Mice
MAPK4 KO mice on a C57BL/6 background were generously provided by Professor Lin Xu from Zunyi Medical University, while C57BL/6 WT mice served as controls. DBA/1 mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice were housed under individually ventilated cage conditions at the Affiliated Hospital of Zunyi Medical University. All experimental protocols involving animals were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals and were approved by the Laboratory Animal Welfare & Ethics Committee of Zunyi Medical University (ZMU21-2302-043).
Patients
Eight newly diagnosed patients with RA and age-matched healthy control individuals were enrolled in this study. Informed consent was obtained from all participants. The study adhered to the principles outlined in the Declaration of Helsinki and received approval from the Ethics Committee of the Affiliated Hospital of Zunyi Medical University (KLLY-2024-075).
Cell culture
B cells were isolated as previously reported [21]. The isolated cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco) at 37 °C with 5% CO2.
Treatment with MAPK4 agonist Vacquinol-1 in vivo and in vitro
For in vitro stimulation, B cells were pretreated with 15 μM of the MAPK4 agonist Vacquinol-1 (5428-80-8, TargetMol) for 1 h before stimulation. For in vivo stimulation, WT mice, aged 6–8 weeks with a 1:1 male-to-female ratio, were randomly assigned to either the agonist group or the control group. Mice in the agonist group were administered intraperitoneal injections of 15 μM Vacquinol-1 every other day for 14 days. Mice in the control group were injected with an equal volume of PBS at the same time points.
Flow cytometry and phos flow
For the analysis of B cell subset in RA patients, PBMCs were stained with antibodies including PerCP-anti-CD3, APC-anti-CD19, Percp/cy5.5-anti-CD19, PB-anti-CD38, PE/CY7-anti-CD27, BV510-anti-IgD, APC-anti-IgM, PE-anti-CD21. For detection of MAPK4 expression, PBMCs from HC and RA patients were stained with PerCP-anti-CD3 and APC-anti-CD19 antibodies. After fixation and permeabilization using the Foxp3 Staining Buffer Set (00-5523-00; eBioscience), the cells were labeled with anti-MAPK4 antibody (bs-1319R; Bioss) and Alexa Fluor™ 488 conjugated donkey anti-rabbit IgG (H + L) (A21206; life technologies), then analyzed by flow cytometry.
For the analysis of B cell in WT and MAPK4 KO mice, bone marrow cells were stained with antibodies including APC-anti-CD43, PE-anti-BP-1, PE/CY7-anti-CD24, BV421-anti-IgM, BV510-anti-B220 and 7AAD. Splenic lymphocytes were stained with FITC-anti-CD19, PE-anti-CD23, Percp-anti-IgD, APC-anti-CD21, PE/CY7-anti-Ki67, APC-anti-GL7, FITC-anti-CD95, and FITC-Annexin V. For B cell intracellular cytokine staining, B cells from WT or MAPK4 KO mice were isolated and stimulated with different conditions: CPG + anti-CD40 for IL-6 detection, CPG alone for IL-10 detection, and PMA + Ionomycin for IFN-γ detection at 37 °C for 5 h. After stimulation, cells were surface-stained with BV510-anti-B220, followed by fixation and permeabilization. The cells were then stained with PE-anti-IL-6, FITC-anti-IL-10, and PE/CY7-anti-IFN-γ antibodies. B1 cells collected from the peritoneal cavity were stained with antibodies including FITC-anti-CD19, PE-anti-CD5, Percp-anti-IgD, APC-anti-CD11b, BV421-anti-IgM.
For phospho-flow analysis, isolated PBMCs or splenic lymphocytes were labeled with APC-anti-CD19 or BV510-anti-B220 antibodies, respectively. Subsequently, the cells were activated using a previously described method [22]. Briefly, cells were incubated with soluble antigen (sAg) comprising Biotin-SP AffiniPure F(ab’)2 Fragment Goat Anti-Human IgG + IgM (H + L) (109-006-127; Jackson) or Alexa Fluor® 594-conjugated AffiniPure F(ab)2 Fragment Goat Anti-Mouse IgG + IgM (H + L) (115-586-068; Jackson) for 30 min, followed by streptavidin (016-000-114; Jackson) for 10 min on ice. BCR signaling activation was then induced at 37 °C for varying time intervals. After fixation, the cells were stained with antibodies specific for phosphorylated tyrosine residues, Alexafluor™ 488 phalloidin, pY, pBTK, BCR, ACTIN, pPI3K(p85), or pWASP. Secondary antibody AF405-conjugated goat anti-rabbit IgG (A31556; Lifetechnology) or Alexa Fluor® 488-conjugated AffiniPure Donkey Anti-Mouse IgG (H + L) (715-545-150, Jackson) was used for detection.
For T cell subpopulation analysis in MAPK4 KO mice, lymphocytes from the thymus, spleen, and lymph nodes were stained with antibodies including Percp-anti-CD44, APC-anti-CD25, APC/CY7-anti-TCRβ, PE/CY7-anti-CD62L, Pacblue-anti-CD4, BV510-anti-CD8, FITC-anti-Foxp3, PE/CY7-anti-Ki67, FITC-anti-IL2. For T cell intracellular cytokine staining, lymphocytes were stimulated for 5 h. at 37 °C, 5% CO2, in the presence of PMA + Ionomycin in combination with GolgiStop (55472; BD Pharmingen). After stimulation, the cells were surface-stained with PE anti-CD4, Percp-anti-CD44 and 7AAD, followed by fixation and permeabilization. The cells were then stained with APC-anti-IL4, PE/CY7-anti-IFN-γ, BV421-anti-IL17.
Subsequently, the stained cells were analyzed using a BD FACS Canto Plus flow cytometer, and data were further processed with FlowJo V10 software (Tree Star). The detailed information for the above-mentioned antibodies is provided in Table S1.
Ex vivo antigen presentation assay
A total of 1 × 106 B cells from WT or MAPK4 KO mice were isolated and incubated with 100 µL of 20 µg/mL Eα52–68 peptide (amino acid sequence: ASFEAQGALANIAVDKA; 13-5741-82, Thermo) at 37 °C for 1 h. The cells were then stained on ice with Biotin-Y-Ae antibody (1:400, Invitrogen, 13-5741-85), PE-SA, and FITC-B220. Finally, the stained cells were analyzed using flow cytometry.
Western blot
Splenic B cells were stimulated with sAg, and the cell lysates were subjected to SDS-PAGE and subsequently transferred onto a PVDF membrane (Millipore, Germany). The membrane was then blocked with a 5% non-fat milk solution before probed with the following primary antibodies: anti-MAPK4, anti-pBTK, anti-BTK, anti-phosphotyrosine, anti-pPI3K(p85), anti-PI3K, anti-pAKT, anti-AKT, anti-pIKKβ, anti-IKKβ, anti-p-mTOR, anti-mTOR, anti-pS6, anti-S6, anti-pFOXO1, anti-FOXO1, anti-pSyk, anti-Syk, anti-pCD19, anti-CD19, anti-pSHIP1, anti-SHIP1, anti-AID, anti-pWASP, anti-pIRF4, and anti-β-ACTIN or anti-GAPDH, which were used as loading controls. Following incubation with HRP-linked secondary antibodies and subsequent washing steps, the blots were visualized using an imaging system (ChemiDoc™ Touch, BIO-RAD). The detailed information for the above-mentioned antibodies is provided in Table S1.
Confocal microscopy
Splenic B cells from WT and MAPK4 KO mice were purified and subsequently incubated with anti-mouse CD16/CD32 to block Fc receptors. Following stimulation as previously described [21], cells were incubated with AF594-AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG + IgM (H + L) (115-586-068; Jackson) and PE-streptavidin (405204; BioLegend) on ice for 30 and 10 min, respectively. BCR signaling was then stimulated at 37 °C for varying time intervals. After fixation and permeabilization, cells were stained with the following antibodies: anti-pBTK (87457S; Cell Signaling Technology), anti-phosphotyrosine (9411S; Cell Signaling Technology), anti-pPI3K(p85) (17366S; Cell Signaling Technology), anti-pWASP (AF7304; Affinity) and Phalloidin-AF488 (R37110; Lifetechnology). Confocal imaging was subsequently performed using a Leica STELLARIS 5 microscope, and data analysis was conducted using the Leica Application Suite X software (Leica, Germany).
Rheumatoid arthritis model
CIA mouse model was established according to previous reports [23]. Male DBA/1 mice aged 8–10 weeks were randomly divided into a model group and a control group. The model group mice were injected with 0.1 mL of collagen (20011, Chondrex) and complete Freund adjuvant (7002, Chondrex) emulsion (1:1) on day 1, followed by a similar injection of collagen and incomplete Freund adjuvant (7002, Chondrex) emulsion (1:1) on day 21. The control group mice were injected with an equal volume of PBS at the same time points. Arthritis manifestation was evaluated every 2 days. On day 33, the mice were euthanized, and splenic B cells were isolated for subsequent flow cytometry and WB analysis.
CO-immunoprecipitation
CO-immunoprecipitation experiments were conducted using a Classic Magnetic Protein A/G IP/Co-IP kit (YJ201; Epizyme) following the manufacturer’s instructions. Briefly, splenic B cell lysates from WT mice were prepared and incubated with anti-IRF4 (DF6198; Affinity), anti-MAPK4 (BS-1319R; Bioss) antibodies, or control IgG overnight at 4 °C, followed by an incubation with Protein A/G-agarose beads for an additional hour. After washing, IRF4 and MAPK4 were measured in all immunoprecipitation (IP) and input samples by Western blotting.
Ca2+ flow
Purified splenic B cells from WT and MAPK4 KO mice were suspended in Ca2+-free HBSS (14175079; Gibco™) containing a final concentration of 0.5 μM calcium-sensitive dye Fluo-4 AM (273221-67-3; MedChemExpress) and incubate for 25 min at 37 °C. Subsequently, the cells were incubated with an BV510-anti-mouse/human CD45R/B220 antibody (103248; BioLegend) for 30 min. Following an initial 30 s recording on a flow cytometer, the cells were briefly mixed with preheated Biotin-SP-AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG + IgM (H + L) (115-066-068; Jackson) to a final concentration of 10 μg/mL, and the recording continued for additional 5 min.
B-Cell proliferation
B-Cell proliferation detection was performed as previously described [24]. Purified B cells were labeled with CFSE (HY-D0938; MedChemExpress) and stimulated with 5 μg/ml LPS (L2880; Sigma) or 10 μg/ml ODN1826 (tlrl-1826-1; InvivoGen) in the culture medium for 4 days. Then the cells were subjected to flow cytometry analysis after staining with BV510-B220 (103248; BioLegend) and 7-AAD (00-6993-50; ebioscience) on ice.
Immunization and ELISA
WT and MAPK4 KO mice were intraperitoneally injected with 40 μg NP-KLH (N-5060-5; Biosearch) or 100 μg NP-FICOLL (sc-396292; SantaCruz) in 200 μL of PBS. After 2 weeks, the splenic lymphocytes were isolated for flow cytometry analysis. Additionally, the NP-specific levels of IgM and IgG1 in serum were determined by ELISA using NP-BSA (N-5050H-10; Biosearch)–coated plates, followed by incubation with anti-IgM (FNSA-0091; Proteintech) and anti-IgG1(FNSA-0086; Proteintech) secondary antibodies. For the detection of anti-double-stranded DNA (dsDNA) antibodies in serum, samples were collected from WT and MAPK4 KO mice, and the levels of anti-dsDNA antibodies were measured using a commercial ELISA kit (CSB-E11194, CUSABIO), following the manufacturer’s instructions.
Bone marrow chimera
Bone marrow cells were extracted from femurs of 8 week-old CD45.2 WT/MAPK4 KO and CD45.1 mice with a 1:1 male-to-female ratio. Bone marrow cell mixtures at a 1:1 ratio of CD45.1 and CD45.2 WT or MAPK4 KO were prepared and randomly intravenously injected into 12–15 week-old CD45.1 mice that had been sublethally irradiated (6 Gy). After 8 weeks, bone marrow and splenic lymphocytes were collected for flow cytometric analysis [25].
BCR internalization
Splenic B cells from WT and MAPK4 KO mice were incubated with BV510-anti-B220 (103248; BioLegend) on ice for 30 min, followed by incubation with Biotin-SP-AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG + IgM (H + L) (115-066-068; Jackson) and PE-Streptavidin (405204; BioLegend) on ice for an additional 30 min. Subsequently, the cells were incubated at 37 °C for 0, 2, 5, 10 or 20 min, then fixed with PFA (final concentration 4%, WI333638, Thermo Fisher Scientific) on ice for 30 min and subjected to flow cytometric analysis. BCR internalization was assessed by comparing the mean fluorescence intensity (MFI) of biotin-conjugated F(ab’)2 Ig-labeled BCR proteins remaining on the cell surface between B cells from WT and MAPK4 KO mice.
H&E staining
The spleens of WT and MAPK4 KO mice were fixed with 4% PFA at room temperature. The tissues were then dehydrated through a gradient of ethanol and embedded in paraffin. Sections of 4 μm were sliced with a paraffin slicer. The slices were then deparaffinized and stained with hematoxylin and eosin. Images were captured using an optical microscope.
Immunofluorescence
The spleens from WT and MAPK4 KO mice were embedded in OCT, frozen in liquid nitrogen, and stored at −80 °C. The frozen samples were sectioned at 5 µm using a cryostat, fixed with acetone for 5 min, air-dried, and stored at −80 °C for subsequent immunofluorescence staining. Sections were incubated with 5% BSA and biotin-IgM (1:50, 13-5790-8; Invitrogen), FITC-CD169 (MA5-28189; Invitrogen), and anti-mouse APC-IgD (17-5993-82; Invitrogen) overnight at 4 °C, followed by incubated with PE-Streptavidin (1:200; 405204; BioLegend). The slides were then mounted after washing with an imaging antifading reagent to prevent fluorescence quenching. Images were captured and analyzed using a confocal microscope (Leica).
Statistical analysis
The number of animals or samples and the number of experimental replicates are specified in the figure legends, with the sample size determined based on previous experience and preliminary data. Two-tailed unpaired t-test and One-way ANOVA were performed using the GraphPad Prism software version 8.0.1 for Windows (San Diego, California USA). A significance level of P < 0.05 was considered indicative of a statistically significant difference between groups.
