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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 5. — PAD4 is important in immune defense against GAS in a mouse model of necrotizing fasciitis. (A) In necrotizing fasciitis assays, M1 GAS but not M1 ΔSda1 GAS induced large lesions in PAD4^+/+ mice (two representative PAD4^+/+ mice are shown on the left panels). In contrast, both M1 GAS and M1 ΔSda1 GAS formed large lesions in PAD4^−/− mice (right). Arrows indicate lesion sites. (B) Lesion size was measured using ImageJ. The size of lesion formed by M1 ΔSda1 GAS in PAD4^−/− mice increased ∼4.03-fold compared with PAD4^+/+ mice (P < 0.001 by a Student’s t test). (C) The number of bacteria recovered from the lesion formation site was analyzed by colony formation assays. The number of M1 ΔSda1 GAS bacteria recovered from PAD4^−/− mice increased ∼3.97-fold compared with PAD4^+/+ mice (P < 0.001 by a Student’s t test). For A-C, three independent experiments were performed, with two pairs of PAD4^+/+ and PAD4^−/− mouse siblings used for each experiment. (D) Representative photomicrographs of skin lesions from PAD4^+/+ and PAD4^−/− mice infected with M1 GAS and M1 ΔSda1 GAS. Arrows indicate neutrophilic infiltrates and the star highlights intact epithelium that is absent in the other sections. Magnification is 100×. Three independent experiments, with one pair of PAD4^+/+ and PAD4^−/− mouse siblings, were formed for each experiment. (E) GST-PAD4 expressed and purified from Escherichia coli was used to treat histone H3 and mononucleosomes. The citrullination of histone H3 was tested by using histone H3 citrullination antibody (H3Cit) in Western blotting. Histone H3 was probed to show the amount of histone H3 in each lane (representative results of three independent experiments). (F) Analyses of the percentages of S. flexneri bacteria killed by histone H3, citrullinated histone H3, mononucleosomes, and citrullinated mononucleosomes at concentrations indicated. Standard deviations (indicated by error bars) are calculated from three independent repeat experiments.