Polymorphonuclear Myeloid-Derived Suppressor Cells Promote Inflammation Progression of Rheumatoid Arthritis by Inducing Age-Associated B Cell Proliferation and Differentiation via the BAFF-Mediated SYK-ERK1/2 Pathway

Polymorphonuclear Myeloid-Derived Suppressor Cells Promote Inflammation Progression of Rheumatoid Arthritis by Inducing Age-Associated B Cell Proliferation and Differentiation via the BAFF-Mediated SYK-ERK1/2 Pathway | 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 Polymorphonuclear Myeloid-Derived Suppressor Cells Promote Inflammation Progression of Rheumatoid Arthritis by Inducing Age-Associated B Cell Proliferation and Differentiation via the BAFF-Mediated SYK-ERK1/2 Pathway Huiming Hong, Fanzhang Yin, Yanan Wang, Yongqi Wang, Jingxue Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6846731/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background In rheumatoid arthritis (RA), both polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and age-associated B cells (ABCs) are significantly elevated in affected joints and are closely linked to disease progression. However, their interaction remains poorly understood. This study aims to explore the regulatory role of PMN-MDSCs on ABCs and the underlying mechanisms, offering new insights into RA pathogenesis and potential therapeutic targets. Methods We conducted flow cytometry analysis to assess PMN-MDSCs and ABCs proportion and their correlation in collagen-induced arthritis (CIA) mice. We used RNA sequencing (RNA-seq) to characterize transcriptomic profiles of CD11c + B cells from CIA mice. In vitro studies examined T-bet expression in CD11c + B cells co-cultured with CIA-derived PMN-MDSCs. We assessed BAFF effects on ABCs proliferation and differentiation through flow cytometry and validated findings using anti-BAFF antibodies in PMN-MDSCs/CD11c + B cells co-cultures. We used western blot and flow cytometry analyses to identify BAFF-mediated signaling pathways in CD11c + B cells. In vivo study evaluated the therapeutic potential of pathway inhibitors in CIA mice. The ratio and regulatory correlation of PMN-MDSCs and ABCs were also assessed by flow cytometry in RA patients. Results Using a CIA mouse model, we observed increased populations of both PMN-MDSCs and ABCs in the joints and spleens, with a strong positive correlation in joint tissues. RNA sequencing of CIA-derived CD11c + B cells revealed distinct pro-inflammatory and chemotactic signatures. PMN-MDSCs enhanced T-bet expression in CD11c + B cells through non-contact-dependent mechanisms, driving ABCs differentiation. BAFF was found to support ABC proliferation and differentiation. Mechanistically, PMN-MDSCs-derived BAFF activated the SYK-ERK1/2 signaling pathway via BAFF receptor (BAFFr), upregulating T-bet expression in CD11c + B cells. In vivo, inhibition of SYK ameliorated arthritis symptoms and reduced ABC populations across tissues. Similarly, patients with RA exhibited elevated levels of PMN-MDSCs and ABCs in both blood and synovial fluid, with a significant correlation between the two. Additionally, RA-derived PMN-MDSCs promoted ABCs differentiation in vitro. Conclusion These findings highlight the role of PMN-MDSCs in driving RA inflammation by promoting ABC proliferation and differentiation via the BAFF-mediated SYK-ERK1/2 pathway. Rheumatoid arthritis Collagen-induced arthritis Polymorphonuclear myeloid-derived suppressor cells B-cell activating factor Age-associated B cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by chronic joint inflammation, leading to cartilage and bone damage and, in severe cases, disability[ 1 ]. Although the exact pathogenesis remains unclear, RA progression involves complex dysregulation, affecting multiple immune cell populations, including T cells, macrophages, and fibroblast-like synoviocytes. Emerging evidence indicates a significant accumulation of myeloid-derived suppressor cells (MDSCs) and age-associated B cells (ABCs) in RA-affected joints[ 2 – 5 ], with both cell types closely linked to disease severity. However, despite their co-localization and strong involvement in RA pathogenesis, the potential regulatory relationship between MDSCs and ABCs remains unexplored. In mice, MDSCs consist of two distinct subpopulations: polymorphonuclear MDSCs (PMN-MDSCs, CD11b + Ly6G + Ly6C low ) and monocytic MDSCs (M-MDSCs, CD11b + Ly6G − Ly6C high )[ 6 ]. These cells have gained increasing attention for their role in RA pathogenesis. Emerging research indicates that MDSCs promote inflammation and exacerbate RA progression, with significantly higher levels in patients with RA compared to healthy controls, a trend also observed in other arthritis-related diseases[ 7 ]. Mouse models with elevated MDSCs exhibit increased interleukin (IL)-1β and tumor necrosis factor-alpha (TNF-α) production, while PMN-MDSCs isolated from these models (spleens) lose their characteristic T cell suppressive function[ 8 ]. In contrast, M-MDSCs inhibit T cell proliferation and interferon-gamma (IFN-γ) production via inducible nitric oxide synthase while simultaneously promoting T helper 17 (Th17) differentiation through IL-1β. The pathogenic role of MDSCs is further supported by the adoptive transfer of MDSCs from the spleens of collagen-induced arthritis (CIA) mice, where CIA-derived MDSCs aggravated disease severity, whereas MDSC depletion alleviated disease progression, reduced Th17 differentiation, and decreased IL-17A secretion[ 4 ]. Additionally, our previous research found that PMN-MDSCs enhance B cell secretion of TNF-α, further exacerbating arthritis in CIA mice[ 9 ]. These findings suggest that MDSCs influence RA etiology through T and B cell regulation. However, their precise roles in RA pathogenesis remain unclear, requiring further investigation. A recently identified B cell subpopulation, known as ABCs, has been found to increase in several autoimmune diseases and mouse models[ 10 – 13 ]. In CIA mice, ABCs are characterized by the expression of B220, CD11c, and T-bet[ 3 ], playing a crucial role in the progression of RA. Qin et al. found that ABC levels were elevated in various tissues of both patients with RA and CIA mice, with a positive correlation to disease activity. Notably, the proportion of ABCs in synovial fluid was found to be 10 times higher than in the blood of the same individual, indicating preferential recruitment to inflammatory sites. Mechanistically, ABCs contribute to RA pathogenesis by activating fibroblast-like synoviocytes through TNF-α signaling[ 3 ]. Our study aimed to explore whether PMN-MDSCs regulate the function of ABCs in RA and to elucidate the molecular mechanisms involved in this potential interaction. Additionally, we sought to examine whether targeting these regulatory pathways could modulate disease progression in both patients with RA and CIA mouse models, offering new therapeutic strategies for RA treatment. Methods Mice Male DBA/1 mice (8–10 weeks old) were obtained from Cavens Experimental Animal Co., Ltd. (Changzhou, China) and maintained under specific pathogen-free conditions. All animal protocols of this study were approved by the Ethics Committee of Nanjing University of Chinese Medicine, Drum Tower Clinical School of Integrated Chinese and Western Medicine, Drum Tower Hospital. Induction of CIA, scoring, and SYK inhibitor treatment Equal volumes of bovine type II collagen and complete/incomplete Freund’s adjuvant were emulsified in a 5 mL syringe using a high-speed homogenizer for 1 min. The emulsion was then cooled down by placing the syringe in ice water for 2 min. This process of mixing and cooling was repeated 10 times. On day 1, DBA/1 mice received a subcutaneous injection of 100 µL of the emulsion containing complete Freund’s adjuvant at the base of the tail for the initial immunization. A second immunization was administered on day 21 using an emulsion containing incomplete Freund’s adjuvant. Starting from day 28 post-immunization, joint inflammation was assessed every 2 days, and arthritis severity was scored using established criteria following a previously published system[ 14 ]. The scoring system was as follows: 0, normal appearance; 1, mild redness or swelling of joints/digits; 2, moderate redness and swelling extending from ankle to midfoot; 3, severe redness and swelling of ankle, foot, and digits; and 4, severe inflammation with joint deformity and functional impairment. The total joint score for each mouse was determined by summing the scores of all four limbs. CIA mice were randomly divided into two groups, with five mice per group, and housed separately in two cages. For the SYK inhibitor treatment, CIA mice were administered an intraperitoneal injection of 5 mg/kg of the SYK inhibitor (PRT062607, Selleck), dissolved in distilled water, on alternate days from day 21 to day 42. Control mice received an equivalent volume of double-distilled water. Histopathological analysis of joint specimens Following Cervical dislocation method to execute mice, the right hindlimb joint was dissected, and the skin was removed. The joint was then washed with 10% phosphate-buffered saline and fixed in 4% paraformaldehyde. Next, decalcification was performed using 10% ethylenediaminetetraacetic acid or 5% formic acid for 30 days. Once decalcified, the joints were embedded in paraffin, sectioned sagittally, and stained with H&E and TRAP staining (Nanjing Youmeng Biotechnology Co., Ltd.). Synovial inflammation and osteoclastogenesis were assessed microscopically. Isolation and co-culture of PMN-MDSCs and CD11c B cells from mice Spleen lymphocytes were isolated under sterile conditions from DBA/1 and CIA mice. The cells were stained with the following antibodies: AF700 anti-mouse CD11b (Biolegend, M1/70), FITC anti-mouse Ly6G (Biolegend, 1A8), PE/Cy7 anti-mouse Ly6C (Biolegend, HK1.4), APC anti-mouse B220 (Biolegend, RA3-6B2), and BV785 anti-mouse CD11c (Biolegend, N418). PMN-MDSCs and CD11c + B cells were sorted using an Aria III flow cytometer (BD Bioscience), with sorted cell purity of > 95%. The collected cells were stored in 15 mL centrifuge tubes for subsequent experiments. CD11c + B cells (2 × 10 5 per well) were cultured in 96-well U-bottom plates in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 ng/mL toll-like receptor 7 (TLR7) (R&D), and 50 ng/mL IL-21 (R&D) for 72 h at 37°C with 5% CO 2 . When co-cultured with PMN-MDSCs, 2 × 10 5 PMN-MDSCs were added to the well, and 5 µg/mL anti-mouse BAFF (R&D) was used to block BAFF function. To examine the role of BAFF in CD11c + B cells culture, 100 ng/mL BAFF (R&D) and 10 µg/mL anti-mouse BAFFr (R&D) were added to inhibit BAFFr. Additionally, SYK and ERK1/2 inhibitors (Selleck) were added to explore the specific mechanism by which BAFF regulates CD11c + B cells. Isolation and co-culture of PMN-MDSCs and B cells from human samples PBMCs were isolated under sterile conditions from patients with RA and healthy controls. For surface staining, cells were incubated for 20 min with the following fluorochrome-conjugated antibodies: anti-human CD11b (Alexa Fluor 700, M1/70), CD33 (Brilliant Violet 605, P67.6), CD15 (FITC, HI98), CD14 (PerCP/Cyanine5.5, 63D3), and CD19 (APC/Cyanine7, HIB19). PMN-MDSCs and B cells were then sorted using a flow cytometer (Aria Ⅲ, BD Bioscience), achieving a purity of > 95%, and collected in 15 mL centrifuge tubes for further experiments. For co-culture experiments, 1×10 5 B cells were seeded into each well of a 96-well U-bottom plate containing RPMI 1640 medium supplemented with 10% FBS, 100 ng/mL TLR7 agonist (R&D), 20 ng/mL IFN-γ (R&D), and 20 ng/mL granulocyte-macrophage colony-stimulating factor (R&D). To a 1:1 cell ratio, 1×10 5 PMN-MDSCs were added to the same wells. The plates were incubated at 37°C in a 5% CO 2 incubator for 72 h. This co-culture system was designed to investigate the regulatory effects of RA-derived PMN-MDSCs on B cell proliferation and differentiation. Flow cytometry analysis Single-cell suspensions were prepared from cultured cells, as well as mouse spleen, joint, peripheral blood, lymph nodes, and human peripheral blood and synovial fluid. For intracellular staining, mouse samples were resuspended in RPMI 1640 medium containing 100 ng/mL PMA (Enzo), 1 µg/mL ionomycin (Enzo), and 5 µg/mL BFA (Enzo) and incubated at 37°C for 4 h. Dead cells were identified using eBioscience™ Fixable Viability Dye eFluor™ 506. Surface marker staining was performed by incubating cells for 20 min with the following anti-mouse antibodies: CD11b (Alexa Fluor 700 M1/70), Ly6G (FITC 1A8), Ly6C (PE/Cy7 HK1.4), B220 (APC RA3-6B2), CD11c (BV785 N418), and CD4 (FITC RM4-5). After fixation and permeabilization, intracellular markers were stained for 45 min using anti-mouse BAFF (PE 121808), TNF-α (PE/Cy7 MP6-XT22), IL-17A (APC TC11-18H10.1), IFN-γ (Alexa Fluor 700 XMG1.2), pERK1/2 (APC 6B8B69), and pSYK (APC MOCH1CT). Nuclear markers, including T-bet (BV421 4B10) and Ki67 (PE/Cy7 16A8), were also stained for 45 min after fixation and permeabilization. For human samples, dead cells were labeled and surface-stained for 20 min with anti-human CD11b (Alexa Fluor 700 M1/70), CD33 (BV605 P67.6), CD15 (FITC HI98), CD14 (PerCP/Cy5.5 63D3), CD19 (APC/Cy7 HIB19), CD21 (APC Bu32), and CD11c (BV650 3.9). All antibodies were obtained from Biolegend and eBioscience. Data were acquired using a BD LSRFortessa (BD Biosciences) and analyzed with the FlowJo software (Tree Star). Unique identifier RNA-seq and transcriptome analysis Total RNA was extracted from CD11c + B cells isolated from the spleens of CIA and DBA/1 mice using Trizol Reagent (Invitrogen, cat. NO 15596026). Following extraction, DNA digestion was performed using DNase I. RNA quality was evaluated by measuring the A260/A280 ratio with Nanodrop ™ OneCspectrophotometer (Thermo Fisher Scientific Inc), and RNA integrity was confirmed via 1.5% agarose gel electrophoresis. Quantification of qualified RNAs was conducted using the Qubit ™ RNA Broad Range Assay kit (Life Technologies, Q10210) on a Qubit3.0 fluorometer. For stranded RNA-seq library preparation, 2 µg of total RNA was processed using the KC-Digital ™ Stranded mRNA Library Prep Kit for Illumina® (Catalog NO. DR08502, Wuhan Seqhealth Co., Ltd. China), following the manufacturer’s instruction. This kit utilizes unique molecular identifiers (UMIs) of eight random bases to label pre-amplified cDNA molecules, minimizing duplication bias during polymerase chain reaction (PCR) amplification and sequencing. Library products corresponding to 200–500 bps were enriched, quantified, and sequenced on a DNBSEQ-T7 sequencer (MGI Tech Co., Ltd. China) using the PE150 model. Clean reads were grouped based on their UMI sequences, clustering reads with identical UMIs together. Within each cluster, pairwise alignment was performed, and reads with > 95% sequence identity were further refined into new sub-clusters. Multiple sequence alignment was then performed to generate a consensus sequence for each sub-cluster, effectively minimizing PCR amplification or sequencing errors and biases. The de-duplicated consensus sequences were used for standard RNA-seq analysis. Reads were mapped to the reference genome using the STAR software (version 2.5.3a) with default settings. Gene expression levels were quantified by counting exon-mapped reads using featureCounts (Subread-1.5.1; Bioconductor), and RPKM values were calculated. Differential gene expression analysis between groups was performed using the edgeR package (version 3.12.1), with statistical significance determined by a p-value of 0.05 and a fold-change threshold of 2. Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis for differentially expressed genes were performed using the KOBAS software (version 2.1.1) with a p-value cutoff of 0.05 to assess statistically significant enrichment. Alternative splicing events were identified using rMATS (version 3.2.5), applying a false discovery rate value cutoff of 0.05 and an absolute value of 0.05. Western blot Denatured protein samples were analyzed using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime) and subsequently transferred to 0.2 µm polyvinylidene fluoride (PVDF) membranes (Merck KGaA). The membranes were blocked with 5% bovine serum albumin (BSA) at 20°C for at least 1 h before overnight incubation at 4°C with primary antibodies. Anti-pERK1/2 and anti-ERK1/2 antibodies were obtained from ABclonal, while anti-GAPDH was sourced from AiFang Biological. Primary antibodies were diluted 1:1000 in TBST with 5% BSA. Following this, the membranes were incubated with HRP-conjugated secondary antibodies (anti-rabbit/anti-mouse IgG, Beyotime catalog A0208/A0216) at a 1:10000 dilution. The blots were visualized using ECL reagents (Beyotime), and immunoblot images were quantified using the ImageJ software (v1.8.0). Enzyme-linked immunosorbent assay Mouse plasma and human synovial fluid samples were collected and analyzed for BAFF levels using a Human/Mouse/Rat BAFF ELISA Kit (ABclonal, RK00402), following the manufacturer’s instructions. Data analysis Statistical analyses were performed using the GraphPad Prism software. Data are expressed as mean ± standard deviation (x̄ ± s). A student’s t-test was used for comparisons between two groups, while a one-way analysis of variance was applied for multiple-group analyses. Statistical significance was set as p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant). Results Increased PMN-MDSCs and ABCs in CIA mice with a significant positive correlation in joints Analysis of PMN-MDSC and ABC levels in various tissues of CIA mice showed a significant increase in both cell types within the joints and spleens compared to controls, with the most notable increase occurring in the joints. No significant differences were observed in peripheral blood mononuclear cells (PBMCs) (Figs. 1 A, B). These results align with our previous findings[ 9 ] and those of Qin et al.[ 3 ]. In the joints of CIA mice, PMN-MDSC and ABC levels increased in parallel with disease progression (Fig. 1 C), indicating a strong association between these cells and joint inflammation. Furthermore, a significant correlation was observed between PMN-MDSC and ABC levels in the joints of CIA mice (Fig. 1 D), suggesting a potential regulatory interaction that contributes to joint inflammation in CIA mice. PMN-MDSCs from CIA mice promote T-bet expression of CD11c + B cells in a non-contact-dependent manner, thereby increasing ABC levels To explore the regulatory effects of PMN-MDSCs on ABCs, we first examined the underlying cause for the increased ABC levels (B220 + CD11c + T-bet + ) in CIA mice. Our analysis showed that while the proportion of CD11c + B cells in the spleens of CIA mice was only slightly different from that of the control group, a significant increase in T-bet expression in CD11c + B cells was observed (Fig. 2 A). T-bet is a key transcription factor that regulates ABC differentiation and function[ 15 ], and its elevated expression suggests that PMN-MDSCs in CIA mice may drive the differentiation of CD11c + B cells into ABCs by upregulating T-bet expression, thereby increasing ABC levels. To further characterize CD11c + B cells in CIA mice, we isolated these cells from the spleens of both CIA and control mice and performed RNA sequencing (RNA-seq) to analyze their transcriptomic profiles. Heatmap analysis revealed 1,092 upregulated and 1,483 downregulated genes in CD11c + B cells from CIA mice compared to controls (Fig. 2 B). Notably, these CIA-derived CD11c + B cells exhibited a distinct pro-inflammatory and chemotactic profile, with a significant increase in T-bet (Tbx21) expression (Fig. 2 C). To validate our hypothesis, we co-cultured PMN-MDSCs from CIA and control mice with CD11c + B cells from control mice. Flow cytometry analysis showed that PMN-MDSCs from CIA mice enhanced T-bet expression in CD11c + B cells (Fig. 2 E), even when cultured in a non-contact-dependent manner using Transwell inserts (Fig. 2 F). These findings indicate that PMN-MDSCs from CIA mice promote T-bet expression in CD11c + B cells through cytokine signaling in a non-contact-dependent manner, leading to an increased proportion of ABCs (Fig. 2 G). PMN-MDSCs promote ABC proliferation and differentiation via BAFF Our previous studies have demonstrated that PMN-MDSCs from CIA mice express high levels of BAFF[ 9 ], also known as BLyS or TNFSF13B. As a member of the TNF superfamily, BAFF plays a crucial role in promoting B cell survival and activation[ 16 ]. We confirmed this finding, observing significantly elevated BAFF expression in PMN-MDSCs from the spleens and joints of CIA mice, particularly in affected joints (Fig. 3 A), along with increased serum BAFF levels (Fig. 3 B). This suggests that PMN-MDSCs and ABCs accumulate in the joints of CIA mice, where PMN-MDSCs may drive ABC proliferation and differentiation through BAFF secretion, thereby exacerbating joint inflammation. To test this hypothesis, we cultured B cells isolated from the spleens of control DBA/1 mice with BAFF to assess its effects on ABCs. BAFF stimulation led to increased expression of pro-inflammatory cytokines (TNF-α, IFN-γ, and IL-17A) in CD11c + B cells, along with upregulated T-bet expression, resulting in higher ABC levels (Figs. 3 C, D). Furthermore, the proportion of Ki67 + ABCs increased significantly (Fig. 3 E), indicating that BAFF promotes ABC proliferation. Next, we directly sorted CD11c + B cells and found that BAFF directly enhanced T-bet expression and increased ABC proliferation, with a significant rise in Ki67 + ABCs (Figs. 3 F, G). To determine whether PMN-MDSCs regulate ABCs via BAFF, we co-cultured PMN-MDSCs and CD11c + B cells in the presence of anti-BAFF antibodies to block BAFF function. Neutralizing BAFF inhibited the ability of PMN-MDSCs from CIA mice to upregulate T-bet expression in CD11c + B cells (Fig. 3 H). These findings indicate that PMN-MDSCs promote T-bet expression and ABC differentiation through BAFF secretion and that BAFF facilitates ABC proliferation and differentiation in vitro. BAFF promotes ABC proliferation and differentiation via the BAFFr-mediated SYK-ERK1/2 signaling pathway Schweighoffer et al. demonstrated that BAFF induces SYK phosphorylation through BAFFr signaling, and SYK subsequently activates the ERK1/2 kinase to support B cell survival and activation[ 17 ]. Therefore, we hypothesized that BAFF activates SYK via BAFFr signaling and that SYK regulates ERK1/2 to promote T-bet expression in CD11c + B cells, thereby driving ABC differentiation (Fig. 4 A). To test this hypothesis, we first sought to identify the receptor responsible for transducing BAFF signaling. BAFF has three receptors: B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI), and BAFFr. Among these, BAFFr is widely expressed on B cells, playing a more crucial role in their survival and activation compared to the other two receptors[ 18 ]. To assess the role of BAFFr, we used anti-BAFFr antibodies to block its function during BAFF stimulation of CD11c + B cells. Neutralizing BAFFr reduced BAFF-induced T-bet expression in CD11c + B cells (Fig. 4 B), indicating that BAFF regulates T-bet expression through BAFFr signaling. Further investigation of the BAFF signaling pathway revealed increased SYK and ERK1/2 phosphorylation levels following 24 h of BAFF stimulation (Figs. 4 C-E). To confirm that BAFF operates through this pathway, we introduced a SYK inhibitor or an ERK1/2 inhibitor into the culture. Inhibition of SYK suppressed ERK1/2 phosphorylation (Fig. 4 F), while both SYK and ERK1/2 inhibitors suppressed BAFF-induced T-bet expression in CD11c + B cells (Figs. 4 G, H). Overall, these findings demonstrate that BAFF promotes ABC proliferation and differentiation via the BAFFr-mediated SYK-ERK1/2 signaling pathway. SYK inhibitor treatment improves CIA-induced arthritis and reduces ABC levels in various tissues To evaluate whether SYK inhibition can suppress ABC proliferation and differentiation in vivo and produce therapeutic effects, we treated CIA mice with SYK inhibitor via intraperitoneal injection every other day, starting from day 21 post-immunization. By the treatment endpoint (day 42), joint swelling was significantly reduced in the SYK inhibitor-treated group compared to the control group (Fig. 5 A). Additionally, beginning on day 32, joint scores were markedly lower in the treated group compared to controls (Fig. 5 B). Histological analysis with Hematoxylin and Eosin (H&E) staining revealed extensive inflammatory cell infiltration, bone destruction, and synovial hyperplasia in the joints of control mice. In contrast, SYK inhibitor treatment demonstrated reduced inflammation, synovial hyperplasia, and bone destruction to some extent (Fig. 5 C). TRAP staining further confirmed fewer osteoclasts in the joints of SYK inhibitor-treated mice, indicating that SYK inhibition effectively mitigates joint destruction in CIA mice (Fig. 5 D). Flow cytometry analysis revealed a significant reduction in ABC levels within the spleens, joints, and lymph nodes of CIA mice following SYK inhibitor treatment. This effect was primarily mediated via the inhibition of T-bet expression in CD11c + B cells (Fig. 5 E), consistent with our in vitro findings. Regulatory correlation between elevated PMN-MDSC and ABC levels in patients with RA To further explore the regulatory relationship between PMN-MDSCs and ABCs in patients with RA, we analyzed their levels in peripheral blood. Our analysis revealed that both PMN-MDSCs and ABCs were significantly elevated in the peripheral blood of patients with RA compared to healthy controls, with a strong positive correlation between their levels (Fig. 6 A). Next, we examined the affected joints of patients with RA by evaluating PMN-MDSC and ABC levels in the synovial fluid. Notably, their levels in RA synovial fluid were approximately 10 times higher than those in peripheral blood (Fig. 6 B), indicating substantial enrichment of these cell types within the affected joints. Additionally, a strong positive correlation was observed between PMN-MDSCs and ABCs in RA synovial fluid (Fig. 6 C). Moreover, BAFF levels in RA synovial fluid were significantly higher than those in the osteoarthritis control group (Fig. 6 D). These findings suggest that, similar to the CIA mouse model, PMN-MDSCs and ABCs accumulate in the affected joints of patients with RA, where PMN-MDSCs may regulate ABC proliferation and differentiation, driving joint inflammation. To validate this hypothesis, we co-cultured PMN-MDSCs isolated from patients with RA and B cells from healthy donors under direct-contact conditions. The results revealed that RA-derived PMN-MDSCs enhanced T-bet expression in CD11c + B cells, thereby increasing ABC levels (Fig. 6 E). These findings are consistent with observations in the CIA mouse model. In summary, our study demonstrates that PMN-MDSCs and ABCs are elevated in patients with RA and exhibit a significant positive correlation. PMN-MDSCs promote ABC differentiation, contributing to RA-associated inflammation. Discussion This study highlights a crucial role of PMN-MDSCs in RA. We demonstrated that both PMN-MDSCs and ABCs are significantly enriched in affected joints, with PMN-MDSCs-derived BAFF promoting the proliferation and differentiation of the inflammatory B cell subset ABCs by upregulating T-bet expression in CD11c + B cells. In RA, the complex interplay between specialized immune cells and effector cells such as ABCs plays a pivotal role in driving inflammation. These findings not only enhance our understanding of RA pathogenesis but also pave the way for innovative therapeutic strategies that target these cellular interactions to modulate RA disease progression. Growing evidence suggests that PMN-MDSCs are pathologically activated neutrophils with distinct immune-regulatory functions[ 19 ]. Numerous studies have highlighted the pathogenic role of MDSCs in autoimmune diseases[ 4 , 20 – 24 ]. For instance, Guo et al. reported that MDSCs in CIA mice produce high levels of inflammatory cytokines, including IL-1β and TNF-α[ 4 ]. Similarly, Yi and Zhang demonstrated that MDSCs in experimental autoimmune encephalomyelitis and CIA mice promote Th17 differentiation via IL-1β, thereby exacerbating disease progression[ 20 , 21 ]. In our study, we found that PMN-MDSCs contribute to RA pathogenesis primarily by regulating B cell subsets. However, research on MDSC-mediated regulation of B cells remains limited. Knier et al. reported that PMN-MDSCs regulate B cell aggregation and cytokine secretion[ 25 ], while other studies have shown that MDSCs promote B cell proliferation and differentiation[ 26 – 28 ]. For instance, Jang[ 27 ] and Dong[ 28 ] found that PMN-MDSCs enhance B cell activation and plasma cell survival in systemic lupus erythematosus (SLE). Consistent with previous research, we observed abnormal elevation of both PMN-MDSCs and ABCs in CIA mice[ 3 , 4 , 9 , 21 ]. To our knowledge, this study is the first to identify a direct regulatory relationship between these two cell types. Flow cytometry analysis revealed significant enrichment of PMN-MDSCs and ABCs in affected joints, with a strong positive correlation between their proportions. Furthermore, co-culture experiments using cells from CIA mice and patients with RA confirmed the regulatory effect of PMN-MDSCs on ABCs. These findings indicate that PMN-MDSCs play a critical role in RA pathogenesis and may serve as a valuable marker for monitoring disease activity. ABCs express both classical B cell markers (CD19 and B220) as well as myeloid cell markers (CD11c and CD11b)[ 29 ]. However, the expression of CD11c and CD11b in ABCs is regulated by their activation and differentiation states[ 30 ], meaning these markers cannot be used as definitive identifiers for ABCs. ABCs also exhibit a distinct transcriptional profile, characterized by increased expression of Itgax, Itgam, and Tbx21 and decreased expression of Il4r, Fcer2a, and Cr2[ 31 ]. Among these, T-bet, a transcription factor upregulated by IFN-γ, plays a central role in regulating ABC differentiation[ 11 , 15 , 32 ]. Our study is the first to assess how PMN-MDSCs regulate T-bet expression in CD11c + B cells, promoting their differentiation into ABCs. Emerging evidence suggests that ABCs are enriched in self-reactive clones that recognize antinuclear antibodies-related antigens and produce IgG2a/c in mice or IgG1 in humans[ 10 , 31 , 33 – 35 ]. Additionally, ABCs function as potent antigen-presenting cells that can activate T cells and promote impaired affinity maturation, contributing to increased susceptibility to infections[ 36 , 37 ]. Beyond aging and infection, ABCs are significantly elevated in various autoimmune diseases, including SLE[ 29 , 38 ], RA[ 29 ], and Sjögren’s syndrome[ 39 ], as well as in autoimmune mouse models such as lupus[ 29 ] and CIA mice[ 13 ]. Consequently, ABCs are recognized as a critical pathogenic B cell subset in autoimmune diseases. In RA specifically, ABCs are central to disease pathogenesis. They are significantly elevated in the blood, spleen, and inflamed joints of CIA mice, as well as in the blood, synovial fluid, and synovial tissue of patients with RA. ABC levels correlate positively with disease activity, and their proportion in the synovial fluid is over 10 times higher than in the blood, suggesting that ABCs preferentially accumulate in inflamed joints, where they exert pro-inflammatory effects[ 3 ]. Our experiments confirmed these findings. Vidal-Pedrola et al. reported that ABCs express high levels of chemokine receptors and adhesion molecules, such as CXCR3 and CD97, indicating their tendency to migrate to inflamed tissues rather than lymph nodes. In synovial fluid, ABCs exhibit a highly activated and proliferative phenotype[ 40 ]. In our study, we observed that CD11c + B cells in CIA mice exhibit a distinct pro-inflammatory and chemotactic profile, potentially enhancing their migration to inflamed tissues and exacerbating joint inflammation. This phenomenon appears to be mediated by PMN-MDSCs, which regulate CD11c + B cells and contribute to the increased ABC levels in CIA mice. However, due to the complexity of the in vivo environment, further research is needed to validate this hypothesis. BAFF, a B cell-activating factor belonging to the TNF family, plays a crucial role in B cell maturation and survival and T cell activation in autoimmune diseases. Research has shown that BAFF levels are significantly elevated in the peripheral blood and synovial fluid of patients with RA[ 16 ], with serum BAFF levels positively correlating with rheumatoid factor titers, highlighting its role in RA progression. BAFF is expressed by various cells. Giordano et al. reported that BAFF produced by dendritic cells, monocytes, and neutrophils is essential for B cell maturation and autoantibody production[ 41 ]. Our previous studies demonstrated that PMN-MDSCs promote RA development in CIA mice by regulating B cells through BAFF overexpression[ 9 ]. In the current study, we further explored the role and mechanism by which PMN-MDSCs modulate ABCs via BAFF in RA. Several aspects of this study require further investigation. Our study primarily focused on the regulation of ABCs by PMN-MDSCs and BAFF. However, as shown in Fig. 3 H, blocking BAFF function did not reduce the regulatory effect of PMN-MDSCs on ABCs to the level observed in the control group, suggesting that PMN-MDSCs may influence ABCs through additional mechanisms. Additionally, our mechanistic analysis was limited to blocking BAFFr to explore the role of BAFF through this pathway. Although BAFF regulates B cell activation primarily through BAFFr, it may also exert a regulatory role through its other receptors, BCMA and TACI. Moreover, it remains unclear whether other cells, such as fibroblast-like synoviocytes, also produce high levels of BAFF and contribute to ABC regulation in RA. Further studies are needed to explore these alternative pathways and identify additional therapeutic targets. Conclusions In conclusion, this study demonstrated elevated levels of PMN-MDSCs and ABCs in both patients with RA and CIA mice, with preferential accumulation in affected joints. PMN-MDSCs from CIA mice enhanced ABC proliferation and differentiation via the BAFF-mediated SYK-ERK1/2 signaling pathway, thereby promoting RA-associated inflammation. The critical involvement of the BAFF/BAFFr/SYK/ERK1/2 axis highlights therapeutic potential for anti-BAFF antibodies and SYK inhibitors in RA treatment. Declarations Acknowledgments This manuscript has not been published in whole or in part nor is it being considered for publication elsewhere. The authors declare that there are no financial or other relationships that might lead to a conflict of interest in the present article. All authors have reviewed the final version of the manuscript and approved it for publication. Author contributions All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. H.Zhang and Tang had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Tang, H.Zhang,Sun Acquisition of data. Hong, Yin,Y.Q.Wang , Y.N.Wang, J.Zhang. Analysis and interpretation of data. Hong, Tang, H.Zhang. Ethics approval Human participants were recruited with informed consent approved by the Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School (2024-160-01) and in compliance with the Helsinki Declaration. The animal use and the experimental protocols were reviewed and approved by the Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School(2021AE01061). Funding The work was supported by the National Natural Science Foundation of China (grant numbers: 81671608 and 81971525) Conflict of interest statement The authors declare no conflict of interest. Consent for publication All authors have reviewed the final version of the manuscript and approved it for publication. References Smolen JS, Aletaha D, McInnes IB. Rheumatoid arthritis. Lancet . 2016;388(10055):2023-2038. Bao W, Xie M, Ye Y. Age-associated B cells indicate disease activity in rheumatoid arthritis. Cell Immunol . 2022;377:104533. 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IL-21 drives expansion and plasma cell differentiation of autoreactive CD11chiT-bet+ B cells in SLE. Nat Commun . 2018;9(1):1758. Rubtsov AV, Rubtsova K, Kappler JW, Jacobelli J, Friedman RS, Marrack P. CD11c-Expressing B Cells Are Located at the T Cell/B Cell Border in Spleen and Are Potent APCs. J Immunol . 2015;195(1):71-79. Zhang W, Zhang H, Liu S, Xia F, Kang Z, Zhang Y, et al. Excessive CD11c+Tbet+ B cells promote aberrant TFH differentiation and affinity-based germinal center selection in lupus. Proc Natl Acad Sci U S A . 2019;116(37):18550-18560. Liu Y, Zhou S, Qian J, Wang Y, Yu X, Dai D, et al. T-bet(+)CD11c(+) B cells are critical for antichromatin immunoglobulin G production in the development of lupus. Arthritis Res Ther . 2017;19(1):225. Saadoun D, Terrier B, Bannock J, Vazquez T, Massad C, Kang I, et al. Expansion of autoreactive unresponsive CD21-/low B cells in Sjogren's syndrome-associated lymphoproliferation. Arthritis Rheum . 2013;65(4):1085-1096. Vidal-Pedrola G, Naamane N, Cameron JA, Pratt AG, Mellor AL, Isaacs JD, et al. Characterization of age-associated B cells in early drug-naïve rheumatoid arthritis patients. Immunology . 2023;168(4):640-653. Giordano D, Kuley R, Draves KE, Elkon KB, Giltiay NV, Clark EA. B cell-activating factor (BAFF) from dendritic cells, monocytes and neutrophils is required for B cell maturation and autoantibody production in SLE-like autoimmune disease. Front Immunol . 2023;14:1050528. Additional Declarations No competing interests reported. Supplementary Files Originalfulllengthwesternblots.docx Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-6846731","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":487761566,"identity":"1b0b7575-8b11-473e-9e89-ead4fef41811","order_by":0,"name":"Huiming Hong","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Huiming","middleName":"","lastName":"Hong","suffix":""},{"id":487761567,"identity":"6e066639-8cad-4028-9b1b-e92ff60d90d0","order_by":1,"name":"Fanzhang Yin","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Fanzhang","middleName":"","lastName":"Yin","suffix":""},{"id":487761568,"identity":"20e03aa8-0588-4c74-b53e-613f7a4c9810","order_by":2,"name":"Yanan Wang","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yanan","middleName":"","lastName":"Wang","suffix":""},{"id":487761569,"identity":"b479e25f-9d86-4219-84dd-f33dfeef627b","order_by":3,"name":"Yongqi Wang","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yongqi","middleName":"","lastName":"Wang","suffix":""},{"id":487761570,"identity":"d7c04ff7-e31b-4e02-a2df-901a3932974a","order_by":4,"name":"Jingxue Zhang","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital Clinical College of Nanjing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jingxue","middleName":"","lastName":"Zhang","suffix":""},{"id":487761571,"identity":"4db2d105-0806-422e-a99e-7cc39cccf8ae","order_by":5,"name":"Xiaojun Tang","email":"","orcid":"","institution":"Affiliated Drum Tower Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Xiaojun","middleName":"","lastName":"Tang","suffix":""},{"id":487761572,"identity":"c40a8ebe-69b3-48c9-a007-8d9dc5eb0716","order_by":6,"name":"Huayong Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYBACAzhLAkRUMDDD2URqOUOyFsY2JO24gLlE8jOJjztqGeRnNz97+HXeHXaDA8wHb/Mw2OXh0mI5I81McuaZ4wyMc46ZG8tue8ZscIAt2ZqHIbkYp8NuJJhJ87YdY2CWADIktx0GauExk+ZhOJDYgFNL+jewFjYJIENyDkgL/zcCWnJAttQw8EjkmEl+bADbwoZfy5k3xZYz2w7wSEjklEkzHDvMLHmYzdhyjkEybi3H0zfe+NhWJyc/I32b5I+aw8l8x5sf3nhTYYdTCxCwAGPhMA+IxQwkkyGRaYBbPUjhBwaGOjCL8QcDgx1etaNgFIyCUTAiAQBn81LlXABTiQAAAABJRU5ErkJggg==","orcid":"","institution":"Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Huayong","middleName":"","lastName":"Zhang","suffix":""},{"id":487761573,"identity":"5665ff83-049d-4cac-af89-1a40e19c7a13","order_by":7,"name":"Lingyun Sun","email":"","orcid":"","institution":"Affiliated Drum Tower Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Lingyun","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2025-06-08 09:53:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6846731/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6846731/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87381225,"identity":"949c6cc9-7a45-4c9a-8b48-ecbe2ddc93a2","added_by":"auto","created_at":"2025-07-23 08:37:14","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1000208,"visible":true,"origin":"","legend":"\u003cp\u003eAbnormal expansion of PMN-MDSCs and ABCs in CIA mice. Cells from joints, spleen, and peripheral blood were stained with CD11b, Ly6G, Ly6C, B220, CD11c, and T-bet antibodies at 2, 4, and 6 weeks post-CIA induction, with DBA/1 mice as controls. (A, B) Representative contour plots, percentages, and absolute counts of PMN-MDSCs (CD11b⁺ Ly6G⁺ Ly6C\u003csup\u003elow\u003c/sup\u003e) were analyzed in the spleen (control, n=5; CIA, n=4), joints (control, n=5; CIA, n=5), and peripheral blood (control, n=4; CIA, n=4) of control and CIA mice. Similarly, ABCs (B220⁺ CD11c⁺ T-bet⁺) were evaluated in these tissues (control, n=4; CIA, n=4). (C) Changes in PMN-MDSC and ABC percentages in joints at 2, 4, and 6 weeks post-CIA induction. (D) Correlation analysis of PMN-MDSC and ABC proportions in the joints (n=23), peripheral blood (n=11), and spleen (n=13) of CIA mice. All data are representative of multiple independent experiments. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001; ns, no significant.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6846731/v1/847316edcca2e050622a8705.jpeg"},{"id":87383980,"identity":"de9e043e-e1ae-4f7c-b5b6-f200bdf9e2ec","added_by":"auto","created_at":"2025-07-23 08:45:14","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":797746,"visible":true,"origin":"","legend":"\u003cp\u003eDistinct pro-inflammatory and chemotactic phenotype of CD11c⁺ B cells in CIA mice, with PMN-MDSCs promoting T-bet expression through a non-contact-dependent mechanism. (A) Representative contour plots and percentages of CD11c⁺ B cells and their T-bet expression in spleens of control and CIA mice. (B, C) RNA-seq analysis of CD11c⁺ B cells from control and CIA mice spleens: (B) volcano plot of differentially expressed genes; (C) clustered heatmap of genes related to inflammation and chemotaxis (n=4 per group). (D, E) PMN-MDSCs from CIA and control mice were co-cultured with CD11c⁺ B cells from control mice in a direct-contact condition, and T-bet⁺ cell percentages were analyzed by flow cytometry (n=4 per group). (F, G) PMN-MDSCs from CIA and control mice were co-cultured with CD11c⁺ B cells from control mice using a Transwell system (non-direct contact), and T-bet⁺ cell percentages were analyzed using flow cytometry (n=5 per group). *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6846731/v1/2770826a1c1bee9102c402de.jpeg"},{"id":87385361,"identity":"ccaecc23-bc7f-46f2-a788-e135f444f1c8","added_by":"auto","created_at":"2025-07-23 08:53:14","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":916110,"visible":true,"origin":"","legend":"\u003cp\u003ePMN-MDSCs from CIA mice promote ABC proliferation and differentiation via high BAFF expression. (A) Representative contour plots, percentages, and numbers of BAFF⁺ PMN-MDSCs in spleen and joints of control and CIA mice (n=4 per group). (B) ELISA quantification of serum BAFF levels in control and CIA mice (n=4 per group). (C) Flow cytometry analysis of TNF-α, IFN-γ, and IL-17A expression in CD11c⁺B cells isolated from the spleen of control DBA/1 mice after in vitro BAFF treatment (n=5 per group). (D, E) In vitro BAFF-induced ABC proliferation and differentiation assessed by flow cytometry analysis of CD11c⁺B cells isolated from the spleen of control DBA/1 mice (n=4 per group). (F, G) In vitro BAFF stimulation effect on ABC proliferation and differentiation assessed via flow cytometry analysis of CD11c⁺B cells isolated from the spleen of control DBA/1 mice (n=4 per group). (H) co-culture experiment of PMN-MDSCs and CD11c⁺B cells isolated from the spleen of control and CIA mice. Control group cells were co-cultured using PMN-MDSCs and CD11c⁺B cells from DBA/1 mice, while CIA group cells were co-cultured using PMN-MDSCs from CIA mice and CD11c⁺B cells from DBA/1 mice. The anti-BAFF group included anti-BAFF to block BAFF function in the CIA group, and T-bet expression in CD11c⁺ B cells was assessed via flow cytometry (n=4 per group). *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6846731/v1/596383dbe5d34e8c8c83dc82.jpeg"},{"id":87383977,"identity":"d6b54db6-84ab-4a4d-aed9-12b939754006","added_by":"auto","created_at":"2025-07-23 08:45:14","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":465000,"visible":true,"origin":"","legend":"\u003cp\u003eBAFF promotes ABC proliferation and differentiation via the BAFFr-mediated SYK-ERK1/2 signaling pathway. (A) Schematic illustration of BAFF-driven T-bet upregulation in CD11c⁺B cells leading to ABC differentiation. (B) Blocking BAFFr reduces BAFF-mediated T-bet expression in CD11c⁺B cells (n=4 per group). (C–E) Flow cytometry and Western blot analysis showing increased SYK and ERK1/2 phosphorylation in CD11c⁺B cells after 24 h of BAFF stimulation. (F) SYK inhibition reduced ERK1/2 phosphorylation (n=3–4 per group). (G, H) SYK and ERK1/2 inhibitors suppressed BAFF-induced T-bet expression in CD11c⁺B cells (n=4 per group). *p \u0026lt; 0.05; **p \u0026lt; 0.01; ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6846731/v1/44b7d1801ba06436a118e9eb.jpeg"},{"id":87381228,"identity":"8b0aa64e-e71d-443e-ae11-4e68e5715f9c","added_by":"auto","created_at":"2025-07-23 08:37:14","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":573936,"visible":true,"origin":"","legend":"\u003cp\u003eSYK inhibitor treatment reduces joint inflammation and ABC differentiation in CIA mice. CIA mice received either double-distilled water (control group) or SYK inhibitor treatment (n=5 per group). (A) Representative composite images of treated and untreated mouse groups. (B) Arthritis score progression in both groups. (C, D) H\u0026amp;E and TRAP staining of ankle joints in treated and untreated groups. (E) ABC proportion in various tissues of the treated and untreated groups. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ****p \u0026lt; 0.0001; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6846731/v1/a412eb9e0447b6d909218fdf.jpeg"},{"id":87381233,"identity":"ba16d2a3-3e43-4614-99eb-630ab487343c","added_by":"auto","created_at":"2025-07-23 08:37:14","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":257090,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased PMN-MDSC and ABC proportions in peripheral blood and synovial fluid of patients with RA, with PMN-MDSCs promoting ABC differentiation. \u003c/strong\u003e(A) Statistical analysis of PMN-MDSC (n=11) and ABC (n=18) percentages in PBMCs from patients with RA and healthy controls, with correlation analysis (n=29). (B) Comparative analysis of PMN-MDSC and ABC levels in the peripheral blood and synovial fluid of patients with RA. (C) Correlation analysis of PMN-MDSC and ABC levels in the synovial fluid of patients with RA (n=9). (D) ELISA quantification of BAFF levels in the synovial fluid from patients with osteoarthritis (n=7) and RA (n=5). (E) PMN-MDSCs isolated from the peripheral blood of four patients with RA were co-cultured with B cells from the peripheral blood of one healthy control. The cells were co-cultured in direct contact for 3 days, and PMN-MDSC effects on ABC proliferation and differentiation were analyzed using flow cytometry (n=4 per group). *p \u0026lt; 0.05; **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6846731/v1/7374ca4f1d2e18fd9eb480d9.jpeg"},{"id":89485656,"identity":"1bbbccd9-d705-400d-ae90-5b5e69330f5a","added_by":"auto","created_at":"2025-08-20 12:47:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5078356,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6846731/v1/f4584247-757c-4bcd-aae1-c190e22bb612.pdf"},{"id":87385362,"identity":"ed7fe3dd-bc2d-421d-a583-f0755d655f3c","added_by":"auto","created_at":"2025-07-23 08:53:14","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1047162,"visible":true,"origin":"","legend":"","description":"","filename":"Originalfulllengthwesternblots.docx","url":"https://assets-eu.researchsquare.com/files/rs-6846731/v1/60b13dd75ec189117d682e35.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Polymorphonuclear Myeloid-Derived Suppressor Cells Promote Inflammation Progression of Rheumatoid Arthritis by Inducing Age-Associated B Cell Proliferation and Differentiation via the BAFF-Mediated SYK-ERK1/2 Pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by chronic joint inflammation, leading to cartilage and bone damage and, in severe cases, disability[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although the exact pathogenesis remains unclear, RA progression involves complex dysregulation, affecting multiple immune cell populations, including T cells, macrophages, and fibroblast-like synoviocytes. Emerging evidence indicates a significant accumulation of myeloid-derived suppressor cells (MDSCs) and age-associated B cells (ABCs) in RA-affected joints[\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], with both cell types closely linked to disease severity. However, despite their co-localization and strong involvement in RA pathogenesis, the potential regulatory relationship between MDSCs and ABCs remains unexplored.\u003c/p\u003e\u003cp\u003eIn mice, MDSCs consist of two distinct subpopulations: polymorphonuclear MDSCs (PMN-MDSCs, CD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003elow\u003c/sup\u003e) and monocytic MDSCs (M-MDSCs, CD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eLy6C\u003csup\u003ehigh\u003c/sup\u003e)[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These cells have gained increasing attention for their role in RA pathogenesis. Emerging research indicates that MDSCs promote inflammation and exacerbate RA progression, with significantly higher levels in patients with RA compared to healthy controls, a trend also observed in other arthritis-related diseases[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Mouse models with elevated MDSCs exhibit increased interleukin (IL)-1β and tumor necrosis factor-alpha (TNF-α) production, while PMN-MDSCs isolated from these models (spleens) lose their characteristic T cell suppressive function[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In contrast, M-MDSCs inhibit T cell proliferation and interferon-gamma (IFN-γ) production via inducible nitric oxide synthase while simultaneously promoting T helper 17 (Th17) differentiation through IL-1β. The pathogenic role of MDSCs is further supported by the adoptive transfer of MDSCs from the spleens of collagen-induced arthritis (CIA) mice, where CIA-derived MDSCs aggravated disease severity, whereas MDSC depletion alleviated disease progression, reduced Th17 differentiation, and decreased IL-17A secretion[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Additionally, our previous research found that PMN-MDSCs enhance B cell secretion of TNF-α, further exacerbating arthritis in CIA mice[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These findings suggest that MDSCs influence RA etiology through T and B cell regulation. However, their precise roles in RA pathogenesis remain unclear, requiring further investigation.\u003c/p\u003e\u003cp\u003eA recently identified B cell subpopulation, known as ABCs, has been found to increase in several autoimmune diseases and mouse models[\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In CIA mice, ABCs are characterized by the expression of B220, CD11c, and T-bet[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], playing a crucial role in the progression of RA. Qin et al. found that ABC levels were elevated in various tissues of both patients with RA and CIA mice, with a positive correlation to disease activity. Notably, the proportion of ABCs in synovial fluid was found to be 10 times higher than in the blood of the same individual, indicating preferential recruitment to inflammatory sites. Mechanistically, ABCs contribute to RA pathogenesis by activating fibroblast-like synoviocytes through TNF-α signaling[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur study aimed to explore whether PMN-MDSCs regulate the function of ABCs in RA and to elucidate the molecular mechanisms involved in this potential interaction. Additionally, we sought to examine whether targeting these regulatory pathways could modulate disease progression in both patients with RA and CIA mouse models, offering new therapeutic strategies for RA treatment.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMice\u003c/h2\u003e\u003cp\u003eMale DBA/1 mice (8\u0026ndash;10 weeks old) were obtained from Cavens Experimental Animal Co., Ltd. (Changzhou, China) and maintained under specific pathogen-free conditions. All animal protocols of this study were approved by the Ethics Committee of Nanjing University of Chinese Medicine, Drum Tower Clinical School of Integrated Chinese and Western Medicine, Drum Tower Hospital.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInduction of CIA, scoring, and SYK inhibitor treatment\u003c/h3\u003e\n\u003cp\u003eEqual volumes of bovine type II collagen and complete/incomplete Freund\u0026rsquo;s adjuvant were emulsified in a 5 mL syringe using a high-speed homogenizer for 1 min. The emulsion was then cooled down by placing the syringe in ice water for 2 min. This process of mixing and cooling was repeated 10 times. On day 1, DBA/1 mice received a subcutaneous injection of 100 \u0026micro;L of the emulsion containing complete Freund\u0026rsquo;s adjuvant at the base of the tail for the initial immunization. A second immunization was administered on day 21 using an emulsion containing incomplete Freund\u0026rsquo;s adjuvant. Starting from day 28 post-immunization, joint inflammation was assessed every 2 days, and arthritis severity was scored using established criteria following a previously published system[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The scoring system was as follows: 0, normal appearance; 1, mild redness or swelling of joints/digits; 2, moderate redness and swelling extending from ankle to midfoot; 3, severe redness and swelling of ankle, foot, and digits; and 4, severe inflammation with joint deformity and functional impairment. The total joint score for each mouse was determined by summing the scores of all four limbs. CIA mice were randomly divided into two groups, with five mice per group, and housed separately in two cages. For the SYK inhibitor treatment, CIA mice were administered an intraperitoneal injection of 5 mg/kg of the SYK inhibitor (PRT062607, Selleck), dissolved in distilled water, on alternate days from day 21 to day 42. Control mice received an equivalent volume of double-distilled water.\u003c/p\u003e\n\u003ch3\u003eHistopathological analysis of joint specimens\u003c/h3\u003e\n\u003cp\u003eFollowing Cervical dislocation method to execute mice, the right hindlimb joint was dissected, and the skin was removed. The joint was then washed with 10% phosphate-buffered saline and fixed in 4% paraformaldehyde. Next, decalcification was performed using 10% ethylenediaminetetraacetic acid or 5% formic acid for 30 days. Once decalcified, the joints were embedded in paraffin, sectioned sagittally, and stained with H\u0026amp;E and TRAP staining (Nanjing Youmeng Biotechnology Co., Ltd.). Synovial inflammation and osteoclastogenesis were assessed microscopically.\u003c/p\u003e\n\u003ch3\u003eIsolation and co-culture of PMN-MDSCs and CD11c B cells from mice\u003c/h3\u003e\n\u003cp\u003eSpleen lymphocytes were isolated under sterile conditions from DBA/1 and CIA mice. The cells were stained with the following antibodies: AF700 anti-mouse CD11b (Biolegend, M1/70), FITC anti-mouse Ly6G (Biolegend, 1A8), PE/Cy7 anti-mouse Ly6C (Biolegend, HK1.4), APC anti-mouse B220 (Biolegend, RA3-6B2), and BV785 anti-mouse CD11c (Biolegend, N418). PMN-MDSCs and CD11c\u003csup\u003e+\u003c/sup\u003e B cells were sorted using an Aria III flow cytometer (BD Bioscience), with sorted cell purity of \u0026gt;\u0026thinsp;95%. The collected cells were stored in 15 mL centrifuge tubes for subsequent experiments.\u003c/p\u003e\u003cp\u003eCD11c\u003csup\u003e+\u003c/sup\u003e B cells (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e per well) were cultured in 96-well U-bottom plates in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 ng/mL toll-like receptor 7 (TLR7) (R\u0026amp;D), and 50 ng/mL IL-21 (R\u0026amp;D) for 72 h at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. When co-cultured with PMN-MDSCs, 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e PMN-MDSCs were added to the well, and 5 \u0026micro;g/mL anti-mouse BAFF (R\u0026amp;D) was used to block BAFF function. To examine the role of BAFF in CD11c\u003csup\u003e+\u003c/sup\u003e B cells culture, 100 ng/mL BAFF (R\u0026amp;D) and 10 \u0026micro;g/mL anti-mouse BAFFr (R\u0026amp;D) were added to inhibit BAFFr. Additionally, SYK and ERK1/2 inhibitors (Selleck) were added to explore the specific mechanism by which BAFF regulates CD11c\u003csup\u003e+\u003c/sup\u003e B cells.\u003c/p\u003e\n\u003ch3\u003eIsolation and co-culture of PMN-MDSCs and B cells from human samples\u003c/h3\u003e\n\u003cp\u003ePBMCs were isolated under sterile conditions from patients with RA and healthy controls. For surface staining, cells were incubated for 20 min with the following fluorochrome-conjugated antibodies: anti-human CD11b (Alexa Fluor 700, M1/70), CD33 (Brilliant Violet 605, P67.6), CD15 (FITC, HI98), CD14 (PerCP/Cyanine5.5, 63D3), and CD19 (APC/Cyanine7, HIB19). PMN-MDSCs and B cells were then sorted using a flow cytometer (Aria Ⅲ, BD Bioscience), achieving a purity of \u0026gt;\u0026thinsp;95%, and collected in 15 mL centrifuge tubes for further experiments. For co-culture experiments, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e B cells were seeded into each well of a 96-well U-bottom plate containing RPMI 1640 medium supplemented with 10% FBS, 100 ng/mL TLR7 agonist (R\u0026amp;D), 20 ng/mL IFN-γ (R\u0026amp;D), and 20 ng/mL granulocyte-macrophage colony-stimulating factor (R\u0026amp;D). To a 1:1 cell ratio, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e PMN-MDSCs were added to the same wells. The plates were incubated at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 72 h. This co-culture system was designed to investigate the regulatory effects of RA-derived PMN-MDSCs on B cell proliferation and differentiation.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e\u003cp\u003eSingle-cell suspensions were prepared from cultured cells, as well as mouse spleen, joint, peripheral blood, lymph nodes, and human peripheral blood and synovial fluid. For intracellular staining, mouse samples were resuspended in RPMI 1640 medium containing 100 ng/mL PMA (Enzo), 1 \u0026micro;g/mL ionomycin (Enzo), and 5 \u0026micro;g/mL BFA (Enzo) and incubated at 37\u0026deg;C for 4 h. Dead cells were identified using eBioscience\u0026trade; Fixable Viability Dye eFluor\u0026trade; 506. Surface marker staining was performed by incubating cells for 20 min with the following anti-mouse antibodies: CD11b (Alexa Fluor 700 M1/70), Ly6G (FITC 1A8), Ly6C (PE/Cy7 HK1.4), B220 (APC RA3-6B2), CD11c (BV785 N418), and CD4 (FITC RM4-5). After fixation and permeabilization, intracellular markers were stained for 45 min using anti-mouse BAFF (PE 121808), TNF-α (PE/Cy7 MP6-XT22), IL-17A (APC TC11-18H10.1), IFN-γ (Alexa Fluor 700 XMG1.2), pERK1/2 (APC 6B8B69), and pSYK (APC MOCH1CT). Nuclear markers, including T-bet (BV421 4B10) and Ki67 (PE/Cy7 16A8), were also stained for 45 min after fixation and permeabilization. For human samples, dead cells were labeled and surface-stained for 20 min with anti-human CD11b (Alexa Fluor 700 M1/70), CD33 (BV605 P67.6), CD15 (FITC HI98), CD14 (PerCP/Cy5.5 63D3), CD19 (APC/Cy7 HIB19), CD21 (APC Bu32), and CD11c (BV650 3.9). All antibodies were obtained from Biolegend and eBioscience. Data were acquired using a BD LSRFortessa (BD Biosciences) and analyzed with the FlowJo software (Tree Star).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eUnique identifier RNA-seq and transcriptome analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from CD11c\u003csup\u003e+\u003c/sup\u003e B cells isolated from the spleens of CIA and DBA/1 mice using Trizol Reagent (Invitrogen, cat. NO 15596026). Following extraction, DNA digestion was performed using DNase I. RNA quality was evaluated by measuring the A260/A280 ratio with Nanodrop\u003csup\u003e\u0026trade;\u003c/sup\u003e OneCspectrophotometer (Thermo Fisher Scientific Inc), and RNA integrity was confirmed via 1.5% agarose gel electrophoresis. Quantification of qualified RNAs was conducted using the Qubit\u003csup\u003e\u0026trade;\u003c/sup\u003e RNA Broad Range Assay kit (Life Technologies, Q10210) on a Qubit3.0 fluorometer.\u003c/p\u003e\u003cp\u003eFor stranded RNA-seq library preparation, 2 \u0026micro;g of total RNA was processed using the KC-Digital\u003csup\u003e\u0026trade;\u003c/sup\u003e Stranded mRNA Library Prep Kit for Illumina\u0026reg; (Catalog NO. DR08502, Wuhan Seqhealth Co., Ltd. China), following the manufacturer\u0026rsquo;s instruction. This kit utilizes unique molecular identifiers (UMIs) of eight random bases to label pre-amplified cDNA molecules, minimizing duplication bias during polymerase chain reaction (PCR) amplification and sequencing. Library products corresponding to 200\u0026ndash;500 bps were enriched, quantified, and sequenced on a DNBSEQ-T7 sequencer (MGI Tech Co., Ltd. China) using the PE150 model.\u003c/p\u003e\u003cp\u003eClean reads were grouped based on their UMI sequences, clustering reads with identical UMIs together. Within each cluster, pairwise alignment was performed, and reads with \u0026gt;\u0026thinsp;95% sequence identity were further refined into new sub-clusters. Multiple sequence alignment was then performed to generate a consensus sequence for each sub-cluster, effectively minimizing PCR amplification or sequencing errors and biases.\u003c/p\u003e\u003cp\u003eThe de-duplicated consensus sequences were used for standard RNA-seq analysis. Reads were mapped to the reference genome using the STAR software (version 2.5.3a) with default settings. Gene expression levels were quantified by counting exon-mapped reads using featureCounts (Subread-1.5.1; Bioconductor), and RPKM values were calculated. Differential gene expression analysis between groups was performed using the edgeR package (version 3.12.1), with statistical significance determined by a p-value of 0.05 and a fold-change threshold of 2. Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis for differentially expressed genes were performed using the KOBAS software (version 2.1.1) with a p-value cutoff of 0.05 to assess statistically significant enrichment. Alternative splicing events were identified using rMATS (version 3.2.5), applying a false discovery rate value cutoff of 0.05 and an absolute value of 0.05.\u003c/p\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\n\u003cp\u003eDenatured protein samples were analyzed using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime) and subsequently transferred to 0.2 \u0026micro;m polyvinylidene fluoride (PVDF) membranes (Merck KGaA). The membranes were blocked with 5% bovine serum albumin (BSA) at 20\u0026deg;C for at least 1 h before overnight incubation at 4\u0026deg;C with primary antibodies. Anti-pERK1/2 and anti-ERK1/2 antibodies were obtained from ABclonal, while anti-GAPDH was sourced from AiFang Biological. Primary antibodies were diluted 1:1000 in TBST with 5% BSA. Following this, the membranes were incubated with HRP-conjugated secondary antibodies (anti-rabbit/anti-mouse IgG, Beyotime catalog A0208/A0216) at a 1:10000 dilution. The blots were visualized using ECL reagents (Beyotime), and immunoblot images were quantified using the ImageJ software (v1.8.0).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEnzyme-linked immunosorbent assay\u003c/h2\u003e\u003cp\u003eMouse plasma and human synovial fluid samples were collected and analyzed for BAFF levels using a Human/Mouse/Rat BAFF ELISA Kit (ABclonal, RK00402), following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using the GraphPad Prism software. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (x̄ \u0026plusmn; s). A student\u0026rsquo;s t-test was used for comparisons between two groups, while a one-way analysis of variance was applied for multiple-group analyses. Statistical significance was set as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; ns, not significant).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eIncreased PMN-MDSCs and ABCs in CIA mice with a significant positive correlation in joints\u003c/h2\u003e\u003cp\u003eAnalysis of PMN-MDSC and ABC levels in various tissues of CIA mice showed a significant increase in both cell types within the joints and spleens compared to controls, with the most notable increase occurring in the joints. No significant differences were observed in peripheral blood mononuclear cells (PBMCs) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). These results align with our previous findings[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and those of Qin et al.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In the joints of CIA mice, PMN-MDSC and ABC levels increased in parallel with disease progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating a strong association between these cells and joint inflammation. Furthermore, a significant correlation was observed between PMN-MDSC and ABC levels in the joints of CIA mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), suggesting a potential regulatory interaction that contributes to joint inflammation in CIA mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePMN-MDSCs from CIA mice promote T-bet expression of CD11c\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eB cells in a non-contact-dependent manner, thereby increasing ABC levels\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo explore the regulatory effects of PMN-MDSCs on ABCs, we first examined the underlying cause for the increased ABC levels (B220\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eT-bet\u003csup\u003e+\u003c/sup\u003e) in CIA mice. Our analysis showed that while the proportion of CD11c\u003csup\u003e+\u003c/sup\u003e B cells in the spleens of CIA mice was only slightly different from that of the control group, a significant increase in T-bet expression in CD11c\u003csup\u003e+\u003c/sup\u003e B cells was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). T-bet is a key transcription factor that regulates ABC differentiation and function[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and its elevated expression suggests that PMN-MDSCs in CIA mice may drive the differentiation of CD11c\u003csup\u003e+\u003c/sup\u003e B cells into ABCs by upregulating T-bet expression, thereby increasing ABC levels. To further characterize CD11c\u003csup\u003e+\u003c/sup\u003e B cells in CIA mice, we isolated these cells from the spleens of both CIA and control mice and performed RNA sequencing (RNA-seq) to analyze their transcriptomic profiles. Heatmap analysis revealed 1,092 upregulated and 1,483 downregulated genes in CD11c\u003csup\u003e+\u003c/sup\u003e B cells from CIA mice compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Notably, these CIA-derived CD11c\u003csup\u003e+\u003c/sup\u003e B cells exhibited a distinct pro-inflammatory and chemotactic profile, with a significant increase in T-bet (Tbx21) expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo validate our hypothesis, we co-cultured PMN-MDSCs from CIA and control mice with CD11c\u003csup\u003e+\u003c/sup\u003e B cells from control mice. Flow cytometry analysis showed that PMN-MDSCs from CIA mice enhanced T-bet expression in CD11c\u003csup\u003e+\u003c/sup\u003e B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), even when cultured in a non-contact-dependent manner using Transwell inserts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These findings indicate that PMN-MDSCs from CIA mice promote T-bet expression in CD11c\u003csup\u003e+\u003c/sup\u003e B cells through cytokine signaling in a non-contact-dependent manner, leading to an increased proportion of ABCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003ePMN-MDSCs promote ABC proliferation and differentiation via BAFF\u003c/h2\u003e\u003cp\u003eOur previous studies have demonstrated that PMN-MDSCs from CIA mice express high levels of BAFF[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], also known as BLyS or TNFSF13B. As a member of the TNF superfamily, BAFF plays a crucial role in promoting B cell survival and activation[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. We confirmed this finding, observing significantly elevated BAFF expression in PMN-MDSCs from the spleens and joints of CIA mice, particularly in affected joints (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), along with increased serum BAFF levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This suggests that PMN-MDSCs and ABCs accumulate in the joints of CIA mice, where PMN-MDSCs may drive ABC proliferation and differentiation through BAFF secretion, thereby exacerbating joint inflammation. To test this hypothesis, we cultured B cells isolated from the spleens of control DBA/1 mice with BAFF to assess its effects on ABCs. BAFF stimulation led to increased expression of pro-inflammatory cytokines (TNF-α, IFN-γ, and IL-17A) in CD11c\u003csup\u003e+\u003c/sup\u003e B cells, along with upregulated T-bet expression, resulting in higher ABC levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). Furthermore, the proportion of Ki67\u003csup\u003e+\u003c/sup\u003e ABCs increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), indicating that BAFF promotes ABC proliferation. Next, we directly sorted CD11c\u003csup\u003e+\u003c/sup\u003e B cells and found that BAFF directly enhanced T-bet expression and increased ABC proliferation, with a significant rise in Ki67\u003csup\u003e+\u003c/sup\u003e ABCs (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G). To determine whether PMN-MDSCs regulate ABCs via BAFF, we co-cultured PMN-MDSCs and CD11c\u003csup\u003e+\u003c/sup\u003e B cells in the presence of anti-BAFF antibodies to block BAFF function. Neutralizing BAFF inhibited the ability of PMN-MDSCs from CIA mice to upregulate T-bet expression in CD11c\u003csup\u003e+\u003c/sup\u003eB cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). These findings indicate that PMN-MDSCs promote T-bet expression and ABC differentiation through BAFF secretion and that BAFF facilitates ABC proliferation and differentiation in vitro.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eBAFF promotes ABC proliferation and differentiation via the BAFFr-mediated SYK-ERK1/2 signaling pathway\u003c/h2\u003e\u003cp\u003eSchweighoffer et al. demonstrated that BAFF induces SYK phosphorylation through BAFFr signaling, and SYK subsequently activates the ERK1/2 kinase to support B cell survival and activation[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, we hypothesized that BAFF activates SYK via BAFFr signaling and that SYK regulates ERK1/2 to promote T-bet expression in CD11c\u003csup\u003e+\u003c/sup\u003e B cells, thereby driving ABC differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To test this hypothesis, we first sought to identify the receptor responsible for transducing BAFF signaling. BAFF has three receptors: B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI), and BAFFr. Among these, BAFFr is widely expressed on B cells, playing a more crucial role in their survival and activation compared to the other two receptors[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To assess the role of BAFFr, we used anti-BAFFr antibodies to block its function during BAFF stimulation of CD11c\u003csup\u003e+\u003c/sup\u003e B cells. Neutralizing BAFFr reduced BAFF-induced T-bet expression in CD11c\u003csup\u003e+\u003c/sup\u003e B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), indicating that BAFF regulates T-bet expression through BAFFr signaling. Further investigation of the BAFF signaling pathway revealed increased SYK and ERK1/2 phosphorylation levels following 24 h of BAFF stimulation (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E). To confirm that BAFF operates through this pathway, we introduced a SYK inhibitor or an ERK1/2 inhibitor into the culture. Inhibition of SYK suppressed ERK1/2 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), while both SYK and ERK1/2 inhibitors suppressed BAFF-induced T-bet expression in CD11c\u003csup\u003e+\u003c/sup\u003e B cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, H). Overall, these findings demonstrate that BAFF promotes ABC proliferation and differentiation via the BAFFr-mediated SYK-ERK1/2 signaling pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eSYK inhibitor treatment improves CIA-induced arthritis and reduces ABC levels in various tissues\u003c/h2\u003e\u003cp\u003eTo evaluate whether SYK inhibition can suppress ABC proliferation and differentiation in vivo and produce therapeutic effects, we treated CIA mice with SYK inhibitor via intraperitoneal injection every other day, starting from day 21 post-immunization. By the treatment endpoint (day 42), joint swelling was significantly reduced in the SYK inhibitor-treated group compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Additionally, beginning on day 32, joint scores were markedly lower in the treated group compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Histological analysis with Hematoxylin and Eosin (H\u0026amp;E) staining revealed extensive inflammatory cell infiltration, bone destruction, and synovial hyperplasia in the joints of control mice. In contrast, SYK inhibitor treatment demonstrated reduced inflammation, synovial hyperplasia, and bone destruction to some extent (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). TRAP staining further confirmed fewer osteoclasts in the joints of SYK inhibitor-treated mice, indicating that SYK inhibition effectively mitigates joint destruction in CIA mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Flow cytometry analysis revealed a significant reduction in ABC levels within the spleens, joints, and lymph nodes of CIA mice following SYK inhibitor treatment. This effect was primarily mediated via the inhibition of T-bet expression in CD11c\u003csup\u003e+\u003c/sup\u003e B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), consistent with our in vitro findings.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eRegulatory correlation between elevated PMN-MDSC and ABC levels in patients with RA\u003c/h2\u003e\u003cp\u003eTo further explore the regulatory relationship between PMN-MDSCs and ABCs in patients with RA, we analyzed their levels in peripheral blood. Our analysis revealed that both PMN-MDSCs and ABCs were significantly elevated in the peripheral blood of patients with RA compared to healthy controls, with a strong positive correlation between their levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Next, we examined the affected joints of patients with RA by evaluating PMN-MDSC and ABC levels in the synovial fluid. Notably, their levels in RA synovial fluid were approximately 10 times higher than those in peripheral blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), indicating substantial enrichment of these cell types within the affected joints. Additionally, a strong positive correlation was observed between PMN-MDSCs and ABCs in RA synovial fluid (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Moreover, BAFF levels in RA synovial fluid were significantly higher than those in the osteoarthritis control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These findings suggest that, similar to the CIA mouse model, PMN-MDSCs and ABCs accumulate in the affected joints of patients with RA, where PMN-MDSCs may regulate ABC proliferation and differentiation, driving joint inflammation. To validate this hypothesis, we co-cultured PMN-MDSCs isolated from patients with RA and B cells from healthy donors under direct-contact conditions. The results revealed that RA-derived PMN-MDSCs enhanced T-bet expression in CD11c\u003csup\u003e+\u003c/sup\u003e B cells, thereby increasing ABC levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These findings are consistent with observations in the CIA mouse model. In summary, our study demonstrates that PMN-MDSCs and ABCs are elevated in patients with RA and exhibit a significant positive correlation. PMN-MDSCs promote ABC differentiation, contributing to RA-associated inflammation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study highlights a crucial role of PMN-MDSCs in RA. We demonstrated that both PMN-MDSCs and ABCs are significantly enriched in affected joints, with PMN-MDSCs-derived BAFF promoting the proliferation and differentiation of the inflammatory B cell subset ABCs by upregulating T-bet expression in CD11c\u003csup\u003e+\u003c/sup\u003e B cells. In RA, the complex interplay between specialized immune cells and effector cells such as ABCs plays a pivotal role in driving inflammation. These findings not only enhance our understanding of RA pathogenesis but also pave the way for innovative therapeutic strategies that target these cellular interactions to modulate RA disease progression.\u003c/p\u003e\u003cp\u003eGrowing evidence suggests that PMN-MDSCs are pathologically activated neutrophils with distinct immune-regulatory functions[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Numerous studies have highlighted the pathogenic role of MDSCs in autoimmune diseases[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For instance, Guo et al. reported that MDSCs in CIA mice produce high levels of inflammatory cytokines, including IL-1β and TNF-α[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Similarly, Yi and Zhang demonstrated that MDSCs in experimental autoimmune encephalomyelitis and CIA mice promote Th17 differentiation via IL-1β, thereby exacerbating disease progression[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In our study, we found that PMN-MDSCs contribute to RA pathogenesis primarily by regulating B cell subsets. However, research on MDSC-mediated regulation of B cells remains limited. Knier et al. reported that PMN-MDSCs regulate B cell aggregation and cytokine secretion[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], while other studies have shown that MDSCs promote B cell proliferation and differentiation[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. For instance, Jang[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and Dong[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] found that PMN-MDSCs enhance B cell activation and plasma cell survival in systemic lupus erythematosus (SLE). Consistent with previous research, we observed abnormal elevation of both PMN-MDSCs and ABCs in CIA mice[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. To our knowledge, this study is the first to identify a direct regulatory relationship between these two cell types. Flow cytometry analysis revealed significant enrichment of PMN-MDSCs and ABCs in affected joints, with a strong positive correlation between their proportions. Furthermore, co-culture experiments using cells from CIA mice and patients with RA confirmed the regulatory effect of PMN-MDSCs on ABCs. These findings indicate that PMN-MDSCs play a critical role in RA pathogenesis and may serve as a valuable marker for monitoring disease activity.\u003c/p\u003e\u003cp\u003eABCs express both classical B cell markers (CD19 and B220) as well as myeloid cell markers (CD11c and CD11b)[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, the expression of CD11c and CD11b in ABCs is regulated by their activation and differentiation states[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], meaning these markers cannot be used as definitive identifiers for ABCs. ABCs also exhibit a distinct transcriptional profile, characterized by increased expression of Itgax, Itgam, and Tbx21 and decreased expression of Il4r, Fcer2a, and Cr2[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Among these, T-bet, a transcription factor upregulated by IFN-γ, plays a central role in regulating ABC differentiation[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our study is the first to assess how PMN-MDSCs regulate T-bet expression in CD11c\u003csup\u003e+\u003c/sup\u003eB cells, promoting their differentiation into ABCs. Emerging evidence suggests that ABCs are enriched in self-reactive clones that recognize antinuclear antibodies-related antigens and produce IgG2a/c in mice or IgG1 in humans[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Additionally, ABCs function as potent antigen-presenting cells that can activate T cells and promote impaired affinity maturation, contributing to increased susceptibility to infections[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Beyond aging and infection, ABCs are significantly elevated in various autoimmune diseases, including SLE[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], RA[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and Sj\u0026ouml;gren\u0026rsquo;s syndrome[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], as well as in autoimmune mouse models such as lupus[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and CIA mice[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Consequently, ABCs are recognized as a critical pathogenic B cell subset in autoimmune diseases. In RA specifically, ABCs are central to disease pathogenesis. They are significantly elevated in the blood, spleen, and inflamed joints of CIA mice, as well as in the blood, synovial fluid, and synovial tissue of patients with RA. ABC levels correlate positively with disease activity, and their proportion in the synovial fluid is over 10 times higher than in the blood, suggesting that ABCs preferentially accumulate in inflamed joints, where they exert pro-inflammatory effects[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Our experiments confirmed these findings. Vidal-Pedrola et al. reported that ABCs express high levels of chemokine receptors and adhesion molecules, such as CXCR3 and CD97, indicating their tendency to migrate to inflamed tissues rather than lymph nodes. In synovial fluid, ABCs exhibit a highly activated and proliferative phenotype[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In our study, we observed that CD11c\u003csup\u003e+\u003c/sup\u003e B cells in CIA mice exhibit a distinct pro-inflammatory and chemotactic profile, potentially enhancing their migration to inflamed tissues and exacerbating joint inflammation. This phenomenon appears to be mediated by PMN-MDSCs, which regulate CD11c\u003csup\u003e+\u003c/sup\u003e B cells and contribute to the increased ABC levels in CIA mice. However, due to the complexity of the in vivo environment, further research is needed to validate this hypothesis.\u003c/p\u003e\u003cp\u003eBAFF, a B cell-activating factor belonging to the TNF family, plays a crucial role in B cell maturation and survival and T cell activation in autoimmune diseases. Research has shown that BAFF levels are significantly elevated in the peripheral blood and synovial fluid of patients with RA[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], with serum BAFF levels positively correlating with rheumatoid factor titers, highlighting its role in RA progression. BAFF is expressed by various cells. Giordano et al. reported that BAFF produced by dendritic cells, monocytes, and neutrophils is essential for B cell maturation and autoantibody production[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Our previous studies demonstrated that PMN-MDSCs promote RA development in CIA mice by regulating B cells through BAFF overexpression[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the current study, we further explored the role and mechanism by which PMN-MDSCs modulate ABCs via BAFF in RA.\u003c/p\u003e\u003cp\u003eSeveral aspects of this study require further investigation. Our study primarily focused on the regulation of ABCs by PMN-MDSCs and BAFF. However, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, blocking BAFF function did not reduce the regulatory effect of PMN-MDSCs on ABCs to the level observed in the control group, suggesting that PMN-MDSCs may influence ABCs through additional mechanisms. Additionally, our mechanistic analysis was limited to blocking BAFFr to explore the role of BAFF through this pathway. Although BAFF regulates B cell activation primarily through BAFFr, it may also exert a regulatory role through its other receptors, BCMA and TACI. Moreover, it remains unclear whether other cells, such as fibroblast-like synoviocytes, also produce high levels of BAFF and contribute to ABC regulation in RA. Further studies are needed to explore these alternative pathways and identify additional therapeutic targets.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, this study demonstrated elevated levels of PMN-MDSCs and ABCs in both patients with RA and CIA mice, with preferential accumulation in affected joints. PMN-MDSCs from CIA mice enhanced ABC proliferation and differentiation via the BAFF-mediated SYK-ERK1/2 signaling pathway, thereby promoting RA-associated inflammation. The critical involvement of the BAFF/BAFFr/SYK/ERK1/2 axis highlights therapeutic potential for anti-BAFF antibodies and SYK inhibitors in RA treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis manuscript has not been published in whole or in part nor is it being considered for publication elsewhere. The authors declare that there are no financial or other relationships that might lead to a conflict of interest in the present article. All authors have reviewed the final version of the manuscript and approved it for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. H.Zhang and Tang had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy conception and design.\u003c/strong\u003e Tang, H.Zhang,Sun\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcquisition of data.\u003c/strong\u003e Hong, Yin,Y.Q.Wang , Y.N.Wang, J.Zhang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis and interpretation of data.\u003c/strong\u003e Hong, Tang, H.Zhang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman participants were recruited with informed consent approved by the Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School (2024-160-01) and in compliance with the Helsinki Declaration. The animal use and the experimental protocols were reviewed and approved by the Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School(2021AE01061).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by the National Natural Science Foundation of China (grant numbers:\u0026nbsp;81671608 and 81971525)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have reviewed the final version of the manuscript and approved it for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSmolen JS, Aletaha D, McInnes IB. Rheumatoid arthritis. 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Arthritis Rheum\u003cem\u003e. \u003c/em\u003e2013;65(4):1085-1096.\u003c/li\u003e\n\u003cli\u003eVidal-Pedrola G, Naamane N, Cameron JA, Pratt AG, Mellor AL, Isaacs JD, et al. Characterization of age-associated B cells in early drug-na\u0026iuml;ve rheumatoid arthritis patients. Immunology\u003cem\u003e. \u003c/em\u003e2023;168(4):640-653.\u003c/li\u003e\n\u003cli\u003eGiordano D, Kuley R, Draves KE, Elkon KB, Giltiay NV, Clark EA. B cell-activating factor (BAFF) from dendritic cells, monocytes and neutrophils is required for B cell maturation and autoantibody production in SLE-like autoimmune disease. Front Immunol\u003cem\u003e. \u003c/em\u003e2023;14:1050528.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rheumatoid arthritis, Collagen-induced arthritis, Polymorphonuclear myeloid-derived suppressor cells, B-cell activating factor, Age-associated B cells","lastPublishedDoi":"10.21203/rs.3.rs-6846731/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6846731/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eIn rheumatoid arthritis (RA), both polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and age-associated B cells (ABCs) are significantly elevated in affected joints and are closely linked to disease progression. However, their interaction remains poorly understood. This study aims to explore the regulatory role of PMN-MDSCs on ABCs and the underlying mechanisms, offering new insights into RA pathogenesis and potential therapeutic targets.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe conducted flow cytometry analysis to assess PMN-MDSCs and ABCs proportion and their correlation in collagen-induced arthritis (CIA) mice. We used RNA sequencing (RNA-seq) to characterize transcriptomic profiles of CD11c\u003csup\u003e+\u003c/sup\u003e B cells from CIA mice. In vitro studies examined T-bet expression in CD11c\u003csup\u003e+\u003c/sup\u003e B cells co-cultured with CIA-derived PMN-MDSCs. We assessed BAFF effects on ABCs proliferation and differentiation through flow cytometry and validated findings using anti-BAFF antibodies in PMN-MDSCs/CD11c\u003csup\u003e+\u003c/sup\u003e B cells co-cultures. We used western blot and flow cytometry analyses to identify BAFF-mediated signaling pathways in CD11c\u003csup\u003e+\u003c/sup\u003e B cells. In vivo study evaluated the therapeutic potential of pathway inhibitors in CIA mice. The ratio and regulatory correlation of PMN-MDSCs and ABCs were also assessed by flow cytometry in RA patients.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eUsing a CIA mouse model, we observed increased populations of both PMN-MDSCs and ABCs in the joints and spleens, with a strong positive correlation in joint tissues. RNA sequencing of CIA-derived CD11c\u003csup\u003e+\u003c/sup\u003e B cells revealed distinct pro-inflammatory and chemotactic signatures. PMN-MDSCs enhanced T-bet expression in CD11c\u003csup\u003e+\u003c/sup\u003e B cells through non-contact-dependent mechanisms, driving ABCs differentiation. BAFF was found to support ABC proliferation and differentiation. Mechanistically, PMN-MDSCs-derived BAFF activated the SYK-ERK1/2 signaling pathway via BAFF receptor (BAFFr), upregulating T-bet expression in CD11c\u003csup\u003e+\u003c/sup\u003e B cells. In vivo, inhibition of SYK ameliorated arthritis symptoms and reduced ABC populations across tissues. Similarly, patients with RA exhibited elevated levels of PMN-MDSCs and ABCs in both blood and synovial fluid, with a significant correlation between the two. Additionally, RA-derived PMN-MDSCs promoted ABCs differentiation in vitro.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThese findings highlight the role of PMN-MDSCs in driving RA inflammation by promoting ABC proliferation and differentiation via the BAFF-mediated SYK-ERK1/2 pathway.\u003c/p\u003e","manuscriptTitle":"Polymorphonuclear Myeloid-Derived Suppressor Cells Promote Inflammation Progression of Rheumatoid Arthritis by Inducing Age-Associated B Cell Proliferation and Differentiation via the BAFF-Mediated SYK-ERK1/2 Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 08:37:09","doi":"10.21203/rs.3.rs-6846731/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1cf24c85-3bc8-4de1-9723-d79a080af07e","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-20T12:39:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-23 08:37:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6846731","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6846731","identity":"rs-6846731","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}