Introduction: Cardiosphere-derived cells (CDCs) have been shown to prevent abnormal heart remodeling, tissue fibrosis, and improve systolic function post myocardial infarction (MI). These effects appear to be largely mediated by paracrine signaling, of which exosomes have been shown to play a role. CDCs have also been shown to mediate cardioprotection via modulation of the macrophage response post MI. The role of CDC-derived exosomes on macrophage polarization, however, is less clear as is the contribution of anti-inflammatory macrophages to a cardioprotective CDC-exosome effect. Identifying the mechanisms of action of CDC-derived exosomes will enable identification of novel therapeutic approaches to augment cardiac repair.
Hypothesis: CDC-derived exosomes polarize cardiac macrophages to a distinct phenotype responsible for mediating cardioprotection and limiting myocardial injury through suppression of inflammation and fibrosis.
Methods: Mouse CD68+ macrophages were isolated by peritoneal lavage, plated at 4x106 cells/ml in RPMI media, and treated with CDC-exosomes or media control. Following a 6 hour incubation, RNA was extracted from macrophages and qPCR performed for a number of M1 and M2 genes. Additionally, to characterize what phenotype was dependent on exosome release, murine peritoneal macrophages were cocultured with either nSMase2 knockdown human CDCs unable to secrete exosomes or a scrambled CDC control. Following 24hrs, macrophage RNA was again extracted and qPCR performed.
Results: Purified CDC-exosomes polarized murine macrophages to a unique phenotype characterized by a downregulation of M1 genes (TNFa, Nos2, Il1b, Il6, and Fpr2) and a dose-dependent upregulation of the M2 gene, Arg1+. Coculture of CDCs with macrophages induced an Arg1high phenotype which was dependent on exosome release, as coculture with nSMase2 KD CDCs was unable to induce Arg1 expression in macrophages.
Conclusions: CDC exosomes polarize macrophages towards an anti-inflammatory phenotype with downregulation of M1 genes and a dose dependent upregulation of Arg1. CDC-exosomes are both necessary and sufficient for macrophage Arg1 expression, a gene previously implicated in suppressing Th2-dependent inflammation and fibrosis.