SLE neutrophils are prone to death and display prominent signatures of type I IFN, TLR signaling, and cell death. (A) Healthy (n = 11) and SLE (n = 11) neutrophils were cultured with 2% autologous serum (AS). Cell viability was evaluated at different time points using trypan blue. ***P < 0.0001, ANOVA (adjusted for Dunn’s test). (B) Microarray analysis of purified blood neutrophils from healthy children (n = 12) and pediatric SLE patients (n = 21). One thousand five hundred and sixty-four transcripts were differentially expressed in SLE neutrophils (P < 0.01, nonparametric t test). Genes representative of the three most prominent pathways according to Ingenuity Pathways Analysis algorithms are shown. (C) Microarray analysis of neutrophils from two healthy donors (HD1 and HD2) incubated for 6 hours with either 20% AS or sera from 10 pediatric SLE patients. Data were normalized to autologous sera cultures. Genes from the three most representative networks according to Ingenuity Pathways Analysis algorithm are depicted. (D) Healthy neutrophils were cultured with 20% AS or sera from four pediatric SLE patients (SLES) with active disease (SLEDAI > 4). TLR7, TLR8, and TLR9 expression was analyzed using real-time PCR. Fold up-regulation was calculated as the difference in cycle threshold after 1- and 6-hour in vitro incubation relative to the expression at time 0. (E) Neutrophils from healthy children (HN, n = 6) and SLE patients (SLEN, n = 7) were cultured overnight in the presence or absence of a TLR7 agonist (R837, 10 µg/ml). IL-8 production was analyzed by Luminex assay. ***P < 0.0001, paired t test. (F) SLE neutrophils (n = 4) were incubated with media, anti-RNP antibody (10 µg/ml), or LPS (25 nM) for 6 hours. Left: IL-8 production was detected by Luminex. Right: cells were stained for CD66b surface expression. Results are displayed as quantification of mean fluorescence intensity (MFI). **P = 0.001; ***P < 0.0001, ANOVA (adjusted for Dunn’s test).

SLE NETs are loaded with LL37 and HMGB1. (A) Activation with anti-RNP antibodies leads SLE, but not healthy neutrophils, to secrete high levels of LL37 (left) and HMGB1 (right) as quantified by ELISA on culture supernatants (P values were obtained by paired t test). (B) LL37 and HMGB1 colocalize following a globular pattern along SLE NETs. NET release was triggered by treating healthy and SLE neutrophils with PMA (25 nM) for 3 hours. Specimens were then fixed and incubated with antibodies against LL37 and HMGB1 followed by the addition of FITC- or TRITC-conjugated secondary Fab fragments, respectively. DNA was counterstained with Hoechst 33342. Magnification, ×40. This experiment is representative of four independent experiments.

Mechanism underlying the generation of anti-RNP antibody–induced SLE NETs and their downstream effects on pDCs. (A) Neutrophils from SLE patients (n = 4) were pretreated for 10 min with FcγRIIa blocker (anti-human CD32), the NADPH oxidase inhibitor diphenylene iodonium (DPI) (left and middle panels), or the TLR7 inhibitor IRS-661 (right panel) before incubation with anti-RNP antibodies. Neutrophil supernatants were then added to pDC cultures. IFN-α secretion (left) and expression of the maturation marker CD83 (middle and right panels) were assessed by Luminex and flow cytometry, respectively. HG IgG, heat-aggregated Ig control. *P = 0.01; ***P < 0.0001, one-way ANOVA with post test Dunn’s correction. (B) Addition of TLR9 inhibitors (ODN-TTAGGG) or DNase I inhibits the capacity of SLE NETs to induce pDC activation as measured by IFN-α secretion (left) and CD83 expression (right), respectively; ODN-TTAGGG did not inhibit NET-induced pDC maturation (middle). **P = 0.001, paired t test.

Anti-RNP antibodies induce NETosis of SLE neutrophils. (A and B) Neutrophils from healthy children (A) and SLE patients (B) were stained with calcein green. Cells were incubated in 2% AS for 3 hours (A and B, a and b) and treated with anti-RNP IgG (10 µg/ml) (A and B, d to f) or with PMA (25 nm) (A and B, g to i). Sytox Red was used to stain DNA and detect cell death. Neutrophils from healthy children display intact membranes after 3 hours of culture with medium (A, a to c). Under the same conditions, SLE neutrophils undergo significant apoptosis (B, a to c). Anti-RNP antibodies induce apoptosis of healthy neutrophils (A, d to f) and NETosis of SLE neutrophils (B, d to f). PMA induces the release of extracellular DNA in both healthy (A, g to i) and SLE (B, g to i) neutrophils. These data are representative of at least six independent experiments. (C) NET DNA quantification. Supernatants from healthy and SLE neutrophils with and without previous activation with anti-RNP antibodies were digested with micrococcal nuclease to dismantle the NET scaffold. Free DNA in the supernatants was quantified by incubation with Sytox Orange followed by relative fluorescence measurement after building a standard curve using λ DNA. As a positive control, NETs were induced by incubating neutrophils with PMA (25 nm) for 12 hours. *P = 0.01, one-way ANOVA with post test Dunn’s correction.

SLE NETs induced by anti-RNP antibodies are potent activators of pDCs. (A) pDCs from healthy donors were cultured with medium (a), CpG A (10 µg/ml) (b), anti-RNP IgG (10 µg/ml) (c), supernatant (20%) from healthy neutrophils without (d) or with (e) activation with anti-RNP IgG, and supernatant (20%) from SLE neutrophils without (f) or with (g) activation with anti-RNP IgG (10 µg/ml). Top panel depicts the light microscopy images after overnight culture under the above-described conditions. Expression of HLA-DR, CD80, CD86, and CD83 was assessed by flow cytometry in the same cultures (gated on live cells). (B) These experiments were repeated using 3 independent healthy pDC donors and 17 SLE sera for induction of CD80 and CD86 expression and 13 sera for induction of CD83 expression. ***P < 0.0001, one-way ANOVA with post test Dunn’s correction. (C) Cytokine measurement in the supernatants of pDCs activated as described in (A). ***P < 0.0001, one-way ANOVA with post test Dunn’s correction.

Treatment of healthy neutrophils with IFN-α renders them susceptible to NETosis upon subsequent activation with anti-RNP antibodies. pDCs were cultured in the presence of medium, CpG A (10 µg/ml), anti-RNP IgG (10 µg/ml), supernatants (20%) from healthy neutrophils (HN/ctrl), healthy neutrophils treated with anti-RNP IgG (HN/RNP), or IFN-α (1000 IU, HN–IFN-α) or from healthy neutrophils treated for 6 hours with IFN-α; washed; and incubated with anti-RNP IgG (HN–IFN-α/RNP). (A) Flow cytometry was used to assess induction of expression of activation markers (CD80, CD86, CD83) after overnight cultures. (B) Luminex assay was used to assess secretion of IFN-α, TNF-α, and IL-6 in the same cultures. *P = 0.01; ***P < 0.0001, one-way ANOVA with post test Dunn’s correction.