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

Abstract

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. 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. 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. 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.