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. 2012 Jan 24;59(4):420-9.
doi: 10.1016/j.jacc.2011.10.863.

Interleukin-17A contributes to myocardial ischemia/reperfusion injury by regulating cardiomyocyte apoptosis and neutrophil infiltration

Yu-Hua Liao  1 , Ni Xia, Su-Feng Zhou, Ting-Ting Tang, Xin-Xin Yan, Bing-Jie Lv, Shao-Fang Nie, Jing Wang, Yoichiro Iwakura, Hong Xiao, Jing Yuan, Harish Jevallee, Fen Wei, Guo-Ping Shi, Xiang Cheng
Affiliations

Interleukin-17A contributes to myocardial ischemia/reperfusion injury by regulating cardiomyocyte apoptosis and neutrophil infiltration

Yu-Hua Liao et al. J Am Coll Cardiol. .

Abstract

Objectives: This study tested whether interleukin (IL)-17A is involved in the pathogenesis of mouse myocardial ischemia/reperfusion (I/R) injury and investigated the mechanisms.

Background: Inflammatory processes play a major role in myocardial I/R injury. We recently identified IL-17A as an important cytokine in inflammatory cardiovascular diseases such as atherosclerosis and viral myocarditis. However, its role in myocardial I/R injury remains unknown.

Methods: The involvement of IL-17A was assessed in functional assays in mouse myocardial I/R injury by neutralization/repletion or genetic deficiency of IL-17A, and its mechanism on cardiomyocyte apoptosis and neutrophil infiltration were further studied in vivo and in vitro.

Results: Interleukin-17A was elevated after murine left coronary artery ligation and reperfusion. Intracellular cytokine staining revealed that γδT lymphocytes but not CD4(+) helper T cells were a major source of IL-17A. Anti-IL-17A monoclonal antibody treatment or IL-17A knockout markedly ameliorated I/R injury, as demonstrated by reduced infarct size, reduced cardiac troponin T levels, and improved cardiac function. This improvement was associated with a reduction in cardiomyocyte apoptosis and neutrophil infiltration. In contrast, repletion of exogenous IL-17A induced the opposite effect. In vitro study showed that IL-17A mediated cardiomyocyte apoptosis through regulating the Bax/Bcl-2 ratio, induced CXC chemokine-mediated neutrophil migration and promoted neutrophil-endothelial cell adherence through induction of endothelial cell E-selectin and inter-cellular adhesion molecule-1 expression.

Conclusions: IL-17A mainly produced by γδT cells plays a pathogenic role in myocardial I/R injury by inducing cardiomyocyte apoptosis and neutrophil infiltration.

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Figures

Fig. 1
Fig. 1
IL17A neutralization and repletion affected mouse myocardial I/R injury. A, Representative images of LV slices from different groups at 1 day after reperfusion. The nonischemic area is indicated in blue, the AAR in red and the infarct area in white. B, Quantification of infarct size of myocardial tissues 1 day or 3 days after reperfusion (n=6–8). C, Representative M-mode echo-cardiography images of the left ventricular 1 day after reperfusion. D, Ejection fraction and LV fractional shortening (n=8). **P<0.01 versus sham; †P<0.05, ‡P<0.01 versus isotype; §P<0.05, §§P<0.01 versus vehicle.
Fig. 2
Fig. 2
IL-17A knockout ameliorated myocardial I/R injury. A, Representative images of LV slices from wild-type and Il17a−/− mice at 1 day after I/R. B, Quantification of infarct size of myocardium 1 day after reperfusion (n=8). C, Representative M-mode images of the LV after sham and myocardial I/R from wild-type and Il17a−/− mice. D, Ejection fraction and LV fractional shortening (n=8). ** P<0.01 versus wild-type.
Fig. 3
Fig. 3
IL-17A mediated cardiomyocyte apoptosis in vivo and in vitro. A, Representative photographs of TUNEL-stained heart sections from different groups after 3 hours reperfusion. Apoptotic nuclei were identified by TUNEL staining (green), cardiomyocyte by anti-sarcomeric actin antibody (red) and total nuclei by DAPI staining (blue). Arrowheads indicate apoptotic cardiomyocytes. Scale bar: 50 mm. B, Percentages of TUNEL-positive nuclei over total number of nuclei (n=4–5). C, Caspase 3 activity in myocardium was assessed after 3 hours of reperfusion and the values were normalized to sham (n=5), **P<0.01 versus sham; †P<0.05, ‡P<0.01 versus control. D, Mouse neonatal cardiomyocyte apoptosis under H2O2 and different doses of IL-17A. Brown staining indicates TUNEL positive cells. Results are representative of three independent assays. Scale bar: 100 mm. *P<0.05, **P<0.01 vs. H2O2(−)IL-17A(−), ‡P<0.01 versus H2O2(+)IL-17A(−). E, Real-time PCR determined mouse neonatal cardiomyocyte Bcl-2 and Bax mRNA levels. The results were expressed as Bax/Bcl-2 ratio. *P<0.05, **P<0.01 versus control. F, Real-time PCR determined mRNA level of Bcl-2 and Bax in ischemia myocardium. The results were expressed as ratio of Bax/Bcl-2. **P<0.01 versus sham, †P<0.05 versus control.
Fig. 4
Fig. 4
IL-17A increased cardiac neutrophil recruitment and migration. Mice were assessed for chemokine expression and neutrophil infiltration at 3 hours after myocardial I/R. A, Cardiac MPO activity in tissue samples. B, The number of CD11b+Gr-1+ neutrophils infiltrated in myocardium were analyzed by flow cytometry. C, The representative flowcytometry dot plots of CD11b+Gr-1+ neutrophils infiltrated in myocardium. D, LIX, KC and MIP-2 mRNA levels were analyzed by real-time PCR. **P<0.01 versus sham; ‡P<0.01 versus control. E, ELISA determined mouse cardiomyocytes KC, MIP-2 and LIX levels in the supernatants. F, Neutrophil migration, means±SEM. The value was normalized relative to medium alone. *P<0.05, **P<0.01 versus medium; ‡P<0.01, versus H2O2; §§P<0.01 versus IL-17A; #P<0.05, ##P<0.01 versus IL-17A/ H2O2. All experiments were repeated three times. n=4–5.
Fig. 5
Fig. 5
IL-17A mediated neutrophil-EC adhesion. A, Mice were assessed for myocardium ICAM-1 and E-selectin expression by real-time PCR at 3 hours after I/R (n=4–5). **P<0.01 versus sham; †P<0.05, ‡P<0.01 versus control. B, Mouse myocardial EC expression of ICAM-1 and E-selectin was measured by a cell-based ELISA, as indicated by the OD450nm. C, Neutrophil adhesion on ECs was determined by fluorescence microscopy. D, Representative photomicrograph of neutrophils adhering to ECs. Neutrophils are identified by green fluorescence. Scale bar: 50 mm. Values are means±SEM; *P<0.05, **P<0.01 versus medium; ‡P<0.01 versus H2O2; §§P<0.01 versus IL-17A; # P<0.05 versus IL-17A/H2O2. All experiments were repeated three times.
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