Rheumatoid arthritis (RA), a chronic autoimmune disease, is characterized by CD4+ T cell-mediated synovial inflammation, with T helper (Th)17 cells being implicated in RA pathogenesis. Nr4a1 is an orphan nuclear receptor functioning as a negative regulator of T cell activation and central tolerance. Cytosporone B (CsnB) is a small-molecule agonist of Nr4a1 and can exert immunomodulatory effects. However, its efficacy in T cell-driven autoimmune arthritis remains unclear. This study aimed to investigate the therapeutic effect of CsnB-mediated Nr4a1 agonization on RA development in SKG mice and evaluate its impact on T cell function.
Methods
The SKG mouse model of T cell-dependent chronic arthritis was constructed via zymosan A induction. The mice were intraperitoneally treated with CsnB, and disease severity and immune cell populations were evaluated by clinical scoring and flow cytometry. In vitro assays were performed to examine T cell antigen receptor (TCR)-induced T cell activation and Th17 differentiation. Additionally, RNA sequencing was performed to profile transcriptomic changes in CD4+ T cells following TCR stimulation.
Results
CsnB markedly attenuated arthritis development and reduced the population of effector memory and Th17 cells in the spleen and synovium. Furthermore, in vitro assay results showed that CsnB suppressed T cell activation, downregulated interleukin (IL)-2 and activation markers, and repressed inflammatory gene expression. CsnB inhibited Th17 differentiation and IL-6–signal transducer and activator of transcription 3 signaling by reducing CD130 (Il6st) expression.
Discussion
Altogether, the findings of this study showed that CsnB, one of the agonists of Nr4a1, suppressed TCR-driven T cell activation and Th17 differentiation, thereby ameliorating autoimmune arthritis in SKG mice. These findings highlight the potential of Nr4a1 as an immunotherapeutic target in T cell-mediated autoimmune arthritis, particularly in RA subsets characterized by TCR signaling dysfunction.
Graphical Abstract
Side-by-side infographic compares immune mechanisms in arthritis without treatment versus with Cytosporone B treatment, showing self-reactive T cells leading to Th17 cells and arthritis in untreated, while Cytosporone B blocks Th17 formation and reduces inflammatory signaling, lowering arthritis development in mice.
Cytosporone Bexperimental arthritisNR4Arheumatoid arthritisSKG miceT cell antigen receptor signalingT cellsThe author(s) declared that financial support was received for this work and/or its publication. This research was supported by AMED under Grant Number JP22ek0109604, JSPS Grant Number JP22K16343, The JCR Grant for Promoting Research for D2T RA, a grant from the Uehara Memorial Foundation, a grant from Takeda Science Foundation, a grant from The Shimizu. Foundation for Immunology and Neuroscience Grant for 2021, and a grant from Eli Lilly Japan KK Innovation Research Grant 2023.section-at-acceptanceAutoimmune and Autoinflammatory Disorders : Autoimmune DisordersIntroduction
Persistent synovial inflammation and progressive joint destruction are characteristic manifestations of rheumatoid arthritis (RA), a chronic inflammatory disease, typically driven by the interplay of various immune cells, including T cells, B cells, and monocytes (1). Among these, the notable association between RA and the human leukocyte antigen-DRB1 locus has been reported, underscoring the pivotal role of CD4+ T cells in the pathophysiology of the disease (2–4). Notably, CD4+ T helper (Th) cells have been classified into different subsets based on their functions, cytokine production, and chemokine receptor expression. Although the precise contributions of each CD4+ T cell subset to RA remain elusive, a pathogenic role of interleukin(IL)-17-producing Th17 cells has been reported in the development and progression of RA (5–7).
Nr4a1, a member of the nuclear receptor subfamily 4A (NR4A) family, is the most abundant among the three NR4A members in T cells (8). Acute stimulation of the T cell antigen receptor (TCR) leads to rapid upregulation of Nr4a1, peaking at 2–6 hours (9–11). Studies have reported that Nr4a1 functions as a negative regulator of T-cell activation, as Nr4a1-deficient T cells exhibit enhanced effector functions and increased cytokine production (12). In addition to its role in peripheral T cell responses, NR4A has also been implicated in central tolerance owing to apoptosis induction in immature thymocytes during thymic negative selection (13–16). Autoreactive T cells are generally derived from those that escape thymic negative selection, suggesting a critical role of Nr4a1 in preventing autoimmunity by controlling both central and peripheral T cell tolerance (17).
Additionally, Nr4a1 is a known orphan receptor, and its functions have been reported in both ligand-dependent and ligand-independent manners (18). Although endogenous ligands have not been conclusively identified, several exogenous ligands have been reported (19–21). Cytosporone B (CsnB), an octaketide fungal metabolite, is a small-molecule agonist that directly binds to the ligand-binding domain of Nr4a1-encoded NUR77. Various studies have used CsnB to investigate the role of Nr4a1 in various murine models of inflammatory diseases (22–25). For instance, in a dextran sulfate sodium-induced colitis model, CsnB was shown to ameliorate disease severity by modulating Toll-like receptor and IL-1 receptor signaling (25). Moreover, CsnB-mediated Nr4a1 agonization has shown therapeutic efficacy in the experimental autoimmune encephalomyelitis model by suppressing the production of interferon (IFN)-γ and IL-17 in the central nervous system (24).
The SKG mouse strain can spontaneously develop chronic arthritis via an autoimmune mechanism, serving as a valuable model for studying human inflammatory arthritis (26). Histopathologically, SKG arthritis has been characterized by symmetrical, pannus-forming synovitis in limb joints, which resembles the joint pathology of RA (26). Although SKG mice can spontaneously develop arthritis under conventional housing environments, they require innate immune stimuli in specific pathogen-free conditions for disease pathogenesis. Reportedly, SKG mice harbor a hypomorphic mutation in Zap70, resulting in attenuated TCR signaling, which impairs thymic negative selection, thereby allowing the escape of autoreactive T cells into the periphery and exacerbating autoimmune pathology. In NUR77-enhanced green fluorescent protein SKG mouse model, the peripheral naïve CD4+ T cells have been shown to exhibit a higher NUR77 expression than that in wild-type mice (27). These studies suggest that SKG mice possess a subset of CD4+ T cells that are chronically stimulated by antigens, including autoantigens.
Many studies have suggested targeting Nr4a1 as a potential approach to mitigate autoimmune arthritis in murine models, such as collagen-induced arthritis (CIA) and K/BxN serum transfer-induced arthritis (STIA) (23, 28, 29). In CIA mouse arthritis model, mice are immunized with type II collagen, which leads to the activation of CD4+ T cells and B cells, thereby initiating arthritis development (30). In contrast, in the STIA model, arthritogenic serum from K/BxN mice is transferred, and the pathogenesis is predominantly mediated by mechanisms independent of adaptive immunity (31). Although studies on these models have expanded on the role of Nr4a1 agonization in inflammatory arthritis, its effects in T cell-dominant autoimmune arthritis remain elusive. Hence, this study aimed to investigate the therapeutic effect of CsnB-mediated Nr4a1 agonization on arthritis development in SKG mice and evaluate the effect on T cell function.
MethodsExperimental animals
SKG mice were obtained from CLEA Japan, Inc. (Osaka, Japan) and bred in specific pathogen-free conditions under a climate-controlled facility with a 12-h light/dark cycle. All animal experiments were approved and performed in accordance with the guidelines of the Institutional Animal Care Committee at Kyoto University (approval numbers MedKyo25238, MedKyo16106–23104).
Induction, scoring, and treatment of arthritis
Female SKG mice (10–14-week-old) were intraperitoneally injected with 2 mg zymosan A (ZyA) (Sigma-Aldrich, Japan) to induce arthritis. Clinical arthritis scores were assessed as previously described (26) and were defined as follows: 0, absence of swelling or erythema; 0.1, presence of swelling or erythema in the digits; 0.5, mild swelling and/or erythema in the wrists or ankle joints; and 1, severe swelling in larger joints. The total score for each mouse was obtained by summing the scores from all affected joints. SKG mice received intraperitoneal injections of 15 mg/kg CsnB (Sigma-Aldrich, Japan) or dimethyl sulfoxide (DMSO) thrice per week, beginning 1 d after ZyA administration.
Flow cytometry
For flow cytometry, single-cell suspensions of splenocytes were harvested from the spleens of SKG mice. Additionally, the synovial tissues from the joints were dissected into small fragments and digested enzymatically for 30 min at 37 °C in Roswell Park Memorial Institute-1640 medium, containing collagenase type I and IV (Worthington Biochemical, US). Following incubation, the digested tissues were mechanically dissociated and filtered through a 70-μm mesh strainer to obtain single-cell suspensions of synovial cells. The monoclonal antibodies used for flow cytometry are presented in Supplementary Table S1. For intracellular staining of transcription factors, the FoxP3 staining buffer set (eBioscience) was used according to the manufacturer’s instructions. For intracellular staining of cytokines, cells were stimulated in Roswell Park Memorial Institute-1640 medium supplemented with 10% serum, 1% ×100 non-essential amino acids, 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffer, 1 mM sodium pyruvate, 2 mM L-glutamine, and 50 μM b-mercaptoethanol for 4 h with 20 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 1 mM ionomycin (Sigma-Aldrich) in the presence of GolgiStop™ (BD Bioscience). After stimulation, cells were fixed and permeabilized using BD Cytofix/Cytoperm™ (BD Bioscience) according to the manufacturer’s instructions. Flow cytometric data were acquired and analyzed using an LSRFortessa™ cell analyzer (BD Biosciences) and the FlowJo software (TreeStar).
<italic>In vitro</italic> T cell stimulation with anti-CD3/anti-CD28 antibodies
For in vitro stimulation, CD4+ T cells were purified using a magnetic-activated cell sorting system with the CD4+ T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Flat-bottom 96-well plates were coated with 1 μg/mL anti-CD3 antibody (clone 2c11, BioLegend) and 2 μg/mL anti-CD28 antibody (clone 37.51, BioLegend) at 4 °C overnight. Following incubation, cells were plated (5 × 105 cells/well) in the culture media containing 10 ng/mL CsnB or DMSO for 2 or 16 h.
RNA sequencing
CD4+ T cells were cultured on anti-CD3/anti-CD28-coated plates for 2 h; following this, the cells were lysed in RLT buffer supplemented with 1% β-mercaptoethanol. Next, the total RNA was extracted using the RNeasy Plus Kit (QIAGEN) according to the manufacturer’s instructions. Messenger RNA (mRNA) libraries were prepared using the Illumina TruSeq Stranded mRNA Library Preparation Kit and sequenced on the NovaSeq X Plus platform by Marogen Japan Co., Ltd. (Tokyo, Japan). Trimmed FASTQ files were aligned to the reference genome using HISAT2, and transcript assembly was performed using StringTie. RNA sequencing was performed using biological triplicates for each experimental condition. Differential gene expression between DMSO- and CsnB-treated CD4+ T cells was analyzed using the DeSeq2 on R ver. 4.4.2. Gene set enrichment analysis (GSEA) was performed using ranked lists of all expressed genes derived from DESeq2 analysis. Genes were ranked according to log2 fold change, and enrichment analysis was conducted using the clusterProfiler package with Gene Ontology biological process gene sets. Gene sets with an adjusted P value < 0.05 were considered significantly enriched.
<italic>In vitro</italic> Th1/17 differentiation
For in vitro Th1/17 differentiation, naïve CD4+ T cells were isolated using the magnetic-activated cell sorting system and the naïve CD4+ T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Purified cells were cultured on anti-CD3/anti-CD28 antibody-coated plates in the presence of 20 ng/mL IL-6, IL-23, and IL-1β (R&D Systems) to induce Th17 differentiation (32). The differentiation medium was supplemented with 10 ng/mL CsnB or DMSO. After 4 days of incubation, flow cytometry was performed to assess the harvested cells for cytokine production.
Statistical analyses
Statistical analyses and data visualizations were performed using Prism v10 (GraphPad Software, Inc.). Data are presented as mean ± standard error of the mean, unless otherwise specified. Unpaired or paired Student’s t-test was used to assess the significance of differences between the two groups, and corrections for multiple comparisons across time points or doses were applied using the Holm–Šídák method. Significance was defined as * P < 0.05, and **P < 0.01.
ResultsCsnB mitigated arthritis development in SKG mice
The therapeutic effect of CsnB on the experimental arthritis model was evaluated in SKG mice, which develop autoimmune arthritis following innate immune stimulation with ZyA, curdlan, or mannan (26). Following 1 day after the intraperitoneal injection of ZyA (200 μg per mouse), 15 mg/kg CsnB was administered intraperitoneally thrice per week (n = 5 each) (Figure 1A). CsnB treatment delayed the onset of arthritis and significantly reduced its severity by day 28 compared with that in the control group (Figures 1B, C). These findings suggest that intraperitoneal treatment with CsnB attenuates the development of experimental arthritis in vivo.
Cytosporone B (CsnB) inhibits arthritis development in SKG mice. (A) CsnB (15 mg/kg, three times weekly) was administered starting 1 day after arthritis induction with a single intraperitoneal injection of 2 mg zymosan (A, B) Clinical scores were significantly reduced in CsnB-treated mice compared with dimethyl sulfoxide (DMSO) controls. Data are representative of three independent experiments with n = 5 mice per group. (C) Representative image of swollen paws from CsnB- and DMSO-treated mice. *P < 0.05, **P < 0.01.
Panel A shows a schematic of arthritis induction and treatment timeline in SKG mice with Zymosan A followed by DMSO or CsnB administration. Panel B presents a line graph comparing arthritis scores over 28 days, indicating higher scores for DMSO-treated mice and significantly lower values in CsnB-treated mice. Panel C displays two photographs showing greater swelling and redness in the paw of the DMSO-treated mouse compared to less severe symptoms in the CsnB-treated mouse.Effector memory T cells and Th17 cells were decreased in CsnB-treated mice
The immunological mechanisms underlying the alleviation of arthritis in CsnB-treated mice were investigated. SKG mice harbor a point mutation in Zap70, which leads to an increase in the number of autoreactive T cells in the periphery. Therefore, flow cytometric analysis of splenic T cell subsets isolated from CsnB-treated mice was performed (26). The total number of splenic CD4+ T cell was not significantly different between the groups, indicating that CsnB did not induce lymphopenia (Supplementary Figure S1A). Notably, the proportion of effector memory CD4+ T cells (CD4+CD62L–CD44+) reduced in the CsnB-treated group, whereas that of naïve CD4+ T cells (CD4+CD62L+CD44–) increased (Figures 2A, B). Furthermore, the frequency of IFNγ+CD4+ T cells remained unchanged, whereas that of IL-17A+CD4+ T cells decreased in the spleen of CsnB-treated mice (Figures 2C, D). When analyzed as absolute counts per spleen, the differences in naïve and effector-memory CD4+ T cells were diminished and did not reach statistical significance (Supplementary Figure S1B), indicating that the observed changes reflect redistribution within the CD4+ compartment rather than true expansion or contraction of these subsets. In contrast, IL-17A+ CD4+ T cells were reduced in both frequency and absolute number, whereas IFNγ+ CD4+ T cell numbers remained unchanged (Supplementary Figure S1C). Additionally, the results showed that the frequencies of regulatory T cells, germinal center B cells, plasma cells, and monocytes did not exhibit significant differences between the CsnB-treated and control groups (Figures 2E–G). In the synovium, the proportion of IL-17A+ CD4+ T cells reduced in the CsnB-treated mice, whereas the proportion of IFNγ+ CD4+ T cells remained unaltered (Figures 2H, I). Conversely, absolute numbers of both IL-17A+ and IFNγ+ CD4+ T cells were diminished in CsnB-treated mice, suggesting that total effector T cell accumulation within the joint was inhibited, with a more pronounced attenuation of Th17 polarization (Supplementary Figure S1D). Overall, these findings suggest that CsnB treatment modulates the population of peripheral CD4+ T cells, particularly by suppressing pro-inflammatory Th17 responses.
Reduction of T helper (Th)17 cells in spleen and synovium after cytosporone B (CsnB) treatment. (A) Representative flow plots showing naïve (CD4+CD62LhiCD44lo) and effector memory (CD4+CD62LloCD44hi) T cells in the spleen. (B) Frequency of naïve and effector memory T cells among CD4+ T cells. (C) Flow plots showing Th1 (CD4+IFN-γ+) and Th17 (CD4+IL-17+) cells in the spleen. (D) Frequency of Th1 and Th17 cells among splenic CD4+ T cells. (E) Frequency of T regulatory (CD4+CD25+FoxP3+) cells among CD4+ T cells. (F) Ratio of plasma cells (CD138+) to total B cells (left) and frequency of germinal center B cells (B220+IgDloGL-7+Fas+) among total B cells (right). (G) Frequency of Ly6Clo and Ly6Chi monocytes. (H) Flow plots of Th1 and Th17 cells in synovium. (I) Frequency of Th1 and Th17 cells among synovial CD4+ T cells. Graphs show mean ± standard error of the mean. Unpaired t-test; *P < 0.05, **P < 0.01. ns means not significant.
Scientific figure with multiple panels displaying flow cytometry plots and bar graphs comparing DMSO and CsnB treatments in mice. Panels A, C, and H show representative flow cytometry plots for CD4 T cell subsets and cytokine expression, comparing distributions between DMSO (blue) and CsnB (red) groups in spleen and synovium. Panels B, D, E, F, G, and I present bar graphs quantifying percentages or ratios of cell subsets, including markers such as CD62L, CD44, Foxp3, IFNγ, IL-17A, and cell types like plasma cells, germinal center B cells, and monocytes. Significance is indicated by asterisks or labelled “ns” for not significant.CsnB inhibits T cell activation following anti-CD3 and anti-CD28 antibody stimulation
In vitro analysis was performed to elucidate the role of CsnB during TCR stimulation. CD4+ T cells were isolated from SKG mice and cultured on anti-CD3/anti-CD28 antibody-coated plates in the presence or absence of CsnB. Quantitative polymerase chain reaction analysis revealed that Nr4a1 mRNA expression peaked at 2 h post-TCR stimulation, and Il2 expression peaked at 4 h (Figure 3A). CsnB treatment substantially suppressed Il2 expression during TCR stimulation and significantly inhibited the upregulation of activation markers post-TCR stimulation, including CD25 and PD-1 (Figure 3B). Based on the observation that Nr4a1 expression peaked at 2 h after stimulation, RNA sequencing was performed at this early time point to capture CsnB-induced modulation of early TCR-driven transcriptional responses. For RNA sequencing, transcriptomic analysis revealed that CsnB downregulated the expression of inflammation-related genes, including Ifng, Csf2, and Il22 (Figures 3C, D). GSEA revealed that pathways related to cytokine-mediated signaling, T cell activation, and JAK–STAT signaling were significantly de-enriched in CsnB-treated samples compared with controls (Figure 3E). Other differentially expressed genes and GSEA results are described in Supplementary Tables 2, 3. Overall, these findings suggest that CsnB attenuates T cell activation in response to TCR signaling.
Cytosporone B (CsnB) attenuates T cell activation induced by anti-CD3/CD28 stimulation. (A) Time-course of Nr4a1 and Il2 expression in CD4+ T cells after anti-CD3/CD28 stimulation. (B) Quantification of CD69, CD25, and PD-1 mean fluorescence intensity in CD4+ T cells after 16 h on anti-CD3/CD28-coated plates. (C) Volcano plot of RNA sequencing results 2 h after stimulation, comparing dimethyl sulfoxide (DMSO) and CsnB conditions (Data were generated from biological triplicates). Genes associated with T cell activation and adjusted P value < 0.05 are shown. (D) Heatmaps showing genes significantly downregulated in CsnB-treated samples (genes with adjusted P value < 0.05 and log2 fold change ≥ 1). (E) Representative immune-related biological processes identified by gene set enrichment analysis using ranked gene lists are shown. Dot size indicates gene set size, and color denotes FDR. **P < 0.01.
Panel A shows two line graphs comparing relative gene expression (Nr4a1, Il2) over time between DMSO and CsnB treatments in T cells; both genes are reduced in CsnB condition. Panel B presents three bar graphs of flow cytometry data indicating significant reductions in CD25 and PD-1, but not CD69, in CD4+ T cells treated with CsnB versus DMSO. Panel C is a volcano plot showing differentially expressed genes; several genes including Il2 and Ifng are labeled as downregulated in CsnB treatment. Panel D is a heatmap comparing expression levels of immune-related genes, with DMSO samples in blue and CsnB in red, showing overall decreased expression in the CsnB group. Panel E displays a gene set enrichment analysis dot plot highlighting significantly altered biological processes, particularly cytokine-mediated signaling and T cell activation, with dot size and color indicating gene set size and FDR q-value respectively.CsnB inhibits Th17 differentiation and IL-6 signals
We hypothesized that CsnB inhibits Th17 differentiation in vivo. Accordingly, the effect of CsnB was evaluated on Th17 differentiation in vitro, and naïve CD4+ T cells isolated from SKG mice were cultured under Th17-polarizing conditions (IL-6, IL-1β, and IL-23) in the presence or absence of CsnB. Strikingly, CsnB treatment significantly reduced the proportion of IL-17A+CD4+ cells (Figures 4A, B) and retinoic acid-related orphan receptor (ROR)γt expression, a key transcription factor for Th17 differentiation, as assessed by flow cytometry (Figure 4C). Th17 differentiation was not inhibited in naïve CD4+ T cells isolated from Nr4a1-knockout mice (Supplementary Figure S2). IL-6 plays a critical role in Th17 cell differentiation; therefore, the effect of CsnB on IL-6 signal transduction was investigated (33). Phospho-flow cytometry results of signal transducer and activator of transcription 3 (STAT3) phosphorylation revealed that CsnB treatment significantly reduced STAT3 phosphorylation during Th17 differentiation (Figures 4D, E). The analysis of IL-6 receptor component expression, including the ligand-binding IL-6Rα-chain (CD126) and signal-transducing subunit gp130 (CD130), showed that CsnB significantly reduced CD130 expression; CsnB did not affect CD126 expression (Figures 4F, G). Furthermore, CsnB treatment reduced relative mRNA expression of Il6st (CD130), whereas Il6ra (CD126) expression remained unchanged (Figure 4H). Overall, these results suggest that CsnB inhibits CD130 transcription and reduces IL-6 signal transduction during Th17 differentiation.
Cytosporone B (CsnB) inhibits T helper (Th)17 differentiation and signal transducer and activator of transcription 3 (STAT3)/CD130 expression in vitro. (A) Representative plots of Th17 cells after 4 d of naïve CD4+ T cell culture under Th17-polarizing conditions (interleukin [IL]-6 + IL-1β + IL-23). (B) Frequency of Th17 cells among CD4+ T cells. (C) Retinoic acid-related orphan receptor-γt expression quantification after 4 d of Th17 culture. (D) Flow plots of phosphorylated STAT3 in CD4+ T cells after 2 d (E) Quantification of phosphorylated STAT3 after 2 d (F) Flow plots of CD130 and CD126 expression after 2 d (G) Quantification of CD130 and CD126 after Th17 culture. (H)Il6st and Il6ra expression in CD4+ T cells after 2 h of culture. *P < 0.05, **P < 0.01.
Flow cytometry and bar graph panels compare DMSO and CsnB treatments in Th17 cell conditions, showing reduced IL-17A production, lower RORγt and pSTAT3 expression, decreased CD130 levels, and reduced Il6st gene expression with CsnB, while CD126 and Il6ra remain unchanged.Discussion
Herein, the therapeutic potential of targeting Nr4a1 with its specific agonist, CsnB, was investigated in a mouse model of autoimmune arthritis. Of note, CsnB treatment significantly attenuated arthritis development in SKG mice (Supplementary Figure S3). Moreover, the frequency of Th17 cells was reduced in both the spleen and synovium of CsnB-treated mice, whereas that of other immune cell populations remained unaffected. In vitro analyses revealed that CsnB modulated T cell activation in response to stimulation with anti-CD3 and anti-CD28 antibodies. Furthermore, CsnB could inhibit Th17 differentiation by blocking the IL-6 signaling pathway.
Targeting Nr4a1 has been reported to ameliorate autoimmune arthritis in both CIA and STIA models (23, 28, 29). However, these models represent immunization/antibody-induced acute forms of arthritis and are self-limiting. In contrast, SKG mice can develop chronic autoimmune arthritis following innate immune stimulation under the specific pathogen-free environment, without spontaneous resolution (26). To the best of our knowledge, the effects of Nr4a1 agonization on T cells in this chronic model of autoimmune arthritis have not been previously characterized. In the present study, CsnB treatment reduced the population of effector memory T cells, with specific inhibition of Th17 cells, as revealed by the subset analysis; Th1 cell population remained unaffected. This finding is consistent with that of previous studies, which showed that Nr4a1 overexpression in T cells did not affect Th1 cytokines in the CIA model; however, the impact on Th17 cytokines was not evaluated in previous studies (28). Considering the predominant role of Th17 cells in arthritis pathogenesis in SKG mice (34), the results of the present study suggest that inhibiting Th17 differentiation is a key mechanism underlying the therapeutic effect of CsnB in this model.
A study on Nr4a1-deficient mice demonstrated that Nr4a1 functions as a negative regulator of T-cell activation (12). Upregulated expression of activation markers such as CD25 and CD69 was observed in the reported mice, along with a high population of effector memory T cells in the absence of exogenous stimulation. Conversely, Nr4a1 overexpression in CD4+ T cells resulted in reduced transcription of Il2, Ifng, and Tbx21 (35). Nr4a1 expression was transiently upregulated within a few hours following TCR stimulation, albeit it returned to the baseline within 24 hours (36). In the present study, CsnB inhibited the expression of T cell activation markers during the acute phase of TCR signaling, thereby suggesting that CsnB enhances Nr4a1 activity and reinforces its role in negative regulation of T cell activation.
In this study, CsnB significantly inhibited the in vitro differentiation of naïve CD4+ T cells into Th17 cells, which is consistent with findings from a previous study (24). Furthermore, CsnB treatment attenuated STAT3 phosphorylation during Th17 differentiation. STAT3 is an essential transcription factor implicated in Th17 differentiation owing to its role in the expression of various downstream genes, including Rorc, which encodes RORγt, the master regulator of the Th17 lineage (37). STAT3 phosphorylation is primarily mediated by IL-6 signaling through its receptor components, such as IL-6α and the common subunit gp130. CsnB could suppress gp130 expression. The IL-6-gp130-Janus kinase signaling axis is the principal pathway for STAT3 activation; hence, gp130 downregulation likely contributed to the STAT3 phosphorylation and subsequent decrease in RORγt expression. A recent study reported that gp130 expression is induced by TCR signaling (38), which suggests that CsnB may act on a downstream component of the TCR pathway to repress gp130 expression. In our analyses, although CsnB treatment mitigated Il6st expression, it remains unclear whether this regulation is direct or indirect. The observed reduction in Il6st expression may occur through indirect mechanisms, including transcriptional repression via Nr4a1-associated regulatory networks, modulation of upstream cytokine signaling, or epigenetic remodeling associated with altered T cell activation states. Collectively, these findings indicate the interference of CsnB with Th17 differentiation via the modulation of both TCR and IL-6 signaling cascades.
Although the findings of this study suggest that CsnB represses Th17 differentiation through Nr4a1 agonization, there are some discrepancies with a previous study reporting that Th17 cell differentiation remained unaffected in Nr4a triple-knockout (Nr4a1/2/3) mice (39). Additionally, enhanced Th17 differentiation has been shown in Nr4a1 single-knockout mice in the experimental autoimmune encephalomyelitis model (40). Nr4a2 knockdown by small interfering RNA has been reported to suppress Th17 differentiation in vitro (41). This functional redundancy among Nr4a family members suggests that the suppressive effect of CsnB on Th17 differentiation may be the result of a cumulative modulation of multiple Nr4a factors and related transcriptional networks. CsnB was originally identified as a naturally occurring agonist of Nr4a1 that directly binds to its ligand-binding domain and activates Nr4a1-dependent transcriptional activity (19). However, although some studies showed the effect of CsnB was impaired in Nr4a1-knockout mice, its functional selectivity toward Nr4a1 versus other Nr4a family members at the doses used in vivo and in vitro experiments in our study and previous studies has not been fully characterized, and potential contributions of Nr4a2 or Nr4a3 cannot be excluded (24, 42). SKG mice harbor a hypomorphic mutation in Zap70, which encodes a cytoplasmic tyrosine kinase essential for initiating proximal TCR signal transduction (26). Consequently, impaired TCR signaling can lead to the escape of autoreactive T cells from negative selection in the thymus, allowing them to persist in the periphery. Upon being chronically exposed to self-antigens, these autoreactive T cells exhibit upregulated Nr4a1 expression in CD4+ T cells (27). IL-6 signaling was enhanced in the Nr4a1hi CD4+ T cells, and Th17 cells were more prevalent in adoptive transfer models using Nr4a1hi CD4+ T cells. CsnB is a naturally occurring Nr4a1 agonist and is presumed to exert greater effects on Nr4a1hi CD4+ T cells, thereby modulating their pathogenic potential.
Potential risks of systemic Nr4a1 activation should be considered. Nr4a family members have been implicated as key regulators of T cell tolerance and exhaustion, restraining effector cytokine programs under chronic antigen stimulation (35). Consistent with this concept, pharmacological activation of Nr4a1 signaling could, in principle, increase susceptibility to infections, dampen vaccine responses, or compromise tumor immune surveillance (35, 43). Importantly, however, the immunological consequences of Nr4a1 agonization appear to be context-dependent. For example, administration of the CsnB improved outcomes in an influenza infection model, reducing lung viral loads and improving pulmonary function (42). Therefore, future studies should define dose- and time-dependent safety profiles of Nr4a1 agonization and evaluate immune competence longitudinally to balance therapeutic benefit with systemic immune risks.
It has been reported that approximately 20% of patients with RA possess antibodies and self-reactive T cells that act against one of the self-antigens identified in SKG mice (44). Moreover, a subset of patients with RA has presented with abnormal TCR signaling, similar to that observed in SKG mice, particularly regarding defective central tolerance and the persistence of autoreactive T cells. Genetic studies implicate altered proximal TCR signaling pathways in RA susceptibility, as indicated by the PTPN22 risk variant, which is linked to reduced TCR signaling (45, 46). In addition, some populations show synovium dominated by lymphoid lineage infiltration, including T cells (47). CsnB could interfere with TCR signaling and suppress effector T cell activation. This mechanism is especially relevant in RA, where both effector memory T cells and Th17 cells contribute to the chronicity of the disease. The findings of this study, showing that CsnB ameliorates arthritis and reduces effector T cell subsets in SKG mice, highlight its potential as a novel immunomodulatory agent for treating autoimmune arthritis, particularly in patients with TCR-signaling-defective or T-cell-dominant mechanisms.
This study has some limitations. First, although the therapeutic efficacy of CsnB in the SKG mouse model of autoimmune arthritis was investigated, the generalizability of these findings to other animal models or humans remains uncertain. Arthritis in SKG mice is induced by innate immune stimulation and is characterized by TCR signaling defects due to the hypomorphic Zap70 mutation, which may not fully recapitulate the diverse immunopathogenic mechanisms of RA across diverse patient subpopulations. Second, although clinical scoring reliably reflects inflammatory disease activity in the SKG model, this study lacked histological analyses. The present findings focus on immunological modulation of arthritis rather than definitive structural joint protection, and future studies incorporating joint histopathology will be necessary to further support these effects. Third, although the results show that CsnB suppressed IL-6 signaling by downregulating CD130 (Il6st), the upstream molecular mechanisms by which Nr4a1 modulates CD130 transcription remain unclear. Hence, further studies involving transcriptional and epigenetic profiling are warranted to delineate the regulatory network involved with CsnB and arthritis.
In conclusion, the findings of this study demonstrate that CsnB, one of the agonists of Nr4a1, can suppress T cell activation and Th17 differentiation, thereby leading to the attenuation of autoimmune arthritis in SKG mice. Altogether, these findings highlight Nr4a1 as a promising therapeutic target for T cell-mediated autoimmune arthritis.
Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/geo/, GSE312091.
Ethics statement
The animal study was approved by Institutional Animal Care Committee at Kyoto University. The study was conducted in accordance with the local legislation and institutional requirements.
We thank Julie Zikherman (University of California, San Francisco) for critical feedback. Graphical abstract are Created in BioRender. Nakayama, Y. (2026) https://BioRender.com/s14e117, https://BioRender.com/t48z465.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The author AM declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.
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Edited by: Minrui Liang, Fudan University, China
Reviewed by: Ana Ramon-Vazquez, University College Cork, Ireland
A. Ezhil Grace, Saveetha University, India
CIA, collagen-induced arthritis; CsnB, cytosporone B; DMSO, dimethyl sulfoxide; GSEA, gene set enrichment analysis; IFN, interferon; IL, interleukin; mRNA, messenger RNA; NR4A, nuclear receptor subfamily 4A; RA, rheumatoid arthritis; ROR, retinoic acid-related orphan receptor; STAT3, signal transducer and activator of transcription 3; STIA, K/BxN serum transfer-induced arthritis; TCR, T cell antigen receptor; Th, T helper; ZyA, zymosan A.