Interleukin-36 is vasculoprotective in both sexes despite sex-specific changes in the coronary microcirculation response to IR injury

Abstract

Aims: Risks and outcomes of myocardial infarction (MI) are different between men and women and some studies have demonstrated that the latter have a higher risk of mortality. Whilst there are many reasons for this, it may also partially be linked to stronger innate and adaptive immune responses mounted by females compared to males. However, little is known about how sex impacts the coronary microvessels, the site where inflammatory processes take place, after an MI. Intravital and laser speckle microscopy was used to image coronary microvessels and ventricular perfusion in vivo in response to myocardial ischaemia-reperfusion (IR) injury in male and female mice. Interleukin-36 (IL-36) is the latest addition to the IL-1 superfamily of pro-inflammatory cytokines and has recently been shown to mediate inflammation in a number of non-cardiovascular diseases. Its role in mediating potential sex-related microcirculatiory pertubations in the heart are unknown. Therefore, the vasculoprotective efficacy of an IL-36 receptor antagonist (IL-36Ra) was also investigated.

Methods and results: Immunostaining and flow cytometry demonstrated higher expression of IL-36 and its receptor in female hearts, an observation confirmed in human samples. Intravital imaging of the anaesthetised mouse beating heart identified significantly greater neutrophil recruitment in female hearts, but a greater burden of thrombotic disease in male hearts. Male mice had reduced functional capillary density and were unable to restore perfusion to baseline values as effectively as females. However, female mice had significantly larger infarcts. Interestingly, IL-36Ra decreased inflammation, improved perfusion, and reduced infarct size in both sexes despite increasing platelet presence in male hearts. Mechanistically, this was explained by IL-36Ra attenuating endothelial oxidative damage and VCAM-1 expression. Importantly, IL-36Ra administration during ischaemia was critical for vasculoprotection to be realised.

Conclusion: This novel study identified notable sex-related differences in the coronary microcirculatory response to myocardial IR injury which may explain why some studies have noted poorer outcomes in women after MI. Whilst contemporary MI treatment focuses on anti-platelet strategies, the heightened presence of neutrophils in female IR injured coronary microvessels necessitates the development of an effective anti-inflammatory approach for treating female patients. We also emphasise the importance of early intervention during the ischaemic period in order to maximise therapeutic effectiveness.

Keywords: coronary microcirculation; interleukin-36; ischaemia-reperfusion injury; myocardial infarction; neutrophils; platelets; sex.

Figure 1

Cardiac expression of IL-36R and IL-36α/β increased with IR injury in both sexes but was generally higher in female mice. (A) Representative images of frozen heart sections from male and female sham and injured mice immunostained with an anti-IL-36R (green) and anti-CD31 (red) antibody. (B) Quantitative analysis of these images for IL-36R expression. N = 4/group. (C,D) Hearts were collected from male and female sham mice, after ischaemia only (0 min) and after reperfusion (30 min and 150 min), collagenase digested and analysed flow cytometrically for IL-36R expression on endothelial cells and cardiomyocytes. N = 3/group. (E) Representative images of frozen hearts sections from male and female sham and injured mice immunostained with an anti-IL-36α/β (green) and anti-CD31 (red) antibody. (F,G) Quantitative analysis of these images for IL-36α and IL-36β expression. N = 4/group. Scale bar = 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined using a two-way ANOVA followed by a Tukey's post-hoc test. All graphs: male = blue; female = pink.

Figure 2

Cardiac expression of IL-36R and IL-36α/β/γ in human heart samples was higher in females. (A) Representative images of heart sections from male and female patients undergoing left ventricular assist device (LVAD) implantation and immunostained with an anti-IL-36R (green) and anti-CD31 (red) antibody. (B) Quantitative analysis of these images for IL-36R expression. N = 3/group (C) representative images of a heart section from a female patient undergoing LVAD implantation and immunostained with anti-IL-36R (green), anti-cTnT (red) and anti-DNA/RNA oxidative damage (blue) antibody. (D) Quantitative analysis of these images for oxidative damage. N = 3/group (E) representative images of heart sections from a female patient undergoing LVAD implantation and immunostained with an anti-IL-36α/β/γ (green) and anti-CD31 (red) antibody. (F–H) Quantitative analysis of these images for IL-36α, IL-36β and IL-36γ expression. Scale bar = 100 µm. N = 3/group. *p < 0.05, **p < 0.01 as determined using an unpaired t-test. All graphs: male = blue; female = pink.

Figure 3

Neutrophil expression of IL-36R and IL-36α/β increased with IR injury in both sexes but was generally higher in male mice. BM-derived neutrophils and monocytes/macrophages were isolated from male and female sham mice, after ischaemia only (0 min) or after reperfusion (150 min), collagenase digested and analysed flow cytometrically for (A,B) IL-36R, (C,D) IL-36α and (E,F) IL-36β expression. N = 3/group. *p < 0.05, **p < 0.01, ***p < 0.001 as determined using a one-way ANOVA followed by a Tukey's post-hoc test. All graphs: male = blue; female = pink.

Figure 4

Sex-related differences in neutrophil and platelet involvement in IR injured hearts with neutrophil presence reduced in both sexes by IL-36R inhibition. An IL-36 receptor antagonist (IL-36Ra; 15 μg/mouse) was injected intra-arterially at 10 min pre-reperfusion and at 60 min post-reperfusion in male and female mice. Representative intravital images of the beating heart showing adherent neutrophils (green) and platelets (red) in the coronary microcirculation at (A) 120 min post-reperfusion and (B) within the first 30 min of reperfusion. (C–F) Quantitative analysis of the intravital data and the AUC analysis for adherent neutrophils and platelets over a time course of 30 min post-reperfusion. (G–J) Quantitative analysis of the intravital data and the AUC analysis for adherent neutrophils and platelets over a time course of 150 min post-reperfusion. Scale bar = 100 µm. IRI N = 5/group; IRI + IL-36Ra N = 6/group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined using a one-way ANOVA followed by a Tukey's post-hoc test. All graphs: male = blue; female = pink.

Figure 5

Neutrophil presence reduced throughout the depth of the IR injured left ventricle in both sexes by IL-36R inhibition. An IL-36 receptor antagonist (IL-36Ra; 15 μg/mouse) was injected intra-arterially at 10 min pre-reperfusion and at 60 min post-reperfusion in male and female mice. The left ventricle was vibratome sectioned into four 300 μm sections and imaged using a multiphoton microscope. (A) Representative Z-stack multiphoton images of neutrophils (green) in the four sections taken from the outermost layer closest to the epicardium (row 1), outer myocardial layer (row 2), inner myocardial layer (row 3) and innermost layer closest to the endocardium (row 4). (B,C) Quantitative analysis of the multiphoton data at various depths and the corresponding AUC analysis for adherent neutrophils. N = 5/group. *p < 0.05, ***p < 0.001, ****p < 0.0001 as determined using a one-way ANOVA followed by a Tukey's post-hoc test. All graphs: male = blue; female = pink.

Figure 6

FCD and ventricular perfusion decreased with IR injury in both sexes but was worse in male mice—improved with IL-36R inhibition in both sexes. An IL-36 receptor antagonist (IL-35Ra; 15 μg/mouse) was injected intra-arterially at 10 min pre-reperfusion and at 60 min post-reperfusion in male and female mice. (A) Representative intravital images of FITC-BSA perfused coronary microvessels (green) at 150 min in male and female sham hearts or at 150 min post-reperfusion. Patchy black areas represent areas devoid of perfusion. N = 3/group. (B) Quantitative analysis of baseline diastolic flux (perfusion) unit readings obtained using LSCI prior to injury. Male N = 8; Female N = 11. (C,D) Quantitative time-course analysis of diastolic flux unit readings as a percentage of baseline values obtained by LSCI and the corresponding AUC curve. (E) Representative LSCI images showing continuous flux data, flux heat maps and a corresponding photo of the beating heart. Arrows highlight the expected decreased ventricular perfusion during ischaemia and the rise in perfusion (or lack of) during reperfusion. IRI = N = 6/group; IRI + IL-36Ra N = 4/group. Scale bar = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001 as determined using a one-way ANOVA followed by a Tukey's post-hoc test. All graphs: male = blue; female = pink.

Figure 7

Infarct size, cardiomyocyte/endothelial cell oxidative damage and VCAM-1 expression decreased in both sexes with IL-36Ra. An IL-36 receptor antagonist (IL-36Ra; 15 μg/mouse) was injected intra-arterially either during ischaemia at 10 min pre-reperfusion (I), 60 min post-reperfusion (R) or at both time points (I + R) in male and female mice. (A) Representative images of TTC-stained infarcts. (B,C) Quantitative analysis of infarct size and area at risk (AAR). N = 4-6/group; IRI + single treatment N = 4/group; Male IRI + double treatment N = 5; Female IRI + double treatment N = 6. (D,E) Hearts were collected from male and female sham, IR injured, and IR injured + IL-36Ra treated mice, collagenase digested and analysed flow cytometrically for oxidative damage in CMs and ECs. N = 3/group. (F) Representative images from these mice immunostained with an anti-CD31 (red), anti-DNA/RNA oxidative damage (green) and anti-VCAM-1 (blue) antibody. (G,H) Quantitative analysis of these immunofluorescent images for endothelial oxidative damage and VCAM-1 expression. N = 4/group. Scale bar = 200 μm. *p < 0.05, **p < 0.01, ***p < 0.001, p < 0.0001 as determined using a one or two-way ANOVA followed by a Tukey's post-hoc test. All graphs: male = blue; female = pink.