Emerging role of METTL3 in inflammatory diseases: mechanisms and therapeutic applications

Despite improvements in modern medical therapies, inflammatory diseases, such as atherosclerosis, diabetes, non-alcoholic fatty liver, chronic kidney diseases, and autoimmune diseases have high incidence rates, still threaten human health, and represent a huge financial burden. N6-methyladenosine (m6A) modification of RNA contributes to the pathogenesis of various diseases. As the most widely discussed m6A methyltransferase, the pathogenic role of METTL3 in inflammatory diseases has become a research hotspot, but there has been no comprehensive review of the topic. Here, we summarize the expression changes, modified target genes, and pathogenesis related to METTL3 in cardiovascular, metabolic, degenerative, immune, and infectious diseases, as well as tumors. In addition to epithelial cells, endothelial cells, and fibroblasts, METTL3 also regulates the function of inflammation-related immune cells, including macrophages, neutrophils, dendritic cells, Th17 cells, and NK cells. Regarding therapeutic applications, METTL3 serves as a target for the treatment of inflammatory diseases with natural plant drug components, such as emodin, cinnamaldehyde, total flavonoids of Abelmoschus manihot, and resveratrol. This review focuses on recent advances in the initiation, development, and therapeutic application of METTL3 in inflammatory diseases. Knowledge of the specific regulatory mechanisms involving METTL3 can help to deepen understanding of inflammatory diseases and lay the foundation for the development of precisely targeted drugs to address inflammatory processes.


Introduction
Inflammation is a protective homeostatic response, characterized by the activation of multiple immune and non-immune cells to eliminate harmful stimuli and promote tissue repair (1)(2)(3).At the cellular and molecular level, inflammation can be understood as a sophisticated regulatory network, composed of inducers, sensors, mediators, and effectors (2).Inducers can be divided into exogenous and endogenous inducers, where exogenous inducers include microbes and microbial products, allergens, foreign bodies, and toxic compounds (4), and endogenous inducers are derived from stressed or dead cells and damaged or dysfunctional tissues (2).Inducers can be detected by transmembrane or cytoplasmic sensors, such as Toll-like receptors (TLRs), C-type lectin receptors, retinoic acid-inducible gene (RIG)-I-like receptors, and NOD-like receptors (NLRs) (5), and trigger inflammatory mediators, including vasoactive amines, vasoactive peptides, lipid mediators, cytokines, chemokines, and proteolytic enzymes (2).These inflammatory mediators eventually affect the functional activities of effectors, including monocytes, macrophages, neutrophils, dendritic cells (DCs), lymphocytes, and mast cells, which are immune cells, as well as non-immune epithelial cells, fibroblasts, and endothelial cells (6).
Timely shutdown of inflammation is crucial for physiological homeostasis (6), and dysregulation of this process leads to chronic inflammation (3,7).Although the regulatory mechanisms underlying chronic inflammation are not completely clear, it is established as associated with aging, chronic infection, unhealthy lifestyle, diet, and adverse environmental conditions (8).At the molecular level, chronic inflammation is usually triggered by endogenous danger signals (9) and causes tissue dysregulation, subsequently leading to various chronic inflammatory diseases, including cardiovascular diseases, type 2 diabetes, non-alcoholic fatty liver, rheumatoid arthritis, and neurodegenerative diseases, among others (3,8,10).
METTL3 was the first m6A methyltransferase to be discovered.In 1997, Boker et al. isolated, purified, and sequenced METTL3 (also referred to as MT-A70) from HeLa cells, and confirmed that it is an important (N6-adenosine)-methyltransferase subunit (17).Together, METTL3, METTL14, and WTAP form the m6A methyltransferase complex (34), in which METTL3 is the core component that provides catalytic activity, while METTL14 functions as an RNA binding platform and WTAP is responsible for recruiting the complex to RNA targets (20,35,36).METTL3 functions in various pathophysiological processes, including the cell cycle, apoptosis, innate immunity, and inflammation, as well as cell proliferation, migration, invasion, and differentiation (37).As the m6A methylase most widely discussed in the context of inflammation and inflammatory diseases, the role of METTL3 in inflammatory diseases has not been systematically elucidated.
In this review, we describe the role of METTL3 in macrophages, neutrophils, DCs, T helper (Th)17 cells, and natural killer (NK) cells which play crucial pathological roles in chronic inflammation.We comprehensively summarize METTL3 expression changes and related pathogenesis in inflammatory diseases, including atherosclerosis, myocardial infarction (MI), non-alcoholic liver disease, hyperuricemia (HUA), diabetes nephropathy, osteoarthritis (OA), intervertebral disc degeneration (IDD), rheumatoid arthritis, psoriasis, inflammatory bowel disease (IBD), sepsis, pneumonia, and tumor.In conclusion, we focus on the therapeutic application of METTL3 in inflammatory diseases.Knowledge of the mechanisms by which METTL3 regulates inflammatory diseases will contribute to the development of improved targeted therapies and broaden clinical perspectives.

METTL3 regulates inflammationrelated immune cell functions 2.1 METTL3 regulates macrophage function
Inflammation involves multiple immune cells, among which macrophages are key participants.Macrophages/monocytes are derived from progenitor cells in the bone marrow and enter the bloodstream (38).After being recruited, these cells can enter tissue through the vascular endothelium and become tissue-resident macrophages.Macrophages are involved in all aspects of the physiological and pathological processes of inflammation, including the perception of stimuli, phagocytosis of pathogens, removal of cell debris, presentation of antigens, secretion of cytokines, and promotion of tissue repair.
Macrophages are plastic and, when stimulated by the microenvironment, can polarize into different phenotypes, which are generally divided into classic activated or inflammatory (M1) and alternate activated or anti-inflammatory (M2) macrophages (39).M1 macrophages are activated by IFN-g and TNF-a, or by lipopolysaccharide (LPS).They secret proinflammatory cytokines, such as TNF-a, IL-1a, IL1-b, IL-6, IL-12, IL-23, nitric oxide (NO), and cyclooxygenase-2 and produce reactive oxygen species (ROS).Functionally, M1 macrophages can remove pathogens and can also contribute to tissue damage (40).M2 macrophages are activated by IL-4, IL-13, and IL-10, and secrete IL-10 and TGF-b.M2 macrophages can clear apoptotic cells, promote angiogenesis and fibrosis, and coordinate tissue remodeling (41).Macrophages play a defensive role in acute inflammation but have strong pathological effects in chronic inflammation and inflammatory diseases (42,43), and macrophage polarization is the pathological basis of many chronic diseases driven by inflammation (39, 40).
Recent studies have indicated that METTL3 can participate in regulating macrophage-mediated inflammation and M1 polarization (Figure 1).Li et al. found that m6A methylation and METTL3 levels were increased in macrophages stimulated by oxidized low-density lipoprotein (oxLDL) (44).Mechanistically, METTL3 promoted signal transducer and activator of transcription 1 (STAT1) expression by increasing the m6A level of the STAT1 transcript in macrophages, thereby elevating the levels of downstream inflammatory factors (44).In another study, Sun et al. found that, although oxLDL could mildly promote expression levels of METTL3 and METTL14, it decreased interaction between METTL3 and METTL14, leading to a reduction in overall m6A levels in macrophages (45).Further, Sun et al. found that Matrin-3 was reduced in macrophages stimulated by oxLDL, and involved in the formation of the METTL3-METTL14 complex, thus affecting its m6A modification of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a) mRNA (45).METTL3 was proven to co-modify PGC-1 mRNA with YTHDF2 and inhibit PGC-1 expression, which reduced ATP production and increased ROS accumulation, eventually aggravating inflammatory responses (46).Guo found that circ_0029589 expression was down-regulated in peripheral blood mononuclear cell-derived macrophages from patients with acute coronary syndrome and that IRF-1 inhibited circ_0029589 through METTL3-mediated m6A modification, thereby facilitating macrophage pyroptosis and inflammation (47).
In another report of macrophage stimulation using LPS, levels of METTL3 and m6A modification were decreased (48).METTL3 knockdown promoted the stability of nucleotide-binding oligomerization domain containing 1 (NOD1) and receptorinteracting serine/threonine kinase 2 (RIPK2) mRNA, mediated by YTHDF2, thereby up-regulating levels of TNF-a, IL-6, and NO (48); however, Tong et al. found that METTL3-deficient macrophages produced less TNF-a and that METTL3 knockout mice had higher bacterial loads in the feces and cecum, indicating that METTL3 promotes the anti-infection activity of macrophages (49).Mechanistically, METTL3 deficiency reduced Irakm (a negative regulatory factor in the TLR4 signaling pathway) mRNA degradation and promoted its expression.Microglia are often referred to as tissue-resident macrophages of the central nervous system (50).When damage or infection is detected, microglia can transform into an activated phenotype with inflammatory functions, which is important in a variety of neuropathological conditions.METTL3 was increased in LPS-stimulated microglia and promoted TRAF6/NF-kB pathway expression by m6A modification of TRAF6, thereby inducing expression of inflammatory cytokines, including IL-1b, IL-6, TNF-a, and IL-18 (51).
METTL3 also promotes proinflammatory M1 polarization.METTL3 was increased in mouse bone marrow-derived M1 macrophages and methylated the mRNA encoding STAT1, a transcription factor that regulated M1 macrophage polarization, promoting its expression (52).Further, METTL3 promoted M1 polarization by regulating macrophage metabolism.METTL3 could upregulate hepatoma-derived growth factor (HDGF) expression in macrophages by enhancing its mRNA stability (53).Proinflammatory M1 macrophages mainly rely on the glycolysis pathway to generate energy (54), while HDGF promotes M1 macrophage polarization by inducing mitochondrial injury and thereby stimulating glycolysis (53).

METTL3 regulates neutrophil activation
Neutrophils are the first innate immune cells recruited to sites of infection.The anti-infection ability of neutrophils depends on migration, phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs).Neutrophils were considered to mainly be involved in acute inflammation, but it is The role of METTL3 in regulating macrophage inflammation and polarization.

METTL3 regulates DC activation
DCs are heterogeneous antigen-presenting cells that connect innate and adaptive immunity.DCs mediate immune defense and immune tolerance and disordered DC migration or activation can have pathological consequences associated with autoimmune and infectious diseases (57).METTL3 is involved in regulating DC activation.m6A modification and METTL3 levels were increased in mature bone marrow DCs from C57BL/6 mice (58), while METTL3 knockout could inhibit DC maturation and cytokine production (58,59).METTL3 increased CD40 and CD80 mRNA m6A modification levels, thereby promoting CD40 and CD80 expression (58); these two membrane co-stimulatory molecules assist in antigen presentation and T-cell activation.In addition, METTL3 promoted Tirap expression by increasing its m6A modification level, which enhanced the activity of the TLR4/NF-kB signaling pathway and cytokine secretion (58).

METTL3 regulates Th17 cell function
Among Th cell subsets, Th17 cells are considered inflammatory, and their differentiation and pathogenic functions are related to various inflammatory and autoimmune diseases, such as psoriasis and rheumatoid arthritis (60).Particular cytokines and environmental stimuli induce Th17 cell differentiation and secretion of the pathogenic cytokine, IL-17 (61, 62).METTL3 was recently identified as involved in regulating the pathogenic function of Th17 cells.METTL3 and m6A modification were reduced in T cells from mice with experimental autoimmune uveitis (63), and METTL3 overexpression inhibited the differentiation of Th17 phenotype cells and reduced IL-17 secretion (63).Mechanistically, METTL3 increased ASH1L mRNA stability, which negatively regulates Th17 cell generation and function (63).In addition, elevated METTL3 and m6A modifications were found in bone samples from patients with periprosthetic joint infection (64); KEGG analysis demonstrated that differentially methylated genes are mainly enriched in Th17 cell differentiation and the IL-17 signaling pathway, indicating that Th17 cells and METTL3 may contribute to the pathogenesis of periprosthetic joint inflammation (64).

METTL3 regulates NK cell function
NK cells play an important role in virus and tumor immunity.NK cells can sense infected and tumor cells through natural cytotoxicity receptors, C-type lectin-like receptors, and coactivating receptors (65).Upon activation, NK cells release lytic granules and cytokines, and collaborate with other immune cells to kill target cells (66).In addition, NK cells can also activate death ligands, which induce target cell apoptosis (67).Recently, METTL3 has been reported to be associated with the function of infiltrating NK cells in tumors.Song et al. found that METTL3 was decreased in NK cells from patients with hepatocellular carcinoma (68).METTL3 knockdown inhibited NK cell infiltration and reactivity to IL-15, which is related to suppressed activation of AKT and MAPK pathways (68).

The role of METTLin inflammatory diseases
METTL3 plays important roles in both inflammation-related immune cells and other cell types, such as epithelial cells, endothelial cells, and fibroblasts.METTL3 expression levels can change under disease conditions, influencing expression of downstream target genes, and thereby participating in inflammatory disease pathogenesis.In the following sections, we elaborate on the mechanism of action of METTL3 and its effects on m6A reading proteins in cardiovascular, metabolic, degenerative, immune, infectious diseases, and tumors.The effects of METTL3 and the m6A reading proteins discussed in this review on RNA modification and metabolism are summarized in Figure 2. Expression changes and pathogenic mechanisms of METTL3 in various inflammatory diseases are shown in Tables 1, 2.

Atherosclerosis
Atherosclerosis is an immunoinflammatory disease featuring the formation of lipid plaques in the intima of arteries, resulting in lumen stenosis (111).The process of atherosclerosis development includes the proliferation of smooth muscle cells and monocytes, increased synthesis and secretion of collagen fibers by smooth muscle cells, and lipid accumulation.Dong et al. found that METTL3 was up-regulated in ox-LDL-induced human umbilical vein endothelial cells and METTL3 knockdown prevented the development of atherosclerosis (69).Functionally, METTL3 m6A modifies JAK2 mRNA and IGF2BP1 enhances JAK2 mRNA stability, while METTL3 knockdown inhibits cell proliferation and angiogenesis by inhibiting the JAK2/STAT3 pathway (69).Stenosis caused by an atherosclerosis plaque will affect blood flow and generate oscillatory stress (OS) in the plaque area.OS causes epithelial activation and deterioration of atherosclerosis.Li et al. found that METTL3 expression was decreased in atherosclerotic regions with OS, leading to reduced m6A modification of epidermal growth factor receptor (EGFR), causing slower EGFR mRNA decay and increased EGFR expression (70).EGFR is associated with atherosclerosis progression (70); however, Chien et al. found that METTL3 was up-regulated under OS (71).METTL3 methylation stabilizes NLRP1 and leads to the degradation of KLF4 mRNA, The effects of METTL3 and m6A reader proteins mentioned in this review on RNA modification and metabolism.Figure Legends: METTL3 adds the m6A motif to RNA.HNRNPA2B1 recognizes m6A in the nucleus and promotes miRNA maturation.IGF2BP1/2 recognizes m6A in the nucleus and promotes mRNA stability.YTHDF1 recognizes m6A in the cytoplasm and enhances mRNA translation.YTHDF2 recognizes m6A in the cytoplasm and promotes mRNA decay.

Myocardial infarction
Microglia inflammation in the paraventricular nucleus after MI results in excessive activation of the sympathetic nerve, leading to ventricular arrhythmia in patients with MI.Patients with MI have high mortality rates from sudden cardiac death caused by ventricular arrhythmia.Increased METTL3 elevates m6A modification of TLR4 and up-regulates its expression, which promotes sympathetic hyperactivity via the TLR4/NF-kB pathway and increases the incidence of ventricular arrhythmias post-MI (72).In another study, Qi et al. found that METTL3 expression increased in rats after MI, and METTL3 targeted TRAF6 and promoted inflammation after MI, through the TRAF6/NF-kB pathway (73).

Hyperuricemia
HUA is a metabolic disease caused by raised serum uric acid (> 6.5-7 mg/dL) (114).Epidemiological evidence indicates that HUA is related to various cardiovascular diseases (115) and cognitive impairment (116).In an in vivo experiment, levels of inflammatory cytokines in the hippocampus of rats in the HUA group were significantly higher than those in the negative control group (117).Further, in a clinical study, serum proinflammatory cytokines and oxidative stress were increased in asymptomatic young patients with primary HUA, who were in a chronic inflammatory state, which may be the reason underlying systemic disease (118).The latest evidence suggests that METTL3 is a bridge linking HUA and neuroinflammation.Chen et al. found that METTL3 was downregulated in HUA mice, while METTL3 knockdown aggravated mouse hippocampal neuronal apoptosis and microglia overactivation through the NF-kB and NLRP3 inflammasome pathway, thus aggravating neuroinflammation (77).

Diabetic complication
Type 2 diabetes mellitus results from the combination of two factors: defective insulin secretion by pancreatic b-cells and insulin resistance of peripheral target tissues (119).Diabetic kidney disease is the most common microvascular complication of diabetes.High glucose levels can elevate the expression of transforming growth factor b1, angiotensin II, cytokines, and advanced glycation end products, with adverse effects on a variety of renal cell types, including lipid oxidation, mitochondrial dysfunction, oxidation, endoplasmic reticulum stress, and fibrosis (120).Several epigenetic mechanisms, including DNA methylation, histone modification, and non-coding RNAs, have been implicated in the pathogenesis of diabetic kidney disease in recent years (120), and m6A modification is no exception.METTL3 is elevated in renal tubules from human biopsies and METTL3 silencing alleviated renal inflammation in tubular epithelial cells via the METTL3/ TAB3 axis (78).METTL3 is also elevated in podocytes from renal biopsies from patients with diabetic nephropathy, while METTL3 knockout reduced the m6A levels of TIMP metallopeptidase inhibitor 2 (TIMP2) and downregulated its expression, which led to clear alleviation of inflammation and apoptosis via the Notch pathway in podocytes (79).Liu et al. found that m6A modification of phosphate and tension homology (PTEN) in podocytes was decreased under high glucose conditions (80).The underlying mechanism involved a high glucose-stimulated decrease in METTL3 expression in podocytes and decreased m6A modification of PTEN, which increased PTEN expression, and PTEN promoted podocyte pyroptosis through PI3K/Akt signaling.METTL3 contributes to the development of renal fibrosis by promoting inflammation.METTL3 facilitates miR-21-5p maturation, and miR-21-5p activates ERK/NF-kB signaling by suppressing sprouty RTK signaling antagonist 1 (SPRY1), which affects renal fibrosis progression (81).

Osteoarthritis
OA is a chronic joint disease characterized by progressive cartilage degeneration, subchondral bone sclerosis, and synovial inflammation (121).Sang et al. found that METTL3 was decreased in patients with OA (83); however, Liu et al. showed that METTL3 was increased in experimental OA (82).METTL3 silencing reduced chondrocyte apoptosis, decreased inflammatory cytokine secretion and promoted extracellular matrix (ECM) degradation (82).METTL3 overexpression increased BCL2 expression through YTHDF1-mediated m6A modification and inhibited chondrocyte apoptosis and autophagy (84).Ribosomal protein L38 (RPL38) can positively regulate METTL3, while RPL38 knockdown increased levels of suppressor of cytokine signaling 2 (SOCS2) through decreasing its m6A level, mediated by METTL3, thereby reducing inflammation and apoptosis of chondrocytes and inhibiting ECM degradation (85).Chen et al. investigated the role of fibroblast-like synoviocyte (FLS) senescence in the development of OA and found that METTL3 knockdown increased the expression of autophagyrelated 7 (ATG7), which inhibited GATA binding protein 4 (GATA4) to ameliorate FLS senescence and OA progression (86).

Intervertebral disc degeneration
IDD is the main cause of back, neck, and radicular pain (122).The incidence rate of IDD rises with age, and its pathogenesis is related to senescence, apoptosis, inflammation levels, and ECM degradation of nucleus pulposus cells (NPCs) (123).Fang et al. found that METTL3 levels were increased in NPCs from patients with IDD (87), while METTL3 depletion resulted in the reduction of siah E3 ubiquitin protein ligase 1 (SIAH1) mRNA stability, which induced apoptosis, inflammation, and senescence, as well as decreasing ECM synthesis in NPCs (87).

Rheumatoid arthritis
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by autoantibody production, synovial inflammation, cartilage damage, and bone erosion (124,125).The etiology of rheumatoid arthritis is very complex.Current evidence shows that genotype, sex, environmental triggers, periodontal microbes, gastrointestinal microbes, and epigenetic modification are involved in the incidence and development of RA (125)(126)(127).Wang et al. found that METTL3 was significantly increased in peripheral blood mononuclear cells from patients with RA, and there were positive correlations between METTL3 levels and the markers of RA activity, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) (88).Another in vivo experiment demonstrated that METTL3 was up-regulated in synovial tissue and FLSs from patients with RA and in an adjuvant-induced arthritis rat model (89).METTL3 silencing suppressed FLS proliferation, migration and invasion, and IL-6 expression, possibly by inhibiting the NF-kB pathway (89).

Psoriasis
Psoriasis is a chronic, immune-mediated disorder, which can be provoked by non-specific triggers, such as mild trauma, sunburn, stress, infection (particularly streptococcal), alcohol, or drugs (128).Briefly, triggers stimulate keratinocytes to produce antimicrobial peptides, which in turn activate Th cells to polarize and release IFNg, IL-17A/IL-17F, and IL-22; these cytokines result in excessive proliferation and abnormal differentiation of keratinocytes, which release pro-inflammatory cytokines, further aggravating psoriasis (129)(130)(131).Recent evidence suggests that m6A epigenetic modification also contributes to the pathogenesis of psoriasis.Lower levels of m6A methylation and METTL3 were detected in psoriatic skin lesions than in healthy human skin samples (90), and levels of m6A methylation and METTL3 were negatively correlated with psoriasis severity (90).Further, compared with mice heterozygous for METTL3 knockout, wild-type control mice showed ameliorated psoriasis-like clinical and pathological manifestations (90).Moreover, KEGG analysis suggests that METTL3 may play a role in the pathogenesis of psoriasis through the WNT pathway (90).

Inflammatory bowel disease
IBD describes chronic inflammatory disorders of the intestine (132).Maialen et al. used an online genome-wide association study and m6A transcription databases to screen five IBD-related single nucleotide polymorphisms in the UBE2L3, SLC22A4, TCF19, C6orf47, and SNAPC4 genes, suggesting that m6A methylation may contribute to IBD pathogenesis (133).Subsequently, METTL3 was found to be elevated in ulcerative colitis tissue from patients with IBD, a dextran sulfate sodium-induced IBD mouse model, and LPS-treated MODE-K cells, while in vivo and in vitro knockdown of METTL3 reduced levels of proinflammatory cytokines and inflammatory enzymes and inhibited p65 phosphorylation (91).

Sepsis
Sepsis is a life-threatening organ dysfunction resulting from dysregulated host responses to infection (134); it can be caused by almost any infectious organism, and the incidence rates of sepsis due to infection by gram-negative and gram-positive bacteria are almost equal (135).The most common infection sites leading to sepsis are the lung, abdomen, blood, kidney, and genitourinary tract (136).According to traditional understanding, sepsis is an overwhelming systemic inflammatory response to infection, accompanied by immunosuppression and secondary infection ( 1 3 7 ) .S e p s i s -i n d u c e d o r g a n d y s f u n c t i o n i n c l u d e s cardiomyopathy, encephalopathy, and cognitive impairment, as well as liver injury, acute kidney injury, and acute respiratory distress syndrome (ARDS).Although many studies have shown that sepsis can cause organ dysfunction by interfering with microcirculation, ischemia, hypoxia, mitophagy, and cell apoptosis, the exact mechanism involved is not fully understood (138).
Aortic injury during sepsis leads to insufficient organ perfusion, but the specific mechanism has not been fully elucidated.Shen et al. found that levels of m6A and METTL3 expression were significantly decreased in aortic tissue from an LPS-induced sepsis rat model (92).According to high-throughput sequencing, KEGG, and GO enrichment analyses, genes affected by m6A modification were related to cation channels, particularly TRP channels, indicating that METTL3 may aggravate vascular endothelial cell inflammation during sepsis by interfering with calcium ion transport (92).
NETs were increased in humans and mice with sepsisassociated ARDS and positively associated with disease severity level, indicating that NETs are harmful in sepsis-associated ARDS (93).Further, Zhang et al. found that NETs upregulated METTL3 expression, leading to methylation of glutathione peroxidase 4 (GPX4) mRNA and decreased GPX4 expression via accelerated degradation of GPX4 mRNA mediated by YTHDF2, with consequent ferroptosis of alveolar epithelial cells (93).Qu et al. showed that NETs could promote METTL3-mediated m6A methylation of sirtuin 1 (SIRT1) mRNA in alveolar epithelial cells, leading to abnormal cell autophagy and deterioration of ARDS (94).Chen et al. found that METTL3 expression was reduced in LPS-induced ARDS mice and LPS-stimulated human pulmonary vascular endothelial cells (95).METTL3 downregulation destroyed endothelial permeability and led to higher inflammatory cytokine expression by reducing the stability of Trim59 mRNA and activating the NF-kB pathway.CircN4bp1 up-regulated the expression of the enhancer of the zeste 2 polycomb repressive complex 2 subunit (EZH2) by combining with miR-138-5p, thus promoting M1 macrophage polarization and aggravating the degree of lung injury in ARDS mice (96).Further, Zhao et al. found that the m6A levels in circN4bp1 were increased in ARDS mice and that the inflammatory response could be blocked by inhibiting METTL3, suggesting that METTL3 might be involved in regulating circN4bp1 expression (96).

Pneumonia
Pneumonia is a common acute respiratory infection caused by many types of microorganisms, including bacteria, respiratory viruses, and fungi, and primarily occurs in children < 5 years old and elderly adults with underlying chronic conditions (139).Yang et al. found that METTL3 was increased in pediatric patients with pneumonia and WI-38 cells, while METTL3 knockdown inhibited the inflammatory response and apoptosis by reducing the expression of EZH2 and the JAK/STAT3 pathway (97).In another study, METTL3 knockdown reduced the translation of STAT2 mRNA mediated by YTHDF1 and alleviated LPS-induced inflammatory damage (98).

Other infectious diseases
When a virus invades, innate immune and epithelial cells can recognize viral structures, such as nucleic acids, peptides, and lipoproteins, leading to primary activation of interferon regulatory factor (IRF) 3/7 and the NF-kB signaling pathway, which induces the production of pro-inflammatory cytokines and interferon (140).Interferon induces interferon-stimulated gene (ISG) transcription by activating the JAK1/STAT1/STAT2 pathway (140).Some viruses can modify host cell genes through epigenetic strategies, to enhance replication and survival.Epstein-Barr virus infection can alter the m6A-modified epitranscriptome of host B cells and upregulate mRNA and protein levels of fas cell surface death receptor (FAS) but downregulate toll-like receptor 9 (TLR9), which are related to apoptosis and virus infection (141).Wang et al. found that METTL3-deficient mice had stronger resistance to rotavirus.METTL3 knockout in intestinal epithelial cells reduced m6A modification of IRF7 and increased its expression, leading to the upregulation of interferons and ISGs (109).Li et al. found that METTL3 depletion of host cells decreased m6A levels of SARS-CoV-2 and the host genome, subsequently increasing innate immune signaling and inflammatory gene expression (99).
Fungal keratitis (FK) is an aggressive infectious corneal disease caused by pathogenic fungi (142).METTL3 was increased in corneas of an FK mouse model and in fungi-infected corneal stromal cells, while METTL3 knockdown attenuated the FK inflammatory response by inhibiting the PI3K/AKT or TRAF6/ NF-kB pathways (100, 101).
In addition, some studies of inflammation have been conducted using LPS-stimulated cells.METTL3 protein expression was elevated in LPS-induced retinal pigment epithelium cells, and silencing METTL3 increased the expression of nuclear receptor subfamily 2 group F member 1 (NR2F1) and promoted IL-6 secretion (102).Further, METTL3 was up-regulated in LPSstimulated human dental pulp cells, and its knockdown reduced inflammatory cytokine secretion through the NF-kB and MAPK pathways (103).

Tumors
Chronic inflammation is a risk factor for tumor development, as it provides a microenvironment conducive to tumorigenesis, tumor development, and metastasis (143).A deep understanding of the r e g u l a t or y m ec h a n i s m s i n t h e i nfl a m m a t o r y t u m o r microenvironment will help to develop more effective immunotherapy.Most tumors recruit tumor-associated macrophages (TAMs) to create a microenvironment favorable for tumor growth.METTL3 has recently been shown to affect the recruitment and polarization of TAMs, with METTL3-deficient mice exhibiting increased infiltration of M1/M2-like TAMs and regulatory T cells into tumors compared with wild-type mice (104).METTL3 knockout promoted M1 and M2 polarization of macrophages by activating the NF-kB/STAT3 pathways (104).Co-culture of monocyte and glioma cells demonstrated that glioma cells overexpressing METTL3 inhibited SOCS2 expression and M1 polarization of monocytes (110), while SOCS2 overexpression could rescue this inhibition.Knockdown of METTL3 in TAMs infiltrating prostate cancer promotes M2 macrophage polarization by activating STAT6 (106).Further, M2-TAMs can promote lung adenocarcinoma immuno-resistance by enhancing METTL3-mediated m6A methylation (107), as well as inducing oxaliplatin resistance by increasing METTL3 in colorectal cancer cells (108).
In addition, METTL3 regulates the infiltration of tumorassociated neutrophils.Silencing METTL3 caused papillary thyroid cancer cells to secrete more pro-inflammatory factors, particularly IL-8, which can recruit infiltration of tumorassociated neutrophils.The NF-kB pathway inhibitor, BAY 11-7082, inhibits IL-8 production by tumor cells and neutrophil infiltration, as well as inhibiting tumor growth (105).

Therapeutic application of METTL3
The study of METTL3 expression changes and related pathogenic processes in inflammatory diseases provides clues for the development of effective drugs and informs novel diagnostic strategies (Table 3).
The therapeutic potential of METTL3 is mainly as a pharmacological target for natural plant medicinal ingredients.Emodin, the main active component of rhubarb, exhibits significant anti-inflammatory activity (149) and can promote METTL3 expression, which downregulates NLRP3 by increasing its m6A levels in LPS-treated 1321N1 cells, thus suppressing pyroptosis and proinflammatory cytokine secretion (144).Cinnamaldehyde (CA) is a natural ingredient extracted from the herb, Ramulus cinnamomi, which has beneficial functions in lipid metabolism and inflammation of the liver (150).Xu et al. found that METTL3 was decreased in mouse hepatocytes stimulated by free fatty acids and aggravated cell steatosis by down-regulating CYP4F40, which could be compensated by CA (145), where CA can promote METTL3 expression and improve steatosis (145).Total flavones isolated from Abelmoschus manihot (TFA) have been used to treat kidney disease (151).Liu et al. found that TFA could upregulate METTL3 expression in podocytes stimulated by high glucose and that METTL3 overexpression promoted PTEN expression via m6A modification of PTEN, thereby inhibiting podocyte pyroptosis mediated by PI3K/Akt signaling (80).Resveratrol is a natural phytoalexin, present in grapes, peanuts, and their derivatives (152), which has a neuroprotective role (153).Izquierdo et al. found that adding resveratrol to the high-fat diet of mice could clearly up-regulate m6A levels in the hippocampus of their offspring and reduce expression of METTL3 and FTO, although the significance and impact of METTL3 reduction have not been studied (146).In another study, resveratrol intake in mice fed a highfat diet could increase METTL3 in the liver tissue, reduce liver fat accumulation, and increase PPARa mRNA levels (147); however, the molecular mechanism underlying METTL3 activity in resveratrolregulated liver lipid homeostasis remains to be determined.
The efficacy of METTL3 inhibitors has also been investigated.Cpd-564, a new METTL3 inhibitor identified by Wang et al., exerted reno-protective effects in cisplatin-treated HK2 cells and an acute kidney injury (AKI) mouse model (78).Cpd-564 inhibited TAB3 expression and inflammatory responses in HK2 cells tubule damage was alleviated in an AKI model after Cpd-564 administration; however, METTL3 inhibition may be harmful to temporomandibular joint (TMJ) OA.The METTL3 inhibitor, Sadenosylhomocysteine, promotes chondrocyte apoptosis and autophagy induced by TNF-a, and aggravated chondrocyte degeneration in TMJ OA mice (84).In addition, the methylation inhibitor, cycloleucine, can reduce IL-8 and IL-6 and promote collagen type II levels in OA cartilage tissue (82).
Moreover, METTL3 may be a target in OA treatment using extracellular vesicles derived from human umbilical cord mesenchymal stem cells (hucMSCs-EVs); miR-1208 in hucMSCs-EVs reduced m6A levels in NLRP3 mRNA by down-regulating METTL3 expression in macrophages, which prevented NLRP3 inflammasome activation and exerted anti-inflammatory effects (148).
In addition, METTL3 may serve as a novel biomarker for RA diagnosis.Wang et al. found that increased METTL3 in RA patients was positively correlated with CRP and ESR (88); however, its sensitivity and specificity require further verification.

Conclusion
The study of METTL3-mediated m6A modification brings new insights into the pathogenesis of inflammatory diseases.METTL3 is upregulated in most inflammatory diseases, indicating that it has pro-inflammatory effects; however, opposite expression trends in METTL3 expression have also been observed in the same diseases, including sepsis-related ARDS and OA, indicating that the effects of its expression may be related to cell type, modeling methods, disease status, or other potential regulatory factors.Moreover, METTL3 can regulate different target genes in the same disease, which challenges the accuracy and safety of drug development.In addition, METTL3 can regulate macrophage M1 polarization, implying that METTL3 is a potential therapeutic target in macrophage-related diseases.As METTL3 can regulate the infiltration and polarization of TAMs and tumor-associated neutrophils, it is meaningful to study the therapeutic application of METTL3 in anti-tumor immune responses.There have been promising studies of natural drugs targeting METTL3 in the treatment of inflammatory diseases, although their detailed pharmacological effects are not yet fully understood.Whether agents targeting METTL3 are natural plant-derived ingredients, chemically synthesized drugs, or biological agents, their therapeutic prospects warrant further research.

TABLE 1
Changes of METTL3 expression in inflammatory diseases.
). METTL3 appears to hinder NASH development.Li et al. showed that METTL3 overexpression alleviated NASH by suppressing the expression of

TABLE 3
Effects of natural products, synthetic compounds, and biological agents on METTL3.