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Extracellular eosinophilic traps in association with Staphylococcus aureus at the site of epithelial barrier defects in patients with severe airway inflammation 

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Presentation on theme: "Extracellular eosinophilic traps in association with Staphylococcus aureus at the site of epithelial barrier defects in patients with severe airway inflammation "— Presentation transcript:

1 Extracellular eosinophilic traps in association with Staphylococcus aureus at the site of epithelial barrier defects in patients with severe airway inflammation  Elien Gevaert, PhD, Nan Zhang, MD, PhD, Olga Krysko, MD, PhD, Feng Lan, MD, PhD, Gabriële Holtappels, BSc, Natalie De Ruyck, MSc, Hans Nauwynck, PhD, Shida Yousefi, PhD, Hans-Uwe Simon, MD, PhD, Claus Bachert, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 139, Issue 6, Pages e6 (June 2017) DOI: /j.jaci Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Eosinophils in nasal mucosal tissue. A-E, Immunohistochemical stain for MBP (red) in the subepithelial region of IT from a control patient (Fig 1, A) and polyp tissue from different patients (Fig 1, B-E) showing localization of eosinophils and different intensities of subepithelial localization. Nuclei (blue) are counterstained with hematoxylin. Scale bar = 100 μm. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Eosinophils and their DNA traps in nasal polyp (NP) tissue. A, Quantification of EETs in the subepithelial region of patient (NP 1-15) and control (IT 1-5) tissues. EETs were expressed as the percentage of eosinophils generating EETs relative to the total amount of eosinophils present. ND, Not determinable. B-E, Immunofluorescent staining of MBP (green) and DNA (red). Fig 2, B, Intact eosinophils in the stroma. Scale bar = 25 μm. Fig 2, C, Increased subepithelial eosinophilic aggregation. Scale bar = 50 μm. Fig 2, D, A single eosinophil is shown generating an EET (indicated by an arrowhead). Scale bar = 5 μm. Fig 2, E, A cluster of eosinophils generating EETs (indicated by arrowheads). Scale bar = 10 μm. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Relevant percentages of eosinophils forming EETs, IL-5 and periostin levels, and S aureus colonization. Tissue levels of IL-5 and periostin were measured (patients with CRSwNP, n = 15) by means of ELISA. S aureus colonization was assessed for all patients by using a PNA-Fish technique. These data were studied for bacterial association with EETs. A, IL-5 levels were increased in patients in whom higher fractions of eosinophils generating EETs were observed. B, Periostin levels were increased when higher fractions of eosinophils generating EETs were detected. C, In patients increased EET formation significantly correlated with higher scores for S aureus colonization. Data were analyzed by using a Mann-Whitney U test, and significance was expressed as follows: *P < .05 and **P < .01. D, Colocalization of S aureus and EETs. Immunofluorescent staining for MBP (red) and DNA (blue) combined with a PNA-Fish stain for S aureus (green). Left image scale bars = 25 μm; right image scale bars = 50 μm. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 EETs in nasal polyp tissue after in vitro exposure to S aureus or S epidermidis. Immunofluorescent staining for MBP (green) and DNA (red) is shown. A, Intact eosinophils after 2 hours of air-liquid culture exposure to TCM (control). Scale bar = 25 μm. B, increased EET formation (arrowheads indicate some, but not all, of the EETs) after 1 hour of air-liquid culture exposure to S aureus. Scale bar = 25 μm. C, Increased subepithelial formation of EETs (dashed line) in the proximity of S aureus (arrow). Scale bar = 25 μm. D, Mainly intact eosinophils after 2 hours of air-liquid culture exposure to S epidermidis. Scale bar = 50 μm. E, Increase in EET formation in the subepithelial region after exposure to S aureus and S epidermidis compared with TCM in nasal polyp tissue (n = 3). Data were analyzed with the Friedman test, followed by the Dunn posttest. Significance is expressed as follows: *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Transepithelial migration and massive EET formation of eosinophils to entrap S aureus. Immunofluorescent staining of MBP (red) and DNA (blue) combined with PNA-Fish staining for S aureus (green) is shown in tissue sections after 2 hours of exposure to S aureus. A, Migration of eosinophils and EETs at sites of epithelial defects where direct contact with S aureus is observed. Scale bar = 25 μm. B, Transepithelial migration of a large fraction of eosinophils and trapping of S aureus in a large EET. Scale bar = 25 μm. C, In vitro migration assay of eosinophils in response to different stimuli: TCM, eotaxin, S aureus (Sa), epithelial cells (ec), epithelial cells with S aureus (ec+Sa), and epithelial cells with S aureus after 4 hours of coincubation (ec+Sa 4h). Data were analyzed with the Mann-Whitney U test (n = 3). Significance is expressed as follows: *P < .05 and **P < .01. D, Eosinophils from a patient with CRSwNP showing EET formation after challenge with S aureus in vitro. Scale bar = 25 μm. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 ROS formation after exposure to S aureus and inhibition of EET formation by diphenyleneiodionium chloride (DPI). A-C, Production of ROS in tissues after 0.5, 1, and 2 hours of in vitro exposure to S aureus (Sa) or S epidermidis (Se; n = 3). MFI, Mean fluorescence intensity. D-G, Immunofluorescent stain for MBP (green) and DNA (red). Fig 6, D, Intact eosinophils after 1 hour of air-liquid culture exposure to TCM (control). Fig 6, E, increased EET formation after 1 hour of air-liquid culture exposure to S aureus. Fig 6, F, Intact eosinophils after 1 hour of DPI treatment and 1 hour of air-liquid culture exposure to TCM (control). Fig 6, G, Mainly intact eosinophils after 1 hour of DPI treatment and 1 hour of air-liquid culture exposure to S aureus. Scale bar = 100 μm. H, Increase in EET formation after exposure to S aureus and inhibition by DPI treatment in nasal polyp tissue (n = 3). Data were analyzed with the Friedman test, followed by the Dunn posttest. Significance is expressed as follows: *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 7 Periostin and TSLP levels after in vitro exposure to S aureus. A, Periostin levels in tissue homogenates were not significantly increased after exposure to S aureus. The black line represents the control value (TCM; n = 4). B, TSLP levels, as determined by means of ELISA, in tissue homogenates were not significantly increased after exposure to S aureus (n = 4). C, Western blot analysis showing no difference in lfTSLP expression in tissue homogenates after exposure to either S aureus or S epidermidis. In addition, the Western blot indicates that sfTSLP was not detected. D, PCR analysis in tissue homogenates indicates an increase in lfTSLP levels after 2 hours exposure to S aureus (n = 3). Sa, S aureus; Se, S epidermidis. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E1 Characterization of the study population. A-C, Tissue levels of IgE (Fig E1, A), IL-5 (Fig E1, B), and eosinophil cationic protein (ECP; Fig E1, C) were measured in all patients (CRSwNP, n = 15) and control subjects (CTR, n = 5) by means of ELISA and were found to be increased in patients with CRSwNP. D, Eosinophils and neutrophils were counted in tissue of control subjects and patients with CRSwNP. E and F, Tissue slides stained for MBP in control subjects (Fig E1, E) and patients with CRSwNP (Fig E1, F) showing the general distribution of eosinophils throughout the tissue. Scale bar = 100 μm. Data were analyzed with the Mann-Whitney U test. Significance is expressed as follows: *P < .05 and ***P < .001. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E2 Subepithelial eosinophils and damaged epithelium. Immunohistochemistry was performed on subsequent tissue slides, and the same region was selected for both panels. The protein of interest is stained in red/purple, and nuclei are stained with hematoxylin (blue). A, Immunohistochemical staining for MBP (red) shows localization of eosinophils in polyp tissue. Scale bar = 200 μm. B, An immunohistochemical stain for caspase-3 (red) demonstrates very few apoptotic cells in the tissue. The inset image (B′) shows no evidence for cell lysis. Fig E2, B, Scale bar = 200 μm; Fig E2, B′, scale bar = 10 μm. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E3 A, Relation between EET formation and eosinophils in tissues. Data were analyzed with the Mann-Whitney U test. Significance is expressed as follows: *P < .05 and ***P < .001. B, Periostin levels in patients with CRSwNP and control subjects. Data were analyzed with the Mann-Whitney U test. Significance is expressed as follows: ***P < .001. C, Scatterplot of periostin and IL-5 levels in patients with CRSwNP with a significant correlation (P = .013, R2 = 0.665), as determined by means of Spearman correlation. D, Scatterplot of TSLP and percentage of eosinophils forming EETs with no correlation, as determined by means of Spearman correlation. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E4 A, Relation between IL-5 levels and percentage of degranulated eosinophils. B, Relation between periostin levels and percentage of degranulated eosinophils. C, Relation between number of eosinophils and percentage of degranulated eosinophils. D, Relation between S aureus colonization and percentage of degranulated eosinophils. Data were analyzed with the Mann-Whitney U test. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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