Katka Szilagyi, MSc, Roel P. Gazendam, MD, John L

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Impaired microbial killing by neutrophils from patients with protein kinase C delta deficiency Katka Szilagyi, MSc, Roel P. Gazendam, MD, John L. van Hamme, BSc, Anton T.J. Tool, PhD, Michel van Houdt, BSc, Wilhelm A.J.W. Vos, BSc, Paul Verkuijlen, BSc, Hans Janssen, BSc, Alexandre Belot, MD, PhD, Laurent Juillard, MD, PhD, Elisabeth Förster-Waldl, MD, PhD, Kaan Boztug, MD, PhD, Georg Kraal, PhD, Menno P.J. de Winther, PhD, Taco W. Kuijpers, MD, PhD, Timo K. van den Berg, PhD Journal of Allergy and Clinical Immunology Volume 136, Issue 5, Pages 1404-1407.e10 (November 2015) DOI: 10.1016/j.jaci.2015.06.016 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Neutrophils of PKCδ-deficient patients show normal extracellular release of H2O2. A, Western blot analysis performed on cell lysates of neutrophils from healthy controls and patients. Freshly isolated neutrophils of healthy donors and patients were stimulated with soluble (B) or bacterial (C) stimuli, and release of H2O2 was measured by Amplex Red assay as described in the Methods section of this article's Online Repository at www.jacionline.org. CGD, Chronic granulomatous disease; fMPL, formyl-Met-Leu-Phe; nd, not determined; ns, not significant; ops, opsonized; PAF, platelet-activating factor; Pt, patient; RFU, relative fluorescence unit; STZ, serum-treated zymosan. ∗P < .05, ∗∗P < .01, and ∗∗∗∗P < .0001. Journal of Allergy and Clinical Immunology 2015 136, 1404-1407.e10DOI: (10.1016/j.jaci.2015.06.016) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Neutrophils deficient in PKCδ have significantly decreased capacity to kill bacterial and fungal pathogens. The bacterial-killing capacity of freshly isolated neutrophils from patient A was assessed with S aureus (A) and E coli (C). The same killing assay was performed with freshly isolated neutrophils from patients B and C and patients with CGD with S aureus (B) or E coli (D). Killing of fungal pathogen by neutrophils of all PKCδ-deficient patients was determined with C albicans conidia (E). Experiments were performed as described in detail in the Methods section of this article's Online Repository. CFU, Colony-forming unit; CGD, chronic granulomatous disease. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. Journal of Allergy and Clinical Immunology 2015 136, 1404-1407.e10DOI: (10.1016/j.jaci.2015.06.016) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Western blot analysis of PKCα and β expression by human neutrophils. Whole-cell lysates were prepared as described in the Methods section, and the expression of PKCα and βII was detected with specific antibodies (A). Quantification of protein expression is depicted relative to α tubulin as a loading control (B). Pt, Patient. Journal of Allergy and Clinical Immunology 2015 136, 1404-1407.e10DOI: (10.1016/j.jaci.2015.06.016) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Intracellular ROS production as determined by the dihydrorhodamine (DHR) assay is not affected in PKCδ-deficient neutrophils. Intracellular production of ROS was measured after purified neutrophils were stimulated with DHR-labeled opsonized E coli (A) or S aureus (B). Figures on the left show examples of kinetics of the fluorescence measurement by flow cytometry. Figures on the right show average ROS production measured by DHR oxidation on the surface of bacteria by freshly isolated neutrophils of healthy donors and PKCδ-deficient patients. Because of high interday variation, values obtained from neutrophils of healthy donors were set at 100% and measurements from neutrophils from patients were calculated as a percentage of a healthy control. Statistics were performed by using the multiple t test with the Holm-Sidak method, in which α = 5.000%. MFI, Mean fluorescence intensity. **P < .01. Journal of Allergy and Clinical Immunology 2015 136, 1404-1407.e10DOI: (10.1016/j.jaci.2015.06.016) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 PKCδ-deficient neutrophils show normal phagocytosis of microbial pathogens. Purified neutrophils of healthy donors and PKCδ-deficient patients were incubated with fluorescently-labeled pathogens and their uptake was quantified by flow cytometry. A-D, Kinetics of the uptake of opsonized S aureus, opsonized E coli, nonopsonized C albicans, and opsonized C albicans, respectively, by neutrophils of patient A and of healthy donors. E, Overview of pathogen uptake at its maximum, in which 100% represents the uptake of healthy donors' neutrophils and the uptake of neutrophils of each patient is expressed as a percentage of day control. Statistical analyses were performed by using the multiple t test with the Holm-Sidak method, in which α = 5.000%. MFI, Mean fluorescence intensity; ns, not significant. Journal of Allergy and Clinical Immunology 2015 136, 1404-1407.e10DOI: (10.1016/j.jaci.2015.06.016) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Degranulation and release of proteases by PKCδ-deficient neutrophils show no defect. A, Surface expression of CD66b (specific granules marker) and CD63 (azurophilic granules marker) was detected by flow cytometry after neutrophils of healthy donors and PKCδ-deficient patients had been triggered to degranulate with various stimuli. Values depicted are corrected for isotype control fluorescence. B, Secretion of proteases from granules of healthy donors and PKCδ-deficient patients was determined using DQ-Green BSA assay as described in the Methods section. Figures show overview of protease release by healthy donors and all PKCδ-deficient patients, depicted first as an example of measurement in patient B in comparison to day control showing measured RFUs and second as a percentage of day controls. Statistical analyses were performed by using the multiple t test with the Holm-Sidak method, in which α = 5.000%. MFI, Mean fluorescence intensity; RFU, relative fluorescence unit. *P < .05. Journal of Allergy and Clinical Immunology 2015 136, 1404-1407.e10DOI: (10.1016/j.jaci.2015.06.016) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Phagosomes of PKCδ-deficient neutrophils show normal fusion and contain both lactoferrin and myeloperoxidase after E coli phagocytosis. Neutrophils of a healthy control and patient A were incubated for 30 minutes to allow phagocytosis and fusion with lysosomes. Immuno-electron microscopy was used to detect lactoferrin (A) and myeloperoxidase (MPO) (B) in phagolysosomes. Phagolysosomes containing E coli with lactoferrin/MPO are marked with an asterisk, and lactoferrin/MPO-positive granules are marked with ►. Journal of Allergy and Clinical Immunology 2015 136, 1404-1407.e10DOI: (10.1016/j.jaci.2015.06.016) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Neutrophils of PKCδ-deficient patients show normal expression of surface epitopes, chemotaxis, and adhesion. A, Flow cytometry analysis of purified neutrophils showed normal phenotype of PKCδ-deficient neutrophils compared with healthy donors. B, Neutrophils of PKCδ patients and healthy donors were used in a transwell migration assay as described in the Methods section showing normal chemotaxis. C, Adhesion of neutrophils to a plastic surface after stimulation with various stimuli was performed as described in the Methods section. Statistical analyses were performed by using the multiple t test with the Holm-Sidak method, in which α = 5.000%. DTT, Dithiothreitol; LBP, LPS-binding protein; ns, not significant; PMA, phorbol 12-myristate 13-acetate; RFU, relative fluorescence unit. *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2015 136, 1404-1407.e10DOI: (10.1016/j.jaci.2015.06.016) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions