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Inflammation in Chronic Granulomatous Disease and Modulation of Human Dendritic Cell Functions by Burkholderia Cenocepacia and Burkholderia Multivorans

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Inflammation in Chronic Granulomatous Disease and Modulation of Human Dendritic Cell Functions by Burkholderia Cenocepacia and Burkholderia Multivorans INFLAMMATION IN CHRONIC GRANULOMATOUS DISEASE AND MODULATION OF HUMAN DENDRITIC CELL FUNCTIONS BY BURKHOLDERIA CENOCEPACIA AND BURKHOLDERIA MULTIVORANS by KELLY LYNN MACDONALD B.Sc., Saint Francis Xavier University, 1997 M.Sc., The University of Guelph, 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Microbiology and Immunology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2009 © Kelly Lynn MacDonald, 2009 Abstract Inflammation and infection are integral to the human diseases cystic fibrosis (CF) and chronic granulomatous disease (CGD). Inflammation was examined in peripheral blood mononuclear cells (PBMCs) from CGD patients. Reactive oxygen species (ROS) generated by the phagocytic NADPH oxidase have been implicated in the activation of the NF-κB, a transcription factor required for proinflammatory cytokine production in response to inflammatory stimuli. Patients with CGD, an immunodeficiency characterized by the inability to produce ROS, frequently develop inflammatory complications indicative of exaggerated inflammatory responses. In the present study, human and murine CGD leukocytes displayed a hyperinflammatory phenotype with increased production of proinflammatory cytokines in response to Toll-like receptor agonists. The major steps involved in NF-κB activation were also intact in human CGD cells. ROS were therefore not required for NF-κB activation and ROS production may instead dampen inflammation. The interaction of Burkholderia cepacia complex (BCC) bacteria with primary human monocyte-derived dendritic cells (DCs) was also explored as a model of infection. B. cenocepacia and B. multivorans, the most clinically important BCC members, are serious opportunistic pathogens infecting CF and CGD patients. The present study investigated whether these pathogens could modulate normal functions of DCs, important phagocytic cells that act as orchestrators of the immune response. DCs co-incubated for 24 h with B. cenocepacia, but not B. multivorans, had reduced expression of co-stimulatory molecules when compared with BCC lipopolysaccharide-matured DCs, as determined using flow cytometry. B. cenocepacia, but not B. multivorans, also induced necrosis in DCs after 24 h, as determined by annexin V and propidium iodide staining. DC necrosis only occurred after phagocytosis of live B. cenocepacia; DCs exposed to heat-killed bacteria, bacterial supernatant, or those pre-treated with cytochalasin D then exposed to live bacteria remained viable. The intracellular lifestyle of BCC bacteria was ii also examined using transmission electron microscopy. After 6 h of co-incubation with DCs, B. cenocepacia occupied the phagosome while B. multivorans resided in the cytoplasm. The ability of B. cenocepacia to modulate DC functions may contribute to its pathogenicity. Understanding the sophisticated mechanisms of infection and inflammation may lead to better treatments for CF and CGD. iii Table of Contents Abstract ........................................................................................................................................... ii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. ix List of Figures .................................................................................................................................. x List of Abbreviations ..................................................................................................................... xi Acknowledgements...................................................................................................................... xiii Co-authorship Statement .............................................................................................................. xvi 1. Introduction .............................................................................................................................. 1 1.1 Cystic fibrosis ................................................................................................................... 2 1.2 Chronic granulomatous disease ........................................................................................ 3 1.3 Phagocytic cell biology ..................................................................................................... 7 1.4 Burkholderia cepacia complex ....................................................................................... 12 1.5 Antibiotic resistance ........................................................................................................ 15 1.6 Virulence factors that may aid in host colonization ........................................................ 15 1.6.1 Adhesins .................................................................................................................. 15 1.6.2 Flagella .................................................................................................................... 16 1.6.3 Siderophores ............................................................................................................ 16 1.6.4 Exopolysaccharide production and biofilm formation ............................................ 17 1.6.5 Quorum sensing ....................................................................................................... 18 1.7 Evasion of host defences by B. cepacia complex ........................................................... 19 1.7.1 Resistance to non-oxidative killing ......................................................................... 19 1.7.2 Resistance to complement ....................................................................................... 20 1.7.3 Intracellular pathogenicity ....................................................................................... 21 iv 1.7.4 Pro-inflammatory potential ...................................................................................... 26 1.7.5 Modulation of apoptosis and necrosis ..................................................................... 27 1.8 BCC genomics ................................................................................................................ 28 1.9 Major thesis themes and hypotheses ............................................................................... 30 1.9.1 Inflammation in CGD .............................................................................................. 30 1.9.2 Dendritic cells and BCC .......................................................................................... 31 1.10 References ....................................................................................................................... 32 2. Enhanced inflammatory responses of chronic granulomatous disease leukocytes involve ROS-independent activation of NF-kappa B. ......................................................................... 44 2.1 Summary ......................................................................................................................... 44 2.2 Introduction ..................................................................................................................... 45 2.3 Materials and methods .................................................................................................... 47 2.3.1 Patients and isolation of cells .................................................................................. 47 2.3.2 Cytokine assays ....................................................................................................... 47 2.3.3 Stimulation and preparation of cellular extracts ...................................................... 48 2.3.4 Western blotting ...................................................................................................... 49 2.3.5 NF-κB p50 ELISA ................................................................................................... 49 2.3.6 Electrophoretic mobility shift assay (EMSA) .......................................................... 49 2.3.7 Mouse strains ........................................................................................................... 50 2.3.8 In vitro stimulation of murine spleen mononuclear cells ........................................ 50 2.3.9 Production of ROS ................................................................................................... 51 2.4 Results ............................................................................................................................. 51 2.4.1 CGD leukocytes produce increased amounts of proinflammatory cytokines after stimulation. ............................................................................................................. 51 2.4.2 Enhanced proinflammatory cytokine responses of murine CGD spleen cells ........ 53 v 2.4.3 ROS production ....................................................................................................... 53 2.4.4 Cytoplasmic phosphorylation and degradation of I-kBα. ....................................... 55 2.4.5 Nuclear translocation of NF-κB binding proteins ................................................... 56 2.4.6 Nuclear translocation of NF-κB p50 ....................................................................... 57 2.4.7 Translocation of p65 is
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