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. 2010 Aug 30;207(9):1853–1862. doi: 10.1084/jem.20100239

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© 2010 Li et al.

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Figure 4. — Antagonism of PAD4-mediated NET formation and bacterial extracellular DNase-mediated NET destruction. (A) Genomic DNA (untreated in lane 1) was incubated with cell culture supernatant from wild-type M1 GAS (lane 2) or M1 ΔSda1 GAS (lane 3). DNA degradation by DNase Sda1 was observed (lane 2; representative results of three independent experiments). (B) Upon incubation of M1 GAS with PAD4^+/+ neutrophils, histone H3 citrullination was detected but NETs were rarely observed by immunostaining (top). In contrast, histone H3 citrullination or NETs were not detected after incubation of M1 GAS with PAD4^−/− neutrophils (bottom). (C) Both histone H3 citrullination and NETs were detected after incubation of M1 ΔSda1 GAS with PAD4^+/+ neutrophils (top, arrows denote NETs). In contrast, histone H3 citrullination or NETs were not detected after incubation of M1 ΔSda1 GAS with PAD4^−/− neutrophils (bottom). (D) Percentages of PAD4^+/+ neutrophils with H3 citrullination staining or with both H3 citrullination and NET formation after incubation with M1 GAS or M1 ΔSda1 GAS were analyzed. The presence of Sda1 decreased NET formation by ∼4.2-fold (P < 0.003 by a Student’s t test). All NETs were positive for the H3 citrullination antibody staining. Error bars indicate standard deviation. (E) Higher-magnification images show NETs formed in PAD4^+/+ neutrophils after incubation with M1 ΔSda1 GAS (arrows denote NETs). For assays in B-E, peripheral blood neutrophils were purified from five PAD4^+/+ or five PAD4^−/− paired mouse siblings in each experiment, and three independent experiments were performed.