Hypoxia and hypoxia-inducible factor 1α modulate the immune response of human dendritic cells against Aspergillus fumigatus Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Mirjam Fließer Geboren in Nürnberg Würzburg 2015 Eingereicht am: 11. August 2015 Mitglieder der Promotionskommission: Vorsitzender: Herr Prof. Dr. Thomas Dandekar Gutachter : Herr Prof. Dr. Jürgen Löffler Gutachter: Herr Prof. Dr. Thomas Rudel Tag des Promotionskolloquiums: 14. Oktober 2015 Doktorurkunde ausgehändigt am: Contents Summary .................................................................................................... 1 Zusammenfassung .................................................................................. 3 1 Introduction........................................................................................ 5 1.1 Aspergillus fumigatus as a human pathogen ..................................... 5 1.1.1 Clinical relevance of A. fumigatus infections .............................................................. 5 1.1.2 Biology and life-cycle of A. fumigatus ........................................................................... 6 1.1.3 A. fumigatus associated diseases .................................................................................... 7 1.2 Host immune responses against A. fumigatus ............................... 11 1.2.1 Components of the anti-fungal immune response................................................. 11 1.2.2 Pattern recognition of A. fumigatus ............................................................................. 14 1.2.2.1 The fungal cell wall is the target for immune recognition ........................................................... 14 1.2.2.2 PRRs are crucial for the recognition of A. fumigatus ...................................................................... 15 1.2.2.3 Dectin-1 is the major PRR for recognition of A. fumigatus .......................................................... 16 1.2.2.4 TLRs play a role in the recognition of A. fumigatus ........................................................................ 18 1.2.2.5 Soluble PRRs involved in anti-A. fumigatus responses ................................................................. 19 1.2.3 Dendritic cells and initiation of acquired immunity ............................................. 20 1.2.3.1 Recent developments in the classification of human DC subsets ............................................. 20 1.2.3.2 Role of DCs in anti-A. fumigatus immune responses ...................................................................... 23 1.2.3.3 T cell responses and adaptive immunity against A. fumigatus .................................................. 25 1.2.4 Model systems for studying human DCs and macrophages ............................... 27 1.3 Hypoxia, HIF-1α and invasive aspergillosis.................................... 29 1.3.1 Hypoxia at sites of A. fumigatus infections ............................................................... 29 1.3.2 Hypoxic adaption by A. fumigatus ................................................................................ 30 1.3.3 HIF-1α as the central regulator of the host hypoxic response .......................... 32 1.3.4 HIF-1α is involved in antimicrobial defense ............................................................ 34 1.3.5 Relevance of HIF-1α and hypoxia for DC functions ............................................... 36 1.4 Aim of Thesis ............................................................................................. 39 2 Material and Methods .................................................................. 41 2.1 Material ....................................................................................................... 41 2.1.1 Equipment and consumables ........................................................................................ 41 2.1.2 Commercially obtained kits ........................................................................................... 43 2.1.3 Buffers, cell culture media and reagents ................................................................... 44 2.1.4 Primers and siRNAs ........................................................................................................... 47 2.1.5 Antibodies ............................................................................................................................. 48 2.1.6 Software ................................................................................................................................. 49 2.2 Methods ....................................................................................................... 51 2.2.1 Cultivation of A. fumigatus .............................................................................................. 51 2.2.2 Human immune cell culture ........................................................................................... 51 2.2.2.1 Isolation of PBMCs ......................................................................................................................................... 51 2.2.2.2 Cryopreservation of PBMCs ...................................................................................................................... 52 2.2.2.3 Magnetic activated cell sorting ................................................................................................................ 52 2.2.2.4 DC generation from CD14+ monocytes ................................................................................................. 54 2.2.2.5 Macrophage generation from CD14+ monocytes ............................................................................. 54 2.2.3 Experimental setups ......................................................................................................... 55 2.2.3.1 Hypoxic cell culture and immune cell stimulation .......................................................................... 55 2.2.3.2 T cell activation assay .................................................................................................................................. 55 2.2.4 Gene expression analysis ................................................................................................ 56 2.2.4.1 RNA isolation ................................................................................................................................................... 56 2.2.4.2 Two-step real-time reverse transcription polymerase chain reaction .................................. 57 2.2.4.3 RNA interference ............................................................................................................................................ 59 2.2.4.4 Gene expression profiling (microarray analysis) ............................................................................ 59 2.2.5 Protein analysis ................................................................................................................... 61 2.2.5.1 Flow cytometry ............................................................................................................................................... 61 2.2.5.2 Cytokine quantification ............................................................................................................................... 61 2.2.5.3 Protein extraction and immunoblotting .............................................................................................. 63 2.2.6 Metabolic quantification .................................................................................................. 65 2.2.7 Statistics ................................................................................................................................. 65 2.2.8 Ethics statement ................................................................................................................. 65 3 Results ............................................................................................... 67 3.1 Generation of monocyte-derived macrophages and DCs ........... 67 3.2 Functional characterization of DCs under hypoxia ...................... 71 3.2.1 DC viability under hypoxic culture conditions ....................................................... 71 3.2.2 Gene expression profiles of DCs under strong hypoxia (0.1 % O2) ................ 73 3.2.2.1 Preparation, performance and validation of the 0.1 % O2 microarrays ................................ 73 3.2.2.2 Analysis of the 0.1 % O2 microarray data ............................................................................................ 77 3.2.3 Gene expression profiles of DCs under moderate hypoxia (1 % O2) .............. 84 3.2.4 Influence of hypoxia (1 % O2) on DC cytokine release ......................................... 92 3.2.5 Effects of hypoxia on T cell related DC functions ................................................... 97 3.2.5.1 Maturation of DCs under hypoxia ........................................................................................................... 97 3.2.5.2 T cell stimulatory capacity of DCs matured under hypoxia ........................................................ 99 3.2.6 Glycolytic activity in DCs under hypoxia ................................................................ 101 3.3 Role of HIF-1α in DC responses against A. fumigatus ................ 103 3.3.1 Analysis of HIF-1α protein in DCs and macrophages ........................................ 103 3.3.1.1 Establishment