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. 2018 Apr;175(8):1329-1343.
doi: 10.1111/bph.13781. Epub 2017 Apr 12.

IL-21 promotes myocardial ischaemia/reperfusion injury through the modulation of neutrophil infiltration

Kejing Wang  1   2 , Shuang Wen  1   2 , Jiao Jiao  1   2 , Tingting Tang  1   2 , Xin Zhao  3 , Min Zhang  1   2 , Bingjie Lv  1   2 , Yuzhi Lu  1   2 , Xingdi Zhou  1   2 , Jingyong Li  1   2 , Shaofang Nie  1   2 , Yuhua Liao  1   2 , Qing Wang  4 , Xin Tu  4 , Ziad Mallat  5 , Ni Xia  1   2 , Xiang Cheng  1   2
Affiliations

IL-21 promotes myocardial ischaemia/reperfusion injury through the modulation of neutrophil infiltration

Kejing Wang et al. Br J Pharmacol. 2018 Apr.

Abstract

Background and purpose: The immune system plays an important role in driving the acute inflammatory response following myocardial ischaemia/reperfusion injury (MIRI). IL-21 is a pleiotropic cytokine with multiple immunomodulatory effects, but its role in MIRI is not known.

Experimental approach: Myocardial injury, neutrophil infiltration and the expression of neutrophil chemokines KC (CXCL1) and MIP-2 (CXCL2) were studied in a mouse model of MIRI. Effects of IL-21 on the expression of KC and MIP-2 in neonatal mouse cardiomyocytes (CMs) and cardiac fibroblasts (CFs) were determined by real-time PCR and ELISA. The signalling mechanisms underlying these effects were explored by western blot analysis.

Key results: IL-21 was elevated within the acute phase of murine MIRI. Neutralization of IL-21 attenuated myocardial injury, as illustrated by reduced infarct size, decreased cardiac troponin T levels and improved cardiac function, whereas exogenous IL-21 administration exerted opposite effects. IL-21 increased the infiltration of neutrophils and increased the expression of KC and MIP-2 in myocardial tissue following MIRI. Moreover, neutrophil depletion attenuated the IL-21-induced myocardial injury. Mechanistically, IL-21 increased the production of KC and MIP-2 in neonatal CMs and CFs, and enhanced neutrophil migration, as revealed by the migration assay. Furthermore, we demonstrated that this IL-21-mediated increase in chemokine expression involved the activation of Akt/NF-κB signalling in CMs and p38 MAPK/NF-κB signalling in CFs.

Conclusions and implications: Our data provide novel evidence that IL-21 plays a pathogenic role in MIRI, most likely by promoting cardiac neutrophil infiltration. Therefore, targeting IL-21 may have therapeutic potential as a treatment for MIRI.

Linked articles: This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

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Figures

Figure 1
Figure 1
IL‐21 and IL‐21 receptors are elevated during the acute phase of MIRI and CD4+ T‐cells are the major source of IL‐21 in the ischaemic myocardium. The time course of changes in the (A) mRNA and (B) protein expression of IL‐21 in the myocardium following MIRI were measured by quantitative real‐time PCR and western blotting, respectively (n = 6 per group). The time course of changes in the (C) mRNA and (D) protein expression of IL‐21 receptors (IL‐21R) in the myocardium following MIRI were measured via quantitative real‐time PCR and western blotting, respectively (n = 6 per group). (E) The infiltrated IL‐21+ leukocytes in myocardial I/R mice after 6 h of reperfusion were analysed by flow cytometry. CD45+ cells were isolated and restimulated. The IL‐21+ CD45+ cells were further analysed for CD3, CD4 and γδTCR expression to detect the cellular source of IL‐21. The proportion of different IL‐21‐secreting cells in the IL‐21+CD45+ cells were quantitatively analysed (n = 5 per group). *P < 0.05 versus sham.
Figure 2
Figure 2
IL‐21 neutralization attenuates, whereas exogenous IL‐21 aggravates, myocardial injury. (A) Representative images of left ventricular slices from different groups 1 day after reperfusion. The non‐ischaemic area is indicated in blue, the area at risk (AAR) in red and the infarct area (I) in white. (B) Quantification of infarct size of myocardial tissues 1 day after reperfusion. (C) Serum cTnT was measured 1 day after reperfusion. (D) Representative M‐mode echocardiographic images of the left ventricular 1 day after reperfusion. (E) Left ventricular EF and FS were measured via echocardiography 1 day after reperfusion. *P < 0.05 versus isotype; # P < 0.05 versus vehicle. Isotype, n = 9; anti‐IL‐21, n = 9; vehicle, n = 11; IL‐21, n = 10.
Figure 3
Figure 3
IL‐21 increases the number of neutrophils infiltrating the myocardium and the myocardial expression of KC and MIP‐2 following MIRI, while anti‐IL‐21 mAb reduced cardiac infiltration of neutrophils and the expression of KC and MIP‐2. (A–D) The number, percentage and representative contour plots of CD11b+Gr‐1+ neutrophils infiltrating the myocardium after 30 min of ischaemia and 3 h of reperfusion were analysed by flow cytometry (n = 5 per group). (E, F) The mRNA expression of KC, MIP‐2 and LIX in the myocardium after 30 min of ischaemia followed by 30 min or 3 h of reperfusion was analysed via real‐time PCR (n = 5 per group). *P < 0.05 versus Sham; #P < 0.05 versus vehicle or isotype.
Figure 4
Figure 4
IL‐21 induces neutrophil migration and the expression of KC and MIP‐2 in CMs and CFs. (A) Neutrophil migration in the presence of conditioned supernatants from stimulated CMs (upper) and CFs (lower) was measured by transwell assay. The values were normalized relative to those of the medium from unstimulated CMs or CFs (control). (B) The mRNA expression of KC and MIP‐2 in response to 1 h of stimulation with IL‐21(100 ng·mL−1) in CMs (left) and CFs (right). (C) ELISA results for KC and MIP‐2 levels in the supernatants of CMs (left) and CFs (right) stimulated by IL‐21(100 ng·mL−1) for 24 h. H2O2 (100 μM) or TNF‐α (5 ng·mL−1) served as positive controls for CMs and CFs respectively. IL‐21‐alone control media was from unstimulated cells ‘spiked’ with IL‐21. Data are representative of five independent experiments. *P < 0.05 versus control.
Figure 5
Figure 5
Activation of the ERK, p38 MAPK, Akt, NF‐κB, STAT1 and STAT3 signalling pathways in the myocardium after the administration of exogenous IL‐21. (A) Representative western blots showing the activation of different signalling pathways 10 min after reperfusion in the myocardium after IL‐21 administration. (B) Quantitative analysis of the levels of phospho‐/total‐ERK, p38 MAPK, Akt, NF‐κB, STAT1 and STAT3 signalling pathways 10 min after reperfusion in different groups (n = 6 per group). (C) Representative western blots showing the activation of different signalling pathways 30 min after reperfusion in the myocardium after IL‐21 administration. (D) Quantitative analysis of the levels of phospho‐/total‐ERK, p38 MAPK, Akt, NF‐κB, STAT1 and STAT3 signalling pathways 30 min after reperfusion in different groups (n = 6 per group). *P < 0.05 versus sham; # P < 0.05 versus vehicle.
Figure 6
Figure 6
Direct effects of IL‐21 on the activation of Akt, p38 MAPK and NF‐κB signalling in isolated CMs and CFs. (A, B) Representative western blots showing the phospho‐/total‐Akt, p38 MAPK and NF‐κB p65 levels in CMs and CFs unstimulated (control) or stimulated with IL‐21 (100 ng·mL−1) for indicated durations. (C, D) Quantitative analysis of the phospho‐/total‐Akt, p38 MAPK and NF‐κB p65 levels in CMs and CFs. Data are representative of five independent experiments. *P < 0.05 versus control.
Figure 7
Figure 7
IL‐21‐induced chemokine expression is Akt/NF‐κB or p38 MAPK/NF‐κB dependent. (A) CMs were pretreated for 1 h with an Akt inhibitor (LY294002, 10 μM) or an NF‐κB inhibitor (BAY11‐7082, 10 μM), followed by IL‐21 (100 ng·mL−1) stimulation for 1 h. The expression of KC and MIP‐2 was measured via real‐time PCR. (B) CMs were pretreated for 1 h with the indicated inhibitors, followed by IL‐21 stimulation for 24 h. Concentrations of KC and MIP‐2 in the culture media were measured by ELISA. (C) CFs were pretreated for 1 h with a p38 MAPK inhibitor (SB203580, 10 μM) or an NF‐κB inhibitor (BAY11‐7082, 10 μM), followed by IL‐21 (100 ng·mL−1) stimulation for 1 h. The expression of KC and MIP‐2 was measured by real‐time PCR. (D) CFs were pretreated for 1 h with the indicated inhibitors, followed by IL‐21 stimulation for 24 h. Concentrations of KC and MIP‐2 in the culture media were measured by ELISA. (E) CMs were pretreated with LY294002, and (F) CFs were pretreated with SB203580 and then cells were stimulated with IL‐21 or vehicle for 1 h. NF‐κB p65 phosphorylation was analysed by western blotting. Representative western blot images (left) and quantitative analyses (right) are shown. Data are representative of five independent experiments. *P < 0.05 versus control; # P < 0.05 versus IL‐21.
Figure 8
Figure 8
Schematic illustration of the IL‐21‐mediated effects on neutrophil recruitment in MIRI. MIRI induces the up‐regulation of IL‐21 in the myocardium, which directly acts on CMs and CFs to promote the mRNA expression and production of KC and MIP‐2 via the activation of Akt/NF‐κB and p38 MAPK/NF‐κB signalling in CMs and CFs respectively. KC and MIP‐2 are potent neutrophil chemoattractants, which recruit neutrophils into the injured myocardium.
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