P4372Augmentation of alternatively activated macrophages by IL-4 for the treatment of myocardial infarction

Abstract

Background: Myocardial infarction (MI) is a leading cause of morbidity and mortality worldwide. Recent research has revealed that alternatively activated (M2) macrophages play an important role in myocardial repair post-MI. Expansion of cardiac M2 macrophages may therefore be a promising therapeutic approach for MI. IL-4 is known to increase M2 macrophages in non-cardiac organs, whilst safety of IL-4 injection has been shown in clinical trials for cancer. This study aimed to evaluate the efficacy of IL-4 as a repurposed drug to increase cardiac M2 macrophages post-MI and the resulting therapeutic benefits for MI. Methods and Results: Long-acting IL-4 complex (IL-4c; 5mug recombinant IL-4 mixed with 25mug anti-IL-4 monoclonal antibody as a stabilizer) or PBS (control) was injected intraperitoneally following left coronary artery ligation in mice. Immunolabeling demonstrated that IL-4c administration increased post-MI accumulation of CD206+F4/80+ M2-like macrophages predominantly in the infarcted myocardium, compared to the PBS control (667+/-54 vs. 437+/-21/mm2 at Day 7; 384+/-38 vs. 205+/-28/mm2 at Day 28 post-treatment, p<0.05). FACSsorted CD206+F4/80+ M2-like macrophages highly expressed a number of antiinflammatory and tissue repair-related genes, including Il10, Il1ra, Igf1, Cdcx12, Hif1a, Tgfb, and Spp1. Consistently these genes were upregulated in the IL- 4c-treated myocardium post-MI compared to PBS injection. Echocardiography and cardiac catheterization showed that IL-4c administration enhanced both systolic and diastolic cardiac function and attenuated ventricular dilatation at Day 28 (LVEF, 46.6+/-0.8 vs. 38.0+/-1.0%, p<0.05; LV max dP/dt, 6,558+/-411 vs. 4,356+/-388 mmHg/s, p<0.05). IL-4c treatment reduced the infarct size whilst increasing the thickness of the infarcted wall, as assessed by histology. Underpinning these therapeutic benefits, we detected improved formation of solid connective tissues, along with increased accumulation and activation of cardiac fibroblasts to myofibroblasts, in the infarct area (but not in the uninjured area). Furthermore, IL-4c treatment enhanced neovascular formation and attenuated cardiomyocyte hypertrophy in the peri-infarct area. In addition, although in-vitro co-culture with CD206+F4/80+ cardiac M2-like macrophages increased activation of primary cardiac fibroblasts, addition of IL-4 (up to 500ng/ml) did not activate cardiac fibroblasts. Together with the lower expression of the IL-4 receptor in cardiac fibroblasts (Il4ra expression was 1/500.000 vs. cardiac M2-like macrophages), activation of fibroblasts detected in-vivo was considered to be induced via M2-like macrophage-mediated effects (i.e. secretion of TGFb and Osteopontin), but not by the direct effect of IL-4. Conclusion(s): IL-4 treatment enhances post-MI cardiac repair through enhancing the M2-like macrophage-mediated intrinsic self-repair mechanism, proposing that IL-4 has great potential as a repurposed biological drug for the treatment of MI.