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The steroidogenic enzyme Cyp11a1 is essential for development of peanut-induced intestinal anaphylaxis  Meiqin Wang, MD, PhD, Julita Ramirez, DVM, PhD,

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Presentation on theme: "The steroidogenic enzyme Cyp11a1 is essential for development of peanut-induced intestinal anaphylaxis  Meiqin Wang, MD, PhD, Julita Ramirez, DVM, PhD,"— Presentation transcript:

1 The steroidogenic enzyme Cyp11a1 is essential for development of peanut-induced intestinal anaphylaxis  Meiqin Wang, MD, PhD, Julita Ramirez, DVM, PhD, Junyan Han, PhD, Yi Jia, PhD, Joanne Domenico, BS, Max A. Seibold, PhD, James R. Hagman, PhD, Erwin W. Gelfand, MD  Journal of Allergy and Clinical Immunology  Volume 132, Issue 5, Pages e8 (November 2013) DOI: /j.jaci Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Cyp11a1 is expressed in mouse jejunum. A, Protocol for induction of peanut allergy. B, Cyp11a1 mRNA expression detected using quantitative RT-PCR in peanut-sensitized and challenged versus sham-sensitized and peanut-challenged mice. C, Representative immunohistochemical staining for Cyp11a1 (magnification ×200). Bar = 100 μm. D, Quantitation of mucosal Cyp11a1-expressing cells. Results are from 3 independent experiments, and each experiment included 4 mice per group (n = 12). *P < .05 and **P < .01. PBS/PE, Sham sensitized and peanut challenged; PE/PE, peanut sensitized and challenged. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Inhibition of Cyp11a1 enzymatic activity does not affect Cyp11a1 protein and mRNA expression in the mouse jejunum. A, Pregnenolone levels were assessed in sera of mice. B, Cyp11a1 mRNA expression in jejunum of mice treated with AMG or vehicle. C, Quantitation of mucosal Cyp11a1-expressing cells. Results are from 3 independent experiments, and each experiment included 4 mice per group (n = 12). *P < .05 and **P < .01. n.s., Not significant; PBS/PE, sham sensitized and peanut challenged; PE/PE, peanut sensitized and challenged; PE/PE/AMG 20 mg/kg, peanut-sensitized and challenged and treated with AMG at 20 mg/kg. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Inhibition of Cyp11a1 enzymatic activity in vivo reduces intestinal responses. A, Kinetics of the development of diarrhea after treatment with AMG (Cyp11a1 inhibitor) versus vehicle. B, Scores based on the severity of clinical signs were assessed 30 minutes after oral challenge. C and D, Quantitation of mucosal mast cell and goblet cell numbers in jejunum. E, Plasma histamine levels were assessed within 30 minutes of the last oral challenge. Results are from 3 independent experiments, and each experiment included 4 mice per group. *P < .05, **P < .01, and #P < .001. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Effects of Cyp11a1 inhibition on cytokine and lineage-specific transcription factor expression in the mouse jejunum. A, IFNG, IL4, IL13, and IL17A mRNA expression in jejunum of mice treated with AMG or vehicle. B, TH1, TH2, and TH17 transcription factors T-bet, GATA3, and RORγt expression in jejunum of mice treated with AMG or vehicle. Results are from 3 independent experiments (n = 12). *P < .05. n.s., Not significant. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Inhibition of Cyp11a1 enzymatic activity suppresses differentiation of naive CD4 T cells into TH2 and TH17 cells without affecting lineage-specific transcription factor and Cyp11a1 expression. A, Relative Cyp11a1 expression in naive CD4 T cells differentiated in vitro into TH1, TH2, and TH17 cells from spleens of naive T-cell receptor transgenic mice (OT II mice) determined using RT-PCR. B, Cyp11a1 mRNA expression in polarized CD4 T cells in the presence of AMG or vehicle. C, Western blot analysis of Cyp11a1 protein in polarized TH1, TH2, or TH17 cells treated with AMG or vehicle. D, Pregnenolone levels were assessed in supernatants of cultured CD4 T cells under TH1-, TH2-, and TH17-polarizing conditions. E, Cytokine levels in supernatants of cultured CD4 T cells treated with inhibitor or vehicle under TH1-, TH2-, and TH17-polarizing conditions. F, TH1, TH2, and TH17 cytokine and lineage-specific transcription factor mRNA expression in polarized TH1, TH2, or TH17 cells treated with inhibitor or vehicle. Data are from 3 independent experiments (n = 12). *P < .05, **P < .01, and #P < n.s., Not significant. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 5 Inhibition of Cyp11a1 enzymatic activity suppresses differentiation of naive CD4 T cells into TH2 and TH17 cells without affecting lineage-specific transcription factor and Cyp11a1 expression. A, Relative Cyp11a1 expression in naive CD4 T cells differentiated in vitro into TH1, TH2, and TH17 cells from spleens of naive T-cell receptor transgenic mice (OT II mice) determined using RT-PCR. B, Cyp11a1 mRNA expression in polarized CD4 T cells in the presence of AMG or vehicle. C, Western blot analysis of Cyp11a1 protein in polarized TH1, TH2, or TH17 cells treated with AMG or vehicle. D, Pregnenolone levels were assessed in supernatants of cultured CD4 T cells under TH1-, TH2-, and TH17-polarizing conditions. E, Cytokine levels in supernatants of cultured CD4 T cells treated with inhibitor or vehicle under TH1-, TH2-, and TH17-polarizing conditions. F, TH1, TH2, and TH17 cytokine and lineage-specific transcription factor mRNA expression in polarized TH1, TH2, or TH17 cells treated with inhibitor or vehicle. Data are from 3 independent experiments (n = 12). *P < .05, **P < .01, and #P < n.s., Not significant. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 6 shRNA-mediated silencing of Cyp11a1 regulates levels of IL-13 without affecting levels of GATA3 transcripts in TH2 cells. A, Cyp11a1 mRNA expression in shRNA-transduced TH2 cells. B, Pregnenolone levels were assessed in supernatants of cultured TH2 cells transduced with Cyp11a1 or luc shRNA. C, Levels of IL4, IL13, and GATA3 mRNA expression in cultured TH2 cells transduced with Cyp11a1 or luc shRNA. D, Levels of IL-4 and IL-13 in supernatants of cultured TH2 cells transduced with Cyp11a1 or luc shRNA. Results are from 3 independent experiments (n = 12). *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig 6 shRNA-mediated silencing of Cyp11a1 regulates levels of IL-13 without affecting levels of GATA3 transcripts in TH2 cells. A, Cyp11a1 mRNA expression in shRNA-transduced TH2 cells. B, Pregnenolone levels were assessed in supernatants of cultured TH2 cells transduced with Cyp11a1 or luc shRNA. C, Levels of IL4, IL13, and GATA3 mRNA expression in cultured TH2 cells transduced with Cyp11a1 or luc shRNA. D, Levels of IL-4 and IL-13 in supernatants of cultured TH2 cells transduced with Cyp11a1 or luc shRNA. Results are from 3 independent experiments (n = 12). *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E1 Decreased mast cell infiltration in the intestinal wall of PE/PE mice treated with AMG. Intestinal mucosal mast cells were quantified in jejunum by using chloroacetate esterase staining. Representative sections of PBS/PE/vehicle mice (A), PE/PE/vehicle mice (B), PE/PE/AMG (5 mg/kg) mice (C), PE/PE/AMG (10 mg/kg) mice (D), and PE/PE/AMG (20 mg/kg) mice (E) are shown (magnification ×400). Bar = 100 μM. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E2 Decreased numbers of goblet cells in the intestinal epithelium of sensitized and challenged mice treated with AMG. Goblet cells were identified by means of periodic acid-Schiff staining 24 hours after the last challenge. Representative sections of PBS/PE/vehicle mice (A), PE/PE/vehicle mice (B), PE/PE/AMG (5 mg/kg) mice (C), PE/PE/AMG (10 mg/kg) mice (D), and PE/PE/AMG (20 mg/kg) mice (E) are shown (magnification ×200). Bar = 100 μM. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E3 Treatment with AMG had no effect on serum immunoglobulin production in peanut-sensitized and challenged mice. Serum peanut-specific IgE, IgG1, and IgG2a levels were assessed by means of ELISA 24 hours after the last challenge and expressed as the OD of diluted serum, as described in the Methods section. Results were obtained from 3 individual experiments with 4 mice per group. #P < n.s., Not significant. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 Fig E4 Treatment with AMG had no effect on plasma estradiol production in peanut-sensitized and challenged mice. Plasma levels of estradiol were assessed by means of ELISA 30 minutes after the last challenge. Results were obtained from 3 individual experiments with 4 mice per group. n.s., Not significant. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

14 Fig E5 Dose-dependent inhibitory effects of AMG on cytokine production in in vitro polarized TH cells. Cytokine levels in supernatants of cultured CD4 T cells treated with different doses of the inhibitor or vehicle under TH1-, TH2-, and TH17-polarizing conditions. *P < .05. n.s., Not significant. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

15 Fig E6 Cell viability in the presence of different concentrations of AMG in in vitro cultures of polarized TH cells. Results were obtained from 3 individual experiments. *P < .05. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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