The role of ATG16L1 in chronic inflammatory bowel disease Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von: Janne Böck Kiel, 2012 Referent/in: Prof. Dr. Rosenstiel Korreferent/in: Prof. Dr. Dr. Bosch Tag der mündlichen Prüfung: 26.06.2012 Zum Druck genehmigt: 02.07.2012 gez. Prof. Dr. Lutz Kipp (Dekan) Table of contents 1 INTRODUCTION ......................................................................................... 1 1.1 The role of autophagy in cell homeostasis and inflammation ....................................... 1 1.2 The role of autophagy in endoplasmic reticulum stress ................................................ 4 1.3 The autophagy protein ATG16L1 ...................................................................................... 6 1.3.1 The role of ATG16L1 in Paneth cell vesicle export ...................................................... 7 1.3.1.1 Paneth cells ......................................................................................................... 7 1.3.1.2 ATG16L1 and Paneth cell vesicle export ............................................................ 8 1.4 Inflammatory bowel disease .............................................................................................. 9 1.4.1 Genetics in inflammatory bowel disease .................................................................... 10 1.4.2 The role of ATG16L1 in Crohn disease ..................................................................... 11 1.4.3 Immunity in inflammatory bowel disease ................................................................... 12 1.4.4 Microbial flora in inflammatory bowel diseases and the link to autophagy ................ 13 1.4.5 Mouse models for intestinal inflammation .................................................................. 14 1.5 Aim of the work ................................................................................................................. 15 2 MATERIAL AND METHODS .................................................................... 17 2.1 Material .............................................................................................................................. 17 2.1.1 Organisms .................................................................................................................. 17 2.1.2 Chemical .................................................................................................................... 17 2.1.3 Media .......................................................................................................................... 18 2.1.4 Buffers and solutions .................................................................................................. 18 2.1.4.1 SDS-polyacrylamide gelelectrophoresis and Western blot ............................... 18 2.1.4.2 Immunohistochemistry ...................................................................................... 19 2.1.4.3 Cell stimulation .................................................................................................. 19 2.1.5 Antibodies................................................................................................................... 20 2.1.6 Oligonucleotides ......................................................................................................... 20 2.1.7 Marker ........................................................................................................................ 21 2.1.8 Kits ............................................................................................................................. 22 2.1.9 Electric devices and other materials .......................................................................... 22 2.1.9.1 Centrifuges ........................................................................................................ 22 2.1.9.2 Incubators .......................................................................................................... 22 2.1.9.3 Electrophoresis devices and power supplies .................................................... 22 2.1.9.4 Microscopes ...................................................................................................... 22 2.1.9.5 Other devices .................................................................................................... 22 2.1.9.6 Consumables ..................................................................................................... 22 2.2 Methods ............................................................................................................................. 23 2.2.1 Isolation of RNA ......................................................................................................... 23 2.2.2 cDNA synthesis .......................................................................................................... 23 2.2.3 Polymerase chain reaction (PCR) .............................................................................. 23 2.2.4 Reverse-transcriptase (RT) PCR ............................................................................... 24 2.2.5 Agarose gel electrophoresis....................................................................................... 24 2.2.6 Protein extraction ....................................................................................................... 24 2.2.7 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ............... 25 2.2.8 Western Blot ............................................................................................................... 25 2.2.9 Animal treatment ........................................................................................................ 25 2.2.10 Induction of DSS-colitis and determination of clinical scores ................................ 26 2.2.11 FITC dextran and BrdU administration .................................................................. 26 2.2.12 Statistical analysis ................................................................................................. 27 2.2.13 Densitometrical analysis with ImageJ .................................................................... 27 2.2.14 Immunohistochemistry ........................................................................................... 27 2.2.14.1 Processing of tissues ........................................................................................ 27 2.2.14.2 Hematoxylin-Eosin (HE) staining ....................................................................... 27 2.2.14.3 BrdU staining ..................................................................................................... 27 2.2.14.4 Immunofluorescence staining ............................................................................ 27 2.2.14.5 Electronmicroscopy and Toluidine blue staining ............................................... 28 2.2.15 Isolation of primary cells ........................................................................................ 28 2.2.15.1 Intestinal epithelial cells (IECs) ......................................................................... 28 2.2.15.2 Bone marrow derived macrophages (BMDMs) ................................................. 28 2.2.16 Stimulation of BMDMs or IECs .............................................................................. 28 2.2.17 Transgenic animals ................................................................................................ 29 2.2.17.1 Generation of Atg16l1 conditional knockout mice (∆IEC) ................................. 29 2.2.17.2 Generation of Atg16l1 knock-in mice (∆WD40) ................................................. 29 3 RESULTS .................................................................................................. 30 3.1 The role of ATG16L1 in DSS induced colitis. ................................................................. 30 3.2 Influence of ATG16L1 truncation or deletion on autophagy ........................................ 38 3.3 Evident morphological alterations in Paneth cells in ∆WD40 and ∆IEC mice ............ 41 3.4 ER-stress in intestinal epithelial cells of ∆IEC and ∆WD40 mice ................................. 56 4 DISCUSSION ............................................................................................ 59 4.1 Influence of ATG16L1 deletion on DSS-induced colitis................................................ 59 4.2 Discussion of the methodology of in vitro studies for autophagy .............................. 61 4.2.1 Estimation of autophagy ............................................................................................. 61 4.2.2 Primary cell culture of intestinal epithelial cells .......................................................... 62 4.2.3 Primary cell culture of bone marrow derived macrophages ....................................... 63 4.3 Influence of ATG16L1 truncation or deletion on autophagy ........................................ 64 4.4 Influence of ATG16L1 truncation or deletion on the secretory pathway .................... 65 4.4.1 Decreased defensin expression by ATG16L1 truncation or deletion ........................ 68 4.5 ER stress induced by truncation or deletion of ATG16L1 ............................................ 70 4.6 Involvement of the autophagy protein ATG16L1 in intestinal inflammation .............. 70 4.7 Conclusion .......................................................................................................................