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Defective nuclear entry of hydrolases prevents neutrophil extracellular trap formation in patients with chronic granulomatous disease  Susana Romao, PhD,

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Presentation on theme: "Defective nuclear entry of hydrolases prevents neutrophil extracellular trap formation in patients with chronic granulomatous disease  Susana Romao, PhD,"— Presentation transcript:

1 Defective nuclear entry of hydrolases prevents neutrophil extracellular trap formation in patients with chronic granulomatous disease  Susana Romao, PhD, Emilio Tejera Puente, PhD, Katarzyna J. Nytko, PhD, Ulrich Siler, PhD, Christian Münz, PhD, Janine Reichenbach, MD  Journal of Allergy and Clinical Immunology  Volume 136, Issue 6, Pages e5 (December 2015) DOI: /j.jaci Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 PI3-kinase inhibition decreases NETosis and ROS production without altering macroautophagy. A and B, SYTOX-based NET assay in PMA-stimulated (Fig 1, A) or Candida albicans–stimulated (Fig 1, B) neutrophils with or without PI3K inhibitors (n = 3, mean ± SD). C, Quantification of macroautophagy in neutrophils from HDs after 1 hour of stimulation (mean ± SD). Neutrophils of 6 HDs (n ≥ 100 cells/well) are shown as triplicates from 2 independent HDs. Scale bars = 5 μm. D, ROS production after 30 minutes of incubation, as indicated (mean ± SD). ***P < .001. E, Quantification of NE signal around nucleus of PMA-stimulated neutrophils with or without 3-MA (mean ± SD). Means ± SDs pooled from 4 HDs are shown. RFU, Relative fluorescence units; SP, spautin-1; WM, wortmannin. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 NE nuclear entry requires ROS for histone H4 degradation during NETosis. A, PMA-stimulated neutrophils from HDs or patients with CGD were analyzed for NE (red; black/white) and DAPI (blue) staining by means of immune fluorescence microscopy. Values from 2 HDs and 3 patients with CGD are shown. Scale bars = 5 μm. B, NE signal around the nucleus of PMA-stimulated neutrophils (mean ± SD). **P < .01, t test. C, Quantification of fold decrease in H4 levels in neutrophils from 9 HDs and 6 patients with CGD with or without PMA. ∗P < .05, t test. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 PI3-kinase inhibition blocks NE release but minimally affects macroautophagy in human activated neutrophils. A, Quantification of extracellular or nuclear NE in neutrophils with or without 3-MA, wortmannin (WM), and spautin-1 (SP) with or without stimulation with opsonized C albicans (1:1 ratio) or PMA for 3 hours (pooled data from 4 HDs; 75 cells/donor; mean ± SD). B, Neutrophils from HDs were treated with or without 3-MA and with or without PMA, and LC3-II levels were assessed by means of Western blotting (1 blot of ≥3). Numbers below the blot represent the fold increase of LC3-II compared with nonstimulated control cells after normalization to corresponding actin levels. C, Neutrophils from HDs were incubated with PMA for 10 minutes, 1 hour, or 3 hours; fixed; permeabilized; and stained for ATG8/LC3 (green), NE (red), and DAPI (blue). Representative confocal pictures of 1 of 50 cells analyzed are shown. Data are from 3 HDs. D, Quantification of fluorescence intensity for ATG8/LC3 and NE signals in the perinuclear region of neutrophils from HDs (n = 50). Cells were left unstimulated or treated with PMA at the indicated time points. Pooled data are from 3 HDs. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Intact macroautophagy in neutrophils from patients with CGD. Quantification of ATG8/LC3+ dots (ATG8/LC3 green, DAPI blue) on neutrophils (n ≥ 100; mean ± SD) from 3 HDs and 4 patients with CGD after 1 hour of stimulation. Scale bars = 5 μm. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 PI3-kinase inhibition prevents NE access to the nucleus of neutrophils. Immunofluorescence analysis of PMA-stimulated neutrophils from HDs (n = 4 HDs) with or without 3-MA, NE (red, merged color signals; black/white, confocal pictures), and DAPI (blue). Quantification is shown in Fig 1, E. Scale bars = 20 μm. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E4 ROS triggers nuclear histone degradation. Western blot analysis (1/6 blots) of histone H4 in neutrophils from HDs (A) or patients with CGD (B) with or without PMA. Numbers indicate the fold increase of histone H4 compared with that nonstimulated control values (normalized to actin). Quantification is shown in Fig 2, C. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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