THE UNIVERSITY OF CHICAGO MICROBIALLY-REGULATED INTESTINAL EPITHELIAL HMGB1: STRESS, CELLULAR ENERGY PRODUCTION AND SURVIVAL A DISSERTATION SUBMITTED TO THE FACULTY OF THE DIVISION OF THE BIOLOGICAL SCIENCES AND THE PRITZKER SCHOOL OF MEDICINE IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY COMMITTEE ON MOLECULAR METABOLISM AND NUTRITION BY NOELLE PATNO CHICAGO, ILLINOIS JUNE 2017 Copyright © 2017 by Noelle Patno All rights reserved DEDICATION I dedicate this to Jeannette Messer, Ph.D., and Candace Cham, Ph.D., without whose tireless effort and constant attention to me and my work, this thesis would not have been completed. Table of Contents List of Abbreviations ........................................................................................................ v List of Figures ..................................................................................................................ix List of Tables ................................................................................................................... x List of Appendices ...........................................................................................................xi Acknowledgements ........................................................................................................ xii Abstract ......................................................................................................................... xiv Chapter 1: Introduction .................................................................................................... 1 Intestinal Structure and Function ................................................................................................................. 1 Intestine and Microbiota .............................................................................................................................. 2 Inflammatory Bowel Disease ........................................................................................................................ 4 Characteristics of Inflammatory Bowel Diseases ...................................................................................... 4 Epidemiology ............................................................................................................................................ 5 Pathogenesis ............................................................................................................................................. 5 Bacterial Associations with IBD Pathogenesis .......................................................................................... 7 IBD Therapies .......................................................................................................................................... 11 IBD Therapies Targeting the Intestinal Bacterial Community ................................................................ 15 The Role of HMGB1 in IBD .......................................................................................................................... 19 Extracellular HMGB1 in IBD .................................................................................................................... 19 Intracellular HMGB1 in IBD ..................................................................................................................... 21 IBD Therapies Exploiting HMGB1’s Intracellular Role ................................................................................. 22 Chapter 2: Methods ....................................................................................................... 28 Mice ............................................................................................................................................................ 28 Organoid Culture ......................................................................................................................................... 29 High Throughput Screening (HTS) Preparation ........................................................................................... 30 HTS Method ................................................................................................................................................ 30 Enteroid Treatment Studies ........................................................................................................................ 31 Western blots/Immunoblots ...................................................................................................................... 33 Chapter 3: Results......................................................................................................... 35 Microbial Regulation of Intestinal Epithelial Cell HMGB1 Expression ........................................................ 35 HMGB1 Cytoprotection Against Bacterially-Stressed Intestinal Epithelium .............................................. 42 HMGB1 Preserves Cellular Energy Production under Bacterial Stress ....................................................... 45 High-Throughput Screening of Approved Drugs for HMGB1-Independent Cellular Energy Production Under Bacterial Stress ................................................................................................................................. 48 Lovastatin Treatment of HMGB1-Deficient Cells Under Bacterial Stress ................................................... 51 Chapter 4: Discussion ................................................................................................... 54 Summary ..................................................................................................................................................... 54 Limitations and Expansion of this Study ..................................................................................................... 55 The Mechanism of AKT Activation .............................................................................................................. 63 The Future of IBD Therapy .......................................................................................................................... 66 References .................................................................................................................... 78 Appendices ..96 iv List of Abbreviations 3-D: three dimensional ACOG: American College of Gastroenterology ADF: Advanced Dulbecco’s Modified Eagle’s Medium Nutrient Mixture F12 Akt: also known as Protein kinase B AMP: Antimicrobial Peptides ANOVA: Analysis of Variance AP: activator protein Atg5: Autophagy protein 5 ATG16L1: Autophagy 16 Like-1 ATP: Adenosine triphosphate B.: Bifidobacteria B. subtilis: Bacillus subtilis CARD15 CD: Crohn’s Disease CDI: Clostridium difficile infection CpG: nucleotide sequence of cytosine followed by guanine CDEIS: Crohn's Disease Endoscopic Index of Severity Colonoids: three dimensional structures grown from stem cells isolated from the colon DCA: deoxycholic acid DNA: Deoxyribonucleic acid DPBS: Dulbecco's Phosphate-Buffered Saline v DSS: Dextran Sodium Sulfate E. Coli: Escherichia Coli EDTA: Ethylenediaminetetraacetic Acid EGF: Epidermal Growth Factor ELISA: Enzyme Linked ImmunoSorbent Assay EMA: European Medicines Agency Enteroids: three dimensional structures grown from stem cells derived from the small intestine f/f or fl/fl: floxed f/f vil-cre or fl/fl vil-cre: floxed villin-cre FDA: Food and Drug Administration (United States Agency) FLA: flagellin FMT: fecal microbial transplantation FODMAP: fermentable oligosaccharide, disaccharide, monosaccharide and polyol FOS: Fructooligosaccharides GAPDH: Glyceraldehyde 3-phosphate dehydrogenase GF: germ-free or gnotobiotic GSK3β: Glycogen synthase kinase 3 beta GWAS: genome wide association studies HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HMGB1: High Mobility Group Box 1 H. pylori: Helicobacter pylori HTS: High-Throughput Screen vi IACUC: Institutional Animal Care and Use Committee IBD: Inflammatory Bowel Disease IEC: Intestinal Epithelial Cells IL: Interleukin L: Lactobacilli LGG: Lactobacillus rhamnosus GG LPS: Lipopolysaccharide LTA: Lipoteichoic Acid MDP: Muramyl DiPeptide mRNA: messenger ribonucleic acid MyD88: myeloid differentiation primary response protein N.A.: North America NF-kB: nuclear factor kappa-light-chain-enhancer of activated B cells NCT: National Clinical Trial NMDA: N-methyl-D-aspartate receptor NOD: Nucleotide- binding oligomerization domain-containing protein ODN: oligodeoxynucleotides Organoids: generic term for three dimensional structures grown from stems cells derived from an organ p62/SQSTM-1: ubiquitin binding protein p62 also known as sequestosome-1 PI3K: Phosphoinositide 3 Kinase PMSF: Phenylmethylsulfonyl fluoride PTEN: phosphatase and tensin homolog vii Organoids: generic term for three dimensional structures grown from stems cells derived from an organ Rep: Replicate S. aureus: Staphylococcus aureus S. typhimurium: Salmonella typhimurium SPF: Specific pathogen-free TLR: toll-like receptors TNF: Tumor Necrosis Factor TNBS: trinitrobenzenesulfonic acid UC: Ulcerative Colitis VSL#3: probiotic formulation of Streptococcus thermophiles, Bifidobacterium breve, Bifidobacterium longum (recently reclassified as B. lactis according to vsl3.com), Bifidobacterium infantis (recently reclassified as B. lactis according to vsl3.com, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus delbrueckii subsp. bulgaricus (recently reclassified as L. helveticus according to vsl3.com) viii List of Figures Figure 1: Various bacterial components