Adenosine A2A receptors induced on iNKT and NK cells reduce pulmonary inflammation and injury in mice with sickle cell disease

Abstract

We showed previously that pulmonary function and arterial oxygen saturation in NY1DD mice with sickle cell disease (SCD) are improved by depletion of invariant natural killer T (iNKT) cells or blockade of their activation. Here we demonstrate that SCD causes a 9- and 6-fold induction of adenosine A(2A) receptor (A(2A)R) mRNA in mouse pulmonary iNKT and natural killer (NK) cells, respectively. Treating SCD mice with the A(2A)R agonist ATL146e produced a dose-dependent reversal of pulmonary dysfunction with maximal efficacy at 10 ng/kg/minute that peaked within 3 days and persisted throughout 7 days of continuous infusion. Crossing NY1DD mice with Rag1(-/-) mice reduced pulmonary injury that was restored by adoptive transfer of 10(6) purified iNKT cells. Reconstituted injury was reversed by ATL146e unless the adoptively transferred iNKT cells were pretreated with the A(2A)R alkylating antagonist, FSPTP (5-amino-7-[2-(4-fluorosulfonyl)phenylethyl]-2-(2-furyl)-pryazolo[4,3-ε]-1,2,4-triazolo[1,5-c]pyrimidine), which completely prevented pro-tection. In NY1DD mice exposed to hypoxia-reoxygenation, treatment with ATL146e at the start of reoxygenation prevented further lung injury. Together, these data indicate that activation of induced A(2A)Rs on iNKT and NK cells in SCD mice is sufficient to improve baseline pulmonary function and prevent hypoxia-reoxygenation-induced exacerbation of pulmonary injury. A(2A) agonists have promise for treating diseases associated with iNKT or NK cell activation.

Figure 1

Lung lymphocytes from NY1DD mice have higher A_2AR mRNA than congenic C57BL/6 controls. Live (DAPI⁻) CD45⁺ pulmonary cells were sorted based on surface antigen staining. (A) iNKT (CD1d-tetramer⁺, CD3⁺), NK (NKp46⁺, CD3⁻), and CD3⁺ T cells (CD1d-tetramer⁻, CD3⁺). Quantitative RT-PCR was used to measure A_2AR transcript levels in sorted populations of pulmonary lymphocytes compared with a housekeeper transcript (cyclophilin). (B) NY1DD mice have increased levels of A_2AR transcripts in iNKT cells and NK cells, but not in CD3⁺ tetramer⁻ T cells compared with C57BL/6 mice. (C) NY1DD iNKT and NK cells have increased A_2AR/cyclophilin mRNA ratios compared with NY1DD CD3⁺ tetramer⁻ T cells.

Figure 2

Dose dependence of ATL146e to reduce lung injury and inflammation in NY1DD mice. NY1DD mice (N = 4) were treated for 3 days with a constant infusion of vehicle or ATL146e (1 ng/kg/minute, 10 ng/kg/minute, and 30 ng/kg/minute, osmotic pump). All parameters were found to be maximally improved by 10 ng/kg/minute. The solid and dashed lines represent the mean ± SEM of parameters from congenic C57BL/6 mice. Data were analyzed by 1-way ANOVA with Neuman-Keuls posttesting. *P < .05 vs day 0.

Figure 3

Time course of ATL146e effects in NY1DD mice. NY1DD mice were treated with osmotic minipumps infusing vehicle (■) or ATL146e (10 ng/kg/minute, osmotic pump, ▾). Pulmonary parameters were measured in NY1DD mice treated for 0, 1, 2, 3, or 7 days after the start of infusion. All parameters were maximally improved by 3 days after the start of ATL146e treatment, because no further changes were noted on day 7. The solid and dashed lines represent the mean ± SEM of parameters from congenic C57BL/6 mice. Data were analyzed by 1-way ANOVA with Neuman-Keuls posttesting. *P < .05 vs day 0.

Figure 4

ATL146e treatment decreases pulmonary dysfunction in NY1DD mice by activation of the A_2AR. C57BL/6, NY1DD, or NY1DD x A_2AR^−/− mice were treated with ATL146e (10 ng/kg/minute, 3 days) or vehicle (saline, 0.2% DMSO). (A-F) Lung sections from NY1DD mice, but not NY1DD x A_2AR^−/− mice have reduced alveolar thickening and reduced vaso-occlusion after ATL146e treatment. (G-H) NY1DD mice, but not NY1DD x A_2AR^−/− mice, had decreased vascular permeability and increased oxygen saturation after ATL146e treatment. (I-J) NY1DD mice, but not NY1DD x A_2AR^−/− mice, displayed improved breathing parameters after ATL146e treatment. Data were analyzed by 1-way ANOVA with Neuman-Keuls posttesting; *P < .05. SO₂: arterial oxygen saturation.

Figure 5

ATL146e treatment decreases pulmonary injury during hypoxia-reoxygenation (H-R) of NY1DD mice. NY1DD mice were subjected to 3 hours of hypoxia (8% oxygen) and 18 hours of reoxygenation. Lungs were then removed, inflation-fixed, and stained with H&E or immunostained for neutrophils. Three hours after reoxygenation, mice were implanted with Alzet minipumps containing either vehicle (A) or ATL146e (10 ng/kg/minute; B). The sections shown are typical of 4 replicates. Bars, 200 μm.

Figure 6

Time course of reversal from pulmonary protection after cessation of ATL146e. NY1DD mice were treated with ATL146e (10 ng/kg/minute, 3 days osmotic pump). Pulmonary parameters were measured on days 3, 4, and 7 after the cessation of ATL146e infusion. All parameters measured (vascular permeability [EBD], breathing measurements [TV, FOB], and pulmonary cell infiltrates) returned toward NY1DD baseline levels on day 4, 1 day after the cessation of ATL146e treatment, and returned to baseline levels by day 7. The solid and dashed lines represent the mean ± SEM of breathing parameters in C57BL/6 mice. Data were analyzed by 1-way ANOVA with Neuman-Keuls posttesting. *P < .05.

Figure 7

Protective effects of ATL146e and anti-CD1d treatments to reduce pulmonary inflammation in NY1DD. NY1DD mice were treated with either ATL146e (10 ng/kg/minute, 3 days), anti-CD1d (10 μg/g/day, 2 days), or a combination of both. All treatments resulted in significantly improved pulmonary parameters. (A-E) Representative H&E staining from sections of lungs at baseline illustrating reduced alveolar thickening and vaso-acclusion as a result of ATL146e or anti-CD1d treatment. (F-H) ATL146e or anti-CD1d treatment improved parameters of pulmonary function in NY1DD mice. Data were analyzed by 1-way ANOVA with Neuman-Keuls posttesting. P < .05, +NY1DD vs C57BL/6; *Different from NY1DD controls; # > anti-CD1d alone.

Figure 8

ATL146e targets A_2ARs on iNKT cells to decrease pulmonary inflammation. NY1DD x Rag1^−/− mice were pretreated with ATL146e (10 ng/kg/minute, 3 days). Two days later, 10⁶ NKT cells pretreated in vitro with vehicle or 200nM concentrations of the A_2AR alkylating agent, FSPTP, were adoptively transferred. Pulmonary parameters were analyzed on day 3 by 1-way ANOVA with Neuman-Keuls posttesting. P < .05: + vs B6; * vs SCD; ** vs SCD - Rag1; # vs SCD-Rag1 + NKT cells; *** vs ATL146e pretreated SCD x Rag1 + NKT cells. B6 = C57BL6; SCD = NY1DD; ViNKT = vehicle-treated iNKT cells; FiNKT = FSPTP-treated iNKT cells.