U UNIVERSITY OF CINCINNATI Date: I, , hereby submit this original work as part of the requirements for the degree of: in It is entitled: Student Signature: This work and its defense approved by: Committee Chair: Approval of the electronic document: I have reviewed the Thesis/Dissertation in its final electronic format and certify that it is an accurate copy of the document reviewed and approved by the committee. Committee Chair signature: Immunobiology of IFRD1, a Novel Genetic Modifier of Cystic Fibrosis Lung Disease A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) In the Graduate Program of Molecular and Developmental Biology of the College of Medicine 2009 by Yuanyuan Gu M.D.-M.S. Nanjing University, China, 2005 Committee Chair: Christopher L. Karp, M.D. Marie-Dominique Filippi, Ph.D. H. Leighton Grimes, Ph.D. David A. Hildeman, Ph.D. Jeffrey A. Whitsett, M.D. Abstract Cystic fibrosis is the most common, lethal autosomal recessive disorder in the United States. Lung disease is the major cause of morbidity and mortality in CF. In the CF lung, chronic infection and dysregulated neutrophilic inflammation lead to progressive airway destruction. Despite the molecular insights afforded by identification of disease-causing gene, CFTR, a clear understanding of the pathogenesis of lung disease in CF remains elusive. There is a poor correlation of genotype with phenotype in lung disease in CF, which strongly suggests that the expression of lung disease in CF is influenced by environmental exposures and/or modifier genes. To search for genes modifying CF lung disease, the Karp lab performed a genome-wide association study in collaboration with GMSG cohort, validating top candidates in collaboration with the CFTSS cohort. Using this approach, genetic variation in IFRD1 was identified and verified as a modifier of lung disease severity in CF. IFRD1 is a HDAC-dependent transcriptional co-activator or co-repressor whose expression is particularly enriched in neutrophils.The goal of my dissertation studies was mechanistic insight into the modulation of CF lung disease by IFRD1. This dissertation research provides evidence in favor of the hypothesis that IFRD1 modulates the course of airway disease in CF through regulation of neutrophil effector function. This study also strongly suggests a mechanism by which IFRD1 modulates neutrophil function in a HDAC-dependent manner to co-suppress the expression of ATF3, a transcriptional repressor of NF-B activity in neutrophils. Finally, this research emphasizes the translational implications for therapeutic targeting of neutrophils in CF. This study suggests that the IFRD1/HDAC axis may provide a tractable therapeutic target in CF, and the plethora of other diseases in which neutrophils play an important pathogenic role. ii iii Acknowledgements I would like to thank my mentor, Dr. Christopher Karp, for his guidance and support during last four years. His passion for science has motivated me throughout my research training. His sense of humor has made the lab a pleasant place to work in. I would also like to thank him for his help in pursuing my career as a surgeon-scientist. I am lucky to have Dr. Jeffrey Whitsett, Marie-Dominique Filippi, Lee Grimes, and David Hildeman on my committee. They have challenged me and, more importantly, have provided valuable insight in my work. I am very thankful to all of my lab members for their help not only in research and but also in my life in the US. Leah Flick, Senad Divanovic, Rajat Madan, and Aurelien Trompette are all great teachers. Jessica Allen and Isaac Harley are the sweetest persons I have ever met. I would also like to thank Dr. Bruce Aronow and Isaac Harley for their help in bioinformatic analysis. I appreciate help from many other fellow graduate students, faculty and staff in the division of Molecular Immunology, Immunobiology and Developmental Biology, Finally, I would like to thank my family for their belief in education, braveness to let me sail in this ―big world‖, and unconditional love all the time. iv Abbreviation ATF3: activating transcription factor 3 CF: Cystic Fibrosis CFTR: CF transmembrane conductance regulator CFTSS: Cystic Fibrosis Twin and Sibling Study ChIP: chromatin immunopreciptation ER: endoplasmic reticulum fMLP: formyl-methionyl-leucyl-phenylalanine GMSG: Modifier Study Group GWAS: genome-wide association studies HDAC: histone deacetylase ICZ: indolo[3,2-b]carbazole IFN-Interferon IFRD1: interferon-related developmental regulator IL-10: interleukin 10 IL-8: interleukin 8 LPS: lipopolysaccharides LTB4: leukotriene B4 MBL2: mannose-binding lectin 2 TAP-MS: Tandem Affinity Purification-Mass spectrometry TGF1: Transforming growth factor beta 1 TLR4: Toll-like receptor 4 TNF-: tumour necrosis factor v Table of Contents Chapter I Introduction .......................................................................................................................... 1 1. Cystic Fibrosis and CFTR ........................................................................................................... 2 2. Neutrophil biology ...................................................................................................................... 8 3. CF modifier genetics ................................................................................................................. 10 4. IFRD1 ....................................................................................................................................... 13 5. Hypothesis ................................................................................................................................. 15 Table 1. Classification of CFTR mutations ................................................................................. 16 References ..................................................................................................................................... 17 Chapter II IFRD1 modifies CF lung disease by regulating neutrophil functions ............................... 27 Identification of IFRD1 as a modifier gene for cystic fibrosis lung disease ................................. 28 Methods Summary ...................................................................................................................... 37 Figure 1. IFRD1-deficient neutrophils exhibit decreases in specific effector functions. ............ 41 Figure 2. Genetic deficiency of IFRD1 is associated with delayed bacterial clearance, but decreased neutrophilic inflammation and ameliorated disease, after airway challenge with mucoid P. aeruginosa. ................................................................................................................................ 43 Figure 3. Association of IFRD1 polymorphisms with variation in human neutrophil effector function. ........................................................................................................................................ 44 Table 1 Transmission analysis of IFRD1 SNPs .......................................................................... 45 Methods ....................................................................................................................................... 46 Supplementary Information ........................................................................................................ 54 Supplementary Table 1. Association of SNPs at the IFRD1 locus with lung disease severity: GMSG cohort genome-wide SNP scan with pooled DNA; Affymetrix 100K array. .................... 54 Supplementary Table 2. Individual genotyping in 779 Caucasian CFTR ΔF508 homozygotes in the GMSG cohort; IFRD1 locus; Illumina SNP beadarray genotyping. ....................................... 56 Supplementary Table 3. Comparison of pooled estimates of allele frequencies with allele frequencies obtained via individual genotyping in 320 CFTR ΔF508 homozygotes GMSG cohort; IFRD1 locus; Affymetrix 100K pooled genotyping versus Illumina SNP beadarray genotyping. 58 Supplementary Table 4. ............................................................................................................... 61 Supplementary Table 5. Association of SNPs at the CEBPA/CEBPG locus with lung disease severity: GMSG cohort genome-wide SNP scan with pooled DNA; Affymetrix 100K array. ..... 62 Supplementary Table 6. Individual genotyping in 779 Caucasian CFTR ΔF508 homozygotes the GMSG cohort; CEBPA/CEBPG locus; Illumina SNP beadarray genotyping. .............................. 63 Supplementary Table 7. Comparison of estimated allele frequencies with allele frequencies obtained via individual genotyping in 320 Caucasian CFTR ΔF508 homozygotes GMSG cohort; CEBPA/CEBPG locus; Affymetrix 100K pooled genotyping versus Illumina SNP bead array genotyping. .................................................................................................................................... 64 Supplementary Table 8. Association of CEBP SNP haplotype rs7253865 G and rs1423062 A with CF lung function derived from family based association testing .......................................... 66 Supplementary Figure 1. Comparison of genome-wide SNP allele frequencies in the CF cohort vi (GMSG) with those from a genome-wide SNP scan in asthma patients and controls (Isle of Wight birth cohort study). .......................................................................................................................