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

Abstract

Objective: Neutrophil 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.

Methods: NETs 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.

Results: We 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.

Conclusions: Our results suggest that NETs have a direct role in RA-associated bone erosion by promoting osteoclast formation.

Keywords: Autoimmune Diseases; Inflammation; Rheumatoid Arthritis.

Figure 1

Neutrophil extracellular traps (NETs) triggers osteoclast formation. Western blot analysis of carbamylated proteins in (A) spontaneously generated RA NETs, PMA-generated NETs in the absence or presence of cyanate (CN). Myeloperoxidase (MPO) was used as loading control. Representative picture of two independent experiments. CD14⁺ monocytes isolated from healthy volunteers were treated with PMA generated-NETs in the absence or presence of cyanate (CN, cNETs), spontaneously generated NETs or 50 ng/mL M-CSF, followed by 100 ng/mL of RANKL. After 24 hours or 120 hours, cells were fixed and stained with TRAP (red) and nuclei were counterstained with Hoechst (blue). (B) TRAP-positive multinucleated cells were quantified. Results are the # of TRAP+cells/per picture, mean±SEM, of four independent experiments. (C) Representative images of CD14⁺ cells that were incubated with NETs or cNETs, Scale bars are 500 μm. (D) Quantification of TRAP-positive multinucleated cells. Results are the mean±SEM of four independent experiments. (E) Representative images of CD14⁺ cells after treatment with cNETs, heat inactivated cNETs or cNETs+ proteinase inhibitor(s). Scale bars are 100 and 500 μm. (F) Representative images of CD14⁺ cells after treatment with cNETs that were digested with 10 U of DNase one or control for 24–48 hours, scale bars are 500 μm. (G) TRAP-positive multinucleated cells were quantified. Results are the mean±SEM of 4–7 independent experiments. (H) Trap-positive multinucleated cells were quantified after CD14⁺ cells were pretreated with TRAF6, Syk, Wnt or PI3K inhibitors in the presence of NETs. Results are the mean±SEM of four independent experiments. *p<0.05, ****p<0.0001. M-CSF, macrophage-colony-stimulating factor.

Figure 2

scRNA sequencing confirms activation of osteoclastogenic pathways. (A) Single cell RNA sequencing on CD14⁺ cells in the absence or presence of cNETs for 24 hours. (B) Relative expression of CD14 and osteoclast genes (SPP1, ACP5, DCSTAMP, OCSTAMP, CA2) from single cell RNA seq data, comparing untreated and cNET treated CD14⁺ cells. (C) Gene ontology (GO) enrichment analysis of upregulated transcripts in CD14+ treated with cNETs. (D) Heatmap analysis of the significantly differentially expressed genes associated with osteoclast formation between CD14⁺ cells and CD14⁺ incubated with cNETs. Each column represents an individual cell. (E, F) Quantitative PCR (qPCR) analysis for osteoclast genes (ACP5, CA2) on CD14⁺ cell in the absence or presence of NETs or cNETs for 24 hours. Results are the mean±SEM of 4–5 independent experiments. (G) Trajectory analysis of scRNA seq (CD14 and CD14+cNETs) displays three clusters based on gene expression that map to CD14 cells (blue), early gene expression (CXCL8, SPP1, CLL3) and late gene expression (CSF1, MMP9, OCSTAMP). *p<0.05.

Figure 3

Elastase regulates IL-8, a cytokine that potentiates cNET-mediated OC. (A) Elastase activity was measured in pairs of NETs generated in the presence of PMA or PMA+Cyanate (cNETs). Results are expressed as relative fluorescent units (RFU). (B) Elastase activity quantified in unmodified and carbamylated recombinant neutrophil elastase. Results are the mean±SEM of three independent experiments and samples were normalised to unmodified elastase. (C) CD14⁺ cells incubated with NETs or cNETs, with and without pretreatment with Sivelestat (50 μM). TRAP-positive multinucleated cells were quantified. Results are the mean±SEM of four independent experiments with (D) representative images. Original magnification ×20 (E, F) Levels of CXCL8 and IL-6 in supernatants of CD14⁺ cells cultured in the presence or absence of cNETs, or cNETs+Sivelestat (n=6–8). (G) CD14⁺ cells incubated with cNETs, with and without CXCL8, TRAP-positive multinucleated cells were quantified. Results are the mean±SEM of four independent experiments with (H) representative images. Original magnification ×20. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. cNETs, carbamylated neutrophil extracellular traps; PMA, phorbol 12-myristate 13-acetate.

Figure 4

Histones mediate NET-induced osteoclastogenesis via TLR4 engagement. (A) TRAP-positive, multinucleated cells (MNCs) were quantified after incubation of CD14⁺ cells treated with cNETs in the absence or presence of a TLR4 inhibitor. (B) Representative picture of CD14⁺ monocytes incubated with cNETs in the absence or presence of anti-histone H3 or H4 neutralising antibodies for 24–48 hours. Scale bars are 100 μm (C) TRAP-positive, MNCs were quantified and for (A, C) results are the mean±SEM of 4–5 independent experiments. (D) SEAP activity of TLR4-reporter HEK cells incubated with spontaneously generated RA NETs, PMA-NETs, cNETs. (E) Pairs of NETs and cNETs from the same donor (n=3) or (F) recombinant histones H3 or carbamylated histone H3 (carH3) for 24 hours (n=9). Results are expressed as SEAP activity. Lipopolysaccharide (LPS) was used as positive control and R848 was used as negative control. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. cNETs, carbamylated neutrophil extracellular traps; PMA, phorbol 12-myristate 13-acetate; RA, rheumatoid arthritis; SEAP, secreted embryonic alkaline phosphatase.

Figure 5

Osteoclasts were increased in DRB1*04:01 (DR4) transgenic mice that receive intra-articular injections of carbamylated NETs. (A) Representative TRAP-stained sections of joint tissues from cNET or vehicle-treated mice (n=6 or 7). TRAP-positive cells (purplish to dark red). (B) TRAP+ osteoclasts were quantified in animals immunised with cNETs (n=4–5 per group). (C) Representative pictures of resorption pit assay of equal number of cNET-generated osteoclasts (OC) in the presence or absence 100 ng of RANKL in a calcium-phosphate plate. (D) Percentage of eroded surface using ImageJ. Results are the mean±SEM of four independent experiments. Mann-Whitney U test was used. (E) RANKL mRNA expression in CD4 T cells in the presence of NET and cNETs after 4 hours incubation. Results are the mean±SEM of four independent experiments. Kruskal-Wallis test was used. (F) Plasma membrane RANKL was quantified by flow cytometry in CD4 T cells treated with NETs or cNETs for 24 hours. *p<0.05, **p<0.01, ***p<0.001. cNETs, carbamylated neutrophil extracellular traps.

Figure 6

Carbamylated histones are associated with bone resorption and inflammation in patients with RA. (A, B) Plasma levels of carbamylated histone H3 (car-H3) and histone H4 (car-H4) were measured in healthy control (n=10) and patients with RA (n=32). Correlation between plasma levels of carbamylated histone H3 (car-H3), carbamylated histone 4 (car-H4) and (C, D) bone resorption activity measured by N-Telopeptide Cross-link (NTx) in patients with RA or (E, F) levels of Rheumatoid factor (RF). (G) Correlation between Rf and N-Telopeptide Cross-link (NTx) in patients with RA. Correlation between plasma levels of carbamylated histone H4 (car-H4) and (H) C reactive protein (CRP) and (I) levels of IL6 measured in patients with RA. R is Pearson correlation coefficient. (J, K) Levels of carbamylated histone H3 (car-H3) and histone H4 (car-H4) were measured in synovial fluids from osteoarthritis (OA, n=10) and patients with RA (n=20). Results are expressed as OD index and are the mean±SEM Mann-Whitney U test was used. **p<0.01, ***p<0.001, ****p<0.0001. RA, rheumatoid arthritis.

Figure 7

Schematic representation of the mechanism of carbamylated NET-induced osteoclastogenesis and bone destruction in RA. Infiltrating neutrophils release NETs containing carbamylated histones and neutrophil elastase. Carbamylated histone H3 engages TLR4 leading to an intracellular signalling that causes osteoclast formation. NET-associated neutrophil elastase regulates the release of IL-8, which potentiates the formation of osteoclasts. Carbamylated NETs activate synovial T cells to release RANKL and activate NET-generated osteoclasts, which leads to increase bone resorption. Diagram was created with BioRender. NETs, neutrophil extracellular traps; RA, rheumatoid arthritis; RANKL, receptor activator of nuclear factor kappa-B ligand.