Extracellular adenosine induces hypersecretion of IL-17A by T-helper 17 cells through the adenosine A2a receptor

Abstract

Extracellular adenosine, produced from ATP secreted by neuronal or immune cells, may play a role in endogenous regulation of inflammatory responses. Studies show that adenosine induces hypersecretion of IL-17A by CD4⁺ T cells upon treatment with an A2aR agonist (PSB0777), and that adenosine-mediated IL-17A hypersecretion is suppressed by the A2aR antagonist (Istradefylline) in humans. However, it is unclear whether A2aR downstream signaling is involved in IL-17A hypersecretion. Here, we show that inhibitors of adenyl cyclase (AC), protein kinase A (PKA), and cAMP response element binding protein (CREB) (which are signaling molecules downstream of the Gs protein coupled to the A2aR), suppress IL-17A production, suggesting that activation of A2aR signaling induces IL-17A production by CD4⁺ T cells. Furthermore, immune subset studies revealed that adenosine induces hypersecretion of IL-17A by T-helper (Th)17 cells. These results indicate that adenosine is an endogenous modulator of neutrophilic inflammation. Administration of an A2aR antagonist to mice with experimental autoimmune encephalomyelitis led to marked amelioration of symptoms. Thus, inhibitors of the novel A2aR-AC-cAMP-PKA-CREB signaling pathway for IL-17A hypersecretion by TCR-activated Th17 cells suppresses adenosine-mediated IL-17A production, suggesting that it may be an effective treatment for Th17-related autoimmune diseases.

Highlights

• •Adenosine upregulates interleukin (IL)-17A secretion in a mixed lymphocyte reaction (MLR) via adenosine A2a receptor (A2aR).
• •Adenosine production is induced in the MLR.
• •Adenosine induces hypersecretion of IL-17A by T-helper (Th)17 cells via A2aR signaling.
• •Adenosine induces hypersecretion of Th17-related cytokines IL-17F and IL-22.
• •Istradefylline, an A2aR antagonist, ameliorates experimental autoimmune encephalomyelitis in SJL mice.

Abbreviations

Th

T-helper

CD

cluster of differentiation

TGF

transforming growth factor

IL

interleukin

APCs

antigen presenting cells

AC

adenyl cyclase

PKA

protein kinase A

CREB

cAMP response element binding protein

MLR

two-way mixed lymphocyte reaction

Ab

antibody

CD3/CD28

agonistic anti-CD3 and CD28 antibodies

TCR

T cell receptor

MHC

major histocompatibility complex

EAE

experimental autoimmune encephalomyelitis

CCPA

2-Chloro-N6-cyclopentyladenosine

AMP-CP

adenosine 5'-(α, β-methylene) diphosphate

BM

bone marrow

BM-DC

bone marrow-derived dendritic cell

Ct

cycle threshold

PLP

myelin proteolipid protein

PLP peptide

I–A^s restricted helper peptide derived from the PLP

MACS

magnetic-activated cell sorting

CCR

C-C chemokine receptor

CFA

complete Freund's adjuvant

FITC

fluorescein isothiocyanate

PE

phycoerythrin

SD

standard deviation

n

number of repeat experiments

1. Introduction

T-helper (Th)17 cells are a subset of T-helper cells induced by stimulation of naïve cluster of differentiation (CD)4⁺ T cells with both transforming growth factor (TGF)-β1 and interleukin (IL)-6 in the presence of T cell receptor signaling. IL-17A production by Th17 cells increases neutrophilic inflammation (Ivanov et al., 2007; Manel et al., 2008; Matsuzaki and Umemura, 2007); however, not all neutrophilic inflammatory diseases are explained by known Th17 responses (Tesmer et al., 2008). Indeed, it is likely that as-yet-unknown Th17 or neutrophilic inflammatory responses occur. Here, we show that an adenosine-mediated signaling pathway induces IL-17A production by CD4⁺ T cells directly. Extracellular adenosine is one of the first “signals” identified during regulation of a large number of physiological and pathological processes, including bulging of an artery (Cronstein and Sitkovsky, 2017), sleep promotion (Jones, 2009), and regulation of nerve action (Boison et al., 2010; Sebastiao and Ribeiro, 2009). Extracellular adenosine is produced from secreted ATP that undergoes rapid stepwise dephosphorylation by ectonucleotidases such as the ecto-nucleoside triphosphate diphosphohydrolase CD39, which converts ATP or ADP to ADP or AMP, respectively, and the 5′-nucleotidase CD73, which dephosphorylates AMP to adenosine (Yegutkin, 2008); both CD39 and CD73 are expressed by activated CD4⁺ T cells and antigen presenting cells (APCs) (Allard et al., 2017; Whiteside et al., 2011). Extracellular adenosine stimulates adenosine receptors (A1R, A2aR, A2bR, and A3R) belonging to a superfamily of membrane proteins called the G protein-coupled receptor family of class A seven-transmembrane domain receptors. A2aR and A2bR signal the Gs protein to trigger cAMP synthesis, which in turn activates adenyl cyclase (AC), protein kinase A (PKA), and cAMP response element binding protein (CREB) (Hasko et al., 2008; van Calker et al., 2019). By contrast, A1R and A3R signal the Gi protein to trigger cAMP degradation. In addition, A2bR and A3R also signals the Gq protein, which in turn activates phospholipase C. In an immunological context, adenosine receptors are expressed by various immune cells, including T cells and APCs (Junger, 2011). With respect to the effect of adenosine on Th cells, previous studies show that adenosine induces hypersecretion of IL-17A by human CD4⁺ T cells treated with an A2aR agonist (PSB0777) (Tokano et al., 2022), as well as inducing Th17 differentiation via activation of A2bR on murine CD4⁺ T cells (Wilson et al., 2011). However, the signaling pathway (downstream of adenosine-mediated IL-17A hypersecretion by CD4⁺ T cells and immune subsets) that induces adenosine-mediated IL-17A hypersecretion is unclear. The aim of this study was to use a two-way mixed lymphocyte reaction (MLR) and agonistic anti-CD3 and CD28 antibody (Ab) (CD3/CD28)-stimulated CD4⁺ T cells to identify the signaling pathway and immune subsets responsible for adenosine-mediated IL-17A hypersecretion. In the MLR, we mixed allogeneic splenic lymphocytes from BALB/c mice (I-A^d MHC class II molecule) and SJL mice (I–A^s MHC class II molecule), leading to selective stimulation of CD4⁺ effector T cells via the T cell receptor (TCR)-allogeneic major histocompatibility complex (MHC) class II molecule interaction (McDevitt, 2000). Agonistic anti-CD3 and CD28 Abs can also stimulate TCRs expressed on the surface of CD4⁺ T cells (Gross et al., 1992; Leo et al., 1987). Moreover, since aberrant IL-17A production correlates with the symptoms of inflammatory autoimmune diseases, we administered an A2aR antagonist (Istradefylline) to mice with experimental autoimmune encephalomyelitis (EAE) (Kleinewietfeld et al., 2013; Nakano et al., 2008), and confirmed amelioration of symptoms (as previously reported) using another A2aR antagonist (SCH58261) (Mills et al., 2008). Taken together, the data presented herein indicate that adenosine-dependent hypersecretion of IL-17A by Th17 cells contributes not only to antibacterial defense but also to neutrophilic autoimmune disease, and that suppressing this process may be an effective therapy for the latter.

2. Materials and methods

2.1. Mice

BALB/c mice were obtained from Japan SLC, Inc. SJL/J mice were obtained from Charles River Laboratories Japan, Inc. Mice were housed in appropriate animal care facilities at Saitama Medical University and handled according to international guidelines for experiments with animals. All experiments were approved by the Animal Research Committee of Saitama Medical University.

2.2. Two-way MLR

Splenic lymphocytes were collected by lyzing tissue in a Dounce homogenizer, followed by layering over Ficoll Paque (GE health care, Chicago, IL, USA), as described previously (Higashi et al., 2011). BALB/c splenic lymphocytes (3 × 10⁶) were mixed with SJL/J splenic lymphocytes (3 × 10⁶) in 2 mL of DMEM medium containing 10% FCS, 100 U/ml penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 μM 2-mercaptoethanol (D10 medium) in 12 well plates in the presence of adenosine (0–1 mM) (Sigma, St. Louis, MO, USA); in the presence of each adenosine receptor agonist (0–10 μM) (A1R: 2-Chloro-N6-cyclopentyladenosine (CCPA), Tocris, Bristol, UK; A2aR: PSB0777, Tocris; A2bR: BAY 60–6583, Tocris; A3R: HEMADO, Tocris); in the presence of A2aR antagonist (0–1 nM) (Istradefylline, Sigma) plus adenosine (100 μM); in the presence of an AC inhibitor (0–1 μM) (MDL-12330A, Enzo Life Sciences, Farmingdale, NY, USA), a PKA inhibitor (0–1 μM) (H-89, Tocris) plus adenosine (100 μM), or a CREB inhibitor (0.1–0.6 μM) (666-15, Cayman Chemical Company, Ann Arbor, MI, USA) plus adenosine (100 μM) (to inhibit A2aR signaling); in the presence of an activator of AC (0.06–0.6 μM) (Forskolin, Sigma); or in the presence of a CD39 inhibitor (0–1 μM) (ARL67156, Tocris) or a CD73 inhibitor (0–1 μM) (adenosine 5'-(α, β-methylene) diphosphate (AMP-CP; Tocris) plus ATP (100 μM) (GE healthcare). After mixing, the plates were incubated for 7 days at 37 °C. The supernatants were collected for use in cytokine ELISAs.

2.3. Isolation of CD4⁺, CD4⁺CD62L⁺ T cells, and B cells

CD4⁺ T cells within the BALB/c and SJL/J splenocyte populations were isolated from the mixture prepared previously after rupturing red blood cells (Kawano et al., 2018). Cells were isolated by positive selection of CD4⁺ T cells using magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's instructions. CD4⁺CD62L⁺ T cells were isolated from BALB/c splenocytes by a combination of negative and positive selection by MACS. During positive selection of CD62L⁺ T cells, negatively isolated CD4⁺ T cells were collected as the flow through fraction. B cells were isolated from BALB/c splenocytes by positive selection using MACS. Cells were resuspended in 1 mL of D10 medium and counted. The purity and viability of CD4⁺ T cells, CD4⁺CD62L⁺ T cells, and B cells were >90% (Sup. Fig. 1). The purity and viability of cells in the flow through fraction collected during isolation of CD4⁺CD62L⁺ T cells are shown in Supplementary Fig. 1.

2.4. Cell sorting

BALB/c CD4⁺ T cells (prepared as described above) were labeled for 30 min at 4 °C with PE-conjugated anti-mouse C-C chemokine receptor (CCR)3, CCR5, CCD6, CD25, or CD62L Abs (BioLegend). The cells were then washed and sorted using a FACS Aria II flow cytometer (BD Biosciences). The purity and viability of the sorted cells are Supplementary Fig. 1.

2.5. CD3/CD28 stimulation

CD4⁺ T cells (1 × 10⁶) were stimulated for 7 days at 37 °C with anti-mouse CD3 (BioLegend; 1 μg/mL) and CD28 (BioLegend; 0.5 μg/mL) Abs in the presence of adenosine (0–1 mM), each adenosine receptor agonist (0–10 μM; A1R: 2-Chloro-N6-cyclopentyladenosine (CCPA), Tocris, Bristol, UK; A2aR: PSB0777, Tocris; A2bR: BAY 60–6583, Tocris; and A3R: HEMADO, Tocris), an A2aR antagonist (Istradefylline; 0–1 nM) plus adenosine (600 μM), an AC inhibitor (0–1 μM; MDL-12330A), a PKA inhibitor (0–1 μM) (H-89), a CREB inhibitor (0.1–0.6 μM; 666-15) plus adenosine (600 μM), or an activator of AC (0.06–0.6 μM; Forskolin) in 500 μL of D10 medium. After cell sorting, cells (3 × 10⁵) were plated in 24 well plates and stimulated for 7 days at 37 °C with anti-mouse CD3 (1 μg/mL) and CD28 (0.5 μg/mL) Abs in the presence of adenosine (600 μM) in 100 μL of D10 medium. After stimulation, the supernatants were collected for use in cytokine ELISAs.

2.6. Adenosine or ATP ELISAs

MLR was performed by mixing BALB/c lymphocytes (6 × 10⁶) with SJL/J lymphocytes (6 × 10⁶) in a 15 mL tube for 0–24 h at 37 °C in the presence of a CD39 inhibitor (ARL67156) (0–1 μM) and a CD73 inhibitor (AMP-CP) (0–1 μM) in 200 μL of D10 medium. CD3/CD28 stimulation was performed for 24 h at 37 °C in a 15 mL tube by incubating BALB/c CD4⁺ T cells (1 × 10⁷) with anti-mouse CD3 (1 μg/mL) and CD28 (0.5 μg/mL) Abs plus ARL67156 (1 μM) or AMP-CP (1 μM) in 200 μL of D10 medium. Simulation with LPS or an anti-mouse CD40 Ab was performed for 24 h at 37 °C in a 15 mL tube; briefly, BALB/c B cells (1 × 10⁷) or BALB/c bone marrow (BM)-derived dendritic cells (BM-DCs) (1 × 10⁷), generated from mouse bone marrow cells as described previously (Lutz et al., 1999), were incubated with LPS (Sigma; 0.5 μg/mL) or anti-mouse CD40 Ab (5 μg/mL; BioLegend) plus ARL67156 (1 μM) or AMP-CP (1 μM) in 200 μL of D10 medium. The purity and viability of BM-DCs were >90% (Sup. Fig. 1). After incubation, the supernatants were collected and tested in adenosine or ATP ELISAs (Biovision, Milpitas, CA, USA).

2.7. Differentiation of naïve CD4⁺ T cells

Naïve CD4⁺ T cells (3 × 10⁵) in 500 μL of D10 medium in 24 well plates were stimulated for 7 days at 37 °C with anti-mouse CD3 (1 μg/mL) and CD28 (0.5 mg/mL) Abs plus mouse IL-6 (20 ng/mL) (Peprotech, Rocky Hill, NJ), and human TGF-β1 (2 ng/mL) (Peprotech) in the presence of an A2aR antagonist (Istradefylline) (0–1 nM). After incubation, the supernatants were collected for use in cytokine ELISAs, and total RNA was extracted from the cells using a Sepasol-RNA I Super G (Nacalai Tesque, Kyoto, Japan) for quantitative PCR. In another experiment, cells in 500 μL of D10 medium were stimulated for another 7 days at 37 °C with anti-mouse CD3 (1 μg/mL) and CD28 (0.5 μg/mL) Abs in the presence of Istradefylline (0.1–1 nM) plus adenosine (600 μM). Supernatants were collected for use in cytokine ELISAs.

2.8. Quantitative PCR

cDNA was synthesized from total RNA obtained in 2.7 using Superscript IV VILO master mix (Invitrogen, Waltham, MA, USA), according to the manufacturer's instructions. Quantitative PCR (qPCR) was performed as described previously (Joseph et al., 2012) using the primer pairs described below, Power SYBR green PCR Master mix (Applied Biosystems, Waltham, MA, USA), and a QuantStudio 12K Flex (Applied Biosystems) equipped with QuantStudio 12K Flex software (Applied Biosystems). Next, relative gene expression was calculated using cycle threshold (Ct) values: ΔCt (Ct of IL-17A – Ct of GAPDH) (Nobles et al., 2010; Robinson et al., 2020; Silver et al., 2006). The following forward and reverse primers, respectively, were used (all from Eurofins Scientific, Luxembourg): GAPDH, 5′-AGCTTGTCATCAACGGGAAG-3′ and 5′-TTTGATGTTAGTGGGGTCTCG-3’; IL-17A, 5′-TGTGAAGGTCAACCTCAAAGTCT-3′ and 5′-GAGGGATATCTATCAGGGTCTTCAT-3’.

2.9. EAE model

EAE was induced as described previously (Nakano et al., 2008). Briefly, SJL/J mice received a subcutaneous inguinal injection (100 μg/mouse) of the proteolipid protein (PLP) peptide (PLP139-151, Tocris) emulsified in complete Freund's adjuvant (CFA) containing mycobacterium tuberculosis H37Ra (100 μg/mouse; Difco, Detroit, MI, USA). Mice also received oral water or an A2aR antagonist (Istradefylline; 6 μg/mouse) once every 2 days from Day −7 to Day +18 after immunization with PLP peptide (Day 0). Mice were examined daily for signs of EAE, which were graded as described (Podojil et al., 2011).

2.10. Histological analysis of spinal cord samples

On Day 18 post-induction, histological analysis was performed by excising the lumbar, thoracal, and cervical parts of the spinal cord. Paraffin-embedded spinal cord sections were stained with hematoxylin and eosin. T cells were labeled with an anti-mouse CD3ε Ab (Cell Signaling technology, Danvers, MA, USA). Tissue sections were observed using the light microscopy mode of an inverted fluorescence phase-contrast microscope (BZ-9000, Keyence, Osaka, Japan).

2.11. Peptide pulse assay

At 7 days post-subcutaneous immunization with PLP peptide emulsified in CFA, splenocytes (2 × 10⁶ in 200 μL of D10 medium) were seeded in 96-well round plates and pulsed for 3 days at 37 °C with PLP peptide (10 μM) in the presence of adenosine (600 μM) and an A2aR antagonist (Istradefylline; 0–1 nM). For the oral administration experiments using the A2a antagonist, splenocytes (2 × 10⁶ in 200 μL of D10 medium) or inguinal lymph node lymphocytes, prepared as described previously (Barthelmes et al., 2016) (1 × 10⁶ in 200 μL of D10 medium) were seeded in 96-well round plates and pulsed for 3 days at 37 °C; this was done at 7 days post-subcutaneous immunization with PLP peptide emulsified in CFA. Mice also received oral water, an A2aR agonist (PSB0777) (6 μg/mouse), or an A2aR antagonist (Istradefylline; 6 μg/mouse) once every 2 days from Day −7 to Day +7 after immunization with PLP peptide (Day 0). At 18 days post-subcutaneous immunization with PLP peptide emulsified in CFA, spinal cord cells prepared as described previously (Barthelmes et al., 2016) (100 μL of resuspended in 500 μL of D10 medium/mouse) were seeded in 96-well round plates and pulsed for 3 days at 37 °C with PLP peptide (10 μM). The supernatants were collected for use in cytokine ELISAs.

2.12. Flow cytometry analysis

The MLR was performed for 7 days in the presence or absence of adenosine (100 μM) (as described above). For the CD4⁺IL-17A⁺ study, cells were blocked with anti-mouse CD16/CD32 Abs (BioLegend, San Diego, CA, USA) and stained for 30 min at 4 °C with a fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 Ab (BioLegend). The cells were then fixed, permeabilized, and stained with a phycoerythrin (PE)-conjugated anti-mouse IL-17A Ab (BioLegend). For the Th subset study, after 0, 3, and 7 days of the MLR assay, cells were blocked with anti-mouse CD16/CD32 Abs (BioLegend) and then stained for 30 min at 4 °C with a FITC-conjugated anti-mouse CD4 Ab (BioLegend) and PE-conjugated anti-mouse CCR5 (a Th1 marker (Takeuchi et al., 2005)), CCR3 (a Th2 marker (Ma et al., 2002)), or CCR6 (a Th17 marker (Acosta-Rodriguez et al., 2007; Annunziato et al., 2007)) Abs (BioLegend). For the MHC, costimulatory molecule, and A2aR expression studies in APCs, BALB/c B cells (3 × 10⁵) or BALB/c BM-DCs (3 × 10⁵) seeded in the wells of a 96-well in 200 μL of D10 medium were incubated with LPS (0.5 μg/mL; Sigma) or an anti-mouse CD40 Ab (5 μg/mL) (BioLegend) for 1 day. Next, supernatants were collected for cytokine ELISAs, or blocked with anti-mouse CD16/CD32 Abs (BioLegend) and then stained for 30 min at 4 °C with FITC-conjugated anti-mouse CD80, CD86, or CD40 Abs and corresponding isotype controls (BioLegend) (for the costimulatory study), with PE-conjugated anti-mouse H-2 or I-A/I-E Ab and corresponding isotype controls (BioLegend) (for the MHC study), or fixed, permeabilized, and stained with a PE-conjugated anti-A2aR Ab (Novus Biologicals, Minneapolis, MN, USA) and corresponding isotype control (for the A2aR expression study). For the A2aR expression study in CD4⁺ T cells, CD4⁺ T cells (1 × 10⁶) were stimulated for 7 days at 37 °C with anti-mouse CD3 (1 μg/mL) and CD28 (0.5 μg/mL) Abs in the presence of adenosine (0–1 mM). At 0–7 days post-stimulation, cells were blocked with anti-mouse CD16/CD32 Abs (BioLegend) and then stained for 30 min at 4 °C with a FITC-conjugated anti-mouse CD4 Ab (BioLegend) before fixation, permeabilization, and staining with a PE-conjugated anti-A2aR Ab (Novus Biologicals). For EAE analysis at 18 days post-subcutaneous immunization with PLP peptide emulsified in CFA, splenocytes (1 × 10⁶), inguinal lymph node lymphocytes (1 × 10⁶), and spinal cord cells (100 μL of resuspended in 500 μL of D10 medium/mouse) were blocked with anti-mouse CD16/CD32 Abs (BioLegend) and then stained for 30 min at 4 °C with a FITC-conjugated anti-mouse CD4 Ab (BioLegend) and PE-conjugated anti-mouse CCR5 (a Th1 marker (Takeuchi et al., 2005)), CCR3 (a Th2 marker (Ma et al., 2002)), or CCR6 (a Th17 marker (Acosta-Rodriguez et al., 2007; Annunziato et al., 2007)) Abs (BioLegend). Finally, the cells were washed and analyzed on a FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ) using FACSDiva acquisition software (BD Biosciences).

2.13. Cytokine ELISAs

The concentrations of IFN-γ (range, 15.6–2000 pg/ml), IL-5 (range, 15.6–2000 pg/ml), IL-6 (range, 15.6 to 1000 pg/ml), IL-17A (15.6 to 1000 pg/ml), IL-17F (11.7 to 1500 pg/ml), IL-22 (15.6 to 2000 pg/ml), IL-23 (9.77 to 2500 pg/ml), and TGF-β1 (15.6 to 2000 pg/ml) in cell supernatants were measured using specific ELISA kits (DuoSet Kit, R&D, Minneapolis, MN, USA). Plates were read in a microplate reader at an absorbance wavelength of 450 nm (Model 550, Bio-Rad, Richmond, CA). Then, the concentration was read off a four-parameter logistic curve constructed using standards as part of the kit (Microplate manager III, Bio-Rad). Any value below the lower limit of detection (15.6 pg/mL) was set to 0. No cytokine cross-reactivity was observed within the detection ranges of the kits. If necessary, samples were diluted appropriately so that the measurements fell within the detection range for each cytokine.

2.14. Statistical analysis

Differences between two groups were analyzed using an unpaired Student's t-tests. Differences between three or more groups were analyzed using one-way ANOVA with Tukey's post-hoc test. Clinical scores were analyzed using a non-parametric Mann-Whitney U test. All calculations were performed using KaleidaGraph software (Synergy software, Reading, PA, USA). A P value < 0.05 was considered statistically significant. We tested each group for outliers using the 3σ test. Any value outside the mean ± three standard deviations (SDs) was classed as an outlier (Hawkins, 1980); this test confirmed that the data analyzed statistically in this study contained no outliers.

3. Results

3.1. Adenosine promotes IL-17A production by CD4⁺ T cells in an MLR

First, we analyzed the effect of adenosine on CD4⁺ T cells during T cell-APC interactions in an MLR (McDevitt, 2000). We found that CD4⁺ T cells exposed to adenosine secreted IL-17A in a dose-dependent manner (Fig. 1A–C). Since both agonist-mediated IL-17A production and antagonist-mediated suppression of adenosine-mediated IL-17A production were observed in the presence of an adenosine A2aR agonist (PSB0777) and an antagonist (Istradefylline), respectively (Fig. 1D and Sup. Fig. 2A), we hypothesized that IL-17A in the MLR was produced by CD4⁺ T cells stimulated via the A2aR. This notion was supported by the finding that AC, PKA, and CREB inhibitors of signaling molecules downstream of the A2aR also suppressed IL-17A production (Fig. 1E). Consistent with this notion, activation of A2aR downstream of AC upregulated IL-17A production (Fig. 1E). In the MLR, the Th1 and Th17 cell populations increased slightly but significantly (by approximately 1.7-fold) in the presence of 100 μM adenosine at Day 7 (Sup. Fig. 2C). Considering that 100 μM adenosine increased IL-17A secretion by approximately 10-fold compared with medium alone (Fig. 1B), it appears that adenosine-mediated upregulation of IL-17A is the main contributor to upregulation of the IL-17A production by CD4⁺ T cells. Furthermore, ATP induced IL-17A production in the MLR, which was suppressed by the A2aR antagonist and by inhibitors of CD39 and CD73 (Fig. 1F), suggesting that adenosine plays a role in IL-17A production. Since the A2aR antagonist and inhibitors of CD39 and CD73 also inhibited production of IL-17A in the MLR (Fig. 1G), we postulate that de novo adenosine production is induced in the MLR.

Fig. 1.

Adenosine induces hypersecretion of IL-17A by CD4⁺ T cells in an MLR. A–C, An MLR was performed for 7 days in the presence of adenosine (0–1 mM). After 7 days, cells were stained with anti-CD4 (x-axis) and IL-17A (y-axis) antibodies, followed by flow cytometry analysis (A, n (number of repeat experiments) = 4). The percentage of IL-17A-producing CD4⁺ T cells within the total CD4⁺ T cell population is shown (B, n = 6–9). Data (n = 4–9) shown in Fig. 1A and B were obtained from 2–3 independent experiments (n = 2–3 mice/group). Cells supernatants were analyzed in an IL-17A ELISA (C, n = 6–9). D, The effects of the A2aR on IL-17A production in the presence of PSB0777 (an A2aR agonist) (left, n = 6–9), Istradefylline (an A2aR antagonist) plus adenosine (100 μM) (right, n = 6–9). E, The effects of A2aR signaling on IL-17A production in the presence of MDL-12330A (an adenyl cyclase inhibitor) plus adenosine (100 μM) (first panel, n = 4–6), H-89 (a protein kinase A inhibitor) plus adenosine (100 μM) (second panel, n = 4–6), 666-15 (a cAMP response element binding protein inhibitor) plus adenosine (100 μM) (third panel, n = 4–6), and Forskolin (an activator of adenyl cyclase) (fourth panel, n = 4–6) were analyzed in an IL-17A ELISA. F, The effects of CD39/CD73 inhibitors on IL-17A production in the presence of ARL67156 (a CD39 inhibitor) plus ATP (100 μM) (left, n = 4–6) or AMP-CP (a CD73 inhibitor) plus ATP (100 μM) (right, n = 4–6) were analyzed in an IL-17A ELISA. G, The effects of an A2aR antagonist and CD39/CD73 inhibitors on basal IL-17A production in the presence of Istradefylline (left, n = 4–7), ARL67156 (center, n = 4–7), or AMP-CP (right, n = 4–7) were analyzed in an IL-17A ELISA. Data (n = 4–9) shown in Fig. 1C–G were obtained from 2–3 independent experiments (n = 2–3 mice/group). Data are expressed as the mean ± standard deviation (SD) and were compared using an unpaired Student's t-test (B) or one-way ANOVA with Tukey's post-hoc test (C–G). *P < 0.05 and **P < 0.01, compared with medium (C, D, left; E, fourth panel; and G), adenosine (100 μM) (D, right, E, first–third panels), or ATP (100 μM) (F).

3.2. Adenosine production in the MLR

CD39 and CD73 expressed on the surface of endothelial cells (Jalkanen and Salmi, 2008; Kaczmarek et al., 1996) and immune cells (Allard et al., 2017; Whiteside et al., 2011), including T cells and DCs, are critical for production of adenosine from ATP. Since we found that inhibitors of CD39 and CD73 inhibited basal IL-17A production in the MLR (Fig. 1G), we next addressed the source of adenosine production in the MLR. As shown in Fig. 2A, production of adenosine and ATP was time-dependent. In accordance with suppression of IL-17A production, inhibitors of CD39 and CD73 suppressed adenosine production at 24 h after the start of the MLR (Fig. 2B and C). Since the MLR induces activation of CD4⁺ T cells by APCs (McDevitt, 2000), we also addressed adenosine production by activated CD4⁺ T cells, B cells, and BM-DCs. Production of both adenosine and ATP by activated CD4⁺ T cells (Fig. 2D), B cells (Fig. 2E), and BM-DCs (Fig. 2F) was observed; inhibitors of CD39 and CD73 suppressed production by activated CD4⁺ T cells (Fig. 2D), although production of both adenosine and ATP by CD4⁺ T cells (Fig. 2D), B cells (Fig. 2E), and BM-DCs (Fig. 2F) was observed in the absence of LPS or anti-CD40 Ab stimulation. This suggests the possibility that adenosine stimulation is mediated by contact between CD4⁺ T cells and other immune cell, and that adenosine produced by CD4⁺ T cells or other immune cells such as B cells may induce adenosine-mediated IL-17A hypersecretion by CD4⁺ T cells in the MLR.

Fig. 2.

Adenosine production in the MLR. A, Concentrations of adenosine or ATP in MLR supernatants were measured in an adenosine (left, n = 4–6) or ATP (right, n = 4–6) ELISA (0–24 h). B and C, The effects of CD39 (B, ARL67156, n = 4–6) and CD73 (C, AMP-CP, n = 4–6) inhibitors on production of adenosine or ATP in the MLR were analyzed in an adenosine (left) or ATP (right) ELISA. D, E and F, Levels of adenosine and ATP in the supernatants of anti-CD3 and CD28 antibody (Ab)-stimulated CD4⁺ T cells (D, n = 4), LPS- or anti-mouse CD40 Ab-stimulated B cells (E, n = 4), and LPS- or anti-mouse CD40 Ab-stimulated BM-DCs (F, n = 4) were analyzed in adenosine and ATP ELISAs at 24 h post-stimulation in the presence of CD39 or CD73 inhibitors. Data (n = 4–6) shown in Fig. 2 were obtained from 2–3 independent experiments (n = 2–3 mice/group). Data are expressed as the mean ± SD and were compared using one-way ANOVA with Tukey's post-hoc test. *P < 0.05 and **P < 0.01, compared with medium (B and C), CD3/CD28 stimulation (D), or LPS or anti-mouse CD40 Ab stimulation (E and F).

3.3. Th17 cells hypersecrete IL-17A in the presence of anti-CD3 and CD28 Abs and adenosine

Next, we tried to identify the Th subset that generated IL-17A in the presence of adenosine. First, we confirmed that CD4⁺ T cells expressed the A2aR and secreted IL-17A by stimulating them with agonistic anti-CD3 and CD28 Abs in the presence of adenosine (Fig. 3A). Time course studies showed that adenosine-mediated IL-17A production was detected from 3 days post-CD3/CD28 stimulation (Fig. 3B and Sup. Fig. 3). To clarify the importance of the timing, we altered the time of adenosine administration post-CD3/CD28 stimulation (0, 6, 24, or 72 h) during the 7 days of CD3/CD28 stimulation (Fig. 3C). During the 7 days of CD3/CD28 stimulation, administration of adenosine within 6 h of Ab stimulation triggered IL-17A production; however, administration at 24 h post-stimulation did not (Fig. 3C). We noticed that A2aR expression by CD4⁺ T cells was upregulated significantly (2–4.7 fold) by anti-mouse CD3 and CD28 Abs at 1–7 days after stimulation, and that this upregulation was further (slightly but significantly) enhanced (by approximately 1.5-fold) by increasing amounts of adenosine (Sup. Fig. 4A). As in the MLR, CD4⁺ T cells also produced IL-17A after stimulation with anti-CD3 and CD28 Abs in the presence of an A2aR agonist (Fig. 3D and Sup. Fig. 2B). Adenosine-mediated IL-17A production by CD3/CD28-stimulated CD4⁺ T cells was suppressed by an A2aR antagonist (Fig. 3D and Sup. Fig. 2B). This was supported by data showing that inhibitors of signaling molecules downstream of the A2R also suppressed IL-17A production, whereas activation of A2aR downstream of AC upregulated the production (Fig. 3E). Production of other Th17-related cytokines (IL-17F and IL-22) was also induced by adenosine and the A2aR agonist (PSB0777), whereas adenosine-mediated production of IL-17F and IL-22 was suppressed by the A2aR antagonist (Istradefylline) in both the MLR (Fig. 4A) and in CD3/CD28-stimulated CD4⁺ T cells (Fig. 4B). This again suggests that activated CD4⁺ T cells produce IL-17A upon A2aR activation. Furthermore, we noticed that CD4⁺CD62L⁻, but not CD4⁺CD62L⁺, cells produced IL-17A after CD3/CD28 stimulation in the presence of adenosine, suggesting that adenosine induces IL-17A production by effector Th cells (Fig. 3F). Therefore, we performed immune subset studies after separating CD4⁺ T cells using anti-CCR Abs. As shown in Fig. 3G, CD4⁺CCR6^hi T cells produced IL-17A upon stimulation of CD3/CD28, and production was strongly upregulated in the presence of adenosine. Since CCR6 is a typical marker of Th17 cells (Acosta-Rodriguez et al., 2007; Annunziato et al., 2007), this suggests that activated Th17 cells hypersecrete IL-17A in the presence of adenosine.

Fig. 3.

Adenosine induces hypersecretion of IL-17A by Th17 cells. A and B, CD4⁺ T cells were stimulated for 1–7 days with anti-CD3 and CD28 antibodies (Abs) in the presence of adenosine (0–1 mM). After stimulation, the supernatants were analyzed in an IL-17A ELISA (n = 4–6). C, Adenosine (600 μM) was added at 0–3 days after CD3/CD28 stimulation. At 7 days post-CD3/CD28 stimulation, supernatants were analyzed in an IL-17A ELISA (n = 4). D, Effects of the A2aR on IL-17A production in the presence of PSB0777 (an A2aR agonist) (left, n = 4–6), Istradefylline (an A2aR antagonist) plus adenosine (600 μM) (right, n = 4–6). E, Effects of A2aR signaling on IL-17A production in the presence of MDL-12330A (an adenyl cyclase inhibitor) plus adenosine (600 μM) (first panel, n = 4–6), or H-89 (a protein kinase A inhibitor) plus adenosine (600 μM) (second panel, n = 4–6), 666-15 (a cAMP response element binding protein inhibitor) plus adenosine (600 μM) (third panel, n = 4–6), or Forskolin (an activator of adenyl cyclase) (fourth panel, n = 4–6) were analyzed in an IL-17A ELISA. F, CD4⁺CD62L⁺ and CD4⁺CD62L⁺FT cells were stimulated for 7 days with anti-CD3 and CD28 Abs in the presence of adenosine (0–1 mM). After 7 days, supernatants were analyzed in an IL-17A ELISA (n = 4). G, Subsets of CD4⁺ T cells were stimulated for 7 days by anti-CD3 and CD28 Abs in the presence of adenosine (600 μM) after isolation of each CCR cell type (high (hi) and low (lo) expression) (n = 4). Data (n = 4–6) shown in Fig. 3 were obtained from 2–3 independent experiments (n = 2–3 mice/group). Data are expressed as the mean ± SD and were compared using an unpaired Student's t-test (G) or one-way ANOVA with Tukey's post-hoc test (A–F). *P < 0.05 and **P < 0.01, compared with CD3/CD28 stimulation (A–C, D, left;E, fourth panel; and F) or CD3/28 stimulation plus adenosine (600 μM) (D, right and E, first–third panels).

Fig. 4.

Effect of adenosine and an A2aR antagonist on production of Th17-related cytokines. A, An MLR was performed for 7 days in the presence of adenosine (0–1 mM) (top row), PSB0777 (an A2aR agonist) (second row), or Istradefylline (an A2aR antagonist) plus adenosine (100 μM) (bottom row). At 7 days post-incubation, the supernatants were analyzed in an IL-17A (left), IL-17F (center), or IL-22 (right) ELISA (n = 6–9). Data (n = 6–9) shown in Fig. 4A were obtained from three independent experiments (n = 2–3 mice/group). B, CD4⁺ T cells were stimulated with an anti-CD3 and CD28 antibodies for 7 days in the presence of adenosine (0–1 mM) (top row), PSB0777 (an A2aR agonist) (second row), or Istradefylline (an A2aR antagonist) plus adenosine (600 μM) (bottom row). After stimulation, supernatants were analyzed in IL-17A (left), IL-17F (center), and IL-22 (right) ELISAs (n = 4–6). Data (n = 4–6) shown in Fig. 4B were obtained from two independent experiments (n = 2–3 mice/group). Data are expressed as the mean ± SD and were compared using one-way ANOVA with Tukey's post-hoc test. *P < 0.05 and **P < 0.01, compared with medium or CD3/CD28 stimulation (top and second row of panels) or adenosine (100 μM) or CD3/CD28 stimulation plus adenosine (600 μM) (third row of panels).

To address the effect of adenosine on APCs, we analyzed production of IL-6, IL-23, and, TGF-β1 by B cells and BM-DCs (Sup. Fig. 4B). Also, we analyzed the effect of adenosine on expression of CD80/CD86/CD40, MHC class I/class II, and A2aR by B cells and BM-DCs (Sup. Fig. 4C). Increasing amounts of adenosine suppressed IL-6 production by LPS-stimulated B cells. and IL-6 and IL-23 production by LPS-stimulated BM-DCs. Expression of CD80/CD86/CD40, MHC class I/class II, and A2aR by LPS or anti-CD40 Ab-stimulated-B cells and -BM-DCs in the presence of 100 μM and 600 μM adenosine was 0.70- and 1.31-fold higher than that by cells stimulated by LPS or anti-CD40 Ab alone. These data indicate that adenosine mainly affects CD4⁺ T cells to activate Th17 function.

3.4. An adenosine A2aR antagonist ameliorates IL-17A-related autoimmune EAE responses

The above results raise the possibility that adenosine-mediated hypersecretion of IL-17A by Th17 cells contributes to Th17-related autoimmune diseases. This hypothesis is supported by a report showing that CD73 knockout mice are resistant to EAE (Mills et al., 2008), a Th17-mediated autoimmune disease (Kleinewietfeld et al., 2013). We expected, therefore, that A2aR antagonist-mediated suppression of Th17 responses should improve EAE. To address this, we examined the efficacy of an A2aR antagonist in EAE model SJL/J mice (Nakano et al., 2008). EAE was induced by immunization of mice with an I–A^s restricted helper peptide derived from a myelin PLP peptide comprising amino acids 139–151 (HSLGKWLGHPDKF). The peptide was emulsified in CFA. First, we confirmed that the A2aR antagonist suppressed adenosine-mediated IL-17A production by CD3/CD28-stimulated CD4⁺ T cells from SJL/J strain mice (Fig. 5A). The A2aR antagonist also significantly suppressed adenosine-mediated IL-17A production after differentiation of Th17 cells from naïve CD4⁺ T cells (Fig. 5B, right). The A2aR antagonist did not suppress IL-17A expression or production during differentiation of Th17 cells from naïve CD4⁺ T cells (Fig. 5B, left and center). By contrast, and in agreement with Fig. 3G, adenosine administration did not induce IL-17A production during and after differentiation of Th1, Th2, and Treg cells from naïve CD4⁺ T cells (data not shown). Next, we pulsed splenocytes with the PLP peptide after immunization to confirm that IL-17A production was induced in a peptide-dependent manner, and that production was upregulated by adenosine. As expected, IL-17A production occurred in a peptide-dependent manner and was upregulated by adenosine (Fig. 5C). Furthermore, the A2aR antagonist suppressed production of IL-17A, suggesting that the A2aR antagonist inhibits IL-17A production by CD4⁺ T cells induced by immunization with the PLP peptide. We next addressed the effect of an oral A2aR antagonist on production of IL-17A by CD4⁺ T cells induced by immunization with the PLP peptide (Fig. 5D). Oral administration of PSB0777 or Istradefylline did not significantly induce or suppress IL-17A production by PLP peptide-pulsed splenocytes and inguinal lymph node lymphocytes. These results indicate that the A2aR antagonist may not inhibit PLP peptide-specific Th17-generation but rather suppress adenosine-mediated IL-17A hypersecretion by PLP peptide-specific Th17 cells. Since oral administration of PSB0777 did not up-regulate IL-17A production, adenosine-mediated IL-17A production may be saturated by de novo extracellular adenosine in the body. Hence, we expect that the A2aR antagonist may suppress adenosine-mediated IL-17A hypersecretion by PLP peptide-specific Th17 cells during migration to the inflammatory site, which may suppress EAE symptoms. Finally, the A2aR antagonist was administered orally to mice before and during EAE induction (Fig. 5E and F). As shown, the clinical scores of mice receiving the A2aR antagonist were markedly lower than those of control mice (receiving water) at 18 days post-immunization with the PLP peptide (Fig. 5E). Accordingly, histological studies showed that the numbers of central nervous system-infiltrating CD3⁺ cells in mice receiving the A2aR antagonist were much lower than those in mice receiving water (Fig. 5F). Consistent with these data, flow cytometry analysis showed that the proportion of CD4⁺ cells in total counts in the spinal cord were reduced significantly by the oral A2aR antagonist; in particular, the percentage of Th17 cells in the spinal cord was reduced significantly (Fig. 5G), suggesting the A2aR antagonist suppressed migration of CD4⁺ T cells to, and retained Th17 cells within, the spinal cord during EAE induction. Accordingly, PLP peptide-dependent production of IL-17A was observed in spinal cord cells after oral administration of water; production was suppressed significantly by the oral A2aR antagonist (Fig. 5H). These results suggest that the A2aR antagonist suppresses hypersecretion of IL-17A by PLP peptide-specific Th17-cells, resulting in impaired migration from the draining lymph nodes to the inflammatory site, and subsequent retention at the inflammatory site. These phenomena may suppress IL-17A-mediated EAE symptoms.

Fig. 5.

Suppression of adenosine-mediated hypersecretion of IL-17A ameliorates EAE. A and B, SJL/J CD4⁺ T cells were stimulated for 7 days by anti-CD3 and CD28 antibodies (Abs) and an A2aR antagonist (A, n = 4). Data (n = 4) shown in Fig. 5A were obtained from three independent experiments (n = 1–2 mice/group). Naïve CD4⁺ T cells were stimulated for 7 days with anti-CD3 and CD28 Abs in the presence of IL-6, TGF-β1, and Istradefylline (an A2aR antagonist) (0–1 nM); expression of mRNA encoding IL-17A was measured by qPCR (B, center, n = 4) and IL-17A protein was measured in a cytokine ELISA (B, left, n = 4). Alternatively, naïve CD4⁺ T cells were stimulated for 7 days by anti-CD3 and CD28 Abs in the presence of IL-6 and TGF-β1, followed by another 7 day incubation with anti-CD3 and CD28 Abs and Istradefylline (0–1 nM) (B, right, n = 4). After the stimulation, supernatants were analyzed in an IL-17A ELISA. Data (n = 4) shown in Fig. 5B were obtained from two independent experiments (n = 2 mice/group). C and D, Mice were immunized subcutaneously with PLP peptide emulsified in CFA (PLP peptide/CFA). Mice received oral water, an A2aR agonist (PSB0777; 6 μg/mouse), or an A2aR antagonist (Istradefylline; 6 μg/mouse) once every 2 days from Day −7 to Day +7 after immunization with PLP peptide/CFA (Day 0) (D). At 7 days post-immunization, splenocytes were incubated for 3 days with PLP peptide in the presence of Istradefylline and adenosine (600 μM) (C, n = 4) or in the presence or absence of PLP peptide (D, n = 4). After the incubation, supernatants were analyzed in an IL-17A ELISA. Data (n = 4) shown in Fig. 5C and D were obtained from two independent experiments (n = 2 mice/group). E, To induce EAE, SJL/J mice were immunized with PLP peptide/CFA. Before and post-immunization with the PLP peptide (Days 0–18), mice received oral Istradefylline (6 μg/mouse) or water once every 2 days. Clinical scores were recorded every day during EAE induction (E, n = 15). Data (n = 15) shown in Fig. 5E were obtained from five independent experiments (n = 3 mice/group). F, At 18 days post-immunization, spinal cord sections from mice administered oral Istradefylline (right) or water (left) were stained with hematoxylin and eosin (upper panels) or with an anti-mouse CD3 Ab (lower panels) (Scale bar, 100 μm) (F, n = 5). Data (n = 5) shown in Fig. 5F were obtained from two independent experiments (n = 2–3 mice/group). Data are representative of two independent experiments. G and H, At 18 days post-immunization, splenocytes (spleen, left), inguinal lymph node lymphocytes (inguinal lymph node, center), and spinal cord cells (spinal cord, right) from mice administered oral water (left of each panel) or Istradefylline (right of each panel) were stained with anti-CD4 (x-axis) and CCR5 (a Th1 marker; top panels, y-axis), CCR3 (a Th2 marker; second panels, y-axis), or CCR6 (a Th17 marker; bottom panels, y-axis) Abs, followed by flow cytometry analysis (G, n = 5). Data (n = 5) shown in Fig. 5G were obtained from two independent experiments (n = 2–3 mice/group). Values shown in the upper and lower right of each panel denote the percentage of CCR-positive counts within the total CD4-positive counts, and the percentage of CD4-positive counts within the total count, respectively. *P < 0.05. Also, spinal cord cells were incubated for 3 days in the presence or absence of PLP peptide (H, n = 4). After the incubation, supernatants were analyzed in an IL-17A ELISA. Data (n = 4) shown in Fig. 5H were obtained from two independent experiments (n = 2 mice/group). Data are expressed as the mean ± SD. Data were compared using one-way ANOVA with Tukey's post-hoc test (A–D, G, and H), or using a non-parametric Mann-Whitney U test (E). *P < 0.05 and **P < 0.01, compared with CD3/CD28 stimulation plus adenosine (600 μM) (A), CD3/CD28 stimulation plus IL-6 and TGF-β1 (B), PLP peptide pulse (C), oral water (G), or PLP peptide pulse (oral water) (H).

4. Discussion

In this study, we confirmed that an A2aR agonist (PSB0777) induced IL-17A hypersecretion by TCR-activated CD4⁺ T cells not only in humans (Tokano et al., 2022) but also in mice; this hypersecretion was suppressed by an A2aR antagonist (Istradefylline). In addition, and to the best of our knowledge, we reveal for the first time that inhibitors of the Gs downstream signaling pathway AC-cAMP-PKA-CREB also inhibit adenosine-mediated IL-17A hypersecretion by TCR-activated Th17 cells in mice (Fig. 6). Because IL-17A plays a central role in the accumulation of neutrophils at inflammatory sites by stimulating stromal cells and epithelial cells to produce neutrophil chemoattractants (e.g., CXCL2 and CXCL8) for antibacterial defense, and because aberrant IL-17A production contributes to the development of neutrophilic autoimmune diseases, suppressing IL-17A production should ameliorate the symptoms of such diseases. As expected, the A2aR antagonist Istradefylline ameliorated the symptoms of EAE, a result that agrees with a previous observation that the A2aR antagonist SCH58261 ameliorates EAE symptoms (Mills et al., 2008).

Fig. 6.

Schematic representation of A2aR downstream signaling leading to IL-17A hypersecretion by TCR-activated Th17 cells.

IL-17A hypersecretion by TCR-activated Th17 cells is induced by the A2aR downstream AC (adenyl cyclase)-cAMP-PKA (protein kinase A)-CREB (cAMP response element binding protein) signaling pathway. To avoid crosstalk between AC-cAMP-PKA-CREB signaling and TCR signaling, A2aR expression is upregulated after TCR signaling.

G, guanine nucleotide-binding regulatory protein; PLC, phospholipase C; DAG, diacylglycerol; IP₃, inositol trisphosphate; PKC, protein kinase C.

Addition of adenosine (1 mM) to a two-way MLR increased IL-17A production to > 25 times the basal level; however, both basal production and increased production of IL-17A were suppressed by an A2aR antagonist, and by CD39/CD73 inhibitors. This indicates that hypersecretion of IL-17A in the presence of adenosine occurs by other mechanisms in addition to T cell-APC interactions. Since endothelial cells and nervous system cells also express CD39/CD73 (Braun et al., 2000; Jalkanen and Salmi, 2008; Kaczmarek et al., 1996; Kulesskaya et al., 2013) and produce adenosine (Gunther and Herring, 1991; Sebastiao and Ribeiro, 2009), and activated CD4⁺ T cells in the present study hypersecreted IL-17A at 6 h post-CD3/CD28 stimulation, it is possible that activated Th17 cells also receive adenosine from endothelial and neuronal cells to induce hypersecretion of IL-17A.

We hypothesize that adenosine affects multiple immunological events as follows: After APCs take up antigen they move to the secondary lymphoid organs. Followed by an innate immune response in the secondary lymphoid organs, APCs induce Th differentiation from naïve CD4⁺ T cells. In particular, APCs induce Th17 differentiation by secreting IL-6 and TGF-β1. Then, the transcription factor RORγt switches on the IL-17A gene by binding to the IL-17A promoter (Ivanov et al., 2006). During this response, adenosine rarely stimulates naïve CD4⁺ T cells due to low levels of A2aR expression (Sup. Fig. 4A). Following an adaptive immune response, APCs in the secondary lymphoid organs stimulates effector Th17 cells and drives expression of the IL-17A gene by recruiting transcription factors such as NF-κB and NFAT downstream of the TCR signaling pathway (Gaud et al., 2018). In this response, A2aR expression is also upregulated (Sup. Fig. 4A), thereby increasing and sensitivity of A2aR signaling in response to adenosine. After stimulation, Th17 cells migrate toward the inflammatory site. During migration, activated Th17 cells receive adenosine from immune cells, neural cells, endothelial cells, or in an autocrine manner. During this process, Th17 cells further up-regulate expression of the IL-17A gene by recruiting transcription factors such as CREB downstream of the A2aR-cAMP-PKA pathway (Hernandez et al., 2015). Then, Th17 cells induce IL-17A hypersecretion at the inflammatory site. In humans, the transcription factor cAMP-responsive element modulator α increases transcription of the human Il-17 gene by binding to the promoter region (Rauen et al., 2011). The A2aR antagonist may suppress adenosine-mediated IL-17A hypersecretion by adenosine-stimulated Th17 cells at the inflammatory site, and subsequently suppress neutrophil-mediated inflammatory responses and Th cell migration. This hypothesis is consistent with a previous EAE study showing that CD73 expression and adenosine receptor signaling are required for efficient entry of lymphocytes into the central nervous system during EAE development (Mills et al., 2008); our own data suggest that CD73 expression and adenosine receptor signaling may be required to induce IL-17A hypersecretion by Th17 cells around the inflammatory site. γδ T cells, which are categorized mainly into two subpopulations (CD8αα⁺ and CD8⁻ cells), express RORγt and are also a source of IL-17A (Kadivar et al., 2016; Khairallah et al., 2018). We hypothesize that the A2aR antagonist will also suppress adenosine-mediated IL-17A production by adenosine-stimulated γδ T cells through A2aR signaling (Liang et al., 2018).

During the sequence of immunological events described above, TCR signaling up-regulates phosphodiesterase activity and suppresses cAMP signaling during the adaptive responses in the secondary lymphoid organs (Bjørgo et al., 2011). The cAMP–PKA signaling pathway plays a major role in regulating immune responses, and cAMP is the most potent and acute inhibitor of T cell activation (Bjørgo et al., 2011). Our data indicate that upregulation of A2aR expression is induced after TCR stimulation (Sup. Fig. 4A); therefore, adenosine-mediated IL-17A hypersecretion is induced after stimulation of TCR signaling as seen in Fig. 3C. Therefore, extracellular adenosine may not block TCR signaling in the body. Also, a specific amount of Forskolin may not block TCR signaling, leading to subsequent upregulation of IL-17A secretion by TCR-activated CD4⁺ T cells (Fig. 1, Fig. 3E). We hypothesize that extracellular adenosine-mediated IL-17A hypersecretion is induced after activation of TCR signaling during migration from the secondary lymphoid organs to the inflammatory site. In the MLR and the PLP peptide pulse experiment, both activated Th17 cells and adenosine-stimulated Th17 cells might produce IL-17A during the T-APC interaction. Thus, inhibitors of CD39/CD73 and an A2aR antagonist may suppress adenosine-mediated hypersecretion by adenosine-stimulated TCR-activated Th17 cells.

It is suggested that physiological concentrations of adenosine are lower than 1 μM; however, they can be increased by stimuli such as high K⁺ levels, electrical stimulation, glutamate receptor agonists, hypoxia, hypoglycemia, and ischemia (Latini and Pedata, 2001). To obtain sufficient adenosine (>100 μM) to trigger hypersecretion of IL-17A, activated Th17 cells may need to make contact with non-immune cells such as adenosine-producing endothelial cells (Carman and Martinelli, 2015) and neuronal cells (Siffrin et al., 2010) to form a microenvironment with a high adenosine concentration (as observed during T cell-APC interactions at immunological synapses) (Monks et al., 1997). Thus, A2aR antagonists, rather than CD39/CD73 inhibitors, might be more effective at inhibiting de novo adenosine-mediated hypersecretion of IL-17 by Th17 cells. A previous study suggests that intracellular adenosine is transported out of cells by efficient equilibrative transporters (Fredholm, 2007); CD39/CD73 inhibitors would not suppress this type of de novo adenosine production.

With regard to the effect of adenosine on other Th subsets, our observations were different from those of previous reports (Csoka et al., 2008; Lappas et al., 2005); here, we observed that adenosine upregulated IFN-γ (a Th1-related cytokine) secretion at 5 and 7 days and had no significant effect on IL-5 (a Th2-related cytokine) secretion by CD4⁺ T cells after CD3/CD28 stimulation with 600 μM of adenosine, although IL-17A production was significant (Sup. Fig. 3). This suggests that adenosine induces hypersecretion of IL-17A by Th17 cell but does not suppress Th1 and Th2 activity. However, previous studies report that adenosine-mediated suppression of IFN-γ and IL-5 was observed 1 day after T cell receptor-mediated stimulation of CD4⁺ T cells (Csoka et al., 2008; Lappas et al., 2005). This may indicate that in the short term adenosine prioritizes stimulation of Th17 cell activity rather than that of Th1 and Th2 cells, and that it does not suppress effector Th activity in the long term.

It is also suggested that the A2aR agonist, CGS21680, suppresses Th17 differentiation (Alchera et al., 2017; Ansari et al., 2017; Wang et al., 2018). This result is opposite to ours; one reason for this may be differences in the source of the A2aR agonist. The A2aR agonist CGS 21680 is much less selective than the A2aR agonist that we used in this study (PSB0777); this is because CGS21680 binds not only to the A2aR but also to A1R and A3R, which are associated with the Gi protein (which has opposite effects to the Gs protein) (El-Tayeb et al., 2011; Klotz, 2000). Therefore, it is probable that CGS21680 may cancel out any agonist effects by activating A1R and A3R. Also, it is suggested that the A2aR antagonist, SCH58261, up-regulates Th17 differentiation in mice (Ansari et al., 2017). We hypothesized that upregulation of Th17 differentiation by SCH58261 may be induced through relative downregulation of A2aR activity compared with that of A2bR; this relative increase in A2bR activity induces Th17 differentiation in mice (Kenakin, 2001; Prather, 2004). Also, it is probable that SCH58261 induces relative increase in the activity of G protein-coupled receptors other than adenosine receptors (e.g., dopamine receptors) to induce Th17 differentiation in mice (Nakano et al., 2011). We also hypothesize that although SCH58261 may induce Th17 differentiation in vivo, it may not stimulate Th17 activity; this is because our data show that an A2a antagonist (Istradefylline) suppressed IL-17A secretion by differentiated Th17 cells but did not suppress Th17 differentiation (Fig. 5B). This hypothesis is supported by previous data showing that SCH58261 markedly suppresses symptoms of EAE, a typical Th17-mediated disease (Mills et al., 2008).

Our data suggest that production of IL-17A is relatively higher after exposure to an A2aR agonist, PSB0777, than after exposure to an A2bR agonist, BAY 60–6583. PSB0777 is a potent adenosine A2aR agonist (Ki = 44.4 nM for rat brain striatal A2aR) (El-Tayeb et al., 2011), and BAY 60–6583 is a potent adenosine A2bR agonist (Ki = 100 nM for rat A2bR) (Alnouri et al., 2015). By assuming that the Ki values of PSB0777 and BAY 60–6583 are comparable, we thought that production of IL-17A mediated by activation of the A2aR might be higher than that mediated by activation of the A2bR. This hypothesis is supported by the notion that the A2aR is a high affinity receptor with activity in the low to mid-nanomolar range, whereas the A2bR has a much lower affinity for adenosine (micromolar) (Cronstein and Sitkovsky, 2017); this suggests that adenosine activates the A2aR rather than the A2bR.

A2aR antagonists have been developed for treatment of Parkinsonism (Kanda et al., 1998) and malignancies (Ohta et al., 2006). In addition, inhibitors of CD39/CD73 have been developed as anti-tumor drugs (Deaglio et al., 2007; Kobie et al., 2006). Regarding the effects of adenosine on tumor immunity, a previous study suggests that adenosine suppresses effector T cell function since tumor cells express both CD39 and CD73 and secrete adenosine (Moesta et al., 2020). By contrast, several reports suggest that IL-17A promotes emergence of pro-tumorigenic neutrophil phenotypes (Coffelt et al., 2015; Tuting and de Visser, 2016). Neutrophils in mouse tumor models promote tumor metastasis (Bald et al., 2014; El Rayes et al., 2015; Wculek and Malanchi, 2015), and observations in cancer patients have linked elevated neutrophil counts in blood with increased risk of metastasis (Templeton et al., 2014). Therefore, it is probable that tumor-produced adenosine induces IL-17A secretion by CD4⁺CCR6^hi T cells followed by neutrophilic inflammation, which promotes tumor metastasis. Cancer vaccines may need to be administered along with an A2aR antagonist to suppress hypersecretion of IL-17A by tumor-specific Th17 cells induced by tumor-produced adenosine, and to inhibit neutrophilic inflammation at the tumor site.

Although we have demonstrated that adenosine-mediated IL-17A hypersecretion by TCR-activated Th17 cells occurs through the A2aR signaling, we mimicked TCR-mediated activation of Th cells in secondary lymph nodes using an MLR and ex vivo stimulation with anti-CD3 and CD28 Abs. Also, we hypothesize that adenosine-mediated IL-17A hypersecretion is induced after migration from the secondary lymph nodes to peripheral sites of inflammation; this is because expression of A2aR by CD4⁺ T cells is upregulated after CD3/CD28 stimulation, and adenosine release by endothelial cells is induced (Jalkanen and Salmi, 2008; Kaczmarek et al., 1996). However, we have not demonstrated that adenosine-mediated IL-17A hypersecretion by TCR-activated Th17 cells occurs via A2aR signaling in vivo. To better understand the physiological importance of IL-17A hypersecretion for neutrophilic inflammation, in vivo experiments should be performed to confirm the results presented herein.

5. Conclusion

Here, we show that adenosine-mediated IL-17A hypersecretion by Th17 cells is induced through the A2aR signaling pathway AC-cAMP-PKA-CREB, and that inhibitors of the pathway suppress IL-17A hypersecretion. Since IL-17A secretion induces neutrophil inflammation by stimulating stromal cells and epithelial cells to produce neutrophil chemoattractants, inhibitors may be effective therapies for antibacterial neutrophilic inflammation. Also, because neutrophilic inflammation plays a role in many Th17-mediated inflammatory autoimmune diseases, inhibitors of the A2aR signaling pathway AC-cAMP-PKA-CREB may also be effective treatments for these diseases (Tesmer et al., 2008), which include psoriasis, neutrophilic bronchial asthma, severe atopic dermatitis, and autoimmune diseases. Moreover, these drugs might be effective treatments for diseases caused by neutrophilic inflammation of unknown etiology in the dermis, including Behcet uveitis (Yazici et al., 2018) and vasculitis caused by adenosine deaminase 2 deficiency (Meyts and Aksentijevich, 2018). This is because endothelial cells express CD39/CD73 and produce adenosine, which may induce hypersecretion of IL-17A by Th17 cells, thereby contributing to inflammation.

Author contributions

M.T., R.T., S.M., and M.K., performed the experiments. M.T., S.M., and M.K., conceived and designed the experiments. M.T., S.M., T.Y., and M.K., wrote the manuscript. All authors discussed the results and commented on the manuscript.

Declaration of competing interest

Sho Matsushita is an employee of iMmno, Inc.

The other authors have no conflicts of interest to declare.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Sup. Fig. 1

Purity and viability of each immune cell subset. A, First row (left): Splenocytes or isolated CD4⁺ T cells were stained with a FITC-conjugated anti-mouse CD4 antibody (Ab) (BioLegend) (x-axis) and a PE-conjugated anti-mouse CD3 Ab (BioLegend) (y-axis). Second row (left): Splenocytes or CD4⁺CD62L⁺ T cells isolated by MACS were stained with a FITC-conjugated anti-mouse CD4 Ab (BioLegend) (x-axis) and a PE-conjugated anti-mouse CD62L Ab (BioLegend) (y-axis). First row (right): Splenocytes and B cells were stained with a FITC-conjugated anti-mouse CD19 Ab (BioLegend) (x-axis) and cell numbers were counted (y-axis). Second row (right): Splenocytes, BM leukocytes, and BM-DCs were stained with a FITC-conjugated anti-mouse CD11c Ab (BioLegend) (x-axis) and cell numbers were counted (y-axis). The number in each panel represents the percentage ± SD of each immune subset within the total cell population (n = 3). B, First row: Isolated CD4⁺ (by MACS)-, CD4⁺CD62L⁻ (by cell sorting)-, or CD4⁺CD62L⁺ (by cell sorting) T cells were analyzed with a PE-conjugated anti-mouse CD62L Ab (BioLegend) (x-axis) and cell numbers were counted (y-axis). The number in each panel represents the percentage of each immune subset (- or +) within the total cell population (- plus +). Data are representative of at least three repeat experiments. Second row: Isolated CD4⁺ (by MACS), CD4⁺CCR3^low(lo) (by cell sorting), or CD4⁺CCR3^high(hi) (by cell sorting) T cells were analyzed with a PE-conjugated anti-mouse CCR3 Ab (BioLegend) (x-axis) and cell numbers were counted (y-axis). Third row: Isolated CD4⁺ (by MACS), CD4⁺CCR5^lo (by cell sorting), or CD4⁺CCR5^hi (by cell sorting) T cells were analyzed with a PE-conjugated anti-mouse CCR5 Ab (BioLegend) (x-axis) and cell numbers were counted (y-axis). Fourth row: Isolated CD4⁺ (by MACS), CD4⁺CCR6^lo (by cell sorting), or CD4⁺CCR6^hi (by cell sorting) T cells were analyzed with a PE-conjugated anti-mouse CCR6 Ab (BioLegend) (x-axis) and cell numbers were counted (y-axis). Fifth row: Isolated CD4⁺ (by MACS), CD4⁺CD25^lo (by cell sorting), or CD4⁺CD25^hi (by cell sorting) T cells were analyzed with a PE-conjugated anti-mouse CD25 Ab (BioLegend) (x-axis) and cell numbers were counted (y-axis). The number in each panel represents the mean percentage ± SD of each immune subset (hi or lo) within the total cell population (hi plus lo) (n = 3). Data (n = 3) shown in Sup.Fig. 1A and B were obtained from three independent experiments (n = 1 mice/group). C, After cell isolation, each immune cell type was mixed with Trypan blue. Viability was calculated as the number of unstained cells/(stained cells + unstained cells) × 100 (n = 3). The percentage represents the mean percentage ± SD.

Sup. Fig. 2

Effect of an adenosine receptor agonist and antagonist on production of IL-17A in an MLR and by CD3/CD28-stimulated CD4⁺ T cells, and the effect of adenosine on proliferation of Th cells. A, An MLR was performed for 7 days in the presence of each adenosine receptor agonist, or each adenosine receptor antagonist plus adenosine (100 μM) (n = 6–9). B, CD4⁺ T cells were stimulated with an anti-CD3 and CD28 antibodies (Abs) for 7 days in the presence of each adenosine receptor agonist, or in the presence of each adenosine receptor antagonist plus adenosine (600 μM) (n = 6–9). After 7 days, the supernatants were analyzed in an IL-17A ELISA. First row: CCPA (an A1R agonist). Second row: PSB0777 (an A2aR agonist, left) and Istradefylline (an A2aR antagonist, right). Third row: BAY 60–653 (an A2bR agonist). Forth row: HEMADO (an A3R agonist). Data (n = 6–9) shown in Sup.Fig. 2A and B were obtained from 2–3 independent experiments (n = 2–3 mice/group). C, An MLR was performed in the presence or absence of adenosine (100 μM). After 0, 3, and 7 days, splenocytes were stained with anti-CD4 (x-axis) and CCR5 (a Th1 marker; top panels, y-axis), CCR3 (a Th2 marker; second panels, y-axis), or CCR6 (a Th17 marker; bottom panels, y-axis) Abs, followed by flow cytometry analysis (n = 3). Values in the upper right of each panel denote the percentage of CCR-positive counts within the CD4 positive counts. Data (n = 3) shown in Sup. Fig. 2C were obtained from three independent experiments (n = 1 mice/group). *P < 0.05 and **P < 0.01. Data are expressed as the mean ± SD and are compared using one-way ANOVA with Tukey's post-hoc test. *P < 0.05 and **P < 0.01, compared with medium (A, left; B, left; and C), adenosine (100 μM) (A, right), CD3/28 stimulation plus adenosine (600 μM) (B, right).

Sup. Fig. 3

Effect of adenosine on production of IFN-γ, IL-5, and IL-17A by CD3/CD28-stimulated CD4⁺ T cells. CD4⁺ T cells were stimulated for 1–7 days with anti-CD3 and CD28 antibodies in the presence or absence of adenosine (600 μM). After stimulation, supernatants were analyzed in IFN-γ (top row), IL-5 (second row), and IL-17A (bottom row) ELISAs (n = 4–6). Data (n = 4–6) shown in Sup.Fig. 3were obtained from 2–3 independent experiments (n = 2–3 mice/group). Data are expressed as the mean ± SD and were compared using one-way ANOVA with Tukey's post-hoc test. *P < 0.05 and **P < 0.01, compared with CD3/CD28 stimulation.

Sup. Fig. 4

Effect of adenosine on A2aR expression by CD4⁺ T cells, and on expression of MHC, costimulatory molecules, and A2aR and production of IL-6, IL-23, and TGF-β by APCs. A, CD4⁺ T cells were stimulated for 0–7 days with anti-CD3 and CD28 antibodies (Abs) in the presence of adenosine (0–1 mM). After stimulation, cells were stained with a FITC-conjugated anti-mouse CD4 Ab (BioLegend) (x-axis) and a PE-conjugated anti-A2aR Ab (Novus Biologicals) (y-axis). First row: Splenocytes (left) or isolated CD4⁺ T cells (right) were stained with anti-CD4 (x-axis) and A2aR (y-axis) Abs. Second row: CD3/CD28-stimulated CD4⁺ T cells were stained with anti-CD4 (x-axis) and A2aR (y-axis) Abs. Third row: CD4⁺ T cells were stimulated with anti-CD3 and CD28 Abs in the presence of adenosine (0–1 mM) and then stained with anti-CD4 (x-axis) and A2aR (y-axis) Abs. The number in each panel represents the percentage ± SD of A2aR^high cells within the CD4⁺ population (n = 3). Data were compared using one-way ANOVA with Tukey's post-hoc test. *P < 0.05 and **P < 0.01, compared with CD3/CD28 stimulation (second and third rows). B, B cells and BM-DCs were stimulated for 1 day with LPS (upper panels) or anti-mouse CD40 Ab (CD40) (lower panels) in the presence of adenosine (0.1–1 mM). After stimulation, the IL-6, IL-23, and TGF-β1 concentrations in the supernatants were analyzed by ELISA (n = 3). Data are expressed as the mean ± SD and were compared using one-way ANOVA with Tukey's post-hoc test. *P < 0.05 and **P < 0.01, compared with LPS or anti-mouse CD40 Ab stimulation. C, B cells (upper panels) and BM-DCs (lower panels) were stimulated for 1 day with LPS or anti-mouse CD40 Ab (CD40) in the presence of adenosine (100 or 600 μM). After stimulation, the cells were stained with a FITC-conjugated anti-mouse CD80 (BioLegend), CD86 (BioLegend), or CD40 (BioLegend) Ab, PE-conjugated anti-mouse H-2 (BioLegend) or I-A/I-E (BioLegend) Ab, or a PE-conjugated anti-A2aR (Novus Biologicals) Ab (x-axis), and cell numbers were counted (y-axis) (n = 3). The number in each panel represents the mean fluorescence intensity ± SD (n = 3). Data were compared using one-way ANOVA with Tukey's post-hoc test. *P < 0.05 and **P < 0.01, compared with LPS or anti-mouse CD40 Ab stimulation. Data (n = 3) shown in Sup.Fig. 4were obtained from three independent experiments (n = 1 mice/group).

Data availability

Data will be made available on request.