Neutrophil extracellular trap-associated carbamylation and histones trigger osteoclast formation in rheumatoid arthritis

Abstract

ObjectiveNeutrophil infiltration into the synovial joint is a hallmark of rheumatoid arthritis (RA), a disease characterised by progressive bone erosion. However, the mechanisms by which neutrophils participate in bone destruction remain unclear. Carbamylation is a posttranslational modification linked to increased bone erosion in RA and we previously showed that carbamylation is present in RA neutrophil extracellular traps (NETs). However, it remains unclear whether NETs and their carbamylated protein cargo directly promote bone destruction and alter osteoclast biology.MethodsNETs and carbamylated NETs (cNETs) were assessed for their capacity to induce osteoclast formation in CD14+ monocytes. Chemical inhibitors and neutralising antibodies were used to elucidate the pathway by which NETs induce osteoclastogenesis. HLA-DRB1*04:01 mice received intra-articular injection of cNETs for 4 weeks. Joints were isolated and assessed for osteoclast formation. Plasma and synovial fluid samples from patients with RA (n=32) were assessed for the presence of carbamylated histone, and correlations to disease specific outcomes were performed.ResultsWe found that NETs, when cNETs, instruct monocytes to undergo rapid osteoclast formation. NET-mediated osteoclastogenesis appears to depend on Toll-like receptor 4 signalling and NET-associated proteins including histones and neutrophil elastase. In vivo, we identified that the number of osteoclasts increased following immunisation with cNETs in HLA-DRB1*04:01 transgenic mice. Furthermore, carbamylated histones are increased in plasma and synovial fluid from patients with RA and correlate with active bone resorption and inflammatory markers.ConclusionsOur results suggest that NETs have a direct role in RA-associated bone erosion by promoting osteoclast formation.

Harnessing osteoimmunity to treat peri-implant inflammatory osteolysis

“…NETs secreted by neutrophils mediate TLR4 signaling and NET-associated proteins, including histones and neutrophil elastase, to induce rapid osteoclastogenesis, especially when NETs are carbamylated. 9 In conclusion, in response to stimulation by inflammatory factors such as the LPS, antigen-presenting cells like DCs activate the proliferation and differentiation of Th1 and Th17 cells. This is followed by clearance of inflammatory stimuli by trending neutrophils and macrophages but also inevitable by tissue destruction due to high expression of RANKL and MMPs.…”

“…NETs secreted by neutrophils mediate TLR4 signaling and NET-associated proteins, including histones and neutrophil elastase, to induce rapid osteoclastogenesis, especially when NETs are carbamylated. 9…”

“…7 In addition to the direct role of the RANKL-RANK-OPG axis, bone homeostasis is also indirectly and synergistically regulated by immune cells, such as macrophages, dendritic cells, and neutrophils. 8,9 In conclusion, inflammatory factors secreted by immune cells aggravate the progression of infectious periimplantitis. The process continues until inflammation subsides and bone homeostasis is restored.…”

Recent advances on neutrophil dysregulation in the pathogenesis of rheumatic diseases

“…A recent study discovered that carbamylated NETs (cNETs) can induce monocytes to undergo rapid osteoclast formation. This process is dependent on Toll-like receptor 4 signaling and NET-associated proteins, including histones and neutrophil elastase [23 ▪▪ ].…”

Neutrophilic activity biomarkers (plasma neutrophil extracellular traps and calprotectin) in established rheumatoid arthritis patients receiving biological or JAK inhibitors: a clinical and ultrasonographic study

“…These in ammatory structures are believed to have both local and systemic effects, potentially contributing to the initiation and perpetuation of the disease through autoantigen exposure [16][17][18][19][20][21]. Furthermore, NETs have been identi ed as a source of speci c autoantibodies in RA, including ACPAs and anti-carbamylated protein (anti-CarP) antibodies [22][23][24][25][26]. RA patients demonstrate elevated NETs formation compared to the general population, revealing NETs to be a promising potential biomarker for the disease [27].…”