Fig. 4.

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.