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The Effect of Western Diet on the Transcriptional Memory of Monocyte-Derived Cells

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The Effect of Western Diet on the Transcriptional Memory of Monocyte-Derived Cells The effect of Western diet on the transcriptional memory of monocyte-derived cells Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Anna-Lena Hardt aus Köln-Porz, Deutschland Bonn, Mai 2020 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn. 1. Gutachter: Prof. Dr. med. Joachim L. Schultze 2. Gutachter: PD. Dr. Marc Beyer Tag der Promotion: 13.11.2020 Erscheinungsjahr: 2020 1 Table of content Table of content ......................................................................................................... 2 Abbreviations ............................................................................................................ 6 Summary .................................................................................................................. 12 1. Introduction ......................................................................................................... 14 1.1 The innate immune system ................................................................................. 14 1.2 Different monocyte subtypes ............................................................................... 15 1.3 Monocyte derived cells ........................................................................................ 16 1.3.1 Pattern recognition and induction of receptor signaling pathways ............. 20 1.4 Trained immunity and tolerance on monocytes and monocyte derived cells ....... 22 1.5 Obesity and diet induced diseases ...................................................................... 24 1.5.1 Dietary fatty acids ...................................................................................... 25 1.5.2 Oleic acid (OA) and palmitic acid (PA) as representatives of unsaturated and saturated fatty acids in diet and their effect on insulin resistance ................ 27 1.5.3 Free fatty acids and chronic inflammation .................................................. 29 1.6 The impact of fatty acid metabolism on myeloid cell differentiation ..................... 29 2. Objectives ............................................................................................................ 32 3. Materials ............................................................................................................... 33 3.1. Chemicals and reagents ..................................................................................... 33 3.2. Cytokines and fatty acids .................................................................................... 34 3.3. Antibodies ........................................................................................................... 34 3.4. PCR primers ....................................................................................................... 35 3.4.1 RT- PCR primers for ChIP quality control .................................................. 36 3.4.2 qPCR primers for fatty acid stimulation quality control ............................... 36 3.5 ATAC primers ...................................................................................................... 37 3.5. Plastic ware ........................................................................................................ 38 3.6. Equipment .......................................................................................................... 39 3.7. Buffers and media .............................................................................................. 40 3.8. Kits ..................................................................................................................... 42 3.9. Software ............................................................................................................. 42 4. Methods ................................................................................................................ 43 4.1 PMBC isolation from buffy coats using ficol ......................................................... 43 2 4.2 Monocyte isolation by CD14+ selection ............................................................... 43 4.3 Monocyte isolation by negative depletion: ........................................................... 43 4.4 Flow cytometry .................................................................................................... 44 4.4.1 Purity check of monocytes: ........................................................................ 44 4.4.2 Annexin-V-PI staining ................................................................................ 44 4.5 Presto-blue cell viability staining .......................................................................... 45 4.6 Cell fixation .......................................................................................................... 45 4.6.1 Formalin fixation (iChIP) ............................................................................ 45 4.6.2 Formalin fixation (ChIP-Nexon) .................................................................. 46 4.7 Immunoprecipitation (IP) ..................................................................................... 46 4.8 ChIP-methods ..................................................................................................... 47 4.8.1 Indexing-first chromatin immunoprecipitation approach (iChIP) ................. 47 4.8.2 NEXSON (nuclei extraction by sonication) - ChIP...................................... 48 4.9 ATAC-sequencing ............................................................................................... 49 4.9.1 ATAC-procedure with in-house Tn5 ........................................................... 49 4.10 Western-blot ...................................................................................................... 50 4.10.1 Wet blotting .............................................................................................. 51 4.11 Trained immunity experiments .......................................................................... 51 4.11.1 Donor information .................................................................................... 51 4.11.2 Fatty acid preparation and culturing of cells ............................................. 52 4.12 RNA isolation from monocytes and cDNA synthesis ......................................... 53 4.12.1 RNA isolation from monocytes for quality control qPCR .......................... 53 4.12.2 cDNA synthesis by reverse transcription ................................................. 53 4.13 Semi-quantitative real-time PCRs ..................................................................... 55 4.13.1 Validation of trained immunity pre-experiments using the LightCycler® 480 probes master .............................................................................................. 55 4.13.2 Validation of ChIP quality using the maxima SYBR green/fluorescein qPCR master ...................................................................................................... 56 4.14 Enzyme linked immunosorbent assay (ELISA) .................................................. 57 4.15 RNA-sequencing ............................................................................................... 57 4.15.1 Isolation of RNA following sequencing ..................................................... 57 4.16 Bioinformatic data analysis ................................................................................ 58 4.16.1 Identification of differentially expressed genes ........................................ 58 4.16.2 Coregulation networks and comparative bioinformatics ........................... 59 3 5. Results ................................................................................................................. 60 5.1 Set-up of trained immunity experiment ................................................................ 60 5.1.1 Set-up of standard conditions .................................................................... 61 5.2. Chromatin immuno-precipitation followed by sequencing (ChIP-seq.) ............... 74 5.2.1 Indexing-first chromatin immunoprecipitation approach (iChIP) ................. 74 5.2.2. Nuclei extraction by sonication (NEXSON) chromatin preparation method for ChIP-seq ........................................................................................................ 78 5.3 Assay for transposase-accessible chromatin with high throughput sequencing (ATAC-Seq.) .............................................................................................................. 81 5.4 Trained immunity experiment on monocytes ....................................................... 89 5.4.1 Main experiment ........................................................................................ 89 5.5 Transcript analysis of monocyte-derived cells after 24 hours of training ............. 98 5.5.1 Data preprocessing .................................................................................... 99 5.5.2 Dimensionality reduction ............................................................................ 99 5.5.3 Variance analysis ..................................................................................... 100 5.5.4 Statistical ANOVA model to describe differentially expressed genes....... 100 5.5.5 Functional interpretation of transcript analysis ......................................... 102 5.5.6 Co-expression network
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