Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2018 The cytokine TGF-฀ promotes the development and homeostasis of alveolar macrophages Yu, Xueyang Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-159840 Dissertation Published Version Originally published at: Yu, Xueyang. The cytokine TGF-฀ promotes the development and homeostasis of alveolar macrophages. 2018, University of Zurich, Faculty of Science. The Cytokine TGF-β Promotes the Development and Homeostasis of Alveolar Macrophages Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Xueyang Yu aus der V. R. China Promotionskommission Prof. Dr. Melanie Greter (Vorsitz und Leitung der Dissertation) Prof. Dr. Burkhard Becher Prof. Dr. med. Markus Manz Zürich, 2018 Disclaimer This thesis is based upon and adapted from the following publication: The Cytokine TGF-β Promotes the Development and Homeostasis of Alveolar Macrophages Xueyang Yu, Anne Buttgereit, Iva Lelios, Sebastian Utz, Dilay Cansever, Burkhard Becher, Melanie Greter Table of Contents 1 Summary ................................................................................................................ 1 2 Zusammenfassung ................................................................................................. 3 3 Abbreviations ......................................................................................................... 5 4 Introduction ........................................................................................................... 8 4.1 The Immune System .................................................................................................. 8 4.1.1 The Innate Immune System .................................................................................. 8 4.1.2 The Adaptive Immune System ........................................................................... 10 4.2 The Ontogeny and Function of Monocytes, Dendritic Cells and Tissue-Resident Macrophages ...................................................................................................................... 13 4.2.1 Dendritic Cells .................................................................................................... 13 4.2.2 Monocyte and Monocyte Derived Cells ............................................................. 15 4.2.3 Tissue-Resident Macrophages ............................................................................ 16 4.3 Alveolar Macrophages ............................................................................................. 21 4.3.1 The Function of Alveolar Macrophages ............................................................. 22 4.3.2 The Developmental Pathway of Alveolar Macrophages .................................... 23 4.4 The TGF-βR Signaling ............................................................................................ 25 4.4.1 The Complexity in TGF-βR Signaling ............................................................... 25 4.4.2 The Role of TGF-βR Signaling in the Immune System ..................................... 29 5 Aims ...................................................................................................................... 31 6 Results ................................................................................................................... 32 6.1 AMs are Dependent on TGF-βR Signaling ........................................................... 32 6.2 Autocrine TGF-β is Required for AMs .................................................................. 37 6.3 TGF-βR Signaling is Essential for the Maintenance of Mature AMs ................. 39 6.4 The Development of Monocyte-Derived ‘AMs’ after Irradiation Requires TGF- βR Signaling ....................................................................................................................... 42 6.5 Embryonic Development of AMs Depends on TGF-β .......................................... 43 6.6 TGF-β Instructs AM Differentiation and Signature Gene Expression .............. 47 6.7 PPAR-γ Agonist can not Rescue Tgfbr2-deficiency Induced AM Loss ............... 50 6.8 Flu-induced AM Loss is not Associated with the Reduced Expression of Pulmonary TGF-β and TGF-βRs. .................................................................................... 52 7 Discussion ............................................................................................................. 54 7.1 TGF-β is Essential for the Differentiation and Homeostasis of AMs. ................. 54 7.2 Regulatory Networks of AM Development ........................................................... 57 7.3 Implications in Pulmonary Function and Diseases ............................................... 60 8 Conclusion ............................................................................................................ 62 9 Methods ................................................................................................................ 63 9.1 Experimental Animals. ............................................................................................ 63 9.2 Method Details ......................................................................................................... 63 9.2.1 Genotyping .......................................................................................................... 63 9.2.2 Bone Marrow Chimeras. ..................................................................................... 64 9.2.3 Tamoxifen Treatment. ........................................................................................ 64 9.2.4 Influenza Virus Infection .................................................................................... 64 9.2.5 Rosiglitazone Administration ............................................................................. 65 9.2.6 Cell Suspension Preparations. ............................................................................. 65 9.2.7 Flow Cytometry .................................................................................................. 65 9.2.8 t-SNE Display. .................................................................................................... 66 9.2.9 Bronchoalveolar Lavage ..................................................................................... 66 9.2.10 Oil Red O Staining ........................................................................................... 66 9.2.11 H&E staining of Lung Tissue Sections ............................................................ 67 9.2.12 Immunofluorescence Staining of Lung Tissue Sections .................................. 67 9.2.13 In vitro Culture of Fetal Monocytes ................................................................. 67 9.2.14 Quantitative RT-PCR (qRT-PCR). ................................................................... 68 9.2.15 ELISA ............................................................................................................... 69 9.2.16 Next Generation Sequencing ............................................................................ 69 9.2.17 Quantification and Statistical Analysis ............................................................. 70 9.3 Data and Software Availability .............................................................................. 70 9.4 Table for Supplementary Materials ....................................................................... 70 10 References .......................................................................................................... 73 11 Acknowledgement ............................................................................................. 89 1 Summary Alveolar macrophages (AMs) are lung resident macrophages that reside in the lumen of small airways including alveoli. They play a critical role in lung homeostasis and maintain respiratory functions via surfactant clearance within the alveolar space. Like most other tissue-resident macrophages, AMs derive from fetal liver monocytes, which colonize the developing lung during embryogenesis and give rise to fully mature AMs perinatally. Recently, AM gene expression profile has been revealed and AM-specific transcription factor PPAR-γ has been identified. However, extracellular signaling mediators involved in AM development and maintenance have not been fully identified and the regulatory mechanisms related to AM genesis are still under investigation. It was previously shown that granulocyte macrophage colony stimulating factor (GM-CSF), an epithelial cell-derived cytokine is required for AM differentiation and Pparg expression, which is also critical for the development of AMs. Whether additional factors are involved in AM regulation is largely not known. Transforming growth factor-β (TGF-β), a multifunctional cytokine, is highly involved in cell fate determination and has been shown centrally involved in the genesis of Langerhans cells (LCs) and microglia activation. This indicates a possible role of TGF-β receptor (TGF-βR) signaling in regulating other myeloid cell populations. Since the role of TGF-βR signaling in the development of other macrophages is largely unknown, we investigated its function across different tissue-resident macrophages. Here we reported that AM differentiation and maintenance, in contrast to most other