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University of Alberta University of Alberta T cell-mediated inflammation is stereotyped: mouse delayed-type hypersensitivity reaction and mouse T cell-mediated rejection of renal allografts share common molecular mechanisms by Jeffery M. Venner A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Experimental Medicine Department of Medicine ©Jeffery M. Venner Fall 2011 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission. Dedicated to those who have inspired me: My family and friends, my teachers and mentors. You laid the foundation – my success is guaranteed ABSTRACT Genome-wide gene expression analysis of diseases has revealed large-scale changes in the expression of thousands of genes (transcripts) representing biological processes. The processes that occur during T cell-mediated rejection (TCMR) of renal allografts in mice and humans have been previously delineated, and they appear to be independent of cytotoxic mechanisms; thus, TCMR is analogous to delayed-type hypersensitivity (DTH). Since TCMR and DTH involve similar mechanisms, we hypothesized the molecular changes in TCMR are stereotyped; thus, they are qualitatively the same as those in DTH. Using microarrays to compare the transcript expression changes of both diseases in mice, we found they share the same processes: T cell and macrophage infiltration, IFNG-effects, alternative macrophage activation, and a coordinated injury-repair response of the tissue parenchyma. Additional analysis revealed IFNG is vital for stabilizing the injury-repair response in TCMR and DTH. We conclude the molecular changes in TCMR and DTH involve stereotyped, coordinated processes. TABLE OF CONTENTS CHAPTER 1: INTRODUCTION PAGE 1.1 Background .......................................................................................... 1 1.2 Transplantation and Allograft Rejection Overview ............................... 2 1.3 Revealing the Herd-Movement in TCMR of Renal Allografts............... 8 1.4 Delayed-Type Hypersensitivity ............................................................. 13 1.5 Project Synopsis and Justification ........................................................ 15 CHAPTER 2: MATERIALS AND METHODS 2.1 Overview .............................................................................................. 17 2.2 Mice ...................................................................................................... 17 2.3 Mouse Experiments .............................................................................. 18 2.4 Tissue RNA Extraction and RNA Expression Analysis ........................ 20 2.5 Mouse Histology and Immunohistochemistry ...................................... 23 2.6 Tables ................................................................................................... 25 CHAPTER 3: COMPARISON OF THE MOLECULAR CHANGES IN MOUSE CONTACT HYPERSENSITIVITY AND T CELL MEDIATED REJECTION IN MOUSE RENAL ALLOGRAFTS 3.1 Overview .............................................................................................. 29 3.2 Establishment of the CHS Model ......................................................... 30 3.3 The Inflammatory Phenotype is Shared Between CHS and Renal Allografts with TCMR ............................................................................ 31 3.4 Transcripts Related to Injury-Repair Processes are Tissue-Selective, but Represent Similar Pathways .......................................................... 34 3.5 Summarizing the Transcript Changes as Transcript Set Scores Reveal Parallel Molecular Changes in CHS and TCMR ...................... 37 3.6 Tables ................................................................................................... 38 3.7 Figures ................................................................................................. 82 CHAPTER 4: INTERFERON-GAMMA-DEPENDENT MOLECULAR CHANGES IN OXAZOLONE-INDUCED CONTACT HYPERSENSITIVITY 4.1 Overview .............................................................................................. 93 4.2 IFNG-Dependent Changes in a Cutaneous Oxazolone-Challenge in Mice ...................................................................................................... 94 4.3 Tables ................................................................................................... 101 4.4 Figures ................................................................................................. 107 CHAPTER 5: DISCUSSION 5.1 Study Summary Part 1: A Comparison of DTH and TCMR ................. 120 5.2 Molecular Changes of T Cell-Mediated Inflammation .......................... 121 5.3 The Active Injury-Repair Response Reflects a Large-Scale Anti- Parallel Shift in the Expression of Transcripts ..................................... 125 5.4 Conclusions Part 1: Molecular Changes are Stereotyped ................... 127 5.5 Study Summary Part 2: IFNG-Dependent Changes ............................ 128 5.6 Conclusions Part 2: IFNG-Dependent Changes in CHS ...................... 131 5.7 Additional Conclusions and Future Directions ..................................... 132 REFERENCES ............................................................................................................ 134 LIST OF TABLES CHAPTER 2: MATERIALS AND METHODS PAGE 2.1 Summary of mice used in the contact hypersensitivity experiments ... 26 2.2 Summary of mice used in the kidney allograft experiments................. 26 2.3 Mouse gene-specific primers and probes used in real-time RT-PCR, using an ABI PRISM 7900 Sequence Detector ................................... 27 2.4 Pathogenesis-based transcript sets (PBTs) ......................................... 28 CHAPTER 3: COMPARISON OF THE MOLECULAR CHANGES IN MOUSE CONTACT HYPERSENSITIVITY AND T CELL MEDIATED REJECTION IN MOUSE RENAL ALLOGRAFTS 3.1 Histological data of mouse renal allografts .......................................... 39 3.2 Unique cytotoxic T cell-associated transcripts found in both oxazolone-challenged skin versus sham-treated skin, and CBA into B6 renal allografts at day 7 versus normal CBA kidneys ..................... 40 3.3 Unique IFNG-induced transcripts (IFNG-effects) found in both oxazolone-challenged skin versus sham-treated skin, and CBA into B6 renal allografts at day 7 versus normal CBA kidneys ..................... 41 3.4 Unique macrophage-associated transcripts found in both oxazolone-challenged skin versus sham-treated skin, and CBA into B6 renal allografts at day 7 versus normal CBA kidneys ..................... 42 3.5 Unique AMA-associated transcripts found in both oxazolone- challenged skin versus sham-treated skin, and CBA into B6 renal allografts at day 7 versus normal CBA kidneys ................................... 43 3.6 691 unique skin-selective injury- and repair-induced transcripts and their annotation ..................................................................................... 44 3.7 Overlapping skin-selective IRITs and the kidney-selective IRITs gene ontology (GO) annotations .......................................................... 58 3.8 465 unique skin-associated transcripts and their annotation ............... 62 3.9 Kidney-associated transcripts (KTs) top gene ontology (GO) annotations ........................................................................................... 74 3.10 Skin-associated transcripts (STs) top gene ontology (GO) annotations ........................................................................................... 77 3.11 Overlapping skin-associated (STs) and kidney-associated transcripts (KTs) gene ontology (GO) annotations .............................. 80 CHAPTER 4: INTERFERON-GAMMA-DEPENDENT MOLECULAR CHANGES IN OXAZOLONE-INDUCED CONTACT HYPERSENSITIVITY 4.1 30 unique IFNG-induced transcripts (IFNG-effects) differentially expressed in oxazolone-challenged GRKO skin over oxazolone- challenged B6 skin ............................................................................... 102 4.2 GRKO enriched skin-selective IRITs top gene ontology (GO) annotations ........................................................................................... 103 4.3 GRKO enriched skin-associated transcripts top gene ontology (GO) annotations ........................................................................................... 105 LIST OF FIGURES CHAPTER 3: COMPARISON OF THE MOLECULAR CHANGES IN MOUSE CONTACT HYPERSENSITIVITY AND T CELL MEDIATED REJECTION IN MOUSE RENAL ALLOGRAFTS PAGE 3.1 Histopathology of sham-treated B6 skin and oxazolone-challenged B6 skin .................................................................................................. 83 3.2 Oxazolone-challenged skin experiences greater inflammation than sham-treated controls..........................................................................
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