4, 7, and 10 post-immunization. EAU was clinically scored by fundoscopy (A). On day 25 post-immunization, when the mice were sacrificed and the eye were taken and subjected to pathological examination (B). The H&E staining of the eye sections from naive, immunized but not BAY 60-6583-treated, and BAY60-6583-treated mice was shown. (C). Serum cytokine (IFN-γ and IL-17) levels measured by ELISA on day 13 post immunization. (D). Cytoplasmic staining assessing the percentage of IL-17+ cells among the in vitro antigen-stimulated IRBP-specific T cells. After 5 days’ of in vitro stimulation, the activated T cells were treated with PMA, ionomycin, and brefeldin, then were intracellularly stained with FITC-conjugated anti-TCR antibodies and PE-conjugated anti-IL-17 (upper panels)or anti-IFN-γ (lower panels) antibodies, followed by FACS analysis. (E). Determination of serum cytokine. 48-hour culture supernatants were assessed for IL-17 and IFN-γ by ELISA. Data are from one single experiment, which are representative of three independent experiments. **p< 0.01.
Mentions: To determine the in vivo effect of an A2BR agonist on EAU development, randomly grouped B6 mice were immunized with a pathogenic dose of IRBP1-20, then received i.p. injections of the A2BR agonist BAY 60–6583 (1.0 mg/kg) or DMSO (vehicle) on days 1, 4, 7, and 10 post-immunization. EAU was monitored by fundoscopic examination for 15 days starting 10 days post-immunization and the mice were sacrificed at day 25 post-immunization and the eyes subjected to pathological examination. As shown in Fig 1A and 1B, the EAU peptide immunized mice treated with BAY 60–6583 showed significantly increased severity of ocular inflammation than those mice treated with vehicle. Fig 1C shows that serum IL-17 levels at day 13 post-immunization were markedly increased in immunized mice compared to naïve mice, and levels were twice as high in the BAY 60-6583-treated mice than the vehicle-treated mice. In contrast, serum IFN-γ levels were increased by immunization, but BAY 60–6583 had no effect (Fig 1C). To determine the effect of treatment on Th1 and Th17 autoimmune responses, CD3+ T cells from BAY 60-6583- or vehicle-treated immunized mice on day 13 post-immunization were prepared using a magnetic sorter and stimulated in vitro for 5 days with the immunizing peptide and APCs in the presence of IL-12 (Th1 polarizing conditions) or IL-23 (Th17 polarizing conditions), then the number of responding T cells expressing IL-17 or IFN-γ was assessed. As shown in Fig 1D, the percentage of IL-17+ T cells among T cells activated under Th17-polarizing conditions was significantly increased in BAY 60-6583-treated mice compared to control mice, whereas the percentage of IFN-γ+ T cells in the Th1 polarized T cells did not differ significantly between the two groups. Measurement of IL-17 and IFN-γ levels in the supernatants of T cells stimulated in vitro for 48 h with the immunizing peptide showed that T cells from BAY 60-6583-treated mice produced significantly higher levels of IL-17 than vehicle-treated mice, whereas similar IFN-γ production was seen in the two sets of cultures.

Bottom Line: We have recently reported that, although adenosine receptor (AR) agonists have a suppressive effect on Th1 autoreactive T cells, their effect on Th17 autoreactive T cells and γδ T cells is stimulatory and this effect is mainly mediated via A2A adenosine receptors (A2ARs).In the present study, we showed that exposure of DCs to A2BR agonist facilitated γδ T cell activation, leading to augmented Th17 responses and progressive EAU development.Our results further support our previous finding that AR agonists have distinct effects on Th1 and Th17 autoimmune responses.

View Article: PubMed Central - PubMed

Affiliation: Doheny Eye Institute and Department of Ophthalmology, University of California, Los Angeles, CA90033, United States of America.

ABSTRACT
We have recently reported that, although adenosine receptor (AR) agonists have a suppressive effect on Th1 autoreactive T cells, their effect on Th17 autoreactive T cells and γδ T cells is stimulatory and this effect is mainly mediated via A2A adenosine receptors (A2ARs). In this study, we further demonstrate that treatment of C57BL/6 (B6) mice with a selective A2B adenosine receptor (A2BR) agonist greatly enhanced the development of experimental autoimmune uveitis (EAU), whereas treatment with an A2BR antagonist significantly ameliorated severity of EAU. The A2BR agonist-treated mice showed augmented Th17, but not Th1, responses. Mechanistic studies showed that the A2BR agonist-induced enhancement of the Th17 response was significantly lower when TCR-δ-/- mice received the same treatment and that transfer of γδ T cells into TCR-δ-/- mice partially restored this effect. We also showed that dendritic cells (DCs) from A2BR agonist-treated mice showed a significantly increased ability to activate γδ T cells and Th17 autoreactive T cells. Thus, our previous studies have shown that, in EAU, activated γδ T cells possess greatly increased ability to enhance Th17 autoimmune responses. In the present study, we showed that exposure of DCs to A2BR agonist facilitated γδ T cell activation, leading to augmented Th17 responses and progressive EAU development. Our results further support our previous finding that AR agonists have distinct effects on Th1 and Th17 autoimmune responses.

No MeSH data available.


Related in: MedlinePlus