N6-Methyladenosine (m6A) Modification in Natural Immune Cell-Mediated Inflammatory Diseases.

m6A and related proteins participate in the development, proliferation, and differentiation of non-specific immune cells and their functions with implications for infectious, autoimmune, liver fibrosis, bone, cancer, neural, and cardiovascular diseases. Recent research has advanced the mechanistic understanding of m6A modification and its role in gene expression. Altered m6A modification-related proteins in immune pathophysiology indicate new targets and directions for immunotherapies, with tremendous therapeutic potential ( Table 1 ). However, there is still much to be learned about dual effects of m6A-related proteins. Caution must be exercised but further research in this field may expedite the development of new tools and therapeutic strategies. Changes and biological outcomes of m6A effector proteins in innate immune cells-mediated inflammatory diseases BMDMs, BMMs, bone marrow-derived macrophages; HMDMs, human monocyte-derived macrophages; LLC, Lewis lung carcinoma; MI, myocardial infarction; PH, pulmonary hypertension; PVR, pulmonary vascular remodeling; VAs, ventricular arrhythmias; PVN, paraventricular nucleus; NASH, nonalcoholic steatohepatitis; NAFLD, nonalcoholic fatty liver disease; TP, treponema pallidum; PEDV, porcine epidemic diarrhea virus; oxLDL, oxidized low-density lipoprotein; CLP, cecal ligation and puncture; TLR4, toll-like receptor 4.

The innate immune system is the first line of defense against pathogen invasion and spread. It encompasses many non-specific defense mechanisms, including barrier, chemical, cellular, and inflammatory, relying on pathogen pattern recognition and requiring no prior contact or memory but facilitating subsequent specific immune response. Defects in the innate immune system are linked to pathogenic immune dysfunction, and an appropriate understanding may facilitate the prevention and treatment of infectious and inflammatory diseases [ 1 , 2 ]. The post-transcriptional N6-methyladenosine (m6A) mRNA modification influences embryonic development, stem cell differentiation, and neuronal growth and has been linked to disease states, such as cancer, diabetes, cardiovascular diseases, and neurological disorders [ 3 ]. m6A is thought to regulate the innate immune system-mediated inflammatory response and is a potential target for immunotherapy. However, controversies remain and the current review summarizes relevant recent research.

The authors have no conflicts of interest to declare.

The authors would like to express their gratitude to EditSprings ( https://www.editsprings.cn ) for the expert linguistic services provided.

This work was supported by funding from the National Natural Science Foundation of China (82302630), the Department of Science and Technology of Sichuan Province Grant numbers (2021ZYD0093, 2022YFS0597), and Central government guides local technology development funds (ZYGX2019J113, ZYGX2021YGLH224).

Pathological damage due to the microglial inflammatory response, in which m6A is involved, contributes to the progression of various neurological diseases [ 70 ] ( Fig. 4 ). Different m6A modification patterns were found in M0, M1, and M2 microglia from MeRIP-seq analysis of untreated microglia (M0) and microglia treated with LPS+IFNγ (M1) or IL-4+IL-13 (M2). M1 microglia showed the greatest change in m6A of pro-inflammatory genes and genes involved in the immune system, signal transduction, and protein degradation were enriched. By contrast, genes involved in genetic information processing, metabolism, and biology of neurodegenerative diseases showed enrichment of m6A modification in M2 microglia. m6A may thus participate in pro- and anti-inflammatory microglial response [ 71 ]. m6A in regulating the immune functions of microglia. Controversies remain, however. Wang et al. [ 72 ] found METTL3 to be upregulated in LPS-stimulated microglia where it bound TNF receptor associated factor 6 (TRAF6) protein and activated the TRAF6/NF-κB pathway, enhancing expression of microglial inflammatory factors, IL-1β, IL-6, TNF-α, and IL-18. Ding et al. [ 73 ] reported the m6A recognition and binding protein, IGF2BP1, to be upregulated in LPS-stimulated microglia where it promoted the inflammatory response by stimulating expression of guanylate binding protein family member 11 ( Gbp11 ) and ceruloplasmin ( Cp ). By contrast, METTL3 inhibited the activity of MyD88-NF-κB and NLRP3-ASC-Caspase1 inflammasomes in BV2 microglia, reducing inflammatory cytokines and oxidative stress mediators. METTL3 thus alleviated BV2 cell overactivation [ 74 ]. Further research is necessary to elucidate mechanisms controlling pro- and anti-inflammatory response in central nervous system microglia and determine whether m6A modification would be an appropriate target for the treatment of neuroinflammatory disorders. m6A modification has also been implicated in microglia-mediated disease, such as sympathetic overactivity, retinopathy, uveitis, and cerebral ischemia/reperfusion injury and may prove to be a therapeutic target for these disorders. m6A modification mediated by METTL3 in small glial cells of the paraventricular nucleus may be associated with ventricular arrhythmias caused by sympathetic overactivity and sudden cardiac death in MI patients. Mettl3 mRNA in small glial cells in the paraventricular nucleus and m6A were shown to be elevated on the 3rd to 7th day after creating a rat model of MI. METTL3-mediated m6A modification stabilized Tlr4 mRNA and activated NF-κB signaling and inflammation. Targeting METTL3 may protect the heart from arrhythmias by reducing sympathetic overactivity and post-MI infarct size [ 75 ]. Diabetic retinopathy is an inflammatory microvascular complication of diabetes and microglial ALKBH5 participates in it, as does the anti-inflammatory, A20. Microglial ALKBH5 was decreased in diabetic retinopathy models and microglial M1 polarization enhanced due to reduction of A20 translation [ 76 ]. Microglial YTHDC1 was found to be downregulated in a mouse model of uveitis which destabilized sirtuin 1 ( Sirt1 ) mRNA, enhancing M1 microglial migration and inflammation. The authors considered the downregulation of YTHDC1 expression to be a potential mechanism underlying the development of uveitis, raising the possibility of using YTHDC1 as a treatment for such autoimmune diseases [ 77 ]. m6A modification promoted cerebral ischemia/reperfusion injury-induced microglial inflammation by increasing the stability of cyclic GMP-AMP synthase ( cGas ) mRNA and aggravating stimulator of interferon response cGAMP interactor (STING)/NF-κB signaling. Inhibition of m6A modification by FTO overexpression in microglia reduced the microglia-mediated inflammatory response and ameliorated brain injury [ 78 ]. m6A reader, YTHDF1, is also involved in regulating the microglial inflammatory response in cerebral ischemia/reperfusion injury. microRNA-421-3p was downregulated, and Ythdf1 expression increased in an in vivo animal model of middle cerebral artery occlusion/reperfusion and an in vitro microglia model of oxygen-glucose deprivation and reoxygenation. Increased YTHDF1 promoted translation of m6A-modified p65 mRNA, enhancing production of pro-inflammatory factors [ 79 ] and giving mechanistic insights into brain ischemia/reperfusion injury.

Neutrophils are traditionally considered “transcriptionally silent” due to their low mRNA content but the neutrophil transcriptome may vary more than that of monocytes and T cells under stress conditions [ 56 ]. Little has been reported regarding neutrophil m6A but some significance has been attached to its role ( Fig. 3 ). m6A in regulating the immune functions of neutrophil, NK cells, and DCs. METTL3-mediated m6A modification was involved in the stimulation of neutrophil Tlr4 expression in LPS-induced sepsis. The consequent activation of the TLR4/Myd88/NF-κB signaling pathway promoted neutrophil activation and cytokine secretion [ 57 ]. The authors found reduced expression of the demethylase, ALKBH5, in bacterially infected neutrophils in vitro and neutrophils from a mouse model of sepsis in vivo. ALKBH5 knockout impaired neutrophil migration and exacerbated infectious symptoms in septic mice, perhaps due to decreased expression of neutrophil migration molecules C-X-C motif chemokine receptor 2 ( Cxcr2 ) and NLR family pyrin domain containing 12 ( Nlrp12 ) and increased expression of prostaglandin E receptor 4 ( Ptger4 ), tenascin C ( Tnc ), and WNK lysine deficient protein kinase 1 ( Wnk1 ) [ 58 ]. Neutrophils also activate other cellular m6A-related signaling pathways through cell-cell interactions. For example, neutrophil extracellular traps (NETs) activated ferroptosis in alveolar epithelial cells by an m6A-dependent mechanism, regulating sepsis-related acute lung injury. NETs upregulated METTL3 in alveolar epithelial cells via the TLR9/MyD88/NF-kB pathway and increased glutathione peroxidase 4 ( Gpx4 ) degradation-induced ferroptosis and alveolar epithelial cell damage [ 59 ]. NETs were also shown to promote METTL3-mediated mRNA methylation and sirtuin 1 (SIRT1)-mediated abnormal cell autophagy in alveolar epithelial cells, causing sepsis-related acute lung injury [ 60 ]. Neutrophils may also promote m6A-dependent tumor development. IL-1β and TNFα secreted by complement C5a receptor 1 (C5aR1)-positive neutrophils may stabilize WTAP via phosphorylation, which promoted breast cancer proliferation in vivo via increased m6A methylation of enolase 1 ( Eno1 ) RNA and enhanced glycolytic activity [ 61 ]. m6A mRNA modification may be involved in the regulation of neutrophil migration and inflammatory response with therapeutic implications [ 57 , 58 ]. Further research is needed to clarify specific pathways and targets.

T.Y. searched and organized literature, wrote and revised the manuscript, provided funding; Y.J. wrote first draft and made the figures; C.J. helped write first draft; Y.L. wrote and revised the manuscript, provided funding.

Dendritic cells (DCs) uptake, process, and present antigens to activate and regulate T cells and B cells [ 67 ]. METTL3-mediated m6A mRNA modification has been shown to activate DCs by promoting translation of YTHDF1-dependent TIR domain containing adapter protein ( Tirap ), a CD40, CD80, and TLR4 signal adapter. DC maturation and activation were thus stimulated via TLR4/NF-κB signaling and cytokine expression [ 68 ]. The T-cell response against new tumor antigens is critical for immunotherapy but tumors expressing new antigens may still evade immune recognition. m6A has also been shown to be involved in DC-mediated tumor escape. DC YTHDF1 stimulated the expression of lysosomal protease genes, allowing degradation of tumor neoantigens engulfed by the DCs. Therefore, YTHDF1 knockout inhibited tumor growth [ 69 ]. Therefore, targeting m6A in DCs may increase the sensitivity to immunotherapy ( Fig. 3 ).

Natural killer cells (NK cells) are involved in antiviral and antitumor immunity and m6A modification is necessary for NK cell homeostasis, affecting development, proliferation, differentiation, survival, and antitumor effects ( Fig. 3 ). METTL3 was reduced in tumor-infiltrating NK cells and METTL3 protein correlated positively with effector molecules. Loss of METTL3 inhibited NK infiltration and function in the tumor microenvironment, accelerating tumor development in mice. METTL3 deficiency was shown to decrease SH2 domain-containing protein tyrosine phosphatase 2 ( Shp-2 ) expression, reducing AKT and MAPK signaling, and the NK cell response to IL-15 [ 62 ]. FTO-deficient NK cells promoted IL-2/15-derived JAK/STAT signaling and prevented melanoma metastasis by destabilization of Socs mRNA. FTO-deficiency also enhanced the anti-leukemia effects of NK cells [ 63 ]. YTHDF2 has been shown to maintain NK cell homeostasis by regulating cell trafficking. The reader protein participated in NK cell terminal maturation by regulating expression of the T-box transcription factor, eomesodermin ( Eomes ). YTHDF2 also stimulated the degradation of TAR DNA-binding protein ( Tardbp ), causing a STAT5-YTHDF2 positive feedback loop, in which STAT5 promoted YTHDF2 expression and YTHDF2 activated STAT5. Cytokine-mediated NK cell survival, proliferation, antiviral, and antitumor effects were thus enhanced [ 64 ]. NKT cells are a subset of T cells that express both the T-cell receptor (TCR) and NK cell receptor. Invariant NKT (iNKT) cells are a subset of NKT cells characterized by constant expression of TCR-Vα14. METTL-dependent m6A modification has been implicated in the development and function of iNKT cells. Deletion of Mettl3 in double-positive thymocytes impaired iNKT cell proliferation, differentiation, and cytokine secretion, resulting in impaired anti-melanocyte activity by regulating the stability of cyclic AMP-responsive element-binding protein 1 ( Creb1 ) transcripts [ 65 ]. Similarly, loss of Mettl14 increased the apoptosis of double-positive thymocytes, and reduced Vα14-Jα18 gene rearrangement and differentiation of iNKT cells. METTL14 also regulated the immune function of mature iNKT cells. Mettl14 knockout inhibited TCR signaling and cytokine production by promoting cytokine inducible SH2-containing protein ( Cish ) expression [ 66 ]. Overall, m6A impacts the development, function, and anti-tumor immunity of NK cells and may be a target for tumor immunotherapy [ 62 – 66 ].

Variations in m6A and related proteins affected immune cell infiltration, tumor development, and autoimmune disease [ 80 – 88 ]. ALKBH5 influenced tumor development in several tumor models by regulating immune cell infiltration and the function of the immune microenvironment. ALKBH5 upregulated IL-8, mediated by MAP3K8, and recruited programmed cell death 1 ligand 1 (PD-L1) + macrophages in the hepatocellular carcinoma (HCC) cell tumor immune microenvironment. ALKBH5 promoted proliferation, migration, and invasion of HCC [ 89 ]. ALKBH5 also regulated Wnt/β-catenin targets, causing accumulation of myeloid-derived suppressor cells and reduction of NK cells to promote colorectal tumorigenesis [ 90 ]. ALKBH5 also recruited hypoxia-induced TAMs and promoted IL-8 secretion, generating an immunosuppressive tumor microenvironment in glioblastoma multiforme [ 91 ]. However, ALKBH5 promoted monocyte/macrophage PD-L1 expression, reducing infiltration of myeloid-derived suppressor-like cells and making intrahepatic cholangiocarcinoma cells more sensitive to anti-PD1 immunotherapy [ 92 ]. METTL3, WTAP, YTHDF1, and IGF2BP1 also regulated innate immune cells in the microenvironments of colorectal cancer [ 93 ], corneal neovascularization [ 94 ], gastric cancer [ 95 ], and HCC [ 96 ], reinforcing the roles of m6A in immune cell infiltration. m6A modification of pathogenic micro-organismal DNA/RNA was also found to influence the host immune microenvironment and immune escape [ 97 – 104 ]. For example, m6A modification of hepatitis B and C viral RNA inhibited virus-activated interferon regulatory factor 3 (IRF3) protein activation and interferon (IFN)-β expression, aiding viral immune escape [ 105 , 106 ]. m6A modification of human immunodeficiency virus-1 allowed the virus to evade the immune response by reducing the interferon expression mediated by transcription factors, interferon regulatory factor 3 (IRF3), and IRF7 [ 107 ].

Macrophages may be divided into phenotypic subtypes of bone marrow-derived macrophages, liver Kupffer cells (KCs), or lung macrophages, but, despite different origins and functions, subtypes have common features, including phagocytosis and toxin clearance, immune regulation, and maintenance of tissue homeostasis [ 5 – 7 ]. The m6A modification has been shown to influence the monocyte/macrophage-mediated antimicrobial response and inflammatory pathways in many organs. Clustered regularly interspaced short palindromic repeat screening was used to demonstrate the involvement of Mettl3 and Mettl14 in the regulation of lipopolysaccharide (LPS)-induced macrophage activation [ 8 ]. METTL3-mediated m6A modifications promote macrophage inflammatory response. M1 macrophages stimulated by interferon γ (IFNγ) had upregulated METTL3 expression which promoted M1 activation by increasing signal transducer and activator of transcription 1 ( Stat1 ) mRNA methylation and inhibited interleukin (IL)-4-induced M2 macrophage polarization [ 9 ]. METTL3 was also shown to enhance the LPS-induced macrophage inflammatory response by promoting toll-like receptor 4 (TLR4) signaling. METTL3 deficiency increased IL-1 receptor-associated kinase M ( Irakm ) mRNA, a negative regulator of TLR4 signaling, and inhibited LPS-induced tumor necrosis factor α (TNFα) production [ 8 ]. Mitochondrial biogenesis and energy metabolism regulate monocyte inflammatory status, and m6A modifications promote the inflammatory response by inhibiting mitochondrial energy metabolism. METTL3 reduced expression of proliferator activated receptor gamma coactivator 1-alpha ( Pgc-1α ), cytochrome c ( Cycs ), and complex I subunit NADH: ubiquinone oxidoreductase subunit C2 ( Ndufc2 ) to decrease ATP production and oxygen consumption. Thus, METTL3 increased monocyte reactive oxygen species and proinflammatory cytokine release [ 10 ]. WTAP has also been shown to promote macrophage IL-6 expression via LPS-induced p65 nuclear translocation [ 11 ]. Some contradictory findings of a negative impact of m6A transferase on macrophage activation have been reported. Cai et al. [ 12 ] found levels of m6A modification and Mettl3 expression to be decreased in LPS-stimulated macrophages, causing upregulation of pro-inflammatory cytokine expression, including TNFα, IL-6, and nitric oxide (NO). The effect was due to reduced degradation of Nod1 and receptor-interacting serine-threonine kinase 2 ( Ripk2 ) mRNA. Similarly, loss of macrophage Mettl14 caused overactivation of the TLR4/nuclear factor κB (NF-κB) signaling pathway and sustained pro-inflammatory cytokine production via m6A modification of suppressor of cytokine signaling 1 ( Socs1 ) and Serine protease inhibitor 2 A ( Spi2a ) [ 13 , 14 ]. Findings regarding the macrophage regulatory functions of m6A modifications have therapeutic implications for inflammatory disorders despite the fact that conflicting results have generated debate. Variations in experimental design, cell types, microbial species, and RNA targets may account for the heterogeneity of results and the complexity of underlying mechanisms may also be a factor [ 8 – 14 ]. Further investigation of the m6A modification in the monocyte/macrophage-mediated antimicrobial response and inflammation is merited. Overexpression of the demethylase, FTO, caused excessive TLR4/NF-κB signaling due to reduced Socs1 expression in a manner analogous to that of METTL14 deficiency. FTO overexpression promoted excessive inflammation and tissue damage [ 13 ]. FTO also enhanced activation of the macrophage stiffness-controlled signal transducer and activator of transcription 1 (STAT1) signaling pathway via YTHDF1-dependent inhibition of Socs1 [ 15 ] and promoted the stability of Stat1 and peroxisome proliferators-activated receptor ( Ppar )-γ mRNA in a YTHDF2-dependent manner, leading to macrophage activation [ 16 ]. The FTO enzyme may have beneficial effects for macrophage activation but excessive activation may cause tissue damage [ 13 , 15 , 16 ]. Findings should be interpreted in a context-dependent light, and FTO impact on various disease states requires exploration. By contrast, ALKBH5 inhibited the anti-microbial inflammatory response by reducing YTHDF2-dependent dual specificity phosphatase 1 ( Dusp1 ) degradation, which inhibited the mitogen-activated protein kinase (MAPK) signaling pathway [ 17 ]. YTHDF1 may stimulate macrophage cytokine expression and contribute to inflammatory damage. It promotes translation of NOD-like receptor protein 3 ( Nlrp3 ) during bacterial infection, stimulating excessive IL-1β expression and inflammatory damage. YTHDF1 knockout extended survival in cecal ligation and puncture-induced polymicrobial sepsis mouse model [ 18 ] and promoted immune paralysis in a rat model of severe sepsis combined with extracorporeal membrane oxygenation. In addition, YTHDF1 knockout reduced macrophage entry into the brain and consequent endothelial damage by inhibiting Janus kinase 2/Stat3 expression in macrophages [ 19 ]. The identification of YTHDF1 involvement in the macrophage inflammatory response indicates this reader protein as a potential therapeutic target for the clinical treatment of inflammatory diseases [ 18 , 19 ]. By contrast, m6A readers, YTHDF2, YTHDC1, and IGF2BP2, may negatively regulate macrophage inflammatory response. YTHDF2 expression was upregulated in LPS-stimulated RAW264.7 cells. YTHDF2 deficiency increased the stability and expression of MAPKs, Map2k4 and Map4k4 , promoting LPS-induced expression of the inflammatory cytokines, IL-6, TNFa, IL-1β, and IL-12 [ 20 ]. YTHDF2 was found to be upregulated in both M1 macrophages, where it suppressed polarization by inhibiting NF-κB, p38, and c-Jun N-terminal kinase signaling, and in M2 macrophages, where polarization was stimulated due to increased degradation of p53 mRNA [ 21 ]. YTHDC1 has been linked to the resolution of inflammation and restoration of colonic epithelial barrier function in patients with inflammatory bowel disease. It inhibited macrophage inflammation by regulating Ras homolog family member H (RHOH) [ 22 ]. IGF2BP2 was also found to convert M1 macrophages to M2 by an action on tuberous sclerosis 1 ( Tsc1 ) and peroxisome proliferator-activated receptor-γ ( Ppar-γ ) during the development of dextran sulfate sodium-induced colitis [ 23 ]. In addition, the macrophage-mediated anti-viral response also depends on m6A modification. Transcriptomic analysis showed that m6A modification influenced the cellular anti-viral immune response in chicken macrophages infected with Newcastle disease virus and THP-1 macrophages infected with treponema pallidum by an effect on gene expression [ 24 , 25 ].The demethylase, ALKBH5 was upregulated in porcine epidemic diarrhea virus-infected pig lung to promote cellular anti-viral activity by stimulating growth arrest-specific protein 6 ( Gas6 ) expression in a YTHDF2-dependent manner [ 26 ]. In addition, YTHDF1 was upregulated and promoted Socs3 translation via an m6A-dependent mechanism to reduce Janus kinase 2/STAT3 signaling and cytokine secretion. The result was decreased treponema pallidum-induced THP-1 macrophage inflammation [ 27 ]. Controversies aside, m6A modification is undisputedly involved in the regulation of the monocyte/macrophage-mediated antimicrobial response and inflammation ( Fig. 1 ). m6A in regulating microbial-induced inflammatory response of monocyte/macrophage. A dysregulated m6A-mediated macrophage inflammatory response has been implicated in the pathogenesis of rheumatoid arthritis (RA), atopic dermatitis, and psoriasis ( Fig. 2 ). m6A in regulating macrophage-related inflammatory diseases. RA is a systemic disease primarily characterized by inflammatory synovitis. m6A regulates macrophage polarization in RA, indicating its diagnostic and therapeutic potential. METTL3 upregulation in peripheral blood mononuclear cells from RA patients positively correlated with C-reactive protein (CRP) and erythrocyte sedimentation rate, two RA disease activity markers. Increased METTL3 is likely to suppress LPS-induced expression of macrophage inflammatory cytokines, IL-6 and TNFa, by inhibiting NF-κB signaling and may thus improve inflammation status [ 28 ]. Circular RNA (circRNA) participates in the development of atopic dermatitis and psoriasis via m6A modification in monocytes/macrophages. Peripheral blood mononuclear cell hsa_circ_0004287 was elevated in atopic dermatitis and psoriasis patients and inhibited LPS-induced macrophage activation by destabilizing metastasis-associated lung adenocarcinoma transcript 1 ( Malat1 ) mRNA in a m6A-dependent manner [ 29 ]. In summary, m6A-mediated macrophage inflammatory response participates in the pathogenesis of autoimmune diseases and further mechanistic research may identify potential therapeutic targets [ 28 , 29 ]. Atherosclerosis (AS) is a systemic chronic inflammatory disease characterized by chronic inflammation of the arterial wall, in which macrophages are involvement. Macrophages engulf excess lipids and become foam cells which accumulate under the endothelium and form raised plaques. Macrophages also interact with vascular smooth muscle cells and secrete pro-inflammatory cytokines and extracellular matrix components, promoting lipoprotein retention [ 30 , 31 ]. METTL3-mediated RNA methylation promoted M1 macrophage inflammation, glycolysis, and lipid accumulation by stabilizing hepatoma-derived growth factor ( Hdgf ) mRNA and protein expression, indicating the regulation of macrophage polarization through energy metabolism reprogramming in AS [ 32 ]. Similarly, METTL3 promoted oxLDL-induced macrophage inflammatory response via the m6A modification of Stat1 and Braf mRNA, explaining the observation that oxLDL enhanced m6A modification of macrophage mRNA. METTL3 also interacted with STAT1 protein to promote transcription of macrophage inflammatory factors [ 33 , 34 ]. However, decreased m6A modification of macrophage RNA in response to oxLDL has been reported, mediated by Matrin-3 (Matr3) which enhanced the formation of the METTL3-METTL14 complex. A reduction in Matr3-mediated m6A modification in oxLDL-stimulated macrophages promoted MAPK signaling, facilitating oxLDL-induced macrophage inflammatory response and AS [ 35 ]. The diverse reported patterns in oxLDL-stimulated macrophage m6A may be attributed to the different cell models (RAW264.7 and THP-1) and detection techniques (dot blot and flow cytometry) used [ 33 – 35 ]. Further investigations, including assessment of cell models and detection techniques, are necessary to establish whether m6A modification promotes or inhibits AS and molecular mechanisms involved. Utilization of a number of different research models and techniques may aid understanding. AS may contribute to acute coronary syndrome (ACS), coronary heart disease, and myocardial infarction (MI), some of which have reported links to m6A [ 36 ] ( Fig. 2 ). Levels of macrophage hsa_circ_0029589 were found to be decreased in ACS patients and METTL3 inhibition significantly reduced macrophage pyroptosis and ameliorated ACS by increasing hsa_circ_0029589 expression [ 37 ]. METTL14 was found to be elevated in patients with coronary heart disease and METTL14 knockout reduced macrophage inflammation and alleviated AS plaque development by regulating the stability of Myd88 mRNA. Thus, increased METTL14 may be responsible for AS development and coronary heart disease [ 38 ]. Ventricular arrhythmias contribute to the high mortality of MI patients by influencing post-MI inflammation-induced sympathetic nerve remodeling, a process which may involve METTL3. Sustained elevation of METTL3 was found in macrophages of a rat MI model. Macrophage METTL3 promoted NF-κB activation, reactive oxygen species production, cytokine release, and nerve growth factor expression by stimulating m6A modification of TNF receptor-associated factor 6 ( Traf6 ) mRNA, leading to excessive post-MI sympathetic nerve reconstruction [ 39 ]. METTL3- and METTL14-mediated m6A modification may thus have a deleterious effect on AS-related diseases [ 37 – 39 ]. METTL3 has been implicated in non-alcoholic fatty liver disease (NAFLD) and liver fibrosis ( Fig. 2 ). Macrophage METTL3 was found to aggravate NAFLD and obesity by decreasing DNA damage inducible transcript 4 ( Ddit4 ) expression, an effect which promoted inflammation and metabolism by stimulating cytokine-stimulated mammalian target of rapamycin (mTOR) and NF-κB pathway activity. METTL3 knockout protected mice against aging-related and diet-induced NAFLD and obesity [ 40 ]. Transforming growth factor β1 (TGF-β1) is secreted by activated KCs and promotes the progression of non-alcoholic steatohepatitis (NASH) to liver fibrosis. NF-κB caused global m6A hypermethylation of RNA by activating the METTL3/METTL14 transmethylase in NASH rat liver and LPS-activated KCs. Hypermethylation upregulated TGF-β1 expression and exacerbated liver fibrosis [ 41 ]. METTL3 was also found to be upregulated in in vivo liver fibrosis models and in vitro M1-polarized macrophages. METTL3 promoted liver fibrosis by upregulating metastasis-associated lung adenocarcinoma transcript 1 ( Malat1 ) expression, ubiquitin-specific peptidase 8 degradation, and transforming growth factor kinase 1 ubiquitination [ 42 ]. In conclusion, METTL3-mediated m6A modification promoted NAFLD and liver fibrosis, and the protein may represent a promising therapeutic target [ 40 – 42 ]. m6A is involved in macrophage and osteoclast differentiation and has been linked to the pathophysiological processes of bone resorption osteoarthritis and osteoporosis ( Fig. 2 ). METTL3 enhanced migration and osteogenesis of bone marrow mesenchymal stem cells by influencing macrophage polarization. Its overexpression promoted IL-6 expression and secretion of inducible NO synthase (iNOS) in M1 macrophages by increasing the m6A modification of dual specificity phosphatase 14 ( Dusp14 ) and histone deacetylase 5 ( Hdac5 ) [ 43 ]. However, METTL3 inhibited iNOS mRNA stability via a YTHDF1-dependent mechanism during inflammation, reducing the mitochondrial dysfunction mediated by iNOS/NO [ 44 ]. Additionally, extracellular vesicles derived from human umbilical cord mesenchymal stem cells exerted anti-inflammatory effects by downregulating macrophage METTL3, resulting in enhanced chondrocyte proliferation/migration and reduced NLRP3-induced inflammation which alleviated osteoarthritis [ 45 ]. METTL14 has been shown to promote T-cell factor 1 ( Tcf1 ) expression, increasing osteogenesis and protecting against osteoporosis by enhancing levels of the bone formation protein, Runt-related transcription factor 2 (RUNX2) [ 46 ]. m6A readers also participate in osteoclast differentiation and bone resorption. YTHDF1 was upregulated during LPS-induced osteoclast differentiation. YTHDF1 overexpression enhanced phosphorylation of proteins in the NF-κB, MAPK, phosphatidylinositol 3-kinase-AKT, and activate transcription factor 6 (ATF6) signaling pathways, by stabilizing the mRNA of TNF receptor superfamily member 11a ( Tnfrsf11a ). Therefore, YTHDF1 promoted osteoclast differentiation and bone resorption [ 47 ]. By contrast, YTHDF2 was upregulated in receptor activator of nuclear factor kappa-B ligand (RANKL)-primed osteoclast precursors induced by LPS where it inhibited pro-inflammatory cytokine secretion to reduce osteoclast formation and bone resorption [ 48 ]. Further research is needed to establish the potential of targeting m6A modification for the treatment of bone disorders. Tumor-associated macrophages (TAMs) infiltrate the tumor microenvironment and promote tumor growth and invasion. TAM m6A modifications have been linked to various types of cancer [ 49 ] ( Fig. 2 ). Conditional Mettl3 knockout reduced m6A modification and degradation of Irakm mRNA, a negative regulator of TLR4 signaling in macrophages. The consequent Irakm upregulation suppressed TLR-mediated macrophage activation and TNFα production, promoting colon adenocarcinoma tumor cell growth in mice [ 8 ]. Conditional macrophage Mettl3 loss also promoted melanoma growth and metastasis, via impaired translation of sprouty-related EVH1 domain protein 2 ( Spred2 ) in YTHDF1-dependent manner. Reduced Spred2 stimulated NF-κB and STAT3 through the ERK pathway and promoted M1/M2-like TAM infiltration of tumor tissue [ 50 ]. The level of METTL14-m6A correlated negatively with numbers of dysfunctional T cells in colorectal cancer patients. Macrophage Mettl14 knockout reduced m6A and increased transcription of cytokine subunit Epstein-Barr virus-induced gene3 ( Ebi3 ) which stimulated CD8 + T-cell differentiation along a dysfunctional trajectory, impairing tumor elimination [ 51 ]. In conclusion, m6A modification may be a promising target for cancer immunotherapy. The involvement of m6A in some other macrophage-mediated inflammatory diseases is shown in Figure 2 . Transcriptomic correlation analysis of LncRNA and m6A-related proteins from diabetic nephropathy patients has implicated the indirect involvement of the demethylase, FTO, in the m6A modification of M1-related lncRNAs, LINC00342, LINC00667, and LNC00963, and WTAP may interact with the M1 immune regulator, RNA-binding motif protein 15 (RBM15) [ 52 ]. Thus, FTO- and ALKBH5-mediated m6A modification in macrophages may be involved in diabetic nephropathy. Endometriosis is characterized by the growth of endometrial-like tissue inside and outside the pelvic cavity, and aberrant M2 macrophage infiltration may be involved in lesion formation. Activated METTL3 and its target gene, tribbles pseudokinase 1 ( Trib1 ), may induce M2 macrophage polarization and activate ERK/STAT3 signaling to promote endometriosis [ 53 ]. Blood-derived bone marrow cells may cross the blood-brain barrier and differentiate into functional macrophages when recruited by central nervous system damage. The cells may be considered to be small glial cells which contribute to neurodegeneration and repair. Defective METTL3 in blood-derived macrophages reduced DNA methyltransferase 3A ( Dnmt3a ) translation in a YTHDF1-dependent manner, downregulating α-tubulin acetylation due to decreased α-tubulin acetyltransferase 1 ( Atat1 ) expression. METTL3 deficiency improved cognitive function in an amyloid β (Aβ)-induced Alzheimer’s disease mouse model by enhancing macrophage migration and Aβ clearance [ 54 ]. Pulmonary hypertension (PH) is characterized by irreversible pulmonary vascular remodeling, leading to right ventricular failure and death. Increased YTHDF2 expression in alveolar macrophages of early hypoxic stage mouse PH model was found which exacerbated PH and pulmonary vascular remodeling. The underlying mechanism involved the promotion of macrophage polarization and oxidative stress, mediated by increased degradation of heme oxygenase 1 ( Hmox1 ) mRNA [ 55 ]. Overall, m6A modification may be a target for the treatment of macrophage-mediated inflammatory diseases.

The m6A modification impacts RNA splicing, stability, ribosome selection, and translation and changes to local RNA structure affect interactions with RNA, DNA, and protein. Three types of proteins, writers, erasers, and readers, affect the modifications. Writers, such as methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms’ tumor 1-associating protein (WTAP), compose the methyltransferase complex to methylate adenosine nucleotides (A) of RNA molecules. The core component is METTL3 which has a nucleic acid binding domain and participates in catalysis. Erasers include fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5) and act to reverse m6A modifications with regulatory impact on RNA transcription, translation, and degradation [ 3 ]. Readers include YTH N6-methyladenosine RNA-binding protein (YTHDF) 1/2/3, YTH domain containing (YTHDC) 1/2, and insulin-like growth factor 2 mRNA-binding proteins (IGF2BP) 1/2/3. The first to be discovered were YTHDFs which have an N-terminal YTH domain to recognize RNA m6A modifications and a C-terminal domain. YTHDCs also have an N-terminal YTH domain with a C-terminal DEAD-box RNA helicase domain. IGF2BP1/2/3 is non-canonical readers, for which RNA binding depends not only on m6A modification domain but also on other RNA domains. Readers regulate RNA stability, translation, degradation, and splicing [ 3 , 4 ].