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D prostanoid receptor 2 (chemoattractant receptor–homologous molecule expressed on TH2 cells) protein expression in asthmatic patients and its effects.

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Presentation on theme: "D prostanoid receptor 2 (chemoattractant receptor–homologous molecule expressed on TH2 cells) protein expression in asthmatic patients and its effects."— Presentation transcript:

1 D prostanoid receptor 2 (chemoattractant receptor–homologous molecule expressed on TH2 cells) protein expression in asthmatic patients and its effects on bronchial epithelial cells  Sally E. Stinson, MSc, Yassine Amrani, PhD, Christopher E. Brightling, PhD  Journal of Allergy and Clinical Immunology  Volume 135, Issue 2, Pages e7 (February 2015) DOI: /j.jaci Copyright © 2014 The Authors Terms and Conditions

2 Fig 1 Images are shown at ×200 magnification. A-D, Examples of DP2 expression (brown staining) on epithelial cells and inflammatory cells within the bronchial submucosa in rabbit immunoglobulin fraction negative control demonstrating a lack of any positive staining (Fig 1, A), a healthy control subject (Fig 1, B), a patient with mild asthma (Fig 1, C), and a patient with severe asthma (Fig 1, D). E, Dot plot of DP2+ inflammatory cells within the submucosa of healthy control subjects and patients with mild, moderate, and severe asthma. P values are based on the Kruskal-Wallis test. Overall P < P values shown in the figure are based on the Dunn post hoc test. F, Numbers of DP2+ mast cells (mast cell tryptase positive), eosinophils (major basic protein positive), and T cells (CD3+), as assessed by means of colocalization of sequential sections. P values are based on 2-way ANOVA. Overall P < P values shown in the figure based on the Tukey post hoc test. G, Dot plot of DP2+ epithelial cells in healthy control subjects and patients with moderate and severe asthma. P values are based on the Kruskal-Wallis test. Overall P < P values shown in the figure are based on the Dunn post hoc test. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

3 Fig 2 A-C, Images (×400 magnification) of serial sections of a severe asthma biopsy specimen after involucrin staining (brown; Fig 2, A), pancytokeratin (Fig 2, B), and DP2 (Fig 2, C). D, Epithelial histology grades for biopsy specimens from healthy control subjects and patients with mild, moderate, and severe asthma. P values are based on 1-way ANOVA. Overall P < P values shown in the figure based on the Tukey post hoc test. E, Grading of involucrin staining for biopsy specimens from healthy control subjects and patients with mild, moderate, and severe asthma. P values are based on Kruskal-Wallis tests. Overall P < P values shown in the figure are based on the Dunn post hoc test. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

4 Fig 3 DP2 expression on cultured submerged epithelial cells. Fluorescent cell staining is shown as follows. A, Green staining for DP2, with blue 4′-6-diamidino-2-pheynylindole dihydrochloride (DAPI) nuclear staining (cells from healthy control subjects). The inset shows a rabbit isotype control with lack of any green staining. B, Green staining for DP2, with blue DAPI nuclear staining (cells from asthmatic patients). Note cells with absence of DP2+ cells (green) staining. C, Green staining for DP2 on ALI culture from healthy control subjects. D, Green staining for DP2 on ALI culture from asthmatic patients. E, Percentage of DP2+ epithelial cells of extracellular expression assessed by means of fluorescence-activated cell sorting. P values are based on unpaired 2-tailed t tests. F, DP2 mRNA expression normalized to the 18S housekeeping gene for epithelial cells. P values are based on unpaired 2-tailed t tests. G, i, Hoechst-positive epithelial cells within the migration zone for vehicle control. ii, Hoechst-positive epithelial cells within the migration zone for 100 nmol/L DK-PGD2 treatment. iii, Hoechst-positive epithelial cells within the migration zone for 100 nmol/L DK-PGD2 plus AZD6430 treatment. H, Dot plot of cell migration fold change over vehicle control for both cells from healthy control subjects and those from asthmatic patients with 100 nmol/L DK-PGD2 and 100 nmol/L DK-PGD2 plus 1 μmol/L AZD6430. P values are based on paired 2-tailed t tests. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

5 Fig 4 A-C, Representative images of healthy control ALI cultures of MUC5AC+ staining (brown, ×200 magnification). Results are shown for untreated conditions (Fig 4, A) and treatment with 100 nmol/L DK-PGD2 for 24 hours (Fig 4, B), and 100 nmol/L DK-PGD2 plus 1 μmol/L AZD6430 for 24 hours (Fig 4, C). D, IL-13 (100 ng/mL) for 24 hours. E, IL-3 (100 ng/mL) plus AZD6430 (1 μmol/L) for 24 hours. F, Dot plot to show fold change in MUC5AC+ cells per millimeter of culture over untreated for cultures with 24-hour treatment. P values are based on 1-way ANOVA. Overall P < P values shown in the figure are based on the Tukey post hoc test. G, Dot plot to show mRNA expression for MUC5AC normalized to 18S expression for cultures with 24-hour treatment. P values are based on Kruskal-Wallis tests. Overall P = P values shown in the figure are based on the Dunn post hoc test. H, Dot plot to show quantitation of MUC5AC staining for cultures with 48 and 72 hours of treatment. P values are based on 1-way ANOVA. Overall P = P values shown in the figure are based on the Tukey post hoc test. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

6 Fig 5 A-E, Representative images of ALI cultures from healthy control subjects of involucrin-positive staining (brown, ×400 magnification). Results are shown for untreated conditions (Fig 5, A) and treatment with 100 nmol/L DK-PGD2 for 48 hours (Fig 5, B), and 100 nmol/L DK-PGD2 plus 1 μmol/L AZD6430 for 48 hours (Fig 5, C). D, TGF-β1 (10 ng/mL) for 72 hours. E, TGF-β1 (10 ng/mL) plus AZD6430 (1 μmol/L) for 72 hours. F, Representative image of involucrin-positive staining for an ALI culture from an untreated asthmatic patient. G, Dot blot to show quantitation of involucrin staining for cultures with 48- and 72-hour treatments. P values are based on 1-way ANOVA. Overall P < P values shown in the figure are based on the Tukey post hoc test. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

7 Fig E1 Images are shown at ×200 magnification. DP2 expression in the presence of corresponding blocking peptide is shown. Note the lack of staining. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

8 Fig E2 Images are shown at ×200 magnification. DP2 expression (brown staining) on a biopsy specimen from a patient with mild asthma with the AstraZeneca-sourced antibody is shown. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

9 Fig E3 Images are shown at ×200 magnification. DP2 expression (brown staining) on the same biopsy specimen as Fig E2 with the Thermo Fisher Scientific OPA antibody is shown. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

10 Fig E4 Spearman correlation graph for DP2+ epithelial cell numbers versus histology scores. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

11 Fig E5 Spearman correlation graph for DP2+ epithelial cell numbers versus involucrin scores. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

12 Fig E6 Calcium response curves (Fura-2 ratio, 340/380 nm) for 3 healthy control donors to 1 μmol/L DK-PGD2 on submerged bronchial epithelial cells. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

13 Fig E7 Dot plot to show change in Fura-2 ratio (340/380 nm [peak/basal]) for 3 healthy control donors to 1 μmol/L DK-PGD2, 1 μmol/L DK-PGD2 plus 1 μmol/L AZD6430, and 1 μmol/L ionomycin on submerged bronchial epithelial cells. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

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