M1-type macrophages respond to danger signals transmitted by bacterial products or interferon-γ (IFN-γ), which attract and activate cells of the adaptive immune system. Additionally, M1-type macrophages are typically activated by lipopolysaccharide (LPS), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor α (TNF-α). Several pathways promote macrophage polarization to the M1 phenotype, including the IRF/STAT, LPS/TLR4, and NF-κB/PI3K pathways. M1-type macrophages are characterized by strong antigen-presenting activity and the secretion of proinflammatory cytokines such as interleukin-1β (IL-1β), IL-6, IL-12, IL-18, IL-23, and TNF-α. Furthermore, M1-type macrophages also secrete chemokines such as CXCL-9 and CXCL-12. Moreover, M1-type macrophages express high levels of major histocompatibility complex II (MHC II), CD68, CD80, and CD86 and so on (2).

M1-type macrophages exert anti-tumor effects by distinguishing tumor cells from normal cells and ultimately eliminating tumor cells. Research indicates that M1-type macrophages utilize two distinct mechanisms to eliminate tumor cells. First, M1-type macrophages directly mediate cytotoxicity against tumor cells by releasing tumor-killing molecules such as reactive oxygen species (ROS) and nitric oxide (NO) (3). This process is relatively slow, typically taking 1 to 3 days. The second mechanism involves antibody-dependent cell-mediated cytotoxicity, which allows for a faster response, generally killing tumor cells within a few hours (4). Antibody-dependent cell-mediated cytotoxicity requires the participation of anti-tumor antibodies.

M2-type macrophages can be activated by parasites, fungal infections, immune complexes, apoptotic cells, and cytokines such as IL-4, IL-13, IL-25, IL-33, and TGF-β. In contrast to the classically activated isoform, the alternatively activated isoform down-regulates IL-1, IL-6, IL-12, IL-23, and TNF-α while up-regulating IL-10. M2-type macrophages express chemokines such as CCL1, CCL17, CCL18, CCL22, and CCL24. The M2 phenotype is characterized by the expression of CD163, CD204, CD206, and CD209. Additionally, M2-type macrophages express numerous scavenger receptors, which are associated with the high-level expression of IL-10, IL-1β, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs) (2).

Four M2 subtypes are known to respond to different stimuli: M2a, M2b, M2c, and M2d (5). These subtypes differ in their cell surface markers, secreted cytokines, and biological functions (6). M2a macrophages are activated by IL-4 or IL-13. IL-4 promotes the expression of CD206 and further up-regulates IL-10, TGF-β, CCL17, CCL18, and CCL22, which are known to promote cell growth, tissue repair, and endocytosis (7). M2b macrophages are activated by immune complexes, TLR ligands, and IL-1β. Upon activation, M2b macrophages release both pro-inflammatory and anti-inflammatory cytokines, including TNF-α, IL-1β, IL-6, and IL-10. M2b macrophages play a critical role in regulating immune responses and inflammation (8). M2c macrophages can be activated by glucocorticoids, IL-10, and TGF-β, and are characterized by high expression of the anti-inflammatory cytokine IL-10, the pro-fibrotic factor TGF-β, CCL16, CCL18, and Mer receptor tyrosine kinase (MerTK), which promotes the endocytosis of apoptotic cells (9). M2d macrophages are activated by TLR antagonists, IL-6, and adenosine, with adenosine promoting the expression of IL-10 and VEGF, thereby exacerbating angiogenesis and tumor progression (10) ( Figure 1 ; Table 1 ). Collectively, M2-type macrophages function to remove debris, promote angiogenesis, facilitate tissue reconstruction and injury repair, and promote tumorigenesis and progression.

M1-type TAMs are generally regarded as tumor-killing macrophages that primarily promote anti-tumor immunity. In contrast, M2-type TAMs are immunosuppressive and facilitate tissue repair and tumor progression. Both M1 and M2 TAMs are present at all stages of tumor progression (11). M1-type TAMs predominate in the early stages, while M2-type TAMs are more prevalent in the middle and late stages. As the tumor progresses, M1-type TAMs gradually polarize into M2-type TAMs, and an increase in the number of M2-type TAMs indicates a poor prognosis. The balance between M1 and M2 TAMs is critical for determining the overall immune response within the TME (12).

M2-type TAMs with the high-level expression of MMPs, such as MMP-2 and MMP-9, degrade the extracellular matrix, facilitating angiogenesis and tumor cell invasion. Similarly, M2d TAMs secrete VEGF, which promotes the formation of new blood vessels, supplying tumors with oxygen and nutrients. VEGF also enhances vascular permeability, facilitating tumor cell invasion and metastasis. Additionally, M2-type TAMs exert immunosuppressive effects in the tumor microenvironment by secreting a series of cytokines and chemokines, as described below: IL-10 is a key immunosuppressive cytokine, which inhibits the activation of dendritic cells and T cells, suppresses the production of pro-inflammatory cytokines, and promotes the differentiation of regulatory T cells (Tregs), leading to immune evasion. TGF-β suppresses the anti-tumor activity of cytotoxic T cells and natural killer (NK) cells, while promoting the differentiation of Tregs. It also contributes to the epithelial-mesenchymal transition, facilitating tumor cell invasion and metastasis. These chemokines (CCL16, CCL17, CCL18, and CCL22) recruit immune-suppressive cells, such as Tregs and myeloid-derived suppressor cells (MDSCs), to the TME, further enhancing immune evasion.

Although M2-type TAMs dominate in most advanced cancers, M1-type TAMs can also be present, especially in the early stages of tumor development. M1-type TAMs secrete factors that exert anti-tumor effects: IL-12, TNF-α, and IL-1β activate cytotoxic T cells and NK cells, and CXCL9 recruits Th1 cells and cytotoxic T cells to the TME, enhancing anti-tumor immunity.

These secretions of TAMs have dual roles depending on the balance between M1 and M2 phenotypes in the TME. For example, while TNF-α can induce tumor cell death, it can also promote tumor progression by activating NF-κB signaling in tumor cells. IL-6 can promote tumor cell survival and proliferation but can also activate immune cells in certain contexts (13). Additionally, lactate produced by tumor cells and TAMs acts as both a metabolic fuel and a signaling molecule. Lactate lowers the pH in the TME, which supports immune evasion by inhibiting the activity of cytotoxic T-cells and NK cells, while promoting Tregs and MDSCs that foster an immunosuppressive environment. Understanding the functional diversity and plasticity of TAMs is essential for developing therapeutic strategies aimed at reprogramming these cells to enhance anti-tumor immunity.

Histone methylation refers to the methylation that occurs on the N-terminal arginine or lysine residues of H3 and H4 histones. Arginine can be methylated once (resulting in monomethyl arginine) or twice. Arginine methyltransferases can transfer two methyl groups to the same nitrogen atom of an arginine residue to form asymmetric dimethylarginine, or they can add a methyl group to each N terminus, resulting in symmetrical dimethylarginine (20). Similarly, lysine can be methylated once, twice, or even three times, catalyzed by lysine methyltransferases. Among histone modifications, methylation is the most extensively studied class. Methylation at different sites on histones can produce diverse biological effects. Methylation of H3K4 and H3K36 occurs in genomic regions with transcriptional activity and is involved in the transcriptional activation process (21). H3K9me3/2 and H3K27me3 are commonly found on transposons and inactive genes, where they repress transcription by recruiting protein complexes that bind to nucleosomes and modify chromatin structure. H3K9me3 is enriched in heterochromatin, while H3K9me2 is frequently associated with gene silencing in euchromatin. In summary, the functions of histone methylation are primarily reflected in heterochromatin formation, gene imprinting, X chromosome inactivation, and transcriptional regulation.

Histone acetylation is another crucial post-translational modification that occurs on histone proteins. In this process, an acetyl group is added to the lysine residues of histone proteins, typically at the N-terminal tails (39). This modification is catalyzed by enzymes called histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs). When histones are acetylated, the positive charge of lysine residues is neutralized, reducing the interaction between histones and the negatively charged DNA. This relaxation of chromatin structure creates a more open and accessible configuration, allowing transcription factors and other regulatory proteins to access DNA more easily, thereby promoting gene transcription. Therefore, histone acetylation and deacetylation work in a dynamic balance to regulate chromatin structure and gene expression.

The progress of lactyl groups adding to histone proteins is called histone lactylation. This modification was first discovered in 2019 and is closely associated with cellular metabolic states, particularly those related to lactate production (54). Lactate metabolism in tumor cells is a key feature of TME, often driven by the “Warburg effect”, where tumor cells preferentially utilize glycolysis over oxidative phosphorylation, even in the presence of oxygen (55). This metabolic reprogramming leads to high production of lactate, which accumulates in the TME and influences both tumor growth and the behavior of surrounding stromal and immune cells (56). Lactate can lead to histone lactylation within TAMs, directly modifying gene expression to support tissue repair and immunosuppressive pathways. In the TME, lactate metabolism by tumor cells promotes TAMs polarization toward a M2-like phenotype, which is partly due to lactate-induced signaling pathways, such as stabilization of hypoxia-inducible factor-1α (HIF-1α) and activation of specific metabolic pathways within TAMs that favor an immunosuppressive state. Jialiang Cai et al. found that lactate produced by tumors induced lactylation of the histone H3K18la site upon transport into macrophages, thereby activating transcription and enhancing M2-like TAMs activity (57). Xia Fang et al. put forward that HIF-1α stabilization and histone lactylation were both required for M2-like TAMs polarization (58). Besides, Xin Wu et al. showed that the secretion of lactate and histone lactylation alterations within tumor cells recruits and polarizes M2-like TAMs through the PI3K/AKT-CXCL14 axis in neuroblastoma (59).

Lactate enhances the immunosuppressive properties of TAMs by promoting the expression of anti-inflammatory cytokines like IL-10 and TGF-β. Alessandra De Leo et al. found inhibition of glycolysis or lactate production in monocyte-derived macrophages (MDMs) impaired IL-10 expression and T cell suppression. Mechanistically, intracellular lactate-driven histone lactylation promoted IL-10 expression to suppress T cell activity. Consistently, abrogated histone lactylation led to the accumulation of intratumoral T cells and tumor growth delay. Besides, inhibition of histone lactylation combination with immunotherapy could block glioblastoma (GBM) progression in vivo ( 60). Xiuming Li et al. reported that tumor-derived lactate fuels H3K18 lactylation prohibited RARγ transcription in macrophages, consequently enhancing IL-6 levels in the TME and endowing macrophages with tumor-promoting functions via activation of signal transducer and activator of transcription 3 (STAT3) signaling in colorectal cancer cells (61).

Given these multifaceted roles, histone lactylation in the TME is considered a promising target for cancer therapies aimed at disrupting the metabolic adaptations of tumors and reshaping the immune landscape to improve anti-tumor immune responses. Kiranj Chaudagar et al. demonstrated that combining androgen deprivation therapy (ADT), PI3K inhibitors (PI3Ki), and PD-1 antibodies (aPD-1) suppressed H3K18 lactylation and fully activated TAMs, achieving significant therapeutic effects in prostate cancer (62) (63).

Phosphorylation usually occurs on serine, threonine and tyrosine residues, and is a reversible modification regulated by phosphorylase or kinase, in which the enzyme transfers the phosphate group on ATP to or removes it from the receptor amino acid residue (64). In addition, phosphorylation modification of arginine and histidine is also present, but the biochemistry is less clear because of their instability. Most histone phosphorylation occurs in the N-terminal tail and mainly in histone H3, but the phosphorylation of histone H1 at multiple sites has been demonstrated as one of the prerequisite steps for gene induction (65). Histone phosphorylation may affect chromatin structure and function through two mechanisms. On the one hand, phosphorylation provides a negative charge for histones, which weakens their ability to bind to DNA and loosens chromatin, similar to acetylation modification. On the other hand, protein complexes that specifically recognize phosphorylation sites can recognize and bind to the surface of phosphorylated histones to exert regulatory functions together (66).

Histone phosphorylation regulates the function of TAMs mainly through the intervention on the interleukin promoter. Sayantan Banerjee et al. reported that both the lack of transcription favorable histone phosphorylation at the Il-12 promoter and the abundance of ERK1/2-dependent histone phosphorylation at the Il-10 promoter led to the polarization of TAMs to M2, although the mechanism underlying the ability of the TME to preferentially change the phosphorylation pattern of histones in TAMs has not been clarified (67). Interestingly, Oakley C Olson et al. reported that TAMs suppressed the duration of Taxol-induced mitotic arrest in breast cancer cells and promote earlier mitotic slippage. This correlates with a decrease in the phosphorylated form of H2AX, decreased p53 activation, and reduced cancer cell death in interphase (68).

Protein ubiquitination is a 76-amino acid ubiquitin molecule (Ub) connected to the Lys of the target protein through the C-terminus. Target proteins can undergo either mono-or polyubiquitination, which is mediated by a combination of E1 activating enzymes, E2 conjugating enzymes, and E3 ligases, while deubiquitinating enzymes (DUBs) can remove Ub from the target protein (69). Histones are among the most abundant mono-ubiquitinated proteins (70). Wenlai Zhou et al. found that mono-ubiquitination of H2A at Lys 119 prevented the recruitment of SPT16 and SSRP1 at the transcriptional promoter region, and blocked RNA polymerase II release at the early stage of elongation, which mediated selective repression of a specific set of chemokine genes modulating migratory responses to TLR activation in macrophages, such as Ccl5, Cxcl0, and Cxcl12 ( 71).

Histone ubiquitination can promote its degradation, participate in DNA damage repair and chromatin remodeling, thereby regulating gene transcriptional activation and silencing. The regulatory effect of histone ubiquitination on TAMs is reflected in its stability to HMTs and HATs. For examples, Peng Wang et al. identified SET and MYND domain-containing protein 3 (SMYD3) as the methyltransferase of EZH2 at K421 residue which accelerates EZH2 ubiquitin proteasome degradation to promote M2 polarization in gastric cancer (72). Lingfang Du et al. revealed that epigenetic regulator KDM6B by virtue of its demethylase activity prevented M2 polarization via promoting the intranuclear degradation of β-catenin (73). Yichang Wang et al. found that USP24 could stabilize p300 to increase the levels of histone-3 acetylation and NF-κB, thereby increasing IL-6 transcription in M2-type TAMs and lung cancer cells, resulting in cancer malignancy finally (48).

SUMOylation is another reversible post-translational modification of proteins, which is also known as small ubiquitin-like modification protein. Similar to ubiquitination, SUMO is a small protein containing 100 amino acids. It is transferred to lysine residues of the target protein by SUMO E1, E2 and E3 enzyme, while SUMO specific protease (SENP) can remove SUMO modification from the target protein (74). Surprisingly, Qi Yang et al. found that HDAC4, belonging to the SUMO E3 ligase family, negatively regulated NF-κB activation for IκBα SUMOylation (75).

Currently, five SUMO isoforms have been identified in humans, namely SUMO-1, SUMO-2, SUMO-3, SUMO-4 and SUMO-5. SUMOylation is functionally different from ubiquitination (76). Ubiquitination mainly promotes the degradation of the target protein, while SUMO modification makes the protein more stable. SUMO modification is essential for biological processes such as gene expression regulation, DNA damage repair, maintenance of genome integrity, cell cycle and apoptosis. Histone SUMOylation was discovered in 2003 (77), but there is still relatively little research and understanding on this aspect. It is known that SUMO can conjugate all histone proteins, H2A variant H2A.X and H3 variant Cse4, and some modification sites have been identified, such as H2BK6, K7, H4K5, K8, etc (78). Histone SUMOylation, which is commonly associated with transcriptional repression, can lead to chromatin condensation and gene silencing by promoting the recruitment of the deacetylase HDAC1 and the heterochromatin-related protein HP1 (79, 80). In addition, acetylated H4 can induce SUMO-1 binding, and enhance SUMOylation through the interaction of acetyltransferase P300, indicating that acetylation may promote SUMOylation (81), and there is a mutual regulatory relationship between SUMOylation and different histone modifications. Gabriel Pascual et al. reported that ligand-dependent SUMOylation of the PPAR-γ ligand-binding domain could target HDAC3 complexes on inflammatory gene promoters in mouse macrophages, thereby antagonizing inflammatory (82).

ADP-ribosylation uses NAD⁺ as a substrate and adp-ribosyltransferase (ARTs) catalyzes the transfer of ADP-ribose groups to the side chain of amino acid residues of target proteins, which regulates the structure and function of proteins transfers. ADP-ribosylation is modified in two forms: mono-ADP-ribosylation and poly-ADP-ribosylation, catalyzed by ecto-ARTs and poly-ARTs.

ADP-ribosylation of all core histones and linker H1 can occur on a variety of amino acid residues such as glutamate, aspartic acid, arginine and lysine in the histone tail, of which 1% occurs on the lysine residue (83). Studies have shown that histone ADP-ribosylation is involved in the regulation of higher chromatin structure and can promote chromatin relaxation (84). Histone ADP-ribosylation directly destabilizes histone-DNA interactions in the nucleosome and increases the site accessibility of the nucleosomal DNA to nucleases (85). Histone H2B and H3 were mainly involved in DNA damage repair, and a small part of H1 and H4 were also involved. DNA damage increases PAR modification of histones by a mechanism that remains unclear. In addition, the level of ADP-ribosylation of histones increases during cell proliferation and continues throughout the proliferation process, contributing to DNA assembly. C Wang et al. found that M-CSF induced ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1) poly-ADP-ribosylation in macrophages (86). Ricardo Martinez-Zamudio et al. reported that LPS stimulation-induced ADP-ribosylation at the nucleosome-occupied promoters of Il-1β, Mip-2, and Csf2 could facilitate the transcription of these genes in macrophages (85). It can be seen that M1-type TAMs are activated in a manner that generally raises the level of histone ADP-ribosylation.

Histone crotonylation is a post-translational modification where a crotonyl group (derived from crotonic acid) is added to lysine residues on histone proteins (87). Discovered in 2011, this modification is structurally distinct from acetylation (88), but Benjamin R Sabari et al. showed that the coactivator p300 has both crotonyltransferase and acetyltransferase activities, and that p300-catalyzed histone crotonylation directly stimulated transcription to a greater degree than histone acetylation (89). Histone crotonylation is associated with active gene promoters and enhancers. Recently, histone crotonylation is investigated in relation to cancer and immune regulation, where it appears to contribute to cellular plasticity and adaptation in the macrophage. For examples, Jing Yang et al. firstly showed the existence of crotonylation in porcine alveolar macrophages (90). Yu Zou et al. reported that inhibition of p300 alleviated partial infraorbital nerve transection-induced macrophage activation and reduced the expression of inflammatory cytokines Tnfα and Il1β, as well as chemokines Ccl2 and Cxcl10. Correspondingly, exogenous crotonyl-CoA induced macrophage activation and the expression of Tnfα, Il1β, Il6, Ccl2 and Ccl7 in trigeminal ganglia (91). Similarly, Lingzhi Li et al. found that a crotonyl-CoA-producing enzyme ACSS2 (acyl-CoA synthetase short chain family member 2) remarkably increased H3K9 crotonylation (H3K9cr) level without influencing H3K9ac in kidneys and tubular epithelial cells. Furthermore, genetic and pharmacologic inhibition of ACSS2 both suppressed H3K9cr-mediated IL-1β expression, which thereby alleviated IL-1β-dependent macrophage activation and tubular cell senescence to delay renal fibrosis (92). Christopher McCrory et al. reported that the short-chain fatty acid crotonate increases histone crotonylation, reduces hyphal formation within macrophages, and slows macrophage lysis and immune escape of C. albicans ( 93). Hao Zhang et al. showed a proteome-wide crotonylation profile of human leukemia monocyte cell line (THP1 cells) infected with methicillin-resistant Staphylococcus aureus and further treated with vancomycin, which pointed to a comprehensive understanding of the biological functions of histone crotonylation in human macrophages (94). Given its distinct and functionally relevant effects, histone crotonylation is a promising focus for epigenetic research and potential therapeutic strategies. Based on the fact that inhibition of histone crotonylation reduces cytokines and chemokines secreted by M2b-type TAMs, we hypothesized that crotonylation promotes M2 polarization of TAMs.

Histone modifications play a key role in physiological and pathological regulation, which is of great value for basic research and clinical exploration. Of course, there are more propionylation, butolylation, hydroxylation and formylation, etc. Interested partners can go to the relevant information to understand. In summary, there are many kinds of histone modifications, and different types have different degrees of crosstalk. Although the role of the aforementioned histone modifications in the function of TAMs has not been thoroughly investigated, based on their impacts on macrophages in other diseases, we believe that they will become potential targets in tumor immunotherapy via regulating TAMs polarization.