Dysregulation of innate immune receptors on neutrophils in chronic granulomatous disease Dominik Hartl, MD, Natalie Lehmann, MD, Florian Hoffmann, MD,

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Dysregulation of innate immune receptors on neutrophils in chronic granulomatous disease Dominik Hartl, MD, Natalie Lehmann, MD, Florian Hoffmann, MD, Annette Jansson, MD, Andreas Hector, Gundula Notheis, MD, Dirk Roos, PhD, Bernd H. Belohradsky, MD, Uwe Wintergerst, MD Journal of Allergy and Clinical Immunology Volume 121, Issue 2, Pages 375-382.e9 (February 2008) DOI: 10.1016/j.jaci.2007.10.037 Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Receptor expression on neutrophils in patients with CGD, patients with pneumonia, and healthy control subjects. TLR (A), complement receptor (B), and IL-8 receptor (C) expression levels were quantified on neutrophils from patients with CGD (n = 15), patients with non-CGD pneumonia (n = 15), and healthy control subjects (n = 15) by means of flow cytometry. For TLR9 detection, neutrophils were permeabilized before antibody staining. Bars represent means. MFI, Mean fluorescence intensity (specific antibody staining minus isotype staining). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001 versus the respective patient group. Single asterisks indicate the 3 patients with CGD with residual NAPDH oxidase activity. Journal of Allergy and Clinical Immunology 2008 121, 375-382.e9DOI: (10.1016/j.jaci.2007.10.037) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Intracellular flow cytometry. Intracellular protein expression was quantified by means of flow cytometry, as described in the Methods section. Neutrophils were fixed and permeabilized before antibody staining. Bars represent means ± SDs. MFI, Mean fluorescence intensity (specific antibody staining minus isotype staining). ∗P < .05, patients with CGD versus healthy control subjects. Journal of Allergy and Clinical Immunology 2008 121, 375-382.e9DOI: (10.1016/j.jaci.2007.10.037) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 mRNA expression. Neutrophils were isolated from freshly drawn peripheral blood. RNA was extracted, and mRNA levels were quantified by means of quantitative RT-PCR, as described in the Methods section. mRNA expression is shown normalized to β-actin expression. Bars represent means ± SDs. ∗P < .05, patients with CGD versus healthy control subjects. Journal of Allergy and Clinical Immunology 2008 121, 375-382.e9DOI: (10.1016/j.jaci.2007.10.037) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Functional relevance. Bars represent means ± SDs. Data are representative for 5 independent experiments. A, Phagocytosis. S aureus bacteria were incubated with the fluorescent ligand Lucifer Yellow for 60 minutes at room temperature. After preopsonization, the bacteria (2 × 107/mL) were incubated at 37°C for 2 hours with neutrophils (2 × 106/mL). After the incubation period, the neutrophils were separated from the free bacteria by means of 3 centrifugations at 200g for 5 minutes. The Lucifer Yellow fluorescence of the isolated neutrophils was analyzed by means of flow cytometry. Phagocytosis by neutrophils from patients with CGD is shown as the percentage related to phagocytosis by neutrophils isolated from peripheral blood of healthy control subjects (= 100%). B, Chemotaxis. Isolated neutrophils were labeled with 4 μg/mL calcein acetomethylester and were placed in the upper compartment of a Transwell filter system (3.0-μm pore size, 12-mm diameter), with IL-8 (10 nmol/L) in the lower compartment. The Transwells were incubated for 60 minutes at 37°C. Chemotaxis by neutrophils from patients with CGD is shown as the percentage related to chemotaxis of neutrophils isolated from peripheral blood of healthy control subjects (= 100%). Journal of Allergy and Clinical Immunology 2008 121, 375-382.e9DOI: (10.1016/j.jaci.2007.10.037) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Clinical correlations. The correlation of TLR5 (A) and CD18 (B) expression levels on neutrophils and the frequency of lymphadenitis (Fig 5, A) and pneumonias (Fig 5, B) per year are shown in patients with CGD. C shows CD35 expression in patients with CGD with (n = 8) or without (n = 7) autoimmune (AI) symptoms. MFI, Mean fluorescence intensity. Correlation analysis was performed by calculating the 2-tailed Pearson correlation coefficient. Journal of Allergy and Clinical Immunology 2008 121, 375-382.e9DOI: (10.1016/j.jaci.2007.10.037) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Receptor stability. Receptor expression levels analyzed were determined by means of flow cytometry by using peripheral blood–isolated neutrophils obtained from 3 representative patients with CGD at 3 different time points within 1 year (mean interval, 3 ± 2 months [mean ± SDs]) to assess receptor expression stability. Each dot represents a single patient with CGD. MFI, Mean fluorescence intensity (specific antibody staining minus isotype staining). Journal of Allergy and Clinical Immunology 2008 121, 375-382.e9DOI: (10.1016/j.jaci.2007.10.037) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

CGD inheritance and complement receptor expression CGD inheritance and complement receptor expression. CD11b and CD18 surface expression levels are shown in patients with CGD with X-chromosomal (X; n = 9) or autosomal-recessive (AR; n = 6) inheritance. Bars represent means ± SDs. MFI, Mean fluorescence intensity (specific antibody staining minus isotype staining); p22, a single autosomal-recessive patient with CGD bearing a mutation in the p22phox subunit. Journal of Allergy and Clinical Immunology 2008 121, 375-382.e9DOI: (10.1016/j.jaci.2007.10.037) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

CD11b/CD18 upregulation. Neutrophils obtained from patients with CGD or healthy control subjects were incubated in RPMI medium at 37°C and 5% CO2 for 1 hour with LPS (100 ng/mL), Pam3CSK4 (1 μg/mL), peptidoglycan (1 μg/mL), CpG-DNA (100 μg/mL), flagellin (1 μg/mL), or zymosan (50 μg/mL). After the incubation period, CD11b (upper panel) or CD18 (lower panel) surface expression was quantified by means of flow cytometry. Bars represent means ± SDs of 3 independent experiments each. MFI, Mean fluorescence intensity (specific antibody staining minus isotype staining). ∗P < .05 versus medium-treated neutrophils. The right panels show CD11b/CD18 expression after medium or Pam3CSK4 stimulation. Journal of Allergy and Clinical Immunology 2008 121, 375-382.e9DOI: (10.1016/j.jaci.2007.10.037) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

TLR5 signaling. Neutrophils isolated from peripheral blood of healthy control subjects or patients with CGD were incubated in RPMI medium at 37°C and 5% CO2 for 1 hour with flagellin (1 μg/mL). TLR5 signaling was inhibited by pretreatment of neutrophils for 30 minutes with anti-TLR5 (αTLR5; 20 μg/mL). As a control, isotype antibodies (20 μg/mL) were used instead of anti-TLR5 antibodies. After the incubation, intracellular calcium levels were measured as described in the Methods section. Bars represent means ± SD of 5 independent experiments. Intracellular calcium levels are presented as a percentage related to medium-treated neutrophils (= 100%). ∗P < .05 versus medium-treated neutrophils. Journal of Allergy and Clinical Immunology 2008 121, 375-382.e9DOI: (10.1016/j.jaci.2007.10.037) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Inhibition of NADPH oxidase Inhibition of NADPH oxidase. Peripheral blood–isolated neutrophils were treated for 6 hours in RPMI with or without DPI (10 μmol/L). Afterward, neutrophils were washed, and TLR (A) or other innate immune receptor expressions (B), as well as TLR5 receptor function (C), were analyzed as described elsewhere in detail. ∗P < .05, DPI versus medium-treated neutrophils. Journal of Allergy and Clinical Immunology 2008 121, 375-382.e9DOI: (10.1016/j.jaci.2007.10.037) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Residual NADPH oxidase. The left panel shows TLR5 expression levels and the right panel shows TLR9 expression levels of neutrophils in patients with CGD with (n = 3) or without (n = 12) residual NADPH oxidase activity. MFI, Mean fluorescence intensity (specific antibody staining minus isotype staining). ∗P < .05. Journal of Allergy and Clinical Immunology 2008 121, 375-382.e9DOI: (10.1016/j.jaci.2007.10.037) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions