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Annale D890ahlfors.Pdf (5.211Mb) TURUN YLIOPISTON JULKAISUJA ANNALES UNIVERSITATIS TURKUENSIS SARJA - SER. D OSA - TOM. 890 MEDICA - ODONTOLOGICA INTERLEUKIN-4 INDUCED LEUKOCYTE DIFFERENTIATION by Helena Ahlfors TURUN YLIOPISTO UNIVERSITY OF TURKU Turku 2009 From Turku Centre for Biotechnology, University of Turku and Åbo Akademi University; Department of Medical Biochemistry and Molecular Biology, University of Turku and National Graduate School of Informational and Structural Biology Supervised by Professor Riitta Lahesmaa, M.D., Ph.D. Turku Centre for Biotechnology University of Turku and Åbo Akademi University Turku, Finland Reviewed by Professor Risto Renkonen M.D., Ph.D. Transplantation laboratory Haartman Institute University of Helsinki Helsinki, Finland and Docent Panu Kovanen, M.D., Ph.D. Haartman Institute Department of Pathology University of Helsinki Helsinki, Finland Opponent Assistant Professor Mohamed Oukka, Ph.D. Seattle Children’s Research Institute Department of Immunology University of Washington Seattle, USA ISBN 978-951-29-4183-4 (PRINT) ISBN 978-951-29-4184-1 (PDF) ISSN 0355-9483 Painosalama Oy – Turku, Finland 2009 Think where mans glory most begins and ends, and say my glory was I had such friends. William Butler Yeats (1865 – 1939) ABSTRACT Helena Ahlfors Interleukin-4 induced leukocyte differentiation Turku Centre for Biotechnology, University of Turku and Åbo Akademi University Department of Medical Biochemistry and Genetics, University of Turku National Graduate School of Informational and Structural Biology, 2009 Monocytes, macrophages and dendritic cells (DCs) are important mediators of innate immune system, whereas T lymphocytes are the effector cells of adaptive immune responses. DCs play a crucial role in bridging innate and adaptive immunity. Naïve CD4+ Th progenitors (Thp) differentiate to functionally distinct effector T cell subsets including Th1, Th2 and Th17 cells, which while being responsible for specific immune functions have also been implicated in pathological responses, such as autoimmunity, asthma and allergy. The main objective of this thesis is to dissect the signaling networks involved in the IL-4 induced differentiation of two important leukocyte subtypes, Th2 cells and DCs. Gene expression profiling lead to identification of over 200 genes which are differentially expressed during cytokine induced differentiation of human monocytes to DCs or macrophages and which are likely to be essential for the proper biological functions of these cell types. Transcriptome analysis demonstrated the dynamic regulation of gene expression by IL-12 and IL-4 during the initiation of Th cell differentiation, which was partly counteracted by an immunosuppressive cytokine, TGFβ, present in the culture media. Results from RNAi mediated gene knockdown experiments and global gene expression analysis elucidated that SATB1 regulates multiple genes important for Th cell polarization or function as well as may compete with GATA3 for the reciprocal regulation of IL-5 transcription. In conclusion, the results obtained have extended our system-level understanding of the immune cell differentiation processes and provide an excellent basis for the further functional studies which could lead to development of improved therapeutic approaches for a range of immunological conditions. Keywords: Dendritic cell, T helper cell differentiation, asthma, cytokine, transcription factor, gene regulation, RNA interference 4 TIIVISTELMÄ Helena Ahlfors Interleukiini-4:n säätelemä valkosolujen erilaistuminen Turun Biotekniikan keskus, Turun Yliopisto ja Åbo Akademi; Lääketieteellisen biokemian ja genetiikan laitos, Turun yliopisto Kansallinen tutkijakoulu ISB - Bioinformatiikka ja biorakenteet, 2009 Monosyytit, makrofagit ja dendriittisolut ovat tärkeitä synnynnäisessä immuunivasteessa, kun taas T-lymfosyyteillä on keskeisiä tehtäviä hankitussa immuunivasteessa. Dendriittisolut esittelevät vieraat mikrobiantigeenit lymfosyyteille ja näin yhdistävät synnynnäisen ja hankitun immuniteetin. Th-solut kehittyvät yhteisestä esiasteesta toiminnallisesti erilaisiksi alatyypeiksi, joista parhaiten tunnetaan Th1-, Th2- ja Th17-solut. Näillä Th-soluilla on spesifiset tehtävänsä immuunivasteessa, kun taas Th-solujen säätelemän immuunivasteen epätasapaino voi johtaa vakavien häiriötilojen syntyyn elimistössä, kuten autoimmuunitauteihin ja atooppisiin sairauksiin. Tämän väitöskirjatutkimuksen päätavoitteena on selvittää IL-4- sytokiinin säätelemät Th2- ja dendriittisolujen erilaistumiseen tarvittavat signalointireitit ja säätelyverkostot. Geeniekspression profiloinnilla löydettiin yli 200 geeniä, jotka ilmenevät eri tavalla sytokiinien avulla monosyyteistä erilaistetuissa dendriittisoluissa ja makrofageissa ja joiden voidaan olettaa olevan tärkeitä näiden solutyyppien biologisille toiminnoille. Th-solujen varhaisen erilaistumisen aikana IL- 12 ja IL-4 -sytokiinien vaikutus osoitettiin olevan dynaamista, ja tämä pystyttiin osittain estämään immunosupressiivisella TGFβ -sytokiinilla. RNA-interferenssillä aikaansaatu geenin hiljeneminen yhdistettynä geeniexpressioanalyysiin osoitti, että SATB1 säätelee useita Th-solujen erilaistumiselle ja toiminnalle tärkeitä geenejä sekä lisäksi saattaa kilpailla GATA3-transkriptiotekijän kanssa IL-5-sytokiinin vastavuoroisesta säätelystä. Tässä tutkimuksessa esitetyt tulokset lisäävät ymmärrystämme immuunisolujen erilaistumisprosesseista sekä tarjoavat erinomaisen perustan toiminnallisiin jatkotutkimuksiin, jotka mahdollisesti johtavat immuunivälitteisten sairauksien hoitomenetelmien kehitykseen. Avainsanat: dendriittisolu, T-auttajasolujen erilaistuminen, astma, välittäjäaine, transkriptiotekijä, geeninsäätely, RNA-interferenssi 5 CONTENTS ABBREVIATIONS ..................................................................................................................... 8 LIST OF ORIGINAL PUBLICATIONS ................................................................................ 11 1 INTRODUCTION.................................................................................................................. 12 2 REVIEW OF THE LITERATURE ...................................................................................... 13 2.1 INTERLEUKIN-4 IN THE IMMUNE SYSTEM........................................................................... 13 2.1.1 Hematopoiesis and interleukin-4 .............................................................................. 13 2.1.2 JAK-STAT signaling ................................................................................................ 14 2.1.3 Monocytes, macrophages and DCs in the immune system....................................... 14 2.1.4 T helper cells in the immune system ........................................................................ 16 2.1.5 T helper cell differentiation ...................................................................................... 18 2.2 REGULATION OF GENE EXPRESSION ................................................................................... 21 2.2.1 Cis-regulatory elements ............................................................................................ 21 2.2.2 Chromatin accessibility ............................................................................................ 23 2.2.3 Regulation of cytokine expression in CD4+ T cells ................................................. 27 3 AIMS OF THE STUDY ......................................................................................................... 37 4 MATERIALS AND METHODS .......................................................................................... 38 4.1 PLASMIDS AND OLIGONUCLEOTIDES ................................................................................. 38 4.1.1 Plasmid constructs and siRNA oligonucleotides (III, unpublished) ......................... 38 4.1.2 Preparation of probes for EMSA (III) ...................................................................... 38 4.2 CELL CULTURE .................................................................................................................. 38 4.2.1 Differentiation of monocytes to DCs and macrophages (I) ...................................... 38 4.2.2 Polarization of primary human T helper cells (II, III, unpublished) ......................... 39 4.3 TRANSFECTIONS ................................................................................................................ 40 4.3.1 Human primary T cell nucleofection (III, unpublished) ........................................... 40 4.3.2 Enrichment of transfected cells (III, unpublished) ................................................... 40 4.3.3 Transactivation assay (III) ........................................................................................ 41 4.4 GENE EXPRESSION ANALYSES ........................................................................................... 41 4.4.1 Oligonucleotide array studies ................................................................................... 41 4.4.2 Quantitative RT-PCR analyses (I-III) ....................................................................... 42 4.5 PROTEIN EXPRESSION ANALYSIS ....................................................................................... 43 4.5.1 Western blotting (I-III) ............................................................................................. 43 4.5.2 Flow cytometry......................................................................................................... 44 4.5.3 Cytokine secretion assay
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