B Cell-specific METTL3 depletion exacerbates experimental autoimmune encephalomyelitis

B Cell-specific METTL3 depletion exacerbates experimental autoimmune encephalomyelitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article B Cell-specific METTL3 depletion exacerbates experimental autoimmune encephalomyelitis XuZhong Pei, Hong Yang, Jie Ding, Haojun Yu, Chunran Xue, Xiying Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7206859/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jan, 2026 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract N6-methyladenosine (m 6 A), the most prevalent RNA modification, plays a pivotal role in regulating mRNA metabolism and cellular processes such as immune responses. Although the m6A methyltransferase METTL3 is known to regulate T-cell homeostasis and influence experimental autoimmune encephalomyelitis (EAE, a model for multiple sclerosis (MS)), its function within B cells remains poorly defined. Crucially, we observed that METTL3 expression is significantly downregulated in peripheral blood mononuclear cells (PBMCs) from MS patients and within B cells isolated from EAE mice. To directly investigate the functional consequences of this B-cell-specific METTL3 reduction in neuroinflammation, we generated B cell-specific METTL3 knockout mice (Mettl3 flox/flox CD19 Cre ). Strikingly, this targeted deletion of METTL3 in B cells markedly exacerbated EAE severity, demonstrated by significantly worsened clinical disease scores, increased spinal cord inflammation, and greater demyelination. Further mechanistic dissection revealed how B-cell METTL3 deficiency drives this exacerbated pathology: it promoted B cell apoptosis, inhibited the differentiation of regulatory B cell (Breg) subpopulations, increased the proportion of pro-inflammatory iNOS+ macrophages, and elevated the production of key inflammatory cytokines (IL-6, BAFF, and BCMA). Collectively, these findings demonstrate that METTL3 functions as a critical negative regulator within B cells, restraining their contribution to neuroinflammation in the EAE model. Importantly, therapeutically relevant overexpression of METTL3 specifically in B cells significantly reduced both the clinical severity and incidence of EAE, underscoring its potential as a novel therapeutic target for MS and similar autoimmune disorders involving pathogenic B-cell responses. m6A modification METTL3 B cells MS EAE inflammatory cytokines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Multiple sclerosis (MS) is a complex autoimmune disease of the CNS that is characterized primarily by inflammatory demyelination and axonal damage and results in neurological dysfunction in affected individuals[1-3]. Although the exact pathogenesis of MS remains incompletely understood, an increasing body of evidence suggests that B cells play critical roles in both the initiation and progression of the disease, for example, anti-CD20 drugs can significantly improve the condition of MS, primarily by targeting the survival and function of B cells[4-7]. Experimental autoimmune encephalomyelitis (EAE) is a classic animal model of MS, capable of mimicking the typical pathological phenotype of MS and commonly used to study the pathological mechanisms of MS. The main pathological features of EAE include inflammation and demyelination in the spinal cord, brainstem, and optic nerves. Research indicates that EAE is primarily mediated by T cells; however, a growing body of evidence suggests that B cells also play a critical role in the pathogenesis of MS and EAE[8,9]. B cells not only are immune cells that produce antibodies but also function as antigen-presenting cells (APCs), activating T cells via the MHC II pathway[10]. Furthermore, B cells secrete various cytokines, such as IL-6, TNF-α, and GM-CSF, which play pivotal roles in the pathogenesis of MS. B cell targeted therapies, such as anti-CD20 monoclonal antibodies (e.g., rituximab and ocrelizumab), effectively alleviate symptoms and delay disease progression in MS patients, thereby underscoring the importance of B cells in this condition[11]. In the EAE model, B-cell activity is closely associated with disease progression. Studies have demonstrated that B cells can influence T-cell responses and other immune responses through antigen presentation and the secretion of proinflammatory cytokines, thereby modulating neuroinflammation in EAE[8,10,11]. N 6 -Adenine methylation (m 6 A) is one of the most common mRNA modifications and is widely found in eukaryotic cells. m 6 A modifications regulate gene expression by affecting processes such as splicing, stability, transport and translation of mRNAs[12]. m 6 A modifications of major methyltransferase complexes include methyltransferase-like 3 (METTL3), METTL14, and Wilms tumor1–associated protein, with METTL3 and METTL14 being the core methyltransferases responsible for adding methyl groups to mRNAs. Numerous studies have shown that m 6 A modifications play important roles in the development and function of a variety of immune cells[13,14]. For example, the regulatory role of METTL3 in T cells has been shown to be involved in maintaining T cell homeostasis and regulating differentiation and signal transduction, and its mechanisms are mediated mainly by m 6 A modifications that affect the stability and degradation of specific mRNAs, which in turn modulate T cell function and immune responses, further influencing the inflammatory response during EAE[15,16]. m 6 A modifications in macrophages are also important for the inflammatory response, with METTL3 being upregulated upon polarization of M1 macrophages, whereas overexpression of METTL3 promotes M1 polarization but inhibits M2 polarization[17], thereby modulating the immune response. However, the role of METTL3 in the B-cell-regulated immune response is currently unknown. In recent years, increasing attention has focused on the role of m 6 A modification in B-cell function. The development and function of B cells are complex and multifaceted and involve processes such as antibody production, antigen presentation, and cytokine secretion[2,11]. As a key enzyme in m 6 A modification, the loss of METTL3 in B cells may impact their immune functions. Previous studies have demonstrated that m 6 A modification plays crucial roles in B-cell maturation and antibody production. The absence of METTL3 in B cells impairs their differentiation process, resulting in a significant reduction in antibody production ability[18]. Additionally, m 6 A modification is involved in regulating the gene expression patterns of B cells, influencing their response to external stimuli. For example, the loss of METTL3 may impair the ability of B cells to effectively express certain proinflammatory factors, thereby affecting their role in autoimmune diseases[19]. However, the specific role of m 6 A modification in B cells and its impact on neuroinflammation in EAE remain inadequately understood. This study elucidates the critical role of B cell-expressed METTL3 in modulating the pathogenesis of experimental autoimmune encephalomyelitis (EAE). We demonstrate that normal METTL3 expression in B cells exerts a protective effect against EAE development, while B cell-specific METTL3 deletion significantly exacerbates disease severity and disrupts B cell function, highlighting the essential regulatory role of B cells in EAE immunopathology. Crucially, to validate this protective function in vivo, we employed transgenic mice with B cell-specific METTL3 overexpression (METTL3-OE). Following EAE induction, these METTL3-OE mice exhibited significantly reduced clinical symptom scores and a markedly lower disease incidence compared to wild-type controls, confirming that enhancing METTL3 expression in B cells mitigates disease progression. Collectively, these findings provide direct in vivo evidence that METTL3 expression within B cells is a key protective factor in EAE, and demonstrate the therapeutic potential of augmenting B cell METTL3 levels to counteract disease progression. Methods Animals Female C57BL/6 mice used in this study were purchased from Charles River, Inc. and were housed at 8-10 weeks of age in the animal house of the Shanghai Institute of Model Organisms. Mettl 3flox/flox mice and Cd19 cre mice were generated by the Southern Model Organisms Research Centre in Shanghai. These Mettl3 flox/flox mice were crossed with Cd19 Cre mice (Shanghai Southern Model Organism Research Centre) to obtain Mettl3 flox/flox Cd19 Cre mice. Similarly, transgenic mice overexpressing METTL3 in B cells and their littermate controls were also purchased from this source. All experimental mice were maintained under SPF conditions with ad libitum access to standard laboratory chow with a 12-hour light-dark cycle, 22 ± 1°C and 55% ± 5% humidity. Construction of the EAE Based on previous strategies [20], Myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) from GL Biochem Co Ltd (Shanghai, China) was dissolved into a homogeneous solution of 3 mg/mL in phosphate buffered saline (PBS), which was subsequently emulsified with 8 mg/mL complete Fuchs adjuvant (CFA) from the H37RA strain (Difco, USA) in a 1:1 ratio. The resulting emulsion was administered via intramuscular injection at the bilateral femoral regions of female mice. Concurrently, pertussis toxin (Millipore, Billerica, MA, USA), utilized as an adjuvant to augment the immunogenicity of the antigen, was administered intraperitoneally at a dosage of 200 ng per mouse on the day of immunization (day 0) and 48 hours postimmunization (day 2). As a control for mice in the EAE group, mice in the CFA group were included for all reagents except MOG 35-55 . The clinical manifestations of mice was assessed in a double-blind manner by two independent researchers based on a scoring system as follows: mild tail weakness was assigned a score of 0.5; complete tail paralysis, 1; ataxia accompanied by hind limb weakness, 2; bilateral hind limb paralysis, 3; forelimb weakness, 4; and a moribund state, 5. All experimental mice were euthanised on day 20 for tissue collection. PBMC extraction The collected peripheral blood samples were centrifuged at room temperature to separate the plasma. The cell fraction was then diluted with phosphate buffered saline (PBS, pH 7.4) at a 1:1 ratio. The diluted suspension was carefully spread on Ficoll-Paque Premium (Cytiva China) at a ratio of 2:1 (sample: Ficoll) and then centrifuged in a gradient for 30 minutes. The plasma-Ficoll interface containing PBMC buffer was aspirated, rinsed twice with PBS, centrifuged and the cells were collected for storage or experiments. HE staining and LFB staining After injection, mouse spinal cords were fixed in 4% paraformaldehyde and tissue sections were taken at the maximum transverse section of the lumbar spine. After dewaxing and rehydration, some sections were placed in haematoxylin-eosin staining solution for HE staining, while others were placed in LFB staining solution and stained overnight at 56°C. Subsequently, differentiation treatment was performed until the boundary between the myelin sheath and white matter was clearly visible. Images were analysed using ImageJ software. Immunofluorescence staining : Paraffin sections were dewaxed and hydrated, soaked in 3% hydrogen peroxide for 10 min at room temperature, then repaired with EDTA antigen repair solution (Solarbio, China) at 100°C for 30 min, cooled to room temperature and then closed, incubated overnight at 4°C with myelin basic protein (MBP) primary antibody (1:200, Abcam, USA), and washed and then incubated with the secondary antibody for 2 h at room temperature. The slices were sealed with DAPI (Beyondi, China) sealer. Sections were examined by fluorescence microscopy (Olympus, USA). Flow cytometry staining After centrifuging the cell suspension at 400g for 5 minutes, discard the supernatant, resuspend in 100μl PBS, add flow cytometry antibody at a ratio of 1:400, and incubate at 4°C away from light for 30 minutes. Wash twice with PBS. For surface staining: fix with 1% PFA at 4°C away from light for 30 minutes, wash twice with PBS, then resuspend in 200μl PBS for testing. For intracellular/nuclear staining: Incubate with membrane-breaking solution at 4°C for 1 hour, wash with membrane-breaking wash solution, centrifuge at 800g, add the same proportion of antibody, incubate at 4°C in the dark for 30 minutes, wash twice with membrane-breaking wash solution, and resuspend in 200 μl PBS. Store samples at 4°C, perform flow cytometry analysis, and analyse using FlowJo software 10.8.1. All centrifugation steps are performed for 5 minutes. Spleen cell extraction and culture Place the mouse spleen in PBS on ice, transfer to a cell culture dish, and mechanically grind through a 40 μm filter. Rinse with PBS to obtain a single-cell suspension. Centrifuge at 400g for 5 minutes, discard the supernatant, add 3 ml of 1× red blood cell lysis buffer (Beyotime, China), and lyse for 5 minutes. Terminate with PBS and centrifuge under the same conditions for washing. Resuspend the cells in PBS and count them. Take 5 × 10⁶ cells in a 1.5 ml EP tube, centrifuge and discard the supernatant. Resuspend in 1640 medium containing stimulants (20 ng/ml phorbol ester + 1 μg/ml ionomycin + 1× Brefeldin A), transfer to a 24-well plate, and incubate at 37°C for 5 hours. Finally, collect the cells for flow cytometric analysis. All centrifugation steps were performed at 400g for 5 minutes. B lymphocyte sorting and culture Isolation of B cells from mouse spleens was performed by employing CD19 MicroBeads (Miltenyi Biotec, Germany). The extracted spleen cells were resuspended and then incubated with CD19 MicroBeads. After ten minutes, the cells were sorted through LS columns, and the cells adsorbed on the magnetic columns were removed, while the purification of the ooze was repeated 2 times. The sorted B cells were resuspended in RPMI 1640 culture medium (Gibco, Thermo Fisher Scientific), and 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin were added to the culture medium. The cells were then plated into a 24-well plate at a density of 1 × 106 cells/mL and cultured at 37°C in a humidified incubator with 5% CO2 for 72 hours. For stimulation and treatment in specific experiments, B cells can be stimulated with lipopolysaccharide (LPS) (Sigma‒Aldrich) at a concentration of 10 μg/mL to induce their activation and proliferation. Cell proliferation Sorted B cells were incubated with the fluorescent dye CFSE (CFDA-SE) prior to culture. The cells were then resuspended in RPMI 1640 culture medium (Gibco, Thermo Fisher Scientific) containing RPMI 1640 with 10% heat-inactivated foetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were then cultured into 24-well plates at a density of 1 × 10 6 cells/mL and incubated in a humidified incubator at 37°C and 5% CO2. Stimulation and treatment In specific experiments, B cells were stimulated with 10 μg/mL concentration of lipopolysaccharide (LPS) (Sigma-Aldrich) to induce activation and proliferation. Analyses were performed by FlowJo software 10.8.1 after 72h of incubation. Annexin V/PI staining Cells cultured by the above method were collected into centrifuge tubes, washed with PBS and then stained using the Membrane Associated Protein V/PI Detection Kit (Multi Sciences, China) according to the instructions. Immediately after staining, data were collected by flow cytometry (BD, USA) and analysed using FlowJo software 10.8.1. Quantitative real -time PCR Total RNA was extracted from the cells using TRIzol (Abconal, China), and the concentration was detected to reach the standard, then reverse transcription and real-time PCR were carried out according to the instructions of the kit (Vazyme, China), and GAPDH was used as an internal reference. The relative expression of the genes was calculated using the 2-ΔΔCt method. The primer sequences are shown in Table S1. Western blot Total protein was extracted from mouse spleen-sorted B cells and then quantified via protein extraction via a BCA kit (Thermo Fisher, USA). Equal amounts of protein were added to SDS polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. The membrane was blocked with blocking solution (Epizyme Biotech, China) and then incubated with primary antibody (METTL3, 1:1000, Abmart, China; GAPDH, 1:10,000, Jackson ImmunoResearch, USA) overnight at 4°C, followed by incubation with secondary antibody for 1 h. After incubation, protein signals were detected using an enhanced chemiluminescence kit (Epizyme Biotech, China) in a Western blot imaging analyzer (Bio-Rad, USA). Proteins were quantified via ImageJ. Cytometric bead array Use the CBA assay kit (Biolegend, USA) to detect cytokine levels in mouse peripheral blood serum. First, capture the microspheres and vortex vigorously (≥1 minute) to ensure uniformity. Lyophilised standards were diluted from the stock concentration (C7) in a series of fourfold dilutions to the lowest concentration (C1). The experiment was conducted in V-bottom 96-well plates: each well was first added with 25 µL of detection buffer, followed by 25 µL of standard or sample. Before use, resuspend the microspheres by vortexing for 30 seconds, then add 25 µL to each well. Seal the plate, incubate in the dark at 800 rpm for 2 hours. Centrifuge at 250 × g for 5 minutes, gently discard the supernatant. After two washes, add 25 µL of detection antibody to each well. Seal the plate, incubate in the dark at 800 rpm for 1 hour, then centrifuge and discard the supernatant. Then, add 25 µL of PE-labelled streptavidin to each well, seal the plate, incubate at 800 rpm for 30 minutes, and wash twice. Finally, resuspend the microspheres in 150 µL of wash buffer, transfer to a flow cytometry tube, and analyse using a flow cytometer (BD LSRFortess X-20, USA). Collect >300 microspheres per region for cytokine quantification. MeRIP-Seq and MeRIP-qPCR This experiment was performed by Cloud Sequence Biologicals, Shanghai, China. As previously reported[21], RNA samples were standardized to defined concentrations and chemically fragmented (70°C, 6 min) to generate ~200-nt fragments. Following the termination of the reaction, the fragmented RNA was pooled, purified via ethanol precipitation, and validated for size via an Agilent Bioanalyzer, with a subset retained as input controls. For immunoprecipitation, m6A-specific antibodies (or IgG controls) were conjugated to PGM magnetic beads and incubated with fragmented RNA at 4°C for 1 h. Nonspecific interactions were eliminated through sequential washes with IP, LB, and HS buffers. The captured RNA was eluted and further purified via MS magnetic beads (RLT buffer binding, 75% ethanol washes) to obtain high-purity RNA for Illumina library construction and sequencing. The immunoprecipitated RNA was reverse-transcribed into cDNA (SuperScript III, 50°C, 60 min). Quantitative PCR was performed on a QuantStudio 5 system using SYBR Green premix and gene-specific primers under optimized conditions: 95°C predenaturation (10 min), followed by 40 cycles of 95°C (10 s) and 60°C (60 s). Methylation levels were quantified via Ct values normalized to input controls. Statistical analysis In this study, we used GraphPad Prism 9 software for statistical analysis, and the error line indicates the standard error of the mean (SEM). For two-group comparisons, a t test was used for normally distributed data, and a nonparametric test was used for nonnormally distributed data. For multiple comparisons, ANOVA (two-way) was performed. p<0.05 was considered statistically significant. Details are provided in the figure legends. Results Decreased METTL3 expression in B cells after EAE induction It has been previously shown that in CD4 + T cells, deletion of the m 6 A methyltransferase METTL3 severely interferes with the homeostatic proliferation of these cells and their ability to differentiate into effector cells, thereby alleviating the pathological features of EAE[ 16 ]. Similarly, research has indicated that specific knockout of the m 6 A demethylase AlkB homologue 5 (ALKBH5) in T cells confers protection against EAE [ 22 ]. However, no study has investigated whether m 6 A regulates the pathological process of EAE through B cells. In this study, we initially assessed the mRNA expression levels of the m 6 A-related enzymes METTL3 and YTH N 6 -methyladenosine RNA binding protein F1 (YTHDF1) throughout disease progression in patients with MS. By detecting the mRNA expression of METTL3 and Y THDF1 in peripheral blood mononuclear cells (PBMCs) from patients with MS and healthy controls, we found that, compared with that in healthy controls, the mRNA expression of METLL3 in MS patients was significantly decreased, whereas that of YTHDF1 was not significantly different (Fig. 1 A and B). We next explored the expression of METTL3 in the B lymphocytes of EAE mice and detected significantly decreased expression of METTL3 mRNA in the splenocytes of EAE mice (Fig. 1 C). B cells in the spleen were sorted via magnetic bead sorting, and the sorting efficiency of the B cells was greater than 90% (Fig. 1 D). Further analysis of the sorted B cells revealed that METTL3 expression was significantly lower at both the mRNA and protein levels in B cells from EAE mice than in those from healthy controls (Fig. 1 E and F). These results suggest that METTL3 may play an important role in the progression of EAE. We subsequently constructed Mettl3 flox/flox Cd19 cre mice with specific deletion of METTL3 in B cells and used their littermate Mettl3 flox/flox mice as controls to further explore the relationship between METTL3 and EAE. By detecting the expression of METTL3 in B cells of Mettl3 flox/flox Cd19 Cre mice, we observed that METTL3 was obviously knocked down (Fig. 1 G-I). In conclusion, METTL3 mRNA expression was decreased in the PBMCs of MS patients; similarly, METTL3 expression was decreased in the B cells of EAE mice. In addition, we successfully constructed Mettl3 flox/flox Cd19 Cre mice for this study. 3.2 Deletion of METTL3 in B cells exacerbates EAE To further clarify the function of METTL3 in the B cells of EAE mice, we induced EAE in Mettl3 flox/flox Cd19 Cre mice and their littermate control Mettl3 flox/flox mice via the myelin oligodendrocyte glycoprotein (MOG) peptide MOG 35 − 55 and assessed their clinical performance via behavioral assessment. The results revealed that Mettl3 flox/flox Cd19 Cre mice presented significantly higher clinical scores than controls group (Fig. 2 A-C). Considering that inflammatory cell infiltration and demyelination are the most typical pathological features of EAE mice, we examined inflammatory infiltration and demyelination in the spinal cord of EAE mice via H&E staining and LFB staining, respectively. The results revealed that the number of inflammatory cells was significantly increased in the spinal cords of mice lacking METTL3 (Fig. 2 D and E). We also observed more pronounced spinal cord demyelination in the mice in the Mettl3 flox/flox Cd19 Cre group (Fig. 2 F and G). In addition, the results of MBP immunostaining revealed a similar trend (Fig. 2 H and I). These results suggest that MTTL3 deficiency in B cells significantly aggravates the clinical symptoms and pathological phenotype of EAE. 3.3 METTL3 deficiency exacerbates proliferation and apoptosis of B cells To further investigate how METTL3 regulates B-cell function after EAE induction, we used flow cytometry to analyse the composition of B cells in the spleen and inguinal lymph node sites, and the proportion of B cells in the spleen of the Mettl3 flox/flox Cd19 Cre mice was significantly reduced compared with that in the Mettl3 flox/flox group (Fig. 3 A and B). Similarly, we found that the proportion of B cells in inguinal lymph nodes was also reduced in Mettl3 flox/flox Cd19 Cre mice (Fig. 3 C and D). These findings suggest that METTL3 knockout may affect the function of B cells in response to inflammation, resulting in their decline. METTL3 was previously shown to collaborate with insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) to regulate the cell cycle response of Germinal Center B cells(GCB) by affecting the stability of Myc mRNA, and a lack of METTL3 resulted in slow progression of the cell cycle and a reduced ability to differentiate into plasma cells in response to half-antigen stimulation[ 18 ]. Similarly, compared with those in the Mettl3 flox/flox group, the numbers of plasma cells in the spleen and lymph nodes of the mice in the Mettl3 flox/flox Cd19 Cre group were also significantly lower (Fig. 3 E-H). In addition, we sorted B cells from the spleen, pertained them with CFSE, cultured them for 72 hours, and detected their fluorescence intensity in response to their degree of proliferation. These results indicate that METTL3-specific deletion promotes B-cell proliferation. Furthermore, we examined the apoptosis of B cells in the two groups of mice and found that the proportion of apoptotic cells was significantly greater in the Mettl3 flox/flox Cd19 Cre group than in the control group. These results suggest that specific METTL3 knockout promotes the functional response of B cells in response to inflammatory stimuli and promotes B-cell proliferation to a certain extent, but the promotion of apoptosis is more pronounced. 3.4 B-cell-specific deletion of METTL3 inhibits the differentiation of regulatory B cells In EAE, B cells regulate the inflammatory response mainly through the secretion of inflammatory factors, including a class of regulatory B-cell subpopulations expressing CD1d and CD5, whose normal expression inhibits the inflammatory response and attenuates the degree of the autoinflammatory response. To further investigate how knockout of METTL3 exacerbates the pathological manifestations of EAE, we examined T-cell and B-cell subsets in the spleens of EAE mice after knockout of METTL3. We found that the proportion of CD1d hi CD5 + B cells among splenic B cells was significantly lower in the Mettl3 flox/flox Cd19 Cre group than in the Mettl3 flox/flox group (Fig. 4 A and E), further testing of the proportion of IL-10 + CD19 + B cells revealed a significant decrease (Fig. 4 B and F). But the Treg subset did not change significantly (Fig. 4 C and G), suggesting that the inhibitory role of B cells in the pathogenesis of EAE is diminished. Previous studies have demonstrated that CD1d hi CD5 + regulatory B cells constitute a crucial immune cell population that performs negative immunomodulatory functions in vivo and influences the progression of EAE by regulating the differentiation of CD4 + T cells. Therefore, we performed flow analyses after stimulating T cells as previously reported in the literature and found that knockout of METTL3 in B cells did not affect the overall proportion of CD4 + T cells (Fig. 4 D and H). In conclusion, the above results suggest that deletion of METTL3 in B cells impedes the suppressive effects of B cells on inflammation, but this pathway is not achieved by regulating CD4 + T cells. 3.5 B cell-specific deletion of METTL3 promotes macrophage inflammatory response and peripheral inflammation To further clarify the cause of the more severe morbidity in the Mettl3 flox/flox Cd19 Cre group of mice, we examined the splenic macrophages of EAE mice and found that macrophages were significantly activated in the spleens of the Mettl3 flox/flox Cd19 Cre group of mice compared with those of the control group (Fig. 5 A and B). Further examination of the iNOS + macrophages revealed that they were significantly elevated (Fig. 5 C and D). Interestingly, we also detected a significant increase in the proportion of CD206 + macrophages (Fig. 5 E and F). In addition, we examined peripheral blood inflammatory factors in EAE mice and found a trend toward increased levels of the proinflammatory factor interleukin 6 (IL-6), a significant increase in the secretion of TNF-family B-cell activating factor (BAFF) and B-cell maturation antigen (BCMA), However, we found that there was no significant difference in IL-10 secretion (Fig. 5 G-N). Considering that we tested peripheral blood serum, we then analysed the transcription level of IL-10 in B cells and found that it had decreased significantly (Fig. 5 O), suggesting that the specific deletion of Mettl3 in B cells exacerbated the EAE-induced peripheral inflammatory response. In conclusion, our study revealed that the deletion of METTL3 in B cells promotes macrophage activation, which exacerbates inflammation, and, it may lead to more severe clinical signs in EAE mice by affecting the secretion of inflammatory factors in the peripheral immune system. 3.6 B cell-specific METTL3 deficiency downregulates key genes in the axon guidance pathway through an m⁶A-dependent mechanism To clarify the molecular targets of METTL3 in EAE mouse B cells, we performed a multiomics joint analysis of RNA-seq and MeRIP-seq data to screen for genes with significantly downregulated levels of m⁶A modification and transcription after METTL3 deletion, including Srgap3, Slit3, Nrp2, Celsr2, Tox2 , and Zfp532 , and KEGG pathway enrichment analysis revealed that METTL3 knockdown resulted in a significant reduction in Axon guidance pathway activity in B cells (Fig. 6 A and B). Among them, Slit3 and Srgap3 play important roles in the normal function of axon guidance. On the basis of these findings, we hypothesized that METTL3 may affect the neural function of EAE mice by regulating the expression of Slit3 and Srgap3 . Next, via IGV visualization, we revealed that Slit3 and Srgap3 are target genes for METTL3 action, and the results revealed that the m6A modification peaks of Slit3 and Srgap3 were significantly decreased in Mettl3 flox/flox Cd19 Cre EAE mice, which was further verified by MeRIP-qPCR. The results revealed that the m6A levels of both target genes were significantly decreased. (Fig. 6 C and D). The overall methylation level of RNA in B cells was examined, and as expected, the deletion of METTL3 in B cells resulted in a decrease in their overall m6A level (Fig. 6 E). In summary, the above results not only confirmed the reliability of the multiomics data but also revealed that METTL3 regulates the expression of Slit3 and Srgap3 through a m⁶A-dependent mechanism, thereby inhibiting the activity of the axon conductance pathway, which may be a key molecular mechanism by which the absence of METTL3 leads to neurological impairments in EAE mice. 3.7 Overexpression of METTL3 in B cells significantly alleviates the symptoms of EAE Finally, to validate these findings in vivo, we employed transgenic mice with B cell-specific overexpression of METTL3. Following induction of experimental autoimmune encephalomyelitis (EAE), these METTL3-overexpressing (OE) mice exhibited significantly reduced clinical symptom scores and a markedly lower disease incidence compared to wild-type (WT) controls (Fig. 7 A-C). Confirmation of successful METTL3 overexpression was obtained by detecting significantly elevated METTL3 levels specifically within B cells of the OE mice (Fig. 7 D). Collectively, these results provide direct in vivo evidence supporting the protective role of B cell-expressed METTL3 in the EAE model. Discussion m 6 A is one of the most common modifications of eukaryotic mRNAs and noncoding RNAs and regulates gene expression by affecting RNA splicing, translation and stability[ 23 ]. By affecting gene expression, in which METTL3 is the most central catalytic subunit of m 6 A, it can regulate the normal function of immune cells[ 24 ]. Although METTL3 controls various aspects of T cell development and activity, thus providing good intervention in autoimmune diseases[ 16 , 25 , 26 ], the role of METTL3 in B cells is not fully understood. In the present study, we observed reduced expression of METTL3 in PBMCs from MS patients and reduced expression of METTL3 in B cells from EAE model mice and further revealed that specific knockout of METTL3 in B cells significantly exacerbated the clinical symptoms of EAE. In addition, studies on B cell function have shown that knockout of METTL3 results in enhanced B-cell proliferation and apoptosis under inflammatory conditions, but apoptosis is more pronounced. Considering that more definitive studies on the regulatory role of B cells in EAE have focused on the Breg subpopulation, our analyses of B cell subpopulations suggest that METTL3 deletion leads to a decrease in CD1d hi CD5 + B cells, which may result in diminished inhibition of the inflammatory response. Furthermore, METTL3 deletion increases the secretion of IL-6, BAFF, and BCMA and decreases IL-13 secretion in the peripheral blood, thereby exacerbating the inflammatory response. A growing body of research suggests that the regulatory role of B cells in the disease process is also critical for EAE. B cells are important components of the adaptive immune system and function in antigen presentation, cytokine secretion, and antibody production[ 27 ]. It has been reported that specific deletion of METTL3 in B cells has little effect on the normal development and growth of mice, whereas functionally, knockout of METTL3 promotes apoptosis in B cells, which is consistent with our observations[ 28 ]. In addition, deletion of Mettl3 at early stages of B cell differentiation via Mb1-Cre blocks B cell differentiation, specifically affecting the transition from pro-B to large pre-B and from large pre-B to small pre-B[ 29 ], and inhibition of Mettl3-mediated m 6 A modification in hematopoietic stem cells (HSCs) during B-cell development leads to impaired HSC differentiation, thereby reducing the proportion of peripheral B cells[ 29 , 30 ]. These findings suggest that knocking down METTL3 in B cells might theoretically provide modest alleviation of B cell-mediated inflammatory responses. However, the reality was the opposite of what was expected. In the EAE mouse model, when B cells are deficient in METTL3, the mice instead show more severe manifestations of the disease. This phenomenon indicates that METTL3 may play a protective role in peripheral B cells during EAE associated inflammation and that its presence may be important for maintaining the balance and stability of B cell associated immune responses. Earlier studies have reported that in EAE mice, gut-derived IgA + PC cells migrate to the CNS, where their number decreases during EAE, and the removal of PC cells exacerbates EAE symptoms, whereas the introduction of gut-sourced IgA + PC cells alleviates symptoms[ 31 ]. In addition, studies have indicated that specific deletion of METTL3 results in slowed cell cycle progression, reduced expression of genes associated with proliferation and oxidative phosphorylation, and a decreased proportion of plasma cells in GCB cells[ 18 ]. Similarly, in our study, we observed a reduction in the number of plasma cells in the spleens and lymph nodes of EAE-induced Mettl3 flox/flox Cd19 Cre mice; however, whether knockout of METTL3 in B cells leads to a decrease in IgA + PC cells in the intestine, thereby exacerbating EAE, deserves further exploration. According to previous studies, EAE is primarily a type of T cell mediated inflammation of the nervous system in which the role of B cells is not particularly clear. Our study provides evidence for the role of METTL3 in B cells during EAE. Specifically, knockout of METTL3 in B cells decreased the proportion of splenic CD1d hi CD5 + cells and increased the proportion of macrophages, suggesting an increased inflammatory response. However, further examination of macrophages revealed an increase in the proportion of both M1-type macrophages and M2-type macrophages, and previous studies have suggested that the activation of M1-type macrophages may lead to chronic inflammation and tissue damage, whereas the activation of M2-type macrophages contributes to inflammation clearance and tissue repair. In addition, knockout of METTL3 in B cells promoted the secretion of the inflammatory factors IL-6, BAFF, and BCMA but had no effect on the secretion of IFN-γ, IL-17 or IL-10. Therefore, the activation of macrophages may contribute to the exacerbation of EAE and profound inflammation, and the increased expression of CD206 may be a secondary response. IL-6 is a key immunomodulatory cytokine that influences the pathogenesis of a variety of diseases, including autoimmune disorders and inflammation[ 32 ]. BAFF is a cytokine belonging to the tumour necrosis factor (TNF) family that is essential for the proliferation and survival of B cells. It influences the regulation of immune responses by binding to specific receptors on the surface of B cells (e.g., BAFF-R, TACI, and BCMA) [ 33 – 35 ] and is expressed in a wide variety of cells, including monocytes, macrophages, dendritic cells, and B and T lymphocytes. In addition, astrocytes from MS patients also express BAFF[ 36 ]. Previous studies have shown that specific variants in the TNFSF13B gene, which encodes the cytokine BAFF, are associated with an increased risk of autoimmune diseases such as multiple sclerosis (MS) and systemic lupus erythematosus (SLE) [ 37 ]. In addition, there is much visual evidence that BAFF is markedly elevated in PBMCs from spinal cord injury (SCI) patients and that BAFF can be largely alleviated in EAE mice by inhibiting BAFF secretion in EAE mice[ 33 , 34 , 38 ]. Moreover, blocking BAFF can inhibit macrophage activation by reducing inflammation, and blocking BAFF reduces the secretion of inflammatory cytokines by macrophages [ 39 ]. However, whether BAFF upregulation directly drives macrophage activation and exacerbates EAE requires further investigation. Moreover, the specific mechanisms by which Mettl3 affects B cell function and the immunological mechanisms that contribute to the pathogenesis of EAE need to be further explored. In addition, previous studies have shown that Slit3 , a key molecule in axon guidance, is involved in peripheral nerve injury repair through the Slit3/Robo signalling pathway[ 40 ], whereas Srgap3 (Slit-Robo GTPase-activating protein 3) dynamically maintains cytoskeletal reorganization by inhibiting the activity of Rac1, a Rho GTPase, and regulating actin remodelling. Notably, Srgap3 deficiency can lead to abnormal synaptic function and cognitive behavioural deficits[ 41 ]. By combined multiomics analysis, we hypothesized that the deletion of METTL3 in B cells caused a decrease in the expression of Slit3 and Srgap3 as well as the downregulation of the axon guidance pathway, which further aggravated neurological damage in EAE mice; however, the specific mechanisms involved also deserve further exploration. At the conclusion of the study, the significantly alleviated clinical symptoms and reduced incidence rate in the transgenic EAE mouse model with B cell-specific overexpression of METTL3 directly confirmed the protective role of METTL3 in vivo. Our subsequent research needs to expand the sample size and further explore the specific mechanisms involved. Conclusion In conclusion, our study describes the critical role of Mettl3 in B cells and its impact on the pathogenesis of EAE. These findings highlight the importance of Mettl3 in regulating B cell function and the immune response and provide new avenues for therapeutic intervention in MS and other autoimmune diseases. Declarations Acknowledgements The authors would like to thank Shanghai Southern Model Biotechnology Co., Ltd. for their valuable assistance in the construction and maintenance of transgenic mice. Authors' contributions XZP: designed and performed the experiments, analysed the data, and wrote the original manuscript; HY: designed and performed the experiments, analysed the data, and revised the manuscript; JD: wrote the original manuscript and analysed the data; HJY, CRX, XYW, and CX: participated in the experiments and revised the manuscript; YC and XYL: participated in the experiments; YTG: Conceptualization, Review and editing, Supervision, Project administration, Funding acquisition. All the authors read and approved the final manuscript. Funding This study was supported by the National Natural Science Foundation of China (No. 82201495), Shanghai Municipal Health Commission (No. 202140414), Municipal Commission of Health and Family Planning Foundation of Shanghai Pudong New Area (No. PW2022E-01), Innovative Research Team of High-Level Local Universities in Shanghai (No. SHSMU-ZDCX20211901), and the New Quality Clinical Specialties of High-end Medical Disciplinary Construction in Pudong New Area (No. 2024-PWXZ-16). Data Availability The raw data has been made available to the public under the registration number PRJNA1255398. http://www.ncbi.nlm.nih.gov/bioproject/1255398. Ethical approval MS patients and healthy human samples were ethically compliant and approved by the Ethics Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine (Grant No. 2023-026-A); for animals, approval for experiments was granted by the Animal Care and Use Committee. (IACUC NO:2023-0056-01). Consent to Participate Not applicable Consent for publication Not applicable. Competing Interests The authors declare no competing interests References Crowley T,Chen J,Rosiewicz K Set al(2025) Mapping CD4+ T cell diversity in CSF to identify endophenotypes of multiple sclerosis.Brain Commun.7:fcaf231.http://doi.org/10.1093/braincomms/fcaf231. Rodríguez Murúa S,Farez M F，Quintana F J(2022) The Immune Response in Multiple Sclerosis.Annu Rev Pathol.17:121-139.http://doi.org/10.1146/annurev-pathol-052920-040318. Mozafari S,Starost L,Manot-Saillet Bet al(2020) Multiple sclerosis iPS-derived oligodendroglia conserve their properties to functionally interact with axons and glia in vivo.Sci Adv.6.http://doi.org/10.1126/sciadv.abc6983. Disanto G,Sacco R,Mallucci G,Zecca C，Gobbi C(2025) Effect of Cumulative Exposure to Ocrelizumab on Memory B-Cell Repopulation Dynamics in Multiple Sclerosis.Ann Neurol.http://doi.org/10.1002/ana.27281. van Puijfelik F,Blok K M,Klein Kranenbarg R A Met al(2024) Ocrelizumab associates with reduced cerebrospinal fluid B and CD20dim CD4+ T cells in primary progressive multiple sclerosis.Brain Commun.6:fcae021.http://doi.org/10.1093/braincomms/fcae021. Polonio C M，Quintana F J(2025) Therapeutic B cell depletion identifies immunoregulatory networks.J Clin Invest.135.http://doi.org/10.1172/JCI189442. Mouat I C,Goldberg E，Horwitz M S(2022) Age-associated B cells in autoimmune diseases.Cell Mol Life Sci.79:402.http://doi.org/10.1007/s00018-022-04433-9. Van Kaer L,Postoak J L,Wang C,Yang G，Wu L(2019) Innate, innate-like and adaptive lymphocytes in the pathogenesis of MS and EAE.Cell Mol Immunol.16:531-539.http://doi.org/10.1038/s41423-019-0221-5. Mouat I C,Allanach J R,Fettig N Met al(2022) Gammaherpesvirus infection drives age-associated B cells toward pathogenicity in EAE and MS.Sci Adv.8:eade6844.http://doi.org/10.1126/sciadv.ade6844. Hua Z，Hou B(2020) The role of B cell antigen presentation in the initiation of CD4+ T cell response.Immunol Rev.296:24-35.http://doi.org/10.1111/imr.12859. Sabatino J J,Pröbstel A-K，Zamvil S S(2019) B cells in autoimmune and neurodegenerative central nervous system diseases.Nat Rev Neurosci.20:728-745.http://doi.org/10.1038/s41583-019-0233-2. Jiang X,Liu B,Nie Zet al(2021) The role of m6A modification in the biological functions and diseases.Signal Transduct Target Ther.6:74.http://doi.org/10.1038/s41392-020-00450-x. Liu C,Yang Z,Li Ret al(2021) Potential roles of N6-methyladenosine (m6A) in immune cells.J Transl Med.19:251.http://doi.org/10.1186/s12967-021-02918-y. Cui L,Ma R,Cai Jet al(2022) RNA modifications: importance in immune cell biology and related diseases.Signal Transduct Target Ther.7:334.http://doi.org/10.1038/s41392-022-01175-9. Li H-B,Tong J,Zhu Set al(2017) m6A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways.Nature.548:338-342.http://doi.org/10.1038/nature23450. Wang X,Chen C,Sun Het al(2023) m6A mRNA modification potentiates Th17 functions to inflame autoimmunity.Sci China Life Sci.66:2543-2552.http://doi.org/10.1007/s11427-022-2323-4. Liu Y,Liu Z,Tang Het al(2019) The N6-methyladenosine (m6A)-forming enzyme METTL3 facilitates M1 macrophage polarization through the methylation of STAT1 mRNA.Am J Physiol Cell Physiol.317:C762-C775.http://doi.org/10.1152/ajpcell.00212.2019. Grenov A C,Moss L,Edelheit Set al(2021) The germinal center reaction depends on RNA methylation and divergent functions of specific methyl readers.J Exp Med.218.http://doi.org/10.1084/jem.20210360. Turner D J,Saveliev A,Salerno Fet al(2022) A functional screen of RNA binding proteins identifies genes that promote or limit the accumulation of CD138+ plasma cells.Elife.11.http://doi.org/10.7554/eLife.72313. Xiang W,Wang K,Han Let al(2024) CD22 blockade aggravates EAE and its role in microglia polarization.CNS Neurosci Ther.30:e14736.http://doi.org/10.1111/cns.14736. Wan Y,Tang K,Zhang Det al(2015) Transcriptome-wide high-throughput deep m(6)A-seq reveals unique differential m(6)A methylation patterns between three organs in Arabidopsis thaliana.Genome Biol.16:272.http://doi.org/10.1186/s13059-015-0839-2. Zhou J,Zhang X,Hu Jet al(2021) m6A demethylase ALKBH5 controls CD4+ T cell pathogenicity and promotes autoimmunity.Sci Adv.7.http://doi.org/10.1126/sciadv.abg0470. Teng Y,Yi J,Chen J，Yang L(2023) N6-Methyladenosine (m6A) Modification in Natural Immune Cell-Mediated Inflammatory Diseases.J Innate Immun.15:804-821.http://doi.org/10.1159/000534162. Geng Q,Cao X,Fan Det al(2023) Potential medicinal value of N6-methyladenosine in autoimmune diseases and tumours.Br J Pharmacol.http://doi.org/10.1111/bph.16030. Guo W,Wang Z,Zhang Yet al(2024) Mettl3-dependent m6A modification is essential for effector differentiation and memory formation of CD8+ T cells.Sci Bull (Beijing).69:82-96.http://doi.org/10.1016/j.scib.2023.11.029. Lu S,Wei X,Zhu Het al(2023) m6A methyltransferase METTL3 programs CD4+ T-cell activation and effector T-cell differentiation in systemic lupus erythematosus.Mol Med.29:46.http://doi.org/10.1186/s10020-023-00643-4. Satitsuksanoa P,Iwasaki S,Boersma Jet al(2023) B cells: The many facets of B cells in allergic diseases.J Allergy Clin Immunol.152:567-581.http://doi.org/10.1016/j.jaci.2023.05.011. Kang X,Chen S,Pan Let al(2022) Deletion of Mettl3 at the Pro-B Stage Marginally Affects B Cell Development and Profibrogenic Activity of B Cells in Liver Fibrosis.J Immunol Res.2022:8118577.http://doi.org/10.1155/2022/8118577. Lee H,Bao S,Qian Yet al(2019) Stage-specific requirement for Mettl3-dependent m6A mRNA methylation during haematopoietic stem cell differentiation.Nat Cell Biol.21:700-709.http://doi.org/10.1038/s41556-019-0318-1. Zheng Z,Zhang L,Cui X-Let al(2020) Control of Early B Cell Development by the RNA N6-Methyladenosine Methylation.Cell Rep.31:107819.http://doi.org/10.1016/j.celrep.2020.107819. Rojas O L,Pröbstel A-K,Porfilio E Aet al(2019) Recirculating Intestinal IgA-Producing Cells Regulate Neuroinflammation via IL-10.Cell.176.http://doi.org/10.1016/j.cell.2018.11.035. Rose-John S,Jenkins B J,Garbers C,Moll J M，Scheller J(2023) Targeting IL-6 trans-signalling: past, present and future prospects.Nat Rev Immunol.23:666-681.http://doi.org/10.1038/s41577-023-00856-y. Saltzman J W,Battaglino R A,Salles Let al(2013) B-cell maturation antigen, a proliferation-inducing ligand, and B-cell activating factor are candidate mediators of spinal cord injury-induced autoimmunity.J Neurotrauma.30:434-440.http://doi.org/10.1089/neu.2012.2501. Huntington N D,Tomioka R,Clavarino Cet al(2006) A BAFF antagonist suppresses experimental autoimmune encephalomyelitis by targeting cell-mediated and humoral immune responses.Int Immunol.18:1473-1485 Damianidou O,Theotokis P,Grigoriadis N，Petratos S(2022) Novel contributors to B cell activation during inflammatory CNS demyelination; An oNGOing process.Int J Med Sci.19:164-174.http://doi.org/10.7150/ijms.66350. Zhang Y,Tian J,Xiao Fet al(2022) B cell-activating factor and its targeted therapy in autoimmune diseases.Cytokine Growth Factor Rev.64:57-70.http://doi.org/10.1016/j.cytogfr.2021.11.004. Steri M,Orrù V,Idda M Let al(2017) Overexpression of the Cytokine BAFF and Autoimmunity Risk.N Engl J Med.376:1615-1626.http://doi.org/10.1056/NEJMoa1610528. Gupta K,Kesharwani A,Rua Set al(2023) BAFF blockade in experimental autoimmune encephalomyelitis reduces inflammation in the meninges and synaptic and neuronal loss in adjacent brain regions.J Neuroinflammation.20:229.http://doi.org/10.1186/s12974-023-02922-7. Wang L,Zhang T,Zhang Zet al(2021) B cell activating factor regulates periodontitis development by suppressing inflammatory responses in macrophages.BMC Oral Health.21:426.http://doi.org/10.1186/s12903-021-01788-6. Chen B,Carr L，Dun X-P(2020) Dynamic expression of Slit1-3 and Robo1-2 in the mouse peripheral nervous system after injury.Neural Regen Res.15:948-958.http://doi.org/10.4103/1673-5374.268930. Bacon C,Endris V，Rappold G A(2013) The cellular function of srGAP3 and its role in neuronal morphogenesis.Mech Dev.130:391-395.http://doi.org/10.1016/j.mod.2012.10.005. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 03 Jan, 2026 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Editorial decision: Major Revision 13 Aug, 2025 Reviewers agreed at journal 28 Jul, 2025 Reviewers invited by journal 27 Jul, 2025 Editor assigned by journal 25 Jul, 2025 First submitted to journal 24 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7206859","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":491645730,"identity":"b34493ca-59e9-4a48-a4ea-04387086ae14","order_by":0,"name":"XuZhong Pei","email":"data:image/png;base64,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","orcid":"https://orcid.org/0009-0005-9336-4018","institution":"Shanghai University School of Medicine, Shanghai 200444","correspondingAuthor":true,"prefix":"","firstName":"XuZhong","middleName":"","lastName":"Pei","suffix":""},{"id":491645731,"identity":"f73c4ba7-7b79-4394-95c8-1352d740bcde","order_by":1,"name":"Hong Yang","email":"","orcid":"","institution":"Department of Neurology,The First Rehabilitation Hospital of Shanghai,School of Medicine,Tongji University,Shanghai","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Yang","suffix":""},{"id":491645732,"identity":"4f4e901f-a3bf-4e9e-9273-f519d54df479","order_by":2,"name":"Jie Ding","email":"","orcid":"","institution":"Department of Neurology,Renji Hospital, Shanghai Jiaotong University School of Medicine,160 Pujian Rd","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Ding","suffix":""},{"id":491645733,"identity":"b98d516b-c0ad-430e-a541-83c9b2fa8011","order_by":3,"name":"Haojun Yu","email":"","orcid":"","institution":"Department of Neurology, Shanghai Sixth People's Hospital, Shanghai Jiaotong University School of Medicine,600 Yishan Rd","correspondingAuthor":false,"prefix":"","firstName":"Haojun","middleName":"","lastName":"Yu","suffix":""},{"id":491645734,"identity":"c8564c0d-9c3b-44af-993d-e1a83ac9866f","order_by":4,"name":"Chunran Xue","email":"","orcid":"","institution":"Department of Neurology,Renji Hospital, Shanghai Jiaotong University School of Medicine,160 Pujian Rd","correspondingAuthor":false,"prefix":"","firstName":"Chunran","middleName":"","lastName":"Xue","suffix":""},{"id":491645735,"identity":"cae3a9d7-74a5-4c07-82b0-c5a3ec492c7f","order_by":5,"name":"Xiying Wang","email":"","orcid":"","institution":"Department of Neurology,Renji Hospital, Shanghai Jiaotong University School of Medicine,160 Pujian Rd","correspondingAuthor":false,"prefix":"","firstName":"Xiying","middleName":"","lastName":"Wang","suffix":""},{"id":491645736,"identity":"0a6352e2-874a-40a6-88cd-600b759b074e","order_by":6,"name":"Chong Xie","email":"","orcid":"","institution":"Department of Neurology,Renji Hospital, Shanghai Jiaotong University School of Medicine,160 Pujian Rd","correspondingAuthor":false,"prefix":"","firstName":"Chong","middleName":"","lastName":"Xie","suffix":""},{"id":491645737,"identity":"dfde3305-1496-4086-b5a1-bfa4437ddf31","order_by":7,"name":"Yi Chen","email":"","orcid":"","institution":"Department of Neurology,Renji Hospital, Shanghai Jiaotong University School of Medicine,160 Pujian Rd","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Chen","suffix":""},{"id":491645738,"identity":"f8861af1-6873-42c2-b04d-c4f482f4f604","order_by":8,"name":"Xinyu Lin","email":"","orcid":"","institution":"Department of Neurology,Renji Hospital, Shanghai Jiaotong University School of Medicine,160 Pujian Rd","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Lin","suffix":""},{"id":491645739,"identity":"1817f686-1e54-4bcb-9908-a3770219278b","order_by":9,"name":"Yangtai Guan","email":"","orcid":"https://orcid.org/0000-0001-7322-6842","institution":"Department of Neurology,Punan Branch,Renji Hospital, Shanghai Jiaotong University School of Medicine,279 Linyi Rd","correspondingAuthor":false,"prefix":"","firstName":"Yangtai","middleName":"","lastName":"Guan","suffix":""}],"badges":[],"createdAt":"2025-07-24 15:02:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7206859/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7206859/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-025-06028-6","type":"published","date":"2026-01-03T15:57:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87893005,"identity":"5bd25567-753e-430c-94ef-56e5caa5ab68","added_by":"auto","created_at":"2025-07-30 07:03:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":145310,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDown-regulation of METTL3 expression in B cells of EAE mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A and B) METTL3 and YTHDF1 mRNA Expression in Peripheral Blood Mononuclear Cells (PBMC) from Healthy People and MS Patients, (\u003cem\u003en\u003c/em\u003e=3 to 4, for Fig1A, \u003cem\u003ep\u003c/em\u003e=0.02684, for Fig1B, \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05, (C) Comparison of METTL3 mRNA expression levels in splenocytes from EAE mice and healthy control mice using real-time quantitative PCR (\u003cem\u003en\u003c/em\u003e=4, \u003cem\u003eP\u003c/em\u003e=0.000097). (D) B lymphocytes were isolated from splenocytes using CD19 magnetic beads, and sorting efficiency and purity were verified by flow cytometry. (E) METTL3 mRNA expression in B cells of EAE and healthy control mice was detected using q-PCR (\u003cem\u003en\u003c/em\u003e=3, \u003cem\u003eP\u003c/em\u003e=0.0022). (F) Detection of METTL3 protein expression in B cells of EAE and healthy control mice using Western blot analysis. (G) Schematic of CRISPR-Cas9-mediated METTL3 knockdown strategy showing disruption of the target locus and METTL3 gene expression. (H) Schematic of the breeding strategy for\u003cem\u003e Mettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre \u003c/sup\u003emice. (I) METTL3 protein expression in B cells of \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre \u003c/sup\u003emice was analyzed to confirm the efficiency of the knockout. (data are presented as mean ±SEM, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, ns represents no significant difference between the two groups)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7206859/v1/b7bb9b7c0859dfb2e9411502.png"},{"id":87893006,"identity":"d37f28bf-5333-41d8-ade5-46b6b6af2d72","added_by":"auto","created_at":"2025-07-30 07:03:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1064242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMETTL3 deficiency in B cells exacerbates EAE.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Behavioral scores obtained on a daily basis in \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice immunized with MOG\u003csub\u003e35-55\u003c/sub\u003e peptide emulsified with complete Freund's adjuvant (n = 22 to 32). (B and C) Statistical analysis of clinical scores and area under the curve on day 17 after EAE modelling in \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice (\u003cem\u003en\u003c/em\u003e = 22 to 32, for Fig2B, \u003cem\u003eP\u003c/em\u003e=0.0080, for Fig2C, \u003cem\u003eP\u003c/em\u003e=0.00036). (D and E) Statistical analysis of H\u0026amp;E staining and inflammatory cell infiltration in intact spinal cord sections from \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice, scale bar = 100 μm, (\u003cem\u003en\u003c/em\u003e = 4 to 6, \u003cem\u003eP\u003c/em\u003e=0.0013). (F and G) Statistical analysis of LFB staining and myelin loss in intact spinal cord sections from \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice, scale bar = 100 μm, (\u003cem\u003en\u003c/em\u003e = 4 to 6, \u003cem\u003eP\u003c/em\u003e=01123). (H and I) Immunofluorescence images of MBP (green) in the lumbar spinal cord of \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice. Cell nuclei were labelled with DAPI staining (blue), (\u003cem\u003en\u003c/em\u003e=5 to 5, \u003cem\u003eP\u003c/em\u003e=0.003815). Zoom bar = 100 μm. (data = mean ±SEM, * represents a significant difference, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, **** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7206859/v1/555f2148e53fc8a86a17645f.png"},{"id":87892594,"identity":"e8771db7-992c-4c8a-9214-65aae40c65ac","added_by":"auto","created_at":"2025-07-30 06:55:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":129483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMETTL3 deletion enhances B cell function after EAE induction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A and B) Proportion of splenic B cells in \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice after EAE induction and their statistical analysis; (\u003cem\u003en\u003c/em\u003e=6 to 7, \u003cem\u003eP\u003c/em\u003e=0.00442). (C and D) Proportion of B cells in lymph nodes of \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice after EAE induction and their statistical analysis; (\u003cem\u003en\u003c/em\u003e=4 to 8, \u003cem\u003eP\u003c/em\u003e=0.01892). (E and F) Representative dot plots showing the proportion of PC cells in the spleen of \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice and their statistical analyses, (\u003cem\u003en\u003c/em\u003e=6 to 7, \u003cem\u003eP\u003c/em\u003e=0.0003). (G and H) Representative dot plots showing the proportion of PC cells in lymph nodes of \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice and their statistical analysis; (\u003cem\u003en\u003c/em\u003e=4 to 8, \u003cem\u003eP\u003c/em\u003e=0.00159). (I) Histogram of CFSE-labelled B cells on a flow cytometer, with the x-axis indicating fluorescence intensity and the y-axis indicating cell number. (J) Flow cytometry detection of B cell apoptotic capacity in \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice after EAE induction. (K) Statistical analysis of B cell proliferation detected by CFSE (\u003cem\u003en\u003c/em\u003e=6 to 4, \u003cem\u003eP\u003c/em\u003e=0.003976). (L) Statistical analysis of B cell apoptosis detected by Annexin V/PI staining (\u003cem\u003en\u003c/em\u003e=4 to 5, \u003cem\u003eP\u003c/em\u003e=0.0010). (data = mean ±SEM, * represents a significant difference, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7206859/v1/5ea9260787ccbfa825c57e1a.png"},{"id":87892593,"identity":"ba92cfed-63e4-45c4-8db0-4187034f4a9e","added_by":"auto","created_at":"2025-07-30 06:55:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":133213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eB cell-specific deletion of METTL3 inhibits differentiation of Breg subpopulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A and E) Detection of splenic CD1d\u003csup\u003ehi\u003c/sup\u003eCD5\u003csup\u003e+ \u003c/sup\u003eB cell subpopulations in \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice after EAE induction by flow cytometry and their statistical analysis (\u003cem\u003en\u003c/em\u003e=5 to 7, \u003cem\u003eP\u003c/em\u003e=0.00092). (B and F) Detection of splenic IL-10\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e B cell subpopulations in \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice after EAE induction by flow cytometry and their statistical analysis (\u003cem\u003en\u003c/em\u003e=4 to 4, \u003cem\u003eP\u003c/em\u003e=0.023709). \u0026nbsp;(C and G) Percentage of Treg cells in CD4+ cells of \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e detected by flow cytometry and their statistical analysis (\u003cem\u003en\u003c/em\u003e=5 to 6, \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05). (D and H) Percentage of CD4\u003csup\u003e+\u003c/sup\u003e T cells in splenocytes of \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e detected by flow cytometry and their statistical analysis (\u003cem\u003en\u003c/em\u003e=5 to 7, \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05). (data = mean ±SEM, ns represents no statistical difference, * represents a significant difference, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ns represents no significant difference between the two groups)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7206859/v1/af80c52e5e7ab2ecd3a4284e.png"},{"id":87893009,"identity":"bfc2afd4-40a9-4bf2-bccc-c43243dcb8fb","added_by":"auto","created_at":"2025-07-30 07:03:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":182979,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eB-cell-specific deficiency of METTL3 promotes peripheral inflammatory responses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A and B) Flow cytometry to detect the proportion of F4/80\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e cells in the spleen of \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice and its statistical analysis (\u003cem\u003en\u003c/em\u003e=5 to 5, \u003cem\u003eP\u003c/em\u003e=0.000113). (C and D) Flow cytometry to detect the spleen iNOS\u003csup\u003e+ \u003c/sup\u003eof \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice macrophage ratio and its statistical analysis (\u003cem\u003en\u003c/em\u003e=5 to 5, \u003cem\u003eP\u003c/em\u003e=0.000121). (E and F) Flow cytometry to detect the spleen CD206\u003csup\u003e+ \u003c/sup\u003eof \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice macrophage ratio and its statistical analysis (\u003cem\u003en\u003c/em\u003e=5 to 5, \u003cem\u003eP\u003c/em\u003e=0.001045). (G-N) Cytometric bead array (CBA) method to detect the secretion of inflammatory factors in peripheral blood of \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice by flow cytometry (\u003cem\u003en\u003c/em\u003e=5 to 5, for Fig5G, p=0.000955, for Fig5H, \u003cem\u003ep\u003c/em\u003e=0.042108, for Fig5I, \u003cem\u003ep\u003c/em\u003e=0.057755, for Fig5J-N, \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05). (O) Transcriptomic analysis of IL-10 in B cells from \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice (\u003cem\u003en\u003c/em\u003e=3 to 3, \u003cem\u003eP=\u003c/em\u003e020081). (data = mean ±SEM, ns represents no statistical difference, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7206859/v1/eb040317eb437f69e87cfe67.png"},{"id":87892596,"identity":"ae4d2fda-8550-4bc2-988a-a8c8f471e079","added_by":"auto","created_at":"2025-07-30 06:55:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":76397,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombined multi-omics analysis of predicted targets of METTL3 action in B cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Combined analysis of differential expression and differential m6A modification. X-axis indicates genes with differential m6A, Y-axis indicates differentially expressed genes, and red dots represent genes with decreased levels of both. All of the above genes are \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.00001. (B) Analysis of the top ten KEGG pathways where both m6A modification levels and transcriptomes are decreased. (C) Visualization of m6A-modified genes \u003cem\u003eSlit3\u003c/em\u003e versus \u003cem\u003eSrgap3\u003c/em\u003e in \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e EAE mice by Integrated Genomics Viewer (IGV). (D) MeRIP-qPCR validation of the top six differentially expressed genes. All of the above genes are \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.00001. (E) Detection of overall levels of RNA methylation in B cells of \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e and \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e EAE mice (\u003cem\u003eP\u003c/em\u003e=0.046245). (\u003cem\u003en\u003c/em\u003e=3, data = mean ±SEM, ns represents no statistical difference, * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7206859/v1/0945f69a2b50404169d3cebf.png"},{"id":87892599,"identity":"d189e01a-97e6-49d1-91c7-c3e70332b741","added_by":"auto","created_at":"2025-07-30 06:55:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":72202,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of METTL3 in B cells alleviates EAE.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Behavioural scores measured daily in WT mice and OE mice after inoculation with MOG\u003csub\u003e35-55\u003c/sub\u003e peptide emulsified with complete Freund's adjuvant (\u003cem\u003en\u003c/em\u003e = 4 to 10). (B and C) Statistical analysis of behavioural scores and incidence rates in WT and OE mice 21 days after EAE modelling (\u003cem\u003en\u003c/em\u003e = 4 to 10, \u003cem\u003eP\u003c/em\u003e=0.070764). (D) Expression of METTL3 in B cells of WT and OE mice (data are presented as mean ± SEM; *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7206859/v1/f4422a6c335c263cbf4e205f.png"},{"id":99545614,"identity":"64465111-f42c-483d-a5a6-feea00259043","added_by":"auto","created_at":"2026-01-05 16:09:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2682023,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7206859/v1/d3d30f92-5e85-4e2c-94da-e530414d5684.pdf"},{"id":87892602,"identity":"d4f04a70-b377-4903-b0ca-50d67e02146f","added_by":"auto","created_at":"2025-07-30 06:55:18","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":96824,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7206859/v1/18d8cbfcb6d9c15312cdefc0.docx"}],"financialInterests":"","formattedTitle":"B Cell-specific METTL3 depletion exacerbates experimental autoimmune encephalomyelitis","fulltext":[{"header":"Background","content":"\u003cp\u003eMultiple sclerosis (MS) is a complex autoimmune disease of the CNS that is characterized primarily by inflammatory demyelination and axonal damage and results in neurological dysfunction in affected individuals[1-3]. Although the exact pathogenesis of MS remains incompletely understood, an increasing body of evidence suggests that B cells play critical roles in both the initiation and progression of the disease,\u0026nbsp;for example, anti-CD20 drugs can significantly improve the condition of MS, primarily by targeting the survival and function of B cells[4-7]. Experimental autoimmune encephalomyelitis (EAE) is a classic animal model of MS, capable of mimicking the typical pathological phenotype of MS and commonly used to study the pathological mechanisms of MS. The main pathological features of EAE include inflammation and demyelination in the spinal cord, brainstem, and optic nerves.\u0026nbsp;Research\u0026nbsp;indicates that EAE is primarily mediated by T cells; however, a growing body of evidence suggests that B cells also play a critical role in the pathogenesis of MS and EAE[8,9]. B cells not only\u0026nbsp;are\u0026nbsp;immune cells that produce antibodies but also function as antigen-presenting cells (APCs), activating T cells via the MHC II pathway[10]. Furthermore, B cells secrete various cytokines, such as IL-6, TNF-α, and GM-CSF, which play pivotal roles in the pathogenesis of MS. B cell targeted therapies, such as anti-CD20 monoclonal antibodies (e.g., rituximab and ocrelizumab), effectively alleviate symptoms and delay disease progression in MS patients, thereby underscoring the importance of B cells in this condition[11]. In the EAE model, B-cell activity is closely associated with disease progression. Studies have demonstrated that B cells can influence T-cell responses and other immune responses through antigen presentation and the secretion of\u0026nbsp;proinflammatory\u0026nbsp;cytokines, thereby modulating neuroinflammation in EAE[8,10,11].\u003c/p\u003e\n\u003cp\u003eN\u003csup\u003e6\u003c/sup\u003e-Adenine methylation (m\u003csup\u003e6\u003c/sup\u003eA) is one of the most common mRNA modifications and is widely found in eukaryotic cells. m\u003csup\u003e6\u003c/sup\u003eA modifications regulate gene expression by affecting processes such as splicing, stability, transport and translation of mRNAs[12]. m\u003csup\u003e6\u003c/sup\u003eA modifications of major methyltransferase complexes include methyltransferase-like 3 (METTL3), METTL14, and Wilms tumor1–associated protein, with METTL3 and METTL14 being the core methyltransferases responsible for adding methyl groups to mRNAs. Numerous studies have shown that m\u003csup\u003e6\u003c/sup\u003eA modifications play important roles in the development and function of a variety of immune cells[13,14]. For example, the regulatory role of METTL3 in T cells has been shown to be involved in maintaining T cell homeostasis and regulating differentiation and signal transduction, and its mechanisms are mediated mainly by m\u003csup\u003e6\u003c/sup\u003eA modifications that affect the stability and degradation of specific mRNAs, which in turn modulate T cell function and immune responses, further influencing the inflammatory response during EAE[15,16]. m\u003csup\u003e6\u003c/sup\u003eA modifications in macrophages are also important for the inflammatory response, with METTL3 being upregulated upon polarization of M1 macrophages, whereas overexpression of METTL3 promotes M1 polarization but inhibits M2 polarization[17], thereby modulating the immune response. However, the role of METTL3 in the B-cell-regulated immune response is currently unknown.\u003c/p\u003e\n\u003cp\u003eIn recent years, increasing attention has focused on the role of m\u003csup\u003e6\u003c/sup\u003eA modification in B-cell function. The development and function of B cells are complex and multifaceted and involve processes such as antibody production, antigen presentation, and cytokine secretion[2,11]. As a key enzyme in m\u003csup\u003e6\u003c/sup\u003eA modification, the loss of METTL3 in B cells may impact their immune functions. Previous studies have demonstrated that m\u003csup\u003e6\u003c/sup\u003eA modification plays crucial roles in B-cell maturation and antibody production. The absence of METTL3 in B cells impairs their differentiation process, resulting in a significant reduction in antibody production ability[18]. Additionally, m\u003csup\u003e6\u003c/sup\u003eA modification is involved in regulating the gene expression patterns of B cells, influencing their response to external stimuli. For example, the loss of METTL3 may impair the ability of B cells to effectively express certain proinflammatory factors, thereby affecting their role in autoimmune diseases[19]. However, the specific role of m\u003csup\u003e6\u003c/sup\u003eA modification in B cells and its impact on neuroinflammation in EAE remain inadequately understood.\u003c/p\u003e\n\u003cp\u003eThis study elucidates the critical role of B cell-expressed METTL3 in modulating the pathogenesis of experimental autoimmune encephalomyelitis (EAE). We demonstrate that normal METTL3 expression in B cells exerts a protective effect against EAE development, while B cell-specific METTL3 deletion significantly exacerbates disease severity and disrupts B cell function, highlighting the essential regulatory role of B cells in EAE immunopathology. Crucially, to validate this protective function in vivo, we employed transgenic mice with B cell-specific METTL3 overexpression (METTL3-OE). Following EAE induction, these METTL3-OE mice exhibited significantly reduced clinical symptom scores and a markedly lower disease incidence compared to wild-type controls, confirming that enhancing METTL3 expression in B cells mitigates disease progression. Collectively, these findings provide direct in vivo evidence that METTL3 expression within B cells is a key protective factor in EAE, and demonstrate the therapeutic potential of augmenting B cell METTL3 levels to counteract disease progression.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale C57BL/6 mice used in this study were purchased from Charles River, Inc. and were housed at 8-10 weeks of age in the animal house of the Shanghai Institute of Model Organisms. \u003cem\u003eMettl\u003c/em\u003e\u003csup\u003e3flox/flox\u003c/sup\u003e mice and \u003cem\u003eCd19\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e mice were generated by the Southern Model Organisms Research Centre in Shanghai. These \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice were crossed with \u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice (Shanghai Southern Model Organism Research Centre) to obtain \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u0026nbsp;\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice. Similarly, transgenic mice overexpressing METTL3 in B cells and their littermate controls were also purchased from this source. All experimental mice were maintained under SPF conditions with ad libitum access to standard laboratory chow with a 12-hour light-dark cycle, 22 \u0026plusmn; 1\u0026deg;C and 55% \u0026plusmn; 5% humidity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEAE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on previous strategies [20], Myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) from GL Biochem Co Ltd (Shanghai, China) was dissolved into a homogeneous solution of 3 mg/mL in phosphate buffered saline (PBS), which was subsequently emulsified with 8 mg/mL complete Fuchs adjuvant (CFA) from the H37RA strain (Difco, USA) in a 1:1 ratio. The resulting emulsion was administered via intramuscular injection at the bilateral femoral regions of female mice. Concurrently,\u0026nbsp;pertussis toxin (Millipore, Billerica, MA, USA), utilized as an adjuvant to augment the immunogenicity of the antigen, was administered intraperitoneally at a dosage of 200 ng per mouse on the day of immunization (day 0) and 48 hours postimmunization (day 2). As a control for mice in the EAE group, mice in the CFA group were included for all reagents except MOG\u003csub\u003e35-55\u003c/sub\u003e. The clinical manifestations of mice was assessed in a double-blind manner by two independent researchers based on a scoring system as follows: mild tail weakness was assigned a score of 0.5; complete tail paralysis, 1; ataxia accompanied by hind limb weakness, 2; bilateral hind limb paralysis, 3; forelimb weakness, 4; and a moribund state, 5. All experimental mice were euthanised on day 20 for tissue collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePBMC extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collected peripheral blood samples were centrifuged at room temperature to separate the plasma. The cell fraction was then diluted with phosphate buffered saline (PBS, pH 7.4) at a 1:1 ratio. The diluted suspension was carefully spread on Ficoll-Paque Premium (Cytiva China) at a ratio of 2:1 (sample: Ficoll) and then centrifuged in a gradient for 30 minutes. The plasma-Ficoll interface containing PBMC buffer was aspirated, rinsed twice with PBS, centrifuged and the cells were collected for storage or experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHE staining and LFB staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter injection, mouse spinal cords were fixed in 4% paraformaldehyde and tissue sections were\u0026nbsp;taken at the maximum transverse section of the lumbar spine. After dewaxing and rehydration, some sections were placed in haematoxylin-eosin staining solution for HE staining, while others were placed in LFB staining solution and stained overnight at 56\u0026deg;C. Subsequently, differentiation treatment was performed until the boundary between the myelin sheath and white matter was clearly visible. Images were analysed using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence staining\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eParaffin sections were dewaxed and hydrated, soaked in 3% hydrogen peroxide for 10 min at room temperature, then repaired with EDTA antigen repair solution (Solarbio, China) at 100\u0026deg;C for 30 min, cooled to room temperature and then closed, incubated overnight at 4\u0026deg;C with myelin basic protein (MBP) primary antibody (1:200, Abcam, USA), and washed and then incubated with the secondary antibody for 2 h at room temperature. The slices were sealed with DAPI (Beyondi, China) sealer. Sections were examined by fluorescence microscopy (Olympus, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter centrifuging the cell suspension at 400g for 5 minutes, discard the supernatant, resuspend in 100\u0026mu;l PBS, add flow cytometry antibody at a ratio of 1:400, and incubate at 4\u0026deg;C away from light for 30 minutes. Wash twice with PBS. For surface staining: fix with 1% PFA at 4\u0026deg;C away from light for 30 minutes, wash twice with PBS, then resuspend in 200\u0026mu;l PBS for testing. For intracellular/nuclear staining: Incubate with membrane-breaking solution at 4\u0026deg;C for 1 hour, wash with membrane-breaking wash solution, centrifuge at 800g, add the same proportion of antibody, incubate at 4\u0026deg;C in the dark for 30 minutes, wash twice with membrane-breaking wash solution, and resuspend in 200 \u0026mu;l PBS. Store samples at 4\u0026deg;C, perform flow cytometry analysis, and analyse using FlowJo software 10.8.1. All centrifugation steps are performed for 5 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpleen cell extraction and culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlace the mouse spleen in PBS on ice, transfer to a cell culture dish, and mechanically grind through a 40 \u0026mu;m filter. Rinse with PBS to obtain a single-cell suspension. Centrifuge at 400g for 5 minutes, discard the supernatant, add 3 ml of 1\u0026times; red blood cell lysis buffer (Beyotime, China), and lyse for 5 minutes. Terminate with PBS and centrifuge under the same conditions for washing. Resuspend the cells in PBS and count them. Take 5 \u0026times; 10⁶ cells in a 1.5 ml EP tube, centrifuge and discard the supernatant. Resuspend in 1640 medium containing stimulants (20 ng/ml phorbol ester + 1 \u0026mu;g/ml ionomycin + 1\u0026times; Brefeldin A), transfer to a 24-well plate, and incubate at 37\u0026deg;C for 5 hours. Finally, collect the cells for flow cytometric analysis. All centrifugation steps were performed at 400g for 5 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003elymphocyte sorting and culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolation of B cells from mouse spleens was performed by employing CD19 MicroBeads (Miltenyi Biotec, Germany). The extracted spleen cells were resuspended and then incubated with CD19 MicroBeads. After ten minutes, the cells were sorted through LS columns, and the cells adsorbed on the magnetic columns were removed, while the purification of the ooze was repeated 2 times. The sorted B cells were resuspended in RPMI 1640 culture medium (Gibco, Thermo Fisher Scientific), and 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin were added to the culture medium. The cells were then plated into a 24-well plate at a density of 1 \u0026times; 106 cells/mL and cultured at 37\u0026deg;C in a humidified incubator with 5% CO2 for 72 hours. For stimulation and treatment in specific experiments, B cells can be stimulated with lipopolysaccharide (LPS) (Sigma‒Aldrich) at a concentration of 10 \u0026mu;g/mL to induce their activation and proliferation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSorted B cells were incubated with the fluorescent dye CFSE (CFDA-SE) prior to culture. The cells were then resuspended in RPMI 1640 culture medium (Gibco, Thermo Fisher Scientific) containing RPMI 1640 with 10% heat-inactivated foetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were then cultured into 24-well plates at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL and incubated in a humidified incubator at 37\u0026deg;C and 5% CO2. Stimulation and treatment In specific experiments, B cells were stimulated with 10 \u0026mu;g/mL concentration of lipopolysaccharide (LPS) (Sigma-Aldrich) to induce activation and proliferation. Analyses were performed by FlowJo software 10.8.1 after 72h of incubation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnnexin V/PI staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells cultured by the above method were collected into centrifuge tubes, washed with PBS and then stained using the Membrane Associated Protein V/PI Detection Kit (Multi Sciences, China) according to the instructions. Immediately after staining, data were collected by flow cytometry (BD, USA) and analysed using FlowJo software 10.8.1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real\u003c/strong\u003e\u003cstrong\u003e-time\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the cells using TRIzol (Abconal, China), and the concentration was detected to reach the standard, then reverse transcription and real-time PCR were carried out according to the instructions of the kit (Vazyme, China), and GAPDH was used as an internal reference. The relative expression of the genes was calculated using the 2-\u0026Delta;\u0026Delta;Ct method. The primer sequences are shown in Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal protein was extracted from mouse spleen-sorted B cells and then quantified via protein extraction via a BCA kit (Thermo Fisher, USA). Equal amounts of protein were added to SDS polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. The membrane was blocked with blocking solution (Epizyme Biotech, China) and then incubated with primary antibody (METTL3, 1:1000, Abmart, China; GAPDH, 1:10,000, Jackson ImmunoResearch, USA) overnight at 4\u0026deg;C, followed by incubation with secondary antibody for 1 h. After incubation, protein signals were detected using an enhanced chemiluminescence kit (Epizyme Biotech, China) in a Western blot imaging analyzer (Bio-Rad, USA). Proteins were quantified via ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytometric\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ebead array\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUse the CBA assay kit (Biolegend, USA) to detect cytokine levels in mouse peripheral blood serum. First, capture the microspheres and vortex vigorously (\u0026ge;1 minute) to ensure uniformity. Lyophilised standards were diluted from the stock concentration (C7) in a series of fourfold dilutions to the lowest concentration (C1). The experiment was conducted in V-bottom 96-well plates: each well was first added with 25 \u0026micro;L of detection buffer, followed by 25 \u0026micro;L of standard or sample. Before use, resuspend the microspheres by vortexing for 30 seconds, then add 25 \u0026micro;L to each well. Seal the plate, incubate in the dark at 800 rpm for 2 hours. Centrifuge at 250 \u0026times; g for 5 minutes, gently discard the supernatant. After two washes, add 25 \u0026micro;L of detection antibody to each well. Seal the plate, incubate in the dark at 800 rpm for 1 hour, then centrifuge and discard the supernatant. Then, add 25 \u0026micro;L of PE-labelled streptavidin to each well, seal the plate, incubate at 800 rpm for 30 minutes, and wash twice. Finally, resuspend the microspheres in 150 \u0026micro;L of wash buffer, transfer to a flow cytometry tube, and analyse using a flow cytometer (BD LSRFortess X-20, USA). Collect \u0026gt;300 microspheres per region for cytokine quantification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeRIP-Seq and MeRIP-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis experiment was performed by Cloud Sequence Biologicals, Shanghai, China. As previously reported[21], RNA samples were standardized to defined concentrations and chemically fragmented (70\u0026deg;C, 6 min) to generate ~200-nt fragments. Following the termination of the reaction, the fragmented RNA was pooled, purified via ethanol precipitation, and validated for size via an Agilent Bioanalyzer, with a subset retained as input controls. For immunoprecipitation, m6A-specific antibodies (or IgG controls) were conjugated to PGM magnetic beads and incubated with fragmented RNA at 4\u0026deg;C for 1 h. Nonspecific interactions were eliminated through sequential washes with IP, LB, and HS buffers. The captured RNA was eluted and further purified via MS magnetic beads (RLT buffer binding, 75% ethanol washes) to obtain high-purity RNA for Illumina library construction and sequencing. The immunoprecipitated RNA was reverse-transcribed into cDNA (SuperScript III, 50\u0026deg;C, 60 min). Quantitative PCR was performed on a QuantStudio 5 system using SYBR Green premix and gene-specific primers under optimized conditions: 95\u0026deg;C predenaturation (10 min), followed by 40 cycles of 95\u0026deg;C (10 s) and 60\u0026deg;C (60 s). Methylation levels were quantified via Ct values normalized to input controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we used GraphPad Prism 9 software for statistical analysis, and the error line indicates the standard error of the mean (SEM). For two-group comparisons, a t test was used for normally distributed data, and a nonparametric test was used for nonnormally distributed data. For multiple comparisons, ANOVA (two-way) was performed. p\u0026lt;0.05 was considered statistically significant. Details are provided in the figure legends.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eDecreased METTL3 expression in B cells after EAE induction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIt has been previously shown that in CD4\u003csup\u003e+\u003c/sup\u003e T cells, deletion of the m\u003csup\u003e6\u003c/sup\u003eA methyltransferase METTL3 severely interferes with the homeostatic proliferation of these cells and their ability to differentiate into effector cells, thereby alleviating the pathological features of EAE[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Similarly, research has indicated that specific knockout of the m\u003csup\u003e6\u003c/sup\u003eA demethylase AlkB homologue 5 (ALKBH5) in T cells confers protection against EAE [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, no study has investigated whether m\u003csup\u003e6\u003c/sup\u003eA regulates the pathological process of EAE through B cells. In this study, we initially assessed the mRNA expression levels of the m\u003csup\u003e6\u003c/sup\u003eA-related enzymes METTL3 and YTH N\u003csup\u003e6\u003c/sup\u003e-methyladenosine RNA binding protein F1 (YTHDF1) throughout disease progression in patients with MS. By detecting the mRNA expression of \u003cem\u003eMETTL3\u003c/em\u003e and Y\u003cem\u003eTHDF1\u003c/em\u003e in peripheral blood mononuclear cells (PBMCs) from patients with MS and healthy controls, we found that, compared with that in healthy controls, the mRNA expression of METLL3 in MS patients was significantly decreased, whereas that of YTHDF1 was not significantly different (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). We next explored the expression of METTL3 in the B lymphocytes of EAE mice and detected significantly decreased expression of METTL3 mRNA in the splenocytes of EAE mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). B cells in the spleen were sorted via magnetic bead sorting, and the sorting efficiency of the B cells was greater than 90% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Further analysis of the sorted B cells revealed that METTL3 expression was significantly lower at both the mRNA and protein levels in B cells from EAE mice than in those from healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F). These results suggest that METTL3 may play an important role in the progression of EAE. We subsequently constructed \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e mice with specific deletion of METTL3 in B cells and used their littermate \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice as controls to further explore the relationship between METTL3 and EAE. By detecting the expression of METTL3 in B cells of \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice, we observed that METTL3 was obviously knocked down (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-I). In conclusion, METTL3 mRNA expression was decreased in the PBMCs of MS patients; similarly, METTL3 expression was decreased in the B cells of EAE mice. In addition, we successfully constructed \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice for this study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2 Deletion of METTL3 in B cells exacerbates EAE\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further clarify the function of METTL3 in the B cells of EAE mice, we induced EAE in \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice and their littermate control \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice via the myelin oligodendrocyte glycoprotein (MOG) peptide MOG\u003csub\u003e35\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e and assessed their clinical performance via behavioral assessment. The results revealed that \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice presented significantly higher clinical scores than controls group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). Considering that inflammatory cell infiltration and demyelination are the most typical pathological features of EAE mice, we examined inflammatory infiltration and demyelination in the spinal cord of EAE mice via H\u0026amp;E staining and LFB staining, respectively. The results revealed that the number of inflammatory cells was significantly increased in the spinal cords of mice lacking METTL3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and E). We also observed more pronounced spinal cord demyelination in the mice in the \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and G). In addition, the results of MBP immunostaining revealed a similar trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH and I). These results suggest that MTTL3 deficiency in B cells significantly aggravates the clinical symptoms and pathological phenotype of EAE.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.3 METTL3 deficiency exacerbates proliferation and apoptosis of B cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further investigate how METTL3 regulates B-cell function after EAE induction, we used flow cytometry to analyse the composition of B cells in the spleen and inguinal lymph node sites, and the proportion of B cells in the spleen of the \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice was significantly reduced compared with that in the \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Similarly, we found that the proportion of B cells in inguinal lymph nodes was also reduced in \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D). These findings suggest that METTL3 knockout may affect the function of B cells in response to inflammation, resulting in their decline. METTL3 was previously shown to collaborate with insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) to regulate the cell cycle response of Germinal Center B cells(GCB) by affecting the stability of \u003cem\u003eMyc\u003c/em\u003e mRNA, and a lack of METTL3 resulted in slow progression of the cell cycle and a reduced ability to differentiate into plasma cells in response to half-antigen stimulation[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Similarly, compared with those in the \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e group, the numbers of plasma cells in the spleen and lymph nodes of the mice in the \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e group were also significantly lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-H). In addition, we sorted B cells from the spleen, pertained them with CFSE, cultured them for 72 hours, and detected their fluorescence intensity in response to their degree of proliferation. These results indicate that METTL3-specific deletion promotes B-cell proliferation. Furthermore, we examined the apoptosis of B cells in the two groups of mice and found that the proportion of apoptotic cells was significantly greater in the \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e group than in the control group. These results suggest that specific METTL3 knockout promotes the functional response of B cells in response to inflammatory stimuli and promotes B-cell proliferation to a certain extent, but the promotion of apoptosis is more pronounced.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.4 B-cell-specific deletion of METTL3 inhibits the differentiation of regulatory B cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn EAE, B cells regulate the inflammatory response mainly through the secretion of inflammatory factors, including a class of regulatory B-cell subpopulations expressing CD1d and CD5, whose normal expression inhibits the inflammatory response and attenuates the degree of the autoinflammatory response. To further investigate how knockout of METTL3 exacerbates the pathological manifestations of EAE, we examined T-cell and B-cell subsets in the spleens of EAE mice after knockout of METTL3. We found that the proportion of CD1d\u003csup\u003ehi\u003c/sup\u003eCD5\u003csup\u003e+\u003c/sup\u003e B cells among splenic B cells was significantly lower in the \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e group than in the \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and E), further testing of the proportion of IL-10\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003eB cells revealed a significant decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and F). But the Treg subset did not change significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and G), suggesting that the inhibitory role of B cells in the pathogenesis of EAE is diminished. Previous studies have demonstrated that CD1d\u003csup\u003ehi\u003c/sup\u003eCD5\u003csup\u003e+\u003c/sup\u003e regulatory B cells constitute a crucial immune cell population that performs negative immunomodulatory functions in vivo and influences the progression of EAE by regulating the differentiation of CD4\u003csup\u003e+\u003c/sup\u003e T cells. Therefore, we performed flow analyses after stimulating T cells as previously reported in the literature and found that knockout of METTL3 in B cells did not affect the overall proportion of CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and H). In conclusion, the above results suggest that deletion of METTL3 in B cells impedes the suppressive effects of B cells on inflammation, but this pathway is not achieved by regulating CD4\u003csup\u003e+\u003c/sup\u003e T cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.5 B cell-specific deletion of METTL3 promotes macrophage inflammatory response and peripheral inflammation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further clarify the cause of the more severe morbidity in the \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e group of mice, we examined the splenic macrophages of EAE mice and found that macrophages were significantly activated in the spleens of the \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e group of mice compared with those of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). Further examination of the iNOS\u003csup\u003e+\u003c/sup\u003e macrophages revealed that they were significantly elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D). Interestingly, we also detected a significant increase in the proportion of CD206\u003csup\u003e+\u003c/sup\u003e macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and F). In addition, we examined peripheral blood inflammatory factors in EAE mice and found a trend toward increased levels of the proinflammatory factor interleukin 6 (IL-6), a significant increase in the secretion of TNF-family B-cell activating factor (BAFF) and B-cell maturation antigen (BCMA), However, we found that there was no significant difference in IL-10 secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-N). Considering that we tested peripheral blood serum, we then analysed the transcription level of IL-10 in B cells and found that it had decreased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO), suggesting that the specific deletion of Mettl3 in B cells exacerbated the EAE-induced peripheral inflammatory response. In conclusion, our study revealed that the deletion of METTL3 in B cells promotes macrophage activation, which exacerbates inflammation, and, it may lead to more severe clinical signs in EAE mice by affecting the secretion of inflammatory factors in the peripheral immune system.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.6 B cell-specific METTL3 deficiency downregulates key genes in the axon guidance pathway through an m⁶A-dependent mechanism\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo clarify the molecular targets of METTL3 in EAE mouse B cells, we performed a multiomics joint analysis of RNA-seq and MeRIP-seq data to screen for genes with significantly downregulated levels of m⁶A modification and transcription after METTL3 deletion, including \u003cem\u003eSrgap3, Slit3, Nrp2, Celsr2, Tox2\u003c/em\u003e, and \u003cem\u003eZfp532\u003c/em\u003e, and KEGG pathway enrichment analysis revealed that METTL3 knockdown resulted in a significant reduction in Axon guidance pathway activity in B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). Among them, \u003cem\u003eSlit3\u003c/em\u003e and \u003cem\u003eSrgap3\u003c/em\u003e play important roles in the normal function of axon guidance. On the basis of these findings, we hypothesized that METTL3 may affect the neural function of EAE mice by regulating the expression of \u003cem\u003eSlit3\u003c/em\u003e and \u003cem\u003eSrgap3\u003c/em\u003e. Next, via IGV visualization, we revealed that \u003cem\u003eSlit3\u003c/em\u003e and \u003cem\u003eSrgap3\u003c/em\u003e are target genes for METTL3 action, and the results revealed that the m6A modification peaks of \u003cem\u003eSlit3\u003c/em\u003e and \u003cem\u003eSrgap3\u003c/em\u003e were significantly decreased in \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e EAE mice, which was further verified by MeRIP-qPCR. The results revealed that the m6A levels of both target genes were significantly decreased. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and D). The overall methylation level of RNA in B cells was examined, and as expected, the deletion of METTL3 in B cells resulted in a decrease in their overall m6A level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). In summary, the above results not only confirmed the reliability of the multiomics data but also revealed that METTL3 regulates the expression of \u003cem\u003eSlit3\u003c/em\u003e and \u003cem\u003eSrgap3\u003c/em\u003e through a m⁶A-dependent mechanism, thereby inhibiting the activity of the axon conductance pathway, which may be a key molecular mechanism by which the absence of METTL3 leads to neurological impairments in EAE mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.7 Overexpression of METTL3 in B cells significantly alleviates the symptoms of EAE\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFinally, to validate these findings in vivo, we employed transgenic mice with B cell-specific overexpression of METTL3. Following induction of experimental autoimmune encephalomyelitis (EAE), these METTL3-overexpressing (OE) mice exhibited significantly reduced clinical symptom scores and a markedly lower disease incidence compared to wild-type (WT) controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). Confirmation of successful METTL3 overexpression was obtained by detecting significantly elevated METTL3 levels specifically within B cells of the OE mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Collectively, these results provide direct in vivo evidence supporting the protective role of B cell-expressed METTL3 in the EAE model.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003em\u003csup\u003e6\u003c/sup\u003eA is one of the most common modifications of eukaryotic mRNAs and noncoding RNAs and regulates gene expression by affecting RNA splicing, translation and stability[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. By affecting gene expression, in which METTL3 is the most central catalytic subunit of m\u003csup\u003e6\u003c/sup\u003eA, it can regulate the normal function of immune cells[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Although METTL3 controls various aspects of T cell development and activity, thus providing good intervention in autoimmune diseases[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], the role of METTL3 in B cells is not fully understood. In the present study, we observed reduced expression of METTL3 in PBMCs from MS patients and reduced expression of METTL3 in B cells from EAE model mice and further revealed that specific knockout of METTL3 in B cells significantly exacerbated the clinical symptoms of EAE. In addition, studies on B cell function have shown that knockout of METTL3 results in enhanced B-cell proliferation and apoptosis under inflammatory conditions, but apoptosis is more pronounced. Considering that more definitive studies on the regulatory role of B cells in EAE have focused on the Breg subpopulation, our analyses of B cell subpopulations suggest that METTL3 deletion leads to a decrease in CD1d\u003csup\u003ehi\u003c/sup\u003eCD5\u003csup\u003e+\u003c/sup\u003e B cells, which may result in diminished inhibition of the inflammatory response. Furthermore, METTL3 deletion increases the secretion of IL-6, BAFF, and BCMA and decreases IL-13 secretion in the peripheral blood, thereby exacerbating the inflammatory response.\u003c/p\u003e\u003cp\u003eA growing body of research suggests that the regulatory role of B cells in the disease process is also critical for EAE. B cells are important components of the adaptive immune system and function in antigen presentation, cytokine secretion, and antibody production[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It has been reported that specific deletion of METTL3 in B cells has little effect on the normal development and growth of mice, whereas functionally, knockout of METTL3 promotes apoptosis in B cells, which is consistent with our observations[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In addition, deletion of Mettl3 at early stages of B cell differentiation via Mb1-Cre blocks B cell differentiation, specifically affecting the transition from pro-B to large pre-B and from large pre-B to small pre-B[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and inhibition of Mettl3-mediated m\u003csup\u003e6\u003c/sup\u003eA modification in hematopoietic stem cells (HSCs) during B-cell development leads to impaired HSC differentiation, thereby reducing the proportion of peripheral B cells[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These findings suggest that knocking down METTL3 in B cells might theoretically provide modest alleviation of B cell-mediated inflammatory responses. However, the reality was the opposite of what was expected. In the EAE mouse model, when B cells are deficient in METTL3, the mice instead show more severe manifestations of the disease. This phenomenon indicates that METTL3 may play a protective role in peripheral B cells during EAE associated inflammation and that its presence may be important for maintaining the balance and stability of B cell associated immune responses. Earlier studies have reported that in EAE mice, gut-derived IgA\u003csup\u003e+\u003c/sup\u003e PC cells migrate to the CNS, where their number decreases during EAE, and the removal of PC cells exacerbates EAE symptoms, whereas the introduction of gut-sourced IgA\u003csup\u003e+\u003c/sup\u003e PC cells alleviates symptoms[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In addition, studies have indicated that specific deletion of METTL3 results in slowed cell cycle progression, reduced expression of genes associated with proliferation and oxidative phosphorylation, and a decreased proportion of plasma cells in GCB cells[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Similarly, in our study, we observed a reduction in the number of plasma cells in the spleens and lymph nodes of EAE-induced \u003cem\u003eMettl3\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e\u003cem\u003eCd19\u003c/em\u003e\u003csup\u003eCre\u003c/sup\u003e mice; however, whether knockout of METTL3 in B cells leads to a decrease in IgA\u003csup\u003e+\u003c/sup\u003e PC cells in the intestine, thereby exacerbating EAE, deserves further exploration.\u003c/p\u003e\u003cp\u003eAccording to previous studies, EAE is primarily a type of T cell mediated inflammation of the nervous system in which the role of B cells is not particularly clear. Our study provides evidence for the role of METTL3 in B cells during EAE. Specifically, knockout of METTL3 in B cells decreased the proportion of splenic CD1d\u003csup\u003ehi\u003c/sup\u003eCD5\u003csup\u003e+\u003c/sup\u003e cells and increased the proportion of macrophages, suggesting an increased inflammatory response. However, further examination of macrophages revealed an increase in the proportion of both M1-type macrophages and M2-type macrophages, and previous studies have suggested that the activation of M1-type macrophages may lead to chronic inflammation and tissue damage, whereas the activation of M2-type macrophages contributes to inflammation clearance and tissue repair. In addition, knockout of METTL3 in B cells promoted the secretion of the inflammatory factors IL-6, BAFF, and BCMA but had no effect on the secretion of IFN-γ, IL-17 or IL-10. Therefore, the activation of macrophages may contribute to the exacerbation of EAE and profound inflammation, and the increased expression of CD206 may be a secondary response. IL-6 is a key immunomodulatory cytokine that influences the pathogenesis of a variety of diseases, including autoimmune disorders and inflammation[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. BAFF is a cytokine belonging to the tumour necrosis factor (TNF) family that is essential for the proliferation and survival of B cells. It influences the regulation of immune responses by binding to specific receptors on the surface of B cells (e.g., BAFF-R, TACI, and BCMA) [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and is expressed in a wide variety of cells, including monocytes, macrophages, dendritic cells, and B and T lymphocytes. In addition, astrocytes from MS patients also express BAFF[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Previous studies have shown that specific variants in the TNFSF13B gene, which encodes the cytokine BAFF, are associated with an increased risk of autoimmune diseases such as multiple sclerosis (MS) and systemic lupus erythematosus (SLE) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In addition, there is much visual evidence that BAFF is markedly elevated in PBMCs from spinal cord injury (SCI) patients and that BAFF can be largely alleviated in EAE mice by inhibiting BAFF secretion in EAE mice[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, blocking BAFF can inhibit macrophage activation by reducing inflammation, and blocking BAFF reduces the secretion of inflammatory cytokines by macrophages [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, whether BAFF upregulation directly drives macrophage activation and exacerbates EAE requires further investigation. Moreover, the specific mechanisms by which Mettl3 affects B cell function and the immunological mechanisms that contribute to the pathogenesis of EAE need to be further explored. In addition, previous studies have shown that \u003cem\u003eSlit3\u003c/em\u003e, a key molecule in axon guidance, is involved in peripheral nerve injury repair through the Slit3/Robo signalling pathway[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], whereas \u003cem\u003eSrgap3\u003c/em\u003e (Slit-Robo GTPase-activating protein 3) dynamically maintains cytoskeletal reorganization by inhibiting the activity of Rac1, a Rho GTPase, and regulating actin remodelling. Notably, \u003cem\u003eSrgap3\u003c/em\u003e deficiency can lead to abnormal synaptic function and cognitive behavioural deficits[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. By combined multiomics analysis, we hypothesized that the deletion of METTL3 in B cells caused a decrease in the expression of \u003cem\u003eSlit3\u003c/em\u003e and \u003cem\u003eSrgap3\u003c/em\u003e as well as the downregulation of the axon guidance pathway, which further aggravated neurological damage in EAE mice; however, the specific mechanisms involved also deserve further exploration. At the conclusion of the study, the significantly alleviated clinical symptoms and reduced incidence rate in the transgenic EAE mouse model with B cell-specific overexpression of METTL3 directly confirmed the protective role of METTL3 in vivo. Our subsequent research needs to expand the sample size and further explore the specific mechanisms involved.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study describes the critical role of Mettl3 in B cells and its impact on the pathogenesis of EAE. These findings highlight the importance of Mettl3 in regulating B cell function and the immune response and provide new avenues for therapeutic intervention in MS and other autoimmune diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Shanghai Southern Model Biotechnology Co., Ltd. for their valuable assistance in the construction and maintenance of transgenic mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXZP: designed and performed the experiments, analysed the data, and wrote the original manuscript; HY: designed and performed the experiments, analysed the data, and revised the manuscript; JD: wrote the original manuscript and analysed the data; HJY, CRX, XYW, and CX: participated in the experiments and revised the manuscript; YC and XYL: participated in the experiments; YTG: Conceptualization, Review and editing, Supervision, Project administration, Funding acquisition. All the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the\u0026nbsp;National Natural Science Foundation of China (No. 82201495), Shanghai Municipal Health Commission (No. 202140414), Municipal Commission of Health and Family Planning Foundation of Shanghai Pudong New Area (No. PW2022E-01), Innovative Research Team of High-Level Local Universities in Shanghai (No. SHSMU-ZDCX20211901), and the New Quality Clinical Specialties of High-end Medical Disciplinary Construction in Pudong New Area (No. 2024-PWXZ-16).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data has been made available to the public under the registration number PRJNA1255398.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003ehttp://www.ncbi.nlm.nih.gov/bioproject/1255398.\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMS patients and healthy human samples were ethically compliant and approved by the Ethics Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine (Grant No. 2023-026-A); for animals, approval for experiments was granted by the Animal Care and Use Committee. (IACUC NO:2023-0056-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCrowley T,Chen J,Rosiewicz K Set al(2025) Mapping CD4+ T cell diversity in CSF to identify endophenotypes of multiple sclerosis.Brain Commun.7:fcaf231.http://doi.org/10.1093/braincomms/fcaf231.\u003c/li\u003e\n\u003cli\u003eRodr\u0026iacute;guez Mur\u0026uacute;a S,Farez M F，Quintana F J(2022) The Immune Response in Multiple Sclerosis.Annu Rev Pathol.17:121-139.http://doi.org/10.1146/annurev-pathol-052920-040318.\u003c/li\u003e\n\u003cli\u003eMozafari S,Starost L,Manot-Saillet Bet al(2020) Multiple sclerosis iPS-derived oligodendroglia conserve their properties to functionally interact with axons and glia in vivo.Sci Adv.6.http://doi.org/10.1126/sciadv.abc6983.\u003c/li\u003e\n\u003cli\u003eDisanto G,Sacco R,Mallucci G,Zecca C，Gobbi C(2025) Effect of Cumulative Exposure to Ocrelizumab on Memory B-Cell Repopulation Dynamics in Multiple Sclerosis.Ann Neurol.http://doi.org/10.1002/ana.27281.\u003c/li\u003e\n\u003cli\u003evan Puijfelik F,Blok K M,Klein Kranenbarg R A Met al(2024) Ocrelizumab associates with reduced cerebrospinal fluid B and CD20dim CD4+ T cells in primary progressive multiple sclerosis.Brain Commun.6:fcae021.http://doi.org/10.1093/braincomms/fcae021.\u003c/li\u003e\n\u003cli\u003ePolonio C M，Quintana F J(2025) Therapeutic B cell depletion identifies immunoregulatory networks.J Clin Invest.135.http://doi.org/10.1172/JCI189442.\u003c/li\u003e\n\u003cli\u003eMouat I C,Goldberg E，Horwitz M S(2022) Age-associated B cells in autoimmune diseases.Cell Mol Life Sci.79:402.http://doi.org/10.1007/s00018-022-04433-9.\u003c/li\u003e\n\u003cli\u003eVan Kaer L,Postoak J L,Wang C,Yang G，Wu L(2019) Innate, innate-like and adaptive lymphocytes in the pathogenesis of MS and EAE.Cell Mol Immunol.16:531-539.http://doi.org/10.1038/s41423-019-0221-5.\u003c/li\u003e\n\u003cli\u003eMouat I C,Allanach J R,Fettig N Met al(2022) Gammaherpesvirus infection drives age-associated B cells toward pathogenicity in EAE and MS.Sci Adv.8:eade6844.http://doi.org/10.1126/sciadv.ade6844.\u003c/li\u003e\n\u003cli\u003eHua Z，Hou B(2020) The role of B cell antigen presentation in the initiation of CD4+ T cell response.Immunol Rev.296:24-35.http://doi.org/10.1111/imr.12859.\u003c/li\u003e\n\u003cli\u003eSabatino J J,Pr\u0026ouml;bstel A-K，Zamvil S S(2019) B cells in autoimmune and neurodegenerative central nervous system diseases.Nat Rev Neurosci.20:728-745.http://doi.org/10.1038/s41583-019-0233-2.\u003c/li\u003e\n\u003cli\u003eJiang X,Liu B,Nie Zet al(2021) The role of m6A modification in the biological functions and diseases.Signal Transduct Target Ther.6:74.http://doi.org/10.1038/s41392-020-00450-x.\u003c/li\u003e\n\u003cli\u003eLiu C,Yang Z,Li Ret al(2021) Potential roles of N6-methyladenosine (m6A) in immune cells.J Transl Med.19:251.http://doi.org/10.1186/s12967-021-02918-y.\u003c/li\u003e\n\u003cli\u003eCui L,Ma R,Cai Jet al(2022) RNA modifications: importance in immune cell biology and related diseases.Signal Transduct Target Ther.7:334.http://doi.org/10.1038/s41392-022-01175-9.\u003c/li\u003e\n\u003cli\u003eLi H-B,Tong J,Zhu Set al(2017) m6A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways.Nature.548:338-342.http://doi.org/10.1038/nature23450.\u003c/li\u003e\n\u003cli\u003eWang X,Chen C,Sun Het al(2023) m6A mRNA modification potentiates Th17 functions to inflame autoimmunity.Sci China Life Sci.66:2543-2552.http://doi.org/10.1007/s11427-022-2323-4.\u003c/li\u003e\n\u003cli\u003eLiu Y,Liu Z,Tang Het al(2019) The N6-methyladenosine (m6A)-forming enzyme METTL3 facilitates M1 macrophage polarization through the methylation of STAT1 mRNA.Am J Physiol Cell Physiol.317:C762-C775.http://doi.org/10.1152/ajpcell.00212.2019.\u003c/li\u003e\n\u003cli\u003eGrenov A C,Moss L,Edelheit Set al(2021) The germinal center reaction depends on RNA methylation and divergent functions of specific methyl readers.J Exp Med.218.http://doi.org/10.1084/jem.20210360.\u003c/li\u003e\n\u003cli\u003eTurner D J,Saveliev A,Salerno Fet al(2022) A functional screen of RNA binding proteins identifies genes that promote or limit the accumulation of CD138+ plasma cells.Elife.11.http://doi.org/10.7554/eLife.72313.\u003c/li\u003e\n\u003cli\u003eXiang W,Wang K,Han Let al(2024) CD22 blockade aggravates EAE and its role in microglia polarization.CNS Neurosci Ther.30:e14736.http://doi.org/10.1111/cns.14736.\u003c/li\u003e\n\u003cli\u003eWan Y,Tang K,Zhang Det al(2015) Transcriptome-wide high-throughput deep m(6)A-seq reveals unique differential m(6)A methylation patterns between three organs in Arabidopsis thaliana.Genome Biol.16:272.http://doi.org/10.1186/s13059-015-0839-2.\u003c/li\u003e\n\u003cli\u003eZhou J,Zhang X,Hu Jet al(2021) m6A demethylase ALKBH5 controls CD4+ T cell pathogenicity and promotes autoimmunity.Sci Adv.7.http://doi.org/10.1126/sciadv.abg0470.\u003c/li\u003e\n\u003cli\u003eTeng Y,Yi J,Chen J，Yang L(2023) N6-Methyladenosine (m6A) Modification in Natural Immune Cell-Mediated Inflammatory Diseases.J Innate Immun.15:804-821.http://doi.org/10.1159/000534162.\u003c/li\u003e\n\u003cli\u003eGeng Q,Cao X,Fan Det al(2023) Potential medicinal value of N6-methyladenosine in autoimmune diseases and tumours.Br J Pharmacol.http://doi.org/10.1111/bph.16030.\u003c/li\u003e\n\u003cli\u003eGuo W,Wang Z,Zhang Yet al(2024) Mettl3-dependent m6A modification is essential for effector differentiation and memory formation of CD8+ T cells.Sci Bull (Beijing).69:82-96.http://doi.org/10.1016/j.scib.2023.11.029.\u003c/li\u003e\n\u003cli\u003eLu S,Wei X,Zhu Het al(2023) m6A methyltransferase METTL3 programs CD4+ T-cell activation and effector T-cell differentiation in systemic lupus erythematosus.Mol Med.29:46.http://doi.org/10.1186/s10020-023-00643-4.\u003c/li\u003e\n\u003cli\u003eSatitsuksanoa P,Iwasaki S,Boersma Jet al(2023) B cells: The many facets of B cells in allergic diseases.J Allergy Clin Immunol.152:567-581.http://doi.org/10.1016/j.jaci.2023.05.011.\u003c/li\u003e\n\u003cli\u003eKang X,Chen S,Pan Let al(2022) Deletion of Mettl3 at the Pro-B Stage Marginally Affects B Cell Development and Profibrogenic Activity of B Cells in Liver Fibrosis.J Immunol Res.2022:8118577.http://doi.org/10.1155/2022/8118577.\u003c/li\u003e\n\u003cli\u003eLee H,Bao S,Qian Yet al(2019) Stage-specific requirement for Mettl3-dependent m6A mRNA methylation during haematopoietic stem cell differentiation.Nat Cell Biol.21:700-709.http://doi.org/10.1038/s41556-019-0318-1.\u003c/li\u003e\n\u003cli\u003eZheng Z,Zhang L,Cui X-Let al(2020) Control of Early B Cell Development by the RNA N6-Methyladenosine Methylation.Cell Rep.31:107819.http://doi.org/10.1016/j.celrep.2020.107819.\u003c/li\u003e\n\u003cli\u003eRojas O L,Pr\u0026ouml;bstel A-K,Porfilio E Aet al(2019) Recirculating Intestinal IgA-Producing Cells Regulate Neuroinflammation via IL-10.Cell.176.http://doi.org/10.1016/j.cell.2018.11.035.\u003c/li\u003e\n\u003cli\u003eRose-John S,Jenkins B J,Garbers C,Moll J M，Scheller J(2023) Targeting IL-6 trans-signalling: past, present and future prospects.Nat Rev Immunol.23:666-681.http://doi.org/10.1038/s41577-023-00856-y.\u003c/li\u003e\n\u003cli\u003eSaltzman J W,Battaglino R A,Salles Let al(2013) B-cell maturation antigen, a proliferation-inducing ligand, and B-cell activating factor are candidate mediators of spinal cord injury-induced autoimmunity.J Neurotrauma.30:434-440.http://doi.org/10.1089/neu.2012.2501.\u003c/li\u003e\n\u003cli\u003eHuntington N D,Tomioka R,Clavarino Cet al(2006) A BAFF antagonist suppresses experimental autoimmune encephalomyelitis by targeting cell-mediated and humoral immune responses.Int Immunol.18:1473-1485\u003c/li\u003e\n\u003cli\u003eDamianidou O,Theotokis P,Grigoriadis N，Petratos S(2022) Novel contributors to B cell activation during inflammatory CNS demyelination; An oNGOing process.Int J Med Sci.19:164-174.http://doi.org/10.7150/ijms.66350.\u003c/li\u003e\n\u003cli\u003eZhang Y,Tian J,Xiao Fet al(2022) B cell-activating factor and its targeted therapy in autoimmune diseases.Cytokine Growth Factor Rev.64:57-70.http://doi.org/10.1016/j.cytogfr.2021.11.004.\u003c/li\u003e\n\u003cli\u003eSteri M,Orr\u0026ugrave; V,Idda M Let al(2017) Overexpression of the Cytokine BAFF and Autoimmunity Risk.N Engl J Med.376:1615-1626.http://doi.org/10.1056/NEJMoa1610528.\u003c/li\u003e\n\u003cli\u003eGupta K,Kesharwani A,Rua Set al(2023) BAFF blockade in experimental autoimmune encephalomyelitis reduces inflammation in the meninges and synaptic and neuronal loss in adjacent brain regions.J Neuroinflammation.20:229.http://doi.org/10.1186/s12974-023-02922-7.\u003c/li\u003e\n\u003cli\u003eWang L,Zhang T,Zhang Zet al(2021) B cell activating factor regulates periodontitis development by suppressing inflammatory responses in macrophages.BMC Oral Health.21:426.http://doi.org/10.1186/s12903-021-01788-6.\u003c/li\u003e\n\u003cli\u003eChen B,Carr L，Dun X-P(2020) Dynamic expression of Slit1-3 and Robo1-2 in the mouse peripheral nervous system after injury.Neural Regen Res.15:948-958.http://doi.org/10.4103/1673-5374.268930.\u003c/li\u003e\n\u003cli\u003eBacon C,Endris V，Rappold G A(2013) The cellular function of srGAP3 and its role in neuronal morphogenesis.Mech Dev.130:391-395.http://doi.org/10.1016/j.mod.2012.10.005.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"m6A modification, METTL3, B cells, MS, EAE, inflammatory cytokines","lastPublishedDoi":"10.21203/rs.3.rs-7206859/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7206859/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eN6-methyladenosine (m\u003csup\u003e6\u003c/sup\u003eA), the most prevalent RNA modification, plays a pivotal role in regulating mRNA metabolism and cellular processes such as immune responses. Although the m6A methyltransferase METTL3 is known to regulate T-cell homeostasis and influence experimental autoimmune encephalomyelitis (EAE, a model for multiple sclerosis (MS)), its function within B cells remains poorly defined. Crucially, we observed that METTL3 expression is significantly downregulated in peripheral blood mononuclear cells (PBMCs) from MS patients and within B cells isolated from EAE mice. To directly investigate the functional consequences of this B-cell-specific METTL3 reduction in neuroinflammation, we generated B cell-specific METTL3 knockout mice (Mettl3\u003csup\u003eflox/flox\u003c/sup\u003eCD19\u003csup\u003eCre\u003c/sup\u003e). Strikingly, this targeted deletion of METTL3 in B cells markedly exacerbated EAE severity, demonstrated by significantly worsened clinical disease scores, increased spinal cord inflammation, and greater demyelination. Further mechanistic dissection revealed how B-cell METTL3 deficiency drives this exacerbated pathology: it promoted B cell apoptosis, inhibited the differentiation of regulatory B cell (Breg) subpopulations, increased the proportion of pro-inflammatory iNOS+ macrophages, and elevated the production of key inflammatory cytokines (IL-6, BAFF, and BCMA). Collectively, these findings demonstrate that METTL3 functions as a critical negative regulator within B cells, restraining their contribution to neuroinflammation in the EAE model. Importantly, therapeutically relevant overexpression of METTL3 specifically in B cells significantly reduced both the clinical severity and incidence of EAE, underscoring its potential as a novel therapeutic target for MS and similar autoimmune disorders involving pathogenic B-cell responses.\u003c/p\u003e","manuscriptTitle":"B Cell-specific METTL3 depletion exacerbates experimental autoimmune encephalomyelitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-30 06:55:13","doi":"10.21203/rs.3.rs-7206859/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-08-13T19:50:56+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-07-28T05:02:36+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-28T02:46:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-25T14:27:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2025-07-24T11:00:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"73518bea-1b4d-4b1e-b0e1-ce3650c43af2","owner":[],"postedDate":"July 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T16:07:27+00:00","versionOfRecord":{"articleIdentity":"rs-7206859","link":"https://doi.org/10.1007/s00018-025-06028-6","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2026-01-03 15:57:58","publishedOnDateReadable":"January 3rd, 2026"},"versionCreatedAt":"2025-07-30 06:55:13","video":"","vorDoi":"10.1007/s00018-025-06028-6","vorDoiUrl":"https://doi.org/10.1007/s00018-025-06028-6","workflowStages":[]},"version":"v1","identity":"rs-7206859","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7206859","identity":"rs-7206859","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}