N6-methyladenosine RNA methylation in relation to the inflammatory response, immunity and the intestinal microbiota in inflammatory bowel disease

Abstract

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the intestine. N6-methyladenosine (m⁶A) RNA methylation, which is the most abundant messenger RNA modification in mammalian cells, plays an important role in IBD by influencing various processes, such as RNA stability, splicing, translation, and degradation, and participating in the pathogenesis of IBD. The key enzymes involved in m⁶A methylation (such as methyltransferase-like 3, methyltransferase-like 4 and fat mass and obesity-associated protein) not only regulate changes in inflammation and immunity in IBD but also bidirectionally regulate the intestinal microenvironment. However, the specific mechanism of m⁶A methylation modification in IBD needs further exploration. Future research should focus on clarifying the molecular network related to m⁶A methylation and IBD to provide new intervention methods for clinical practice and improve the outcomes of IBD patients. This article reviews the research progress on m⁶A RNA methylation in IBD, aiming to improve the understanding of IBD pathogenesis at the epigenetic level and provide new directions for exploring treatments targeting m⁶A-modified related proteins for IBD.

Key Words: N6-methyladenosine; RNA methylation; Immunity; Intestinal microbiota; Inflammatory bowel disease

Core Tip: Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the intestine. N6-methyladenosine (m⁶A) RNA methylation, which is the most abundant messenger RNA modification in mammalian cells, plays an important role in IBD by influencing various processes, such as RNA stability, splicing, translation, and degradation, and participating in the pathogenesis of IBD. The key enzymes involved in m⁶A methylation (such as methyltransferase-like 3, methyltransferase-like 4 and fat mass and obesity-associated protein) not only regulate changes in inflammation and immunity in IBD but also bidirectionally regulate the intestinal microenvironment. However, the specific mechanism of m⁶A methylation modification in IBD needs further exploration. Future research should focus on clarifying the molecular network related to m⁶A methylation and IBD to provide new intervention methods for clinical practice and improve the outcomes of IBD patients. This article reviews the research progress on m⁶A RNA methylation in IBD, aiming to improve the understanding of IBD pathogenesis at the epigenetic level and provide new directions for exploring treatments targeting m⁶A-modified related proteins for IBD.

INTRODUCTION

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the intestine that frequently recurs. IBD has several characteristic symptoms, such as abdominal pain, diarrhea, and mucus-purulent stools. IBD includes Crohn’s disease and ulcerative colitis and involves factors related to genetics, the environment, intestinal microorganisms, and the immune system[1]. IBD is diagnosed mainly in Western countries. In recent years, with changes in dietary structure and the fast pace of life, the incidence of IBD has gradually increased in Asian, South American, and African countries[2]. With the continuous in-depth exploration of the pathogenesis of IBD, N6-methyladenosine (m⁶A) RNA methylation has been shown to play important roles in the occurrence and development of IBD[3].

At present, more than 100 types of RNAs, including messenger RNAs (mRNAs), transfer RNAs, ribosomal RNAs, and non-coding RNAs, have been discovered. m⁶A is the most common and abundant internal modification[4] and is involved in almost all stages of the RNA cycle, including mRNA transcription, maturation, translation, degradation, and stability regulation[5]. Although m⁶A was discovered as early as 50 years ago[6], its biological function is still largely unknown. m⁶A modification plays important roles in the inflammatory response, immune response, and intestinal microorganisms in the pathogenesis of IBD[7]. This article reviews the mechanism of m⁶A RNA methylation in IBD, aiming to elucidate the potential value of targeting m⁶A for individualized treatment strategies for IBD patients and to provide new directions and individualized choices for the treatment of IBD patients.

OVERVIEW OF M⁶A RNA METHYLATION

RNA modification refers to a chemical change that occurs in the chemical components of an RNA molecule after transcription that may alter the RNA function[8]. Commonly modified RNA bases are located primarily on noncoding RNAs, such as transfer RNAs and ribosomal RNAs[9]. Studies have shown that the most common reversible internal base modification in mRNA is m⁶A[10]. m⁶A was discovered in 1970. m⁶A is “written” by methyltransferases, “erased” by demethylases and recognized by reading proteins to modulate various processes, such as mRNA translation, splicing, nuclear export, and degradation, thereby altering gene expression and regulation[11].

m⁶A methylation is a dynamic and reversible type of posttranscriptional RNA modification mediated by m⁶A regulators or m⁶A RNA methylation regulators[12]. These enzymes are classified as m⁶A methyltransferases (writers), m⁶A demethylases (erasers), and m⁶A reading proteins (readers), which establish complex interactions for m⁶A incorporation, degradation, and recognition[13]. m⁶A methylation affects the abundance, localization, and potential splicing of mRNAs, all of which are related to protein expression[14]. The protein level is the main factor determining the biological phenotype[15]. As research has progressed, m⁶A modifications, which are diverse and reversible, are gradually being regarded as widely applicable regulatory mechanisms that can control gene expression in various physiological and pathological processes, thereby influencing the development of diseases, including IBD.

m⁶A methyltransferases

The main m⁶A methyltransferases are methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms tumor-associated protein (WTAP), which form a complex to methylate mRNAs[16].

METTL3 is an important catalytic enzyme that transfers methyl groups from S-adenosyl-L-methionine to the N6 atom of adenine, promoting the methylating reaction[17]. The catalytic domain of METTL3 is the only domain with a cavity that can accommodate the necessary methyl donor S-adenosyl-L-methionine. The catalytic center of METTL3 is likely used for writing m⁶A modifications[18]. METTL14 provides structural support for METTL3 near the active site; although METTL14 lacks a catalytic subunit, it binds tightly to METTL3 to stabilize its conformation so that METTL3 can perform its catalytic function and plays a key role in substrate RNA recognition[19]. METTL14 and METTL3 can form a stable complex, and this synergistic effect allows the generation of m⁶A markers on homologous RNA oligonucleotides[20].

WTAP is the third component of the m⁶A methyltransferase complex. Although the methyltransferase activity of WTAP has not been demonstrated in vitro, WTAP interacts and forms heterodimers with various proteins, such as METTL3 and METTL14, thereby regulating the m⁶A methylation level[21]. In the absence of WTAP, the ability of the m⁶A methyltransferase complex to bind mRNA targets is significantly reduced[22]. The m⁶A methyltransferase complex also includes RBM15/15B, ZC3H13, and VIRMA subunits. ZC3H13 plays a key role in anchoring the methyltransferase complex in the cell nucleus to carry out m⁶A modification, and RBM15/15B and VIRMA have been suggested to enable more efficient completion of this modification[23]. Posttranscriptional m⁶A modifications usually occur in the cell nucleus, but the methylation of RNA viruses in the cytoplasm has also been proven to be an m⁶A modification, indicating that other methyltransferase complexes may also exist outside the nucleus[24].

m⁶A demethylases

In 2011, a new demethylase, fat mass and obesity-associated protein (FTO), was discovered, indicating that methylation is a reversible and regulatable modification process[25]. The FTO protein is localized in the cell nucleus, and its main function is to reduce the m⁶A level through its oxidative demethylase activity[26]. FTO is an RNA demethylase that can combine with various RNAs and remove methyl groups from mRNAs, including those at the mRNA chain termini, while also reducing the stability of demethylated mRNA[27].

In 2013, a second m⁶A demethylase, namely, Alk B homolog 5 (ALKBH5), was discovered, and m⁶A-modified mRNAs were confirmed to be substrates for ALKBH5 in cells. ALKBH5 catalysis does not require the oxidative demethylase activity, and it directly removes the methyl group from m⁶A-adenine[28]. The ALKBH5 and FTO proteins are distributed mainly in the cell nucleus, but they may translocate to the cytoplasm under certain conditions[29].

Removal of the RNA m⁶A modification is performed mainly by two demethylases, namely, FTO and ALKBH5. The incorporation and removal of m⁶A from mRNA is a dynamic and reversible process[30]. FTO exhibits m⁶A demethylase activity and demethylates m⁶A-modified residues in mRNA. ALKBH5 expression is significantly tissue-specific, with its highest expression in the testis and relatively low expression in the heart and brain. ALKBH5 functions include the regulation of nuclear RNA output, metabolic processes, and gene expression[31]. ALKBH5 affects mouse spermatogenesis, suggesting that m⁶A is a dynamic and reversible modification of mRNA in mice[32]. A metabolic labeling study in HeLa cells revealed that the m⁶A level is largely stable during the mRNA life cycle[33]. m⁶A demethylation may occur only under certain specific conditions, but its specific physiological effects still need to be further studied.

m⁶A reading proteins

RNA-binding proteins that recognize and specifically bind to m⁶A are called m⁶A reading proteins. Most of these proteins contain YTH domains. These proteins bind to RNA in an m⁶A-dependent manner to mediate the regulatory function of m⁶A on the modified RNA[34]. m⁶A reading proteins regulate mRNA nuclear export, splicing, degradation, translation, and stability. The most well-known m⁶A readers are members of the YTHDF family and the IGF2BP family[35]. The YTHDF family includes mainly YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2. YTHDC1 is widely expressed in the cell nucleus and can directly affect mRNA splicing. In HeLa cells, the RNA-binding protein YTHDC1 can mediate the export of methylated mRNA from the cell nucleus to the cytoplasm[36]. YTHDC2 is located mainly in germ cells and can be expressed in both the nucleus and the cytoplasm where it regulates the stability of methylated mRNA and promotes its effective translation[37]. The amino acid sequences encoded by the YTHDF1, YTHDF2, and YTHDF3 genes are highly similar, and these proteins belong to the YTHDF protein family, which exists mainly in the cytoplasm. YTHDF1 recruits the eIF3 complex of eukaryotic translation initiation factors to increase the translation efficiency of targeted mRNAs[38]. YTHDF2 destabilizes m⁶A RNA and accelerates its degradation[39]. YTHDF3 not only accelerates the translation of target mRNAs and promotes protein synthesis through cooperation with YTHDF1 but also affects the decay of methylated mRNA through YTHDF2[40]. In addition to m⁶A readers with YTH domains, other RNA-binding proteins, including FMR1, IGF2BP, and heterogeneous nuclear ribonucleoproteins superfamily proteins, can also specifically bind to m⁶A-containing RNA[41]. The functions and mechanisms of these RNA-binding proteins still need further study[42]. The “reader” IGF2BP family is responsible for recruiting RNA stabilizers to promote mRNA stability, thereby affecting disease progression[43].

M⁶A AND INTESTINAL EPITHELIAL HOMEOSTASIS

The surface of mature intestinal epithelium serves as a physical barrier between the body and the external environment. Intestinal epithelial cells need to be constantly renewed to maintain normal intestinal homeostasis. Similarly, the intestinal epithelium has a strong ability to regenerate, with complete renewal occurring every 5-7 days[44]. This regeneration is maintained by a population of rapidly dividing intestinal stem cells. In the colon tissue of mice, METTL14 plays an important role in reducing the apoptosis of colon epithelial cells. METTL14 regulates the stability of NFKBIA mRNA and the nuclear factor kappa-B (NF-κB) pathway to reduce the apoptosis of intestinal epithelial cells[45]. This regulation leads to damage to colon stem cells and the occurrence of colitis, thereby initiating intestinal mucosal barrier dysfunction. Although these findings have been confirmed in mice, it is still uncertain whether this process is the same in humans[46].

In individuals with IBD, the expression levels of IGF2BP2, HNRNPA2B1 (a guide factor), and ZCCHC4 (a writer factor) decrease, although the specific remains unclear. Notably, IGF2BP2 (also known as IMP2), an m⁶A reading factor directly involved in glucose, lipid, protein, and energy metabolism, plays a key role in the pathogenesis of IBD and may influence its progression[47]. HNRNPA2B1 (a guide factor) promotes effective interferon (IFN) production through cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-STING32, and cGAS-STING plays a key role in the viral immune signaling pathway related to intestinal microbiota dysbiosis[48]. ZCCHC4 plays an important role in regulating core cytokines related to IBD[49].

m⁶A RNA modification is involved in the regulation of intestinal epithelial homeostasis in the context of IBD. The m⁶A recognition protein YTHDF1 is highly expressed in intestinal stem cells; the absence of YTHDF1 does not affect the normal development of the mouse intestine but reduces stem cell stemness and blocks Wnt signaling-driven intestinal epithelial regeneration and the release of inflammatory factors[50]. YTHDF1 promotes the translation of Wnt signaling effectors, such as TCF7 L2/TCF4, increasing the protein activity of β-catenin to maintain the homeostasis of intestinal stem cells[51]. After specific knockout of METTL14 in intestinal epithelial cells, mice exhibit growth retardation and emaciation at 2 weeks of age, with a shortened colon, loss of goblet cells, deformed crypt structures, inflammatory cell infiltration, and epithelial ulcers, whereas the length of the small intestine, the length of the villi, and the integrity of the crypts do not significantly change[52]. METTL14 deficiency also leads to the apoptosis of colon stem cells, as METTL14 maintains the homeostasis of intestinal epithelial cells by regulating the NF-κB-mediated antiapoptotic pathway[53]. These research results indicate that m⁶A RNA modification can affect intestinal epithelial homeostasis by regulating intestinal stem cells (Figure 1).

Figure 1 N6-methyladenosine and the intestinal immune balance in inflammatory bowel disease. m⁶A: N6-methyladenosine; IL: Interleukin-6; TNF: Tumor necrosis factor; IBD: Inflammatory bowel disease; Th17: T helper cell 17; Treg: Regulatory T.

M⁶A AND THE INFLAMMATORY RESPONSE IN IBD

Inflammatory changes occur throughout the pathogenesis of IBD. METTL3 knockout inhibits the inflammatory response mediated by lipopolysaccharides (LPS) and reduces the abnormal absorption of long-chain fatty acids in vitro[54]. This is because METTL3 knockout decreases the m⁶A level of Traf6 mRNA, thereby retaining its transcript in the nucleus. A reduction in Traf6 expression inhibits the activation of the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways[54]. METTL3-mediated m⁶A modification stabilizes tumor necrosis factor (TNF)-α mRNA and promotes its translation, exacerbating inflammation[55]. Conversely, under specific conditions, m⁶A modification may indirectly inhibit the production of TNF-α by inducing the expression of anti-inflammatory factors. Moreover, METTL3 increases the stability of interleukin-6 (IL-6) and IL-6 receptor mRNAs, increases STAT3 phosphorylation, drives T helper (Th) 17 cell differentiation and maintains persistent inflammation[56]. The absence of METTL3 reduces the translation of key regulatory factors of NF-κB (such as inhibitor of kappa B kinase-β), thereby inhibiting the inflammatory response in macrophages and IBD intestinal epithelial cells[57].

METTL3 promotes LPS-induced inflammation in microglia through the TRAF6/NF-κB pathway. In human dental pulp cells stimulated by LPS, the levels of m⁶A and METTL3 increase[58]. METTL3 knockdown reduces the accumulation of inflammatory cytokines and inhibits the activation of the NF-κB and MAPK signaling pathways[59]. Therefore, the key m⁶A methyltransferase METTL3 promotes the development of inflammation in the pathogenesis of IBD through the NF-κB/MAPK signaling pathway[60]. Additionally, the knockout of YTHDF2 activates the MAPK and NF-κB signaling pathways in macrophages under inflammatory conditions to induce the expression of proinflammatory cytokines, thereby exacerbating the stress-induced inflammatory response[61].

Changes in the expression of another key m⁶A enzyme, namely, METTL14, also affect the development of inflammation[62]. A lack of METTL14 in T cells leads to regulatory T (Treg) cell dysfunction, resulting in increased levels of Th1 and Th17 cytokines and the induction of spontaneous colitis in mice[63]. In an endothelial cell inflammation model induced by TNF-α, METTL14 directly binds to FOXO1 mRNA and increases its m⁶A level[64]. After binding to YTHDF1, the rate of translation is accelerated by METTL14, increasing the expression of FOXO1 and thereby exacerbating endothelial cell inflammation[65]. In IBD, however, FOXO1 inhibits the differentiation of pathogenic Th17 cells and downregulates the expression of IL-17A, thereby reducing the severity of colitis in mouse models. The expression level of FOXO1 is significantly reduced in peripheral blood mononuclear cells and the mucosa of IBD patients[66]. Therefore, METTL14 regulates the development of inflammation in the context of IBD by modulating the m⁶A level of FOXO1 mRNA; however, further research is needed.

M⁶A AND IMMUNITY IN IBD

The immune response in IBD is divided into innate immunity and adaptive immunity. Numerous cytokines are involved in immune activity, such as IFN-I, which has a protective effect during the acute phase of IBD at onset and is a proinflammatory factor during the chronic recovery period[67]. In the innate immune response against pathogens, ALKBH5 can demethylate its substrates, and antiviral mRNA transcripts with m⁶A modifications remain in the nucleus, preventing their translation and inhibiting the production of IFN-1[68]. In the innate immune response induced by DNA virus infection, specific types of mRNAs involved in the antiviral response, such as cGAS, p204 and Sting, undergo m⁶A modification in a hnRNPA2B1-dependent manner[69]. hnRNPA2B1 interacts with FTO and modulates the m⁶A level of specific types of mRNAs, regulating their nuclear cytoplasmic transport and translation and thus playing important roles in the host innate immune response against viruses[70]. Moreover, knockout of FTO leads to increased IFN-1 expression in macrophages, and METTL3 participates in the m⁶A modification of hnRNPA2B1-bound mRNA in macrophages, thereby inducing the expression of IFN-β and a better response to DNA virus infection[46]. TNF-α is a major participant in intestinal inflammation in the pathogenesis of IBD. TNF-α inactivates FOXO3 in the nucleus and cytoplasm of colonic crypt epithelial cells through the phosphatidylinositol 3-kinase and inhibitor of kappa B kinase pathways, leading to Treg cell dysplasia and the induction of severe intestinal inflammation[5]. Mice lacking METTL3-expressing Treg cells develop severe autoimmune diseases because the absence of METTL3 prevents effective m⁶A-mediated IL-2 receptor signaling to activate STAT5, thereby decreasing the ability of Treg cells to inhibit inflammation[71-74]. Additionally, METTL14 deficiency impairs the function of induced Treg cells induced from naive T cells, and dysfunctional Treg cells accelerate the development of colitis[10]. As a member of the m⁶A reading protein family, YTHDF3 cooperates with PABP1 and eIF4G2 under physiological conditions to bind to the translation initiation region of FOXO3 mRNA, which induces FOXO3 translation, thereby maintaining host antiviral immune function and preventing inflammatory responses[75].

The key to adaptive immunity, namely, the stability of T cells, is important in IBD. m⁶A RNA modifications play important roles in the stability of T cells. Studies have shown that the mRNAs of genes, such as SOCS1, undergo m⁶A modification, and METTL3 deficiency increases their mRNA stability and protein expression levels, thereby inhibiting IL-7-mediated STAT5 activation and T-cell proliferation and differentiation[76]. Unlike FTO, the m⁶A demethylase ALKBH5 maintains the ability of initial cluster of differentiation (CD) 4-positive T cells to induce adoptive transfer-type colitis[77,78]. ALKBH5 deficiency in lymphocytes specifically induces the expansion of γδ T cells through the expression of the Notch signaling pathway-related genes Jagged1 and Notch2, improving protection against Salmonella Typhimurium infection in the gastrointestinal tract of mice[13]. Moreover, the WTAP component of the m⁶A methyltransferase complex regulates the m⁶A modification of the mRNAs Orai1 and Ripk1, which alters their mRNA stability, thus affecting the activation of calcium signaling and the survival of T cells[79]. Specific knockout of METTL14 in mouse T cells leads to damage to Treg cells, resulting in spontaneous colitis; moreover, antibiotic treatment significantly alleviates colitis progression[12].

In a lymphoid-sensitive mouse adoptive transfer model, METTL3 knockout in naive T cells prevents the development of colitis because m⁶A regulates T-cell homeostasis by marking and inducing the degradation of SOCS gene family mRNAs in vivo[80,81]. METTL3 deficiency leads to a slower rate of SOCS mRNA degradation and an increase in SOCS protein levels, thereby inhibiting the activation of STAT5 mediated by IL-7 and the proliferation and differentiation of T cells[82]. Specific deletion of METTL3 also impairs the phenotypic and functional maturation of dendritic cells, resulting in decreased expression of the costimulatory molecules CD40 and CD80 and the cytokine IL-12 and a reduced ability to stimulate T-cell responses both in vitro and in vivo; the expression of these costimulatory molecules is reduced through short hairpin RNA-mediated knockdown of YTHDF1[83]. ALKBH5 and FTO reduce the efficiency of the immune response in the context of IBD, and the deletion of METTL3 and METTL14 induces immune disorders and accelerates the development of colitis; in addition, YTHDF3 stimulates the immune system to inhibit inflammation[67].

Dendritic cells are antigen-presenting cells that play a key role in immune balance. m⁶A modification is involved in the immune response of dendritic cells. METTL3 mediates m⁶A modification of CD40, CD80, and Toll-like receptor 4 signaling adapter transcripts, inducing their translation in dendritic cells and promoting their activation[84,85]. Therefore, RNA m⁶A modification regulates multiple aspects of intestinal immune homeostasis and may become a new target for treating intestinal immune-related diseases.

M⁶A AND THE GUT MICROBIOTA IN IBD

The trillions of microorganisms that inhabit the gut are important factors influencing intestinal diseases. The symbiotic microbial communities in the gut affect host metabolism, organ development, and immune system regulation, among other processes[86]. The interaction between host cells and microorganisms in the gut is strictly regulated to distinguish symbiotic microorganisms from pathogens and maintain gut microbial homeostasis[87,88]. Microbial metabolites (such as short-chain fatty acids) may affect the m⁶A modification pattern of host cells; conversely, changes in m⁶A modification may affect the composition of the microbiota by regulating antimicrobial peptide or mucin expression, resulting in the formation of a feedback loop that influences inflammation[67].

m⁶A modification is involved in the cross-talk between host cells and gut microorganisms[89]. RNA activated by Toll-like receptors activates the innate immune system in mammals[90]. However, when RNA is modified by RNA 5-methylcytosine, m⁶A, 5-methyluridine, 2-thiouridine or pseudouridine, fewer cytokines and activation markers are expressed in dendritic cells, leading to weakened innate immune activation[91]. Mammalian RNA is rich in modified nucleotides, whereas bacterial and mitochondrial RNA have fewer modified nucleotides, and only the latter can effectively activate the innate immune system. These findings indicate that the innate immune system can distinguish bacteria and necrotic tissues by monitoring RNA modifications[92]. Additionally, multiple research teams have reported that changes in m⁶A modification-related enzyme levels affect the types of gut microbiota. For example, METTL3 gene knockout mice exhibit gut microbiota dysregulation and chronic intestinal inflammation at 3 months of age[93]. Compared with those in normal mice, the abundances of the Bacteroidetes S24-7 family and Lactobacillaceae in the gut microbiota are decreased at 24 weeks of age in METTL14-knockout mice, whereas the abundances of Firmicutes, Helicobacteraceae, Desulfobulbaceae and Enterobacteriaceae are increased in the gut microbiota in METTL14-knockout mice[94]. Deletion of the FTO gene resulted in decreases in the abundance of Streptococcus and Helicobacter but an increase in the abundance of Lactobacillus. These changes can inhibit or promote inflammation[95].

m⁶A, as the most abundant mRNA modification in mammalian cells, is also involved in the metabolism of intestinal microorganisms. The intestinal microbiota regulates host cell responses to external stimuli by altering the host epigenome, thereby influencing the corresponding gene transcription mechanisms[96]. Compared with those in normal mice, m⁶A levels in multiple organs, such as the intestine, liver, and brain, are significantly increased in germ-free mice, which lack microorganisms. These findings indicate that the microbiota strongly affects the modification of host m⁶A mRNA[97]. Acinetobacter, Escherichia coli, Shigella, Lactobacillus plantarum, other Lactobacillus species, and Bacteroides are intestinal microorganisms that have certain functions in the mouse cecum, and their presence or absence causes differences in m⁶A methylation levels between the ceca of normal mice and germ-free mice, respectively, by regulating m⁶A modifications in inflammatory pathways[98]. The use of antibiotics to inhibit intestinal microorganisms also alters m⁶A RNA modification levels in host cells[99]. Additionally, intestinal microbial metabolites, such as LPS, directly regulate m⁶A methylation and can significantly increase the m⁶A level in intestinal epithelial cells[100]. The absence of maternal intestinal microbiota in mice leads to alterations in m⁶A RNA modification levels in the intestinal tissues of offspring and regulates the stability and expression of genes related to intestinal development; in addition, the sensitivity of the fetus to the maternal intestinal microbiota is reduced in METTL3 heterozygous mutant mice[101]. These findings suggest that the metabolic products of the maternal intestinal microbiota directly or indirectly affect m⁶A RNA modification levels in the fetus. METTL3-knockout mice develop chronic intestinal inflammation at 3 months after birth, which is accompanied by intestinal microbiota dysbiosis[102]. Therefore, m⁶A modification may selectively modulate the function of the microbial community by regulating the inflammatory response in the intestine. Anxiety and depressive behaviors are reduced in FTO-deficient mice because of changes in the intestinal microbial community, such as an increase in the abundance of lactic acid bacteria and decreases in the abundances of Porphyromonas and Helicobacter[103]. These studies suggest that the feedback between m⁶A modification levels and intestinal bacteria is not one way but rather two-way regulation is involved. Currently, many challenges associated with studying tandem m⁶A RNA methylation, intestinal nutrition, and metabolic physiology exist[104]. The interaction between the intestinal microbiota of IBD patients and m⁶A methylation still needs further in-depth study (Figure 2).

Figure 2 N6-methyladenosine and the gut microbiota in inflammatory bowel disease. m⁶A: N6-methyladenosine.

PROSPECTS AND CHALLENGES FOR CLINICAL TRANSLATION

Biomarkers: Progress from tissue to body fluids

Studies have shown that the expression levels of the methyltransferases METTL3 and METTL14, as well as the demethylase FTO, significantly differ in the intestinal mucosa of IBD patients[105]. A cohort study of 156 IBD patients and 80 healthy controls revealed that METTL3 expression in the mucosa of active Crohn’s disease patients was upregulated 2.3-fold (P < 0.001) and was positively correlated with the simplified Crohn’s disease endoscopy score (r = 0.61)[105]. Notably, there are differences in the expression profiles of m⁶A regulatory factors between ulcerative colitis and Crohn’s disease patients: Patients with ulcerative colitis exhibit decreased expression of FTO, while patients with Crohn’s disease exhibit increased expression of METTL3, suggesting that the m⁶A regulatory factor profile can serve as an auxiliary indicator of disease subtype.

The detection of m⁶A regulatory factors in peripheral blood mononuclear cells and plasma overcomes the limitations of invasive tissue biopsy. Two independent studies (n = 112 and n = 89) confirmed that the FTO mRNA level in the peripheral blood mononuclear cells of IBD patients was approximately 40% lower than that in healthy controls and was negatively correlated with the C-reactive protein level[9]. Additionally, the plasma free m⁶A modification level has shown potential as an emerging biomarker: A preliminary study (n = 45) revealed that the total m⁶A modification level in the plasma of ulcerative colitis patients was 1.8 times greater than that in the control group, and detecting both plasma m⁶A levels and fecal calprotectin levels increased the area under the receiver operating characteristic curve for IBD diagnosis to 0.91[106].

Drug development targeting m⁶A: From proof of concept to preclinical studies

Currently, no drugs targeting m⁶A regulatory factors have entered clinical trials for IBD, but multiple preclinical studies have demonstrated the therapeutic potential of this strategy[107].

FTO inhibitors: FTO, an m⁶A demethylase, is expressed at relatively low levels in IBD patients. Theoretically, FTO agonists have greater therapeutic value. However, the main focus in the field of drug chemistry is on FTO inhibitors (for tumor treatment), and the development of FTO-specific agonists has not yet been explored[108]. A previous animal experiment using a small-molecule inhibitor of FTO, FB23-2, revealed that FB23-2 (5 mg/kg, intraperitoneal injection) could alleviate colitis induced by dextran sulfate sodium (DSS) in mice, as indicated by a 52% reduction in the disease activity index, colon length recovery, and decreased levels of proinflammatory cytokines (TNF-α, IL-6, and IL-1β). Mechanistically, FTO inhibition leads to increased m⁶A levels and promotes the degradation of downstream anti-inflammatory target mRNAs. Notably, in this study, systemic administration was used, and the intestinal selectivity of FB23-2 was not optimized, indicating the potential for systemic toxicity risks.

METTL3 regulation: The role of METTL3 in IBD is cell type dependent: In epithelial cells, METTL3 deficiency exacerbates colitis; In macrophages, METTL3 promotes their polarization toward the M1 phenotype. This bidirectional characteristic poses challenges for direct targeting of METTL3. Previous studies have employed cell-specific intervention strategies: An epithelial-specific adeno-associated virus (AAV) vector (AAV2-E-cadherin-METTL3) administered rectally alleviated DSS-induced colitis in mice, and macrophage-specific METTL3 knockdown also showed protective effects[109]. These results indicate that targeting specific cell types is a key prerequisite for the development of METTL3-targeted drugs.

Other targets: Small-molecule inhibitors targeting the m⁶A reading proteins YTHDF1 and YTHDF2 are still in the early screening stage, and their application has not been reported in IBD models[110]. The function of METTL4, a recently discovered methyltransferase, in IBD is still limited to correlation analyses, and no drug intervention studies have been conducted[82].

Transformation bottlenecks: The three core challenges from the laboratory to clinical application

Although basic research on m⁶A modification in IBD is becoming increasingly in depth, the transformation from laboratory discoveries to clinical application still faces three core bottlenecks[73].

Cell/tissue type specificity: m⁶A regulatory factors may perform opposite functions in different cell types. As mentioned above, METTL3 plays a protective role in intestinal epithelial cells but promotes inflammation in macrophages. This cell type-dependent function implies that systemic administration of a drug may counteract its therapeutic effect or even cause harm because the wrong cell type may be targeted. Recent preclinical studies have adopted mostly systemic administration, and the safety window has not been clarified. Future transformation strategies must be based on cell type-specific delivery systems, such as epithelial-targeted AAVs, macrophage-targeted nanoparticles or local intestinal administration formulations.

Technical barriers to intestinal-specific delivery systems: Oral administration is an ideal approach for IBD treatment, but m⁶A-targeted drugs (mostly small-molecule inhibitors/activators or nucleic acid drugs) face multiple obstacles, such as gastrointestinal degradation, the mucus layer barrier, and differences in epithelial permeability. Recent research on delivery systems has the following deficiencies: (1) A lack of active targeting to the inflammatory mucosa of IBD models; (2) Failure to consider the significant differences in intestinal permeability among IBD patients; and (3) Most studies stop at animal model validation and lack large animal models and pharmacokinetic data. Potential of hydrogen-responsive hydrogels, inflammation-targeted nanoparticles (such as those targeting the mannose receptor on the macrophage surface) and probiotic carrier systems are promising delivery methods currently under investigation, but all of these are at the laboratory stage.

Complex interference from the intestinal microenvironment: The intestinal microenvironment in individuals with IBD exhibits a complex network of bacterial community-metabolite-immune cell interactions. This environmental factor may significantly affect the therapeutic effect of m⁶A-targeted drugs. Previous studies have shown that: (1) Bacterial metabolites in the intestine (such as butyrate and secondary bile acids) can regulate the expression of m⁶A-related enzymes; and (2) m⁶A modification itself can affect the production of antimicrobial peptides, thereby reshaping the bacterial community structure. This bidirectional interaction implies that the therapeutic effect of m⁶A-targeted drugs may depend on the specific background bacterial communities in different individuals and that drug intervention may result in unforeseen off-target effects through the m⁶A bacterial community axis. Currently, no studies have systematically evaluated the regulatory effect of the bacterial community background on the therapeutic effect of m⁶A-targeted drugs, which is a research gap that must be filled before clinical transformation. How m⁶A modification mediates the regulation of immune cells by intestinal microbiota metabolites (such as short-chain fatty acids) and identifying the transformation bottlenecks of intestinal-specific delivery systems targeting m⁶A regulatory factors.

CONCLUSION

Although the potential of m⁶A as a biomarker is vast, related research still faces certain challenges. How to accurately and reliably measure the level of m⁶A modification and combine these data with clinical indicators are key to future research. Given the differences in the level and function of m⁶A in different types of cells, exploring its specificity and sensitivity in specific diseases will help reveal the practical value of m⁶A as a biomarker. Future research will clarify the specific mechanisms of m⁶A modification in the pathogeneses of ulcerative colitis and Crohn’s disease, providing a new basis for individualized treatment. By precisely analyzing the RNA regulatory effects of m⁶A modification, its specific roles in the inflammatory response and immune regulation in the context of IBD can be studied. Determining the different cell type-specific functions of METTL3 and FTO in different subtypes of IBD and how m⁶A modification mediates the regulation of immune cells by intestinal microbiota metabolites (such as short-chain fatty acids) and identifying the transformation bottlenecks of intestinal-specific delivery systems targeting m⁶A regulatory factors can thus lay a foundation for the development of new intervention strategies. In the future, large-scale clinical trials and multicenter studies should be conducted to verify the application prospects of m⁶A in IBDs and other related diseases, thereby promoting its translation as a practical clinical detection method.

ACKNOWLEDGEMENTS

Thank you very much to all the team members for your contributions to this article.