Investigating a Novel Role for the IL-36 / IL-36R Pathway in Mediating Inflammation in the Adult and Aged Murine Heart after Ischaemia-Reperfusion Injury

Introduction: Whilst blood flow restoration is critical following myocardial infarction (MI), ischaemia-reperfusion injury (IRI) accounts for ~50% of the final infarct size. The newly discovered cytokine, interleukin-36 (IL-36), could potentially mediate these disturbances. However, its role in myocardial IRI is not known. Although several anti-inflammatory therapies have been successful in pre-clinical models of MI, they have failed in the clinical setting. This translational failure may be linked to a lack of inclusion of comorbidities and/or risk factors, as well as an early benefit at the level of the coronary microcirculation. We firstly investigated if IL-36 cytokines and its receptor (IL-36R) were present in the heart, and whether their expression varied in an injury-, age-, and sex-related manner. We then determined whether coronary microcirculatory disturbances and infarct size post-IRI were modified by age and sex, and whether IL-36Ra could confer vasculoprotection. Methods: Myocardial IRI was induced in adult (3-months) and aged (>18-months) female mice, with sex differences assessed in adult male and female mice. Myocardial IL-36R/α/β, VCAM-1 expression, and oxidative stress were investigated by immunostaining, flow cytometry, and/or using Western blot. Expression on human heart tissue samples of varying ages was also determined. IL-36R/α/β expression was also determined on stimulated vena cava endothelial cells (VCECs). The beating heart coronary microcirculation was imaged in vivo intravitally for neutrophils, platelets, and functional capillary density, and also ex vivo using multiphoton microscopy. Laser speckle contrast imaging was used to determine overall left ventricular perfusion. The effects of topical IL-36 cytokine application was also observed intravitally. In some studies, recombinant mouse IL-36Ra (15μg/mouse) was injected intra-arterially at 5 minutes pre-reperfusion and 60 minutes post-reperfusion. Infarct size was measured using dual TTC/Evans Blue staining. Results: Expression of IL-36 cytokine and its receptor was predominantly on the vasculature and cardiomyocytes of both the murine and human hearts, and their expression increased with age and injury. Expression was significantly higher in healthy and injured adult female hearts compared to male hearts. Basal VCEC surface expression of IL-36R increased after cytokine stimulation in a concentration-dependent manner. Intravital imaging of the beating heart demonstrated heightened basal and injury-induced neutrophil recruitment and poorer blood flow in the aged coronary microcirculation when compared with adult hearts. These events were mirrored in deeper myocardial layers when imaged using multiphoton microscopy. A greater burden of thrombotic disease was noted in adult injured male coronary microvessels, whilst a greater neutrophil presence was identified in adult injured female coronary microvessels. Infarct size was significantly larger in injured adult female hearts when compared to males. All IL-36 cytokines were able to induce an inflammatory response when topically applied to the adult and aged hearts. An IL-36R antagonist (IL-36Ra) decreased neutrophil recruitment, improved blood flow and ventricular perfusion, 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. Conclusion: These novel results are the first to demonstrate myocardial presence of IL-36 and its receptor and how expression changes with age and sex. Our findings of enhanced coronary microcirculatory perturbations associated with age may explain the poorer outcomes in elderly MI patients. The cellular nature of the thromboinflammatory response may explain the sex-related differences in outcomes after MI. Importantly, we are the first to demonstrate that targeting IL-36/IL-36R pathway may be a potential novel therapy for treatment of myocardial IRI

Monitoring coronary blood flow by laser speckle contrast imaging after myocardial ischaemia reperfusion injury in adult and aged mice

Introduction: Investigating coronary microvascular perfusion responses after myocardial infarction (MI) would aid in the development of flow preserving therapies. Laser speckle contrast imaging (LSCI) is a powerful tool used for real-time, non-contact, full-field imaging of blood flow in various tissues/organs. However, its use in the beating heart has been limited due to motion artifacts. Methods: In this paper, we report the novel use of LSCI, combined with custom speckle analysis software (SpAn), to visualise and quantitate changes in ventricular perfusion in adult and aged mice undergoing ischaemia-reperfusion (IR) injury. The therapeutic benefit of inhibiting the actions of the pro-inflammatory cytokine interleukin-36 (IL-36) was also investigated using an IL-36 receptor antagonist (IL-36Ra). Results: Imaging from uncovered and covered regions of the left ventricle demonstrated that whilst part of the LSCI flux signal was derived from beating motion, a significant contributor to the flux signal came from ventricular microcirculatory blood flow. We show that a biphasic flux profile corresponding to diastolic and systolic phases of the cardiac cycle can be detected without mathematically processing the total flux data to denoise motion artifacts. Furthermore, perfusion responses to ischaemia and postischaemia were strong, reproducible and could easily be detected without the need to subtract motion-related flux signals. LSCI also identified significantly poorer ventricular perfusion in injured aged mice following IR injury which markedly improved with IL-36Ra. Discussion: We therefore propose that LSCI of the heart is possible despite motion artifacts and may facilitate future investigations into the role of the coronary microcirculation in cardiovascular diseases and development of novel therapies

Inflammation as the nexus: exploring the link between acute myocardial infarction and chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD), particularly following acute exacerbations (AE-COPD), significantly heightens the risks and mortality associated with acute myocardial infarction (AMI). The intersection of COPD and AMI is characterised by a considerable overlap in inflammatory mechanisms, which play a crucial role in the development of both conditions. Although extensive research has been conducted on individual inflammatory pathways in AMI and COPD, the understanding of thrombo-inflammatory crosstalk in comorbid settings remains limited. The effectiveness of various inflammatory components in reducing AMI infarct size or slowing COPD progression has shown promise, yet their efficacy in the context of comorbidity with COPD and AMI is not established. This review focuses on the critical importance of both local and systemic inflammation, highlighting it as a key pathophysiological connection between AMI and COPD/AE-COPD

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

AimsRisks 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 resultsImmunostaining 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.ConclusionThis 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

Video1_Monitoring coronary blood flow by laser speckle contrast imaging after myocardial ischaemia reperfusion injury in adult and aged mice.mp4

IntroductionInvestigating coronary microvascular perfusion responses after myocardial infarction (MI) would aid in the development of flow preserving therapies. Laser speckle contrast imaging (LSCI) is a powerful tool used for real-time, non-contact, full-field imaging of blood flow in various tissues/organs. However, its use in the beating heart has been limited due to motion artifacts.MethodsIn this paper, we report the novel use of LSCI, combined with custom speckle analysis software (SpAn), to visualise and quantitate changes in ventricular perfusion in adult and aged mice undergoing ischaemia-reperfusion (IR) injury. The therapeutic benefit of inhibiting the actions of the pro-inflammatory cytokine interleukin-36 (IL-36) was also investigated using an IL-36 receptor antagonist (IL-36Ra).ResultsImaging from uncovered and covered regions of the left ventricle demonstrated that whilst part of the LSCI flux signal was derived from beating motion, a significant contributor to the flux signal came from ventricular microcirculatory blood flow. We show that a biphasic flux profile corresponding to diastolic and systolic phases of the cardiac cycle can be detected without mathematically processing the total flux data to denoise motion artifacts. Furthermore, perfusion responses to ischaemia and postischaemia were strong, reproducible and could easily be detected without the need to subtract motion-related flux signals. LSCI also identified significantly poorer ventricular perfusion in injured aged mice following IR injury which markedly improved with IL-36Ra.DiscussionWe therefore propose that LSCI of the heart is possible despite motion artifacts and may facilitate future investigations into the role of the coronary microcirculation in cardiovascular diseases and development of novel therapies.</p