N6-methyladenosine (m6A) modification in inflammation: a bibliometric analysis and literature review

Date collection

The Web of Science is one of the most widely utilized academic databases, serving as the primary research platform for hard sciences, social sciences, arts, and humanities information, as well as functioning as an independent global citation database for the most reputable publishers (Liu et al., 2022b). Additionally, the Web of Science is regarded as the optimal database for conducting bibliometric analyses (Ding & Yang, 2022). To enhance the representativeness and accessibility of the data, the Web of Science Core Collection (WOSCC) was chosen as the data source for this investigation. Furthermore, to ensure the precision of the retrieved data, the index selected was the Science Citation Index Expanded (SCI-expanded). The search terms employed were: (TS = (inflammatory OR inflammation OR inflammations)) AND (TS = (N-methyladenosine OR N(6)-methyladenosine OR N(6)mAdo OR N6-methyladenosine OR N6-methyladenosine (m6A) OR 6-methyladenosine)). The retrieved data were collected on December 31, 2023, to mitigate any potential discrepancies arising from daily updates. Following the aforementioned search strategy, 417 documents were initially retrieved from WoSCC. After excluding certain publication types, including meeting abstracts, editorial materials, early access, and retracted publications, 409 publications were retained, comprising 336 original research articles, and 73 review articles. The comprehensive screening process is illustrated in Fig. 1.

Date analysis

The acquired text was processed using VOSvivewer software (version 1.6.19) and CiteSpace software (version 6.2.4) for subsequent analysis. VOSviewer facilitates the construction and visualization of bibliometric networks, including countries/regions, institutions, authors, journals, and keywords. CiteSpace was employed to investigate the clustering and bursting of keywords. In addition, the keyword timeline view was generated through CiteSpace, effectively illustrating the evolution of knowledge and the historical breadth of literature within a specific cluster over time, thereby enhancing our understanding of the developmental trajectory and trends within the field. For a more precise representation of the data, Scimago Graphica Beta 1.0.36 and GraphPad Prism 8.3.0 were also utilized for literature visualization.

Analysis of annual publications distribution

The volume of publications serves as an indicator of the developmental trajectory and level of interest within the research domain. As depicted in Fig. 2, the literature pertaining to m6A and inflammation-related studies has been present since 1996. However, research in this domain remained sparse until 2018. Beginning in 2019, there has been a notable exponential increase in both the number of publications and citations, culminating in a total of 155 publications in 2022. This trend signifies a growing interest in this research area over the past 5 years.

Bibliometric analysis of countries/regions and institutions

These publications originated from 23 countries and 442 organizations/institutions. The leading five countries were China (n = 362, 88.51%), the United States (n = 40, 9.78%), Germany (n = 7, 1.71%), Italy (n = 5, 1.22%), and Canada (n = 4, 0.98%) (Table 1). The contributions from China and the United States constituted a significant majority of the total (98.29%). We subsequently visualized these 23 countries and developed collaborative networks based on the volume of publications and inter-country relationships (Fig. 3A).

The 10 institutions with the highest publication counts are detailed in Table 2. The top ten institutions were all situated in China, with the five institutions producing the most publications being Central South University (n = 26, 6.36%), Sun Yat-sen University (n = 26, 6.36%), Nanjing Medical University (n = 22, 5.38%), Shanghai Jiao Tong University (n = 22, 5.38%), Fudan University (n = 16, 3.91%). We visualized and constructed a collaborative network of 38 institutions that had a publication volume of five or more (Fig. 3B). While Central South University holds the record for the most publications, Shanghai Jiao Tong University is recognized as the most cited institution (n = 1,456) and demonstrates the closest collaborative ties with other institutions (total link strength = 18).

Bibliometric analysis of authors and co-cited authors

These publications involved 2,891 authors, with the top 10 authors ranked by article production listed in Table 3. Chuan He from The University of Chicago, Wei Wang from Sun Yat-sen University, and Qiong Xu from Sun Yat-Sen University Hospital of Stomatology emerged as the most prolific authors (n = 7). Qiong Xu is also the most cited author (n = 590). Among the 12,720 co-cited authors, seven authors were co-cited more than 100 times. The most frequently cited author was Xiang Wang (n = 266), followed by Kate D Meyer (n = 165) and Dan Dominissini (n = 125) (Table 4). Figures 4A and 4B illustrate the collaborative networks of authors and co-cited authors, respectively.

Bibliometric analysis of journals and co-cited journals

A total of 225 journals published the documents. As illustrated in Table 5, Frontiers in Immunology emerged as the leading journal with the highest volume of articles pertaining to m6A and inflammation, featuring 18 published papers. This was followed by Frontiers in Genetics (n = 15), Frontiers in Cell and Developmental Biology (n = 14), International Journal of molecular sciences (n = 10), Nature communications (n = 8), Frontiers in pharmacology (n = 7). The citation network of journals is depicted in Fig. 5A. Table 6 shows the top 20 journals with the most significant of co-citations, with the top 5 being Nature (n = 1,040), Cell (n = 728), Nucleic Acids Research (n = 578), Molecular Cancer (n = 559) and Molecular Cell (n = 544). The co-citation journals network is illustrated in Fig. 5B.

Bibliometric analysis of co-cited references

The 10 most frequently referenced sources are presented in Table 7. The most cited reference was a dissertation authored by Professor Wang et al. (2014) (n = 111), followed by (n = 103) Dominissini et al. (2012), and Wang et al. (2015) (n = 89). Among the top 10 most cited publications, nine were original research articles and one was a review article.

Bibliometric analysis of keywords

Initially, we examined the frequency and co-occurrence of keywords utilizing VoSviewer. The top 20 keywords are presented in Table 8. The most prevalent keyword identified was inflammation (n = 127), followed by messenger-RNA (n = 99), N6-methyladenosine (n = 95), methylation (n = 91), and expression (n = 83). The keyword co-occurrence network is illustrated in Fig. 6A. Subsequently, we performed a cluster analysis of the keywords (Fig. 6B). Modularity q-value = 0.5046, Silhouette s-value = 0.7688. The results showed that all the keywords could be categorized into 12 distinct groups, which include FOXO3, m6A modification, immune system, immune infiltration, metabolism, activation, IPEC-J2, DNA methylation, presynaptic, ischemia-reperfusion (i/r), polymyositis and agonist. Additionally, we created a timeline viewer of the keywords by CiteSpace (Fig. 7A). Furthermore, we aggregated all the data from 1996 to 2023 to identify burst keywords through CiteSpace. Burst keywords are defined as those that exhibit a significant frequency of occurrence within a specific timeframe. This analysis not only highlighs the evolution of research hotspots over time but also reflects recent research trends and may forecast future directions. Figure 7B shows the top 13 keywords with the highest incidence of outbreaks. The prominent terms that emerged in the last 5 years include “promotes”, “microRNAs”, “METTL3”, “hepatocellular carcinoma”, and “obesity”. However, the intensity of these burst keywords remains below 2. We interpret this as indicative of the field’s nascent status, with a substantial volume of literature primarily published post-2019. Notably, the role of m6A in inflammatory regulation has garnered significant scholarly interest since 2019 and is expected to continue to be a focal point in future research.

Database cross-validation

To substantiate our findings, bibliometric analyses were were performed utilizing the PubMed and Scopus databases. A total of 372 publications were extracted from the PubMed database, while 581 publications were sourced from Scopus. In the PubMed database, publications released after 2019 represented a substantial majority, comprising 97.84% (364/372) of all entries. The authors with the highest publication counts were Chuan He (n = 8) and Wei Wang (n = 7). The most prevalent keywords in this dataset were “humans”, “animals”, “inflammation”, “mice”, and “adenosine.” The Scopus database provides a more comprehensive compilation of publications, with works published post-2019 accounting for 91.39% (531/581) of the total. The countries with the most significant contributions to these publications were China (n = 477), the United States (n = 66), Germany (n = 17), the United Kingdom (n = 14), and Japan (n = 12). Notably, journals with the highest publication counts comprised Frontiers in Immunology (n = 30), Frontiers in Genetics (n = 19), Frontiers in Cell and Developmental Biology (n = 14), International Journal of Molecular Sciences (n = 12), and Aging (n = 9). The most common keywords within this context included “human”, “article”, “6 N methyladenosine”, “controlled study”, and “nonhuman”. It is crucial to acknowledge that bibliometric analyses concerning institutions may not be directly comparable due to inherent discrepancies in data characteristics between PubMed and Scopus. Despite some observed variations across different databases, overarching trends regarding annual publication rates, participating countries/regions, leading authors, prominent journals, and recurring keywords displayed analogous patterns. Detailed results from these bibliometric analyses are accessible in the Supplemental Materials.

The mechanism of the m6A modification involvement in the inflammatory response

As the most widespread internal mRNA modification, m6A plays a significant role in the inflammatory response to disease. Some mechanisms are summarized and elucidated by bibliometric analysis and systematic organization of keywords. The analysis highlighted that the key mechanisms through which m6A regulates inflammation encompass apoptosis, autophagy, oxidative stress, immune cell dysfunction, and dysregulation of signaling pathways.

m6A and apoptosis

Apoptosis is the first recognized form of programmed cell death, typically occurring during developmental processes and aging, and it functions as a homeostatic mechanism to maintain cellular populations within tissues (Elmore, 2007). The apoptotic mechanism is intricate, with current studies indicating the existence of two primary apoptotic pathways: the extrinsic and intrinsic pathways. Notably, there is an intersection between the two pathways. In addition, there exists an additional pathway involving T cell-mediated cytotoxicity and perforin-granzyme-dependent cell killing. The extrinsic, intrinsic, and perforin/granzyme pathways converge on a common execution pathway (Xu, Lai & Hua, 2019; Lopez & Tait, 2015; Zhang & Xia, 2023; Kashyap, Garg & Goel, 2021). During the process of apoptosis, several proteins are crucial in modulating the apoptotic response, including both anti-apoptotic proteins and pro-apoptotic proteins. Anti-apoptotic proteins include B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl), while pro-apoptotic proteins include Bcl-2-associated X protein (Bax) and Bcl-2 antagonist/killer (Bak) (Zhang & Xia, 2023). Dysregulation of apoptosis has been implicated in various inflammatory diseases (Vallejo-Garcia et al., 2012; Littlewood & Bennett, 2003; Zhan et al., 2024). Furthermore, m6A modifications influence apoptosis by directly or indirectly modulating apoptosis-related factors or pathways (Xu et al., 2021). Figure 8 illustrates the primary mechanisms by which m6A regulates apoptosis.

METTL3 serves different functions in apoptosis through various mechanisms. In the prostate cancer cell lines LNCaP and PC3, the depletion of METTL3 results in increased expression levels of the pro-apoptotic proteins Bak and Bax, alongside heightened activity of caspase3 and caspase7, while simultaneously decreasing the expression levels of the anti-apoptotic proteins Bcl-2 and Bcl-xl (Cai et al., 2019). This study does not address the m6A modification levels in mRNAs of apoptosis-related proteins; thus, investigating the m6A modification levels of target genes may provide deeper insights into the mechanistic role of METTL3. In human myeloid leukemia (MOLM13) cells with METTL3 depletion, the level of m6A modification of BCL-2 is reduced, correlating with decreased protein expression. The upregulation of METTL3 inhibits apoptosis by enhancing the translation of MYC, BCL2, and PTEN mRNAs through increased methylation (Vu et al., 2017). The expression of METTL3 was diminished in both in vivo temporomandibular joint osteoarthritis (TMJ OA) mouse models and in vitro TNF-α-stimulated mouse chondrocytes (ATDC5). MeRIP analysis indicated that METTL3 overexpression significantly increased the level of m6A methylation of Bcl-2 mRNA. The m6A modification installed by METTL3 on target mRNAs was dependent on reader recognition. Further experiments showed that YTHDF1 mediated Bcl-2 mRNA stability in an m6A-dependent manner. In conclusion, overexpression of METTL3 increases Bcl-2 mRNA stability through YTHDF1-mediated m6A modification, thereby inhibiting TNF-α-induced apoptosis and autophagy in ATDC5 (He et al., 2022). Furthermore, METTL3 overexpression increased the m6A modification level of Bax mRNA in a cardiomyocyte cell line (HL1) following in vitro hypoxia/reperfusion injury (iH/R), potentially safeguarding cardiomyocytes from iH/R-induced apoptosis by downregulating pro-apoptotic gene expression (Su et al., 2021). Conversely, another study indicated that METTL3 had an opposing effect on apoptosis. METTL3 knockdown significantly mitigated inflammation and apoptosis in renal podocytes from diabetic patients, and its overexpression significantly exacerbated these responses in vitro. Further studies revealed that METTL3 regulates Notch signaling and exerts pro-inflammatory and pro-apoptotic effects through the m6A modification of TIMP2 in an insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2)-dependent manner (Jiang et al., 2022).

Moreover, significantly increased expression levels of METTL14 were observed in the kidneys of mice suffering from diabetic nephropathy. METTL14-dependent RNA m6A modification contributes to podocyte damage by regulating Sirt1 mRNA post-transcriptionally. The knockdown of METTL14 resulted in the inhibition of apoptosis and inflammation in podocytes (Lu et al., 2021).

In myocardial ischemia-reperfusion injury (IRI) mice and hypoxia/reoxygenation (H/R)-induced cardiomyocytes (CMs), the m6A eraser fat mass and obesity-associated protein (FTO) is downregulated. Further studies indicate that FTO target Yes-associated protein (YAP1) mRNA in CMs. As a transcriptional co-activator, YAP1 was shown to prevent CM apoptosis by promoting AKT signaling (Del Re et al., 2013). Overexpression of FTO enhances the stability of YAP1 mRNA by demethylating it, thereby attenuating H/R-induced apoptosis and inflammation in CMs (Ke et al., 2022). Furthermore, FTO overexpression reduces cleaved caspase-3 and Bax levels and increased Bcl-2 expression (Shen et al., 2021). Silencing of ALKBH5 inhibits apoptosis and suppresses ultraviolet B (UVB)-induced cellular pyroptosis and inflammatory responses in patients with chronic actinic dermatitis (CAD) (He et al., 2023b).

The m6A reader YTHDF3 binds to the 3′UTR m6A modification site of mitochondrial calcium unidirectional transport protein (MCU), a key protein in human cytomegalovirus (HCMV)-induced apoptosis in vascular endothelial cells, enhancing MCU translation and promoting apoptosis (Zhu, Zhang & Wang, 2022). YTHDF3 or IGF2BP2 knockdown suppresses H/R-induced apoptosis in BEAS-2B cells by deactivating the p38, ERK1/2, AKT, and NF-κB pathways (Xiao et al., 2022).

m6A and autophagy

Autophagy is a highly conserved process of eukaryotic cell death and recycling. Through the degradation of cytoplasmic organelles, proteins, and macromolecules, and the recycling of catabolic products, autophagy plays a critical role in cell survival and maintenance. Numerous autophagy-related (ATG) genes regulate this process (Parzych & Klionsky, 2014). Disruption of autophagy affects various inflammatory conditions, including infectious and non-infectious diseases such as autoimmune disorders, tumors, and neurodegenerative diseases (Deretic, 2021). The m6A modification may influence the transcriptional regulation of ATG genes, thereby affecting autophagy. Figure 9 illustrates the primary mechanisms by which m6A modulates autophagy.

The effects of m6A modifications on autophagy may are primarily inhibitory, with METTL3 shown to negatively regulate autophagy in a variety of contexts. In H/R-induced cardiomyocytes, METTL3 methylates transcription factor EB (TFEB), a key regulator of lysosomal biosynthesis and autophagy genes, at the 3′-UTR. The m6A modification promotes the binding of the RNA-binding protein HNRNPD to the TFEB precursor mRNA, subsequently promoting the degradation of the TFEB mRNA, which results in decreased expression levels and reduced autophagy (Song et al., 2019). In primary hepatocellular carcinoma (HCC), METTL3 adds m6A modification to the 3′-UTR of FOXO3 mRNA, a negative regulator of autophagy, increasing its mRNA stability in a YTHDF1-dependent manner (Lin et al., 2020). Conversely, some studies report that METTL3 overexpression increased m6A levels on ATG5 and ATG7 transcripts, promoting their expression and enhancing autophagy (Chen et al., 2021; Liu et al., 2020a).

The m6A eraser FTO is also a crucial regulator of autophagy, functioning as a positive regulator. FTO promotes autophagy initiation by specifically upregulating the protein levels of Unc-51-like kinase 1(ULK1), a key kinase in autophagy initiation (Jin et al., 2018). FTO silencing leads to higher m6A levels on ATG5 and ATG7 transcripts, causing their degradation and reduced protein expression, thereby inhibiting autophagy (Wang et al., 2020). ALKBH5 similarly promotes autophagy by demethylating TFEB mRNA (Song et al., 2019).

The m6A reader YTHDC1 regulates autophagy by stabilizing the nuclear mRNA of the autophagy receptor SQSTM1. YTHDC1 knockdown leads to SQSTM1 mRNA degradation, reducing autophagic flux (Liang et al., 2022). YTHDF1 enhances the translation of ATG2A and ATG14 in an m6A-dependent manner, regulating hypoxia-induced autophagy and autophagy-related HCC progression (Li et al., 2021). YTHDF3 promotes autophagy by recognizing the m6A modification site near the FOXO3 mRNA termination codon and facilitates FOXO3 translation by recruiting eIF3a and eIF4B (Hao et al., 2022).

m6A and oxidative stress

Oxidative stress arises from the excessive generation of reactive oxygen species (ROS) in cells or tissues, which disrupts redox homeostasis due to an imbalance between ROS generation and scavenging mechanisms. This disruption may be a significant mechanism in the development of chronic inflammation (Hussain et al., 2016). The m6A modification plays a critical role in regulating cellular responses to oxidative stress. Figure 10 illustrates the primary mechanisms by which m6A participates in the oxidative stress response.

Oxidative stress triggered by agents such as hydrogen peroxide and arsenite elevates intracellular levels of m6A methyltransferases (Li et al., 2017). Zhao et al. (2019) demonstrated that arsenite-induced oxidative stress increased the level of m6A modification by promoting WTAP and METTL14 protein expression. Conversely, when arsenite-induced oxidative stress was inhibited by antioxidant N-acetylcysteine (NAC), m6A modification and methylase levels returned to baseline (Zhao et al., 2019; Pedre et al., 2021). Furthermore, METTL3 may serve a protective function in oxidative stress. Wang et al. (2019a) found that overexpression of METTL3 in mouse renal tubular epithelial cells (mRTECs) led to significantly lower ROS levels and lipid peroxidation marker malondialdehyde (MDA), along with increased activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX). Further investigation revealed that METTL3 interacts with the microprocessor protein DiGeorge syndrome critical region gene 8 (DGCR8), which regulates miRNA biogenesis, and positively regulated the miR-873-5p maturation process in an m6A-dependent manner. miR-873-5p regulated the Keap1-Nrf2 pathway, thereby counteracting fucoxanthin-induced oxidative stress and cellular apoptosis (Wang et al., 2019a).

The m6A eraser FTO is also associated with oxidative stress. A significant increase in palmitate-induced ROS production was observed in human myotubular cells overexpressing FTO (Bravard et al., 2011). Zhuang et al. (2019) observed specific upregulation of genes related to mitochondrial biosynthesis (PGC-1α, NRF1, and TFAM) and oxidative phosphorylation (Cox5a, Atp5 g1, Atp5a1) in FTO overexpressing cells. Mitochondria, as key regulators of apoptosis and oxidative stress in mammalian cells, are affected by FTO overexpression, which alters the mitochondrial respiratory chain and intensifies oxidative stress (Guo et al., 2013). This further elucidates the functional mechanism of FTO in oxidative stress.

m6A readers exhibit diverse functions in oxidative stress. In endothelial cells, reduced expression of YTHDC1 correlates with increased ROS levels and decreased SOD2 expression (Yin et al., 2023). Translation of Peroxiredoxin 3 (PRDX3), a major regulator of mitochondrial oxidative stress, is directly regulated by YTHDF3 in an m6A-dependent manner. Overexpression of PRDX3 prevents mitochondrial oxidative stress-induced injury in a variety of diseases (Arkat et al., 2016; Hwang et al., 2019). PRDX3 expression was suppressed when YTHDF3 was knocked down (Sun et al., 2022).

m6A and immune cell dysregulation

The bibliometric analysis of keywords indicates that m6A is involved in modulating the inflammatory response by regulating the function of innate immune cells such as neutrophils, macrophages, and NK cells, in addition to adaptive immune cells including T cells and B cells (Fig. 11).

Neutrophil

Neutrophils represent a crucial element of the innate immune response and constitute the predominant leukocyte population in peripheral blood. Upon pathogen invasion, neutrophils are mobilized from the bone marrow into the bloodstream and migrate to the infection site in the early stages (Shen et al., 2017). The migration and aggregation of neutrophils at the site of infection are critical for the effective innate immune. A thorough understanding of the molecular mechanisms governing neutrophil migration is essential for enhancing the body’s anti-inflammatory response while preventing inflammatory tissue damage. Neutrophil migration and aggregation are mediated by extracellular signals such as chemokines and cytokines, as well as chemokine receptors, cytoskeletal proteins, and intracellular signaling pathways (Park et al., 2019). METTL3 mediates m6A modification of Toll-like receptor 4 (TLR4) mRNA to promote its protein expression. In lipopolysaccharide (LPS)-induced endotoxemia, METTL3 controls neutrophil release from the bone marrow into the circulation in a TLR4-signaling-dependent mechanism involving surface expression of CXC chemokine receptor 2 (CXCR2) (Luo et al., 2023). ALKBH5 demethylates neutrophil migration-associated molecules and alters the fate of target RNAs. ALKBH5 increases the expression of the neutrophil migration-promoting molecules CXCR2 and NLR family pyrin domain containing 12 (NLRP12), while reducing the expression of the neutrophil migration-suppressing molecules prostaglandin E receptor 4 (PTGER4), tenascin-C (TNC), and with-no-lysine kinase 1 (WNK1). This intrinsic epigenetic mechanism enables the accumulation of neutrophils at infection sites and, promotes effective bacterial clearance, thereby preventing an excessive inflammatory response (Qian & Cao, 2022).

Macrophage

Macrophages initiate immune response by recognizing pathogen-associated molecular patterns (PAMPs) from invading pathogens and damage-associated molecular patterns (DAMP) from stress or tissue injury. Upon activation by external stimuli, macrophages trigger complex cascade of responses, demonstrating plasticity (M1 and M2 polarization) and pluripotency (pro-inflammatory and anti-inflammatory) (Shapouri-Moghaddam et al., 2018). Pooled CRISPR screening has identified METTL3 as a positive regulator in the innate immune responses of macrophages. METTL3 deficiency in macrophages impairs their resistance to pathogens and tumors. METTL3 mediates the m6A-modified of interleukin 1 receptor-associated kinase 3 (IRAK3) mRNA, accelerating its degradation and thereby promoting macrophage activation. In METTL3-deficient macrophages, elevated IRAK3 expression reduces TLR4 signaling, which negatively regulates macrophage activity (Tong et al., 2021). METTL3 is upregulated in M1-polarized macrophages, and overexpression of METTL3 promotes M1 polarization while inhibiting M2 polarization. The regulation of macrophage polarization by METTL3 involves multiple signaling pathways. Further studies have shown that METTL3 directly catalyzes the m6A modification of STAT1 mRNA, a transcription factor critical for M1 macrophages activation, thereby enhancing mRNA stability and expression (Liu et al., 2019b). However, some studies have found that METTL3 exhibits an inhibitory effect on macrophage inflammation. For instance, METTL3 reduces LPS-induced inflammatory responses in macrophages via the NF-κB signaling pathway (Wang et al., 2019b). Additionally, METTL3 inhibits M1 macrophage polarization by degrading m6A-modified SOCS2 mRNA (Zhong et al., 2021). The differing functions of METTL3 in macrophages may result from variations in the cellular microenvironment. In addition, macrophages deficient in METTL14 or YTHDF1 exhibit impaired SOCS1 induction, leading to increased pro-inflammatory cytokine and chemokine production, which exacerbates responses to bacterial infections (Du et al., 2020). FTO knockdown inhibited NF-κB signaling pathway and decrease the mRNA stability of signal transducer and activator of transcription 1 (STAT1) and peroxisome proliferator-activated receptor-γ(PPAR-γ) through YTHDF2, thereby obstructing macrophage activation (Gu et al., 2020). ALKBH5 promotes the activation of JNK and ERK pathways by upregulating MAP3K8, which regulates IL-8 expression and promotes macrophage recruitment (You et al., 2022). YTHDF2 facilitates M2 macrophage polarization by promoting p53 mRNA degradation and inhibits M1 macrophage polarization by suppressing NF-κB, p38, and JNK signaling pathways (Cai et al., 2022a).

NK cell

Natural killer (NK) cells, a core component of the innate immune system, are granular lymphocytes with inherent cytotoxicity, enabling them to target and kill foreign, transformed, or infected cells (Pfefferle et al., 2020). Recent findings indicate that m6A serves as a positive regulator of NK cell-mediated antitumor immunity. METTL3 expression correlates positively with NK cell effector molecule levels and functional. Research by Song et al. (2021) highlighted a decrease in METTL3 expression within tumor-infiltrating NK cells. The depletion of METTL3 in NK cells disrupts their homeostasis and hampers both their infiltration and function in the tumor microenvironment. The gene encoding src homology-2-containing protein tyrosine phosphatase 2 (SHP-2) undergoes m6A modification, with METTL3-mediated m6A methylation enhancing its expression. In NK cells, SHP-2 facilitates the activation of the AKT-mTOR and MAPK-ERK signaling pathways in response to IL-15 stimulation. METTL3 deficiency thus diminishes NK cell effector function (Song et al., 2021). FTO acts as a negative regulator of IL-2/15-driven JAK/STAT signaling by stabilizing the mRNA of SOCS family suppressor genes, thereby inhibiting NK cell activation and function (Kim et al., 2023). NK cells exhibit higher levels of YTHDF2 compared to other immune cells, and YTHDF2 expression is significantly upregulated in NK cells upon activation by tumors, viral infections, and cytokines, such as IL-15. YTHDF2 maintains NK cell homeostasis, maturation, and IL-15-mediated survival and proliferation, while also promoting NK cell effector functions through the establishment of a STAT5-YTHDF2 positive feedback loop. In contrast, YTHDF2 deficiency impairs NK cells’ antitumor and antiviral activities (Ma et al., 2021b).

T-lymphocyte

T lymphocytes originate from bone marrow progenitors, mature in the thymus, and then become activated, proliferated, and differentiated before being transported to periphery sites for immune function (Du et al., 2020). Double-positive (DP, CD4⁺CD8⁺) cells expressing the αβ T-cell antigen receptor in the thymus differentiated into either CD4⁺T helper cells or CD8⁺T cytotoxic cells (Collins & Littman, 2005). Under different cytokines stimulations, naïve CD4⁺T cells may differentiate into diverse subpopulations, including T helper cells (Th1, Th 2, Th 9, Th 17, Th 22, etc.) and T regulatory cells (Treg) (Zhu, Yamane & Paul, 2010). The m6A methylation modification plays a significant role in the differentiation and functionality of CD4⁺ T cells. Li et al. (2017) demonstrated that deletion of METTL3 in mouse T cells disrupts T cell homeostasis and differentiation. METTL3-deficient T cells remained in their initial state for 12 weeks and failed to undergo stable proliferation. Furthermore, METTL3-deficient naïve T cells exhibited decreased methylation of SOCS family gene transcripts, leading to an increased SOCS family mRNA and protein expression. Elevated SOCS activity suppresses interleukin-7 (IL-7)-mediated STAT5 activation, thereby inhibiting homeostatic T cell proliferation and differentiation. Moreover, METTL3-deficient naïve T cells differentiated into fewer Th1 and Th17 cells and more Th2 cells than wild-type (WT) naïve T cells, without affecting Treg induction (Li et al., 2017). Research also suggests that METTL14 deficiency affects invariant NKT (iNKT) development through cell-intrinsic mechanisms, leading to impaired iNKT cell maturation and differentiation. Mechanistically, the disruption of METTL14-dependent m6A modification alters expression of key molecules involved in the p53-mediated apoptotic pathway, potentially increasing apoptosis in DP thymocytes and iNKT cells. In addition, METTL14 knockdown in mature iNKT cells results in increased Cish expression and diminished TCR signaling, which in turn reduces their cytokine production (Cao et al., 2022). Ding et al. (2022) discovered that ALKBH5 depletion in lymphocytes decreased Jag1/Notch2 signaling by increasing m6A RNA modification in thymocytes, which specifically induces γδ T cell expansion (Zhao, Ding & Li, 2023).

B-lymphocyte

B-lymphocytes play a central role in adaptive humoral immunity by producing antigen-specific immunoglobulins (Ig) that target invasive microorganisms (Vaughan, Roghanian & Cragg, 2011). In humans, B cells also originate from bone marrow hematopoietic stem cells. Hematopoietic stem cells differentiate into multipotent progenitor cells (MPPs), which lack self-renewal capacity, and lymphoid-induced multipotent progenitor cells (LMPPs), which can give rise to common lymphoid progenitor cells (CLPs). The B cell-biased lymphoid progenitor cells (BLPs) within the CLPs will further develop into B cells. The B-cell development process follows an intrinsic program, passing through several intermediary stages, including progenitor cells (Pro-B cells) and precursor B cells (large Pre-B cells, Small Pre-B cells), before entering the peripheral blood. Subsequently, immature B cells migrate to the spleen for further maturation (Eibel et al., 2014; Rolink et al., 1999). The m6A modification is crucial in regulating early B cell proliferation and development (Zhao et al., 2022). The deletion of both METTL14 and YTHDF2 in B cells significantly blocked IL-7-induced Pro-B cell proliferation and development. Loss of METTL14 hinders Pro-B cell proliferation and cell growth in vitro and leads to a reduced viability of large Pre-B cells in vivo. In addition, METTL14 deficiency disrupts IL-7-driven transition from Pro-B cell to Small Pre-B cell, resulting in aberrant gene expression patterns crucial for B cell development and significantly impeding early B cell maturation.YTHDF2-mediated mRNA degradation is also critical for the transition from Pro-B to small Pre-B, with METTL14 deletion in B cells reducing YTHDF2 binding to its target mRNAs, thereby affecting early development (Zheng et al., 2020). METTL14 also plays a vital role in germinal center (GC) B cell responses. In a YTHDF2-dependent manner, METTL14-mediated m6A modification positively regulates GC B cell responses. Mechanistically, METTL14-mediated m6A promotes mRNA decay of genes encoding negative immunomodulatory factors, such as Lax1 and Tipe2, thereby indirectly upregulating genes essential for positive selection and proliferation of GC B cells. METTL14 deficiency results in elevated Myc proteins levels in GC B cells and vitro-stimulated B cells (Huang et al., 2022). By contrast, Grenov et al. (2021) found that no significant alterations in B cell development in the METTL3-deficient mouse model. However, METTL3-deficient GC B cells exhibited reduced cell cycle progression and lower expression of genes related to proliferation and oxidative phosphorylation, resulting in a marked reduction in B cell capacity to mount an effective immune response (Grenov et al., 2021).

m6A and dysregulation of inflammatory signaling pathways

Keyword analysis indicates that m6A primarily influences the inflammatory response by regulating the NF-κB pathway, a classic pro-inflammatory signaling pathway. Pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), activate NF-κB, which in turn drives the expression of additional pro-inflammatory genes, including cytokines, chemokines, and adhesion molecules (Lawrence, 2009). In the chondrogenic cell line ATDC5, IL-1β treatment increased both overall m6A methylation levels and METTL3 mRNA expression. Silencing METTL3 reduced IL-1β-induced apoptosis and suppressed IL-1β-mediated increases in inflammatory cytokines and NF-κB activation in chondrocytes (Liu et al., 2019a). METTL3 overexpression promotes TRAF6-NF-κB pathway activation in an m6A-dependent manner, enhancing LPS-induced microglial cell inflammation (Wen et al., 2022). METTL14 regulates IL-6 transcription through the NF-κB/p65 pathway. METTL14 knockout mice reduced atherosclerotic plaque formation and inflammatory responses (Zheng et al., 2022a). FTO was reduced in LPS-induced human normal chondrocyte (C28/I2) cells. Overexpression of FTO attenuated LPS-induced C28/I2 cell injury. miR-515-5p is involved in IL-1β-induced chondrocyte apoptosis, inflammation, and extracellular matrix degradation, highlighting its critical role in OA progression (Wu et al., 2021). Further studies revealed that FTO interacts with DGCR8 and regulates pri-miR-515-5p processing in an m6A-dependent manner, and miR-515-5p inactivates the MyD88/NF-κB pathway via TLR4 targeting. Consequently, FTO overexpression suppressed synovial inflammation and alleviated osteoarthritis by inhibiting the miR-515-5p/TLR4/MyD88/NF-κB axis in an m6A-dependent manner (Cai et al., 2023b). Single-cell sequencing (scRNA-seq) and validation studies showed significantly reduced FTO expression in retinal microglia from uveitis mice and in microglia clone 3 (HMC3) cells from inflammatory patients. Glypican 4 (GPC4), a glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycan (HSPG), serves as a target of FTO and modulates the TLR4/NF-κB pathway by anchoring CD14 (He et al., 2023a).