Human synovial fluid samples were obtained under a protocol for discarded specimens approved by the Stanford University School of Medicine Institutional Review Boards that does not require consent. The samples were stored frozen at -80 °C until the time of analysis. Patients with RA that met the American College of Rheumatology criteria for the disease were included in the study (22). The sample donors were patients with knee osteoarthritis (OA) and over 45 years of age with radiographic Kellgren/Lawrence grade ≥ 3 changes (23). The levels of H2A, H2B, H3, and H4 were determined using the enzyme-linked immunosorbent assay (ELISA) kit for each histone (antibodies-online, Aachen, Germany) according to the manufacturer’s protocol.

Eight-week-old male C57BL/6 mice (Koatech, Pyeongtaek, Korea) were housed under controlled conditions at 23 ± 2°C and 50 ± 10% humidity on a 12 h light/dark cycle and had free access to a standard chow diet and water. STA was induced via intraperitoneal injection of 150 μL of K/BxN serum into C57BL/6 mice on day 0 and day 2. The mice were intraperitoneally injected with 200 μg of calf histone (Roche, Basel, Switzerland) or phosphate-buffered saline on day 1 and day 3. We used native histones purified by calf thymus. It is a mixture of histones H1, H2A, H2B, H3, and H4, and we used it as it is without any additional procedures. The histone treatment dose for intraperitoneal injection was determined based on the previously published method in a sepsis model, which demonstrated that the intravenously injected calf thymus histones were lethal at a concentration of 75-100 mg/kg and sublethal at 50 mg/kg in C57BL/6 mice (12). To investigate the effect of histones in arthritis, we injected 200 μg of calf thymus histones (equivalent to 10 mg/kg) intraperitoneally into each mouse based on our preliminary dose titration study (data not shown). The clinical arthritis score was determined as previously described (19): 0, no swelling or erythema; 1, mild swelling and erythema or digit inflammation; 2, moderate swelling and erythema confined to the region distal to the mid-paw; 3, pronounced swelling and erythema with extension to the ankle; and 4, severe swelling, erythema, and joint rigidity of the ankle, foot, and digits. The severity of arthritis in each limb was graded on a scale of 0–4 (a maximum possible score of 16 for each mouse) by a nonblinded examiner. Hind paw thickness was measured with a dial thickness gauge. The mouse joint tissues were fixed with 10% formalin, decalcified in 5% formic acid, and embedded in paraffin. Sections of the paraffin-embedded tissue were stained with hematoxylin and eosin (H&E), and scored by a blinded examiner for synovitis, pannus formation, and erosion of bone and/or cartilage based on a previously described scoring system (24). For each experimental condition, the joints of ten mice were evaluated and scored. The sample size was determined based on our pilot experiments. Mice were assigned to each group at random, and the order of treatments was randomized. All mice were included in the analysis. All animal experiments and protocols were approved by the Institutional Animal Care and Use Committee at Pusan National University (Miryang, Korea) and performed in accordance with the institutional and national guidelines for the care and use of laboratory animals.

Human synovial fibroblast cell line MH7A was cultured in RPMI-1640 (Hyclone, Logan, UT, USA) containing 25 mM HEPES and L-glutamine (Hyclone), supplemented with heat-inactivated 10% fetal bovine serum (FBS; Hyclone), 100 units/mL penicillin (Hyclone), and 100 μg/mL streptomycin (Hyclone). THP1-HMGB1-Lucia cells (THP-1 cells, InvivoGen, San Diego, CA, USA) were cultured in RPMI-1640, supplemented with 25 mM HEPES, L-glutamate, heat-activated 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 100 μg/mL Normocin (InvivoGen). For all assays, THP-1 cells were seeded at a density of 1 × 10⁵ cells/well in 96-well plates and differentiated into adherent macrophages with 25 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, MO, USA) overnight. Cells were treated with calf thymus histones (Abnova, Taipei, Taiwan) or recombinant histones, H2A, H2B, H3, and H4 (New England BioLabs, Cambridge, MA, USA) or histone peptides (Peptron, Daejeon, Korea, Table 1 ).

The level of LDH released into the medium from cells was measured using an LDH assay kit (DoGenBio, Seoul, Korea). This kit measures the amount of a water-soluble tetrazolium salt (WST) to determine the cytotoxicity of cells. Cells were seeded onto 96-well plates at a density of 1 × 10⁵ cells/well in serum-free media. After incubation with different concentrations of histones, 10 μL supernatant of each sample and 100 μL LDH Reaction Mix (WST substrate mix, LDH assay buffer) were incubated at 25°C in the dark for 30 min, and the optical density of soluble WST was then measured at 450 nm using FlexStation3 microplate reader (Molecular Devices, San Jose, CA, USA).

MH7A cells and THP-1 cells were placed at a density of 1 × 10⁵ cells/well and 5 × 10⁵ cells/well, respectively. The cells were treated with 5 μM Hoechst 33342 stain (ThermoFisher Scientific, Waltham, MA, USA) for 1 h, then treated with various concentrations of histones and/or lipopolysaccharide (LPS, Sigma-Aldrich) and stained with a plasma membrane-impermeable DNA-binding dye, SYTOX Green (Invitrogen), according to the manufacturer’s protocol, in a serum-free condition. Before histone addition, the cells were treated with or without 10 mM sodium perchlorate (Sigma-Aldrich) for 24 h. The live images of the cells were obtained under a fluorescence microscope (Eclipse Ti2, Nikon Instruments, Tokyo, Japan) or by an automatic cell imaging system (Operetta CLS™, PerkinElmer, Waltham, MA, USA). For images obtained from the Operetta cell imaging system, image analysis and cell counting were performed according to the manufacturer’s assay protocol using Harmony^® software (Version 4.9, PerkinElmer).

The level of HMGB1::Lucia luciferase released from THP-1 cells was measured with a luminometer (FlexStation3) using luciferin. A sample from each well was mixed with QUANTI-Luc (InvivoGen) according to the manufacturer’s instructions, and the luciferase reading was measured.

ATPlite (PerkinElmer) is a luciferase-based assay used for the quantitative evaluation of cell proliferation and cytotoxicity. For this assay, cells were seeded onto a 96-well plate at a density of 5 × 10⁵ cells/well. The cells were treated with different concentrations of histones for 1 h. Then, the ATPlite buffer and the lyophilized substrate solution were mixed. Finally, cells in each well were incubated with 50 μL of this mixture for 10 min, followed by a luminescence measurement.

?A3B2 tlsb .2pt?>To measure the cellular binding of FITC labeled poly-L-lysine (Sigma-Aldrich), THP-1 cells were seeded at a density of 2 × 10⁵ cells/well and treated with 100 ng/mL LPS for 24 h. Then, 10 μg/mL poly-L-lysine-FITC was added to the cells and incubated for 30 min at 4°C. Cells were observed under a fluorescence microscope (Eclipse Ti2) to obtain fluorescence images. Quantification of fluorescence intensity of images was performed using ImageJ (https://imagej.nih.gov).

To detect cell membrane damage in flow cytometry, a DNA-staining fluorescent marker 7-AAD (Invitrogen) was used. MH7A and THP-1 cells were seeded at a density of 5 × 10⁵ cells/well and treated with various concentrations of histones for 1 h. Subsequently, 1 μg/mL 7-AAD was added to the cells and incubated for 30 min. Cells were analyzed on BD LSRII SORP (BD Biosciences, San Jose, CA, USA) to detect 7-AAD.

IL-1β (eBioscience, San Diego, CA, USA), TNF-α (Invitrogen, San Diego, CA, USA), and CCL4 (R&D Systems, Minneapolis, MN, USA) levels were measured using ELISA kits as per the manufacturer’s instructions.

Total RNA was isolated from THP-1 cells treated with H2B-α1 peptide or cit-H2B-α1 peptide, using TRIzol reagent (Invitrogen). Samples of 3 μg of total RNA were prepared from each condition with three experimental replicates, and gene expression profiling was performed using the QuantSeq 3′ mRNA-Seq service (Ebiogen, Seoul, Korea). After quality checks using Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA), an oligo-dT primer set containing Illumina-compatible sequences at its 5′ end was hybridized to the prepared RNA, and reverse transcription was performed. High-throughput sequencing was performed as single-end sequencing using Illumina NextSeq 500 (San Diego, CA, USA). The resulting reads were aligned using Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2) with the human reference genome hg19 (25). The DEGs were determined based on the counts from alignments using coverage in Bedtools (https://bedtools.readthedocs.io). The read count data were processed based on the quantile normalization method using EdgeR (https://bioconductor.org). Gene enrichment analysis was performed by MATLAB (MathWorks, Natick, MA, USA) using the Reactome pathway database (https://reactome.org) and Gene Ontology Consortium database (http://geneontology.org).

Statistical analysis was performed using Prism 9 software (GraphPad, San Diego, CA, USA). Data are presented as mean ± standard error of the mean or median ± interquartile range. In addition, we performed an unpaired t-test or Mann–Whitney test to analyze the statistical differences between the two groups. One-way or two-way ANOVA with Tukey–Kramer multiple comparison tests were used for comparisons between multiple groups. Statistical significance level was set at P < 0.05.

The levels of histones H2A, H2B, H3, and H4 were higher in RA compared to those in OA synovial fluid samples, though the difference in H2B was not statistically significant ( Figure 1A ). We tested whether extracellular histones could aggravate inflammatory arthritis. When mice were induced for STA, co-injection of histone aggravated arthritis ( Figure 1B ). Histopathological analyses also showed that histones increased synovitis, pannus formation, and erosion of bone and cartilage ( Figures 1C, D ). These results showed that histones have a deleterious effect on arthritis.

We investigated if the extracellular histones could induce cell injury and death in synoviocytes and macrophages. We observed that histones increased cytotoxicity in MH7A (synoviocytes) and PMA-differentiated THP-1 (macrophages) cells, as visualized by SYTOX Green dead cell staining ( Figures 2A, B ; Supplementary Movies 1A and B ). Notably, histone-induced lytic cell death was more rapid and prominent in THP-1 cells than in MH7A cells ( Supplementary Figures S1, S2 ).

Synovial macrophages are one of the most abundant inflammatory cell types in the synovium of RA patients, and activated macrophages produce various cytokines and chemokines to recruit immune cells (26, 27). To investigate the mechanism of histone-induced cytotoxicity and its consequence in inflamed joints, we determined the level of ATP, one of the molecules released in cell damage. Extracellular ATP, which can act as a DAMP, was increased by histones in both MH7A cells and THP-1 cells ( Figure 2C ). In contrast, IL-1β was induced by histones only in THP-1 cells, not in MH7A cells ( Figure 2D ). The level of HMGB1, a well-studied DAMP, was also increased by histones in THP-1 cells ( Figure 2E ). We also measured the level of TNF-α when THP-1 cells were treated by histones with or without LPS. The increased level of TNF-α under histone-treatment demonstrated that histones cause a pro-inflammatory effect that is additive with LPS ( Figure 2F ).

Histones exhibit cytotoxicity by binding to cells through their strong positive charge (28). Since anionic polysaccharides and peptides neutralize the cytotoxicity of histones (29), we tested if chondroitin sulfate, one of the charged molecules in synovial fluid, could attenuate histone-induced cytotoxicity. Histone-induced cytotoxicity and HMGB1 production were strongly inhibited by chondroitin sulfate ( Figures 3A, B ).

We also analyzed the effect of histones on cells treated with LPS. SYTOX Green staining of THP-1 cells revealed that histone-induced cytotoxicity increased when cells were treated with LPS and histones than with histones alone ( Figure 3C ). To verify the electrostatic interaction during the inflammatory process of THP-1 cells, we evaluated the binding of positively charged poly-L-lysine to cells when they were stimulated by LPS. When treated with LPS, increased poly-L-lysine binding was observed ( Figure 3D ), and treatment with sodium perchlorate, a sulfation inhibitor, reduced the cytotoxicity ( Figure 3C ).

We investigated the classes of histones to reveal those that induce cytotoxicity. When THP-1 cells were treated with each histone, all histones induced cytotoxicity ( Figure 4A ), and all significantly increased HMGB1 production except for H4 ( Figure 4B ). As the recombinant histones were purified from E. coli, the levels of LPS in histones were measured and a significant amount of LPS was detected in H2B and H3 ( Supplementary Figure S3 ). Since the LPS content of each histone was not proportional to the levels of LDH or HMGB1, LPS is not the major source of the cytotoxicity of the histones. However, the additive effect of the contaminated LPS in varying amounts should be considered. Referring to the previously studied histone structure (30, 31), we tested the cytotoxicity of α1 domain of each protein, H2A, H2B, H3, and H4 ( Table 1 ). It has been suggested that histones bind to phospholipid-phosphodiester bonds using their DNA-binding α1 helices (32). When cells were treated with the α1 domain peptide of each histone, H2B-α1 peptide strongly induced cytotoxicity and HMGB1 production in THP-1 cells ( Figures 4C, D ). Since the binding of histones can be altered by modification of their positive charge, we replaced the positively charged arginines in H2B-α1 with citrullines ( Table 1 ). Citrullination of H2B-α1 inhibited H2B-α1-induced cytotoxicity and HMGB1 production ( Figures 4E, F ).

To examine the cellular effects of histones, we analyzed the RNA-seq data of THP-1 cells treated with native or citrullinated H2B-α1 peptide. The clustergram representing the normalized expression of DEGs is shown ( Figure 5A ). Compared to THP-1 cells treated with H2B-α1 peptide, the transcriptome of the cells treated with citrullinated H2B-α1 peptide did not shift considerably from the expression pattern of the non-treated control cells. Applying principal component analysis (PCA) on 1420 DEGs, we verified two principal components, PC1 and PC2, explaining 76.9% and 14.5% of the variances, respectively ( Figure 5B ). In the two-dimensional PC space, representations of three replicates of each treatment group were exclusively distributed and completely separated. Transcriptome data was summarized as a Venn diagram ( Figure 5C ).

Gene ontology and pathway analysis were performed to examine the molecular pathways related to the effect of native histones. The most enriched Reactome pathways are listed in Figure 5D , and for each pathway, the top DEGs are shown based on the false discovery rate (FDR) and expression fold change ( Figure 5E and Supplementary Figures S4A–K ) (33). H2B-α1 peptide stimulation changed the gene expression pattern of THP-1 cells involving chemokines, IL-1, -4, -10, -13 signaling, and TLR signaling pathways. Citrullination of positively charged arginines of cytotoxic H2B-α1 peptide decreased histone-induced proinflammatory gene expression. The histone-induced expression of CCL4, one of the DEGs with the largest fold change, was demonstrated in THP-1 cells ( Figure 5F ). This result showed that histone toxicity was related to inflammatory and apoptosis pathways and that arginine residues in the H2B-α1 peptide play an important role in histone toxicity.