Anti‐Carbamylated Protein Antibodies Stabilize Carbamylated Histone H3 to Promote Synovial Activation and Neutrophil Extracellular Trap‐Mediated Bone Loss in Rheumatoid Arthritis

Abstract

Objective

Carbamylation, a nonenzymatic post‐translational modification, contributes to rheumatoid arthritis (RA) pathogenesis. Anti‐carbamylated protein antibodies (anti‐CarP) occur in around 50% of patients with RA and associate with greater joint damage. Neutrophil extracellular traps (NETs) are a major source of carbamylated autoantigens. We sought to study how carbamylation and anti‐CarP antibodies promote joint pathology.

Methods

NETs were generated with calcium ionophore and analyzed for carbamylation by immunofluorescence, flow cytometry, and mass spectrometry. Enzymatic pathways were interrogated using pharmacologic inhibitors and peptidyl arginine deiminase (PAD)2/4 deficient mice. Degradation kinetics of carbamylated histone H3 (carH3) were evaluated with neutrophil elastase (NE) and anti‐CarP IgG from patients with RA. Toll‐like receptor 4 (TLR4) activation was quantified using a HEK293 reporter line. RA fibroblast‐like synoviocytes (FLS) were stimulated with carH3 or carH3‐IgG immune complexes (ICs) and profiled by RNA‐sequencing and enzyme‐linked immunosorbent assay.

Results

Histone carbamylation, especially of H3, occurred during NETosis and was independent of peptidylarginine deiminase‐4, myeloperoxidase, NE, or oxidative pathways. CarH3 was rapidly degraded by NE, but anti‐CarP antibodies protected it from proteolysis. NETs exposed to anti‐CarP IgG triggered stronger TLR4 activation and enhanced osteoclastogenesis. In FLS, carH3 and particularly carH3‐ICs induced robust up‐regulation of pro‐inflammatory genes and cytokine secretion (interleukin [IL‐]6, IL‐8, MCP‐1, GM‐CSF). Transcriptomic analysis revealed enrichment of immune activation, chemotaxis, and angiogenesis pathways, most pronounced with carH3‐IC.

Conclusion

Anti‐CarP antibodies stabilize carH3 within NETs, amplifying its pro‐inflammatory and osteoclastogenic activity. The resulting carH3‐IgG ICs potently activate FLS, linking humoral immunity to tissue inflammation and bone destruction in RA. These findings identify carH3 stabilization as a potential therapeutic target in antibody‐mediated joint damage.

INTRODUCTION

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation, cartilage destruction, and periarticular bone erosion that affects approximately 1% of the global population. ¹ A hallmark of RA is the presence of autoantibodies targeting post‐translationally modified (PTM) proteins, most notably anti‐citrullinated protein antibodies (ACPAs), which can precede clinical onset by years and correlate with disease severity. ¹ , ² In addition to citrullination, carbamylation, which is a nonenzymatic modification of lysine residues mediated by cyanate, has emerged as another critical PTM in RA. Approximately 40% to 50% of patients with RA develop anti‐carbamylated protein (anti‐CarP) antibodies, and their presence associates with greater joint damage, bone erosion, and death. ² , ³ , ⁴ , ⁵

Neutrophils, which are abundant in RA synovial tissue, play a central role in both inflammation and tissue injury. ⁶ , ⁷ Through the formation of neutrophil extracellular traps (NETs), which are web‐like structures composed of DNA, histones, and granule enzymes such as myeloperoxidase (MPO) and neutrophil elastase (NE), neutrophils externalize modified autoantigens, including citrullinated and carbamylated proteins. ⁸ , ⁹ Excessive NET formation or defective clearance has been implicated in autoimmune pathology, including RA.

We and others have shown that NETs are enriched in RA synovial lesions and contain carbamylated proteins capable of stimulating both innate and adaptive immune responses. ³ , ⁴ , ⁷ , ⁹ Carbamylated NETs can promote osteoclastogenesis, driving bone resorption independently of the canonical RANKL pathway through Toll‐like receptor 4 (TLR4)‐dependent activation by NET‐derived DNA, histones, and NE. ¹⁰

Despite these insights, key questions remain regarding how carbamylated proteins within NETs are generated, stabilized, and rendered immunogenic. Although MPO‐mediated cyanate production likely contributes to protein carbamylation, the role of NE in processing and degrading carbamylated NET proteins, particularly histones, remains unclear. We hypothesized that anti‐CarP antibodies may stabilize carbamylated histones by shielding them from NE‐mediated degradation, thereby sustaining proinflammatory and osteoclastogenic signaling in RA.

In this study, we demonstrate that anti‐CarP antibodies protect carbamylated histone H3 (carH3) from elastase–mediated degradation, promoting the persistence of carH3–IgG immune complexes (ICs) that enhance fibroblast activation and osteoclast differentiation. These findings define a novel mechanism linking neutrophil‐derived PTMs, humoral immunity, and bone destruction in RA, and reveal previously unrecognized mechanisms by which anti‐CarP antibodies amplify osteoimmune pathology.

MATERIALS AND METHODS

Human study population

Participants diagnosed with RA fulfilled the 2010 classification criteria established by the American College of Rheumatology. ¹¹ , ¹² All participants provided written informed consent and were enrolled in a study protocol approved by the Ethics Committee of the Instituto Nacional de Ciencias Médicas y de la Nutrición Salvador Zubirán (INCMNSZ, reference number 1243). Healthy control participants were recruited through public advertisement. Peripheral blood was collected into EDTA tubes and samples were fractionated by Ficoll‐Paque Plus (GE Healthcare) density gradient centrifugation. Neutrophils were subsequently isolated by dextran sedimentation followed by hypotonic lysis of erythrocytes, as described in past protocols. ⁶ , ⁹

Isolation of mouse neutrophil

Mouse neutrophils were isolated from bone marrow extracted from femurs and tibias using a negative selection approach with the Mouse Neutrophil Isolation Kit (Miltenyi Biotec). Breeding pairs of PAD2/4 double‐knockout (DKO) mice were provided by Dr Scott Coonrod (Cornell University), whereas wild‐type (WT) (C57BL/6J) and MPO‐deficient mice were obtained from The Jackson Laboratory. All animals were maintained under specific pathogen‐free conditions. Experimental procedures adhered to the guidelines set forth by the National Institutes of Health (NIH) and were approved by the Animal Care and Use Committee of the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) under protocol number A022‐08‐03.

Histone purification

Neutrophil histones were purified using the Histone Purification Mini Kit (Active Motif) following the manufacturer's protocol.

Immunofluorescence analysis

Immunofluorescence analysis was performed as previously described. ⁹ In brief, neutrophils were fixed overnight in 4% paraformaldehyde at 4°C, washed, permeabilized by 0.2% Triton‐X100 for 10 minutes (for intracellular carbamylated protein staining), and blocked with 0.2% porcine gelatin for 30 minutes. Cells were then incubated with primary antibodies for 1 hour at 37°C in a humid chamber, which was followed by secondary antibody incubation for 30 minutes. Nuclei were counterstained with Hoechst (1:1000) and after final washes, coverslips were mounted using ProLong Gold (Invitrogen). Images were captured using a Leica microscope.

Mass spectrometry

Detection of carbamylation was done as previously described. ⁹ Briefly, extracted histones from neutrophils treated with calcium ionophore were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE), stained with Coomassie blue, and processed for in‐gel trypsin digestion. The resulting peptides were analyzed by nanoLC‐MS/MS using an Ultimate 3000 system coupled to an Orbitrap Lumos Tribrid mass spectrometer. Peptides were separated on an EASY‐Spray C18 column with a 65‐minute acetonitrile gradient. MS data were acquired in data‐dependent mode and processed using Proteome Discoverer (v2.4) with Byonic for identification of carbamylated and citrullinated peptides, applying strict search parameters and an false discovery rate (FDR) threshold of less than 2%. Neutral loss of isocyanic acid from carbamylated peptides was also examined.

Statistical analysis

Statistical analyses and graph generation were performed using GraphPad Prism 9.5.1 (GraphPad Software). Data are expressed as mean ± SEM. Normality was assessed with the Shapiro‐Wilk test. Depending on data distribution, comparisons were made using two‐tailed unpaired t‐tests or nonparametric tests (Mann‐Whitney for two groups; one‐way analysis of variance or Kruskal‐Wallis with post hoc analysis for multiple groups). Exact P values are provided in the figure legends. Data will be shared upon request.

RESULTS

Neutrophil histone carbamylation during NETosis is independent of PAD, MPO, and oxidative pathways

We previously demonstrated that NETs from activated neutrophils of patients with RA contain carbamylated proteins. ⁹ Here, we examined whether carbamylation occurs in resting neutrophils, how it changes after activation, and which enzymatic pathways contribute to this process.

Immunofluorescence of unstimulated peripheral blood human neutrophils revealed a nuclear carbamylation signal predominantly localized to euchromatin (decondensed) regions (Figure 1A). Following calcium ionophore (A23187) stimulation, nuclear carbamylation increased markedly and colocalized with DNA throughout the nucleus (Figure 1B). Time‐course imaging showed that carbamylation peaked before full NET release and declined as chromatin was expelled (Figure 1C), indicating that carbamylation is an intrinsic neutrophil feature that diminishes during NET extrusion.

Figure 1.

Intrinsic carbamylation of histones in neutrophils. Immunofluorescence of purified neutrophils from healthy donors at steady state (A) and after two hours of stimulation with calcium ionophore A23187 (B). Images were acquired at 63× original magnification. (C) Time‐course analysis of histone carbamylation in neutrophils stimulated with calcium ionophore. Red, carbamylated lysines; blue, DNA. Images are representative of four independent experiments and were acquired at 40× original magnification. (D) Mass spectrometry of isolated neutrophil nuclei following calcium ionophore stimulation. Red marks indicate carbamylated lysines and their peptide positions. (E) Flow cytometry quantification of carbamylation‐positive neutrophils at baseline and after stimulation with calcium ionophore. Data represent mean ± SEM from five independent experiments; statistical significance assessed by Mann‐Whitney U test. (F) Flow cytometric analysis of neutrophils pretreated with inhibitors of PAD (Cl‐amidine), MPO (4‐ABAH), or NE (alvelestat) followed by stimulation with calcium ionophore. Data represent the mean ± SEM from five independent experiments. Mann‐Whitney U test was used for statistical analysis. (G) and (H) Immunofluorescence of bone marrow neutrophils from WT, PAD2/PAD4 DKO, or MPO KO mice following stimulation with calcium ionophore. Histone carbamylation and/or citrullination were assessed. Images are representative of three independent experiments. Images were acquired at 20× original magnification. (I) Representative immunofluorescence images of human neutrophils stimulated with calcium ionophore in the presence or absence of Cl‐amidine, 4‐ABAH, or alvelestat, showing histone carbamylation. 4‐ABAH, 4‐aminobenzoic acid hydrazide; carLys, carbamylated lysine; DKO, double knockout; MPO, myeloperoxidase; NE, neutrophil elastase; PAD, peptidylarginine deiminase; WT, wild‐type.

Mass spectrometry identified multiple carbamylated histone peptides, particularly histone H3, confirming histones as major carbamylation targets in NETs (Figure 1D). Flow cytometry demonstrated that approximately 10% of neutrophils were carbamylation‐positive at baseline, rising to around 20% after ionophore stimulation (Figure 1E).

To determine whether certain peptidyl arginine deiminases (PAD2/PAD4) mediate this modification, we used both pharmacologic and genetic approaches. Treatment with the pan‐PAD inhibitor Cl‐amidine did not affect carbamylation frequency (Figure 1F). Likewise, bone‐ marrow neutrophils from PAD2/PAD4 DKO mice exhibited robust carbamylation after ionophore stimulation. In contrast, citrullinated histone H3 was increased in WT but absent in PAD2/4‐deficient neutrophils, confirming antibody specificity and the distinct nature of the two PTMs (Figure 1G). Thus, histone carbamylation occurs independently of PAD activity and is mechanistically distinct from citrullination.

Because MPO and NE are key drivers of NET formation, we next tested whether they influence carbamylation. Inhibiting MPO (4‐aminobenzoic acid hydrazide [4‐ABAH]) or NE (alvelestat) during calcium ionophore stimulation did not reduce carbamylation (Figure 1F and I). Similarly, neutrophils from MPO‐deficient mice retained carbamylation capacity (Figure 1H), indicating that MPO is dispensable. Inhibition of NADPH oxidase with diphenyleneiodonium or the antioxidant treatment with N‐acetylcysteine likewise had no effect on carbamylation (Supplementary Figure 1). Collectively, these results demonstrate that nuclear histone carbamylation is an intrinsic neutrophil process that increases during NET formation and occurs independently of PAD enzymes, MPO, NE, and oxidative pathways.

Anti‐CarP antibodies protect carbamylated histone H3 from neutrophil elastase–mediated degradation

Because histone H3 emerged as a principal target of carbamylation and carbamylation levels declined during NET formation, we next examined the stability of carH3. Neutrophils were stimulated with calcium ionophore, and purified histones were analyzed by Western blot. Immunoblotting with an anti‐carbamylation antibody revealed a loss of intact carH3 (15–20 kDa) and the appearance of a lower MW fragment (~10 kDa), consistent with proteolytic cleavage. Probing for total histone H3 confirmed this degradation pattern (Figure 2A).

Figure 2.

Anti‐CarP antibodies protect carbamylated histone h3 from neutrophil elastase–mediated degradation. (A) Western blot analysis of total histone H3 and carbamylated histone H3 (carH3) from neutrophils stimulated with calcium ionophore in the presence or absence of MPO (4‐ABAH) and/or NE (alvelestat) inhibitors. A ~10 kDa band indicates degraded histone fragments, whereas bands around 15–20 kDa represent intact histone H3. (B) Time‐course degradation assay of recombinant histone H3 and carH3 in vitro, with or without anti‐carbamylated lysine antibodies and/or recombinant NE. (C) Representative Western blot showing degradation of in vitro carH3 by recombinant NE in the presence of purified IgG from anti‐CarP⁺ (n = 7) or anti‐CarP⁻ (n = 6) patients with RA. Densitometric quantification of histone remnants is shown in the panel to the right. Data represent the mean ± SEM. Mann‐Whitney U test was used for statistical analysis. *P < 0.05. (D) Western blot analysis of histones from calcium ionophore‐induced NETs incubated with purified IgG from anti‐CarP⁺ (n = 7) or anti‐CarP⁻ (n = 6) patients with RA. Densitometry of histone remnants is shown to the right. Data represent the mean ± SEM. Mann‐Whitney U test was used for statistical analysis. *P < 0.05. (E) Representative immunofluorescence images of NETs generated with calcium ionophore in the presence or absence of IgG from anti‐CarP⁺ or anti‐CarP⁻ patients with RA. Histones are stained green; DNA is stained in blue. Images are representative of three independent experiments and acquired at 40× original magnification. 4‐ABAH, 4‐aminobenzoic acid hydrazide; anti‐CarP, anti‐carbamylated protein; carH3, carbamylated histone H3; carLys, carbamylated lysine; MPO, myeloperoxidase; NE, neutrophil elastase; NETs, neutrophil extracellular traps; RA, rheumatoid arthritis.

To identify the protease responsible, we inhibited MPO and NE using 4‐ABAH and alvelestat, respectively. MPO inhibition had no effect, whereas NE inhibition markedly reduced carH3 degradation, preserving the intact 20–15 kDa band and reducing the 10 kDa fragment (Figure 2A), implicating NE in carH3 proteolysis during NET formation.

A time‐course comparison of unmodified and carH3 exposed to NE further demonstrated that carH3 was degraded rapidly, within one minute, whereas unmodified histone H3 persisted for up to 60 minutes (Figure 2B). Pre‐incubation of carH3 with an anti‐carbamylated lysine (carLys) antibody delayed degradation, with intact protein detectable up to 10 minutes, suggesting that masking carbamylated lysines can partially block NE recognition (Figure 2B). Similarly, carbamylated histone H4 was more susceptible to NE‐mediated degradation than its unmodified counterpart (Supplementary Figure 2).

To determine whether anti‐CarP antibodies from patients with RA exert similar protective effects, recombinant carH3 was incubated with IgG purified from anti‐CarP⁺ or anti‐CarP⁻ RA sera. CarH3 was significantly protected from degradation in the presence of anti‐CarP⁺ IgGs (Figure 2C). Finally, when neutrophils were stimulated with calcium ionophore in the presence of IgGs from anti‐CarP⁺ or anti‐CarP⁻ in patients with RA, NETs generated with anti‐CarP⁺ IgGs contained higher levels of intact histone H3 than those incubated with anti‐CarP⁻ IgG (Figure 2D and E). IgGs isolated from anti‐CarP⁻ patients exhibited elevated ACPA titers (see Supplementary Table 1), indicating that the effects observed in these experiments are unlikely to be confounded by ACPAs.

Collectively, these findings demonstrate that carbamylation increases histone susceptibility to NE‐mediated degradation and that anti‐CarP antibodies can stabilize carbamylated histone H3 both in vitro and during NET formation, preserving it as a potential autoantigen within the RA inflammatory environment.

Anti‐CarP antibodies enhance the pro‐osteoclastogenic and pro‐inflammatory effects of carbamylated histone H3

We previously showed that levels of carH3 are elevated in RA and correlate with anti‐CarP antibody titers. ⁹ , ¹⁰ Carbamylated NETs (cNETs) promote rapid osteoclastogenesis through carH3‐dependent TLR4 activation. ¹⁰ Based on these observations, we hypothesized that anti‐CarP antibodies stabilize carH3, protecting it from degradation and enhancing its ability to activate TLR4 and drive bone resorption.

To test this, purified NETs were incubated with IgG from patients with RA who were positive or negative for anti‐CarP antibodies. NETs exposed to anti‐CarP⁺ IgG induced significantly greater TLR4 activation in a HEK‐Blue reporter assay compared with NETs treated with anti‐CarP⁻ IgG (Figure 3A), consistent with antibody‐mediated protection of carH3 from elastase degradation.

Figure 3.

Carbamylated histone H3–IgG ICs trigger osteoclastogenesis and inflammatory responses in FLS. (A) SEAP‐TLR4 reporter assay measuring TLR4 activation by calcium ionophore‐induced NETs incubated with purified IgG from anti‐CarP⁺ or (n = 7) or anti‐CarP⁻ (n = 6) patients with RA. Results are shown as mean ± SEM. Mann‐Whitney U test was used; P < 0.01. (B) Quantification of TRAP‐positive osteoclasts by colorimetric assay (OD at 540 nm) following incubation of CD14⁺ monocytes with cNETs and purified IgG from anti‐CarP⁺ or anti‐CarP⁻ patients with RA (n = 4–8). Data are presented as mean ± SEM. Mann‐Whitney U test was used; *P < 0.05, ***P < 0.001. (C) Quantitative PCR analysis of IL‐6 and IL‐8 mRNA levels in OA FLS treated for 24 hours with carH3 or carH3–IgG ICs (n = 4 per group). Kruskal‐Wallis test was used; *P < 0.05, **P < 0.01. (D) Heatmap showing differentially expressed genes in OA FLS following treatment with carH3 or carH3–IgG ICs for 24 hours. (E) Gene Ontology enrichment analysis of differentially expressed genes in OA FLS treated with carH3. (F) Dot plot showing significantly enriched biologic pathways in OA FLS treated with carH3–IgG ICs (adjusted P value <0.05). (G) Dot plot comparing enriched biologic pathways between OA FLS treated with carH3 alone vs carH3–IgG ICs (adjusted P value <0.05). (H) Network diagram representing up‐regulated genes and pathways in OA FLS stimulated with carH3–IgG ICs. (I) ELISA quantification of IL‐8, IL‐6, MCP‐1, and GM‐CSF levels in the supernatant of OA FLS treated with carH3 or carH3–IgG ICs (n = 4 per group). Kruskal‐Wallis test was used; *P < 0.05, **P < 0.01, ***P < 0.001. anti‐CarP, anti‐carbamylated protein; cNETs, carbamylated neutrophil extracellular traps; ELISA, enzyme‐linked immunosorbent assay; FLS, fibroblast‐like synoviocytes; ICs, immune complexes; IL, interleukin; mRNA, messenger RNA; NETs, neutrophil extracellular traps; OA, osteoarthritis; OD, optical density; PCR, polymerase chain reaction; RA, rheumatoid arthritis; SEAP, secreted embryonic alkaline phosphatase; TLR4, Toll‐like receptor 4; TRAP, tartrate‐resistant acid phosphatase.

We next evaluated the functional consequence of this effect on osteoclastogenesis. CD14⁺ monocytes cultured with cNETs differentiated into osteoclasts, an effect significantly amplified by the presence of purified IgG from anti‐CarP⁺ patients with RA and reduced with exposure to anti‐CarP⁻ IgG (Figure 3B). These findings suggest that anti‐CarP antibodies potentiate cNET‐driven osteoclast differentiation, likely through stabilization of carH3 and enhanced TLR4 signaling.

To assess the impact of carH3 on fibroblast‐like synoviocytes (FLS) activation, we stimulated osteoarthritis‐derived FLS (OA FLS), chosen for their immunologically naïve baseline, with carH3 or carH3–IgG ICs. Quantitative polymerase chain reaction analysis showed induction of IL6 and IL8 by carH3, which was further potentiated by carH3‐IgG ICs (Figure 3C). Similar changes were also observed in RA‐derived FLS at 24 hours, although with a lesser magnitude, which we believe reflects their pre‐activated or “primed” state (Supplementary Figure 3A). RNA‐sequencing profiling confirmed broad transcriptional activation by both stimuli relative to unstimulated controls (Figure 3D). CarH3 up‐regulated pro‐inflammatory (IL6, TNFAIP3, NFKBIZ), chemotactic (CXCL1, CXCL5, and CXCL8) and matrix‐remodeling (MMP1, PTGS2) genes, whereas carH3‐IC elicited a stronger response, further enhancing expression of IL6, CXCL13, TNFAIP6, and additional CXCL/CCL chemokines (Figure 3D).

Gene ontology analysis revealed that carH3 stimulation enriched for pathways related to innate immune activation and granulocyte migration (Figure 3E), whereas carH3‐IC additionally activated antimicrobial, inflammatory, leukocyte migration, and lipid‐response pathways (Figure 3F). Direct comparison showed that IC formation amplified networks linked to immune modulation, angiogenesis, and antiviral responses (Figure 3G and H). These transcriptional changes translated into enhanced cytokine secretion, with enzyme‐linked immunosorbent assay demonstrating increased release of interleukin (IL)‐6, IL‐8, MCP‐1, and GM‐CSF in OA FLS, most prominently in response to carH3–IC (Figure 3I). IL‐6 and IL‐8 were also significantly elevated in RA‐derived FLS treated with carH3–IgG complexes, indicating that the effect extends to pre‐activated, disease‐relevant synovial fibroblasts (Supplementary Figure 3B).

Together, these results indicate that anti‐CarP antibodies amplify the inflammatory and osteoclastogenic effects of carH3. By stabilizing carH3 and promoting the formation of carH3–IgG ICs, they potentiate TLR4‐dependent signaling, FLS activation, and cytokine production, thereby linking humoral autoimmunity to joint inflammation and bone destruction in RA.

DISCUSSION

RA is characterized by chronic synovial inflammation and progressive bone erosion, with neutrophils playing a central role, particularly in the early stages of disease. ¹ Although neutrophils have traditionally been viewed as transient amplifiers of inflammation, their direct contribution to bone destruction has been less clearly defined. Our findings reveal a previously unrecognized mechanism linking NETs, PTMs, and humoral autoimmunity in RA. Specifically, we show that anti‐CarP antibodies stabilize carH3 within NETs, protecting it from NE‐mediated degradation and thereby amplifying its proinflammatory and osteoclastogenic functions.

This stabilization mechanism expands the functional repertoire of autoantibodies beyond their classical role in IC formation. By preserving their cognate antigen, anti‐CarP antibodies sustain innate immune activation and extend the lifespan of NET‐derived autoantigens. In doing so, they transform NET components from transient byproducts of neutrophil death into persistent inflammatory drivers, thereby linking antibody specificity directly to the perpetuation of joint damage.

We previously identified carH3 as a prominent NET‐associated antigen elevated in patients with RA that promotes osteoclast differentiation through TLR4 activation. ¹⁰ Here, we uncover an additional layer of regulation, showing that anti‐CarP antibodies functionally potentiate this process by preventing NE‐mediated degradation. As a result, carH3 persists longer within the inflammatory microenvironment, maintaining its capacity to engage TLR4 and promote osteoclast differentiation. This suggests that anti‐CarP antibodies not only serve as biomarkers of autoimmune reactivity but also act as amplifiers of joint inflammation and bone destruction by stabilizing immunostimulatory PTMs within NETs.

Functionally, this stabilization may have key consequences for RA pathogenesis. We previously demonstrated that carbamylated NETs induce a rapid, RANKL‐independent form of osteoclastogenesis driven by TLR4 signaling. ¹⁰ Here, we show that protection of carH3 by anti‐CarP antibodies further enhances TLR4 activation and osteoclast differentiation. Monocytes exposed to NETs in the presence of anti‐CarP⁺ IgG undergo more robust osteoclastogenesis, whereas FLS stimulated with carH3–IgG ICs up‐regulate IL‐6, IL‐8, MCP‐1, and GM‐CSF, adopting a proinflammatory, chemotactic, and matrix‐remodeling phenotype. Together, these findings position anti‐CarP antibodies not only as disease markers but also as functional effectors that sustain and amplify NET‐driven inflammation and tissue destruction.

Our findings also underscore the broader immunogenic complexity of PTM within NETs. ² , ⁵ The co‐existence of native, citrullinated, and carbamylated proteins likely contributes to spreading epitope and the diversity of autoantibody responses in RA. Although anti‐CarP and ACPA antibodies display limited cross‐reactivity, they may cooperate to intensify inflammation and tissue damage, particularly when localized within the same NET structures. ⁴ , ⁵ Unlike ACPAs, which promote osteoclastogenesis primarily via Fcγ receptor– and RANKL‐dependent pathways, ² anti‐CarP antibodies appear to act through Fc‐independent stabilization of NET antigens and TLR4‐mediated signaling. These complementary mechanisms may help explain the additive pathogenicity observed in patients harboring both ACPA and anti‐CarP responses.

This work also highlights the technical and conceptual challenges in dissecting PTM biology in vivo. Unlike citrullination, which is enzymatically mediated by PADs, carbamylation is a nonenzymatic process with no known specific catalyst or inhibitor. The absence of selective tools to modulate carbamylation or neutralize carbamylated histones has limited functional dissection of these pathways. Future approaches such as site‐specific PTM mapping, advanced proteomics, and targeted cyanate scavenging strategies could clarify the temporal and spatial interplay of PTMs in RA and other autoimmune settings.

Beyond the neutrophil compartment, the concurrent activation of other synovial components suggests that anti‐CarP–carH3 interactions may orchestrate multicellular inflammatory circuits within the RA synovium. These interactions may contribute to persistent inflammation, angiogenesis, and tissue remodeling, which are all hallmarks of chronic joint pathology. The persistence of carbamylated antigens stabilized by autoantibodies provides an additional mechanistic explanation for the chronicity and self‐sustaining nature of synovial inflammation.

Taken together, our study identifies a novel immune–bone interface in RA in which anti‐CarP antibodies preserve the inflammatory potential of NETs by stabilizing carbamylated histones, prolonging TLR4 activation, monocyte‐driven osteoclastogenesis, and synovial inflammation. This model positions NETs and their modified cargo not only as triggers of autoimmunity but also as direct effectors of joint damage and tissue injury. Therapeutically, these insights suggest that disrupting the stabilization of carbamylated histones or blocking their recognition by anti‐CarP antibodies may attenuate downstream inflammatory and osteoclastogenic cascades. Similar to the development of PAD inhibitors targeting citrullination, strategies that limit cyanate formation, neutralize carbamylated proteins, or selectively degrade pathogenic NETs may hold promises for mitigating both systemic inflammation and local bone erosion in RA.

AUTHOR CONTRIBUTIONS

All authors contributed to at least one of the following manuscript preparation roles: conceptualization AND/OR methodology, software, investigation, formal analysis, data curation, visualization, and validation AND drafting or reviewing/editing the final draft. As corresponding author, Dr Carmona‐Rivera confirms that all authors have provided the final approval of the version to be published and takes responsibility for the affirmations regarding article submission (eg, not under consideration by another journal), the integrity of the data presented, and the statements regarding compliance with institutional review board/Declaration of Helsinki requirements.

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