Eosinophil and neutrophil extracellular DNA traps in human allergic asthmatic airways Ryszard Dworski, MD, PhD, Hans-Uwe Simon, MD, PhD, Aimee Hoskins,

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Eosinophil and neutrophil extracellular DNA traps in human allergic asthmatic airways Ryszard Dworski, MD, PhD, Hans-Uwe Simon, MD, PhD, Aimee Hoskins, RN, BSN, Shida Yousefi, PhD Journal of Allergy and Clinical Immunology Volume 127, Issue 5, Pages 1260-1266 (May 2011) DOI: 10.1016/j.jaci.2010.12.1103 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Eosinophil and EET counts per 10 hpfs in airway biopsy specimens from asthmatic and control subjects. Values are presented as single numbers, as well as mean levels ± SEMs. Journal of Allergy and Clinical Immunology 2011 127, 1260-1266DOI: (10.1016/j.jaci.2010.12.1103) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Representative endobronchial biopsy specimens from atopic asthmatic subjects showing EETs and NETs. A, EETs generated by eosinophils contain DNA and MBP. Biopsy specimens were from 3 different patients. In the left panels infiltrating eosinophils expressing MBP are circled by dashed lines. In the middle panels examples of extracellular DNA are indicated by arrows. In the right panels colocalization of extracellular DNA and MBP is circled by dashed lines. B, NETs generated by neutrophils contain DNA and elastase. Representative results from 1 patient are shown. In the left panel infiltrating neutrophils expressing elastase are circled by dashed lines. In the middle panel examples of extracellular DNA are indicated by arrows. In the right panel colocalization of extracellular DNA and elastase is circled by dashed lines. Bars = 10 μmol/L. Control antibodies showed no staining (data not shown). Journal of Allergy and Clinical Immunology 2011 127, 1260-1266DOI: (10.1016/j.jaci.2010.12.1103) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Extracellular DNA is released from viable eosinophils. A, Positive TdT-mediated dUTP-biotin nick end-labeling (TUNEL) analysis of an esophageal biopsy specimen obtained from a patient with eosinophilic esophagitis (EoE; left panel) and an asthmatic airway biopsy specimen showing a negative TUNEL assay (right panel). Eosinophils and EETs were identified by using anti-ECP antibody (arrows). Lower panels represent magnifications of the indicated fields within the upper panels. B, Fluorescent in situ analysis of asthmatic biopsy specimens. Positive signals within the extracellular DNA (arrows) were seen with an Atp6 but not with a Gapdh probe. The data in each panel are representative of at least 3 independent experiments. Bars = 10 μmol/L. DAPI, 4′-6-Diamidino-2-phenylindole dihydrochloride. Journal of Allergy and Clinical Immunology 2011 127, 1260-1266DOI: (10.1016/j.jaci.2010.12.1103) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 No effect of segmental allergen challenge on numbers of EETs and NETs in airway biopsy specimens of atopic asthmatic subjects. A, Eosinophils and EETs before and 24 hours after allergen challenge. B, Neutrophils and NETs before and 24 hours after allergen challenge in asthmatic subjects with neutrophil infiltration. Values are presented as single numbers, as well as mean levels ± SEMs per 10 hpfs. Changes are not significant. Journal of Allergy and Clinical Immunology 2011 127, 1260-1266DOI: (10.1016/j.jaci.2010.12.1103) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Effect of segmental allergen challenge on cytokine/chemokine concentrations in BAL fluid of atopic asthmatic subjects. The indicated cytokines/chemokines were measured before and 24 hours after allergen challenge. Values are presented as single numbers, as well as mean levels ± SEMs. P values are indicated. Journal of Allergy and Clinical Immunology 2011 127, 1260-1266DOI: (10.1016/j.jaci.2010.12.1103) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions