Targeting IL-36 improves age-related coronary microcirculatory dysfunction and attenuates myocardial ischemia/reperfusion injury in mice
Juma El-Awaisi, Dean P.J. Kavanagh, Marco R. Rink, Chris J. Weston, Nigel E. Drury, Neena Kalia
Juma El-Awaisi, Dean P.J. Kavanagh, Marco R. Rink, Chris J. Weston, Nigel E. Drury, Neena Kalia
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Research Article Aging Inflammation

Targeting IL-36 improves age-related coronary microcirculatory dysfunction and attenuates myocardial ischemia/reperfusion injury in mice

Abstract

Following myocardial infarction (MI), elderly patients have a poorer prognosis than younger patients, which may be linked to increased coronary microvessel susceptibility to injury. Interleukin-36 (IL-36), a newly discovered proinflammatory member of the IL-1 superfamily, may mediate this injury, but its role in the injured heart is currently not known. We first demonstrated the presence of IL-36(α/β) and its receptor (IL-36R) in ischemia/reperfusion-injured (IR-injured) mouse hearts and, interestingly, noted that expression of both increased with aging. An intravital model for imaging the adult and aged IR-injured beating heart in real time in vivo was used to demonstrate heightened basal and injury-induced neutrophil recruitment, and poorer blood flow, in the aged coronary microcirculation when compared with adult hearts. An IL-36R antagonist (IL-36Ra) decreased neutrophil recruitment, improved blood flow, and reduced infarct size in both adult and aged mice. This may be mechanistically explained by attenuated endothelial oxidative damage and VCAM-1 expression in IL-36Ra–treated mice. Our findings of an enhanced age-related coronary microcirculatory dysfunction in reperfused hearts may explain the poorer outcomes in elderly patients following MI. Since targeting the IL-36/IL-36R pathway was vasculoprotective in aged hearts, it may potentially be a therapy for treating MI in the elderly population.

Authors

Juma El-Awaisi, Dean P.J. Kavanagh, Marco R. Rink, Chris J. Weston, Nigel E. Drury, Neena Kalia

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Figure 1

Age increases expression of IL-36R in healthy and IR-injured hearts, and expression can also be induced in endothelial cells with TNF-α or IL-36 cytokine stimulation.

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Age increases expression of IL-36R in healthy and IR-injured hearts, and...
Hearts were assessed for IL-36R using immunostaining, Western blot analysis, and flow cytometry. Representative images of IL-36R (green) staining of (A) frozen heart sections and (B) Western blots. The molecular weight of IL-36R is about 65 kDa, but it migrates to the position of about 85 kDa in denaturing protein gels. Hence, 2 bands were observed corresponding to 65 kDa, the more active protein due to cleavage of its signaling peptide, and 85 kDa, the less potent glycosylated form. Quantitative analysis of the (C) immunofluorescence images (n = 4/group) and (D) Western blots (n = 4/group). P = 0.0146 adult sham vs. adult IR immunofluorescence. P = 0.0003 aged sham vs. aged IR immunofluorescence. P = 0.001 aged sham vs. aged IR Western blot. Hearts were also collagenase digested and analyzed flow cytometrically for IL-36R expression. Flow cytometry analysis confirmed that IR injury and age induced a significant increase in IL-36R on (E) coronary endothelial cells and (F) cardiomyocytes (n = 3/group). (G and H) Murine vena cava endothelial cells (VCECs) were cultured and stimulated for 4 hours with experimental media (control), an IL-36 cytokine (α, β, or γ), or TNF-α. (G) Representative images of IL-36R (red) expression on stimulated, nonpermeabilized cells (Hoechst 33342–stained nuclei in blue). (H) Quantitative analysis of IL-36R expression on VCECs following stimulation (n = 3/group). Scale bar indicates 200 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as determined using a 1-way ANOVA followed by a Tukey’s post hoc test.