Điều hòa sinh học macrophage và đáp ứng miễn dịch bởi lipid mycobacterium

MODULATION OF MACROPHAGE BIOLOGY AND HOST IMMUNE RESPONSE BY MYCOBACTERIAL LIPIDS A Dissertation Submitted to the Graduate School of the University of Notre Dame in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Sanchita Bhatnagar, M. Schorey, Director Graduate Program in Biological Sciences Notre Dame, Indiana November 2006 UMI Number: 3243285 UMI Microform 3243285 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.

ProQuest Information and Learning Company 300 North Zeeb Road P. Box 1346 Ann Arbor, MI 48106-1346 MODULATION OF MACROPHGAE BIOLOGY AND HOST IMMUNE RESPONSE BY MYCOBACTERIAL LIPIDS Abstract By Sanchita Bhatnagar Members of the Mycobacterium genus present a serious health threat which account for significant morbidity and mortality in humans and other species. Many pathogenic mycobacterial species maintain an intracellular life style within macrophages serving as the primary host cell. The mycobacterium’s ability to modulate the macrophage function requires the expression of specific mycobacterial surface components.

Moreover, a number of studies have shown that various mycobacterial surface components can promote macrophage activation. This dissertation aims to define the role of glycopeptidolipids (GPL), which are highly antigenic glycolipids, in the immune-modulation of the host and thus establish its function as an important virulence factor. To address the role of GPL as a virulence factor, we utilized M. avium strains either naturally lacking GPL expression or genetically altered to express modified GPL.

Our studies showed that the loss of GPL or altering its structure leads to an M. avium Sanchita Bhatnagar strain which induces increased macrophage activation compared to GPL sufficient M. Additional analysis showed attenuated virulence of GPL- deficient/modified M. avium strains in a mouse infection model.

To further understand the mechanism of action of GPL, we investigated the trafficking of GPL in primary macrophages infected with M. Our studies revealed significant release and trafficking of GPL from the mycobacteria containing phagosome to a distinct endocytic compartment called the multivesicular bodies (MVB). Additional analysis showed that mycobacterial components including GPL are trafficked outside the infected cells on “exosomes”. Further, we determined that exosomes purified from the culture supernatants of macrophages infected with M.

tuberculosis H37Rv or M. bovis BCG stimulate a pro-inflammatory response in uninfected macrophages. Our studies are first to establish GPL’s importance as a virulence factor in the M. The data demonstrate a significant shedding of glycopeptidolipids in M.

avium infected macrophages and its transport to the neighboring “bystander cells”. This transport of mycobacterial components occur through release of exosomes extracellularly which can also act to stimulate uninfected macrophages both in vitro and in vivo. Together, our studies put forward a novel mechanism of intercellular communication mediated by exosomes which can carry mycobacterial components to neighboring cells. DEDICATION For my loving husband, Jogender Singh Tushir and my parents, Satish and Sunita Bhatnagar, my little sister Anchita.

With great admiration and thanks. ii CONTENTS FIGURES…………………………………………………………………………….xii CHAPTER 1: INTRODUCTION 1.2 Classification of mycobacteria…………………………………………….3 Mycobacterial cell wall…………………………………………………………6 1.4 Biology of macrophages…………………………………….5 Mycobacterial entry in macrophage………………….6 Mycobacterial invasion of macrophage……………………………………….7 Mycobacterial lifestyle inside macrophages………………………………….8 Immunity to mycobacteria…………………………………………………….9 Trafficking of Mycobacterial lipids……………………………………………24 1.10 Macrophage endocytic pathway……………………………………………….25 CHAPTER 2: Mycobacterium avium 104 deleted of the methyltransferase D gene by allelic replacement lacks serotype-specific glycopeptidolipids and shows attenuated virulence in mice.2 Materials and Methods…………………………………………………………36 2.1 Bacterial strains and growth conditions……….2 Gene exchange by homologous recombination.3 BMMΦ isolation and culture………………………………………………….5 Cytokine profile of macrophages infected with M.6 In vivo mycobacterial infections…………………………………………….1 Disruption of the M. avium 104 mtfD gene by homologous recombination………………………………………………………………….2 In vitro macrophage infections with WT, mtfD mutant and complemented M.3 Mouse infections with the WT, mtfD mutant and complemented M.49 CHAPTER 3: Elevated MAP kinase signaling and increased macrophage activation in cells infected with a glycopeptidolipid-deficient Mycobacterium avium.2 Materials and Methods……………………………………….1 BMMΦ isolation and culture………………………….3 Preparation of surface-exposed material from M.4 Extraction and purification of GPLs………………………………………….6 Western blot analysis……………………………….8 Chemokine/Cytokine profile of macrophages infected with M.9 Bacterial killing assay……………………………….1 Differential activation of the MAPK in macrophages infected with Rg and SmT M.2 Calmodulin kinase is upstream of ERK1/2 MAPK in the macrophages infected with M.3 MAPK activation is differentially required for TNF-α production in macrophages infected with Rg compared to SmO and SmT M.4 TNF-α production is NF-κB dependent…………….5 Differential activation profile for BMMΦs following infection with Rg and SmT M.6 The Rg 2151 is killed by BMMΦs…………………………………………….75 CHAPTER 4: Release and trafficking of glycopeptidolipids in macrophages infected with M.2 Materials and Methods…………………………………….1 BMMφ isolation and culture……………………….4 Antibodies and immunofluorescence staining…….5 Thin layer chromatography immunostaining…….7 Isolation of exosomes…………………………….8 Sucrose density gradient centrifugation…………….9 Labeling with N-Rh-PE………………………….10 Analysis of un-infected bystander cells………….11 Coupling of exosomes to latex beads…………….12 Flow cytometric analysis of exosomes…………….13 Macrophage infection with exosomes…………….1 Glycopeptidolipid trafficking in M. avium 2151 infected macrophages.2 Intracellular sorting of released glycopeptidolipids………………………….3 Rab11 is transiently associated with GPL positive MVBs…………………….4 Role of exosomes in intercellular communication…………………………….5 Macrophage activation by the exosomes from mycobacteria infected macrophages………….6 TNF-α production is toll-like receptor dependent…………………………….7 Exosome stimulatory activity is not dependent on GPLs…………………….…………………………………………………………………110 CHAPTER 5: Macrophage activation by exosomes isolated from mycobacteria infected macrophages.2 Materials and Methods……………………………………………………….1 BMMφ isolation and culture……………………………………………….5 Isolation of exosomes…………………………………………………………121 5.6 Sucrose density gradient centrifugation…………………………………….7 Coupling of exosomes to latex beads……………………………….8 Flow cytometric analysis of exosomes……………………………….9 Macrophage infection with exosomes……………………………………….11 Western blot analysis………………………………….1 Exosomes isolated from mycobacteria infected cells induce pro-inflammatory response in non-infected macrophages…………………………………….2 Toll-like receptor dependent production of TNF-α in exosomes treated cells………………………………………………………………………….3 Exosomes from activated macrophages are not enough to induce pro-inflammatory response……………………………………………………132 5.4 Activated exosomes from other intracellular pathogen……………………….5 In vivo induction of pro-inflammatory response by the mycobacterial exosomes………………………….140 CHAPTER 6: Summary and future perspectives…………………………………….1 Mycobacterial species known to cause human disease……………….2 A chemical model of the mycobacterial cell wall……….3 List of some common mycobacterial lipids present on mycobacterial cell wall……………………………………………….4 Structure of glycopeptidolipids……………………………………….5 Different serovars showing oligosaccharides attached to the core structure of GPLs…………………………………………….6 List of pattern recognition receptors and some of their respective pathogen associated molecular patterns……………………19 Figure 1.7 Schematic of formation of MVB and its subsequent fate in the cell…………………………………………………………….1 Schematic organization of the mtfD disruption construct…………….2 BMMΦs infected with M.

avium mtfD mutant show increased production of TNF-α and RANTES compared to BMMΦs infected with M. avium 104 WT or mtfD mutant complemented with intact mtfD…………………………………….3 Bacterial loads in the livers or spleens or lungs following an infection of 129/Sv mice with M. avium104 WT, mtfD deficient mutant or mtfD complemented mutant……………………………….4 Cytokine profile in lung homogenates of mice infected with M. avium104 WT, mtfD deficient mutant or mtfD complemented mutant…………………………………………………………………48 Figure 3.1 MAPK activation upon infection of murine bone-marrow derived macrophages with M.

avium 2151 isogenic strains……………………63 vi Figure 3.2 Calmodulin Kinase activity is required for ERK1/2 activation in both Rg 2151 and SmT 2151 infected macrophages………………….3 Differential requirement for MAPK activation in TNF-α production by macrophages infected with Rg or SmT 2151……….4 TNF-α production by macrophages infected with SmO M. avium 2151 is dependent on p38 and ERK 1/2 activation…………….5 TNFα production by macrophages infected with either Rg or SmT M. avium 2151 is dependent on NF-κB activation……………….6 Macrophage produce more inflammatory mediators upon infection with Rg M. avium 2151 compared to SmT 2151 infected cells………………………………………………………………….7 RANTES and IL-6 production by Rg M.

avium 2151 infected macrophages is dependent on p38 but not ERK activation…….8 Killing of Rg M. avium 2151 by Balb/c bone marrow-derived macrophages………………………………………………………….9 Schematic illustration of the signal pathways activated in bone marrow-derived macrophages upon infection with Rg or SmT M.1 Intracellular trafficking of glycopeptidolipids in macrophages infected with Mycobacterium avium serovar 2……………………….2 Comparison of staining pattern of lipoarabinomannan (LAM) and glycopeptidolipids in macrophages infected with M.3 Glycopeptidolipids localizes to multi-vesicular bodies in Mycobacterium avium 2151 infected macrophages…………….4 Rab11 co localizes with the GPL loaded vesicles at later times…….5 Trafficking of mycobacterial components outside the infected cells via exosomes……………………………………………………103 Figure 4. avium 2151 infected macrophages induce a pro-inflammatory response in primary macrophages but not entirely dependent on GPL…………………………………….7 TNF-α production by macrophages infected with exosomes from M. avium 2151 SmT morphotype is dependent on toll-like receptor……………………………………………………………….8 Balb/c BMMΦs were infected with exosomes from SmT Rg morphotype of M.1 Characterization of exosomes isolated from non-infected, M.

bovis BCG-infected and M. tuberculosis H37Rv-infected J774 cells…….2 Exosomes isolated from M. bovis BCG and M. tuberculosis H37Rv infected THP-1 induce a pro-inflammatory response in human macrophages………………………………………………….3 TLR dependent TNF-α induced by exosomes isolated from mycobacterial infected cells………………………………………….4 Macrophage activation by exosomes derived from THP-1 macrophage activated with LPS (200ng/ml) or infected with heat- inactivated M.

bovis BCG (HI-BCG) or Salmonella typhimurium SL1344…………………………………………………………………135 Figure 5.5 Mycobacterial exosomes induced pro-inflammatory response in the lungs of Balb/c mice…………………….1 Schematic illustration of the signal pathways activated in bone marrow-derived macrophages upon infection with Rg or SmT M.154 viii ABBREVIATIONS Ab: Antibody AraLAM: Arabinofuranosyl-terminated LAM BCA: Bicinchoninic acid assay BCG: Mycobacterium bovis bacillus-Calmette Guerin BMMΦ: Bone marrow derived macrophages C: Celsius CaM: Calmodulin CAPE: Caffeic Acid Phenylethyl Ester CFU: Colony forming unit CaMK: Ca2+- calmodulin dependent protein kinase DC-SIGN: Dendritic cell-specific ICAM-grabbing non-integrin DMSO: Dimethyl sulfoxide DMEM: Dulbecco’s modified eagle’s medium ECL: Enhanced chemilumnescence ELISA: Enzyme-linked immunosorbent assay ERK1/2: Extracellular signal-regulated kinase1/2 EM: Electron microscopy FITC: Fluorescein isothiocyanate ix FBS: Fetal bovine serum GM-CSF: Granulocyte-macrophages colony stimulating factor GPL: Glycopeptidolipid HIV: Human immunodeficiency virus ICAM: Intracellular adhesion molecule IgG: Immunoglobulin G IFN-γ: Interferon gamma ILV: Intra-luminal vesicles iNOS: Inducible nitric oxide synthase 2 LAM: Lipoarabinomannan LAMP1: Lysosome associated membrane protein 1 LAMP2: Lysosome associated membrane protein 2 LPS: Lipopolysaccharide MAC: Mycobacterium avium complex ManLAM: Mannosylated-lipoarabinomannan MAPK: Mitogen activated protein kinase mAb: Monoclonal antibody MR: Mannose receptor mtfD: Methyl transferase D MVB: Multi-vesicular body NF-κB: Nuclear factor kappa B nsGPL: non-serovar specific glycopeptidolipids OADC: Oleic acid, dextrose, catalase x PAMP: Pathogen associated molecular pattern PBS: Phosphate-buffered saline PD98059: MEK1/2 specific inhibitor PILAM: Phosphoinositide-capped lipoarabinomannan PIM: Phosphatidylinositol mannoside PMA: Phorbol 12-myristate 13-acetate RC: Resting Cell Rg: Rough RNI: Reactive nitrogen intermediates SB203580: p38 specific inhibitor SD: Standard deviation SDS: Sodium dodecyl sulfate TC: Tissue culture grade TH: T helper cells TNF-α: Tumor necrosis factor-alpha TLR: Toll like receptor PRR: Pattern recognition receptor ssGPL: serovar specific glycopeptidolipids SmT: Smooth transparent SmO: Smooth opaque WT: Wild type xi ACKNOWLEDGMENTS First, I would like to thank my advisor Dr. Jeff Schorey for his help and guidance throughout my project. He has been a great mentor these past 4 years and I am thankful for the opportunity to have worked with him. I appreciate Jeff’s scientific enthusiasm and his creative ideas.

I am forever grateful to Jeff for being supportive of my project and letting me follow my whims. Many thanks to my thesis committee members, Dr Crislyn D’Souza-Schorey, Dr Holly Goodson and Dr Mary Ann Mcdowell for their guidance, advice and tremendous support along the way. It has been a pleasure for me to get to know you all and I thank you for your valuable insights that helped me put together this thesis. I will also like to thank the present and past members of the Schorey lab for their cooperation and providing exciting working environment in the lab.

I will also like to acknowledge Crislyn D’Souza lab members for their help and insightful discussions. Special thanks to Severson lab and Mary Ann McDowell lab for providing many reagents and the equipment. Finally, I will like to thank my loving husband, Jogi for his help and support. He has been a great inspiration for me and was the only reason of my pursuing the PhD.

He has been my biggest critic and his advice has always helped me to move ahead. I want to thank you for being with me at every step and helping me in everything. He has taught xii me to be strong and gave me strength to overcome some of the hard times in the life. I will also like to thank my parents, who believed in me and supported my every decision in life.

I would not have been here without the support of my younger sister, Anchita who is the joy of our life.