Molecular Mechanisms of Synergy Between IL-13 and IL-17A in Severe Asthma A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Immunology Graduate Program of the College of Medicine by Sara L. Hall M.S. University of Cincinnati 2011 Committee Chair: Ian P. Lewkowich, Ph.D. 1. Abstract Increased IL-17A production has been associated with more severe asthma; however, the mechanisms whereby IL-17A can contribute to IL-13-driven pathology in asthmatic patients remain unclear. In this thesis, we sought to elucidate the molecular mechanisms by which IL- 17A enhances IL-13-dependent airway pathology in patients with severe asthma using in vivo and in vitro systems. We have found that compared to mice given intratracheal (i.t.) IL-13 alone, those co-exposed to IL-13 + IL-17A demonstrate enhanced airway hyperresponsiveness (AHR), mucus production, airway inflammation, and IL-13-induced gene expression. In vitro, IL-17A directly enhanced IL-13-induced gene expression in asthma-relevant murine and human cells. In contrast to the exacerbating effect of IL-17A on IL-13-driven responses, co-treatment with IL-13 diminished IL-17A-driven gene expression in vivo and in vitro. Mechanistically, in vivo and in primary human and murine cells, the IL-17A mediated increase in IL-13-induced gene expression was associated with a rapid increase in IL-13-driven signal transducer and activator of transcription (STAT)6 phosphorylation. Disrupting protein-tyrosine phosphatase function using Na3VO4 abrogated IL-17A- mediated enhancement of IL-13-driven STAT6 phosphorylation, suggesting that the ability of IL-17A to augment IL-13 activity was driven by changes in protein-tyrosine phosphatase activity. Consistent with this, co-exposure to IL-13 + IL-17A triggered a rapid decrease in the phosphorylation of Src homology region 2 domain-containing phosphatase (SHP)-1 and SHP-2, negative regulators of IL-13 signal transduction. Pharmacologic inhibition of SHP-1 but not SHP-2 similarly abrogated IL-17A-mediated enhancement of IL-13-driven STAT6 phosphorylation. However, the ability of IL-17A-driven alterations in protein-tyrosine ii phosphatase activity to enhance cytokine signaling was specific for IL-13, as IL-17A had no effect on IL-6- and IFN-γ-dependent activation of STAT3 and STAT1, respectively. Surprisingly, the enhancement of STAT6 phosphorylation was not sufficient to explain IL-17A-mediated increases in IL-13-driven gene expression. Although IL-13 and IL-17A activate distinct cellular signaling pathways, we have identified specific transcription factors downstream of the IL-17A/Act1/TRAF6 signaling axis that influence transcriptional enhancement between IL-13 and IL-17A. Inhibition of NF-κB or C/EBPβ and C/EBPδ transcription factors partially attenuated IL-17A-mediated enhancement of IL-13-induced gene expression, while the inhibition of p38 mitogen-activated protein kinase (MAPK) completely abrogated the effect of IL-17A in cells co-exposed to IL-13 + IL-17A. However, the inhibition of p38 MAPK did not diminish IL-17A-mediated enhancement of STAT6 phosphorylation, implying that IL-17A signaling differentially regulates the accumulation of IL-13-induced STAT6 activation and gene expression. Collectively, our data suggest that IL-17A contributes to asthma pathophysiology by: 1) Negatively regulating protein-tyrosine phosphatase activity to increase the capacity of IL-13 to activate the STAT6 signaling axis, and 2) Transcriptional cooperation between IL-13-driven STAT6 and IL-17A-induced transcription factors. These data represent the first mechanistic explanation of how IL-17A can directly contribute to the pathogenesis of IL-13-driven pathology in asthma. Further, our results identify multiple novel regulators of IL-13 activity, which may be targeted in future therapies. iii iv 2. Acknowledgements Foremost, I would like to thank my mentor, Dr. Ian Lewkowich, for his patience, support, and guidance during this dissertation. It was an honor to be his first Ph.D. student. I would also like to acknowledge my committee members, Dr. Fred Finkelman, Dr. Senad Divanovic, Dr. Matthew Weirauch, and Dr. Tim Le Cras, for their thoughtful feedback and encouragement over the last five years. I would also like to thank my friends and family. Their contributions to my success, and sanity, cannot be overstated. Last, but never least, I must thank my loving and encouraging husband, James. This dissertation could not have been accomplished without his support, and I look forward to discovering the next chapter together! v Table of Contents 1. Abstract ii 2. Acknowledgements v 3. List of Original Communications xi 4. Aim of the Study 1 4.1 Statement of the Problem 2 4.2 Background and Rationale 2 4.3 Objective 4 5. Review of the Literature 5 5.1 Public Health Implications of Asthma 6 5.1.1 Asthma Pathogenesis and Epidemiology 6 5.1.2 Asthma Treatment 7 5.1.3 Severe Asthma and Corticosteroid Resistance 8 5.2 The Immunopathology of Asthma 8 5.2.1 The Development of Allergic Asthma 8 5.2.2 Innate Immunity in Allergic Inflammation 11 5.2.3 Th2 Cells and Allergic Asthma 12 vi 5.2.4 Th17 Cells and Severe Asthma 14 5.3 The Th2/Th17 Cytokine Milieu 15 5.3.1 Distinct Roles for IL-13 and IL-4 in Allergic Inflammation 15 5.3.2 IL-13 Effector Functions in Asthma 17 5.3.3 IL-13 Signal Transduction and Regulation 19 5.3.4 Distinct Features of IL-17A 22 5.3.5 IL-17A Effector Functions in Asthma 24 5.3.6 IL-17A Signal Transduction and Regulation 26 6. Materials and Methods 30 6.1 Mice 31 6.2 In Vivo Cytokine Treatment Protocol and Analysis of AHR 31 6.3 Determination of Th2 Cytokine and IgE Concentration 32 6.4 Cell Culture 32 6.4.1 Culture Conditions and In Vitro Cytokine Treatment 32 6.4.2 Lung Fibroblast Isolation and Culture 33 6.4.3 CD11c Enrichment of Dendritic Cells 34 6.4.4 Human Subjects and Nasal Epithelial Cell Sampling 34 6.5 DNA Constructs, Promoter Cloning, and Mutagenesis 35 6.6 Luciferase Assay 35 6.7 mRNA Stability Assay 36 6.8 Inhibitor Assays 36 vii 6.8.1 Cycloheximide Assay 36 6.8.2 Protein-Tyrosine Phosphatase Inhibitor Assays 37 6.8.3 NF-κB, C/EBP, and MAPK Inhibitor Assays 37 6.9 Flow Cytometry 38 6.10 Immunoblotting and Immunoprecipitation 38 6.11 RNA Purification and Real-Time PCR 39 6.12 Statistical Analysis 39 7. IL-17A Enhances IL-13 Activity by Enhancing IL-13-Induced 41 STAT6 Phosphorylation 7.1 Introduction 42 7.2 Results 43 7.2.1 IL-17A Increases IL-13-Induced Lung Pathology In Vivo 43 7.2.2 Reciprocal Co-Regulation of IL-13- and IL-17A-Induced Genes 44 In Vivo 7.2.3 IL-13Rα2 is not Required for IL-17A-Mediated Enhancement of 45 IL-13 Pathology 7.2.4 Reciprocal Co-Regulation of IL-13- and IL-17A-Induced Genes 46 In Vitro 7.2.5 IL-17A-Mediated Enhancement of IL-13-Induced Gene Express- 46 ion Requires Functional IL-13 and IL-17A Signaling Complexes in the Same Cell 7.2.6 IL-17A does not Enhance the Stability of IL-13-Induced Tran- 47 viii scripts 7.2.7 IL-17A Enhances IL-13-Driven STAT6 Phosphorylation 48 7.2.8 Reciprocal Co-Regulation by IL-13 and IL-17A in Human Cells 50 8. IL-17A-Mediated Inhibition of SHP-1 Enhances IL-13 Signal 69 Transduction 8.1 Introduction 70 8.2 Results 72 8.2.1 TRAF6 and TRAF3 are not Required for IL-17A-Mediated 72 Enhancement of IL-13-Induced STAT6 Phosphorylation 8.2.2 Treatment with IL-13 and IL-17A Augments Tyrosine Kinase 74 Activity Upstream of STAT6 8.2.3 Co-Stimulation with IL-13 and IL-17A Inhibits the Phosphor- 74 ylation of SHP-1 and SHP-2 8.2.4 SHP-1 is Required for IL-17A-Mediated Enhancement of STAT6 76 Phosphorylation 8.2.5 IL-17A does not Enhance STAT3 or STAT1 Phosphorylation 77 9. Identification of Transcriptional Regulators Controlling 85 IL-17A-Mediated Enhancement of IL-13 Activity 9.1 Introduction 86 9.2 Results 86 ix 9.2.1 TRAF6 does not Contribute to the Enhancement of pSTAT6, but 87 is Required for IL-13/IL-17A Transcriptional Synergy 9.2.2 5’-Upstream Elements Mediate Enhanced Arg1 and C3 88 Promoter Activity by IL-13 and IL-17A 9.2.3 Inhibition of C/EBP Transcription Factors Attenuates Transcrip- 89 tional Cooperation Between IL-13 and IL-17A 9.2.4 NF-κB also Contributes to Transcriptional Cooperation Between 91 IL-13 and IL-17A 9.2.5 Erk MAPK Upregulates Gene Expression but not IL-13/IL-17A 91 Synergy 9.2.6 p38 MAPK is Required for Transcriptional Synergy Between 93 IL-13 and IL-17A 10. Discussion 104 10.1 Major Findings of the Study 105 10.2 Directions for Future Research 113 10.3 Conclusion 116 11. Glossary 118 12. Bibliography 121 x 3. List of Original Communications This thesis is based on the following original communications. Some related unpublished results are also included in this thesis. Chapter 7 – Hall SL, Baker T, Lajoie S, Richgels PK, Yang Y, McAlees JW, Van Lier A, Wills-Karp M, Sivaprasad U, Acciani TH, LeCras TD, Biagini Myers J, Butsch Kovacic M, and Lewkowich IP: IL-17A Enhances IL-13 Activity by Enhancing IL-13-Induced Signal Transducer and Activator of Transcription 6 Activation, J Allergy Clin Immunol, 2017 Feb; 139(2):462-471.e14.