Aus der Kinderklinik und Kinderpoliklinik im Dr. von Haunerschen Kinderspital der Ludwig-Maximilians-Universität München Direktor: Prof. Dr. Dr. med. Christoph Klein Mechanisms of immune regulation during development of atopic diseases in childhood: Analysis of T cell subpopulations considering genetic and epigenetic influences Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München vorgelegt von Diana Rädler aus Wangen im Allgäu 2013 Gedruckt mit Genehmigung der Medizinischen Fakultät der Ludwig-Maximilians-Universität München Betreuer: Prof. Dr. rer. nat. Thomas Illig Zweitgutachter: Prof. Dr. rer. nat. Ludger Klein Dekan: Prof. Dr. med. Dr. h.c. M. Reiser, FACR, FRCR Tag der mündlichen Prüfung: 24.02.2014 STATUTORY DECLARATION I hereby assure that I wrote this scientific work without the inadmissible help of others and without the use of aids other than the ones listed. Thoughts directly and indirectly taken from sources are indicated as such. Munich, 31.07.2013 Diana Rädler 1. INTRODUCTION INDEX 1. Introduction………………………………………………….…………………………….…...1 1.1 Atopic diseases in childhood………...............................................................................................1 1.2 Asthma in childhood.......................................................................................................................3 1.3 T cell subsets and function..............................................................................................................4 1.4 Role of regulatory T cells and Th17 cells in the development of atopic diseases..........................8 1.4.1 Regulatory T cells..................................................................................................................8 1.4.2 Th17 cells..............................................................................................................................9 1.5 Genetic and epigenetic changes associated with atopic diseases..................................................10 1.6 Aims of the study..........................................................................................................................12 2. Materials and Methods……..………………………………..…………………………....….14 2.1 Materials.......................................................................................................................................14 2.1.1 Reagents and chemicals....................................................................................................14 2.1.2 Solutions and buffers........................................................................................................15 2.1.3 Reagent systems (Kits)......................................................................................................16 2.1.4 Consumables.....................................................................................................................16 2.1.5 Laboratory equipment......................................................................................................16 2.1.6 Software............................................................................................................................16 2.2 Methods........................................................................................................................................18 2.2.1 Study population...............................................................................................................18 2.2.1.1 PAULINA/PAULCHEN............................................................................................18 2.2.1.2 CLARA......................................................................................................................19 2.2.2 Isolation and culture of CBMCs and PBMCs...................................................................20 2.2.3 Quantitative analysis of regulatory T cells and Th17 cells..............................................21 2.2.4 Qualitative analysis of regulatory T cells.........................................................................22 2.2.5 Cytokine secretion............................................................................................................23 2.2.6 DNA and RNA extraction..................................................................................................23 2.2.7 Microarrays......................................................................................................................23 2.2.8 Quantitative RT-PCR........................................................................................................24 2.2.9 Genetics: Polymorphism selection and genotyping..........................................................25 2.2.10 Epigenetic regulation of IFN- and FOXP3………………….……………………...........26 2.2.10.1 IFN- promoter and FOXP3 TSDR methylation.………..…………………….…...26 2.2.10.2 IFN- and FOXP3 acetylation.………......................................................................27 2.2.11 Statistical analyses............................................................................................................27 I 1. INTRODUCTION 3. Results…………………………………………..…………….……………………………..…30 3.1 Immune regulation early in life (Cord blood)..............................................................................30 3.1.1 Role of TLR SNPs in innate and adaptive immunity in cord blood..................................30 3.1.1.1 TLR SNP characteristics.............................................................................................30 3.1.1.2 Impact of TLR SNPs on gene expression of Treg-associated genes and Th1, Th2 and TNF- cytokine secretion...................................................................................31 3.1.1.3 Influence of TLR2 rs4696480 and rs1898830 on TLR2 mRNA expression…….......36 3.1.2 Role of IL-10 SNPs in adaptive immunity in cord blood..................................................37 3.1.2.1 IL-10 SNP characteristics...........................................................................................38 3.1.2.2 Impact of IL-10 SNPs on cytokine secretion and mRNA expression of Treg- associated genes and IL-10.........................................................................................40 3.2 Immune regulation during the manifestation of atopic diseases in early childhood……………43 3.2.1 Prevalence of atopic diseases and wheeze in carriers of TLR SNPs.................................43 3.2.2 Prevalence of atopic dermatitis and wheeze in carriers of IL-10 SNPs............................45 3.3 Immune regulation during the manifestation of childhood asthma in school-age………...........48 3.3.1 Characterization of the CLARA study population.............................................................48 3.3.2 Immune signature of allergic and non-allergic asthmatics in regards to Treg and Th17 cells, gene expression, protein secretion and epigenetic regulation………..……..50 3.3.2.1 Immune signature of allergic asthmatics....................................................................51 3.3.2.2 Immune signature of non-allergic asthmatics............................................................59 3.3.2.3 Shared immunological profile of allergic and non-allergic asthmatics......................63 3.4 Allergic and non-allergic childhood asthma: Identification of novel differentially regulated biomarker genes and pathways by microarray analysis...............................................................67 3.4.1 Differentially expressed genes within allergic asthmatics, non-allergic asthmatics and healthy children….......................................................................................................67 3.4.2 Differentially regulated pathways within allergic asthmatics, non-allergic asthmatics and healthy children..........................................................................................................74 4 Discussion……………………………………………………………………….…….……….81 5 Summary………………………………………………………………………………….…...99 6 Zusammenfassung………………………………………..……………………………..…...102 7 References………………………………………………………………………………..…..105 8 Table Index………………………………………………………………………………......114 9 Figure Index…………………………………………………………………………….…....115 10 Abbreviations……………………………………………………………………………...…117 11 Acknowledgement……………………………………………………………………….......119 II 1. INTRODUCTION 1. INTRODUCTION 1.1 Atopic diseases in childhood The term atopy describes an inherited predisposition towards the development of a certain allergic hypersensitivity. Atopic subjects suffer from diseases like atopic eczema (atopic dermatitis), allergic asthma and allergic rhinitis (hay fever) (Biedermann and Rocken, 1999). The underlying pathophysiology is mediated by different immunoregulatory pathways and interaction of the innate and adaptive immune system. These two major parts of immune defence have to be efficiently regulated by itself and closely connected in their regulation in order to keep a healthy immune balance. Besides T cell mediated immune responses, the humoral and the mucosal immune system play a prominent role in the adaptive immune defence (Raedler and Schaub 2013, in press). A dysregulation of this balance is present in atopic individuals, with less robust cell-mediated innate immune responses to most antigens, but increased humoral (IgE-mediated) responses to allergens (Umetsu et al. 2002). Thymus-derived T cells play a critical role in cell-mediated immunity and in atopic diseases. T cells can differentiate into several