B cell-specific METTL3 depletion exacerbates experimental autoimmune encephalomyelitis

Xuzhong Pei; Xiying Wang; Jie Ding; Haojun Yu; Chunran Xue; Chong Xie; Yi Chen; Xinyu Lin; Hong Yang; Yangtai Guan

doi:10.1007/s00018-025-06028-6

. 2026 Jan 3;83(1):63. doi: 10.1007/s00018-025-06028-6

B cell-specific METTL3 depletion exacerbates experimental autoimmune encephalomyelitis

Xuzhong Pei ^1,², Xiying Wang ³, Jie Ding ³, Haojun Yu ⁴, Chunran Xue ³, Chong Xie ³, Yi Chen ³, Xinyu Lin ³, Hong Yang ^5,^✉, Yangtai Guan ^2,^✉

PMCID: PMC12819949 PMID: 41484580

Abstract

N6-methyladenosine (m⁶A), 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/floxCD19^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 IL-6, BAFF, and BCMA. In the central nervous system, it promotes neurological damage by affecting axonal function and facilitating the loss of neurons. 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.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-025-06028-6.

Keywords: m⁶A modification, Neuroinflammation, Breg cells, Macrophage polarization, Axon guidance, Slit3

Background

Multiple sclerosis (MS) is a complex autoimmune disease of the central nervous system (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].

N⁶-Adenine methylation (m⁶A) is one of the most common mRNA modifications and is widely found in eukaryotic cells. m⁶A modifications regulate gene expression by affecting processes such as splicing, stability, transport and translation of mRNAs [12]. m⁶A 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 m⁶A 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 m⁶A 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]. m⁶A 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 m⁶A 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 m⁶A modification, the loss of METTL3 in B cells may impact their immune functions. Previous studies have demonstrated that m⁶A 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, m⁶A 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 m⁶A 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. Mettl3^flox/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-h 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 (MOG_35–55) 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 h 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 min. The plasma-Ficoll interface containing PBMC buffer was aspirated, rinsed twice with PBS, centrifuged and the cells were collected for storage or experiments.

Histochemical 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 and Silver 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, antibodies diluted in PBS such as anti-MBP (Abcam, 1:200,), anti-IBA1 (Abcam, 1:400), anti-GFAP (Cell Signaling Technology, 1:500), Anti-NeuN (Abcam, 1:400), were left on slices for incubation at 4℃ overnight. 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 400 g for 5 min, discard the supernatant, resuspend in 100 μl PBS, add flow cytometry antibody at a ratio of 1:400, and incubate at 4 °C away from light for 30 min. Wash twice with PBS. For surface staining: fix with 1% PFA at 4 °C away from light for 30 min, wash twice with PBS, then resuspend in 200 μl PBS for testing. For intracellular/nuclear staining: Incubate with membrane-breaking solution at 4 °C for 1 h, wash with membrane-breaking wash solution, centrifuge at 800 g, add the same proportion of antibody, incubate at 4 °C in the dark for 30 min, 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 min.

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 400 g for 5 min, discard the supernatant, add 3 ml of 1 × red blood cell lysis buffer (Beyotime, China), and lyse for 5 min. 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 h. Finally, collect the cells for flow cytometric analysis. All centrifugation steps were performed at 400 g for 5 min.

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 h. 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 × 10⁶ 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 72 h of incubation. For the co-culture with macrophages, B cells and RAW 264.7 cells were plated at a 4:1 ratio. Following the adherence of 2 × 10⁶ B cells seeded in the lower chamber for 4 h, 5 × 10⁵ RAW 264.7 cells were added to the upper chamber. The cells were then co-cultured for 68 h under the described conditions.

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; SLIT3, 1:500, Abmart, China; SRGAP3, 1:500, bioss, China) 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 min) 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 s, then add 25 µL to each well. Seal the plate, incubate in the dark at 800 rpm for 2 h. Centrifuge at 250 × g for 5 min, 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 h, 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 min, 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). Comparisons between two groups were performed as follows: data from animal experiments were analyzed using Student’s t-test, whereas comparisons of EAE clinical scores were assessed using the Mann–Whitney U test. Comparisons among multiple groups were analysed by one-way ANOVA followed by Bonferroni post-hoc analysis. In all cases, p values < 0.05 were considered as statistically significant.

Results

Decreased METTL3 expression in B cells after EAE induction

It has been previously shown that in CD4⁺ T cells, deletion of the m⁶A 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 m⁶A demethylase AlkB homologue 5 (ALKBH5) in T cells confers protection against EAE [22]. However, no study has investigated whether m⁶A regulates the pathological process of EAE through B cells. In this study, we initially assessed the mRNA expression levels of the m⁶A-related enzymes METTL3 and YTH N⁶-methyladenosine RNA binding protein F1 (YTHDF1) throughout disease progression in patients with MS. By detecting the mRNA expression of METTL3 and YTHDF1 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. 1A 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. 1C). B cells in the spleen were sorted via magnetic bead sorting, and the sorting efficiency of the B cells was greater than 90% (Fig. 1D). 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. 1E and F). These results suggest that METTL3 may play an important role in the progression of EAE. We subsequently constructed Mettl3^flox/floxCd19^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/floxCd19^Cre mice, we observed that METTL3 was obviously knocked down (Fig. 1G-I). In conclusion, METTL3 mRNA expression was decreased in the PBMCs of MS patients; similarly, MET TL3 expression was decreased in the B cells of EAE mice. In addition, we successfully constructed Mettl3^flox/floxCd19^Cre mice for this study.

Down-regulation of METTL3 expression in B cells of EAE mice. (A and B) *METTL3* and *YTHDF1* mRNA Expression in Peripheral Blood Mononuclear Cells (PBMC) from Healthy People and MS Patients, (n = 3 to 4, for Fig. 1 A, p = 0.02684, for Fig. 1B, p > 0.05, (C) Comparison of *Mettl3* mRNA expression levels in splenocytes from EAE mice and healthy control mice using real-time quantitative PCR (n = 4, p = 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 (n = 3, p = 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 *Mettl3*^flox/floxCd19^Cre mice. (I) METTL3 protein expression in B cells of *Mettl3*^flox/floxCd19.^Cre mice was analyzed to confirm the efficiency of the knockout. (data are presented as mean ± SEM, Statistics employedStudent’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns represents no significant difference between the two groups)

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/floxCd19^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/floxCd19^Cre mice presented significantly higher clinical scores than controls group (Fig. 2A-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. 2D and E). We also observed more pronounced spinal cord demyelination in the mice in the Mettl3^flox/floxCd19^Cre group (Fig. 2F and G). In addition, the results of MBP immunostaining revealed a similar trend (Fig. 2H and I). These results suggest that MTTL3 deficiency in B cells significantly aggravates the clinical symptoms and pathological phenotype of EAE.

METTL3 deficiency in B cells exacerbates EAE. (A) Behavioral scores obtained on a daily basis in *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre mice immunized with MOG_35–55 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 *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre mice (n = 22 to 32, for Fig. 2B, P = 0.0080, for Fig. 2 C, p = 0.00036). (D and E) Statistical analysis of H&E staining and inflammatory cell infiltration in intact spinal cord sections from *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre mice, scale bar = 100 μm, (n = 4 to 6, p = 0.0013). (F and G) Statistical analysis of LFB staining and myelin loss in intact spinal cord sections from *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre mice, scale bar = 100 μm, (n = 4 to 6, p = 0.01123). (H and I) Immunofluorescence images of MBP (green) in the lumbar spinal cord of *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19.^Cre mice. Cell nuclei were labelled with DAPI staining (blue), (n = 5 to 5, p = 0.003815). Zoom bar = 100 μm. (data = mean ± SEM, Statistics were performed with Mann–Whitney U test (A) and Student's t–test (B-I). * represents a significant difference, *p < 0.05, ** p < 0.01, **** p < 0.0001)

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/floxCd19^Cre mice was significantly reduced compared with that in the Mettl3^flox/flox group (Fig. 3A and B). Similarly, we found that the proportion of B cells in inguinal lymph nodes was also reduced in Mettl3^flox/floxCd19^Cre mice (Fig. 3C 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/floxCd19^Cre group were also significantly lower (Fig. 3E-H). In addition, we sorted B cells from the spleen, pertained them with CFSE, cultured them for 72 h, 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/floxCd19^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.

Fig. 3 — METTL3 deletion enhances B cell function after EAE induction. (A and B) Proportion of splenic B cells in *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre mice after EAE induction and their statistical analysis; (n = 6 to 7, p = 0.00442). (C and D) Proportion of B cells in lymph nodes of *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre mice after EAE induction and their statistical analysis; (n = 4 to 8, p = 0.01892). (E and F) Representative dot plots showing the proportion of PC cells in the spleen of *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre mice and their statistical analyses, (n = 6 to 7, p = 0.0003). (G and H) Representative dot plots showing the proportion of PC cells in lymph nodes of *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre mice and their statistical analysis; (n = 4 to 8, p = 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 *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19.^Cre mice after EAE induction. (K) Statistical analysis of B cell proliferation detected by CFSE (n = 6 to 4, p = 0.003976). (L) Statistical analysis of B cell apoptosis detected by Annexin V/PI staining (n = 4 to 5, p = 0.0010). (data = mean ± SEM, Statistics employed Student’s t-test. * represents a significant difference, *p < 0.05, ** p < 0.01, ***p < 0.001)

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^hiCD5⁺ B cells among splenic B cells was significantly lower in the Mettl3^flox/floxCd19^Cre group than in the Mettl3^flox/flox group (Fig. 4A and E), further testing of the proportion of IL-10⁺CD19⁺B cells revealed a significant decrease (Fig. 4B and F). But the Treg subset did not change significantly (Fig. 4C and G), suggesting that the inhibitory role of B cells in the pathogenesis of EAE is diminished. Previous studies have demonstrated that CD1d^hiCD5⁺ 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. 4D 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.

Fig. 4 — B cell-specific deletion of METTL3 inhibits differentiation of Breg subpopulations. (A and E) Detection of splenic CD1d^hiCD5⁺ B cell subpopulations in *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre mice after EAE induction by flow cytometry and their statistical analysis (n = 5 to 7, p = 0.00092). (B and F) Detection of splenic IL-10⁺CD19⁺ B cell subpopulations in *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre mice after EAE induction by flow cytometry and their statistical analysis (n = 4 to 4, p = 0.023709). (C and G) Percentage of Treg cells in CD4 + cells of *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre detected by flow cytometry and their statistical analysis (n = 5 to 6, p > 0.05). (D and H) Percentage of CD4⁺ T cells in splenocytes of *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19.^Cre detected by flow cytometry and their statistical analysis (n = 5 to 7, p > 0.05). (data = mean ± SEM, Statistics employed Student’s t-test. ns represents no statistical difference, * represents a significant difference, ***p < 0.001, ns represents no significant difference between the two groups)

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/floxCd19^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/floxCd19^Cre group of mice compared with those of the control group (Fig. 5A and B). Further examination of the iNOS⁺ macrophages revealed that they were significantly elevated (Fig. 5C and D). Interestingly, we also detected a significant increase in the proportion of CD206⁺ macrophages (Fig. 5E 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) (Fig. 5G-I), However, we found that there was no significant difference in IL-10 secretion (Fig. S2G). 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. S2H), suggesting that the specific deletion of METTL3 in B cells exacerbated the EAE-induced peripheral inflammatory response. To assess the influence of B cells on macrophage polarization, we co-cultured macrophages with B cells isolated from EAE mice. METTL3-deficient B cells markedly promoted M1 macrophage polarization compared to controls, with a concurrent but modest trend toward M2 polarization (Fig. 5J-M). We also observed a significant upregulation of Tnfsf13b (encoding BAFF) in these METTL3-deficient B cells following co-culture (Fig. 5N). Collectively, our data indicate that METTL3 loss in B cells potentiates pro-inflammatory macrophage polarization, likely through enhanced BAFF expression. 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.

B-cell-specific deficiency of METTL3 promotes peripheral inflammatory responses. (A and B) Flow cytometry to detect the proportion of F4/80⁺CD11b⁺ cells in the spleen of *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre mice and its statistical analysis (n = 5 to 5, p = 0.000113). (C and D) Flow cytometry to detect the spleen iNOS⁺ of *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre mice macrophage ratio and its statistical analysis (n = 5 to 5, p = 0.000121). (E and F) Flow cytometry to detect the spleen CD206⁺ of *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre mice macrophage ratio and its statistical analysis (n = 5 to 5, p = 0.001045). (G-I) Cytometric bead array (CBA) method to detect the secretion of inflammatory factors in peripheral blood of *Mettl3*^flox/flox mice and *Mettl3*^flox/floxCd19^Cre mice by flow cytometry (n = 5 to 5, for Fig. 5G, p = 0.000955, for Fig. 5H, p = 0.042108, for Fig. 5I, p = 0.057755). (J) Scatter plots and statistical analysis of the effects of B cells on macrophage polarization in a co-culture system using B cells from *Mettl3*^flox/flox and *Mettl3*^flox/floxCd19.^Cre EAE mice. (n = 5 to 5, for Fig. 5L, p = 0.005850, for Fig. 5 M, p = 0.129162). (N) mRNA expression of *Tnfsf13b* in B cells after co-culture with macrophages. (n = 5 to 5, p = 0.000329.) (data = mean ± SEM, Statistics employed Student’s t-test. ns represents no statistical difference, *p < 0.05, ** p < 0.01, ***p < 0.001)

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. 6A 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/floxCd19^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. 6C 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. 6E). We further observed that METTL3 deficiency in B cells exacerbated the loss of spinal cord neurons in EAE mice. Silver staining revealed more severe axonal and neurofilament damage (Fig. 6F and G). Consistent with a heightened neuroinflammatory response, we detected increased microglial activation and astrocyte loss in Mettl3^flox/floxCd19^Cre EAE mice (Fig. 6H). Finally, we identified a specific and significant decrease in SLIT3 protein expression within the spinal cord of mutant mice (Fig. 6I), pointing to a region-specific suppression that may underlie the observed neural damage. In summary, the above results not only confirmed the reliability of the multiomics data but also revealed that METTL3 regulates the expression of Slit3through 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.

Fig. 6 — Combined multi-omics analysis of predicted targets of METTL3 action in B cells. (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 p < 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 *Slit3* versus *Srgap3* in *Mettl3*^flox/flox and *Mettl3*^flox/floxCd19^Cre EAE mice by Integrated Genomics Viewer (IGV). (D) MeRIP-qPCR validation of the top six differentially expressed genes. All of the above genes are p < 0.00001. (E) Detection of overall levels of RNA methylation in B cells of *Mettl3*^flox/flox and *Mettl3*^flox/floxCd19^Cre EAE mice (n = 3, p = 0.046245). (F and H) Immunofluorescence staining of spinal cord neurons (purple), astrocytes (green), and microglia (red) in *Mettl3*^flox/flox and *Mettl3*^flox/floxCd19^Cre EAE mice, with nuclei counterstained by DAPI (blue). (n = 5 to 5, p < 0.05). (G) Silver staining of spinal cords from *Mettl3*^flox/flox and *Mettl3*^flox/floxCd19.^Cre EAE mice. (n = 4 to 4, p < 0.05). (I) Western blot analysis of SLIT3 and SRGAP3 protein expression in the spinal cords of two EAE mouse groups. (data = mean ± SEM, Statistics employed Student’s t-test. ns represents no statistical difference, * p < 0.05, ***p < 0.001)

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. 7A-C). Confirmation of successful METTL3 overexpression was obtained by detecting significantly elevated METTL3 levels specifically within B cells of the OE mice (Fig. 7D). Collectively, these results provide direct in vivo evidence supporting the protective role of B cell-expressed METTL3 in the EAE model.

Fig. 7 — Overexpression of METTL3 in B cells alleviates EAE. (A) Behavioural scores measured daily in WT mice and OE mice after inoculation with MOG_35–55 peptide emulsified with complete Freund's adjuvant (n = 4 to 10). (B and C) Statistical analysis of behavioural scores and incidence rates in WT and OE mice 21 days after EAE modelling (n = 4 to 10, p = 0.070764). (D) Expression of METTL3 in B cells of WT and OE mice (data are presented as mean ± SEM, Statistics were performed with Mann–Whitney U test (A) and Student's t–test (B, C). *p < 0.05)

Discussion

m⁶A 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 m⁶A, it can regulate the normal function of immune cells [24]. Indeed, METTL3 exerts anti-inflammatory and neuroprotective effects in multiple autoimmune and neurological diseases. For example, METTL3 expression is reduced in macrophages from pediatric allergic asthma patients, and its myeloid-specific deletion aggravates allergic airway inflammation [25]. Similarly, METTL3 downregulation in CD4⁺ T cells from systemic lupus erythematosus (SLE) patients correlates with disease severity, and its pharmacological inhibition exacerbates lupus-like phenotypes in a cGVHD mouse model [26]. Moreover, a selective METTL3 activator improved motor behavior and preserved dopaminergic neurons in a Parkinson’s disease rat model [27]. Furthermore, although the function of METTL3 in T cells within the EAE context has been progressively clarified, its role in B cells remains relatively unexplored, and emerging evidence suggests that its effects are highly dependent on cellular context. In T cells, METTL3 is generally regarded as a positive regulator of activation and differentiation [26, 28]. For example, METTL3-mediated m⁶A modification is essential for T cell homeostasis, and its conditional deletion in T cells results in impaired proliferative capacity and effector functions, ultimately attenuating EAE severity. This is consistent with the generally pro-inflammatory role of T cells in EAE pathogenesis [15, 16, 29]. 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^hiCD5⁺ B cells, which may result in diminished inhibition of the inflammatory response. Furthermore, METTL3 deletion increases the secretion of IL-6, BAFF, and BCMA secretion in the peripheral blood. Although no significant change was observed in the secretion level of IL-10, its transcriptome level was markedly reduced.

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 [30]. 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 [31]. 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 [32], and inhibition of METTL3-mediated m⁶A modification in hematopoietic stem cells (HSCs) during B-cell development leads to impaired HSC differentiation, thereby reducing the proportion of peripheral B cells [32, 33]. 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 [34]. 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/floxCd19^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^hiCD5⁺ 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 [35, 36]. During the progression of multiple sclerosis (MS), both M1 and M2 macrophage/microglia subtypes are frequently activated concurrently. Their polarization states are highly dynamic and strictly dependent on the local microenvironment, under the coordinated regulation of various cytokines, duration of stimulation, and multiple signaling pathways. In the acute phase, although the M1-type response predominates, it is often accompanied by synchronous M2-type activity that may attempt to modulate inflammation and promote tissue repair. Despite the overall inflammatory response remaining dominant during this stage, a shift toward M2-polarization becomes apparent during the recovery phase. Throughout this transition, intermediate phenotypes co-expressing both M1 and M2 characteristics are commonly observed, reflecting the continuous and plastic nature of functional repolarization [36–38]. 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 IL-10. Therefore, the activation of macrophages may contribute to the exacerbation of EAE and profound inflammation, and the increased expression of CD206⁺ macrophages 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 [39]. 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) [40–42] 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 [43]. 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 MS and SLE [44]. 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 [40, 41, 45]. Moreover, blocking BAFF can inhibit macrophage activation by reducing inflammation, and blocking BAFF reduces the secretion of inflammatory cytokines by macrophages [46]. 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 [47], 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 [48]. 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. The pathogenesis of neuroimmune diseases involves disruption of the blood–brain barrier, accompanied by infiltration of activated B cells into the CNS, This phenomenon is further supported by the presence of extensive B cell aggregates within the CNS of patients with MS [2, 49]. Notably, in addition to producing antibodies and pro-inflammatory cytokines, these infiltrating B cells contribute to the pathology of MS by releasing exosomes that promote oligodendrocyte and neuronal cell death [50, 51]. As an evolutionarily conserved glycoprotein, SLIT3 may function through secretion by centrally infiltrating B cells. This hypothesis is further supported by our observed axonal damage and neuronal loss. 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.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 340 KB)^{(340.1KB, docx)}

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; XYW: performed the experiments and analysed the data; JD: wrote the original manuscript and analysed the data; HJY, CRX, and CX: participated in the experiments and revised the manuscript; YC and XYL: participated in the experiments; HY: designed and performed the experiments, analysed the data, and revised the manuscript; 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.

Declarations

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.

Footnotes

Xuzhong Pei, Xiying Wang and Jie Ding contributed equally to this work and share the first authorship.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Hong Yang, Email: jinuoyuxizi@163.com.

Yangtai Guan, Email: yangtaiguan@sina.com.

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38.Vogel DYS, Vereyken EJF, Glim JE et al (2013) Macrophages in inflammatory multiple sclerosis lesions have an intermediate activation status. J Neuroinflammation 10:35. 10.1186/1742-2094-10-35 [DOI] [PMC free article] [PubMed] [Google Scholar]
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42.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. 10.7150/ijms.66350 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Zhang Y, Tian J, Xiao F et al (2022) B cell-activating factor and its targeted therapy in autoimmune diseases. Cytokine Growth Factor Rev 64:57–70. 10.1016/j.cytogfr.2021.11.004 [DOI] [PubMed] [Google Scholar]
44.Steri M, Orrù V, Idda M et al (2017) Overexpression of the cytokine BAFF and autoimmunity risk. N Engl J Med 376:1615–1626. 10.1056/NEJMoa1610528 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Gupta K, Kesharwani A, Rua S et 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. 10.1186/s12974-023-02922-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
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48.Bacon C, Endris V, Rappold GA (2013) The cellular function of srGAP3 and its role in neuronal morphogenesis. Mech Dev 130:391–395. 10.1016/j.mod.2012.10.005 [DOI] [PubMed] [Google Scholar]
49.Bar-Or A, Li R (2021) Cellular immunology of relapsing multiple sclerosis: interactions, checks, and balances. Lancet Neurol 20:470–483. 10.1016/S1474-4422(21)00063-6 [DOI] [PubMed] [Google Scholar]
50.Benjamins JA, Nedelkoska L, Touil H et al (2019) Exosome-enriched fractions from MS B cells induce oligodendrocyte death. Neurol Neuroimmunol Neuroinflamm 6:e550. 10.1212/NXI.0000000000000550 [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Lisak RP, Nedelkoska L, Benjamins JA et al (2017) B cells from patients with multiple sclerosis induce cell death via apoptosis in neurons in vitro. J Neuroimmunol 309:88–99. 10.1016/j.jneuroim.2017.05.004 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (DOCX 340 KB)^{(340.1KB, docx)}

Data Availability Statement

The raw data has been made available to the public under the registration number PRJNA1255398.

http://www.ncbi.nlm.nih.gov/bioproject/1255398.

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[CR49] 49.Bar-Or A, Li R (2021) Cellular immunology of relapsing multiple sclerosis: interactions, checks, and balances. Lancet Neurol 20:470–483. 10.1016/S1474-4422(21)00063-6 [DOI] [PubMed] [Google Scholar]

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PERMALINK

B cell-specific METTL3 depletion exacerbates experimental autoimmune encephalomyelitis

Xuzhong Pei

Xiying Wang

Jie Ding

Haojun Yu

Chunran Xue

Chong Xie

Yi Chen

Xinyu Lin

Hong Yang

Yangtai Guan

Abstract

Supplementary Information

Background

Methods

Animals

Construction of the EAE

PBMC extraction

Histochemical staining

Immunofluorescence staining

Flow cytometry staining

Spleen cell extraction and culture

B lymphocyte sorting and culture

Cell proliferation

Annexin V/PI staining

Quantitative real-time PCR

Western blot

Cytometric bead array

MeRIP-seq and MeRIP-qPCR

Statistical analysis

Results

Decreased METTL3 expression in B cells after EAE induction

Fig. 1.

Deletion of METTL3 in B cells exacerbates EAE

Fig. 2.

METTL3 deficiency exacerbates proliferation and apoptosis of B cells

Fig. 3.

B-cell-specific deletion of METTL3 inhibits the differentiation of regulatory B cells

Fig. 4.

B cell-specific deletion of METTL3 promotes macrophage inflammatory response and peripheral inflammation

Fig. 5.

B cell-specific METTL3 deficiency downregulates key genes in the axon guidance pathway through an m⁶A-dependent mechanism

Fig. 6.

Overexpression of METTL3 in B cells significantly alleviates the symptoms of EAE

Fig. 7.

Discussion

Conclusion

Supplementary Information

Acknowledgements

Authors' contributions

Funding

Data availability

Declarations

Ethical approval

Consent to participate

Consent for publication

Competing interests

Footnotes

Contributor Information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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