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Prominent role of IFN-γ in patients with aspirin-exacerbated respiratory disease  John W. Steinke, PhD, Lixia Liu, MD, Phillip Huyett, MD, Julie Negri,

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Presentation on theme: "Prominent role of IFN-γ in patients with aspirin-exacerbated respiratory disease  John W. Steinke, PhD, Lixia Liu, MD, Phillip Huyett, MD, Julie Negri,"— Presentation transcript:

1 Prominent role of IFN-γ in patients with aspirin-exacerbated respiratory disease 
John W. Steinke, PhD, Lixia Liu, MD, Phillip Huyett, MD, Julie Negri, BS, MBA, Spencer C. Payne, MD, Larry Borish, MD  Journal of Allergy and Clinical Immunology  Volume 132, Issue 4, Pages e3 (October 2013) DOI: /j.jaci Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 TH1/TH2 cytokine signature in sinus tissue from control subjects, patients with CHES, and patients with AERD, as determined by using qPCR. Tissue samples were homogenized after surgical removal and RNA isolated. Transcript levels for IL-4, IL-5, IL-13, and IFN-γ were quantified by using PCR with SYBR Green detection. Data (mean ± SEM) reflect relative expression of each gene in comparison with β-actin (2ΔCT). Control samples (n = 9) are depicted in black bars, CHES samples (n = 30) are depicted in gray bars, and AERD samples (n = 15) are depicted in white bars. *P < .05 compared with control samples. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Intracellular cytokine staining of CCR3+ eosinophils in patients with CHES and those with AERD. NPs were digested, and cells were separated from collagenous support material. Cells were stained with appropriate markers and analyzed by means of flow cytometry. Initial gating was performed to include only CD45+ cells. This was followed by extracellular staining for CCR3, CD4, CD8, or CD19 and intracellular staining for cytokines. A, Representative staining of CCR3+ NP cells from patients with CHES, showing intracellular levels of IL-4, IL-5, and IFN-γ. B, Representative staining of CCR3+ NP cells from patients with AERD, showing intracellular levels of IL-4, IL-5, and IFN-γ. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Immunofluorescence for IFN-γ in polyps from patients with AERD and those with CHES. Sections from paraffin-embedded NPs from patients with AERD (A) and patients with CHES (B) were stained with an APC-labeled antibody directed against IFN-γ and counterstained with 4′,6-diamidino-2-phenylindole to display nuclei. Each grouping displays an isotype control, specific IFN-γ staining, and an enlarged region to show specific cells. In these figures red represents IFN-γ staining, and blue represents nuclear staining. White arrows show eosinophils, and yellow arrows show mononuclear cells. i, Insert. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 IFN-γ potentiation of expression of markers for mature eosinophils. CD34+-enriched hematopoietic stem cells were cultured for 3 days with stem cell factor, thymopoietin, Flt3L, IL-3, and IL-5, after which they were cultured for 3 additional weeks with just IL-3 and IL-5 with or without the additional presence of IFN-γ. Media were changed weekly. Maturation status was assessed by means of flow cytometry for CCR3 and Siglec-8. A, Cells matured with IL-5 and IL-3 only. B, Cells matured in the additional presence of IFN-γ. C, Summary data for all 5 samples. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Enhanced secretion by eosinophils differentiated in the additional presence of IFN-γ. Eosinophils matured from CD34+ progenitors, as described, were activated with or without calcium ionophore and phorbol 12-myristate 13-acetate for 20 minutes. Supernatants were collected and EDN (A) and CysLT (B) levels were quantified. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E1 ImageStream flow cytometry of CD45+CCR3+ leukocytes in patients with CHES, patients with AERD, or both. We used ImageStream flow cytometry, as described in the text, to confirm that the CCR3+ cells were eosinophils. DAPI, 4′,6-Diamidino-2-phenylindole; FITC, fluorescence isothiocyanate. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E2 IFN-γ potentiation of eosinophilopoiesis. CD34+-enriched hematopoietic stem cells were cultured for 3 days with stem cell factor, thymopoietin, Flt3L, IL-3, and IL-5, after which they were cultured for 3 additional weeks with just IL-3 and IL-5 with or without the additional presence of IFN-γ. Media were changed weekly. After 3 weeks, aliquots were cytospun and stained (H&E). A, Cells matured with only IL-5 and IL-3 (×200 magnification). B, Cells matured in the additional presence of IFN-γ (×200 magnification). C, Cells matured in the additional presence of IFN-γ (×400 magnification). Viable cells with bilobed nuclei and granules are demonstrated along with numerous apoptotic bodies. All viable cells have the appearance of granulocytes. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E3 Absence of basophil markers on CD34+-derived eosinophils. CD34+-enriched hematopoietic stem cells were cultured for 3 days with stem cell factor, thymopoietin, Flt3L, IL-3, and IL-5, after which they were cultured for 3 additional weeks with just IL-3 and IL-5 with or without the additional presence of IFN-γ. Media were changed weekly. After 3 weeks, aliquots were collected and analyzed by means of flow cytometry for expression of CD203c and FcεRI. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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