INTERLEUKIN 17 AND SENESCENCE REGULATE THE FOREIGN BODY RESPONSE by Liam Chung A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland October, 2019 © 2019 Liam Chung All rights reserved Abstract Synthetic biomaterials and medical devices suffer to varying levels from fibrosis via the foreign body response (FBR). To explore mechanistic connections between the immune response and fibrosis from the FBR, we first analyzed fibrotic capsule surrounding human breast implants and found increased numbers of interleukin (IL)17-producing γδ+ T cells and CD4+ TH17 cells as well as senescent cells. Further analysis in a murine model demonstrated an early innate IL17 response to synthetic implants, mediated by innate lymphoid cells and γδ+ T cells, was followed by a chronic adaptive antigen dependent CD4+ TH17 cell response. Mice deficient in IL17 signaling established that IL17 was required for the fibrotic response to materials and the development of p16INK4a senescent cells. Treatment with a senolytic agent reduced IL17 expression and fibrosis. Discovery of a feed-forward loop between the TH17 and senescence response to synthetic materials introduces new targets for therapeutic intervention in the foreign body response. Primary Reader: Jennifer H Elisseeff, Department of Biomedical Engineering, Johns Hopkins University Secondary Readers: Drew M Pardoll, Franck Housseau, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University ii Acknowledgments The following people had a significant influence on this thesis: Dr. Jennifer Elisseeff, Dr. Drew M Pardoll, and Dr. Franck Housseau for all guidance and outstanding advices. I’d also like to thank all members of the Elisseeff, Pardoll, and Sears lab for all your contribution and support: Andriana Lebid, David Maestas Jr, Ashlie Mageau, Xinqun Wu, Isabel Vanderzee, Hongni Fan, Ada Tam, Radhika Narain, Dr. Xiaokun Wang, Dr. James I Andorko, Dr. Gedge D. Rosson, Dr. Kaitlyn Sadtler, Dr. Matthew T Wolf, Dr. Daniela Cihakova, and Dr. Claude Jourdan Le Saux. iii Content Abstract .......................................................................................................................................ii Acknowledgements ......................................................................................................................iii List of Abbreviations ...................................................................................................................vi List of Figures ..............................................................................................................................vii Chapter 1: Introduction ................................................................................................................1 1.1 Thesis Statement ..................................................................................................1 1.2 Motivation ............................................................................................................1 1.3 Key Problems .......................................................................................................2 Chapter 2: Interleukin 17 secreted by T cells is associated with fibrosis in tissue surrounding human breast implant ...................................................................................................................5 2.1 Overview ..............................................................................................................5 2.2 Results ..................................................................................................................6 2.2 Discussion ............................................................................................................7 2.2 Figures..................................................................................................................8 Chapter 3: Innate and adaptive lymphocytes sequentially contribute to chronic IL17 production in response to synthetic materials ................................................................................................11 3.1 Overview ..............................................................................................................11 3.2 Results ..................................................................................................................11 3.3 Discussion ............................................................................................................14 3.4 Figures..................................................................................................................15 Chapter 4: Chronic IL17 response to synthetic implants promotes fibrosis ................................19 4.1 Overview ..............................................................................................................19 4.2 Results ..................................................................................................................19 4.3 Discussion ............................................................................................................21 4.4 Figures..................................................................................................................24 Chapter 5: p16INK4a senescent cells develop during the FBR and senolysis reduces fibrosis ..29 5.1 Overview ..............................................................................................................29 iv 5.2 Results ..................................................................................................................29 5.3 Discussion ............................................................................................................31 5.4 Figures..................................................................................................................32 Chapter 7: Conclusion..................................................................................................................35 Chapter 6: Methods ......................................................................................................................37 6.1 Clinical samples handling and processing procedures.........................................37 6.2 Surgical procedures and implantation ..................................................................37 6.3 Bone marrow chimera ..........................................................................................39 6.4 Gene expression analysis .....................................................................................39 6.5 Histopathology .....................................................................................................41 6.6 Flow cytometry ....................................................................................................42 6.7 Treadmilling test ..................................................................................................43 6.8 IL-17 neutralization and senolytic treatment .......................................................44 6.9 Antibody titer .......................................................................................................44 6.10 TH17 in vitro polarization assay ...........................................................................45 6.11 Cell proliferation assay ........................................................................................45 6.12 Statistical analysis ................................................................................................45 References ....................................................................................................................................47 Curriculum Vitae .........................................................................................................................53 v List of Abbreviations FBR Foreign body response VML Volumetric muscle loss ILC Innate lymphoid cell DAMP Damage associated molecular pattern CD Cluster of differentiation IL Interleukin PCL Poly(caprolactone) PEG Polyethylene glycol PE Poly(ethylene) SnC Senescent cell SASP Senescent associated secretory phenotype αSMA Alpha smooth muscle actin TGFβ Transforming growth factor beta TNFα Tumor necrosis factor alpha VEGF Vascular endothelial growth factor IFNγ Interferon gamma STAT Signal transducer and activator of transcription PSR Picrosirius red Pdgfα Platelet derived growth factor alpha Vegfα Vascular endothelial growth factor alpha MHC Major histocompatibility complex Navi Navitoclax vi List of Figures Figure 1.1: IL-17 signature in human breast implants .................................................................8 Figure 1.2: CD3+ T cells are highly upregulated in human fibrotic capsule tissue .....................10 Figure 2.1 Synthetic materials induce an IL17 immune response ...............................................15 Figure 2.2: IL17 is unique signature to synthetic material but not IFNγ and IL4 .......................17 Figure 2.3: Expression prolife of immune cells from synthetic implant is proinflammatory ......18 Figure 3.1: IL17 inhibition reduces the fibrotic response to synthetic materials.........................24 Figure 3.2: IL17KO mice improve muscular recovery and regain functional outcome quicker than WT mice .............................................................................................................26 Figure 3.3: Synthetic material shows adjuvant effect upon OVA activation ..............................27 Figure 3.4: Type 17 response regulates