A Novel Role Of IL-21 In Myocardial Ischemia Reperfusion Injury And Its Mechanisms

Part I Expression and pathological role of IL-21 in MIRIObjectives:The immune system plays important roles in myocardial ischemia/reperfusion injury (MIRI). Interleukin-21 (IL-21) is a pleiotropic cytokine that modulates multiple immune responses. However, its role in MIRI remains unknown. This part of study aims to investigate the expression patterns and the pathological role of IL-21 in a mouse model of MIRI.Methods:A murine MIRI model was established by temporal occlusion of the left anterior descending (LAD) coronary artery. Mice were subjected to a sham procedure or to 30 minutes (min) of ischemia followed by 30 min or 1,6,12, or 24 hours (h) of reperfusion. Real-time PCR and western blot were performed to measure the expression patterns of IL-21 and its specific receptor IL-21 R. To elucidate the causative role of IL-21 in this process, mice were pretreated with anti-IL-21 neutralizing mAb or recombinant mouse-IL-21 before the procedure to block or strengthen the effects of IL-21. The MIRI indexes were examined 24 h after reperfusion, including the following:area of the infarct was evaluated by Even's blue/TTC staining, the serum cardiac troponin T (cTnT) level, as well as cardiac function evaluated by ejection fraction (EF) and fractional shortening (FS) via echocardiograph.Results:In comparison to the sham group, the mRNA and protein expression of IL-21 increased rapidly within 30 min after reperfusion, and remained elevated for up to 24 h. The mRNA and protein expression of IL-21 R elevated 12 h after reperfusion and maintained for 24 h. In comparison to isotype-treated mice, IL-21 neutralization reduced infarct size, decreased cTnT levels and restored cardiac function, whereas exogenous IL-21 administration exerted the opposite effects.Conclusions:The expression of IL-21 is early elevated during the acute phase following MIRI. Neutralization of endogenous IL-21 protects against myocardial injury, whereas exogenous IL-21 administration aggravates myocardial injury.Part II IL-21 promotes MIRI through the modulation of neutrophil infiltrationObjectives:Neutrophil infiltration is a critical event in MIRI. This part of study aims to investigate the effect of IL-21 on neutrophil infiltration as well as the relative mechanisms.Methods:C57BL/6 mice were randomly assigned to sham group, MIRI group (Control) and recombinant mouse-IL-21 treatment MIRI group (IL-21). At 3 hours after reperfusion, the number of cardiac infiltrating neutrophils was detected by flow cytometry. Meanwhile, mRNA expression of ELR+CXC chemokines KC, MIP-2 and LIX in the myocardium was measured by real-time PCR. Isolated neonatal cardiomyocytes (CMs) and cardiac fibroblasts (CFs) were stimulated by IL-21 for 1 h, the mRNA expression of KC and MIP-2 was measured via real-time PCR. Meanwhile, CMs and CFs were exposed to IL-21 for 24 h, and KC and MIP-2 secretion in the supernatant were quantified using commercial ELISA kit. In neutrophil migration assays, the isolated neutrophils were added to the upper chambers of Transwell inserts in 24-well tissue culture plates, and conditioned supernatants from CMs and CFs were added to the lower chambers. After 90 min of incubation, neutrophils migrated to the lower chamber were counted using an inverted microscope.Results:The neutrophils infiltrating the ischemic myocardium 3 h after reperfusion were quantitatively analized via flow cytometry. In comparison to sham-operated mice, neutrophil infiltration was increased in the reperfused myocardium, and was further increased by exogenous IL-21 administration. Relative to the vehicle-treated mice, IL-21 administration rapidly up-regulated KC and MIP-2 mRNA expression after reperfusion. At a cellular level, IL-21 increased both mRNA expression and secretion of CMs and CFs. The neutrophil migration assay indicated enhanced migration of neutrophils in the presence of conditioned supernatants from stimulated CMs or CFs.Conclusions:IL-21 induces expression of chemokines KC and MIP-2 in CMs and CFs, consequently promoting neutrophil infiltration and MIRI.Part III Signaling mechanisms underlying the IL-21-mediated enhancement of neutrophil infiltraionObjectives:IL-21-mediated cellular effects were reported to be associated with JAK-STAT, PI3K/Akt, ERK, p38 MAPK and NF-?B signaling pathways, which are also involved in MIRI. This part of study aims to examine the effects of IL-21 on the above-mentioned signaling pathways.Methods:A murine MIRI model was established as previously described. Mice were subjected to a sham procedure or to 30 minutes of ischemia followed by 10,30,60 or 120 min of reperfusion. Western blot was performed to measure the activation patterns of ERK, p38 MAPK, Akt, NF-?B p65, STAT1 and STAT3 signaling pathways. Subsequently, activation of the studied pathways in the myocardium after the administration of exogenous IL-21 were examined at both 10 and 30 min after reperfusion. Isolated neonatal CMs and CFs were stimulated by IL-21 for indicated time points (10,30,60,120 min), and the activation of the above-mentioned signaling pathways was examined via western blot. Moreover, CMs and CF were pre-treated by relevant signaling inhibitors before IL-21 stimulation to confirm whether IL-21-induced up-regulation of chemokines depended on these signaling pathways.Results:Reperfusion increased the phosphorylation of ERK, p38 MAPK and NF-?B p65 as early as 10 min, while the activation of Akt, STAT1 and STAT3 occurred at 30 min after reperfusion. Administration of exogenous IL-21 further increased the phosphorylation of p38 MAPK at both 10 and 30 min after reperfusion and enhanced NF-?B p65 activation at 30 min after reperfusion. In CMs, the phosphorylation of Akt was enhanced by IL-21 administration at early time points (10-30 min), and NF-?B p65 phosphorylation was induced within 30 min of IL-21 administration. Stimulation with IL-21 in CFs induced the activation of p38 MAPK signaling within 30 min, and NF-?B p65 phosphorylation was induced at 1 h. There were no significant changes in the activity of the ERK, STAT1 or STAT3 signaling pathways following stimulation with IL-21 in either CMs or CFs. Pre-incubation with either the Akt or NF-?B inhibitor abolished the IL-21-induced KC and MIP-2 mRNA expression in CMs, whereas KC and MIP-2 secretions in the presence of IL-21 were partially inhibited. In CFs, the inhibition of cither p38 MAPK or NF-?B significantly reduced the IL-21-mediated up-regulation of KC and MIP-2 mRNA expression. Likewise, the IL-21-induced KC and MIP-2 secretions were also partially inhibited.Conclusions:IL-21-induced up-regulation of CXC chemokines is associated with the activation of Akt/NF-KB signaling in CMs and p38 MAPK/NF-?B signaling in CFs.