Aus dem Bereich Klinische Medizin der Medizinischen Fakultät der Universität des Saarlandes, Homburg/Saar Influence of Vitamin D on Genome-Wide Expression in Natural Killer Cells Dissertation zur Erlangung des Grades eines Doktors der Medizin der Medizinischen Fakultät der UNIVERSITÄT DES SAARLANDES 2021 vorgelegt von: Konstantinos Christofyllakis geb. am.: 06.05.1988 in Athen, Griechenland Tag der Promotion: 19. April 2021 Dekan: Univ.-Prof. Dr. med- Michael D. Menger Berichterstatter: PD Dr. Frank Neumann Prof. Dr. Markus Hoth The experiments for the present doctoral thesis were conducted at the Clinic for Internal Medicine I - Oncology, Hematology, Clinical Immunology and Rheumatology, as well as at the José Carreras Institute of Saarland University in Homburg (Saar). Konstantinos Christofyllakis Saarland University Medical Center – IMED (bldg. 41) Department of Internal Medicine I Oncology, Hematology, Rheumatology and clinical Immunology Director: Prof. Dr. Stephan Stilgenbauer Kirrberger Strasse 100 66421 Homburg/Saar, Germany Mail: [email protected] TABLE OF CONTENTS Table of Contents ................................................................................................................... i List of Figures ........................................................................................................................iv List of Tables ......................................................................................................................... v 1 Summary ........................................................................................................................ 1 2 Introduction ..................................................................................................................... 2 2.1 Natural Killer Cells ................................................................................................... 2 2.1.1 Subsets of NK Cells .......................................................................................... 2 2.1.2 NK-Cell-mediated Cytotoxicity .......................................................................... 3 2.1.3 Regulation of NK-Cell Activity – NK-Cell Receptors .......................................... 5 2.1.4 Toll-Like Receptors ........................................................................................... 7 2.1.5 Antibody-Dependent Cytotoxicity ...................................................................... 8 2.2 Vitamin D ................................................................................................................. 9 2.2.1 Synthesis and Metabolism of Vitamin D .......................................................... 10 2.2.2 Role of Vitamin D in Humans .......................................................................... 10 2.2.3 Vitamin D Deficiency ...................................................................................... 11 2.2.4 Skeletal Effects of Vitamin D Supplementation ............................................... 11 2.2.5 Non-Skeletal Effects of Vitamin D Supplementation ....................................... 11 2.2.6 Vitamin D and Cancer..................................................................................... 12 2.2.7 Vitamin D and the Immune System ................................................................. 12 2.2.8 Vitamin D and Natural Killer Cells ................................................................... 13 2.3 Aims of this Thesis ................................................................................................ 14 3 Materials and Methods ................................................................................................. 15 3.1 Study Design ......................................................................................................... 15 3.1.1 Inclusion and Exclusion Criteria ...................................................................... 15 3.1.2 Data Handling and Ethical Aspects ................................................................. 15 3.1.3 Vitamin D Supplementation ............................................................................ 15 3.1.4 Sample Labeling ............................................................................................. 16 3.1.5 Participant Characteristics and Vitamin D Levels ............................................ 16 i 3.2 Materials ................................................................................................................ 17 3.2.1 Chemicals, Reagents, Buffers, Mediums and Solutions .................................. 17 3.2.2 Antibodies, Enzymes, Primers ........................................................................ 19 3.2.3 Consumables ................................................................................................. 19 3.2.4 Equipment ...................................................................................................... 20 3.3 Methods................................................................................................................. 21 3.3.1 Cell Counting .................................................................................................. 21 3.3.2 Isolation of PBMC ........................................................................................... 22 3.3.3 NK-Cell Enrichment by Magnetic Cell Sorting ................................................. 22 3.3.4 Flow Cytometry............................................................................................... 23 3.3.5 RNA Extraction ............................................................................................... 24 3.3.6 Spectrophotometry ......................................................................................... 24 3.3.7 RNA Electrophoresis ...................................................................................... 25 3.3.8 Microarray Assay ............................................................................................ 25 3.3.9 Microarray Quality Control .............................................................................. 26 3.3.10 Quantitative Real Time Polymerase Chain Reaction Validation ...................... 33 3.4 Statistical Analysis ................................................................................................. 35 3.4.1 Data Analysis Strategy ................................................................................... 35 3.4.2 Comparisons .................................................................................................. 35 3.4.3 Differentially Expressed Genes ....................................................................... 36 3.4.4 Pathway Analysis ........................................................................................... 37 4 Results ......................................................................................................................... 39 4.1 Quantitative Real Time Polymerase Chain Reaction Validation ............................. 39 4.2 Comparison I: Deficiency – Substitution................................................................. 41 4.2.1 Overview of Differentially Expressed Genes ................................................... 41 4.2.2 Differentially Expressed Genes ....................................................................... 47 4.3 Comparisons Based on Vitamin D Status und Sex ................................................ 59 4.3.1 Comparison II: Male – Female ........................................................................ 59 4.3.2 Comparison III: Differentially Expressed Genes – Female Subjects................ 61 4.3.3 Comparison IV: Differentially Expressed Genes – Male Subjects ................... 64 ii 4.3.4 Comparison V: Female vs. Male in the Vitamin D Deficient State ................... 66 4.3.5 Comparison IV: Female vs. Male after Vitamin D Supplementation ................ 69 4.4 Pathway Analysis .................................................................................................. 70 4.4.1 NK-Cell Cytotoxicity Pathway ......................................................................... 72 4.4.2 Immune System-Related Pathways – Upregulated ......................................... 74 4.4.3 Immune System-Related Pathways – Downregulated .................................... 76 4.4.4 Unsupervised Pathway Analysis ..................................................................... 81 5 Discussion .................................................................................................................... 83 5.1 Role of Pathway Analysis in Genomic Research ................................................... 83 5.2 Altered Cytokine - mRNA Profile After Vitamin D Supplementation ........................ 84 5.3 Altered Pathways After Vitamin D Supplementation .............................................. 86 5.4 Indirect Influence of Vitamin D on NK Cells ........................................................... 89 5.5 Role of Sexual Dimorphism ................................................................................... 89 5.6 Vitamin D in the Context of Autoimmunity and Chronic Inflammation ..................... 91 5.7 Critical View and Outlook ....................................................................................... 92 5.8 Conclusion ............................................................................................................. 93 6 References ..................................................................................................................