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Severe glucose-6-phosphate dehydrogenase deficiency leads to susceptibility to infection and absent NETosis  Ulrich Siler, PhD, Susana Romao, PhD, Emilio.

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Presentation on theme: "Severe glucose-6-phosphate dehydrogenase deficiency leads to susceptibility to infection and absent NETosis  Ulrich Siler, PhD, Susana Romao, PhD, Emilio."— Presentation transcript:

1 Severe glucose-6-phosphate dehydrogenase deficiency leads to susceptibility to infection and absent NETosis  Ulrich Siler, PhD, Susana Romao, PhD, Emilio Tejera, PhD, Oleksandr Pastukhov, PhD, Elena Kuzmenko, PhD, Rocio G. Valencia, PhD, Virginia Meda Spaccamela, MD, Bernd H. Belohradsky, MD, Oliver Speer, PhD, Markus Schmugge, MD, Elisabeth Kohne, MD, Manfred Hoenig, MD, Joachim Freihorst, MD, Ansgar S. Schulz, MD, Janine Reichenbach, MD  Journal of Allergy and Clinical Immunology  Volume 139, Issue 1, Pages e3 (January 2017) DOI: /j.jaci Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 G6PD activity. G6PD activity in MNCs, granulocytes, and erythrocytes (total protein extract: granulocytes/MNCs, 20 μg; erythrocytes, 200 μg). The NADP+-dependent conversion of glucuronate-6-phosphate to ribulose-5-phosphate by G6PD activity was inhibited in the presence of maleimide, and the increase in NADPH production by G6PD was detected by following absorbance at 340 nm. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Reduced/oxidized NADP levels. Reduced NADPH and oxidized NADP+ concentrations in protein extract normalized to protein content (white and black bars, respectively). Asterisks indicate statistically significant differences (P < .05, 2-tailed unpaired t test) of concentrations of NADPH and NADP+ in granulocytes and MNCs from patients compared with concentrations in corresponding control samples (n = 8). C, Healthy control subject. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Detection of ROS in blood granulocytes of patients with G6PD deficiency and their carrier mother. Detection of ROS on PMA stimulation by using DHR (A) or NBT (B) assays. White arrows, ROS-positive cells; black arrows, ROS-negative cells. C, Control; M, mother. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Detection of ROS in blood granulocytes of patients with G6PD deficiency. A, ROS quantification by using a cytochrome C assay. B, Detection of ROS upon PMA stimulation by using a chemiluminescence-based assay. C, Higher magnification of gray-highlighted area in Fig 4, B. C, Control. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 NETosis. A, NET kinetics (control subject, G6PD-carrier mother, G6PD deficiency, CGD carrier [18.8% normal and 81.2% cells without ROS], CGD [residual ROS15], and CGD [without ROS]). GO, Glucose oxidase-treated. B, Immunofluorescence (red, NE; blue, 4′,6-diamidino-2-phenylindole [DAPI]) of control or G6PD-deficient neutrophils. Black/white, NE; merged: NE/DAPI. C, Percentage of NETotic cells (panel B; n ≥ 250 cells). D, Percentage of cells with perinuclear NE signal (panel B; n ≥ 300 cells). *P < .05. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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