Exogenous Ubiquitin Differentially Modulates the Phenotype and Function of M1 and M2 Macrophages

Background: Unresolved inflammation is a major predictor of heart failure following myocardial infarction. Exogenous ubiquitin (eUB) is shown to decrease inflammatory response and confer cardioprotection in mice 3 days post-ischemia/reperfusion (I/R) injury. Here, we hypothesized that eUB differentially modulates the phenotype and function of M1 and M2 macrophages. Methods and Results: Peritoneal macrophages, pretreated with UB for 30 min, were exposed to IFN-gamma (M1 polarization) or IL-4 (M2 polarization) for 72 h. Cytokine/chemokine levels were measured in conditioned media, while cells were used for functional and biochemical assays. eUB reduced TNF-alpha secretion in M1, and TNF-alpha and IL-10 secretion in M2 macrophages. eUB induced cytoskeletal reorganization and reduced surface area in M1 macrophages. eUB enhanced M1 migration; however, it decreased M2 macrophage migration and efferocytosis. It decreased STAT1 and FAK phosphorylation in M1, while increasing STAT6 and FAK phosphorylation in M2 macrophages. Total protein ubiquitination remained unchanged. In non-activated macrophages, eUB altered morphology, suppressed IL-1 beta, IL-2, and IL-5 secretion, and enhanced efferocytosis. Conclusion: eUB modulates macrophage polarization, reduces pro-inflammatory cytokine secretion, and alters functional parameters and intracellular signaling. These effects may contribute to the cardioprotective potential of eUB 3 days post-I/R injury.