Serum DNase1 degrades NETs. Human neutrophils were activated to form NETs and incubated in the indicated conditions before measurement of the digested and released NET DNA using the fluorescencent DNA dye Picogreen. (A) In the absence of serum (open bars) NETs are stable for at least 90 h in vitro. NET degradation with MNase for 10 min at each time point (black bars) represents the total NETs recovered. (B) Serum-mediated degradation of NETs is concentration and (C) time dependent when exposed to 10% serum, suggesting an enzymatic activity (open bars are medium controls). (D) Activated neutrophils that formed NETs were incubated in media with 10% serum for the indicated time points, fixed, and immunostained for myeloperoxidase (green) and histones (red). DNA (blue) was stained with Draq5. (Scale bar, 10 μm.) (E) Serum NET degradation requires calcium. NETs were incubated with 10% serum for 6 h. EGTA, a calcium chelator, inhibited degradation. Calcium, but not magnesium ions, restored the NET-degrading activity. (F) Inhibition of NET degradation by G-actin, a specific inhibitor of DNase1, is dose dependent. NETs were incubated with 10% serum for 6 h in the presence of the indicated concentrations of G-actin, and NET degradation was measured as described. (G) To control for the specificity of G-actin for DNase1, we incubated NETs with 20 μM of G-actin with either commercially available purified DNase1 or MNase to digest the NETs. G-actin blocked degradation by DNase1 but not MNase. (H) NETs were incubated with serum as described above and with the indicated concentrations of polyclonal anti-DNase1 (black bars) or irrelevant control Abs (open bars). Inhibition of NET degradation by anti-DNase1 Abs was specific and dose dependent. (I) Anti-DNase1 antibodies are specific. We incubated NETs in the presence of 40 μg/mL of anti-DNase1 Abs and incubated with purified DNase1 or MNase. The data shown are representative of experiments performed in triplicate and are presented as mean ± SD.

NET degradation is impaired in a subset of SLE patients. (A) We activated neutrophils isolated from healthy donors to make NETs, incubated them with 10% sera from our cohort for 6 h, and quantified NET degradation. The cohort is described in Materials and Methods and in Table S1. Each circle corresponds to one individual donor. The samples are grouped into healthy donors, SLE patients, and RA patients as indicated. One hundred percent NET degradation was determined using the serum from the healthy donor of the neutrophils. We arbitrarily considered sera that degrade at least 60% of the NETs within 6 h as normal (horizontal line). Sera from all healthy donors (n = 54, black circles) degraded NETs normally; 36.1% of SLE patient sera (n = 61, open circles) and 3.3% of the RA sera (n = 30, gray circles) degraded NETs poorly. ***P < 0.001; Kruskal-Wallis test with Dunn's post hoc comparisons. (B–D) NETs were exposed to representative sera (labeled B, C, or D in A), fixed and immunostained for myeloperoxidase (green) and histones (red). DNA (blue) was stained with Draq5. Representative micrographs show efficiency of NET degradation, with serum from a healthy donor (B), from an SLE patient who degraded NETs (C), and from an SLE patient who did not disassemble NETs (D). (Scale bar in D, 25 μm for B–D.)

Inhibitory mechanisms of NET degradation. (A) A subset of SLE sera contains DNase1 inhibitor(s). NETs incubated with sera from healthy donors (n = 5) or SLE patients who did not degrade NETs (n = 22) were spiked with exogenous DNase1 or MNase, and then we quantified NET degradation. Degradation of NETs by healthy sera was unaffected by the addition of the exogenous nucleases. The SLE nondegrader sera fell into two groups: in group 1, addition of MNase but not DNase1 fully restored NET degradation activity, suggesting the presence of specific DNase1 inhibitor(s). In group 2, neither DNase1 nor MNase completely restored NET degradation, suggesting a mechanism of NET protection. ***P < 0.001; **P = 0.0013; *P < 0.05; P > 0.05; ns, nonsignificant compared by Friedman's test with Dunn's post hoc comparison. The bar denotes the median of the group. Protecting Abs impair NET degradation. (B) Sera from NET degraders and nondegraders were depleted of antibodies with protein A/G Sepharose beads. The Ab-depleted sera were incubated with NETs for 6 h before quantification of NET degradation. Depletion of Abs increased the NET degradation of sera from group 1 only marginally. In contrast, sera from patients in group 2 degraded NETs efficiently after depletion. This indicates that sera from patients of group 2 contain Abs shielding NETs from degradation. ***P < 0.0001; **P = 0.0056; ns, nonsignificant using parametric paired t test, because the data followed a Gaussian distribution. Each circle in A and B represents the activity of a single serum and is the value of the mean in an experiment performed in triplicate. Bars denote the mean of the group.

Defective NET degradation correlates with high anti-NET Abs anti-dsDNA, ANA titers and increased risk of lupus nephritis. (A) Abs binding to NETs were quantified with a NET ELISA, whereby NETs are used as antigen, patient sera as primary Abs, and anti-human Cy3 coupled Abs as secondary Abs. Sera from healthy donors, from SLE degraders, or from patients with other autoimmune diseases did not contain anti-NET Abs. Most of the sera in group 2 contained high levels of anti-NET Abs. Sera in group 1, however, were heterogeneous, but as a group the concentrations of anti-NET antibodies were significantly higher than in the NET degraders. In A and B each circle represents the activity of a single serum and is the mean of an experiment performed in triplicate. Bars show the median of the group. (B) The concentrations of anti-dsDNA Abs were significantly higher in SLE nondegraders compared with degraders. (C) The titers of ANA detected by indirect immunofluorescence on fixed Hep2 cells were significantly higher in nondegraders compared with degraders. There was a significant difference between SLE degraders and group 2 but not with group 1. For A: ***, **, *P < 0.05; for B and C: ***P < 0.001; *P < 0.05; ns, P > 0.05 using Kruskal Wallis test with post hoc Dunn's multiple comparison test. Each circle in A represents the mean of a triplicate experiment with patient serum. Bar denotes the median of the group. (D) We retrospectively analyzed the association between ineffective NET degradation with nephritis. There was a higher incidence of nephritis in SLE nondegraders than in degraders. Group 1 and group 2 showed a significantly higher risk of nephritis when compared with NETs degraders. The statistics for D are based on Fisher's exact test. The odds ratio with 95% confidence interval between nondegraders and degraders is 6.79 (2.108–21.86), **P = 0.0012; between degraders and group 1 is 5.73 (1.457–22.52), *P = 0.0188; and between degraders and group 2 is 8.909 (1.596–49.74), (**)P = 0.0091.