Integrated Metabolomics, Transcriptomics, and Signal Protein Profile Define the Effector Function and Associated Metabotype of Five Polarized Macrophage Phenotypes

Abstract

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 Immunometabolism and Inflammaging: Roles of Mitochondrial Dysfunction, Cellular Senescence, CD38, and NAD

“…Traditionally, they have been polarized with IL-4 and IL-13 to the M2a subdivision and most studies generally refer to this subtype when mentioning M2 macrophages. Since macrophages are heterogeneous and the various phenotypes are still being characterized, it is currently unknown exactly how the macrophage phenotypic landscape changes with age, but new metrics from various omics technologies may be able to help elucidate this in the near future [ 25 ]. Regardless, it does seem that macrophages increase in number in most, but not all, studied tissue types with age and display an altered physiology that often contribute to pathology [ 19 , 22 – 24 , 26 , 27 ].…”

Implication of metabolism in the polarization of tumor-associated-macrophages: the mass spectrometry-based point of view

“…M1 macrophages express high levels of inducible nitric oxide synthetase (iNOS) that promote the release of nitric oxide, reactive oxygen species and pro-inflammatory cytokines (IL-1β, IL-6, IL-12) promoting Th1 response ( 2 , 13 ). The high production of anti-microbial and anti-tumoral molecules gives to M1 macrophages the capability to kill intracellular pathogens ( 5 , 18 ). Classically activated macrophages express specific surface proteins, such as CD68, CD80, and CD86 ( Figure 1A ).…”

“…M2 macrophages have a poorly glycolytic profile with higher arginine metabolism and preferential fatty acid oxidation (oxidative glucose metabolism), glutamine metabolism and OXPHOS ( Figure 1B ) ( 5 , 18 , 19 , 24 ). Glycolysis is not essential for M2 phenotype, and glucose is mainly employed to sustain OXPHOS ( 25 – 27 ).…”