Fig. 3.

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.