Aims:
Macrophages have been appreciated as an attractive cell therapy candidate based on their important role in tissue repair from inflammation to fibrosis. More importantly, macrophages can acquire memory-like characteristics in response to stimulation. Therefore, re-education of these macrophages has been actively studied in diverse disease models as a therapeutic strategy. Recent studies showed that hypoxic preconditioning or hypoxic tolerance drives endogenous protective mechanism against ischemic injury. Especially, hypoxia-primed stem cells showed protective effects on survival or regenerative capabilities in several diseases.  However, the effect of the hypoxic preconditioning on the physiology of macrophages and the impact of the respective changes on cardiac remodeling has not yet been visualized and studied. Our aim is to investigate whether hypoxia-primed macrophages can be applied as a novel therapeutic tool to regulate excessive inflammation and fibrosis in cardiac diseases.

Methods & Results:
Hypoxia-primed macrophages showed a significant downregulation in the level of inflammation and fibrosis compared to macrophages exposed to normoxic condition. These phenotypic changes were reversed upon inhibiting hypoxia signal, HIF-1a, indicating protective effects of hypoxia preconditioning on macrophages activation. Further, to investigate the impact of hypoxia on the polarization of the macrophages, we subjected the cells to hypoxic or normoxic conditions, followed by polarization towards the M2 (anti-inflammatory and pro-fibrotic) phenotype. Interestingly, hypoxia-primed macrophages amplified anti-fibrotic phenotypes following polarization to M2, suggesting regulatory effects of hypoxia priming on macrophages phenotype changes. Next, to test the effect of hypoxia-primed macrophages on cardiac remodeling after injury, we implemented a co-culture of macrophages on a novel ex vivo model, called living myocardial slices (LMS). LMS are ultrathin (100 - 400 um) sections of multicellular living cardiac tissue and more importantly, we could mimic ischemic injury by subjecting them to cryo-injury. Co-cultured macrophages were recruited into border zone of the cryo-injury area while they were dispersed globally on LMS without cryo-injury. Furthermore, we examined the effects of hypoxic- or normoxic-primed macrophages on the functional parameters of LMS with or without cryo-injury. Here, native LMS were used as negative control. Interestingly, LMS with cryo-injury showed an improved contractility when co-cultured with hypoxia-primed macrophages.

Conclusions:
Hypoxia-priming empowered macrophages toward anti-inflammatory and anti-fibrotic phenotypes. Importantly, hypoxia-primed macrophages presented protective effects on cardiac function in pathological ex vivo model. Altogether, our results highlighted that hypoxia priming of macrophages could be a cell therapy option for ischemic cardiac injuries.