Arthritis & rheumatology (Hoboken, N.J.) · 2026
Anti-Carbamylated Protein Antibodies Stabilize Carbamylated Histone H3 to Promote Synovial Activation and Neutrophil Extracellular Trap-Mediated Bone Loss in Rheumatoid Arthritis.
СтатьяНазвание и аннотация: Перевод в очереди
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Nakabo Shuichiro, Hanata Norio, Patino-Martinez Eduardo, Holewinski Ronald, Barrera-Vargas Ana, Merayo-Chalico Javier, Coonrod Scott A, Fox David A, Kaplan Mariana J, Carmona-Rivera Carmelo
Аннотация
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.
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.