ANNI KLEINO The Imd Pathway-mediated Immune Response in Drosophila ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Medicine of the University of Tampere, for public discussion in the Auditorium of Finn-Medi 1, Biokatu 6, Tampere, on June 23rd, 2010, at 12 o’clock. UNIVERSITY OF TAMPERE ACADEMIC DISSERTATION University of Tampere, Institute of Medical Technology Tampere Graduate School of Biomedicine and Biotechnology (TGSBB) Tampere University Hospital, Department of Pediatrics Finland Supervised by Reviewed by Professor Mika Rämet Professor Seppo Meri University of Tampere University of Helsinki Finland Finland Professor Håkan Steiner Stockholm University Sweden Distribution Tel. +358 40 190 9800 Bookshop TAJU Fax +358 3 3551 7685 P.O. Box 617 [email protected] 33014 University of Tampere www.uta.fi/taju Finland http://granum.uta.fi Cover design by Juha Siro Acta Universitatis Tamperensis 1527 Acta Electronica Universitatis Tamperensis 966 ISBN 978-951-44-8098-0 (print) ISBN 978-951-44-8099-7 (pdf) ISSN-L 1455-1616 ISSN 1456-954X ISSN 1455-1616 http://acta.uta.fi Tampereen Yliopistopaino Oy – Juvenes Print Tampere 2010 To my family 3 Contents CONTENTS...................................................................................................4 LIST OF ORIGINAL COMMUNICATIONS ..............................................7 ABBREVIATIONS .......................................................................................8 ABSTRACT ................................................................................................11 TIIVISTELMÄ ............................................................................................13 1. INTRODUCTION ...................................................................................15 2. REVIEW OF THE LITERATURE .........................................................16 2.1 Innate immunity...............................................................................................16 2.2 NF-κB signaling cascades................................................................................17 2.2.1 TNFR signaling ...................................................................................19 2.3 Drosophila as a model organism .....................................................................21 2.4 Overview of the Drosophila immune system ..................................................23 2.5 Cellular response..............................................................................................24 2.6 Humoral response ............................................................................................26 2.6.1 Antimicrobial peptides ........................................................................27 2.6.2 The Toll pathway.................................................................................28 2.6.3 The Imd pathway.................................................................................32 2.6.4 The JNK pathway................................................................................34 2.6.5 Negative regulation of the Imd pathway .............................................35 2.6.6. Tissue-specific immune response.......................................................39 3. AIMS OF THE STUDY ..........................................................................41 4 4. MATERIALS AND METHODS............................................................ 42 4.1 Cells and cell culture (I-III) .............................................................................42 4.2 dsRNA synthesis (I-III)....................................................................................42 4.2.1 Generation of the dsRNA library ........................................................42 4.2.2 Generation of targeted dsRNAs...........................................................43 4.3 Oligonucleotide microarrays (I, II)..................................................................43 4.4. Sequencing (III) ..............................................................................................43 4.5 Western blotting (II, III) ..................................................................................44 4.6 Epistasis analysis .............................................................................................44 4.7 Assays for measuring Imd pathway activation ................................................45 4.7.1 Reporter assays (I-III)..........................................................................45 4.7.2 Semi-quantitative RT-PCR (I) and quantitative RT-PCR (qRT- PCR) (II).......................................................................................................45 4.7.3 Immunocytochemistry (III) .................................................................46 4.8 In vivo experiments..........................................................................................46 4.8.1 Drosophila stocks (I-III)......................................................................46 4.8.2 RNA extraction from flies (II).............................................................46 4.8.3 DNA extraction from flies (II) ............................................................47 4.8.4 Protein extraction from flies (II)..........................................................47 4.8.5 Infection assays (I-III) .........................................................................47 5. RESULTS ............................................................................................... 48 5.1 Gram-negative bacteria-induced changes in the gene expression profiles in S2 cells (I).............................................................................................48 5.2 Identification of CG15678/Pirk as a negative regulator of the Imd pathway (I, II) ........................................................................................................49 5.2.1 Pirk suppresses Imd pathway activation in vivo (II)...........................51 5.2.2 Characterization of Pirk’s mechanism of action (II) ...........................55 5.3 Identification of novel Imd pathway components by RNAi screening in S2 cells (III) ...........................................................................................................56 5.3.1 RNAi-based screening and validation of the screen in S2 cells (III) ...............................................................................................................56 5.4 CG7417/Tab2 as a component of the Imd pathway (III, I)..............................57 5.5 Iap2 as a regulator of the Imd pathway activation (III, I, II) ...........................59 5 5.5.1 Iap2 is required for resistance towards Gram-negative bacteria in vivo (I, III)................................................................................................60 6. DISCUSSION..........................................................................................62 6.1 Methodological aspects....................................................................................62 6.1.1 Drosophila as a model in studying innate immunity...........................62 6.1.2 Drosophila cell lines............................................................................63 6.1.3 Microarrays..........................................................................................63 6.1.4 In vitro RNAi screening ......................................................................64 6.1.5 Drosophila in vivo assays....................................................................67 6.2 Pirk as an Imd pathway negative regulator......................................................69 6.3 Iap2 and Tab2 as novel components of the Imd pathway................................72 7. CONCLUSIONS AND FUTURE PERSPECTIVES..............................76 8. ACKNOWLEDGEMENTS.....................................................................78 9. REFERENCES ........................................................................................81 10. ORIGINAL COMMUNICATIONS................................................... 102 6 List of original communications The thesis is based on the following original publications, which are referred to accordingly by their roman numerals: I Valanne S, Kleino A, Myllymäki H, Rämet M. Iap2 is required for a sustained response in the Drosophila Imd pathway. Dev Comp Immunol 31:991-1001 (2007). II Kleino A, Myllymäki H, Kallio J, Vanha-aho L-M, Oksanen K, Ulvila J, Hultmark D, Valanne S, Rämet M. Pirk is a negative regulator of the Drosophila Imd pathway. J Immunol 180:5413- 5422 (2008). III Kleino A1, Valanne S1, Ulvila J, Kallio J, Myllymäki H, Enwald H, Stöven S, Poidevin M, Ueda R, Hultmark D, Lemaitre B, Rämet M. Inhibitor of apoptosis 2 and TAK1-binding protein are components of the Drosophila Imd pathway. EMBO J 24:3423-3434 (2005).2 1 Equal contribution. 2 The publication No III was also used in the doctoral thesis of Johanna Ulvila. The original publications are reproduced in this thesis with the permission of the copyright holders. 7 Abbreviations aa amino acid Ab antibody AMP antimicrobial peptide Att Attacin BIR baculovirus IAP repeat Bc black cells bp base pair bsk basket, Drosophila JNK CARD caspase recruitment domain Cec Cecropin cDNA complementary DNA C-terminal carboxy-terminal da daughterless DAP diaminopimelic acid DD death domain Dif Dorsal-related immunity factor DNA deoxyribonucleic acid Dnr1 Defence repressor 1 Dpt Diptericin Dredd Death-related ced-3/Nedd2-like protein Dro Drosocin Drs Drosomycin Dscam