Anti-Neutrophil Extracellular Trap Antibodies and Impaired Neutrophil Extracellular Trap Degradation in Antiphospholipid Syndrome

Abstract

Objective: The release of neutrophil extracellular traps (NETs) by hyperactive neutrophils has recently been recognized to play an important role in antiphospholipid syndrome (APS). This study was undertaken to evaluate autoantibodies targeting NETs in patients with primary APS, and to determine their potential functions and clinical associations.

Methods: We measured global anti-NET activity in 76 patients with primary APS, 23 patients with systemic lupus erythematosus without antiphospholipid antibodies (aPL), 11 patients with a history of unprovoked venous thrombosis without aPL, and 44 healthy controls. The ability of APS sera to degrade NETs was also assessed.

Results: We found markedly elevated levels of anti-NET IgG and IgM in patients with primary APS compared with healthy controls (for IgG, mean ± SD optical density 0.55 ± 0.34 versus 0.33 ± 0.17; for IgM, mean ± SD optical density 0.76 ± 0.51 versus 0.26 ± 0.23). This anti-NET activity did not correlate with levels of traditional aPL and was relatively stable over time. Mechanistically, anti-NET antibodies (especially of the IgG isotype) impaired the ability of patient sera to degrade NETs (r = 0.4, P = 0.003). Levels of anti-NET IgM inversely correlated with complement C4 (r = 0.4, P = 0.019). Clinically, anti-NET antibodies associated with certain APS clinical manifestations, and in particular recurrent venous thrombosis (odds ratio 4.3; P = 0.002). Interestingly, anti-NET antibody levels also appeared to be associated with unprovoked venous thrombosis in the general population (for IgM, mean ± SD optical density 0.67 ± 0.34 versus 0.26 ± 0.23).

Conclusion: Our data indicate high levels of anti-NET antibodies in patients with primary APS, which may impair NET clearance and activate the complement cascade. These findings may ultimately enable more effective risk stratification.

Figure 1:. High levels of anti-NET antibodies among patients with primary APS.

A-B, Anti-NET IgG and IgM were measured in individuals with primary APS, SLE without antiphospholipid antibodies, unprovoked venous thrombosis without antiphospholipid antibodies, or no known disease (healthy controls). Levels of anti-NET IgG and IgM at 450-nm optical density (OD) were compared by Kruskal–Wallis test **p<0.01, ***p<0.001, and ****p<0.0001 as compared with the control group. C, The relationship between anti-NET IgG and anti-β₂GPI IgG was assessed by Spearman’s correlation test and linear regression. D, Anti-NET IgG levels were measured in six patients with primary APS who had two to four years of follow-up in our clinic.

Figure 2:. Anti-NET antibodies decorate NETs.

Control neutrophils were stimulated with PMA to generate NETs, which were then incubated with plasma from patients with high (top panels) or low (bottom panels) anti-NET IgG; scale bars=200 microns.

Figure 3:. Anti-NET antibodies correlate with impaired NET degradation.

A, NET release was triggered from control neutrophils by PMA. Fresh NETs were then incubated with sera from patients with primary or secondary APS, or from healthy controls. Some samples were treated with Micrococcal nuclease as a positive control. Percentage of NET degradation was compared to the control group by one-way ANOVA; **p<0.01 and ****p<0.0001. B, Correlation between NET degradation and anti-NET IgG was assessed by Spearman’s correlation and linear regression. C, APS serum samples (six unique patients) were depleted of total IgG, and compared with mock-depleted samples. Paired t test was used to assess the difference in NET degradation before and after depletion; **p<0.01. D, Correlation between anti-NET IgM and complement C4 was assessed by Spearman’s correlation and linear regression. E-F, Univariate logistic regression was performed to assess the association between positive anti-NET antibodies and various clinical manifestations in 154 individuals with primary APS, SLE without antiphospholipid antibodies, unprovoked venous thrombosis without antiphospholipid antibodies, or no known disease. Odds ratio and 95% confidence intervals are presented.