N⁶-methyladenosine, m⁶A, RNA translation, RNA stability, sepsis, inflammation, and oxidative stress.

N⁶-methyladenosine, m⁶A, RNA translation, RNA stability, sepsis, inflammation, and oxidative stress.

m⁶A methylation is a ubiquitous mRNA modification in eukaryotes, which can regulate mRNA processing and stability. According to different functions, m⁶A modification proteins can be divided into methyltransferase (writer), demethylase (eraser), and m⁶A-binding protein (reader). For example, methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and WT1 proteins can induce RNA methylation, while alkB homolog 5 (ALKBH5) functions as an RNA demethylase that can erase RNA methylation and reverse methylation (13). Moreover, YTH domain family (YTHDF) 1-3 and YTH domain-containing family (YTHDC) 1-2 are m⁶A-binding proteins that recognize m⁶A methylation modification of RNA and thus affect the fate of mRNA (14).

m⁶A methylation plays a role in transcription by affecting RNA splicing, export, translation, and stability. Firstly, mRNA is spliced into mature transcripts and exported from the nucleus to the cytoplasm before translation. The process of splicing and export is regulated by m⁶A. Zheng et al. (15) have reported that ALKBH5 can affect mRNA export and metabolism at the same time. After ALKBH5 demethylation, mRNA is mainly concentrated in the nucleus, while in ALKBH5-deleted cells, mRNA is mainly located in the cytoplasm, indicating that ALKBH5 demethylation affects mRNA export. However, knockdown of ALKBH5 can increase the synthesis rate of nascent RNA. Another study has also demonstrated that YTHDC1 is involved in mRNA splicing and export by interacting with exonuclear transporters (16). Secondly, mRNA translation is regulated by m⁶A methylation. YTHDF1 has been reported to promote mRNA translation by binding to m⁶A, while YTHDF1 knockdown leads to a decrease in translation efficiency. YTHDF1 interacts with m⁶A-modified p65 mRNA and facilitates its translation, thereby regulating the inflammatory response (17). Another study by Zhang et al. (18) has shown that YTHDF1 regulates cell stability by promoting the translation of m⁶A-modified Robo3.1 mRNA. In addition, Weng et al. (19) have demonstrated that METTL14 and YTHDF1 participate in neurogenesis by promoting protein translation, implying the pivotal role of m⁶A in mRNA translation. All these findings make m⁶A a new target for inhibiting inflammation. Finally, m⁶A methylation is negatively correlated with RNA stability. Wang et al. (20) have found that the expression of target mRNA is decreased after METTL3 and METTL14 knockdown. YTHDF2 selectively binds m⁶A-methylated mRNA to regulate mRNA degradation (21). In addition, the stability of target mRNA is positively correlated with the number of binding sites after YTHDF2 knockout. Briefly, m⁶A methylation regulates gene expression by affecting RNA metabolism, including splicing and export, degradation, translation, and stability. m⁶A-mediated gene regulation plays a vital role in the pathogenesis of many diseases. Next, the regulatory effects of m⁶A on sepsis progression and septic inflammatory response were discussed.

In the pathophysiology of sepsis, abnormal inflammation and immune response can aggravate sepsis, leading to shock or myocardial dysfunction (22). Sepsis-induced myocardial injury is a notable factor resulting in poor clinical outcomes in sepsis patients. The heart is one of the most vulnerable target organs in sepsis. Sepsis-induced myocardial injury is mainly manifested by cardiomyocyte degeneration and necrosis, as well as myocardial systolic and diastolic dysfunction (23). The pathophysiological mechanism of sepsis-induced myocardial injury is complex, involving both pathogens and the host immune system. It has also been demonstrated that m⁶A methylation plays a significant regulatory role in sepsis-induced myocardial injury (24). Shen et al. (25) have shown that after endotoxin (0, 0.5, 1, 2, 5, and 10 μg/mL) is applied to the lipopolysaccharide (LPS)-induced cardiomyocyte model for 24 h, the expression of m⁶A methyltransferase METTL3 is elevated with the increase of the dose. Endotoxin can up-regulate the levels of pro-inflammatory cytokines such as interleukin (IL)-6, IL-8, and tumor necrosis factor-α (TNF-α), while silencing METTL3 reduces the levels of pro-inflammatory cytokines. By detecting several m⁶A methylation readers, it is found that the METTL3/insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1)/histone deacetylase 4 (HDAC4) axis regulates the inflammatory injury of endotoxin-induced cardiomyocytes. After LPS injection, m⁶A methylation is increased in mouse myocardial tissues, while the expression of fat mass and obesity-associated protein (FTO, an m⁶A demethylase) is decreased correspondingly. These changes are related to the significant increase in the expressions of myocardial inflammatory cytokines, such as IL-6, TNF-α, and IL-1β, as well as the decrease in left ventricular function. In addition, rat myoblasts (H9c2) also present similar changes (enhanced m⁶A RNA methylation, increased expressions of inflammatory cytokines, and decreased FTO gene expression) under LPS treatment (26). In addition, methylated RNA immunoprecipitation assays show that the expressions of IL-6 and TNF-α in endotoxin-treated H9c2 cells are increased compared with those in untreated cells. Rats are allocated to the control group and LPS-induced sepsis group. The m⁶A level of rat left ventricular tissues is determined by liquid chromatography-tandem mass spectrometry (LC-MS), and the transcription level of m⁶A is analyzed by transcriptional exon microarray (mRNAs and lncRNAs). LC-MS-based mRNA modification analysis indicates that after intraperitoneal injection of LPS, the overall m⁶A level in rat aortic tissues is significantly reduced (27). Microarray analysis hints that 40 transcripts (31 mRNAs and 9 lncRNAs) are hypermethylated and 223 transcripts (156 mRNAs and 67 lncRNAs) are hypomethylated in the aortic tissues of LPS-treated rats. The overall m⁶A level in left ventricular myocardial tissues of rats treated with endotoxin is significantly decreased. Similarly, another study unveils the hypermethylation of 27 transcripts (23 mRNAs and 4 lncRNAs) and hypomethylation of 46 transcripts (39 mRNAs and 7 lncRNAs) in the LPS group (28). The mRNA expressions of writers and readers in the LPS group are notably decreased. The m⁶A modification of C-type lectin domain family 1 member B (CLEC1B), serine/threonine kinase 38 like (STK38L), and tumor necrosis factor receptor superfamily member 26 (Tnfrsf26) is related to platelet activation and apoptosis pathways (29). In general, macrophages are activated after the onset of sepsis resulting in the production of large amounts of LPS in the plasma and stimulating the upregulation of m6 A methyltransferase METTL3 expression. Further, METTL3 stimulates enhanced m6A RNA methylation in cardiomyocytes, while increased expression of inflammatory cytokines within the microenvironment leads to cardiomyocyte degeneration and necrosis. The above studies suggest that m⁶A methylation is implicated in the regulation of cardiovascular system damage induced by sepsis.

SAE is a serious brain dysfunction mainly caused by sepsis in the non-central nervous system, with clinical manifestations of consciousness disorder, mild cognitive impairment, insanity, and coma. The mortality rate of SAE is as high as 30%-70%, which seriously compromises the health of patients (30). Currently, electroencephalogram, transcranial Doppler, and a series of serum markers including intercellular adhesion molecule-5 (ICAM-5) and S-100β have a certain value in the early diagnosis, evaluation, and prognosis of SAE (31). The specific pathogenesis of SAE has not been fully elucidated. The occurrence of SAE is thought to be related to nonspecific inflammation and non-inflammatory responses of brain cells. Changes in the metabolic function of brain cells after brain injury underlie the pathogenesis of SAE. Moreover, neuroinflammation is the main mechanism of m⁶A methylation recently reported to be associated with the occurrence of SAE (32). Previous studies have shown that the levels of white blood cell (WBC), neutrophil (Neut), platelet (PLT), and IL-6 in the SAE group are higher than those in the non-SAE group. Spearman correlation coefficient analysis has indicated a significant correlation between intestinal flora/serum metabolites and SAE. The number of Acinetobacter is positively correlated with the expression of METTL3. Thus, these findings further reveal the regulatory axial relationship of intestinal tract-methylation-inflammation-SAE (33).

Due to the increasing number of studies based on the gut-brain axis, existing mechanistic studies have introduced metabolic changes induced by the gut microbiome into the genesis of SAE. The gut-brain axis includes the central nervous system, the central endocrine system and the central immune system, which includes the hypothalamic-pituitary-adrenal axis (HPA axis), the sympathetic, parasympathetic (vagal) and enteric nervous systems of the autonomic nervous system, and the microbiota in the gut. The gastrointestinal surface is mainly governed by the autonomic nervous system, but the stimulation of the gastrointestinal tract’s bacteriophage also induces the secretion of physiological regulatory messages by the gut epidermal cells, which not only induce local immune responses, but also transmit physiological messages to the brain center via the autonomic nervous system, which in turn affects the central endocrine system and the central immune system. The pathogenic route based on the intestinal flora has been first investigated, namely the expression of METTL3 in immune cells stimulated by abundant immobile metabolites. However, studies on SAE-induced methylation are still in their infancy and further in-depth studies and explorations are still needed to determine the specific mechanisms.

The lung is particularly susceptible to sepsis injury (34). About 50% of patients with severe sepsis may develop acute respiratory distress syndrome (ARDS), with a mortality rate of 30%-40%. Unfortunately, no specific treatment has been proven to be effective against sepsis-caused ARDS so far (35). Further investigation on the pathogenesis of SA-AKI can provide novel treatment strategies for this urgent clinical issue, thus reducing ICU mortality. Many genetic and epigenetic alternations are related to the maintenance of endothelial function (36). There is increasing evidence that sepsis-related pulmonary microvascular leakage and organ damage/dysfunction are associated with changes in epigenetics and gene expression (37). Chemical modification of RNA molecules is crucial for the post-transcriptional regulation of gene expression. m⁶A is the most abundant form of mRNA and lncRNA methylation in eukaryotic cells, affecting a variety of physiological and pathological processes. YTH family proteins, IGF2BP, and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) have been confirmed to affect mRNA stability and translation (38). Abnormal m⁶A methylation is responsible for the occurrence and development of human diseases, suggesting the vital role of m⁶A modification in different genomic backgrounds. Chen et al. (39) have found that in sepsis, the reduction of m⁶A modification is associated with the down-regulation of METTL3. METTL3 knockout can accelerate barrier dysfunction and inflammatory response. In addition, tripartite motif containing 59 (TRIM59) is considered to be a key m⁶A effector, and its deletion exacerbates lung injury. Mechanistically, m⁶A reader YTHDF1 recognizes and stabilizes m⁶A-modified TRIM59 mRNA to protect vascular endothelial cells from barrier dysfunction and inflammatory response (40).

Wang JN et al. reported that METTL3 expression was elevated in renal tubules from different AKI models as well as in human biopsied and cultured tubular epithelial cells (TECs) (41). The researchers evaluated the specific function of METTL3 in HK2 cells in response to inflammatory stimuli. The results showed that silencing METTL3 attenuated renal inflammation and programmed cell death in TECs in response to TNF-α (tumor necrosis factor-α), cisplatin and LPS (lipopolysaccharide) stimulation, whereas METTL3 overexpression had the opposite effect. Conditional knockout of METTL3 in mouse kidneys attenuated cisplatin and ischemia/reperfusion (I/R)-induced renal dysfunction, injury and inflammation. This experiment reveals the mechanism of action by which genetic and pharmacological inhibition of METTL3 attenuates TAB3 m6A modification through an IGF2BP2-dependent mechanism, thereby reducing renal injury and inflammation, suggesting the METTL3/TAB3 axis as a potential target for the treatment of AKI.

Neutrophils are the primary components of innate immune response and also the main population of leukocytes in peripheral blood, functioning as important innate defense cells of the host against invading pathogens. During the bacterial attack, a large number of neutrophils are mobilized and released from the bone marrow into the peripheral circulation and migrate to the infection site at a very early stage (42). The rapid and effective migration and aggregation of neutrophils at the site of infection are essential for the innate defense against bacteria and the subsequent regression of inflammation ( Figure 1 ). The migration and function of neutrophils can be realized by extracellular signals such as chemokines and cytokines, and also by the intrinsic elements of neutrophils including chemokine receptors, cytoskeleton proteins, intracellular signal transduction modes, and cell metabolism (43). As the first line of the innate immune response, once the migration of neutrophils is impaired due to the imbalance of the above processes, the host cannot initiate effective innate defense against bacterial infection in a timely manner, resulting in failure of bacterial clearance, systemic inflammation, and even sepsis. On the other hand, bacterial infection may hijack the migration of neutrophils to escape the innate defense of the host, leading to persistent infection and unresolved inflammation (44). Therefore, the identification of new intracellular molecules that essentially determine the migration ability of neutrophils can not only enhance the innate defense against bacteria in the early stage of infection but also appropriately induce inflammatory resolution to avoid tissue damage after the removal of invading bacteria. ALKBH5 deficiency increases the mortality of mice with polybacterial sepsis induced by cecal ligation and perforation (CLP), and ALKBH5-deficient CLP mice exhibit higher bacterial load and more pro-inflammatory cytokines in the peritoneal and blood due to reduced neutrophil migration (45). ALKBH5-deficient neutrophils have lower C-X-C motif chemokine receptor 2 (CXCR2) expression and thus exhibit impaired migration to C-X-C motif chemokine ligand 2 (CXCL2). ALKBH5-mediated m⁶A demethylation targets neutrophil migration-related molecules by altering RNA decay to enhance the migration ability of neutrophils, such as increasing the expressions of neutrophil migration-promoting CXCR2 and NLR family pyrin domain containing 12 (NLRP12), but decreasing the expressions of neutrophil migration-suppressive prostaglandin E receptor 4 (PTGER4), tenascin-C (TNC), and with-no-lysine kinase 1 (WNK1) (46).

Xuetao Cao’s group at Peking Union Medical College reported that ALKBH5-mediated m6A demethylation enabled neutrophils to have high migratory capacity by altering RNA decay, thereby regulating the protein expression of their targets, neutrophil migration-related molecules, including increased neutrophil migration promoting CXCR2 and NLRP12 expression, but decreased neutrophil expression migration inhibiting PTGER4, TNC and WNK1 (47). In conclusion, the results of this study reveal an unknown role for ALKBH5 in imprinting neutrophil migration-promoting transcriptome features and intrinsically promoting neutrophil migration for antimicrobial defense, highlighting the potential application of targeting neutrophil m6A modifications in the control of bacterial infections.

The generation of neutrophil extracellular traps (NETs) is the main strategy of polymorphonuclear neutrophils (PMN) against microorganisms. NETs are closely related to the pathogenesis of various lung injuries, but relatively little is known about the role of NETs in SA-AKI (37). Elevated levels of NETs and ferroptosis can be observed in infected ARDS patients and SA-AKI mice. Blocking the formation of NETs by peptidyl arginine deiminase 4 (PAD4) inhibitor and PAD4 knockdown in mice, or inducing the depletion or degradation of NETs by anti-Ly6G and DNase, can significantly reduce sepsis-induced ferroptosis and protect mice from SA-AKI. Moreover, the cellular damage caused by NETs to human epithelial cells is weakened when ferroptosis is blocked. RNA-seq and qPCR analysis have shown that the expression of METTL3 is significantly increased during the ferroptosis of alveolar epithelial cells induced by NETs (47). In vitro models of METTL3 knockout and overexpression elucidate that METTL3-mediated m⁶A modification of glutathione peroxidase 4 (GPX4) is involved in the ferroptosis of human alveolar epithelial cells induced by NETs. The in vivo model of CLP-induced sepsis and the murine model of SA-AKI using METTL3 KO mice further demonstrate the critical role of METTL3-mediated m⁶A modification in NET-induced ferroptosis and protection of mice from SA-AKI (48).

Recent studies have established the critical role of macrophages in the pathogenesis of sepsis-caused ARDS (49) ( Figure 1 ). Macrophages can change from the initial pro-inflammatory M1 phenotype at the onset of lung injury to the anti-inflammatory M2 phenotype at the stage of initiating lung repair (50). Direct evidence has been provided to demonstrate the ability of polarized M2 phenotype in vivo and in vitro to prevent lung injury, inflammation, and subsequent fibrosis in ARDS (51). Zhao et al. have explored the differential expression pattern of circular RNAs (circRNAs) in pulmonary macrophages of sepsis-induced ARDS mice using microarray and found that circN4bp1 affects macrophage differentiation by binding to miR-138-5p, thereby regulating enhancer of zeste homolog 2 (EZH2) expression in vivo and in vitro. Subsequently, elevated m⁶A levels of circN4bp1 are found in ARDS mice, and inhibition of m⁶A methyltransferase METTL3 reverses this trend in vitro (52). Therefore, circN4bp1 can act as a sponge of miR-138-5p and modulate the polarization of macrophages by regulating the expression of EZH2. YTHDF1 knockdown improves the immune paralysis of macrophages, Th1/Th17 cells, and cytotoxic T lymphocytes (CTLs) and also alleviates macrophage-caused endothelial damage in severe sepsis rats with extracorporeal membrane oxygenation (ECMO) by suppressing the high mobility group box-1 (HMGB1)/receptor for advanced glycation end products (RAGE) axis and YTHDF1 and decreasing the m⁶A RNA methylation of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and pJAK2/pSTAT3 proteins in macrophages (53).

There is also compelling evidence that epigenetic regulation coordinates dynamic macrophage polarization. m6A methylation is the most abundant epigenetic modification in species mammalian mRNAs, but its role in macrophage polarization remains completely unknown. m6A catalase METTL3 was shown to be specifically upregulated by Vu LP et al. following M1 polarization in mouse macrophages (54). Furthermore, knockdown of METTL3 by siRNA transfection significantly inhibited M1 but enhanced M2 macrophage polarization. Conversely, its overexpression by plasmid transfection greatly promoted M1 but attenuated M2 macrophage polarization. Further immunoprecipitation of methylated RNA and in vitro m6A methylation analysis showed that METTL3 directly methylates mRNA encoding STA T1, the master transcription factor controlling M1 macrophage polarization, located in its CDS and 3’UTR regions. Furthermore, METTL3-mediated methylation of STAT1 mRNA significantly increased mRNA stability and subsequently upregulated STAT1 expression. In conclusion, METTL3 drives M1 macrophage polarization through direct methylation of STAT1 mRNA and may serve as an anti-inflammatory target.

In recent years, more and more scientists have focused on the role of m6A methylation modifications in the immune system. However, the effect and regulation of this methylation on NK cells is not yet clear. Recently, a researcher has studied this. The results showed that the expression of the m6A “writer” METTL3 was decreased in tumor-infiltrated NK cells, and the protein expression level of METTL3 was positively correlated with the effector molecules in NK cells.

METTL3 deficiency in NK cells alters NK cell homeostasis and inhibits NK cell infiltration and function in the microenvironment. The gene encoding SHP-2 is modified by m6A and its protein expression is reduced in METTL3-deficient NK cells, which is associated with the suppression of AKT and MAPK signaling pathway activation in mettl3-deficient NK cells (55).

Overall, macrophages are activated after the onset of sepsis resulting in the production of large amounts of LPS in the plasma and stimulating the upregulation of m6 A methyltransferase METTL3 expression. Further, METTL3 stimulates enhanced m6A RNA methylation in cardiomyocytes, while increased expression of inflammatory cytokines within the microenvironment leads to cardiomyocyte degeneration and necrosis.