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Nuclear factor (erythroid-derived 2)-like-2 pathway modulates substance P–induced human mast cell activation and degranulation in the hair follicle  Laura.

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Presentation on theme: "Nuclear factor (erythroid-derived 2)-like-2 pathway modulates substance P–induced human mast cell activation and degranulation in the hair follicle  Laura."— Presentation transcript:

1 Nuclear factor (erythroid-derived 2)-like-2 pathway modulates substance P–induced human mast cell activation and degranulation in the hair follicle  Laura Jadkauskaite, BSc, PhD, Rajia Bahri, BSc, PhD, Nilofer Farjo, MB, ChB, BAO, LRCPSI, Bessam Farjo, MB, ChB, BAO, LRCPSI, Gail Jenkins, BSc, PhD, Ranjit Bhogal, BSc, PhD, Iain Haslam, BSc, PhD, Silvia Bulfone-Paus, MD, PhD, Ralf Paus, MD, FRSB  Journal of Allergy and Clinical Immunology  Volume 142, Issue 4, Pages e8 (October 2018) DOI: /j.jaci Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Nrf2 activity within human MCs. A, Nrf2 and MCT immunofluorescent staining in isolated human HFs. MCT+ cells (green) showed low expression of Nrf2 immunofluorescence (red) with infrequent Nrf2/MCT double-positive immunofluorescence. B, The number of Nrf2/MCT double-positive cells did not increase after 2 and 4 hours (the graph presents combined 2-hour and 4-hour treatment of SFN). C and D, Treatment with SFN and SP significantly increased expression of MCT/HO-1 double-positive cells. MCT (purple) detected in the CTS and HO-1 (red) activity was found in the HF epithelium and vasculature of CTS. (n = 3 donors; data are mean ± SEM; 1-way ANOVA; significance indicated by *P < .05; **P < .01; ***P < .001). E and F, Expression of phoshoprylated nuclear Nrf2 (red) in human primary MCs is increased after treatment with SFN (n = 3 donors; ± SD). HO-1 (G), NQO1 (H), and IL-1β (I) in PBDMCs were measured by quantitative RT-PCR and data reported as fold changes in normalized expression (n = 8 donors; data are mean ± SEM; 1-way ANOVA; significance indicated by *P < .05; **P < .01; ***P < .001). DAPI, 4′-6-Diamidino-2-phenylindole, dihydrochloride; NQO1, NADP(H) dehydrogenase-quinone 1. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Stimulation with SP induces MC degranulation and upregulates secretion of mediators. A, Gating strategy identifying expression of CD107a and CD63 in PBDMCs. B and C, Decrease in CD63 and CD107a PBDMCs after pretreatment with SFN. D, Pretreatment with SP suppressed SP-induced β-hexosaminidase release (n = 7 donors; data are mean ± SEM; paired t test; significance indicated by *P < .05). E and F, Secretion of lipid derivatives after treatment with SFN and SP (n = 4-5 donors; data are mean ± SEM; 1-way ANOVA; significance indicated by *P < .05). G-I, Toluidine blue staining indicated MCs degranulation and number within CTS of human HFs (n = 3 donors; data are mean ± SEM; 1-way ANOVA; significance indicated by *P < .05; **P < .01; ***P < .001). Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 LAD2 cells were treated with SFN to measure nuclear (DAPI) Nrf2 (green) translocation. In both, control and SFN conditions, the expression of Nrf2 (green) was cytoplasmic, with no nuclear detection (DAPI). DAPI, 4′-6-Diamidino-2-phenylindole, dihydrochloride. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Expression of MC target genes in PBDMCs. A, Expression of CD69 mRNA in PBDMCs did not change after treatment with SFN or SP. B, The mRNA levels of TNF-α were increased after pretreatment with SFN before SP (n = 8 donors; data are mean ± SEM; 1-way ANOVA; significant indicate by *P < .05). Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 The expression of Nrf2 and MCs target genes in human LAD2. HO-1 (A), NQO1 (B), CD69 (C), and IL-1β (D) in LAD2 were measured by quantitative RT-PCR and data reported as fold changes in normalized expression (n = 9 repeats; data are mean ± SEM; 1-way ANOVA; significance indicated by *P < .05; **P < .01; ***P < .001). NQO1, NADP(H) dehydrogenase-quinone 1. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E4 LAD2 degranulation after treatment with SP and SFN (1 hour). A, SP alone increased CD63 expression on LAD2 surface. Pretreatment with SFN did not reduce SP-induced activity. B, Treatment with SP induced CD107a expression on LAD2, but SFN pretreatment did not have an effect against SP. C, Pretreatment with SFN before SP exposure did not reduce release of β-hexosaminidase (n = 3-4 repeats; data are mean ± SEM). Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E5 PBDMCs degranulation after treatment with SFN (24 hours) and SP (1 hour). The expression of both MC degranulation markers (A), CD63 (B), and CD107a (C) was not reduced after pretreatment with SFN for 24 hours (n = 4 donors; data are mean ± SEM). FSC-A, Forward scatter-area. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E6 Measurement of secretion of PGD2 and PGE2 and mRNA levels of PTGDS and PTGES in PBDMCs. A, Pretreatment with SFN for 24 hours reduced SP-induced PGD2 release. B, Stimulation with SFN for 24 hours had a minimal suppression effect of PGE2 release during SP exposure. (n = 4 donors; data are mean ± SEM; 1-way ANOVA; significance indicated by **P < .01). C, The expression of PTGD mRNA was not affected by SFN and SP treatment. D, The PTGE mRNA levels were variable between 2 donors and no effect of SP and SFN was observed. (n = 2-5 donors; data are mean ± SD). Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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