It is believed that macrophages belong to the mononuclear phagocytic system, mainly derived from bone marrow progenitor cells. When macrophages are within the bloodstream, they are called mononuclear cells. When inflammation or injury occurs, monocytes are recruited to tissues and induced into macrophages based on the condition of the local environment (1, 2). As a matter of fact, most adult tissue-resident macrophages are seeded during embryonic development, have self-renewal capacity and are maintained without depending on monocytes (2–4). The most majority of tissue-resident macrophages are derived from erythro-myeloid progenitors that develop in vitellus capsule embryos. However, the existence of tissue-specific macrophage progenitors that can promote the maintenance of adult macrophages has not been clarified (4). When the quantity of monocytes in the blood is reduced, those in the tissues are largely unaffected. However, monocytes and their progenitors can supplement classical tissue mononuclear phagocytes as needed (2). Embryonic macrophages are involved in tissue remodeling while monocyte-derived macrophages mainly play a role in host immune response (2). The activation status and the function of tissue macrophages under different stimuli are largely dependent on the local tissue microenvironment (3, 5). The importance of the local microenvironment has been identified through studies based on transcriptomics, epigenetics and open chromatin regions (3).

Currently, comparative studies of gene expression have been conducted in macrophages from the most commonly used cell sources: murine bone marrow, human peripheral monocytes, and human leukemic monocytic cell line THP-1, as well as those derived from induced pluripotent stem cells (iPSCs) which were generated from differentiated cells by upregulating pluripotency factors (Oct3/4, Sox2, c-Myc, and Klf4) (6). MCP1, IL6, TNF, IL10, CXCL12, IL1β and IL6 are expressed in human blood-derived macrophages; meanwhile, IL1β, TNFα, iNOS, IL12β, Arg1, VEGFA and IL10 are expressed in murine bone marrow-derived M1 and M2 macrophages (7). IL1 and CD36 are expressed in THP-1 cells in vitro ( 7), which can be treated as a model of human macrophages when studing relatively simple biological processes, but cannot be used in more comprehensive immunopharmacology and drug screening programs (8). Human peripheral blood-derived macrophages and human induced pluripotent stem cell (iPSC)-derived macrophages showed similar gene expression patterns, reminding that iPSC-derived monocytes can be used as a credible cell source of human macrophages for in vitro studies (7, 9). Further studies are needed to determine how the heterogeneity of macrophages is reflected, whether the functions and activities of tissue-specific macrophages are different from those of peripheral migration-induced cells, and the concordance between in vitro and in vivo experiments.

Diversity and plasticity are hallmarks of macrophages. In response to IFN-γ/lipopolysaccharide(LPS) (10) or IL-4/IL-13, macrophages undergo M1 or M2 activation (11), which represent two extremes of a continuum of functional states. M1 macrophages have proinflammatory abilities and are able to initiate and maintain inflammatory reactions (12, 13), induce Th1 response activation (14), activate endothelial cells, amplify antigen presenting capacity (12, 14, 15), secrete proinflammatory cytokines and recruit other immune cells into inflammatory tissue. Nevertheless, M2 macrophages release anti-inflammatory mediators (16), support angiogenesis, induce adaptive Th2 immunity, refurnish and repair of scavenge debris and damaged tissue, take part in tumor progression, allergic reactions, and response to helminths (15). Some molecules are relative to both M1 and M2 macrophage polarization, such as NF‐κB (17), IRF, AP1, PPAR‐γ, AMPK and SIRPα (18). Some pathways are mainly involved in one polarization process of macrophages, JAK/STAT1 (12), JAK/STAT5 (19), extracellular signal-regulated kinase (ERK) (19), and Notch-RBP-J signaling pathway (18) take part in M1 polarization. JAK/STAT6 (12) and IL-4-JNK-c-Myc pathway (20) participate in M2 polarization. The common transcription factors and the differences in M1 and M2 macrophages are described in Figure 1 .

Recent studies have found that the M2 macrophages are further sub-categorized into M2a, M2b, M2c, and M2d subtypes (21). M2a macrophages can be induced by IL-4 or IL-13, expressing high levels of arginase 1 (Arg1), FIZZ1 and Ym1 (22), playing a role in anti-inflammatory activity and tissue remodeling. M2b macrophages are activated by IL‐1 receptor ligand, immune complexes (ICs) and Toll-like receptor (TLR) agonists and produce both anti-inflammatory and proinflammatory cytokines, including IL-6, IL-10, CCL1, CD86, TNF-α and SpHK1 (23), playing a role in immunoregulation. The M2c subset of macrophages is induced by IL-10, TGF-β or glucocorticoids (24), secreting high levels of IL-10, TGF-β, CCL16, CCL18, MMP7, MMP8, and TIMP1, playing crucial roles in the phagocytosis of apoptotic cells (2) and wound healing (25). M2d macrophages, also called tumor-associated macrophages (TAMs), can be induced by IL-6 or combined exposure to adenosine 2A receptor (A2AR) agonists and TLR agonists. M2d macrophages secrete high levels of TGF-β, VEGF and IL-10, and low levels of IL-1β, IL-12 and TNF-α, taking part in tumor growth, angiogenesis and metastasis (26). It has been demonstrated that several stimuli in the tumor microenvironment, such as hypoxia, may contribute to the tumoral heterogeneity of TAMs. The integrated use of new technologies, such as single-cell RNA-seq, spatial transcriptomics, mass cytometry, and systems biology approaches, is promising to strongly reveal the tumoral heterogeneity of TAMs, potentially redefining TAMs with new valuable biomarkers (27). Excessive activity of either polarized phenotype is responsible for tissue damage, inflammatory disease, fibrosis, or tumor growth. Fortunately, macrophages sustain plasticity after activation and can change phenotypes according to the microenvironment (15).

Functional macrophage polarization has been reported in vivo, under physiological conditions (embryogenesis and pregnancy) and pathological conditions (chronic inflammation, infection and cancer). In some conditions, such as infection or pregnancy, macrophage polarizations express mixed or unique phenotypes. in vitro Because of the complexity of tissue macrophages, the in vitro-based M1/M2 dichotomous classification does not fully capture the classification of them. Some researchers have hypothesized that an M3 switch phenotype exists under the background of M1 and M2 phenotypes based on studies in lung diseases (28). M3 switch phenotype reprograms toward the anti-inflammatory M2 phenotype with proinflammatory stimuli, contrarily, M3 switches to proinflammatory M1 phenotype with anti-inflammatory stimuli. Therefore, understanding the role of coexisting phenotypes of macrophages and the mechanisms driving their dynamic modulation to adjust to the microenvironmental changes will be a key challenge in the coming years. The effort might provide a great prospective for designing macrophage-centered diagnostic and therapeutic strategies.

The maternal-fetal interface consist of various types of immune cells, such as decidual macrophages (dMϕs), natural killer (dNK) cells, T cells, dendritic cells, B cells and NKT cells. dMϕs account for a proportion of ~20% of decidual immune cells, and is the second most common immune cells during early human pregnancy (29). dMϕs are present in all stages of pregnancy and cannot be simply distinguished according to the M1 or M2 types. dMϕs constantly switch in the spectrum of continuous changes between the M1 and M2 types throughout the pregnancy. It has been found that dMϕs around the blastocyst are inclined toward M1 polarization before implantation. While trophoblast cells begin to invade the myometrium, dMϕ are inclined toward M1/M2 mixed-type polarization. M2 phenotype predominates during the late first trimester and maintenances of pregnancy during the second trimester. Then during the third trimester and parturition, M2 phenotype begins to decline and M1 phenotype again increases. Successful pregnancy requires that the dMϕs activation states remain regulated throughout pregnancy. Imbalanced M1/M2 dynamics are associated with complications, such as fetal growth restriction, preeclampsia and preterm delivery.

Different from dMϕs, which are primarily recruited from the maternal peripheral circulation, there is another homogeneous population of macrophages of fetal origin at the maternal-fetal interface, which are called Hofbauer cells (HBCs) (30). HBCs exist in the placental villi starting from Day 11 in mice and Day 18 in humans, persisting until parturition and representing a highly pleiomorphic cell population (31). HBCs phenotypically and functionally resemble M2 macrophages and are thought to have broad roles in immune regulation, placental morphogenesis, stromal water content and ion transport across the maternal–fetal barrier (32). In vitroAlterations of HBCs have been associated with several pregnancy disorders, such as Villitis and preterm delivery (33).

HBCs may be DC-SIGN+/CD163+, which may be connected with the relation between the high expression of IL-10 in the chorionic villi and the maintenance of immune regulation (34). CD28 expression has also been found in Hofbauer cells, which may adjust the immune function of leukocytes positively or negatively, and may have a very important influence on physiological or pathological processes (35). HBCs expressd higher levels of SPP1, PLIN2, HMOX1, CD36, and LYVE1, whereas dMϕs showed higher expression of HLA genes (HLA-DRA, HLA-DPA1, HLA-DRB1, HLA-DPB1, HLA-DQA1, and HLA-DMA) and invariant chain CD74, indicating strong antigen-presenting capacity of dMϕs. What’s more, dMϕ sspecifically expressed MS4A4A, STAB1, SEPP1, and MS4A7 (36). In a recent study, CD74 was regarded as a critical marker in Hofbauer cells. CD45⁺CD68⁺CD74^- cells were determined as HBC-like-1 cells and CD45⁺CD68⁺CD74⁺ cells were determined as HBC-like-2 cells (37). The expression of these markers is regulated by environmental signals, such as cytokines and hormones,. Meanwhile, it is suggested that differential epigenetic regulatory patterns might be critical for the functional and characteristic differences between dMϕ and HBCs (38). Epigenetic patterns will give clues for further understanding of the immunological characteristics of dMϕ and HBCs during human pregnancy.

Fatty acid synthesis (FAS) takes part in the inflammatory reaction and signaling in macrophages (57). The proinflammatory response in macrophages can be inhibited through restraining fatty acid synthase (FASN), which is a key enzyme of FAS that catalyzes the production of long-chain fatty acids (58). Lipid droplets (LDs) is a feature of foamy macrophages that have been found in many diseases. In addition, polyunsaturated fatty acid (PUFA) metabolism in macrophages also influences pathological processes. In cardiovascular diseases, purified n-3 FA supplementation may be a potential strategy fortreatment and prevention (109). Therefore, understanding how immune cells handle and synthesize PUFAs is important. During macrophages activating, SREBPs and LXRs, the two master transcription factors of FAS, are upregulated. Upregulation in SREBPs enhances the maturation of pro-inflammatory precursors and causes macrophages to differentiate toward the pro-inflammatory M1 phenotype. In contrast, LXRα promotes cells towards an M2 phenotype (110).

FAO is essential in functional M2 macrophages by enhancing the secretion of IL-1β (59). Treatment with LPS attenuates the expression of inflammatory genes in macrophages when FAO is suppressed (111). FAO plays a crucial role in activation of NLRP3 inflammasome of M1 macrophages (112). It is implied that enhancing FAO in macrophages may be an underlying therapeutic method for patients with obesity, type 2 diabetes (113) or cancers (114). However, it remains unclear whether the increase in FAO correlates with M2 polarization (115). IL-4-induced polarization is not affected by the inhibition of FAO (116). The two subtypes of carnitine palmitoyltransferase (CPT) system, CPT1 and CPT2, are essential to the β-oxidation of long-chain aliphatic acid in mitochondria. Lacking CPT2, macrophages are unable to finish β-oxidation of fatty acids, but they can be polarized to M2 phenotype after stimulated by IL-4 in vitro and in vivo (117). Moreover, the CPT-1 inhibitor etomoxir in low concentrationcould suppress CPT-1 without changing M2 polarization.i However, high concentration of etomoxir can inhibit M2 polarization without affecting the activity of CPT-1 (56). Macrophage FAO likely plays a correlative rather than causative role in systemic metabolic dysfunction (118). Further exploration is needed to determine which is the dominant factor, the switchof cell phenotypes or the changes of metabolic transformation.

Mills et al. first proposed the notion that arginine can be used to determine the functions of macrophages (119). The M1 macrophages are a product of the iNOS pathway, while the M2 macrophages come from the arginase pathway (60). L-Arginine takes part in initiating of intracellular signaling pathways in macrophages, such as triggering inflammatory responses and accelerating the sensitivity to bacterial endotoxin (120). Arginine deprivation attenuates osteoclastogenesis and also dampens generation of IL-4 induced multinucleated giant cells. Strikingly, in the absence of extracellular arginine, osteoclasts and IL-4-induced multinucleated giant cells display flexibility, since their formation can be restored by supplementation with select arginine precursors in vitro ( 121). The production of α-Ketoglutarate (αKG) is important for activation of M2 macrophages, including engagement of FAO and epigenetic reprogramming of M2 genes. The potential M2-promoting mechanism is demonstrated by the high αKG/succinate ratio,Whereas a low αKG/succinate ratio strengthens M1 polarization in macrophages (61). Nontargeted metabolomic analysis revealed that tryptophan metabolism is involved in M1 polarization (122). Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, leads to an increase in putrescine levels and imparis the transcription of M1 genes. In M1 macrophages, the translation of proinflammatory mediators can be regulated by spermidine and spermine (15). In applied research, there are no clear results that using amino acid auxotrophy to decrease the growth of cancerous lymphocyte, though attempting for decades (123). As for macrophages, there is also a long way to go. The detailed metabolic pathways in macrophages are shown in Figure 3 .

In addition, the classic metabolic pathways, some bypasses, and the enzymes involved have also come into view in recent years. Glucose is converted to sorbitol by aldose reductase (AR) in Polyol pathway. When activating the Polyol pathway, the production of proinflammatory cytokines was increased in kidney cortex of diabetic mice (124). What’s more, upregulation of AR in human macrophages is proinflammatory in foam cells in atherosclerosis (125). When inhibiting the activity of AR, the phenotype was transferred to M2 (126). Ribulose 5-phosphate 3-epimerase (RPE) is an important enzyme for cellular response against oxidative stress and takes part in PPP, contributing to the reversible conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate (127). The gene coding RPE has exposed to be relative to poor outcome in cancer patients (128). Some metabolites also affect the function and polarization of macrophages. Lactate-derived lactylation of histone serves as an epigenetic modification and could directly stimulates M2 gene expression during M1 macrophage polarization (129). Ketone body, especially, β-hydroxybutyrate (BHB) promotes M2 macrophage polarization, contributing to the resolution of intestinal inflammation and providing ideas for the treatment of inflammatory bowel disease (130). Metabolites in shaping the phenotypes and functions of macrophages were summarized in Table 2 .