Macrophage polarization is a dynamic process driven by a complex interplay of cytokine signaling, metabolism, and epigenetic modifications mediated by pathogens. Upon encountering specific environmental cues, monocytes differentiate into macrophages, adopting either a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype, depending on the cytokines present. M1 macrophages are induced by interferon-gamma (IFN-γ) and are characterized by their reliance on glycolysis and their role in host defense. In contrast, M2 macrophages, stimulated by interleukin-4 (IL-4) and interleukin-13 (IL-13), favor oxidative phosphorylation and participate in tissue repair and anti-inflammatory responses. Metabolism is tightly linked to epigenetic regulation, because key metabolic intermediates such as acetyl-coenzyme A (CoA), α-ketoglutarate (α-KG), S-adenosylmethionine (SAM), and nicotinamide adenine dinucleotide (NAD+) serve as cofactors for chromatin-modifying enzymes, which in turn, directly influences histone acetylation, methylation, RNA/DNA methylation, and protein arginine methylation. These epigenetic modifications control gene expression by regulating chromatin accessibility, thereby modulating macrophage function and polarization. Histone acetylation generally promotes a more open chromatin structure conducive to gene activation, while histone methylation can either activate or repress gene expression depending on the specific residue and its methylation state. Crosstalk between histone modifications, such as acetylation and methylation, further fine-tunes macrophage phenotypes by regulating transcriptional networks in response to metabolic cues. While arginine methylation primarily functions in epigenetics by regulating gene expression through protein modifications, the degradation of methylated proteins releases arginine derivatives like asymmetric dimethylarginine (ADMA), which contribute directly to arginine metabolism—a key factor in macrophage polarization. This review explores the intricate relationships between metabolism and epigenetic regulation during macrophage polarization. A better understanding of this crosstalk will likely generate novel therapeutic insights for manipulating macrophage phenotypes during infections like tuberculosis and inflammatory diseases such as diabetes.

Mycobacterium tuberculosis (Mtb) is responsible for approximately 1.5 million deaths each year. Though 10% of patients develop tuberculosis (TB) after infection, 90% of these infections are latent. Further, mice are nearly uniformly susceptible to Mtb but their M1-polarized macrophages (M1-MΦs) can inhibit Mtb in vitro, suggesting that M1-MΦs may be able to regulate anti-TB immunity. We sought to determine whether human MΦ heterogeneity contributes to TB immunity. Here we show that IFN-γ-programmed M1-MΦs degrade Mtb through increased expression of innate immunity regulatory genes (Inregs). In contrast, IL-4-programmed M2-polarized MΦs (M2-MΦs) are permissive for Mtb proliferation and exhibit reduced Inregs expression. M1-MΦs and M2-MΦs express pro- and anti-inflammatory cytokine-chemokines, respectively, and M1-MΦs show nitric oxide and autophagy-dependent degradation of Mtb, leading to increased antigen presentation to T cells through an ATG-RAB7-cathepsin pathway. Despite Mtb infection, M1-MΦs show increased histone acetylation at the ATG5 promoter and pro-autophagy phenotypes, while increased histone deacetylases lead to decreased autophagy in M2-MΦs. Finally, Mtb-infected neonatal macaques express human Inregs in their lymph nodes and macrophages, suggesting that M1 and M2 phenotypes can mediate immunity to TB in both humans and macaques. We conclude that human MФ subsets show unique patterns of gene expression that enable differential control of TB after infection. These genes could serve as targets for diagnosis and immunotherapy of TB.

Although the BCG vaccine offers partial protection, tuberculosis remains a leading cause of infectious disease death, killing ∼1.5 million people annually. We developed mucosal vaccines expressing the autophagy-inducing peptide C5 and mycobacterial Ag85B-p25 epitope using replication-defective human adenovirus (HAdv^85C5) and bovine adenovirus (BAdv^85C5) vectors. BAdv^85C5-infected dendritic cells (DCs) expressed a robust transcriptome of genes regulating antigen processing compared to HAdv^85C5-infected DCs. BAdv^85C5-infected DCs showed enhanced galectin-3/8 and autophagy-dependent in vitro Ag85B-p25 epitope presentation to CD4 T cells. BCG-vaccinated mice were intranasally boosted using HAdv^85C5 or BAdv^85C5 followed by infection using aerosolized Mycobacterium tuberculosis (Mtb). BAdv^85C5 protected mice against tuberculosis both as a booster after BCG vaccine (>1.4-log₁₀ reduction in Mtb lung burden) and as a single intranasal dose (>0.5-log₁₀ reduction). Protection was associated with robust CD4 and CD8 effector (T_EM), central memory (T_CM), and CD103⁺/CD69⁺ lung-resident memory (T_RM) T cell expansion, revealing BAdv^85C5 as a promising mucosal vaccine for tuberculosis.

Macrophages (MФ) are an essential immune cell for defense and repair that travel to different tissues and adapt based on local stimuli. A critical factor that may govern their polarization is the crosstalk between metabolism and epigenetics. However, simultaneous measurements of metabolites, epigenetics, and proteins (phenotype) have been a major technical challenge. To address this, we have developed a novel triomics approach using mass spectrometry to comprehensively analyze metabolites, proteins, and histone modifications in a single sample. To demonstrate this technique, we investigated the metabolic-epigenetic-phenotype axis following polarization of human blood–derived monocytes into either ‘proinflammatory M1-’ or ‘anti-inflammatory M2-’ MФs. We report here a complex relationship between arginine, tryptophan, glucose, and the citric acid cycle metabolism, protein and histone post-translational modifications, and human macrophage polarization that was previously not described. Surprisingly, M1-MФs had globally reduced histone acetylation levels but high levels of acetylated amino acids. This suggests acetyl-CoA was diverted, in part, toward acetylated amino acids. Consistent with this, stable isotope tracing of glucose revealed reduced usage of acetyl-CoA for histone acetylation in M1-MФs. Furthermore, isotope tracing also revealed MФs uncoupled glycolysis from the tricarboxylic acid cycle, as evidenced by poor isotope enrichment of succinate. M2-MФs had high levels of kynurenine and serotonin, which are reported to have immune-suppressive effects. Kynurenine is upstream of de novo NAD⁺ metabolism that is a necessary cofactor for Sirtuin-type histone deacetylases. Taken together, we demonstrate a complex interplay between metabolism and epigenetics that may ultimately influence cell phenotype.

Arginine and tryptophan are pivotal in orchestrating cytokine-driven macrophage polarization and immune activation. Specifically, interferon-gamma (IFN-γ) stimulates inducible nitric oxide synthase (iNOS) expression), leading to the conversion of arginine into citrulline and nitric oxide (NO), while Interleukin-4 (IL4) promotes arginase activation, shifting arginine metabolism toward ornithine. Concomitantly, IFN-γ triggers indoleamine 2,3-dioxygenase 1 (IDO1) and Interleukin-4 induced 1 (IL4i1), resulting in the conversion of tryptophan into kynurenine and indole-3-pyruvic acid. These metabolic pathways are tightly regulated by NAD⁺-dependent sirtuin proteins, with Sirt2 and Sirt5 playing integral roles. In this review, we present novel insights that augment our understanding of the metabolic pathways of arginine and tryptophan following Mycobacterium tuberculosis infection, particularly their relevance in macrophage responses. Additionally, we discuss arginine methylation and demethylation and the role of Sirt2 and Sirt5 in regulating tryptophan metabolism and arginine metabolism, potentially driving macrophage polarization.

The nucleoprotein (NP) is a vital target for the heterosubtypic immunity of CD8⁺ cytotoxic T lymphocytes (CTLs) due to its conservation among influenza virus subtypes. To further enhance the T cell immunity of NP, autophagy-inducing peptide C5 (AIP-C5) from the CFP10 protein of Mycobacterium tuberculosis was used. Mice were immunized intranasally (i.n.) with human adenoviral vectors, HAd-C5-NP(H7N9) or HAd-NP(H7N9), expressing NP of an H7N9 influenza virus with or without the AIP-C5, respectively. Both vaccines developed similar levels of NP-specific systemic and mucosal antibody titers; however, there was a significantly higher number of NP-specific CD8 T cells secreting interferon-gamma (IFN-γ) in the HAd-C5-NP(H7N9) group than in the HAd-NP(H7N9) group. The HAd-C5-NP(H7N9) vaccine provided better protection following the challenge with A/Puerto Rico/8/1934(H1N1), A/Hong Kong/1/68(H3N2), A/chukkar/MN/14951-7/1998(H5N2), A/goose/Nebraska/17097/2011(H7N9), or A/Hong Kong/1073/1999(H9N2) influenza viruses compared to the HAd-NP(H7N9) group. The autophagy transcriptomic gene analysis of the HAd-C5-NP(H7N9) group revealed the upregulation of some genes involved in the positive regulation of the autophagy process. The results support further exploring the use of NP and AIP-C5 for developing a universal influenza vaccine for pandemic preparedness.