Histone lactylation promotes rheumatoid arthritis progression by increasing NFATc2 expression and the production of anti-lactylated histone autoantibodies

Wu, Gan; Yang, Chenglin; Huang, Yuan; Liu, Bin; Zhai, Haige; Cao, Zelin; Lu, Siyue; Ji, Jijian; Yin, Xinyue; Xin, Xianming; Jin, Shengwei; Cai, Youzhi; Wang, Jianguang

doi:10.1038/s41467-025-64096-5

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Published: 10 October 2025

Histone lactylation promotes rheumatoid arthritis progression by increasing NFATc2 expression and the production of anti-lactylated histone autoantibodies

Gan Wu^1,2,3^na1,
Chenglin Yang⁴^na1,
Yuan Huang²^na1,
Bin Liu²,
Haige Zhai²,
Zelin Cao ORCID: orcid.org/0000-0002-5425-1353²,
Siyue Lu²,
Jijian Ji^1,3,
Xinyue Yin²,
Xianming Xin²,
Shengwei Jin^1,3,
Youzhi Cai⁴ &
…
Jianguang Wang ORCID: orcid.org/0000-0003-0597-021X^1,2,3

Nature Communications volume 16, Article number: 9034 (2025) Cite this article

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Abstract

Elevated lactate in the joint microenvironment of rheumatoid arthritis patients is crucial for disease progression, though the mechanism remains unclear. This study shows significantly increased global lactylation levels within fibroblast-like synoviocytes from RA patients compared to healthy controls, with lactylated proteins being enriched in histones. Furthermore, we find anti-lactylated histone autoantibodies present in RA patients that positively correlate with Disease Activity Score 28. Using CUT&Tag and RNA-seq, we identify NFATc2 as a key target gene regulated by histone H3 lysine 9 lactylation. Functional studies reveal that NFATc2 promotes migration of RA-FLSs. Additionally, using collagen antibody-induced arthritis and collagen-induced arthritis mouse models, we demonstrate that NFATc2 exacerbates RA disease progression through enhancing the cartilage invasive function of FLS. Here, we show that upregulated target gene NFATc2 by lactate-dependent histone lactylation, can be used as a potential therapeutic target for intervention, anti-lactylated histone autoantibodies is promising as a diagnostic marker for RA.

Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by autoantibody (autoAb) production, synovitis, and bone destruction. The synovium is the main lesion site of RA, which forms an invasive pannus induced by the excessive proliferation of fibroblast-like synoviocytes (FLSs)^1,2,3,4,5. Proteomics and metabolomics studies have been conducted to clarify the material changes in the joint microenvironment of synovium and synovial fluid from RA patients, revealing that the hypoxic and hyperlactated joint microenvironment plays an important role in RA^{6,7,8,9,10,11}. It has been reported that the high lactate environment of RA joints is mainly due to the enhanced glycolysis of FLSs and the metabolic reprogramming of activated immune cells infiltrating the synovium^{10,12,13,14,15}. High intra-articular lactate further promotes the proliferation and migration of FLSs, which form invasive pannus and damage cartilage and bone tissue^10,12,16,17, however, the specific mechanism remains unclear.

Previously, lactate was thought to be a waste byproduct of metabolic processes. However, as a signaling molecule, it plays an essential role in regulating the immune response of tumor cells, affecting immune surveillance and escape-related behaviors^18,19,20. In addition, Zhao identified lactylation, a novel protein post-translational modification that regulates gene expression and is regulated by the concentration of the substrate of lactate²¹. Recent studies have demonstrated that protein lactylation plays an important role in the occurrence and development of many diseases; for example, Li reported that High mobility group box-1 (HMGB1) lactylation increases endothelial cell permeability and exacerbates sepsis²², and Lu reported that moesin lactylation promotes hepatocellular carcinoma tumorigenesis²³. Therefore, it is critical to analyze the regulatory mechanism of lactylation and characterize its function to understand RA pathogenesis and discover new therapeutic targets and strategies.

At present, it is believed that protein post-translational modification is of profound importance not only for the impact such changes have on immune responses but also in terms of immunogenicity and possible involvement in autoantibody (autoAb) production^24,25,26,27. Citrullination of protein arginine leads to the production anti-citrullinated protein antibodies (ACPAs) and is listed as an important serological diagnostic indicator of RA in the 2010 ACR/EULAR classification criteria. Studies have demonstrated that anti-citrullinated histone autoAb levels are significantly increased in the serum of RA patients and can promote RA progression^25,26,27. Since histone post-translational modifications exert epigenetic effects by transcriptionally regulating the expression of downstream target genes, histone modifications have received increasing attention^28,29. Classical histone modifications include acetylation³⁰, methylation³¹, and ubiquitination³². In recent years, many studies have also demonstrated various histone modifications using cellular metabolites as substrates, including succinylation³³ crotonylation³⁴, and lactylation²¹. Histone lactylation has been gradually confirmed to play a regulatory role in many diseases, such as cancer immunity³⁵, intestinal inflammation³⁶, sepsis³⁷, and repair after myocardial infarction³⁸. However, as a new post-translational modification, the effect of histone lactylation on the production of autoAbs and the RA process requires further clarification.

In this study, we first determine the increased level of histone lactylation in RA patients and screen the downstream genes by CUT&Tag and transcriptomics. Most importantly, we determine the role of histone lactylation and corresponding autoAbs in the onset and progression of RA, providing new ideas and targets for the diagnosis and treatment of this disease.

Results

The level of H3K9la is increased in FLSs from the synovium of RA patients

FLSs are the main lesion cells of RA, and glycolysis is the main energy metabolism mode, which produces a large amount of lactate, and lactate is the direct substrate of lactylation²¹. In this study, we first detected the level of global lactylation in FLSs from RA patients, HC (healthy controls) and gouty arthritis (GA, a form of inflammatory arthritis) patients by western blotting and immunofluorescence staining. The results showed that the level of global lactylation was increased in RA-FLSs, and the lactylated proteins were enriched at the 15 kDa band, localized in the nuclei of FLSs (Fig. 1A–C and Supplementary Fig. S1A). Given that synovial fluid is the microenvironment of FLSs, a hypoxic, hyperinflammatory, and hyperlactate environment is observed in RA patients (Supplementary Fig. S1B–D). Next, we investigated the influence of the joint microenvironment on the global lactylation of FLSs. The results showed that lactate (10 mM) increased the global lactylation level, while hypoxia (3% oxygen), TNF-α (10 ng/mL), or IL-1β (1 ng/mL) had no effect (Supplementary Fig. S1E). Considering the acidity of the hyperlactate environment, we investigated the influence of pH on global lactylation. The results showed that hydrochloric acid, with the same pH value as lactate (10 mM), did not affect the lactylation level (Fig. 1D). In addition, lactate treatment with the same concentration (10 mM) at different pH values (pH3.2, pH5.2 and pH7.2) could not cause the change of lactylation level (Supplementary Fig. S1F). However, sodium lactate and lactate increased global lactylation in RA-FLSs, and it was further confirmed that the lactylated proteins were enriched at the 15 kDa band and localized in the nuclei (Fig. 1D, E). Histones are the main proteins in the nucleus with a molecular weight of 15 kDa, and histone lactylation is a newly discovered epigenetic modification²¹. We detected the lactylation level of eight identified histone lactylation sites (H2BK16la, H3K9la, H3K14la, H3K18la, H4K5la, H4K8la, H4K12la, and H4K16la), revealing that the level of H3K9la was significantly increased in the FLSs and synovium of RA patients (Fig. 1F–I and Supplementary Fig. S1G). Lactate markedly elevated H3K9la levels in RA-FLSs (Fig. 1J). Overall, these data suggest that H3K9 lactylation is increased in the synovium and FLSs of RA patients.

**Fig. 1: The global lactylation and histone lactylation increased in FLSs and synovial tissues from RA patients.**

NFATc2 was screened and verified as the target gene of H3K9la

Histone modification is believed to directly regulate the transcriptional activity of target genes^28,29. To explore the potential function of H3K9la, we performed a CUT&Tag assay using an H3K9la antibody. More H3K9la binding reads were enriched in the promoter region of genes from RA-FLSs than in those from HC (Fig. 2A), and 7333 differential peaks were screened for binding to H3K9la (Fig. 2A). A motif analysis revealed that the most frequently H3K9la binding motif is different in RA-FLS and HC-FLS, it is “GKACTGTA” in RA-FLS and “SKGTATAC” in HC-FLS, respectively. (Fig. 2B). H3K9la binds to these motifs and thus regulates the expression of the target genes that have these motifs on the promoter (the genes and relative motifs are detailed in Supplementary Table S2), we found that NFATc2 and the inflammation-related genes of CXCL5, TNFSF13B and IL12A were included in H3K9la binding motif of RA. Subsequently, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the genes with H3K9la binding motif of RA and found that it was enriched in “Rheumatoid arthritis” pathway (Supplementary Fig. S2A). Additionally, a KEGG analysis showed that differential peaks were enriched in pathways such as “Tight junction” and “Rheumatoid arthritis” (Fig. 2C). These results suggest that H3K9la may play an important role in RA development. Transcriptome sequencing was performed to further clarify the biological functions of the target genes. A total of 3908 differentially expressed genes (DEGs) with q values less than 0.05 and fold changes greater than 2 were screened out. A Gene Ontology (GO) analysis showed that the DEGs were enriched in the functions of “cell adhesion” and “positive regulation of cell migration” (Fig. 2D). Next, we combined CUT&Tag data with RNA-seq data, and 68 target genes were screened that were both overexpressed in RA-FLSs and highly enriched in H3K9la binding DNA sequences (Supplementary Table S7, Fig. 2E). Subsequently, GO analysis revealed that the “cell motility” function was highly enriched (Fig. 2F), which is a critical functional phenotype associated with the disease progression mediated by RA-FLS. Seven genes (NFATc2, TGFB2, IL12A, ATRNL1, GPC1, TNS3, ITGBL1) were found to be enriched in this function. Given that transcription initiation is a fundamental regulatory point for gene expression, and the binding ability of the promoter to histones is crucial for gene expression. Therefore, we further screened the seven genes for those with H3K9la binding motifs in their promoters, and identified NFATc2 and IL12A (Supplementary Fig. S2C). Among these two genes, NFATc2 exhibited the highest differential expression (NFATc2 Log2FC = 2.1423 and IL12A Log2FC = 1.2922 in RNA-seq data). Furthermore, we observed that H3K9la is enriched in the promoter region of NFATc2 (Fig. 2G), which contains an H3K9la-binding motif (Supplementary Table S2), suggesting that H3K9la regulates the expression of NFATc2. To confirm that H3K9la transcriptionally enhances the expression of NFATc2, we assessed the high enrichment of H3K9la in the NFATc2 promoter region by ChIP‒qPCR (Fig. 2H) and further demonstrated the overexpression of NFATc2 in RA-FLSs by RT-qPCR (Fig. 2I).

**Fig. 2: Screening the target genes regulated by H3K9la in RA-FLSs using CUT&Tag and RNA-seq.**

Overexpression of NFATc2 promotes RA progression by enhancing FLS migration and invasion

The role of NFATc2, a nuclear factor that promotes cell migration, has not been reported in RA-FLSs. We first compared the expression of NFATc2 in FLSs and synovium from RA and HC by WB and immunohistochemistry, and the results showed that NFATc2 expression was increased in RA (Fig. 3A, B). Phosphorylated NFATc2 is inactive and located in the cytoplasm, while dephosphorylated NFATc2 is activated by calcineurin, which undergoes nuclear translocation and plays a transcriptional regulatory role³⁹. In this study, WB and immunofluorescence revealed that the expression, dephosphorylation, and nuclear localization of NFATc2 were increased in RA-FLSs (Fig. 3C), indicating that NFATc2 is highly activated in RA-FLSs. To further explore the function of NFATc2, we knocked down the expression of NFATc2 in RA-FLS using short hairpin RNA (shRNA) (Fig. 3D), which decreased the expression, dephosphorylation, and nuclear localization of NFATc2 (Supplementary Fig. S3A) and weakened the invasive migration ability of RA-FLSs (Supplementary Fig. S3B–G). The severe combined immune-deficiency (SCID) mouse co-implantation model was constructed to further verify the effect of NFATc2 on the migration and invasion of RA-FLSs, and knocking down NFATc2 in RA-FLSs reduced the number of eroded FLSs in the contralateral implanted cartilage (Supplementary Fig. S3H, I). Then we explored the function of NFATc2 in RA by intra-articular injection of lentiviral vector carrying shNFATc2 (LV-shNFATc2) in CIA mice to knock down the expression of NFATc2. Reduced joint swelling and clinical scores were observed in LV-shNFATc2-infected versus LV-shNC (control vector)-treated mice (Supplementary Fig. S3J, K). Decreases in histological scores (Supplementary Fig. S3L) and improvements in cartilage destruction were also observed by HE staining and safranin O fast green staining (Supplementary Fig. S3J). Further, we constructed mice with FLS-specific NFATc2 knockout for CAIA modeling, and the results also showed improvement in arthritis symptoms in mice (Fig. 3E–L). Lastly, in order to further clarify the mechanism by which NFATc2 plays a role in promoting RA FLS migration and invasion, we performed RNA-seq on RA-FLS knocking down NFATc2, and the results showed that some cartilage destruction and inflammation-related genes (MMP9, MMP13 and IL-6) (Supplementary Fig. S4A) were down-regulated after knocking down, GO analysis showed that DEGs were enriched in “cell adhesion”, “positive regulation of cell migration” and “calcium−dependent cell−cell adhesion via plasma membrane cell adhesion molecules” and other functions, and KEGG analysis showed that DEGs were enriched in “cell adhesion molecules” (Supplementary Fig. S4B, C). As a transcription factor, NFATc2 can regulate gene expression, we used JASPA to predict the presence of NFATc2 binding motif in the promoter regions of MMP9, MMP13 and IL-6, and proved that NFATc2 could promote their transcription by dual luciferase assay (Supplementary Fig. S4D, E). These results indicate that NFATc2 promote the invasive migration ability of RA-FLSs and exacerbate the progression of RA.

**Fig. 3: Overexpression of NFATc2 promotes RA disease progression by enhancing FLSs migration and invasion.**

Lactate-dependent H3K9la enhances the expression of NFATc2

Lactate levels are increased in the synovial fluid and synovium of RA patients^7,8. Lactate is the direct substrate of histone lactylation²¹, and both intracellular and extracellular lactate can affect this process. Intracellular lactate is derived from pyruvic acid catalyzed by lactate dehydrogenase. In this study, we investigated the effect of intracellular lactate on H3K9la through knockdown or inhibition (FX-11) of the expression or activity of lactate dehydrogenase (LDH) (Fig. 4A). LDH, a key enzyme that catalyzes pyruvate to lactate, is a tetramer comprising LDHA and LDHB subunits; LDHA catalyzes the conversion of pyruvate to lactate, and LDHB catalyzes the conversion of lactate to pyruvate⁴⁰. WB results showed that both LDHA knockdown and inhibitor intervention could significantly reduce the level of H3K9la (Fig. 4B). LDHA knockdown or inhibition decreased the enrichment of H3K9la on the promoter of NFATc2 and the expression of NFATc2, as determined by ChIP‒qPCR and RT‒qPCR (Fig. 4C, D) and decreased the migration and invasion of RA-FLSs, as determined by scratch and Transwell assays (Fig. 4E, F). Meanwhile, the secretion levels of inflammatory cytokines (IL-6, IL-8) and matrix metalloproteinases (MMP9, MMP13) were decreased (Supplementary Fig. S5E, F).

**Fig. 4: Inhibition of endogenous and exogenous lactate reduces H3K9la levels and cell migration of RA-FLS.**

Previous results showed that extracellular lactate intervention could increase the level of H3K9la in RA-FLSs (Fig. 1G), and extracellular lactate could act by binding the receptor of lactate (G protein-coupled receptor 81, GPR81) directly or transmembrane transport into cells by the lactate transporter (Monocarboxylate transporter 1, MCT1). WB and nucleic acid electrophoresis revealed that MCT1 but not GPR81 is expressed in RA-FLSs (Fig. 4H, I). MCT1 promotes the extracellular to intracellular transportation of lactate in a concentration-dependent manner⁴¹. In this study, lactate intervention increased the level of H3K9la (Fig. 4J) and upregulated the expression of NFATc2 in FLSs (Fig. 4K, L), promoting FLS migration, invasion and cytokine secretion (Fig. 4M, N and Supplementary Fig. S5G, H). However, these effects were inhibited by AZD-3965, an inhibitor of MCT1. These results indicate that extracellular lactate is transported into the cell through the lactate transporter MCT1, promoting histone lactylation in FLSs.

In summary, decreasing the production of intracellular lactate and uptake of extracellular lactate could inhibit the expression of NFATc2 by decreasing the lactylation of H3K9, thereby inhibiting the invasive migration of FLSs.

Lactate-dependent H3K9la alleviates arthritis progression in CIA mice by inhibiting NFATc2 expression

To further clarify the role of lactate-dependent H3K9la in RA disease progression, we constructed a CIA mouse model, a classic model of RA, to investigate the effect of endogenous and exogenous lactate on RA disease progression by upregulating H3K9la (Fig. 5A). The results showed that the CIA mice treated with the lactate dehydrogenase inhibitor FX-11 (2 mg/kg) or the lactate transporter MCT1 inhibitor AZD-3965 (100 mg/kg) exhibited milder joint swelling, lower arthritis scores (Fig. 5B, C), improved cartilage destruction (Fig. 5C), and reduced histological scores (Fig. 5D). Immunofluorescence showed higher H3K9la levels and NFATc2 expression in the synovium of CIA mice, which were reduced by FX-11 or AZD-3965 treatment (Fig. 5C). Meanwhile, FX-11 or AZD-3965 treatment reduced the concentration of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), increased the concentration of anti-inflammatory cytokines (IL-10) in the serum of CIA mice (Fig. 5E–H). Finally, in order to evaluate the efficacy of both treatments in combination with existing treatments, we treated CIA mice with FX-11 or AZD-3965 combined with methotrexate (MTX), and the results showed that the arthritis symptoms of CIA mice were further improved after the combination treatment compared with MTX treatment alone (Supplementary Fig. S6A–H).

**Fig. 5: Reducing H3K9la and NFATc2 levels alleviates joint destruction in CIA mice.**

In conclusion, the reduction in H3K9la levels by FX-11 and AZD-3965 intervention alleviated arthritis disease progression in CIA mice and combined with MTX had a better therapeutic effect.

AutoAbs that specifically recognize lactylated histones are present in the serum of RA patients

We found that the lactylated histone antigens present in the serum of RA patients were detected by WB (Fig. 6A). Then, lactylated histone polypeptides were synthesized to investigate the existence of autoAbs specifically recognizing lactylated histone in the serum of RA patients, including H2B (lactylated K15 + K16), H3 (lactylated K9 + K14 + K18), and H4 (lactylated K5 + K8 + K12 + K16) peptides. A dot blot showed that autoAbs against lactylated histones were present in the serum of 10 (100%) RA patients, compared to only 1 (10%) in HC and none in PsA patients (Supplementary Fig. S7A). AutoAbs against H2B, H3, and H4 were present in 2 (20%), 8 (80%), and 4 (40%) RA patients, respectively (Supplementary Fig. S7B–D). Further experiments showed that 7 of 10 RA patients were positive for H3K9la autoAbs (70%), but only one (10%) of the 10 healthy volunteers (Supplementary Fig. S7E). ELISA revealed a significantly increased level of anti-lactylated histone autoAbs, including H3, H4, and H3K9, in RA patients (Fig. 6B, D–F). However, the level of anti-lactylated H2B autoAbs was slightly higher than that of HC and PsA patients (Fig. 6C), but it was still statistically significant. By contrast, no significant difference was observed in unmodified histones. Further, we found that only anti-H3K9la autoAbs levels were positively correlated with DAS28 in RA patients (Fig. 6G–K) and were higher in RA patients with high activity (Fig. 6L). Meanwhile, we also found that the levels of anti-H3K9la autoAb tended to rise as the disease advanced in individual RA patients (Fig. 6M). Finally, we found that anti-H3K9la autoAbs is present in both seropositive and seronegative RA patients (Fig. 6N) and is not associated with anti-CCP levels (Fig. 6O). In conclusion, we confirmed the presence of anti-lactylated histone autoAbs in the serum of RA patients, and the level of anti-H3K9la autoAbs were correlated with disease activity in RA patients.

**Fig. 6: Detected the levels of anti-lactylated histone autoAbs in serum of HC, PsA patients and RA patients.**

Discussion

Joint damage is the main pathological change in RA, and changes in the joint microenvironment play an important role^9,10,11. A previous metabolomics study revealed that the concentration of lactate is increased in the synovial fluid of RA patients, which positively correlates with DAS28^7,8. Numerous subsequent studies have confirmed the acidic microenvironment of joints in RA patients^42,43,44. Increased lactate is thought to be produced by enhanced glycolysis in FLSs and inflammatory cells in the RA joint microenvironment^12,13,14. Here, the level of lactylation in FLSs was investigated for the first time to further explore the mechanism by which high lactate levels promote RA progression. The results revealed increased global lactylation in RA-FLSs, and both endogenous and exogenous lactate could increase the level of lactylation in FLS. The lactylation level in RA-FLSs was confirmed to increase in a concentration-dependent manner. Lactylation is a newly discovered post-translational protein modification that plays an important role in numerous diseases, including inflammatory disease and tumorigenesis^22,23. However, the biological role of lactylation in RA pathogenesis requires further exploration.

Further experiments showed that the increased lactylation proteins in RA-FLSs were enriched in histones. Histone lactylation is a recently discovered post-translational modification of histones²¹ that epigenetically regulates the balance between transcriptional activation and transcriptional repression and plays an important role in the occurrence and development of many diseases, such as tumor immunosuppression, sepsis, and intestinal inflammation. In the present study, we found that interfering with lactate production and uptake attenuated the invasion ability of RA-FLSs by reducing the level of histone lactylation. The elevation of histone lactylation in RA was also confirmed in the CIA model. Reducing the level of histone lactylation by inhibiting the production and uptake of lactate could significantly inhibit synovial invasion and cartilage destruction, alleviate joint swelling, and reduce serum inflammatory cytokine levels. These findings indicate that histone lactylation plays an important role in RA disease progression, and inhibiting the level of histone lactylation may be a potential therapeutic intervention for RA.

To further identify the downstream target gene of histone lactylation, we performed CUT&Tag combined RNA-seq and screening and verified that NFATc2 was the downstream target gene of histone lactylation. NFATc2 participates in T-cell development⁴⁵ and promotes the invasive migration of tumor cells⁴⁶. The biological function of NFATc2 in RA progression has not been reported. We first found that elevated histone lactylation promoted NFATc2 expression in RA-FLSs and that overexpression of NFATc2 promoted the migration and invasion ability of FLSs. It was further confirmed that knocking down NFATc2 could alleviate joint damage in CIA mice. These results indicate that NFATc2 plays an important role in promoting RA progression.

Numerous autoAbs have been detected in RA patients, such as anti-CCP, anti-perinuclear factor, anti-RA33, and anti-keratin antibodies, which are often used in the clinical diagnosis of RA^47,48. Anti-CCP antibodies against citrullinated protein antigens are recommended by the 2010 ACR/EULAR guidelines for their high specificity in RA diagnostics⁴⁹. It has also been reported that anti-citrullinated histone autoAbs are specifically elevated in RA patients and can promote the disease process^25,26,27. In this study, we demonstrate for the first time that anti-lactylated histone autoAbs are present in RA patients and found that anti-H3K9la antibodies are associated with disease activity in patients.

In summary, we found that the level of histone lactylation is elevated in RA-FLSs, which promoted RA progression by enhancing the expression of the downstream target gene NFATc2. In addition, anti-lactylated histone antibodies were present in RA patients. In the future, mass spectrometry and site mutation will be used to discover and evaluate the function of specific lactylation proteins and further explore the effect of non-histone lactylation in RA. Meanwhile, numerous studies have demonstrated that lactylation influences the differentiation and function of immune cells^{21,36,50,51,52,53}. Research by Zhang indicated that H3K18la can facilitate the transition of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages²¹. In RA, the high-lactate microenvironment within joints is also characterized by significant infiltration of macrophages, which contribute to disease progression. However, the specific effects of lactylation on macrophages and its role in RA remain unclear; further investigation is needed to determine whether it acts synergistically with or inhibits FLS. Additionally, this study is limited by the small sample size, and future studies on a larger sample are required to further verify the diagnostic value and mechanism of anti-lactylated histone antibodies in RA.

Methods

Study approval

This study does not involve sex-based analysis, and sex was not considered in the study design, since RA involves both. This study was approved by the Ethics Committee of Wenzhou Medical University. Informed consent was obtained from patients before inclusion in the study. The human studies were adhered to the Declaration of Helsinki. Animal experiment procedures were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University. All animal experiments in this study abide by the ARRIVE guidelines.

Patients and samples

Serum and synovial fluid samples were obtained from active RA patients who were newly diagnosed in the outpatient department before disease-modifying antirheumatic drug (DMARDs) treatment, serum from RA patients in remission who were treated with DMARDs. And synovial tissues were obtained from RA patients underwent knee arthroplasty who have not been given biological agents. Seropositive refers to RA patients who test positive for RF or ACPA, and seronegative refers to RA patients who test negative for both RF and ACPA. All RA patients fulfilled the 2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria. Synovial tissues were obtained from GA patients fulfilled the 2015 ACR/EULAR gout classification criteria. Serum obtained from PsA patients fulfilled the Classification Criteria for Psoriatic Arthritis. Serum of HC from healthy volunteers, and the synovial tissues, synovial fluid and cartilage were used to be controls from patients of non-inflammatory knee joint diseases underwent knee operations. FLSs were isolated from synovial tissues as previously described⁵⁴. In brief, after obtaining the synovial tissue, large pieces of adipose tissues were removed, the blood was washed off, and in a sterile environment, the synovial tissue was repeatedly cut into a paste-like consistency and centrifuged to remove adipose tissues. Finally, FLS were isolated through enzymatic digestion using type II collagenase. All FLS from passage 3 to 5 were used for the experiments. This study was approved by the Ethics Committee of Wenzhou Medical University. Informed consent was obtained from patients before inclusion in the study. The clinical details of all participants are shown in Supplementary Table S1.

Cells culture, intervention and transfection

FLSs from HC, GA patients and RA patients were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% Fetal Bovine Serum (FBS, 11011-8611, Ever Green, China) at 37 °C in 5% CO₂.

In the intervention experiments, RA-FLSs were treated with 10 mM lactate (L1750, Sigma-Aldrich, Germany), 3% oxygen, 10 ng/mL human TNF-α (10291-TA, R&D Systems, USA), 1 ng/mL human IL-1β (201-LB, R&D Systems, USA), 10 mM sodium lactate (L7022, Sigma-Aldrich, Germany), and hydrochloric acid, respectively. After 24 h incubation, the cells were collected for western blotting. In the inhibitor intervention experiments, RA-FLSs were pre-treated with 10 µM lactate dehydrogenase inhibitor (FX-11, 213971-34-7, MCE, USA) or 10 mM lactate+100 nM MCT1 inhibitor (AZD-3965, 1448671-31-5, MCE, USA) for 24 h, and the cells were used for western blotting, ChIP-qPCR, RT-qPCR, scratch migration and transwell assay. Cell viability of FLSs were detected using Calcein/PI Cell Viability/Cytotoxicity Assay Kit (C2015M, Beyotime, China) according to the manufacturer’s instructions. Finally, images of the live (green fluorescence) and dead (red fluorescence) cells were captured using an inverted fluorescence microscope (Ti-S, Nikon, Japan).

In the siRNA transfection experiment, the cells were cultured into 6-well plates (2.5 × 10⁵ cells/well) for 24 h. Then, siRNA diluted with 250 µL Opti-MEM medium (11058021, Gibco, USA) was mixed with lipofectamine 3000 (L3000015, Thermo Fisher Scientific, USA) diluted with 250 µL DMEM. Then, the mixture was added to each well. After 6 h culture, the supernatant was replaced by basal medium and cultured for additional 48 h. The cells were collected for western blotting, ChIP-qPCR, RT-qPCR, scratch migration and transwell invasion assay. The siRNA sequence was described in Supplementary Table S3.

Western blotting

FLSs or synovial tissues were lysed with RIPA lysis buffer. The protein was quantitated by BCA Protein Assay Kit (P0012, Beyotime, Shanghai, China). 20 μg protein were loaded onto Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels for separation and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and incubated with the primary antibodies at 4 °C overnight, and then incubated with the HRP-conjugated secondary antibodies. The bands were detected by electrochemiluminescence (ECL), and the ChemiScope 6000 imaging system (Clinx, China) was applied for photography. Protein gray value detection was analyzed by Image J software. The following antibodies used were available from Supplementary Table S4.

Immunofluorescence staining and immunohistochemistry

FLSs were inoculated on the round coverslip and fixed with 4% paraformaldehyde after adhesion, then were blocked with 5% BSA and incubated with primary antibody overnight at 4 °C. On the second day, incubated with fluorescein-conjugated secondary antibody for 1 h in the dark at room temperature, nuclei were counterstained with DAPI.

The mouse joint sections or synovial tissue sections were deparaffinized, hydrated, antigenically repaired, blocked, and incubated with primary antibody overnight at 4 °C. For immunofluorescence analysis, then incubated with fluorescein-conjugated secondary antibody for 1 h in the dark at room temperature, nuclei were counterstained with DAPI. Imaging with laser scanning confocus (C2si, Nikon, Japan). For immunohistochemistry analysis, incubated with HRP-conjugated secondary antibody after incubated with primary antibody, then visualization with the 3,3′-Diaminobenzidine (DAB) substrate and imaging with photomicroscope (CI-L, Nikon, Japan). The following antibodies used were available from Supplementary Table S4.

Lactate measurement

The concentration of lactate in synovial fluid was detected using L-Lactate Assay Kit (ab65330, Abcam, USA) according to the manufacturer’s instructions. The quantification was performed with Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, USA) at 570 nm.

ELISA

The concentrations of TNF-α, IL-1β in synovial fluid and the concentrations of IL-1β, TNF-α, IL-6, IL-10 in the serum of mice were detected using ELISA kits (R&D Systems, USA). The concentrations of IL-6, IL-8, MMP9 and MMP13 in supernatant from RA-FLS were detected using ELISA kits (Abcam, UK). As per the manufacturer’s instructions and measured using Varioskan LUX multimode microplate reader at 450 nm.

To detect anti-lactylated histone in serum, the Costar ELISA plates (Corning, USA) were coated with peptides (5 µg/mL) in PBS at 4 °C overnight. Subsequently blocked by 2% BSA (1 h, room temperature) and following incubation with human serum (diluted 1:100) for 1 h at room temperature. After three times washed with PBST, followed by HRP-Goat anti-human IgG (AS002, ABclonal, Wuhan, China) for 1 h at room temperature. Bound antibodies were visualized using tetramethylbenzidine and hydrogen peroxide. Measured using Varioskan LUX multimode microplate reader at 405 nm.

CUT&Tag assay and bioinformatics analysis

To explore the candidate target genes of H3K9la in FLSs, CUT&Tag assay was conducted using Hieff NGS G-Type In-Situ DNA Binding Profiling Library Prep Kit for Illumina (12598, Yeasen, China), following the manufacturer’s instruction (three independent samples of each group). Briefly, cells were harvested and bound to concanavalin A-coated magnetic beads, reacted with primary antibody against H3K9la, secondary antibody of anti-rabbit IgG, and pA/G-Transposome Mix to activate transposase. FLSs were lysed by proteinase K and DNA was extracted, amplified and purified for library construction using Hieff NGS Tagment Index Kit (12416, Yeasen, China), and the library quality was assessed on the Agilent Bioanalyzer 2100 system. Then the library was sequenced on Illumina Novaseq platform at Novogene Science and Technology Co., Ltd (Beijing, China). The peaks with fold change of RPM more than 2, p < 0.05 were considered as differential peaks. The position of peak summit around transcript start sites of genes can predict the interaction sites between protein and gene. ChIPseeker⁵⁵ was used to retrieve the nearest genes around the peak and annotate genomic region of the peak. Peak-related genes can be confirmed by ChIPseeker, and subjected to enrichment analysis of KEGG. Motif analysis was performed using findMotifsGenome.pl program in HOMER v4.11 software.

RNA-seq and bioinformatics analysis

The total RNA of FLSs (three independent samples of each group) was extracted using Trizol reagent (15596026, Invitrogen, USA) following the manufacturer’s procedure. The RNA libraries were sequenced on the Illumina NovaseqTM 6000 platform by LC Bio-Technology CO.,Ltd (Hangzhou, China). Differentially expressed genes (DEGs) were screened by DESeq2 software between RA-FLS and HC-FLS. The genes with the parameter of q value < 0.05 and |log2FC|> = 1 were considered DEGs. Then DEGs were subjected to enrichment analysis of GO and KEGG. The critical value of significant gene enrichment was set as p < 0.05.

Chromatin immunoprecipitation (ChIP)-qPCR

The assay was performed using ChIP Kit (ab500, Abcam, USA) following the standard protocol. Briefly, FLS were fixed with 1% formaldehyde and quenched with 125 mM glycine, then the cells were lysed and sonicated. For immunoprecipitation, the diluted chromatin was incubated with H3K9la antibodies overnight at 4 °C with constant rotation. The precipitated chromatin DNA was purified with the QIAquick PCR Purification Kit (Qiagen). The qPCR was performed using Taq Pro Universal SYBR qPCR Master Mix (Q712, Vazyme, China) and analyzed using the QuantStudio 6 Flex Real-Time PCR system (Thermo Fisher Scientific, USA). The primer sequences were described in Supplementary Table S5.

RT-qPCR

Total RNA was isolated from FLSs using Trizol reagent (15596026, Invitrogen, USA). Reverse transcription was performed using HiScript III RT SuperMix for qPCR (R323, Vazyme, China). Real-time quantitative PCR (qPCR) was performed in QuantStudio 6 Flex Real-Time PCR system using Taq Pro Universal SYBR qPCR Master Mix. Primer sequences of NFATc2 and β-tubulin genes were described in Supplementary Table S4. The relative mRNA expression normalized to β-tubulin was calculated using the 2^−ΔΔCt method.

Construction of short hairpin RNA-expression lentivirus and infection

The lentivirus-based shRNA vectors were designed and constructed by Tsingke Biotech Co., Ltd. (Beijing, China). The lentivirus of shRNA targeting NFATc2 and the control lentivirus were generated for the NFATc2-knockdown experiment. Briefly, pLKO.1 was constructed as the lentivirus transfer vector, which contained a puromycin resistant gene. The co-transfection of HEK 293T cells with shRNA-pLKO.1 and helper plasmids, psPAX2, and pMD2.G vectors. During transfection, transfected cells were screened using puromycin, and lentiviral particles were collected. For infection, RA-FLS were treated with lentiviruses and polybrene (10 µg/mL) for 12 h at 37 °C. Subsequently, the virus-containing medium was replaced with fresh medium, the cells were collected for the follower experiments. The transfection and infection efficiency were observed under an inverted fluorescent microscope, and the knockdown efficiency of shRNA was evaluated by WB. The sequences of shRNA were described in Supplementary Table S3.

Scratch migration assay

RA-FLSs were seeded in six-well plates (1 × 10⁵ cells/well). the scratch was made along the diameter of the well using a 10 μL pipette tip (Axygen, USA) as the cells reached to 95% confluency. Images were captured at 0 h and 24 h by a camera under the microscope (×40 magnification). Image J was used to calculate the mobility ratio, and the mobility ratio was calculated using the following equation: migrated cellular area/scratched area × 100%.

Transwell assay

A 24-well transwell chamber with 8-μm pores (Corning, USA) was used for Transwell assays. The bottoms of the transwells (on top of the membrane) are coated with a thin layer of Matrigel (356234, Corning, USA). FLSs were trypsinized and re-suspended with serum-free DMEM medium at a final concentration of 1 × 10⁵/mL, 200 μL cell suspension was seeded at the top of matrigel, the cells are cultured in the upper chamber of the transwell insert. 500 μL DMEM with 10% FBS as chemoattractant was added to the lower chamber. The plate was incubated at 37 °C under 5% CO₂ for 24 h. Then, the migrated cells on the underside of the membrane were fixed with methanol, and stained with crystal violet. Pictures were taken under the microscope (TS100-F, Nikon, Japan) and the cell number was quantified by the software Image-Pro.

Humanized synovitis in vivo model using SCID mice

The cartilage from HC was cut into 5–8 mm³ pieces (one piece) and implanted into the left flanks of SCID mice wrapped with a sterile sponge infiltrated with RA-FLS (5 × 10⁵ cells per 100 μL). In the right flanks, the cartilage (one piece) was implanted only. In order to observe the migration of FLSs on the left flanks to the cartilage on the right flanks, the mice were sacrificed on day 60 after implantation, the right implants were removed and embedded in Tissue-Tek OCT compound (4583, Sakura Finetek, Japan) for frozen section and hematoxylin-eosin (H&E) staining. Then the invasiveness of RA-FLS into cartilage was observed. The level of invasiveness was scored as follows: 0 = no or minimal invasion, 1 = visible invasion (two-cell depth), 2 = invasion (five-cell depth), and 3 = deep invasion (more than ten-cell depth).

Construction of CAIA model in NFATc2^fl/fl × Thy1-CreERT2^−/− and NFATc2^fl/fl × Thy1-CreERT2^+/− mice

All mice were raised in a specific-pathogen-free (SPF) room at the Laboratory Animal Center of WMU and housed in cages (five per cage) kept at 22–26 °C and 60–65% humidity on a regular 12-h light/dark cycle (light, 8:30–20:30) and had ad libitum access to water and food, except where noted. Rodent chow diet (corn, soybean meal, flour, bran, fish meal, salt, various vitamins and mineral elements, etc) were purchased from Keao Xieli Feed Co (Beijing, China). This study does not involve sex-based analysis, and sex was not considered in the study design. Therefore, in order to control for the variable, we used only male mice in this study. NFATc2^fl/fl × Thy1-CreERT2^−/− and NFATc2^fl/fl × Thy1-CreERT2^+/− mice (5 weeks old) were treated with tamoxifen (10540-29-1, Sigma-Aldrich, Germany) (Administer 175 mg/kg tamoxifen in corn oil via intraperitoneal injection once every 48 h for 3 times). Mice were tail intravenously day 18 with 5 mg anti-type II collagen monoclonal antibody cocktail (Chondrex, USA), according to the manufacturer’s instructions. The mean clinical score (0–4) was assigned as follows: 0 = no symptoms, 1 = erythema and slight swelling limited to the ankle joint and toes, 2 = erythema and slight swelling spreading from the ankle to the midfoot, 3 = erythema and severe swelling spreading from the ankle to the metatarsal joints, and 4 = ankylosing deformity with joint swelling. On day 25, The mice were euthanized, and collect serum and joint tissues for further study.

CIA model and histological analysis

DBA/1 mice were obtained from Shanghai SLAC Laboratory Animal. All experiment procedures were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University. All mice were raised in a SPF room at the Laboratory Animal Center of WMU and housed in cages (five per cage) kept at 22–26 °C and 60–65% humidity on a regular 12 h light/dark cycle (light, 8:30–20:30) and had ad libitum access to water and food, except where noted. Rodent chow diet (corn, soybean meal, flour, bran, fish meal, salt, various vitamins and mineral elements, etc) were purchased from Keao Xieli Feed Co (Beijing, China). This study does not involve sex-based analysis, and sex was not considered in the study design. Therefore, in order to control for the variable, we used only male mice in this study. On day 0, the mice were intradermally injected with 100 µL of type II bovine collagen (2 mg/mL) (20021, Chondrex, USA) emulsified in equal volumes of Freund’s complete adjuvant (7001, Chondrex). On day 21, the mice were injected with 100 µL of type II bovine collagen (2 mg/mL) emulsified in equal volumes of Freund’s incomplete adjuvant (7002, Chondrex) for booster immunization. Meanwhile, mice were given weekly intra-articular injections of shRNA (2 × 10⁸ TU/mL) or daily intraperitoneally with FX-11 (2 mg/kg) or AZD-3965 (100 mg/kg). On day 49, the mice were euthanized, joint tissues and serum samples were harvested for analysis. The knee joints of mice were dissected and fixed in 4% paraformaldehyde and then decalcified in 50 nM EDTA and embedded in paraffin. The sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) or safranine O-fast green stain for histological analysis. Histology scores were assessed as previously described⁵⁶.

Peptides synthesis

The peptides of lactylated histone and non-lactylated histone (10–20 aa) corresponding to the whole sequence of the human histone H2B, H3 and H4 were synthesized by KS-V Peptide Biotechnology Co., Ltd (Hefei, China). Peptides were purified by high-performance liquid chromatography (HPLC) (purity >95%) and characterized by electrospray ionization mass spectrometry (ESI-MS). The sequences of peptides were described in Supplementary Table S6.

Dot blot assay

The peptides were diluted into 1 µg/mL, then spotted (1 µL/dot) severally onto the nitrocellulose membrane (1 cm × 1 cm) using narrow-mouth pipette tip, then dried naturally and blocked by 10% BSA (2 h, room temperature), three times washed with PBST. Subsequently, the membrane was incubated overnight with human serum (200 µL) at 4 °C, three times washed with PBST, followed by HRP-Goat anti-human IgG (AS002, ABclonal, Wuhan, China) for 1 h at room temperature. The dots were detected by electrochemiluminescence (ECL), and the ChemiScope 6000 imaging system (Clinx, China) was applied for photography. Exposure time is controlled at 10 seconds.

Statistical analysis

Statistical analysis was conducted in GraphPad Prism 8 (GraphPad Software, La Jolla, CA), and the data were presented as the mean ± SD. The Shapiro–Wilk method was used to determine whether the data were normally distributed and the homogeneity of variance was tested by the Levene method. If the measurements between two groups were normally distributed, the unpaired Student’s t test was used, otherwise the Mann–Whitney U test was used. One-way analysis of variance test with post hoc contrasts by Tukey test was applied to compare the means of multi groups. ANOVA of repeated measurements was used for inference the effects of treatment and time on experimental objects (clinical scores of mice).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data associated with this study are present in the paper and the Supplementary Information or the corresponding author on request. The RNA-seq data and CUT&Tag data used in this study are available in the Gene Expression Omnibus (GEO) database under accession code GSE284403, GSE285734 and GSE284763. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China to J.W. and S.J. (Grant No. 82272451 and 82270098); the National Key R&D Program of China to S.J. (Grant No. 2022YFC2504404); the National Natural Science Foundation of China Joint Fund Project to S.J. (Grant No. U22A20273); and the Key Research and Development Program of Wenzhou to J.W. (Grant No. ZY2023028).

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These authors contributed equally: Gan Wu, Chenglin Yang, Yuan Huang.

Authors and Affiliations

Department of Anesthesia and Critical Care, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
Gan Wu, Jijian Ji, Shengwei Jin & Jianguang Wang
Institute of Autoimmune Diseases, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China
Gan Wu, Yuan Huang, Bin Liu, Haige Zhai, Zelin Cao, Siyue Lu, Xinyue Yin, Xianming Xin & Jianguang Wang
Key Laboratory of Pediatric Anesthesiology, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, China
Gan Wu, Jijian Ji, Shengwei Jin & Jianguang Wang
Sports Medical Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
Chenglin Yang & Youzhi Cai

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Contributions

Conceptualization, G.W., S.J., Y.C., and J.W.; Methodology, G.W., C.Y., B.L., and H.Z.; Software, G.W., Y.H., and X.X.; Validation, C.Y., Y.H., B.L., H.Z., Z.C., S.L., and X.X.; Formal Analysis, G.W., C.Y., Y.H., and B.L.; Investigation, C.Y., Y.H., H.Z., Z.C., J.J., and J.W.; Resources, S.J., Y.C., and J.W.; Data Curation, G.W. and C.Y.; Writing – Original Draft, G.W., C.Y., Y.H., and X.Y.; Writing – Review & Editing, G.W., S.J., Y.C., and J.W.; Visualization, G.W., Y.H., S.L., J.J., and X.Y.; Supervision, Y.C. and J.W.; Project Administration, S.J., Y.C., and J.W.; Funding Acquisition, S.J. and J.W.

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Correspondence to Shengwei Jin, Youzhi Cai or Jianguang Wang.

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Wu, G., Yang, C., Huang, Y. et al. Histone lactylation promotes rheumatoid arthritis progression by increasing NFATc2 expression and the production of anti-lactylated histone autoantibodies. Nat Commun 16, 9034 (2025). https://doi.org/10.1038/s41467-025-64096-5

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Received: 13 August 2024
Accepted: 04 September 2025
Published: 10 October 2025
DOI: https://doi.org/10.1038/s41467-025-64096-5