Adenosine 5'-diphosphate is a key endogenous cell-signaling molecule that can activate P2 purinergic receptor family members. ADP-P2Y signaling is reported to be associated with inflammation, but its function in T cell differentiation and autoimmune diseases pathogenesis is unclear. In this study, we found that the P2Y12 receptor was upregulated in the peripheral immune tissues of experimental autoimmune encephalomyelitis (EAE) mice. Deficiency of P2Y12 led to a reduced peak severity and cumulative disease score in EAE mice, followed by a dramatic reduction of leukocyte infiltration and less extensive demyelination. The percentage of Th17, one of the main pathogenic T cells in EAE, was sharply decreased in P2Y12 knockout mice, accompanied by decreased IL-17A production and a low mRNA level of Th17-related genes. In vitro culture assay further verified that P2Y12 directly regulated Th17 differentiation. More interestingly, clopidogrel and ticagrelor, two P2Y12-specific antagonists, effectively alleviated the disease severity of EAE and inhibited Th17 differentiation both in vivo and in vitro. Further study demonstrated that blocking the P2Y12 receptor also ameliorated the symptoms of 2,4,6-trinitrobenzenesulfonic acid–induced colitis and multiple low-dose streptozocin-induced type 1 diabetes. Our findings not only revealed the critical role of P2Y12 in Th17 differentiation and EAE pathogenesis, but also suggested the promising potential of P2Y12 antagonists in the treatment of autoimmune diseases.

Multiple sclerosis (MS) is a common autoimmune inflammatory disease of the CNS that is characterized by immune-mediated demyelination and neurodegeneration. It is thought to be initiated by an acute autoimmune inflammatory reaction to myelin components, and then progresses into a chronic phase in which oligodendrocytes, myelin, and axons degenerate (1, 2). Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell–mediated demyelinating disease that shares many pathological and histological similarities with MS (3, 4). It is generally accepted that over activation of CD4+ T cell subsets, especially the Th1 and Th17 subpopulations, is the major cause of this disease (5). No treatments modified the MS disease course until the early 1990s, when IFN-β was introduced. Injection and infusion drugs remained the mainstay of MS treatments for almost two decades when, finally, oral therapies, such as fingolimod, teriflunomide, and BG-12, were developed (6). Several oral treatments are under development for relapsing remitting MS and progressive forms of MS. However, these treatments are only partially effective in halting the MS disease process. Although the list of MS therapies continues to progress, treatment of MS remains a huge challenge. Exploration of the pathogenesis and new therapeutic approaches for EAE and MS are key.

G protein–coupled receptors (GPCRs) are the largest receptor superfamily, with more than 1000 members. They broadly participate in mediating biological processes and are involved in many human diseases (7). GPCRs are thought to be the most commonly used drug target. Many GPCRs have been reported to mediate the pathogenesis of MS and EAE (8, 9). Purinergic receptors are comprised of two classes: P1 and P2 receptors. P2 receptors can be subdivided into P2X and P2Y subtypes (10). P2Y GPCRs include the P2Y1,2,4,6,11,12,13,14 subtypes (11). Previous studies have proved that the P2X7 and P2Y6 receptors were expressed in murine CD4+ T cells, and activation of P2Y6 and P2X7 receptors contributes to T cell activation via TCRs (12). The P2Y14 receptor, similar to the P2Y12 receptor, is highly expressed in human monocyte–derived immature dendritic cells (DCs), and its agonist treatment can upregulate the expression of costimulatory molecules CD86, showing that P2Y14 is involved in the maturation of DCs (13).

P2Y12, a Gαi-coupled ADP receptor, is expressed on platelets and microglia in humans and rodents, and acts on platelet aggregation and microglial chemotaxis (1418). Activating the receptor on microglial cells by ADP induces membrane ruffling and chemotaxis (19, 20). The P2Y12 receptor is also essential for ADP-induced thrombus growth and stability (21, 22). The thienopyridine compounds such as clopidogrel and prasugrel are first converted to an active metabolite in the liver, which irreversibly inactivates the P2Y12 receptor. In addition, the P2Y12 receptor is expressed in murine DCs, which are the most potent APCs of the immune system (23, 24), and P2Y12 activation enhanced Ag endocytosis by DCs with subsequent enhancement of specific T cell activation (25). The P2Y12 receptor is required for the proinflammatory actions of the stable abundant mediator LTE4 and is a novel potential therapeutic target for asthma (26), but its functions in MS pathogenesis and T cell differentiation are still unclear.

In this study, we found that the P2Y12 receptor was upregulated in the peripheral lymphoid tissues of EAE mice. Inhibition of P2Y12 with the selective antagonists, clopidogrel and ticagrelor, or genetic deletion of P2Y12 attenuated the clinical symptoms of EAE and suppressed Th17 differentiation both in vivo and in vitro. The promising therapeutic potential of P2Y12 antagonists was also demonstrated in mouse models of 2,4,6-trinitrobenzenesulfonic acid (TNBS)–induced colitis and multiple low-dose streptozocin (STZ)–induced type 1 diabetes (T1D).

C57BL/6 mice were purchased from Shanghai Laboratory Animal Center (Shanghai, China). P2Y12-knockout (KO) mice on a C57BL/6 background were described in a previous report (27). All mice were maintained in pathogen-free conditions with standard laboratory chow and water ad libitum. All experiments were approved and conducted in accordance with the guidelines of the Animal Care Committee of Tongji University.

Myelin oligodendrocyte glycoprotein (MOG)35–55 (MEVGWYRSPFSRVVHLYRNGK) was purchased from GL Biochem (Shanghai, China) with a purity >95%. SYBR Green JumpStart Taq Ready-Mix kit and sodium fluorescein were from Sigma-Aldrich. Percoll was from GE Healthcare. CFA was purchased from Sigma-Aldrich. PE-Cy7 anti-mouse CD4, PE anti-mouse IL-17A, APC anti-mouse IFN-γ, and BD Cytofix/Cytoperm kit were purchased from BD Biosciences. Dynal Mouse CD4 Cell Negative Isolation Kit was purchased from Invitrogen (Carlsbad, CA). TNBS and STZ were purchased from Sigma. Clopidogrel was purchased from Sanofi (Hangzhou) Pharmaceuticals. Ticagrelor was purchased from AstraZeneca.

Female mice at 10–12 wk of age were immunized with 200 μg MOG35–55 in CFA containing 5 mg/ml heat-killed Mycobacterium tuberculosis H37RA. Pertussis toxin (200 ng per mouse) was injected i.p. on days 0 and 2. Mice were assessed daily for clinical signs by researchers blinded to experimental conditions and were assigned scores as follows: 0, no clinical signs; 1, paralyzed tail; 2, paresis; 3, paraplegia; 4, paraplegia with forelimb weakness or paralysis; and 5, moribund or death. Clopidogrel was given via oral administration (5–50 mg/kg body weight) once daily from day 3 postimmunization (PI) until the end of the study. Water was given as vehicle control (200 μl per mouse).

For bone marrow chimeras, 6-wk old female wild type (WT) or P2Y12−/− mice were irradiated with a lethal dose of 8 Gy. The bone marrow cells from 6-wk old WT or P2Y12−/− mice from the same litter were then transferred i.v. into the irradiated mice (1 × 107 cells per mouse) on the same day, and antibiotics were given for the following 2 wk. Six weeks later, the bone marrow chimeras were subjected to EAE induction with MOG immunization.

Female or male mice at 10–12 wk of age were immunized with 150 μl TNBS presensitization solution (mixture solvents: acetone and olive oil 4:1 volume, with 5% TNBS diluted to 1% by this mixture) on the back of the mice on day 0. On day 8, 100 μl 5% TNBS was administered intrarectally 4 cm proximal to the anus, and the mice were sacrificed 4 d later. Clopidogrel was given via oral administration (15 mg/kg body weight) once daily from day 0 until the end of the study. Water was given as vehicle control (200 μl per mouse).

Male mice at 10–12 wk of age were given daily i.p. injections of 50 mg/kg STZ dissolved in 0.1 M sodium citrate (pH 4.5) after 4–6 h fasting for five consecutive days. The blood glucose and body weights were measured. Clopidogrel was given via oral administration (15 mg/kg body weight) once daily from day 0 until the end of the study. Water was given as vehicle control (200 μl per mouse).

For histological staining, mice were anesthetized and perfused with PBS (pH 7.4), followed by 4% (w/v) paraformaldehyde. Tissue samples were then fixed in 4% (w/v) paraformaldehyde overnight. Paraffin-embedded sections of spinal cord were stained with H&E or Luxol fast blue for analysis of inflammation or demyelination, respectively.

Total RNA was extracted from mouse tissues using TRIzol (Invitrogen). The TaqMan Array is designed for two-step RT-PCR. In the reverse-transcription step, the RNA was subjected to reverse transcription with a random hexamer primer and Moloney murine leukemia virus reverse transcriptase. In the PCR step, PCR products were amplified from cDNA samples using the TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays. The TaqMan assays were preloaded in each reaction well of the TaqMan Array. Real-time PCR was conducted in the Light Cycler quantitative PCR (QPCR) apparatus (Stratagene) using the SYBR Green JumpStart Taq ReadyMix kit (Sigma-Aldrich). The expression value was normalized to β-actin in the same sample and then normalized to the control.

Naive CD4+ T cells were prepared by magnetic cell separation (Invitrogen) from the spleens of female C57BL/6 mice, 6–8 wk of age. Cells were incubated with anti-CD3 (2 mg/ml) and anti-CD28 (2 mg/ml) and induced to differentiate into Th1 cells by supplementation with IL-12 (10 ng/ml) and anti–IL-4 (10 mg/ml). For Th17 differentiation, cells received anti–IL-4 (10 mg/ml) and anti–IFN-γ (10 mg/ml) plus a Th17 mixture containing TGF-β1 (3 ng/ml), IL-6 (30 ng/ml), TNF-α (10 ng/ml), and IL-1β1 (10 ng/ml). Compounds were added to the cytokine mixture to assess their influence on T cell differentiation.

Lymphocytes were incubated for 5 h at 37°C with PMA (50 ng/ml; Sigma-Aldrich), ionomycin (750 ng/ml; Sigma-Aldrich), and brefeldin A (10 mg/ml; Sigma-Aldrich). Surface markers were first stained with relevant Abs. Cells were then resuspended in fixation/permeabilization solution (Cytofix/Cytoperm kit; BD Pharmingen), and intracellular cytokines were stained with appropriate Abs. Guava easyCyte 8HT System and Guava Soft software were used for the analysis.

Leukocytes isolated from the lymph nodes of WT and P2Y12-KO EAE mice or EAE mice treated with vehicle or clopidogrel were seeded in 96-well plates at a density of 2 × 105 per well per 200 μl and restimulated with MOG35–55 (20 mg/ml) at 37°C for 72 h. Cytokines (IL-17A, IFN-γ, TGF-β1) in the culture supernatants after stimulation were quantified by ELISA, according to the manufacturer’s instructions. IL-17A in the culture supernatants of T cell differentiation in vitro was also quantified by ELISA.

Data are presented as mean ± SEM. The statistical significance of the EAE clinical scores between treatments was analyzed with a two-way ANOVA test. Other analyses were assessed by the Student-t test. A p value <0.05 was considered statistically significant.

To explore the role of GPCRs in MS pathogenesis, EAE was induced in C57BL/6 mice with MOG35–55, and the mRNA levels of GPCRs were analyzed PI by gene microarray analysis. The results revealed interesting expression changes in the P2Y family members (Fig. 1A, 1B). In the spleen, P2Y1, P2Y12, and P2Y14 were remarkably upregulated at day 9, in the preclinical stage of the disease, and the upregulation was maintained for the duration of the study (Fig. 1A). However, the expression of P2Y4, P2Y5, P2Y6, P2Y10, and P2Y13 were downregulated on day 9, and gradually restored on day 18 and day 27, with no obvious change of P2Y2 observed (Fig. 1A). In the spinal cords, the expression levels of these P2Y receptors were also analyzed by gene microarray. Most of them showed relatively very low expression levels, and few changes were revealed except a 10-fold upregulation of P2Y10 observed on day 18 (Fig. 1B). Previous reports have shown that P2Y12 receptor–expression on lymphocytes is essential for controlling the severity of the inflammatory response (26), and we speculated that P2Y12 might participate in the initiation and progression of the disease. mRNA expression of P2Y12 in the spleen, draining lymph nodes, spinal cord, and brain was further verified by QPCR. The results demonstrated that P2Y12 was significantly increased 4-fold in the spleen, and its level remained upregulated until the end of EAE (Fig. 1C). In the draining lymph nodes, mRNA expression of P2Y12 also significantly increased to 7–10-fold PI (Fig. 1D), whereas in the spinal cord and brain, no significant changes were observed (Fig. 1E, 1F). Then we performed Western blot to detect the protein levels of P2Y12 during EAE pathogenesis. The results showed that the protein levels of P2Y12 were upregulated on day 9 and 18, but few changes were observed in the spinal cord (Fig. 1G, 1H). Both the proinflammatory role and the constant upregulation of P2Y12 indicated that P2Y12 might be functionally involved in EAE pathogenesis.

FIGURE 1.

Expression changes of P2Y receptors during EAE pathogenesis. Total RNA was isolated from the spleens, lymph nodes, spinals cords, and brains of EAE mice. TaqMan PCR Array was performed to analyze gene expression. (A and B) Gene expression of P2Y1, P2Y2, P2Y4, P2Y5, P2Y6, P2Y10, P2Y12, P2Y13, and P2Y14 in spleens (A) and spinal cords (B) analyzed by TaqMan PCR Array. (CF) Gene expression of P2Y12 in the spleen (C), lymph nodes (D), spinal cords (E), and brains (F) was verified by QPCR. Results were normalized to β-actin expression in the same sample, then normalized to the expression level on day 0. Data are mean ± SEM (n = 6) and are representative of two independent experiments. **p < 0.01, ***p < 0.001 versus EAE mice at day 0. (G and H) Western blot analysis of the protein levels of P2Y12 at day 0, day 9, and day 18 in spleens (G) and spinal cords (H).

FIGURE 1.

Expression changes of P2Y receptors during EAE pathogenesis. Total RNA was isolated from the spleens, lymph nodes, spinals cords, and brains of EAE mice. TaqMan PCR Array was performed to analyze gene expression. (A and B) Gene expression of P2Y1, P2Y2, P2Y4, P2Y5, P2Y6, P2Y10, P2Y12, P2Y13, and P2Y14 in spleens (A) and spinal cords (B) analyzed by TaqMan PCR Array. (CF) Gene expression of P2Y12 in the spleen (C), lymph nodes (D), spinal cords (E), and brains (F) was verified by QPCR. Results were normalized to β-actin expression in the same sample, then normalized to the expression level on day 0. Data are mean ± SEM (n = 6) and are representative of two independent experiments. **p < 0.01, ***p < 0.001 versus EAE mice at day 0. (G and H) Western blot analysis of the protein levels of P2Y12 at day 0, day 9, and day 18 in spleens (G) and spinal cords (H).

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To verify the hypothesis that P2Y12 might regulate EAE development, EAE was induced with MOG35–55 immunization in P2Y12-KO mice and their WT littermate controls. Compared to the WT controls, the P2Y12-KO mice exhibited significantly alleviated EAE symptoms, including a significantly reduced peak severity and cumulative disease score (Fig. 2A). Histological examination of the spinal cords at day 23 PI revealed a dramatic reduction of leukocyte infiltration (Fig. 2B, 2C), and less extensive demyelination in P2Y12-KO mice (Fig. 2D, 2E). To explore how P2Y12 could take part in EAE pathogenesis, bone marrow chimeras were generated by transferring WT or P2Y12-KO bone marrow cells to lethally irradiated WT mice, and transferring WT cells to lethally irradiated P2Y12-KO mice. EAE was induced after immune system reconstitution. P2Y12-KO deletion in the bone marrow cells decreased the EAE score in these chimeras. No significant changes were observed in the disease score between transferring WT cells to lethally irradiated WT mice and to lethally irradiated P2Y12-KO mice (Fig. 2F). Histological examination of the spinal cords at day 23 PI revealed a dramatic reduction of leukocyte infiltration (Fig. 2G, 2H) and less extensive demyelination (Fig. 2I, 2J) in the P2Y12-KO bone marrow cells to lethally irradiated WT mice. These data demonstrate that in the immune system, P2Y12 plays a critical role in controlling the disease severity, whereas in the CNS P2Y12 is not involved in EAE pathogenesis.

FIGURE 2.

P2Y12-KO alleviates clinical symptoms of EAE mice. (A) WT and P2Y12-KO mice were immunized with MOG33–35 and clinical scores were assessed daily. (B) H&E staining and (D) Luxol fast blue staining of paraffin sections of spinal cords isolated from WT and P2Y12-KO EAE mice on day 23. (C) Quantitation of the total infiltrates and (E) the demyelination were presented relatively. Mice from each group were sacrificed, and 10 sections from each mouse were analyzed. Data are mean ± SEM. #p < 0.05 (two-way ANOVA test), *p < 0.05, **p < 0.01 versus WT EAE group. (F) EAE scores of lethally irradiated WT or P2Y12-KO mice reconstituted with WT or P2Y12-KO bone marrow cells and immunized with MOG35–55. (G) H&E staining and (I) Luxol fast blue staining of paraffin sections of spinal cords isolated from WT to WT, WT to P2Y12-KO, and P2Y12-KO to WT EAE mice on day 23. (H) Quantitation of the total infiltrates and (J) the demyelination were presented relatively. Mice from each group were sacrificed, and 10 sections from each mouse were analyzed. Data are mean ± SEM. ##p < 0.01 (two-way ANOVA test), *p < 0.05, **p < 0.01 versus WT to WT EAE group.

FIGURE 2.

P2Y12-KO alleviates clinical symptoms of EAE mice. (A) WT and P2Y12-KO mice were immunized with MOG33–35 and clinical scores were assessed daily. (B) H&E staining and (D) Luxol fast blue staining of paraffin sections of spinal cords isolated from WT and P2Y12-KO EAE mice on day 23. (C) Quantitation of the total infiltrates and (E) the demyelination were presented relatively. Mice from each group were sacrificed, and 10 sections from each mouse were analyzed. Data are mean ± SEM. #p < 0.05 (two-way ANOVA test), *p < 0.05, **p < 0.01 versus WT EAE group. (F) EAE scores of lethally irradiated WT or P2Y12-KO mice reconstituted with WT or P2Y12-KO bone marrow cells and immunized with MOG35–55. (G) H&E staining and (I) Luxol fast blue staining of paraffin sections of spinal cords isolated from WT to WT, WT to P2Y12-KO, and P2Y12-KO to WT EAE mice on day 23. (H) Quantitation of the total infiltrates and (J) the demyelination were presented relatively. Mice from each group were sacrificed, and 10 sections from each mouse were analyzed. Data are mean ± SEM. ##p < 0.01 (two-way ANOVA test), *p < 0.05, **p < 0.01 versus WT to WT EAE group.

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We then tried to find out which kind of immune cells is regulated by P2Y12 during EAE pathogenesis. It is widely accepted that Th1 and Th17 are the main pathogenic T cells involved in EAE development. The populations of Th1 and Th17 were assessed at day 10 PI in both P2Y12-KO and WT EAE mice, and the data revealed a slight decrease in the Th1 population in both the spleen and draining lymph nodes of P2Y12-KO mice (Fig. 3A, 3B, left panels). More interestingly, a significant decrease of the Th17 population was observed in the draining lymph nodes of P2Y12-KO mice (Fig. 3B, right panels). However, no obvious changes of the Th17 population were observed in the spleen, which might be attributed to the low percentages of Th17 detected (Fig. 3A, right panels). Furthermore, mRNA expression of Th1- and Th17-related genes were analyzed in the lymphocytes. Genes specific to the Th17 lineage, including il17a, il17f, and rorγt, were downregulated significantly in P2Y12-KO mice versus the WT controls, and no significant changes were observed in the expression levels of two Th1-related genes, ifnγ and t-bet (Fig. 3C). We also detected cytokine production by the lymphocytes after in vitro MOG restimulation. We found that IL-17A was significantly decreased in P2Y12-KO lymphocytes from EAE mice (Fig. 3D), and IFN-γ exhibited no significant changes in P2Y12-KO lymphocytes (Fig. 3F). Regulatory T cell markers were also analyzed and exhibited no significant changes (Fig. 3C, 3F). All the above data demonstrated that P2Y12-KO alleviates the clinical symptoms of EAE and inhibits Th17 development in vivo.

FIGURE 3.

P2Y12-KO inhibits Th17 differentiation in vivo. Splenocytes and lymphocytes were isolated from P2Y12-KO or WT EAE mice on day 10 PI and analyzed with flow cytometry after Ab staining. (A and B) Th1 and Th17 cells were analyzed by intracellular staining of IFN-γ and IL-17A, respectively, in the CD4+ gate. (C) QPCR analysis of Th1- and Th17-related gene expression in lymph nodes. (D and F) Lymphocytes from WT and P2Y12-KO EAE mice were restimulated in vitro with MOG35–55 for 72 h, and (D) IL-17A, (E) IFN-γ, and (F) TGF-β1 in supernatants were detected by ELISA. Data are mean ± SEM (n = 6). *p < 0.05, **p < 0.01 versus WT EAE mice.

FIGURE 3.

P2Y12-KO inhibits Th17 differentiation in vivo. Splenocytes and lymphocytes were isolated from P2Y12-KO or WT EAE mice on day 10 PI and analyzed with flow cytometry after Ab staining. (A and B) Th1 and Th17 cells were analyzed by intracellular staining of IFN-γ and IL-17A, respectively, in the CD4+ gate. (C) QPCR analysis of Th1- and Th17-related gene expression in lymph nodes. (D and F) Lymphocytes from WT and P2Y12-KO EAE mice were restimulated in vitro with MOG35–55 for 72 h, and (D) IL-17A, (E) IFN-γ, and (F) TGF-β1 in supernatants were detected by ELISA. Data are mean ± SEM (n = 6). *p < 0.05, **p < 0.01 versus WT EAE mice.

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We then tested whether the P2Y12 receptor can regulate T cell differentiation directly, using an in vitro assay. We performed in vitro T cell differentiation with ADP, an agonist of P2Y1, P2Y12, and P2Y13 (2830). We found that treatment with ADP significantly enhanced Th17 differentiation, although the enhancement of Th17 differentiation was abolished in P2Y12 receptor knockout cells (Fig. 4A, 4B). These data indicated an important role of P2Y12 in regulating Th17 differentiation (Fig. 4A). To further verify that P2Y12-KO inhibits Th17 differentiation in vitro, we measured the IL-17A protein level in the supernatant during Th17 differentiation with ELISA analysis. Consistent with the results of Th17 intracellular staining, IL-17A protein levels in the supernatant were significantly increased by ADP treatment; however, this increase was abolished in P2Y12-KO cells (Fig. 4D). Clone figures of Th17 also presented consistent results (Fig. 4C). As the percentage of Th1 also decreased in P2Y12-KO EAE mice, in this study we tested the role of P2Y12 in Th1 differentiation in vitro. The results showed no significant changes upon ADP administration or P2Y12-KO (Fig. 4E, 4F). These data indicated that blocking the P2Y12 receptor inhibits differentiation of Th17 cells in vitro, and such an effect is mediated by P2Y12 on T cells.

FIGURE 4.

P2Y12-KO inhibits Th17 differentiation in vitro. Naive CD4+ T cells were induced to differentiate into Th17 and Th1 cells in vitro. (A) Th17 differentiation was assessed by intracellular staining of IL-17A. (B) FACS analysis. (C) Clone of Th17 cells and (D) ELISA detection of IL-17A production in supernatants. (E) Th1 differentiation was assessed by intracellular staining of IFN-γ and (F) clone formation. Data are mean ± SEM (n = 3). *p < 0.05, ***p < 0.001 versus WT (ADP = 0 μM), ###p < 0.001 versus WT (ADP = 15 μM).

FIGURE 4.

P2Y12-KO inhibits Th17 differentiation in vitro. Naive CD4+ T cells were induced to differentiate into Th17 and Th1 cells in vitro. (A) Th17 differentiation was assessed by intracellular staining of IL-17A. (B) FACS analysis. (C) Clone of Th17 cells and (D) ELISA detection of IL-17A production in supernatants. (E) Th1 differentiation was assessed by intracellular staining of IFN-γ and (F) clone formation. Data are mean ± SEM (n = 3). *p < 0.05, ***p < 0.001 versus WT (ADP = 0 μM), ###p < 0.001 versus WT (ADP = 15 μM).

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The above data demonstrated the important role of the P2Y12 receptor in regulating EAE pathogenesis. We hypothesized that blockage of the P2Y12 receptor may serve as a therapeutic strategy for EAE. Clopidogrel and ticagrelor, two selective P2Y12 receptor antagonists, were used for the treatment of MOG-induced EAE mice. The drugs were given via oral administration once daily from day 3 PI until the end of the experiment, and the control mice were given double distilled (dd) H2O. Mice given 5 and 15 mg/kg clopidogrel developed alleviative EAE. However, when given 50 mg/kg clopidogrel, mice suffered severe EAE, which may be due to the side effects of high-dose clopidogrel treatment (Fig. 5A–C). When given at 15 mg/kg, clopidogrel significantly reduced the peak severity and cumulative clinical score of EAE (Fig. 5B). To exclude the off-target effects of clopidogrel, we also tested another P2Y12 antagonist, ticagrelor, and found that ticagrelor treatment also significantly decreased the severity of EAE when given at 30 mg/kg (Fig. 5D). Further histological examination of the spinal cords was performed at day 23 PI. Compared to vehicle control, clopidogrel (15 mg/kg) caused a dramatic reduction of leukocyte infiltration in the spinal cord (Fig. 5E, Supplemental Fig. 1A). Luxol fast blue staining also revealed less demyelination in clopidogrel-treated mice than in controls (Fig. 5F, Supplemental Fig. 1B). As we demonstrated that P2Y12-KO inhibits Th17 differentiation, we then checked if clopidogrel treatment can also inhibit Th17 differentiation in vivo. Lymph node cells were collected and restimulated with MOG35–55. We found that IL-17A production was significantly reduced in the lymphocytes from clopidogrel (15 mg/kg)-treated mice compared with vehicle controls (Fig. 5G). We also tested the levels of IFN-γ and TGF-β1. The results showed no significant changes between clopidogrel (15 mg/kg)-treated mice and vehicle controls (Fig. 5H, 5I). In conclusion, blockage of the P2Y12 receptor alleviated the symptoms of EAE and inhibited Th17 differentiation in vivo, which is the same effect as P2Y12-KO.

FIGURE 5.

P2Y12 antagonist treatment alleviates clinical symptoms of EAE mice and inhibits Th17 differentiation in vivo. (AD) EAE mice were treated with (A–C) clopidogrel (5, 15, or 50 mg/kg/d) or (D) ticagrelor (30 mg/kg/d), once daily via oral administration from day 3 PI to the end of the study. Water was given as vehicle control. ***p < 0.001 versus vehicle (two-way ANOVA test). On day 23 mice from each group were sacrificed, and 10 sections from each mouse were analyzed. (E and F) Quantitative analysis of the numbers of total infiltrates (E) and the amount of demyelination measured (F) in paraffin sections of spinal cords isolated from vehicle and clopidogrel (15 mg/kg)-treated EAE mice. (GI) Lymphocytes from vehicle and clopidogrel (15 mg/kg)-treated EAE mice were restimulated in vitro with MOG35–55 for 72 h, and IL-17A (G), IFN-γ (H), and TGF-β1 (I) in supernatants were detected with ELISA. Data are mean ± SEM (n = 6). *p < 0.05 versus vehicle.

FIGURE 5.

P2Y12 antagonist treatment alleviates clinical symptoms of EAE mice and inhibits Th17 differentiation in vivo. (AD) EAE mice were treated with (A–C) clopidogrel (5, 15, or 50 mg/kg/d) or (D) ticagrelor (30 mg/kg/d), once daily via oral administration from day 3 PI to the end of the study. Water was given as vehicle control. ***p < 0.001 versus vehicle (two-way ANOVA test). On day 23 mice from each group were sacrificed, and 10 sections from each mouse were analyzed. (E and F) Quantitative analysis of the numbers of total infiltrates (E) and the amount of demyelination measured (F) in paraffin sections of spinal cords isolated from vehicle and clopidogrel (15 mg/kg)-treated EAE mice. (GI) Lymphocytes from vehicle and clopidogrel (15 mg/kg)-treated EAE mice were restimulated in vitro with MOG35–55 for 72 h, and IL-17A (G), IFN-γ (H), and TGF-β1 (I) in supernatants were detected with ELISA. Data are mean ± SEM (n = 6). *p < 0.05 versus vehicle.

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We then tested if blocking the P2Y12 receptor with antagonists affects T cell differentiation in vitro. Th17 differentiation was enhanced significantly by the administration of ADP in a dosage-dependent manner (Fig. 6A). However, increased Th17 differentiation by ADP administration was abolished by further administration of ticagrelor (Fig. 6B). Measurement of the IL-17A production in the supernatant further verified that inhibition of P2Y12 by ticagrelor abolished the enhancement of Th17 differentiation by ADP (Fig. 6C). Clone figures and flow cytometry analysis of Th17 also showed similar results (Fig. 6D, 6E). Taken together, these data demonstrated that blocking the P2Y12 receptor with antagonists inhibits differentiation of Th17 cells in vitro.

FIGURE 6.

Blockage of P2Y12 receptor with antagonists inhibits Th17 differentiation in vitro. (A and B) Th17 cells were induced from naive CD4+ T cells. The influences of receptor agonist (A) and antagonist (B) on Th17 differentiation were analyzed by flow cytometry. (C) IL-17A in supernatants was detected by ELISA. (D) Clone and (E) flow cytometry analysis of Th17 cells. Data are mean ± SEM (n = 3). *p < 0.05, ***p < 0.001 versus WT (ADP = 0 μM), ##p < 0.01 versus WT (ADP = 15 μM).

FIGURE 6.

Blockage of P2Y12 receptor with antagonists inhibits Th17 differentiation in vitro. (A and B) Th17 cells were induced from naive CD4+ T cells. The influences of receptor agonist (A) and antagonist (B) on Th17 differentiation were analyzed by flow cytometry. (C) IL-17A in supernatants was detected by ELISA. (D) Clone and (E) flow cytometry analysis of Th17 cells. Data are mean ± SEM (n = 3). *p < 0.05, ***p < 0.001 versus WT (ADP = 0 μM), ##p < 0.01 versus WT (ADP = 15 μM).

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TNBS-induced colitis is another well-known autoimmune disease. We tried to explore whether P2Y12 is involved in colitis pathogenesis. At first, WT and P2Y12-KO mice were induced to develop colitis by TNBS. P2Y12-KO mice showed less body weight loss than WT mice (Fig. 7A). P2Y12-KO mice also developed an ameliorated severe disease appearance with decreased weight per unit length of intestine (Fig. 7B–E). The severity of colon inflammation was further evaluated by histological examinations; histological sections were examined microscopically after H&E staining, WT mice showed more immune cell infiltration in the lamina propria and more severe goblet cell destruction in the colon than P2Y12-KO mice (Supplemental Fig. 1C). Finally, mRNA expression of Th1- and Th17-related genes and some colitis-related proinflammatory cytokines were detected in the colon. Il17a, il17f, tnfα, il1β, rorγt, ifnγ, t-bet, and il12 were downregulated significantly in P2Y12-KO mice (Fig. 7F), indicating that P2Y12-KO mice developed less severe disease.

FIGURE 7.

Deleting P2Y12 or clopidogrel treatment alleviates clinical symptoms of colitis. (AF) WT and P2Y12-KO mice were induced to develop colitis by TNBS and the body weight of the mice was measured daily (A). At day 5, the mice were sacrificed and the colon picture (B), length (C), weight (D), and weight/length (E) were measured. (F) QPCR analysis of major inflammation-related gene expression in colons. (GL) Mice were induced to develop colitis by TNBS. ddH2O (vehicle) and clopidogrel (15 mg/kg) were given once daily via oral administration and body weight loss of the mice was measured daily (G). At day 4, the mice were sacrificed and the colon pictures (H), length (I), weight (J), and weight/length (K) were measured. (L) QPCR analysis of major inflammation-related gene expression in colons. Data are mean ± SEM, *p < 0.05, **p < 0.01.

FIGURE 7.

Deleting P2Y12 or clopidogrel treatment alleviates clinical symptoms of colitis. (AF) WT and P2Y12-KO mice were induced to develop colitis by TNBS and the body weight of the mice was measured daily (A). At day 5, the mice were sacrificed and the colon picture (B), length (C), weight (D), and weight/length (E) were measured. (F) QPCR analysis of major inflammation-related gene expression in colons. (GL) Mice were induced to develop colitis by TNBS. ddH2O (vehicle) and clopidogrel (15 mg/kg) were given once daily via oral administration and body weight loss of the mice was measured daily (G). At day 4, the mice were sacrificed and the colon pictures (H), length (I), weight (J), and weight/length (K) were measured. (L) QPCR analysis of major inflammation-related gene expression in colons. Data are mean ± SEM, *p < 0.05, **p < 0.01.

Close modal

We also explored P2Y12 function in TNBS-induced acute colitis with clopidogrel. We observed ∼25% weight loss in the vehicle-treated mice (Fig. 7G). Interestingly, the weight loss was prevented by clopidogrel administration. This effect was statistically significant at day 4 after the disease induction. Clopidogrel treatment also ameliorated TNBS-induced acute colitis from the shape variation of the intestine (Fig. 7H–K), notably, the weight per unit length of intestine decreased compared with the vehicle mice, showing a less severe disease appearance. H&E staining of the colon showed fewer infiltrating cells in the lamina propria after clopidogrel administration (Supplemental Fig. 1D). Furthermore, mRNA expression of Th1- and Th17-related genes and some colitis-related proinflammatory cytokines were also detected in the colon. These genes were downregulated significantly in mice treated with clopidogrel versus the vehicle (Fig. 7L).

We also tested the therapeutic potentials of blocking P2Y12 by clopidogrel treatment in a multiple low-dose STZ-induced T1D mouse model. In diabetes induced by STZ, blood glucose levels and body weight loss were the main indicators. P2Y12-KO mice developed a lower blood glucose level and showed less body weight loss than WT mice (Fig. 8A, 8B). Further histological analysis of the pancreas showed that P2Y12-KO mice displayed alleviative damage to the islets of Langerhans (Supplemental Fig. 1E).

FIGURE 8.

Deleting P2Y12 or clopidogrel treatment alleviates clinical symptoms of multiple low-dose STZ-induced T1D. (A and B) WT and P2Y12-KO mice were developed to multiple low-dose STZ-induced T1D. Blood glucose (A) and body weight (B) of the mice were measured every week. (C and D) Mice were induced to develop T1D by multiple low-dose STZ. ddH2O (vehicle), and clopidogrel (15 mg/kg) were given once daily via oral administration. Blood glucose (C) and body weight (D) of the mice were measured every week.

FIGURE 8.

Deleting P2Y12 or clopidogrel treatment alleviates clinical symptoms of multiple low-dose STZ-induced T1D. (A and B) WT and P2Y12-KO mice were developed to multiple low-dose STZ-induced T1D. Blood glucose (A) and body weight (B) of the mice were measured every week. (C and D) Mice were induced to develop T1D by multiple low-dose STZ. ddH2O (vehicle), and clopidogrel (15 mg/kg) were given once daily via oral administration. Blood glucose (C) and body weight (D) of the mice were measured every week.

Close modal

Blockage of the P2Y12 receptor with clopidogrel also significantly restored blood glucose levels and body weights (Fig. 8C, 8D). Further histological analysis of the pancreas showed that clopidogrel relieved the damage to the islets of Langerhans (Supplemental Fig. 1F). These data indicate that P2Y12 plays an important role in pathogenesis of T1D.

Understanding of the pathogenesis of MS is still limited, and the disorder remains devastating. Drugs currently used in MS patients include those treating attacks, such as corticosteroids, and those modifying the disease course, such as IFN-β and glatiramer acetate (31, 32). However, as these treatments are only partially effective in halting the MS disease process and are frequently associated with side effects and suboptimal patient adherence, new therapeutic drug targets for MS are necessary.

A recent paper indicated that P2Y12 expression is modulated during neuroinflammation in MS brain lesions, which might indicate the beneficial effects of P2Y12 receptor antagonists in EAE (33). ADP is an endogenous ligand for both the purinergic P2Y1 receptor and P2Y12 receptor (15, 34). P2Y12, but not P2Y1, receptor antagonists, clopidogrel and ticagrelor, are available in the clinic and used for the treatment of platelet aggregation (35, 36). Inflammation is widely recognized as a contributor to the pathology of MS. In the P2Y family, P2Y1, a Gαq-coupled receptor, acts in possible autocrine or paracrine control of the microglia-mediated initiation and progression of an inflammatory response with leukotriene systems (37). Although P2Y1 was also upregulated in the peripheral lymphoid tissues of EAE mice, a more direct link between the purinergic P2Y1 receptor and MS remains to be found. P2Y13 and P2Y14 receptors, a potential novel therapeutic target for allergic conditions, were characterized during a systematic study of orphan receptors (38, 39). The P2Y14 receptor appears to be functionally expressed in murine spleen-derived T lymphocytes. These observations suggest that the P2Y14 receptor may play an important role in modulating immune function (40).

In this study, we supplied direct evidence demonstrating the critical role of the P2Y12 receptor in regulating Th17 differentiation and EAE pathogenesis. In EAE mice, blocking the P2Y12 receptor with antagonists and knocking out the receptor can ameliorate EAE severity and histological pathology. At the same time, the percentage of Th17 both in vitro and in vivo sharply decreased upon P2Y12 receptor blockage. The antagonists of P2Y12 treatment can relieve the clinical symptoms of EAE and are more likely to be used in MS. We not only used P2Y12-KO mice but also chose two antagonists of the P2Y12 receptor in case there were off-target effects.

Previous studies show that P2Y12 plays a key role in arteriosclerosis (41), pulmonary inflammation and asthma (26), inflammatory erosive arthritis (42, 43), and other diseases, suggesting it has a potential therapeutic value. Recent studies have demonstrated that CD4+ lymphocytes play an important role in the development of ulcerative colitis, a form of inflammatory bowel disease (44, 45). CD4+ lymphocytes are increased and activated in the intestinal lamina propria of inflammatory bowel disease patients and in several colitis models (46). The development of TNBS-induced colitis is thought to be related to Th1 and Th17 (47, 48). STZ is a d-glucopyranose derivative of N-methyl-N-nitrosourea, endowed with potent alkylating properties (49). Although at high doses this agent has diabetogenic potential, due to its capacity to selectively promote death of insulin-producing β cells by apoptosis or necrosis, at low doses STZ generates hydrogen peroxide (50), and induces expression of the glutamic acid decarboxylase autoantigen (51). T1D is an autoimmune condition traditionally associated with Th1 populations (52), but in recent years, multiple low doses of STZ-induced T1D demonstrated the involvement of Th17 cells in the pathogenesis of T1D (53), and therapeutic strategies designed to inhibit these cells are likely to be applicable in T1D (54). In EAE, we found that clopidogrel and ticagrelor affect the development of Th17 but not Th1, so we investigated the model of TNBS-induced colitis and multiple low-dose STZ-induced T1D with the antagonists. We found clopidogrel treatment alleviated the clinical symptoms of colitis and diabetes, suggesting that P2Y12 plays an important role in colitis and T1D.

In summary, our results demonstrated that the P2Y12 receptor plays critical regulating roles in Th17 differentiation and MS pathogenesis. Most interestingly, our results not only revealed part of the mechanisms underlying the onset of MS, but also provided new therapeutic targets for the clinical intervention of other autoimmune diseases.

This work was supported by grants from the Ministry of Science and Technology of China (2014CB541903 and 2012CB910404), the National Natural Science Foundation of China (31171348 and 31371414), the Shanghai Municipal Education Commission (14zz042), the State Key Laboratory of Drug Research (SIMM1302KF-09), and Fundamental Research Funds for the Central Universities.

The online version of this article contains supplemental material.

Abbreviations used in this article:

DC

dendritic cell

dd

double distilled

EAE

experimental autoimmune encephalomyelitis

GPCR

G protein–coupled receptor

KO

knockout

MOG

myelin oligodendrocyte glycoprotein

MS

multiple sclerosis

PI

postimmunization

QPCR

quantitative PCR

STZ

streptozocin

T1D

type 1 diabetes

TNBS

2,4,6-trinitrobenzenesulfonic acid

WT

wild type.

1
Compston
,
A.
,
A.
Coles
.
2002
.
Multiple sclerosis.
Lancet
359
:
1221
1231
.
2
Hauser
,
S. L.
,
J. R.
Oksenberg
.
2006
.
The neurobiology of multiple sclerosis: genes, inflammation, and neurodegeneration.
Neuron
52
:
61
76
.
3
Krishnamoorthy
,
G.
,
A.
Saxena
,
L. T.
Mars
,
H. S.
Domingues
,
R.
Mentele
,
A.
Ben-Nun
,
H.
Lassmann
,
K.
Dornmair
,
F. C.
Kurschus
,
R. S.
Liblau
,
H.
Wekerle
.
2009
.
Myelin-specific T cells also recognize neuronal autoantigen in a transgenic mouse model of multiple sclerosis.
Nat. Med.
15
:
626
632
.
4
Harkiolaki
,
M.
,
S. L.
Holmes
,
P.
Svendsen
,
J. W.
Gregersen
,
L. T.
Jensen
,
R.
McMahon
,
M. A.
Friese
,
G.
van Boxel
,
R.
Etzensperger
,
J. S.
Tzartos
, et al
.
2009
.
T cell-mediated autoimmune disease due to low-affinity crossreactivity to common microbial peptides.
Immunity
30
:
348
357
.
5
Matsui
,
M.
2013
.
[Immunology for understanding the pathogenesis of multiple sclerosis]
.
Rinsho Shinkeigaku
53
:
898
901
.
6
Haghikia
,
A.
,
R.
Hohlfeld
,
R.
Gold
,
L.
Fugger
.
2013
.
Therapies for multiple sclerosis: translational achievements and outstanding needs.
Trends Mol. Med.
19
:
309
319
.
7
Jalink
,
K.
,
W. H.
Moolenaar
.
2010
.
G protein-coupled receptors: the inside story.
BioEssays
32
:
13
16
.
8
Yanachkov
,
I. B.
,
H.
Chang
,
M. I.
Yanachkova
,
E. J.
Dix
,
M. A.
Berny-Lang
,
T.
Gremmel
,
A. D.
Michelson
,
G. E.
Wright
,
A. L.
Frelinger
III.
2016
.
New highly active antiplatelet agents with dual specificity for platelet P2Y1 and P2Y12 adenosine diphosphate receptors.
Eur. J. Med. Chem.
107
:
204
218
.
9
Du
,
C.
,
X.
Xie
.
2012
.
G protein-coupled receptors as therapeutic targets for multiple sclerosis.
Cell Res.
22
:
1108
1128
.
10
Fredholm
,
B. B.
,
M. P.
Abbracchio
,
G.
Burnstock
,
G. R.
Dubyak
,
T. K.
Harden
,
K. A.
Jacobson
,
U.
Schwabe
,
M.
Williams
.
1997
.
Towards a revised nomenclature for P1 and P2 receptors.
Trends Pharmacol. Sci.
18
:
79
82
.
11
Abbracchio
,
M. P.
,
J. M.
Boeynaems
,
E. A.
Barnard
,
J. L.
Boyer
,
C.
Kennedy
,
M. T.
Miras-Portugal
,
B. F.
King
,
C.
Gachet
,
K. A.
Jacobson
,
G. A.
Weisman
,
G.
Burnstock
.
2003
.
Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family.
Trends Pharmacol. Sci.
24
:
52
55
.
12
Tsukimoto
,
M.
,
A.
Tokunaga
,
H.
Harada
,
S.
Kojima
.
2009
.
Blockade of murine T cell activation by antagonists of P2Y6 and P2X7 receptors.
Biochem. Biophys. Res. Commun.
384
:
512
518
.
13
Skelton
,
L.
,
M.
Cooper
,
M.
Murphy
,
A.
Platt
.
2003
.
Human immature monocyte-derived dendritic cells express the G protein-coupled receptor GPR105 (KIAA0001, P2Y14) and increase intracellular calcium in response to its agonist, uridine diphosphoglucose.
J. Immunol.
171
:
1941
1949
.
14
Takasaki
,
J.
,
M.
Kamohara
,
T.
Saito
,
M.
Matsumoto
,
S.
Matsumoto
,
T.
Ohishi
,
T.
Soga
,
H.
Matsushime
,
K.
Furuichi
.
2001
.
Molecular cloning of the platelet P2T(AC) ADP receptor: pharmacological comparison with another ADP receptor, the P2Y(1) receptor.
Mol. Pharmacol.
60
:
432
439
.
15
Hollopeter
,
G.
,
H. M.
Jantzen
,
D.
Vincent
,
G.
Li
,
L.
England
,
V.
Ramakrishnan
,
R. B.
Yang
,
P.
Nurden
,
A.
Nurden
,
D.
Julius
,
P. B.
Conley
.
2001
.
Identification of the platelet ADP receptor targeted by antithrombotic drugs.
Nature
409
:
202
207
.
16
Haynes
,
S. E.
,
G.
Hollopeter
,
G.
Yang
,
D.
Kurpius
,
M. E.
Dailey
,
W. B.
Gan
,
D.
Julius
.
2006
.
The P2Y12 receptor regulates microglial activation by extracellular nucleotides.
Nat. Neurosci.
9
:
1512
1519
.
17
Diehl
,
P.
,
C.
Olivier
,
C.
Halscheid
,
T.
Helbing
,
C.
Bode
,
M.
Moser
.
2010
.
Clopidogrel affects leukocyte dependent platelet aggregation by P2Y12 expressing leukocytes.
Basic Res. Cardiol.
105
:
379
387
.
18
Moers
,
A.
,
B.
Nieswandt
,
S.
Massberg
,
N.
Wettschureck
,
S.
Grüner
,
I.
Konrad
,
V.
Schulte
,
B.
Aktas
,
M. P.
Gratacap
,
M. I.
Simon
, et al
.
2003
.
G13 is an essential mediator of platelet activation in hemostasis and thrombosis.
Nat. Med.
9
:
1418
1422
.
19
Honda
,
S.
,
Y.
Sasaki
,
K.
Ohsawa
,
Y.
Imai
,
Y.
Nakamura
,
K.
Inoue
,
S.
Kohsaka
.
2001
.
Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors.
J. Neurosci.
21
:
1975
1982
.
20
Sasaki
,
Y.
,
M.
Hoshi
,
C.
Akazawa
,
Y.
Nakamura
,
H.
Tsuzuki
,
K.
Inoue
,
S.
Kohsaka
.
2003
.
Selective expression of Gi/o-coupled ATP receptor P2Y12 in microglia in rat brain.
Glia
44
:
242
250
.
21
Kim
,
S.
,
S. P.
Kunapuli
.
2011
.
P2Y12 receptor in platelet activation.
Platelets
22
:
56
60
.
22
Bhavaraju
,
K.
,
A.
Mayanglambam
,
A. K.
Rao
,
S. P.
Kunapuli
.
2010
.
P2Y(12) antagonists as antiplatelet agents - recent developments.
Curr. Opin. Drug Discov. Devel.
13
:
497
506
.
23
Shortman
,
K.
,
S. H.
Naik
.
2007
.
Steady-state and inflammatory dendritic-cell development.
Nat. Rev. Immunol.
7
:
19
30
.
24
Alvarez
,
D.
,
E. H.
Vollmann
,
U. H.
von Andrian
.
2008
.
Mechanisms and consequences of dendritic cell migration.
Immunity
29
:
325
342
.
25
Ben Addi
,
A.
,
D.
Cammarata
,
P. B.
Conley
,
J. M.
Boeynaems
,
B.
Robaye
.
2010
.
Role of the P2Y12 receptor in the modulation of murine dendritic cell function by ADP.
J. Immunol.
185
:
5900
5906
.
26
Paruchuri
,
S.
,
H.
Tashimo
,
C.
Feng
,
A.
Maekawa
,
W.
Xing
,
Y.
Jiang
,
Y.
Kanaoka
,
P.
Conley
,
J. A.
Boyce
.
2009
.
Leukotriene E4-induced pulmonary inflammation is mediated by the P2Y12 receptor.
J. Exp. Med.
206
:
2543
2555
.
27
Andre
,
P.
,
S. M.
Delaney
,
T.
LaRocca
,
D.
Vincent
,
F.
DeGuzman
,
M.
Jurek
,
B.
Koller
,
D. R.
Phillips
,
P. B.
Conley
.
2003
.
P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries.
J. Clin. Invest.
112
:
398
406
.
28
Daniel
,
J. L.
,
C.
Dangelmaier
,
J.
Jin
,
B.
Ashby
,
J. B.
Smith
,
S. P.
Kunapuli
.
1998
.
Molecular basis for ADP-induced platelet activation. I. Evidence for three distinct ADP receptors on human platelets.
J. Biol. Chem.
273
:
2024
2029
.
29
Jin
,
J.
,
S. P.
Kunapuli
.
1998
.
Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation.
Proc. Natl. Acad. Sci. USA
95
:
8070
8074
.
30
Chhatriwala
,
M.
,
R. G.
Ravi
,
R. I.
Patel
,
J. L.
Boyer
,
K. A.
Jacobson
,
T. K.
Harden
.
2004
.
Induction of novel agonist selectivity for the ADP-activated P2Y1 receptor versus the ADP-activated P2Y12 and P2Y13 receptors by conformational constraint of an ADP analog.
J. Pharmacol. Exp. Ther.
311
:
1038
1043
.
31
Milne
,
R.
,
A.
Clegg
,
J.
Bryant
.
2001
.
Drug treatment of multiple sclerosis. Clinical review was unsystematic.
BMJ
322
:
299
.
32
Tabansky
,
I.
,
M. D.
Messina
,
C.
Bangeranye
,
J.
Goldstein
,
K. M.
Blitz-Shabbir
,
S.
Machado
,
V.
Jeganathan
,
P.
Wright
,
S.
Najjar
,
Y.
Cao
, et al
.
2016
.
Erratum to: advancing drug delivery systems for the treatment of multiple sclerosis.
Immunol. Res.
64
:
640
.
33
Gustafsson
,
A. J.
,
L.
Muraro
,
C.
Dahlberg
,
M.
Migaud
,
O.
Chevallier
,
H. N.
Khanh
,
K.
Krishnan
,
N.
Li
,
M. S.
Islam
.
2011
.
ADP ribose is an endogenous ligand for the purinergic P2Y1 receptor.
Mol. Cell. Endocrinol.
333
:
8
19
.
34
Amadio
,
S.
,
C.
Parisi
,
C.
Montilli
,
A. S.
Carrubba
,
S.
Apolloni
,
C.
Volonté
.
2014
.
P2Y(12) receptor on the verge of a neuroinflammatory breakdown.
Mediators Inflamm.
2014
:
975849
.
35
Dorsam
,
R. T.
,
S.
Murugappan
,
Z.
Ding
,
S. P.
Kunapuli
.
2003
.
Clopidogrel: interactions with the P2Y12 receptor and clinical relevance.
Hematology
8
:
359
365
.
36
Capodanno
,
D.
,
K.
Dharmashankar
,
D. J.
Angiolillo
.
2010
.
Mechanism of action and clinical development of ticagrelor, a novel platelet ADP P2Y12 receptor antagonist.
Expert Rev. Cardiovasc. Ther.
8
:
151
158
.
37
Ballerini
,
P.
,
P.
Di Iorio
,
R.
Ciccarelli
,
F.
Caciagli
,
A.
Poli
,
A.
Beraudi
,
S.
Buccella
,
I.
D’Alimonte
,
M.
D’Auro
,
E.
Nargi
, et al
.
2005
.
P2Y1 and cysteinyl leukotriene receptors mediate purine and cysteinyl leukotriene co-release in primary cultures of rat microglia.
Int. J. Immunopathol. Pharmacol.
18
:
255
268
.
38
Gao
,
Z. G.
,
Y.
Ding
,
K. A.
Jacobson
.
2010
.
UDP-glucose acting at P2Y14 receptors is a mediator of mast cell degranulation.
Biochem. Pharmacol.
79
:
873
879
.
39
Communi
,
D.
,
N. S.
Gonzalez
,
M.
Detheux
,
S.
Brézillon
,
V.
Lannoy
,
M.
Parmentier
,
J. M.
Boeynaems
.
2001
.
Identification of a novel human ADP receptor coupled to G(i).
J. Biol. Chem.
276
:
41479
41485
.
40
Scrivens
,
M.
,
J. M.
Dickenson
.
2005
.
Functional expression of the P2Y14 receptor in murine T-lymphocytes.
Br. J. Pharmacol.
146
:
435
444
.
41
Li
,
D.
,
Y.
Wang
,
L.
Zhang
,
X.
Luo
,
J.
Li
,
X.
Chen
,
H.
Niu
,
K.
Wang
,
Y.
Sun
,
X.
Wang
, et al
.
2012
.
Roles of purinergic receptor P2Y, G protein-coupled 12 in the development of atherosclerosis in apolipoprotein E-deficient mice.
Arterioscler. Thromb. Vasc. Biol.
32
:
e81
e89
.
42
Boilard
,
E.
,
P. A.
Nigrovic
,
K.
Larabee
,
G. F.
Watts
,
J. S.
Coblyn
,
M. E.
Weinblatt
,
E. M.
Massarotti
,
E.
Remold-O’Donnell
,
R. W.
Farndale
,
J.
Ware
,
D. M.
Lee
.
2010
.
Platelets amplify inflammation in arthritis via collagen-dependent microparticle production.
Science
327
:
580
583
.
43
Garcia
,
A. E.
,
S. R.
Mada
,
M. C.
Rico
,
R. A.
Dela Cadena
,
S. P.
Kunapuli
.
2011
.
Clopidogrel, a P2Y12 receptor antagonist, potentiates the inflammatory response in a rat model of peptidoglycan polysaccharide-induced arthritis.
PLoS One
6
:
e26035
.
44
Langrish
,
C. L.
,
B. S.
McKenzie
,
N. J.
Wilson
,
R.
de Waal Malefyt
,
R. A.
Kastelein
,
D. J.
Cua
.
2004
.
IL-12 and IL-23: master regulators of innate and adaptive immunity.
Immunol. Rev.
202
:
96
105
.
45
Monteleone
,
I.
,
P.
Vavassori
,
L.
Biancone
,
G.
Monteleone
,
F.
Pallone
.
2002
.
Immunoregulation in the gut: success and failures in human disease.
Gut
50
(
Suppl. 3
):
III60
III64
.
46
Kappeler
,
A.
,
C.
Mueller
.
2000
.
The role of activated cytotoxic T cells in inflammatory bowel disease.
Histol. Histopathol.
15
:
167
172
.
47
Lim
,
S. M.
,
J. J.
Jeong
,
H. S.
Choi
,
H. B.
Chang
,
D. H.
Kim
.
2016
.
Mangiferin corrects the imbalance of Th17/Treg cells in mice with TNBS-induced colitis.
Int. Immunopharmacol.
34
:
220
228
.
48
Jin
,
Y.
,
Y.
Lin
,
L.
Lin
,
C.
Zheng
.
2012
.
IL-17/IFN-γ interactions regulate intestinal inflammation in TNBS-induced acute colitis.
J. Interferon Cytokine Res.
32
:
548
556
.
49
Weiss
,
R. B.
1982
.
Streptozocin: a review of its pharmacology, efficacy, and toxicity.
Cancer Treat. Rep.
66
:
427
438
.
50
Friesen
,
N. T.
,
A. S.
Büchau
,
P.
Schott-Ohly
,
A.
Lgssiar
,
H.
Gleichmann
.
2004
.
Generation of hydrogen peroxide and failure of antioxidative responses in pancreatic islets of male C57BL/6 mice are associated with diabetes induced by multiple low doses of streptozotocin.
Diabetologia
47
:
676
685
.
51
Choi
,
S. E.
,
H. L.
Noh
,
H. M.
Kim
,
J. W.
Yoon
,
Y.
Kang
.
2002
.
Streptozotocin upregulates GAD67 expression in MIN6N8a mouse beta cells.
J. Autoimmun.
19
:
1
8
.
52
Wilson
,
S. B.
,
S. C.
Kent
,
K. T.
Patton
,
T.
Orban
,
R. A.
Jackson
,
M.
Exley
,
S.
Porcelli
,
D. A.
Schatz
,
M. A.
Atkinson
,
S. P.
Balk
, et al
.
1998
.
Extreme Th1 bias of invariant Vα24JαQ T cells in type 1 diabetes. [Published erratum appears in 1999 Nature 399: 84.]
Nature
391
:
177
181
.
53
Wang
,
M.
,
L.
Yang
,
X.
Sheng
,
W.
Chen
,
H.
Tang
,
H.
Sheng
,
B.
Xi
,
Y. Q.
Zang
.
2011
.
T-cell vaccination leads to suppression of intrapancreatic Th17 cells through Stat3-mediated RORγt inhibition in autoimmune diabetes.
Cell Res.
21
:
1358
1369
.
54
Emamaullee
,
J. A.
,
J.
Davis
,
S.
Merani
,
C.
Toso
,
J. F.
Elliott
,
A.
Thiesen
,
A. M.
Shapiro
.
2009
.
Inhibition of Th17 cells regulates autoimmune diabetes in NOD mice.
Diabetes
58
:
1302
1311
.

The authors have no financial conflicts of interest.

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