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IL-17A enhances IL-13 activity by enhancing IL-13–induced signal transducer and activator of transcription 6 activation  Sara L. Hall, MS, Theresa Baker,

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Presentation on theme: "IL-17A enhances IL-13 activity by enhancing IL-13–induced signal transducer and activator of transcription 6 activation  Sara L. Hall, MS, Theresa Baker,"— Presentation transcript:

1 IL-17A enhances IL-13 activity by enhancing IL-13–induced signal transducer and activator of transcription 6 activation  Sara L. Hall, MS, Theresa Baker, MS, Stephane Lajoie, PhD, Phoebe K. Richgels, MS, Yanfen Yang, MS, Jaclyn W. McAlees, PhD, Adelaide van Lier, Marsha Wills-Karp, PhD, Umasundari Sivaprasad, PhD, Thomas H. Acciani, PhD, Timothy D. LeCras, PhD, Jocelyn Biagini Myers, PhD, Melinda Butsch Kovacic, MPH, PhD, Ian P. Lewkowich, PhD  Journal of Allergy and Clinical Immunology  Volume 139, Issue 2, Pages e14 (February 2017) DOI: /j.jaci Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Journal of Allergy and Clinical Immunology 2017 139, 462-471
Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 1 Dose-dependent effects of IL-13 and IL-17A in vivo. A-I, AHR in A/J mice treated intratracheally with IL-13 or IL-17A (Fig 1, A and B); total cell counts from BAL fluid of cytokine-treated animals (Fig 1, C and D); frequency of macrophages, lymphocytes, neutrophils, and eosinophils in the BAL fluid (Fig 1, E and F); CLCA3 and α-tubulin expression and densitometry in total lung homogenates (Fig 1, G); and total lung expression of IL-13–induced (Fig 1, H) and IL-17A–induced (Fig 1, I) transcripts after cytokine treatment. #P < .05, ##P < .01, and ###P < .001 versus PBS. +P < .05, ++P < .01, and +++P < .001 versus IL-13. Means + SEMs of 4 to 9 mice per group (pooled over 3 independent experiments) are shown. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 2 IL-13Rα2 is not required for IL-17A–mediated enhancement of IL-13–induced pathology. A-F, AHR in WT or IL-13Rα2−/− mice treated intratracheally with IL-13 or IL-17A (Fig 2, A); total cell counts from BAL fluid of cytokine-treated animals (Fig 2, B); frequency of macrophages, lymphocytes, neutrophils, and eosinophils in BAL fluid (Fig 2, C and D); and total lung expression of IL-13–induced (Fig 2, E) and IL-17A–induced (Fig 2, F) transcripts after cytokine treatment. #P < .05 and ##P < .01 versus PBS. +P < .05 and +++P < .001 versus IL-13 (Fig 2, A and B) or IL-17A (Fig 2, F). Means + SEMs of 5 to 11 mice per group (pooled over 3 independent experiments) are shown. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 3 Reciprocal coregulation of IL-13– and IL-17A–induced genes occurs in vitro. A-D, Real-time PCR analysis of C3 (Fig 3, A), Il13ra2 (Fig 3, B), Cebpd (Fig 3, C), and Lcn2 (Fig 2, D) expression in NIH/3T3 cells stimulated with IL-13, IL-17A, or both cytokines over 24 hours. ##P < .01 and ###P < .001 versus medium. +P < .05, ++P < .01, and +++P < .001 versus IL-13 (Fig 3, A and B) or IL-17A (Fig 3, C). Means ± SEMs of 4 to 8 replicates per condition are shown. Results show 1 of 3 independent experiments. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 4 IL-17A directly signals on IL-13–responsive cells to enhance gene expression. A-C, Real-time PCR analysis of C3 (Fig 4, A), Il13ra2 (Fig 4, B), and Lcn2 (Fig 4, C) expression in cocultured WT or IL-17RA−/− lung fibroblasts stimulated with IL-13, IL-17A, or both cytokines for 24 hours. #P < .05 and ###P < .001 versus medium. ++P < .01 and +++P < .001 versus IL-13 (Fig 4, A and B) or IL-17A (Fig 4, C). Means + SEMs of 3 replicates per condition are shown. Results show 1 of 3 independent experiments. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 5 IL-17A does not stabilize IL-13–induced transcripts. A and B, Percentage mRNA remaining of IL-13–induced C3 (Fig 5, A) and Il13ra2 (Fig 5, B) in NIH/3T3 cells stimulated with IL-13 or IL-13 plus IL-17A for 16 hours and incubated with actinomycin D (ActD). Real-time PCR analysis of transcript levels was evaluated over 24 hours, and t½ was calculated. Means ± SEMs of 4 to 8 replicates per condition shown. Results show 1 of 3 independent experiments. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 6 IL-17A directly enhances IL-13–driven pSTAT6. A-G, pSTAT6/STAT6 levels in NIH/3T3 cells pretreated with dimethyl sulfoxide (DMSO; Fig 6, A) or cycloheximide (Fig 6, B) and then stimulated with cytokines for 5 minutes (Fig 6, C); densitometry of pSTAT6/STAT6 expression shown in Fig 6, A and B; geometric mean fluorescence intensity of pSTAT6 by using phosphoflow in NIH/3T3 cells stimulated with cytokines over 1 hour (Fig 6, D); pSTAT6 and STAT6 expression and associated densitometry in whole lung homogenates from cytokine-treated A/J mice (means + SEMs of n = 7-10 mice per group, pooled from 3 independent experiments (Fig 6, E); or real-time PCR analysis of C3 and Il13ra2 (Fig 6, F) or Lcn2 (Fig 6, G) expression in WT and STAT6−/− lung fibroblasts stimulated with cytokines for 24 hours. #P < .05, ##P < .01, and ###P < .001 versus PBS or medium. +P < .05, ++P < .01, and +++P < .001 versus IL-13. Cyclohexamide experiments display 1 of 3 independent experiments performed. Phosphoflow cytometry and real-time PCR results are shown. Means + SEMs of 4 to 6 replicates per condition shown. Results show 1 of 2 independent experiments. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig 7 Reciprocal coregulation by IL-13 and IL-17A is conserved in human cells. A and B, Real-time PCR analysis of SERPINB4 or IL13RA2 (Fig 7, A) and LCN2 (Fig 7, B) expression in primary human NECs (n = 7) stimulated with IL-13, IL-17A, or both cytokines for 24 hours. C-E, pSTAT6 and STAT6 levels and associated densitometric analysis in Caco-2 (Fig 7, C) or A549 (Fig 7, D) cells stimulated with cytokines for 5 minutes or NECs (n = 2) stimulated with cytokines for 15 minutes (Fig 7, E). Caco-2 and A549 immunoblots are representative of 2 and 3 independent experiments, respectively. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E1 A and B, TH2 cytokine levels in BAL fluid (Fig E1, A) and total serum IgE levels (Fig E1, B) of A/J mice after intratracheal cytokine treatment. #P < .05, ##P < .01, and ###P < .001 versus PBS. Means + SEMs of 4 to 9 mice per group (pooled over 3 independent experiments) are shown. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E2 A-D, Total lung expression of IL-13 (Fig E2, A and B) and IL-17A (Fig E2, C and D) receptor subunits in A/J mice after intratracheal cytokine treatment. Means + SEMs of 4 to 9 mice per group (pooled over 3 independent experiments) are shown. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E3 Fold change of Airway Pressure Time Index (APTI) data shown in Fig 2, A (Fig E3, A), and total serum IgE levels (Fig E3, B) in BALB/c WT or IL-13Rα2−/− mice after intratracheal cytokine treatment. ##P < .01 versus PBS. +P < .05 and +++P < .001. The numbers above the IL-13 plus IL-17A–induced AHR bars indicate the fold difference in AHR between IL-13– and IL-13 plus IL-17A–exposed WT or IL-13Rα2−/− mice. Means + SEMs of 5 to 11 mice per group (pooled over 3 independent experiments) are shown. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 Fig E4 A and B, Real-time PCR analysis of C3 (Fig E4, A) and Il13ra2 (Fig E4, B) expression in NIH/3T3 cells stimulated with IL-13 (0-100 ng/mL) for 24 hours. #P < .05, ##P < .01, and ###P < .001 versus medium. Results display means ± SEMs of 4 replicates from 1 of 2 experiments performed. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

14 Fig E5 A-D, Real-time PCR analysis of -IL-13 (Fig E5, A and B) and IL-17A (Fig E5, C and D) receptor subunits in NIH/3T3 cells stimulated with IL-13, IL-17A, or both over 24 hours. Data are representative of at least 3 experiments. Results display means ± SEMs of 4 replicates from 1 of 3 experiments performed. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

15 Fig E6 A-F, Real-time PCR analysis of C3 (Fig E6, A and D), Il13ra2 (Fig E6, B and E), and Lcn2 (Fig E6, C and F) expression in cocultured C57BL/6 WT/WT or IL-17RA−/−/IL-17RA−/− lung fibroblasts stimulated with IL-13, IL-17A, or both cytokines for 24 hours. #P < .05, ##P < .01, and ###P < .001 versus medium. +P < .05 and +++P < .001 versus IL-13. Results display means + SEMs of 3 replicates from 1 of 3 experiments performed. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

16 Fig E7 A and B, Percentage of mRNA remaining of Csf3 (Fig E7, A) and Lcn2 (Fig E7, B) in NIH/3T3 cells stimulated with TNF-α or TNF-α plus IL-17A for 12 hours and incubated with actinomycin D (ActD). Real-time PCR analysis of transcript levels was evaluated over 2 hours, and t½ was calculated. Results display means ± SEMs of 4 replicates from 1 of 2 experiments performed. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

17 Fig E8 A and B, Real-time PCR analysis of C3 (Fig E8, A) and Il13ra2 (Fig E8, B) expression in NIH/3T3 cells stimulated with IL-13, IL-17A, or both cytokines before and 24 hours after incubation with actinomycin D (ActD). ##P < .01 and ###P < .001 versus medium. +P < .05 and +++P < .001 versus IL-13. Data are representative of at least 3 experiments. Results display means + SEMs of 4 replicates from 1 of 3 experiments performed. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

18 Fig E9 pSTAT6 phosphoflow staining in NIH/3T3 cells stimulated with IL-13, IL-17A, or both over 30 minutes. Results display means + SEMs of 6 replicates from 1 of 2 experiments. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

19 Fig E10 Real-time PCR analysis of IL13RA2 (A) and SERPINB4 (B) expression in HBEC3-KT cells stimulated with IL-13 (0-100 ng/mL) for 24 hours. ##P < .01 versus medium. Means ± SEMs are shown of 4 replicates per condition. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

20 Fig E11 A-J, Frequency of IL-13+IL-17A+CD4+ T cells (Fig E11, A and B), IL-13+CD4+ T cells (Fig E11, C and D), IL-17A+CD4+ T cells (Fig E11, E and F), IL-13+ γδ T cells (Fig E11, G and H), and IL-17A+ γδ T cells (Fig E11, I and J) in the lungs of cytokine-treated A/J mice. Results display means + SEMs of 4 to 6 replicates from 1 of 2 experiments performed. Journal of Allergy and Clinical Immunology  , e14DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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