Infarct-Sparing Effect of Adenosine A2B Receptor Agonist Is Primarily Due to Its Action on Splenic Leukocytes Via a PI3K/Akt/IL-10 Pathway

Abstract

Background: Adenosine A2B receptor (A_2BAR) agonist reduces myocardial reperfusion injury by acting on inflammatory cells. Recently, a cardiosplenic axis was shown to mediate the myocardial postischemic reperfusion injury. This study aimed to explore whether the infarct-squaring effect of A_2BAR agonist was primarily due to its action on splenic leukocytes.

Methods: C57BL6 (wild type [WT]) mice underwent 40 min of left coronary artery occlusion followed by 60 min of reperfusion. A_2BAR knockout (KO) and interleukin (IL)-10KO mice served as donors for splenic leukocytes. Acute splenectomy was performed 30 min before ischemia. The acute splenic leukocyte adoptive transfer was performed by injecting 5 × 10⁶ live splenic leukocytes into splenectomized mice. BAY 60-6583, an A_2BAR agonist, was injected by i.v. 15 min before ischemia. The infarct size (IS) was determined using 2,3,5-triphenyltetrazolium chloride and Phthalo blue staining. The expression of p-Akt and IL-10 was estimated by Western blotting. Immunofluorescence staining assessed the localization of IL-10 expression.

Results: BAY 60-6583 reduced the myocardial IS in intact mice but failed to reduce the same in splenectomized mice, which had a smaller IS than intact mice. BAY 60-6583 reduced the IS in splenectomized mice with the acute transfer of WT splenic leukocytes; however, it did not protect the heart of splenectomized mice with the acute transfer of A_2BRKO splenic leukocytes. Furthermore, BAY 60-6583 increased the levels of p-Akt and IL-10 in the WT spleen. Moreover, it did not exert any protective effect in IL-10KO mice.

Conclusions: A_2BAR activation before ischemia stimulated the IL-10 production in splenic leukocytes via a PI3K/Akt pathway, thereby exerting anti-inflammatory effects that limited the myocardial reperfusion injury.

Keywords: Adenosine A2B receptor; Myocardial reperfusion injury; PI3K/Akt; Splenic leukocytes.

Figure 1.. Experimental protocol.

SPAT, splenic-leukocyte reconstituted mice; SPLX, splenectomy.

Figure 2.. Myocardial IS measured using TTC and Phthalo blue staining.

BAY 60–6583 significantly reduced the myocardial IS as compared to the IR group but failed to reduce that of the splenectomized mice, which had a smaller IS than the intact mice. The reconstitution of splenic leukocytes from either WT or A2bARKO mice to splenectomized mice restored the IS to the level of the IR group. BAY 60–6583 reduced the IS of the WT splenic-leukocyte reconstituted mice but failed to protect the heart of A_2BARKO splenic- leukocyte reconstituted mice. The risk regions were not different among all groups. IR, myocardial ischemia and reperfusion injury; SPATA2B, A_2BARKO splenic-leukocyte reconstituted mice; SPATWT, wild-type splenic-leukocyte reconstituted mice; SPLX, splenectomy.

Figure 3.. Western blotting results of p-Akt levels in BAY 60–6583-treated non- injured mice.

BAY 60–6583 significantly increased the p-Akt levels in the spleen but did not affect the same in the myocardium. *p<0.05 compared to the vehicle group. BAY, BAY 60–6583.

Figure 4.. Western blotting results of IL-10 levels in BAY 60–6583-treated non- injured mice.

BAY 60–6583 significantly increased the IL-10 levels in spleen, which could be attenuated by wortmannin, a specific PI3K inhibitor, but did not affect IL-10 in the myocardium. *p<0.05 compared to the other groups. BAY, BAY 60–6583; Wort, wortmannin.

Figure 5.. Immunofluorescence staining.

Confocal imaging results showed that majority of IL-10 elevated in the spleen was co-localized with CD-11b⁺ cells. Red staining presented IL-10, and green staining presented CD11b.

Figure 6.. Western blotting results in the post-ischemic reperfused heart.

BAY 606583 significantly increased myocardial IL-10 levels after reperfusion. However, the IL- 10 levels were not increased in the reperfused myocardium in splenectomized mice. *p<0.05 compared to the other groups. BAY, BAY 60–6583; SPLX, splenectomy.

Figure 7.. Myocardial IS measured using TTC and Phthalo blue staining.

IL-10KO mice had higher IS after IR injury as compared to the WT mice. BAY 60–6583 did not exert a protective effect on IL-10KO mice. Furthermore, BAY 60–6583 failed to reduce the IS in splenectomized mice with IL-10KO splenic-leukocyte reconstitution. *p<0.05 compared to the other groups. IR, myocardial reperfusion injury; SPLX, splenectomy; SPATIL-10KO, IL-10 knockout splenic leukocytes reconstituted mice; WT, wild-type.

Figure 8.. Schematic representation of A2BAR activation-mediated cardioprotection.