Metabolic Immunomodulation, Transcriptional Regulation, and Signal Protein Expression Define the Metabotype and Effector Functions of Five Polarized Macrophage Phenotypes

As of 2013, there were nearly 30 million Americans with diabetes or one in ten adults, but by 2050, estimates from the American Diabetes Association indicate that one in three adults (~350 million Americans) will be diabetic. At current rates of amputation (~25%) due to non-healing wounds, this 2050 population will have to absorb the social and economic burden of over 85 million diabetic amputees. While normal wound healing proceeds through a well-described iterative process, in non-healing wounds this process stalls at the transition from inflammation to tissue repair. Our central hypothesis is that macrophage plasticity is key to this transition and directly facilitates shifting the wound environment from pro-inflammatory to promoting tissue repair. While more established signaling molecules of immunity (such as cytokines and chemokines) remain essential to driving functional phenotype in the immune system, recent advances in metabolomics profiling have revealed a pivotal role for metabolites in immunomodulation. Presented herein, an ex vivo macrophage culture model produces five macrophage functional phenotypes compared to the parent resting macrophage phenotype and is a systems-level model for understanding the contribution of metabolism to macrophage functional plasticity. Specifically, CD14+ peripheral blood monocytes are isolated, differentiated into M0 macrophages, and polarized into the M1 (IFN-gamma;/LPS), M2a (IL-4/IL-13), M2b (IC/LPS), M2c (IL-10), and M2d (IL-6/LIF) phenotypes. Each phenotype is then characterized by a bioanalyte matrix of four cell surface markers, ~50 secreted immunomodulating proteins (cytokines, chemokines, and growth factors), ~800 myeloid genes, and ~450 identified metabolites (including lipids). Signal protein profiles and pathway enrichment analysis of expressed genes generally groups the phenotypes into pro-inflammatory (M1 and M2b) and tissue repair/regenerative (M2a, M2c, and M2d); however, clear distinctions between each phenotype can be made. For example, M1 macrophages activate processes that facilitate acute inflammatory responses through control of nitric oxide (NO) and reactive oxygen species (ROS), whereas M2b macrophages activate processes that regulate chronic inflammation and chemotaxis of lymphocytes, endothelial cells, and epithelial cells. Furthermore, both M2c and M2d macrophages activate angiogenesis processes, but M2c macrophages promote extracellular matrix (ECM) assembly while M2d macrophages activate processes that drive ECM degradation. Finally, metabolomics profiles further validate recent findings that shifts between aerobic glycolysis, the pentose phosphate pathway, and oxidative phosphorylation distinguish pro-inflammatory macrophages and tissue repair/regeneration macrophages; however, our model provides further evidence to support the association between metabolism (such as decoupling of the TCA cycle, fatty acid synthesis, and -oxidation) and functional phenotype. Metabolomics is fundamentally changing our understanding of immunomodulation in diverse macrophage populations and that deeper understanding is informing our long-term goals of developing novel diagnostic and therapeutic approaches to wound healing. By integrating metabolomics into our systematic characterization of these phenotypes, we have developed quantifiable metrics that not only define metabolic and functional phenotype, but also provide insight into the cellular functions fundamental to macrophage plasticity.