Neutrophil extracellular trap formation in anti-neutrophil cytoplasmic antibody-associated and large-vessel vasculitis

Abstract

Levels of neutrophil extracellular traps (NETs) were measured in plasma of healthy controls (HC, n = 30) and patients with granulomatosis with polyangiitis (GPA, n = 123), microscopic polyangiitis (MPA, n = 61), Takayasu's arteritis (TAK, n = 58), and giant cell arteritis (GCA, n = 68), at times of remission or activity and correlated with levels of the platelet-derived thrombospondin-1 (TSP-1). Levels of NETs were elevated during active disease in patients with GPA (p < 0.0001), MPA (p = 0.0038), TAK (p < 0.0001), and GCA (p < 0.0001), and in remission for GPA, p < 0.0001, MPA, p = 0.005, TAK, p = 0.03, and GCA, p = 0.0009. All cohorts demonstrated impaired NET degradation. Patients with GPA (p = 0.0045) and MPA (p = 0.005) had anti-NET IgG antibodies. Patients with TAK had anti-histone antibodies (p < 0.01), correlating with presence of NETs. Levels of TSP-1 were increased in all patients with vasculitis, and associated with NET formation. NET formation is a common process in vasculitides. Targeting NET formation or degradation could be potential therapeutic approaches for vasculitides.

Keywords: Anti-neutrophil cytoplasmic antibody associated vasculitis; Histone; Large vessel vasculitis; Neutrophil extracellular traps; Neutrophils; Platelet activation; Thrombospondin-1.

Figure 1.. Levels of neutrophil elastase (NE)-DNA complexes in patients with AAV and LVV.

Plasma levels of NE-DNA complexes (A) were measured by ELISA in healthy controls (HC), and patients with granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), Takayasu’s arteritis (TAK), and giant cell arteritis (GCA), both in remission (rem) and at a time of active disease (active). Plasma levels of NE-DNA complexes (B) were related to disease activity in patients in remission (rem) and matching patients with active disease (active) as assessed by physician global assessment (PGA) in MPA and TAK. Plasma levels of NE-DNA complexes correlated with CRP in GPA (C), MPA (D), TAK (E), and GCA (F). Statistical analyses were done using Mann-Whitney U test (A), Wilcoxon signed-rank test (B), Spearman’s correlation test (C-F) with * p < 0.05, **p < 0.01, ***p < 0.001. Unless otherwise indicated, all analyses are compared to healthy controls. Each circle represents an individual sample, with the bar representing the median of the group.

Figure 2.. Plasma-mediated NET degradation in patients with AAV and LVV.

(A) NETs were incubated with plasma from either healthy controls (HC, n=30) or patients with granulomatosis with polyangiitis (GPA, n=53), microscopic polyangiitis (MPA, n=49), Takayasu’s arteritis (TAK, n=51), and giant cell arteritis (GCA, n=48) in remission. Residual NETs were determined by dividing DNA content in plasma-treated NETs with untreated NETs (B) DNA degradation ability was analyzed in plasma from healthy controls and patients with vasculitis. Statistical analyses were done using Mann-Whitney U test, with * p < 0.05, **p < 0.01, ***p < 0.001. All analyses are compared to healthy controls. Each circle represents an individual sample, with the bar representing the median of the group.

Figure 3.. Levels of anti-NET and anti-histone antibodies in patients with AAV and LVV.

Plasma levels of anti-NET antibodies (A) were measure by ELISA in healthy controls (HC, n=30), in patients with granulomatosis with polyangiitis (GPA, n=50), microscopic polyangiitis (MPA, n=50), Takayasu’s arteritis (TAK, n=51) and giant cell arteritis (GCA, n=49). Immunofluorescence microscopic images showing DNA (green) and IgG (red) staining of stimulated neutrophils incubated with plasma from patients with MPA (B) and TAK (C). Circulating levels of anti-histone antibodies (D) were assessed by ELISA in HC, in patients with GPA, MPA, TAK, and GCA both in remission and at a time of active disease. Plasma levels of anti-histone antibodies were associated with NE-DNA levels in patients with TAK (E). Statistical analyses were done using Mann-Whitney U test (A, D), and Spearman’s correlation test (E) with * p < 0.05, **p < 0.01, ***p < 0.001. All analyses are compared to healthy controls. Each circle represents an individual sample, with the bar representing the median of the group. Scale bars, 400 μm.

Figure 4.. Levels of thrombospondin-1 in patients with AAV and LVV.

Plasma levels of thrombospondin-1 (TSP-1) (A) were measured by ELISA in healthy controls (HC), and patients with granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), Takayasu’s arteritis (TAK), and giant cell arteritis (GCA) both in remission and at a time of active disease. Plasma levels of TSP-1 (B) were related to disease activity in patients in remission (rem) and matching patients with active disease (active) as assessed by physician global assessment (PGA) in GPA, MPA, TAK and GCA. Plasma levels of TSP-1 correlated with NETs in GPA (C), MPA (D), TAK (E), and GCA (F). Plasma levels of TSP-1 (G) were compared among patients with GPA with and without thromboembolism (VTE). Statistical analyses were done using Mann-Whitney U test (A, G), Wilcoxon signed-rank test (B), Spearman’s correlation test (C-F) with * p < 0.05, **p < 0.01, ***p < 0.001. Unless otherwise indicated, all analyses are compared to healthy controls. Each circle represents an individual sample, with the bar representing the median of the group.