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The role of CD43 in the growth and pathogenesis of Mycobacterium tuberculosis within the mammalian host Randhawa, April Kaur 2007

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THE ROLE OF CD43 IN THE GROWTH AND PATHOGENESIS OF MYCOBACTERIUM TUBERCULOSIS WITHIN THE MAMMALIAN HOST  by  APRIL KAUR RANDHAWA  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES  (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA December 2007  © April Kaur Randhawa, 2007  Abstract  Mycobacterium tuberculosis exploits various molecules on host cells to gain entry and establish a niche for survival and replication. We characterized the role of the glycoprotein CD43 in the pathogenesis of M. tuberculosis. Using gene-deleted mice (CD43-/-), we assessed association of the bacterium with macrophages and found that CD43 was required for optimal binding of M. tuberculosis strain Erdman by splenic, peritoneal, alveolar, and bone marrow-derived macrophages. Macrophages from heterozygote (CD43+/-) mice, which express 50% less CD43 than wild type (CD43+/+) mice, bound more bacteria than CD43-/- but less than CD43+/+ indicating that the surface expression of CD43 correlates with binding of M. tuberculosis. The role of CD43 in binding bacteria may be restricted to mycobacterial species as CD43-/- macrophages also bound less Mycobacterium avium and Mycobacterium tuberculosis H37Rv, but there was no observed role in the binding of Salmonella typhimurium or Listeria monocytogenes. Although absence of CD43 resulted in decreased binding of M. tuberculosis, the subsequent growth of the bacterium within CD43-/- macrophages was enhanced as illustrated by increased bacterial numbers and decreased doubling times, indicating that that the mechanism of entry may influence subsequent. To elucidate mechanisms by which CD43 controls of growth of M. tuberculosis, we examined the induction of antimycobacterial activities.  In response to M.  tuberculosis, CD43-/- macrophages were deficient in the production of nitric oxide, TNF-α, and IL-12. Furthermore, M. tuberculosis induced less apoptosis, but more necrosis, in CD43-/- macrophages compared to CD43+/+. The enhanced growth of M.  ii  tuberculosis was abrogated by IFN-γ-stimulation with whereas addition of TNF-α restored both the intracellular growth rates and amounts of apoptosis to wild type levels. To investigate the role of CD43 in vivo, we infected CD43-/- and CD43+/+ mice with M. tuberculosis and assessed bacterial loads and organ pathology. Absence of CD43 resulted in increased bacterial loads in lungs and spleens during both acute and chronic stages of infection, and formation of granulomas occurred more quickly in CD43-/- mice. These data point to a dual role for CD43 in the uptake and subsequent growth of M. tuberculosis in macrophages and mice.  iii  Table of Contents Page  Abstract ………………………………………………………………….……………… ii Table of Contents …………………………………………………………….…………. iv List of Tables ……………………………………………………………………………viii List of Figures …………………………………………………………………………... ix List of Abbreviations …………………………………………………………………… xi Acknowledgements ……………………………………………………………………. xiv Dedication ……………………………………………………………………………… xv Chapter 1 1.1  Introduction Tuberculosis ……………………………………………………………... 1 1.1.1  Prevalence & global impact of Tuberculosis …………………….. 3  1.1.2  Factors contributing to the resurgence of Tuberculosis worldwide …………………………………………. 3  1.1.3  Prevention of Tuberculosis ……………………………………… 5  1.1.4  Treatment of Tuberculosis ………………………………………. 6  1.2  Mycobacterium tuberculosis biology …………………………………… 8  1.3  The immune response to Mycobacterium tuberculosis …………………. 9  1.4  1.3.1  Mycobacteria:Mφ interactions ………………………………….. 10  1.3.2  Adaptive immunity ……………………………………………... 13  1.3.3  Latency/persistence …………………………………………….. 14  CD43 …………………………………………………………………… 15  iv  1.4.1  CD43 Expression ……………………………………………….. 16  1.4.2  CD43 Structure …………………………………………………. 17  1.4.3  Described functions of CD43 …………………………………... 19  1.4.4  CD43 & disease ………………………………………………… 22  1.5  Introduction to the thesis ……………………………………………….. 26  1.6  Literature cited …………………………………………………………. 28  Chapter 2  CD43 is required for optimal growth inhibition of Mycobacterium tuberculosis in macrophages and in mice  2.1  Summary ………………………………………………………………... 48  2.2  Introduction …………………………………………………………….. 49  2.3  Materials and methods …………………………………………………. 51  2.4  CD43–/– Mφ of different origins bind M. tuberculosis less readily than CD43+/+ Mφ at various multiplicities of infection ………………… 56  2.5  Opsonization of bacteria overcomes the impaired ability of CD43-/- Mφ to bind M. tuberculosis ………………………… 57  2.6  CD43 is involved in binding other mycobacteria, but its absence does not abrogate binding of S. typhimurium or L. monocytogenes by BMMφ …………………………………………… 58  2.7  CD43 deficiency does not affect Mφ-nonspecific uptake or phagocytosis via FcγRs and complement receptors but does enhance binding via lectin-like receptors ………………………………. 58  v  2.8  The level of CD43 surface expression differs between Mφ phenotypes …………………………………………………………. 59  2.9  CD43 gene dose correlates to the ability of BMMφ to bind M. tuberculosis …………………………………………………. 59  2.10  The survival and replication of M. tuberculosis within CD43-/- Mφ is enhanced ……………………………………………….. 60  2.11  CD43-deficient mice have a reduced ability to control M. tuberculosis growth during the acute and chronic phases of infection following aerosol inhalation of bacteria …………………… 61  2.12  Organ pathology is exacerbated in CD43-deficient mice ……………… 62  2.13  Discussion ……………………………………………………………… 63  2.14  Literature cited ………………………………………………………… 80  Chapter 3  CD43 controls intracellular growth of Mycobacterium tuberculosis through the induction of TNF-α α mediated apoptosis  3.1  Summary ………………………………………………………………... 86  3.2  Introduction …………………………………………………………….. 87  3.3  Materials and methods …………………………………………………. 89  3.4  IFN-γ activation abrogates the enhanced growth of M. tuberculosis in CD43-/- Mφ ………………………………………… 95  3.5  CD43 is required for greatest induction of pro-inflammatory  vi  mediators by M. tuberculosis-infected Mφ …………………………….. 96 3.6  Enhanced growth of M. tuberculosis in CD43-/- Mφ is associated with increased levels of necrosis but decreased induction of apoptotic pathways ……………………………….………. 98  3.7  Enhanced growth of M. tuberculosis and dysregulation of apoptosis in CD43-deficient Mφ is partially regulated by induction of TNF-α …………………………………………………… 100  3.8  Discussion ……………………………………,……………………….. 102  3.9  Literature cited ……………………………………,………………….. 116  Chapter 4  Discussion & future directions  4.1  Discussion ………………………………………………………………124  4.2  Future directions ………………………………………………………. 127  4.3  Literature cited ………………………………………………………… 129  vii  List of Tables  Table  Title  2.1  Intracellular survival and replication of M. tuberculosis  Page  is enhanced in CD43-/- BMMφ ………………………………………… 69 2.2  Granuloma formation in CD43-/- mice is more severe and has altered morphology …………………………………………….. 70  3.1  M. tuberculosis grows more readily in resting but not IFN-γ activated CD43-/- Mφ compared to CD43+/+ Mφ ………… 107  viii  List of Figures  Figure  Title  2.1  M. tuberculosis has a reduced ability to associate  Page  with CD43 deficient Mφ ……………………………………………….. 71 2.2  Heat-labile serum opsonins overcome the reduced binding of M. tuberculosis to CD43-/- Mφ …………………………….. 72  2.3  CD43 is involved in Mφ binding of other mycobacterial species but not other intracellular bacteria ………………….……….…. 73  2.4  The absence of CD43 on Mφ does not affect phagocytosis via complement receptors, FcγR, or non-specific uptake, but enhances uptake of zymosan ……………………………………….. 74  2.5  Surface expression of CD43 varies on different Mφ phenotypes ………. 75  2.6  M. tuberculosis binding to BMMφ is dependent on CD43 gene dose ……………………………………………………………….. 76  2.7  CD43 is necessary for the control of M. tuberculosis growth during both the acute and chronic phase of infection in mice ………….. 77  2.8  CD43 deficient mice infected intravenously with M. tuberculosis do not show impaired control of bacterial growth during the acute phase of infection …………………………….. 78  2.9  Lung pathology is exacerbated in CD43 deficient mice infected with M. tuberculosis ……………………………………………………. 79  ix  3.1  IFN-γ stimulation overcomes enhanced growth of M. tuberculosis in CD43-/- Mφ ……………………………………….. 108  3.2  CD43-/- Mφ are deficient in pro-inflammatory cytokine production in response to M. tuberculosis infection …………………... 109  3.3  CD43-/- Mφ infected with M. tuberculosis produce less reactive nitrogen intermediates than CD43+/+ Mφ ………………….…………. 110  3.4  M. tuberculosis infection causes greater cytoxicity in CD43-/- Mφ than in CD43+/+ Mφ …………………………………….. 111  3.5  CD43 is required for optimal induction of apoptosis in M. tuberculosis-infected Mφ ………………………………………….. 112  3.6  Absence of CD43 results in decreased activity of Caspase-3 in M. tuberculosis-infected Mφ ……………………………………….. 113  3.7  Enhanced growth of M. tuberculosis in CD43-/- Mφ is due to deficient TNF-α production …………………………………………… 114  3.8  M. tuberculosis-induced apoptosis in CD43-/- Mφ is TNF-α dependent ……………………………………………………... 115  x  List of Abbreviations  AIDS  acquired immune deficiency syndrome  AMφ  alveolar macrophage  BCG  Bacille Calmette-Guerin  BMMφ  bone marrow-derived macrophage  CFU  colony forming unit  DC  dendritic cell  DC-SIGN  DC-specific intercellular adhesion molecule-3-grabbing non-integrin  DOT  directly observed therapy  EIgG  IgG-coated erythrocytes  EIgMC’  IgM & complement-coated erythrocytes  ELISA  enzyme-linked immunosorbant assay  ESAT-6  early secreted antigenic target-6  EMB  ethambutol  GVHD  graft versus host disease  H&E  hematoxylin & eosin  HIV  human immunodeficiency virus  ICAM  intercellular adhesion molecule  IEL  intraepithelial lymphocytes  IFN  interferon  INH  isoniazid  LAM  lipoarabinomannan  xi  LDH  lactate dehydrogenase  LPS  lipopolysaccharide  Mφ  macrophage  mAb  monoclonal antibody  MDR  multiple drug-resistant  MHC  major histocompatibility complex  MOI  multiplicity of infection  MTB  Mycobacterium tuberculosis  NO  nitric oxide  OADC  oleic acid-albumin-dextrose complex  PIM  phosphatidylinositol  PMφ  peritoneal macrophage  PPD  purified protein derivative  PZA  pyrazinamide  RA  rheumatoid arthritis  RIF  rifampin  RNI  reactive nitrogen intermediates  ROI  reactive oxygen intermediates  SpMφ  splenic macrophage  STR  streptomycin  Th1  T-helper cell type 1  TLR  toll-like receptor  xii  TB  Tuberculosis  TST  Tuberculin skin test  TNF  tumour necrosis factor  WAS  Wiskott-Aldrich syndrome  WT  wild type  WHO  World Health Organization  XDR  extensively drug-resistant  xiii  Acknowledgements  This work would not have been possible without the support of several individuals. My research supervisor, Dr Richard Stokes, whose guidance and expertise were invaluable in all aspects of this project and throughout my development as a scientist. I would like to recognize members of my supervisory committee, Drs. Hermann Ziltener, Alice Mui and Rob McMaster, and the members of my laboratory who always provided thoughtful insight and facilitated meaningful discussions. I would also like to thank the academic and support staff at the University of British Columbia and the Child & Family Research Centre who provided assistance throughout the duration of my graduate program, with special thanks to Dr. Vincent Duronio, Patrick Carew and the Experimental Medicine Program. In addition to the support by those at my institution and within my department, I must acknowledge how grateful I am for the unwavering encouragement and assistance provided by my friends and family. I would particularly like to thank my sisters Sharney and Shanon whose confidence in me served as great motivation throughout the past five years, and my niece India who has unknowingly provided inspiration since the day she was born. Most importantly, I must thank my parents Kuljit Kaur and Rajinder Singh Randhawa. Words cannot express how much their support has meant throughout my lifetime, but especially now.  xiv  Dedication  In loving memory of Balwant Singh Randhawa, who taught me the value of education, inspired me to pursue my goals, and instilled in me the drive and determination needed to achieve them.  xv  Chapter 1  1.1  Introduction  Tuberculosis  Tuberculosis (TB), a chronic bacterial infection caused by Mycobacterium tuberculosis, is the leading cause of death due to an infectious agent (1). Active TB infection is characterized by fatigue, weight loss, a chronic productive cough, fever, and night sweats. The majority of TB infections occur after inhalation of droplet nuclei expelled from an infectious host. It has been estimated that a single respiratory event, such as a cough, generates thousands of droplets containing the causative bacterium and that inhalation of less than 10 bacilli may lead to infection in a susceptible host (2). This route of infection will most often lead to deposition of the bacteria within the lungs, potentially causing pulmonary TB. Although this is the most common route of transmission, TB infection can also occur in other organs including the skin, eyes, lymph nodes, genitourinary tract, bones and joints, meninges and the gastrointestinal tract. The TB bacterium is highly contagious with a single TB case causing up to 15 new infections per year (3, 4). Infection of immune competent individuals without prior exposure to TB results in a mild self-limiting disease in the majority of cases. Approximately 90% of individuals exposed to TB will not develop active disease, but the bacteria will remain in a latent state. Reactivation disease or secondary TB can occur years or even decades later with the same characteristics as primary TB. 5-10% of infected individuals will go on to  1  develop active TB; those with depressed immunity may succumb rapidly to severe systemic disease causing death within weeks. Conventional tests for diagnosing TB have major limitations and, until recently, no major advances in diagnostics had been made for many years. A widely used tool for diagnosing clinically undeclared TB is the Tuberculin Skin Test (TST). TST measures delayed type hypersensitivity response to the purified protein derivative (PPD), a crude mixture of several mycobacterial antigens (5, 6). A major drawback of this diagnostic is the high rate of false positives due to prior BCG vaccination or previous exposure to nontuberculous mycobacteria (7). In addition, the administration and reading of the TST results in variability and inaccuracies. Sputum smears have also been used for many decades as a reliable method of diagnosis, but not without drawbacks such as the inability to distinguish between strains. Culture of the organism has been regarded as the gold standard but has historically proven inefficient due to the slow growth of M. tuberculosis, although newer methods such as the BACTEC biphasic culture systems have provided faster recovery of organisms than traditional culture on solid media (8, 9). New methods for diagnosis include molecular biological tests based on nucleic acid amplification, automated sequencing analysis, and immunological assays for the rapid detection of M. tuberculosis antigens and antibodies to these antigens (10). In addition to challenges in diagnosing TB, there are several barriers surrounding prevention and treatment efforts, which will be explored in sections 1.1.3 and 1.1.4.  2  1.1.1  Prevalence & global impact of Tuberculosis  Every second of every day, someone is newly infected with Mycobacterium tuberculosis - the causative bacterium of TB (11). 1/3 of the world’s population, or 2 billion people, are currently infected with M. tuberculosis and each year over 8 million new infections occur (12). With over 2 million deaths annually, TB is the most common cause of death due to an infectious agent (13, 14). The highest mortality rate due to TB infection occurs in Africa, where the epidemic is growing most rapidly due to the HIV infection rates in the region. In sub-Saharan Africa, the rates are as high as 350 cases per 100 000 people – even greater than in South-East Asia, which had the largest number of new cases in 2005 (11). TB, along with HIV and malaria are the deadliest human pathogens today, but research funding for these devastating diseases is not proportionate to their impact on global health. Although TB affects a significant portion of the world’s population, there is more funding spent on smallpox and anthrax, which have not proven to be global health threats in recent years (15). With the tremendous global impact of this disease, and the complexities of the causative organism that will be discussed in upcoming sections, it is clear that further dedication of research funding is required, and warranted.  1.1.2  Factors contributing to the resurgence of Tuberculosis worldwide  TB, once known as the white plague or consumption, became the principal cause of death during the 19th century. The incidence of TB began to decline rapidly in the  3  early 20th century in developed countries with improved living conditions and these trends were accelerated by the implementation of vaccination and by the development of antibiotics such as streptomycin and isoniazid. The number of reported cases of TB continued to decline until the mid 1980’s when an increase in reported cases was observed and led to the World Health Organization (WHO) declaring TB a global public health emergency in 1993. The resurgence in TB cases worldwide can be attributed to a number of factors including prevention and treatment complexities, and social conditions. Social factors that have led to the resurgence of TB include increasing populations, poverty, and homelessness, as TB thrives in crowded living conditions where people are in close contact with others with infectious TB. Individuals living in such conditions have poor access to light, fresh air, sufficient food, and clean water, predisposing them to a variety of infections. Moreover, the increase in world travel has allowed the rapid spread of infectious agents across borders and has exposed all populations to the threat of this disease. One of the most important contributing factors to the resurgence of TB is the HIV/AIDS pandemic. The increase in TB rates began in the 1980s along with the emergence of HIV, and rates have continued to rise with the increased percentage of HIV positive individuals worldwide (1, 11, 14). It has been estimated that the risk of developing active TB among HIV/TB co-infected persons is 8% per year, compared to a 10% lifetime risk for those latently infected with M. tuberculosis alone (16). In addition, AIDS patients are also susceptible to infection with opportunistic mycobacteria such as the M. avium complex and other atypical mycobacteria (17). TB has proven to be the  4  principal cause of mortality among HIV positive persons in hospitals and HIV is more prevalent among TB patients than within the whole populations, indicating a tragic synergy between the two diseases (18). According to the WHO, more than 30% of the increase in TB cases since 2000 can be associated with HIV co-infection (19). The recent emergence of multiple drug-resistant (MDR) and extensively drugresistant (XDR) strains of M. tuberculosis are further complicating treatment of this disease. These strains require lengthy treatment with second line anti-TB drugs which are not only more expensive than first-line chemotherapeutics, but produce more severe sideeffects. TB treatment and prevention efforts are both extremely complex and advancement has been confounded by a number of factors that will be explored in the next section.  1.1.3  Prevention of Tuberculosis  History has shown that TB can be controlled to some extent by improving standards of living, such as sanitation, clean drinking water and nutrition. However, these advancements are not always possible, especially in developing nations where additional measures must be taken to prevent new infections. The currently available TB vaccine, Bacille Calmette-Guerin (BCG), originated from a virulent strain of Mycobacterium bovis that was passaged extensively over 13 years causing loss of virulence in animal models (20, 21). BCG has been used since the 1920s in attempts to prevent TB infection, but has demonstrated a wide range of efficacy from 0-80% in randomized control trials so is no longer in use in some parts of the world, including Canada (22). Despite  5  variability in protecting against pulmonary TB in adults, BCG has been proven effective, and is still used for protection, against childhood TB, TB meningitis, and miliary TB with over 100 million infants vaccinated per year (23). The range of observed efficacy of BCG against TB can be attributed to a number of factors including the great global variation in clinical TB strains, altered immunity due to previous exposure to environmental mycobacteria, and the absence of TB antigens in the vaccine strain (24-26). An ideal vaccine would prevent disease in the absence of prior exposure to M. tuberculosis, prevent reactivation of latent infection, and would not interfere with TB diagnosis (3, 27). Current strategies for vaccine development include attempts to boost the T-helper type 1 (Th1) immune response by using immunodominant antigens of M. tuberculosis such as Antigen 85A and the 6 kDa early secreted antigenic target (ESAT-6) as protein subunit or DNA vaccines (28-31). Alternatively, the current BCG vaccine could be improved by inducing expression of such immunodominant antigens (32, 33). Several groups are also attempting to produce live, attenuated M. tuberculosis virulence mutants, or auxotrophic mutants although the safety of live vaccines is controversial (34-36). Recently, the role of apoptosis in immunity to M. tuberculosis has lead to the development of pro-apoptotic mutants that elicit an enhanced immune response and may be considered as potential vaccine candidates (37, 38).  1.1.4  Treatment of Tuberculosis  Before the development of effective chemotherapy, nearly half of TB patients died within two years of developing active pulmonary disease (39). There are now five  6  first-line antibiotics for use in treating TB; streptomycin (STR), isoniazid (INH), ethambutol (EMB), rifampin (RIF), and pyrazinamide (PZA). STR, which works by inhibiting protein synthesis, was first discovered in the mid 1900s and was demonstrated to be effective against pulmonary TB in one of the first ever randomized clinical trials (40-42). INH was the next drug added to the treatment regimen, and was found to function by inhibiting cell wall synthesis, similarly to EMB which was discovered soon after INH (43-45). The addition of RIF, an inhibitor of RNA synthesis in M. tuberculosis, in 1970 allowed for a decrease in treatment duration to 9 months (46, 47). Although discovered in the 1950s, it wasn’t until 1986 that PZA, an inhibitor of fatty acid synthesis, was added to the INH/RIF regimen, further reducing treatment time to 6 months (48-50). The worldwide resurgence of TB since the 1980s and the emergence of drugresistance prompted the WHO to implement Directly Observed Therapy (DOT), combining the use of standardized diagnostics and therapeutics along with case management in efforts to improve detection and treatment (51). The current drug regimen used in areas with high TB incidence is a two month induction phase of combined therapy of INH/RIF/PZA and either EMB or STR, followed by a continuation phase of RIF/INH for up to four additional months (47, 52). This regimen results in a success rate of greater than 85% among drug-sensitive strains compared to greater than 50% mortality if active TB is left untreated (53). Successful treatment of TB is highly dependent upon strict adherence to prescribed drug therapy. Compliance is often compromised due to the length of treatment, drug side effects and, especially in the developing world, access to adequate  7  heath care services. These obstacles are even further confounded by the increased rates of multi-drug resistant and extensively drug resistant strains of M. tuberculosis.  1.2  Mycobacterium tuberculosis biology  Mycobacterium tuberculosis is a member of the genus Mycobacterium and the order Actinomycetales. Other members of the genus include non-pathogenic environmental strains such as M. smegmatis, opportunistic pathogens such as members of the M. avium-intracellulare complex, and pathogenic strains such as M. leprae, the cause of leprosy, and M. ulcerans, the cause of buruli ulcers. Other pathogenic strains that do not normally affect humans, but are important in the agricultural industry include M. bovis, which causes a TB-like disease in cattle and many other species, and M. avium ssp. paratuberculosis, the causative agent of Johne’s disease in several species of ungulates (54). The mycobacteria are slim, rod-shaped organisms 0.2 to 0.4 x 2 to 10 µM in size. M. tuberculosis, the most notorious member of the Mycobacteria, was first isolated and cultured by Robert Koch in 1882. It is a slow growing, non-motile, non-spore-forming intracellular pathogen that is highly resistant to drying and chemical disinfection. The complex cell wall of M. tuberculosis includes mycolic acid, a β-hydroxy fatty acid that makes the organism difficult to visualize with commonly used stains. Specialized stains such as Ziehl-Neelson, carbol fuchsin, and Auromine O must be used to stain mycobacteria which, once stained, resist decolourization with up to 3% hydrochloric acid, 95% ethanol, or both; this characteristic is referred to as acid-fastness (2, 54, 55).  8  The cell wall of M. tuberculosis is highly intricate and a major determinant of virulence for the pathogen. The plasma membrane is surrounded by the cell wall core: a layer of peptidoglycan covalently linked to arabinogalactan, which is in turn attached to the long mycolic acids. The upper segment of the cell wall structure is composed of free lipids, and fatty acid chains interspersed with the cell wall proteins. These proteins include the phosphatidylinositols (PIMs), the phthiocerol-containing lipids, lipomannan, and lipoarabinomannan (LAM) (56). One of the cell wall lipids, cord factor (trehalose 6,6'-dimycolate, TDM) is a mycolic acid containing glycolipid and is responsible for the characteristic serpentine cording often visible when M. tuberculosis is grown on solid media, and the clumps formed in liquid culture (56).  The outer layer of the  mycobacterial cell wall, or the capsule, consists of polysaccharides, proteins, and lipids and has been shown to play a role in host cell interactions The complex cell wall is a major factor contributing to the very slow growth of this organism which has a doubling time of approximately of 10-20 hours in liquid media, or 24 hours on solid media. Slow growth poses a challenge for diagnosis and treatment of Tuberculosis; clinical specimens such as sputum samples must be incubated for several weeks to confirm diagnosis, and testing of antibiotic susceptibility is also affected, leading to severe consequences for patients infected with drug-resistant strains.  1.3  The immune response to Mycobacterium tuberculosis  Although infection with M. tuberculosis can occur at virtually any tissue site, the lung represents the chief site of infection. When droplets containing TB bacilli are  9  expelled by an infectious host, they can be taken up by others in the vicinity. The organisms enter the lungs where they are taken up by phagocytes, mainly alveolar macrophages (Mφ). Mφ and dendritic cells (DC), the main mediators of the innate response to M. tuberculosis, can then stimulate adaptive immunity through antigen presentation and the production of cytokines. Specific T cells stimulated in the draining lymph nodes can travel to the site of infection and induce the formation of granulomas, which contain the infection. However, eradication of the bacteria does not occur and the infection may persist or enter a state of dormancy. Several components of the immune system are involved in the response to this pathogen and will be discussed in following sections.  1.3.1  Mycobacteria:Mφ φ interactions  Mφ are most often the first cells of the immune system to encounter inhaled M. tuberculosis bacilli. It is well-known that these cells serve as a niche for M. tuberculosis and are exploited during the establishment of successful infection. The interaction of M. tuberculosis with Mφ can occur in the presence or absence of serum opsonins (57, 58). In healthy individuals, the lungs are relatively free of serum opsonins so it is thought that the initial uptake of M. tuberculosis occurs non-opsonically; this inference is supported by the finding that alveolar Mφ do not express high levels of receptors for serum opsonins (59, 60). Several studies have been devoted to characterizing the Mφ surface molecules involved in mediating the binding and phagocytosis of M tuberculosis (61). To date,  10  mycobacteria have been reported to be detected or bound by: the complement receptors, CR1, CR3 and CR4 (57, 58, 62, 63); Fcγ receptor (64); the mannose receptor (62, 65-67); scavenger receptors (61); surfactant proteins A and D (68-71); dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) (72, 73); CD14 (74); CD43 (75, 76); CD44 (77); and possibly dectin-1 (78, 79) and the toll-like receptors (80-82). The relative contribution of each of these moieties to mycobacterial recognition is not completely understood, though the complement receptors appear to have a major role in binding M. tuberculosis in both opsonic and non-opsonic environments (58, 83). The possible ligands on mycobacteria that bind the aforementioned Mφ surface molecules remain unidentified, although several studies point to potential candidates. For example, lipoarabinomannan is thought to bind the mannose receptor (65, 84) and phosphatidylinositols have been shown to bind CR3 at a site distinct from the iC3b binding domain (83, 85, 86). It is well established that Mφ can mediate killing or inhibition of intracellular pathogens. The antimicrobial activities of Mφ have proven to be important elements of immunity, but are not completely understood. In response to M. tuberculosis, Mφ employ several effector mechanisms including the production of reactive nitrogen (RNI) and oxygen (ROI) intermediates, phagolysosome fusion leading to phagosome acidification, and the induction of apoptosis (87, 88). The intracellular survival of M. tuberculosis depends on the organism’s ability to inhibit such antimycobacterial functions, and the bacterium has evolved several strategies to do so. Mycobacteria have the capacity to arrest the development of the phagosome at the early endosome stage, and to hinder the fusion of the phagosome with lysosomes (64, 89,  11  90). This property, displayed by virulent strains of M. tuberculosis (91) prevents acidification of the phagosome and allows access to the extracellular milieu, thereby giving the pathogen to readily access nutrients such as iron and facilitating intracellular survival (92, 93). Residing in the early endosome may also reduce antigen processing, impeding activation of other immune cells (94). Phagosome maturation is promoted by activation of Mφ by IFN-γ, which also stimulates other antimycobacterial functions including the production of ROI and RNI (92, 95). The activation of Mφ by cytokines such as TNF-α and IFN-γ results in the initiation of intracellular signaling pathways that lead to the activation of NADPH oxidase and the generation of ROI such as superoxide, hydrogen peroxide, hydroxyl radicals and singlet oxygen, and the production of nitric oxide from L-arginine by inducible nitric oxide synthase (96). In the murine model, RNI have been shown to have a protective role in both the acute and chronic phases of M. tuberculosis infection (97, 98). Moreover, high levels of nitric oxide synthase expression has been detected in alveolar lavage fluid from patients with active TB (99). The importance of ROI, however, is not as as clearly described (100), although hydrogen peroxide produced by activated Mφ has been shown to be mycobacteriocidal (101). Mycobacteria are capable of evading these toxic metabolites by mechanisms such as the production of a catalase peroxidase, KatG, which has been shown to be required for growth of M. tuberculosis in vivo (102) and counteracts the toxic effects of hydrogen peroxide in vitro (103). An additional innate defense mechanism employed by Mφ is the induction of apoptosis, which allows for controlled killing of the infected cell without eliciting an inflammatory response (104). A role for apoptosis in restricting the growth of M.  12  tuberculosis has been well-evidenced and is thought to function by depriving the bacterium of a niche and leading to enhanced cross-priming of antigen-specific CD8+ T cells (37, 105-108). Evasion of apoptosis by mycobacteria has also been described as an additional means of intramacrophage survival (105, 109, 110) and is thought to occur by the upregulation of anti-apoptotic pathways by the pathogen (111).  1.3.2  Adaptive immunity  Cell-mediated immunity to M. tuberculosis develops 2 to 6 weeks after infection following the secretion of cytokines and chemokines by infected phagocytes including Mφ and DCs. These cells can also travel to the draining lymph nodes where they will present mycobacterial antigens to circulating T cells (88, 112). The T cell subsets involved in mounting a protective response against M. tuberculosis include primarily CD4+ and CD8+ T cells (88, 113, 114), although there is increasing evidence of a role for CD1 restricted T cells (108, 115, 116) and αβ/γδ T cells (117-119) may also play a role (120). Expansion of antigen-specific T cells is followed by the development of the granulomas at the site of infection; such lesions contain activated B and T lymphocytes, monocytes, fibroblasts, and multi-nucleated giant cells (88, 121). Granuloma formation occurs in an attempt to sequester and prevent dissemination of the infection, but mycobacteria exploit this environment to establish a latent infection that can eventually re-activate. Such re-activation of infection will cause increased bacterial growth and caseation of the granulomas, leading to eruption of the lesion and release of the bacteria into the airways thereby facilitating transmission to a new host (88, 121, 122).  13  1.3.3  Latency/persistence  A feature of M. tuberculosis central to its pathogenesis is its unique ability to persist in the granulomas of an asymptomatic host. In these situations, the immune response is able to restrict and contain the pathogen, but not completely eradicate it. Viable M. tuberculosis have been isolated from granulomatous lesions in the lungs of individuals with clinically inactive TB, suggesting that the bacteria can survive in a latent state for several years (123, 124). It is not known whether the bacteria are actually metabolically dormant or if they continue to replicate during inactive disease. Attempts to investigate these questions are made difficult by the lack of animal models that accurately represent human latent TB, though in vitro and in vivo systems have been developed and have contributed to the current knowledge (125-129). Further investigation is necessary, given the difficulties surrounding treatment of latent TB.  14  1.4  CD43  CD43 (gpL115; leukosialin; sialophorin) is a sialoglycoprotein that is abundantly expressed on cells of haematopoietic origin including monocytes/macrophages, granulocytes and T cells (130-132). The molecule is a member of the mucin-type Oglycoprotein family, which are characterized by the linkage of N-acetylgalactosamine to serine or threonine residues. CD43 has been shown to have a role in number of biological functions including cell activation, regulation of apoptosis, lymphocyte homing, and cell signaling (133, 134). It is proposed that CD43 has dual function in cell-cell interaction as there is evidence for it acting as both an adhesion and a barrier molecule (133, 135-142). CD43 knockout mice (CD43-/-) have been generated by homologous recombination via embryonic stem cell chimeras. Genetic deletion of CD43 results in a mild phenotype with normal T cell development and normal haematopoiesis (135, 143). Although preliminary attempts at knocking out the CD43 gene in mice resulted in a hyperproliferative T cell phenotype (135), this was likely due the 129 genetic background of the mice used, as it has since been shown that CD43-/- mice backcrossed onto the C57Bl/6 background do not display this hyper-responsive phenotype (143).  15  1.4.1  CD43 expression  The human CD43 gene is found on chromosome 16p11.2, while in mice it is located on chromosome 7 (144-146). In both species, the gene is encoded by two exons, the first encoding for a 5’-untranslated sequence whereas the second exon encodes the entire polypeptide backbone (145, 147, 148). Furthermore, CD43 in lymphoid cell lines has been found to be encoded by two mRNA species with diverse polyadenylation signals, that are encoded by the same CD43 exon and translated to a single gene product (144, 147). It is thus evident that the molecular weight heterogeneity of the CD43 molecule is not due to alternative gene splicing, but post-translational modifications (131). As previously stated, CD43 expression has been found on most haematopoietic cells, with the exception of mature B cells. CD43 was originally described on K562 human erythroleukemic cells (149), but has since been found to be expressed on a number of cell types in humans, mice, rats and rabbits (131, 132, 138, 150-152). Tissue specific expression patterns of CD43 isoforms have been observed on granulocytes, monocytes, platelets, pre-cursor and mature T cells, B cell pre-cursors and antibodysecreting plasma cells (131, 153-155). It is proposed that the level of CD43, and the prevalent gycoforms expressed on these cell types varies with cell lineage, or maturation/activation state of the cells (156). Although CD43 is thought to be a lymphohaematopoietic cell surface marker, it has also been found on colon cell adenomas carcinomas, even though normal epithelial  16  cells in the colon do not express CD43 (157, 158). The kinetics of increased CD43 expression in these studies suggests a role for the molecule in tumour development. The regulation of CD43 expression is thought to occur by several mechanisms. The mucin-like extracellular region of CD43 has been found in human plasma so there appears to be some means of shedding (159). There is evidence for the molecule being proteolytically cleaved from neutrophils, T cells and mast cells (160-162), and in neutrophils this can be induced by antibody ligation or by stimulatory molecules such as TNF-α (163). In dendritic cells, antibody ligation of CD43 causes internalization of the molecule by endocytosis (164).  1.4.2  CD43 structure  CD43 is a single chain type I transmembrane glycoprotein of 400 amino acids in humans or 395 in mice including a leader peptide (19aa) followed by the extracellular region (234aa in humans, 228aa in mice), a transmembrane domain (22aa) and an intracytoplasmic domain (123aa in humans, 124aa in mice) (145, 165). The overall composition of the molecule has been found to be 60% carbohydrate and 40% protein (165, 166). CD43 has a significant number of sialic acid residues, which impart a net negative charge on the molecule and may be responsible for some of its anti-adhesive properties (167). The extracellular mucin-like region extends 45nm from the cell surface and is a rigid carbohydrate covered polypeptide backbone rich in serine, threonine, and proline residues (144, 168). Proteolytic cleavage of the extracellular region of CD43 can  17  occur, and has been found in a soluble form known as galactoglycoprotein in blood plasma (169). There are two major isoforms of CD43 that differ in the glycosylation of the extracellular portion leading to molecular weight heterogeneity. The 115kDa glycoform has been shown to be predominant on resting T cells and monocytes and is characterized by tetrasaccharide side chains, whereas neutrophils and activated T cells carry mainly hexasaccharide side chains and express the 135kDa CD43 glycoform. Some cell types, including macrophages, are thought to express a mixture of both isoforms (170, 171). The branching of the O-glycans on CD43 is attributed to the activity of the glycosyltransferase Core 2-β−1,6-N-acetylglucosaminyl transferase (C2GnT), which catalyzes the transfer of N-acetylglucosamine residues on O-glycans (171-174). Monoclonal antibodies (mAb) can be used to distinguish the two CD43 glycoforms. For example, the rat mAb 1B11 recognizes only the 135kDa hexasaccharide form, whereas the lower molecular weight tetrasaccharide glycoform can be identified by the rat mAb S7. In contrast, the mAb S11 has been shown to recognize CD43 independent of glycosylation and can thus be utilized as a pan-CD43 antibody (174-176). It has been demonstrated that the molecular weight heterogeneity of CD43 is due to post-translational modifications and not alternative exon splicing (131). Furthermore, heterogeneity among species is largely due to differences in the extracellular regions, as the transmembrane and cytoplasmic domains are highly conserved among humans, mice and rats. Mouse and human CD43 transmembrane and cytoplasmic domains share, respectively, 65% and 72% sequence identity while the extracellular region shows only  18  42% identity (146, 152, 177). Despite differences in the extracellular region, the heavily glycosylated mucin characteristics remain similar across species.  1.4.3  Described functions of CD43  Several distinct functions have been described for CD43 including roles in cell activation and proliferation, and signal transduction. In cell-cell interactions, CD43 is proposed to have a dual function as there is evidence for both adhesive and anti-adhesive properties (133). Moreover, CD43 has been implicated in regulating cell death through its role in apoptosis Evidence for these suggested functions of CD43 will be discussed in this section.  Cell Activation CD43 has been shown to be involved in the activation of T cells, monocytes and neutrophils. Several studies showed that anti-CD43 mAb (L10) could induce proliferation in T cells comparable to that induced by anti-CD3 (178, 179). It was later shown that additional anti-CD43 antibodies could amplify T cell proliferative responses induced by the known T cell mitogens Concanavalin A and leukoagglutinin (138). Sperling et al. found that an anti-CD43 mAb (R2/60) could co-stimulate T cells in the absence of accessory cells in a fashion similar to CD28, suggesting a role for CD43 as a T cell receptor (180). Anti-CD43 mAb L10 has also been implicated in activation of monocytes, as measured by hydrogen-peroxide producing capacity, at levels comparable to activation  19  by IFN-γ (181), and in activation of neutrophils leading to increased adhesiveness and aggregation (182).  Cell signaling Although there are no definitive ligands identified for CD43, antibody ligation of the molecule has been shown to induce cell signaling events. It is likely this process is mediated by the cytoplasmic tail of CD43, as this region displays properties which indicate signaling capabilities, such as the presence of conserved serine and threonine residues that could potentially be phosphorylated, and has been found to be required for CD43 to generate intracellular signals (183-186). Park et al. found the cytoplasmic domain of CD43 to be hyper-phosphorylated during T cell activation, and others have shown constitutive phosphorylation in response to tumour promoting phorbol esters and ligation of the molecule with anti-CD43 antibodies (183, 186, 187). CD43 engagement in human T cells leads to tyrosine phosphorylation and recruitment of Src family kinases to the cytoplasmic tail of CD43, and phosphorylation of the adapter proteins Shc and SLP76 leading to several downstream signaling events (180, 188, 189). Furthermore, CD43mediated activation of T cells can result in activation of the mitogen-activated protein kinase pathway, causing recruitment of transcription factors including AP-1, NF-AT, and NF-κB, ultimately leading to regulation of gene expression (189, 190). Despite the evidence for signaling capabilities, and identification of some components of CD43mediated signaling cascades, the mechanisms by which CD43 leads to these events are not completely understood. It should be noted that CD43 has also been implicated in  20  signaling events leading to cell death by apoptosis, evidence for which will be discussed in the proceeding section.  Apoptosis Numerous experimental findings have pointed to a role for CD43 in the regulation of apoptosis. Both pro- and anti-apoptotic roles have been described as ligation of CD43 on T cells and haematopoietic progenitor cells can induce cell death (191-194), but high levels of CD43 expression on T cells can inhibit T cell receptor (TCR)/CD3 mediated apoptosis (195). Recently, it was found that CD43 can modulate Fas-induced cell-death in a human T lymphoblastic cell line pointing to a potential role in activated-induced cell death (196). CD43 expression decreases during neutrophil apoptosis and has been shown to parallel, but not result from, membrane blebbing, with the molecule being released on microvesicles distinct from blebs (197, 198). Moreover, transient capping of CD43 has been observed on Jurkat cells undergoing apoptosis and that this correlated with the ability of macrophages to recognize these cells, suggesting that CD43 capping is responsible for the generation of ligands for recognition of dying cells by the immune system (199). The mechanisms by which CD43 modulates apoptosis are unclear, but it could possibly occur by direct receptor-ligand interactions during adhesion, or CD43 itself may be a transducer of apoptotic signals.  Adhesion vs. Anti-adhesion The role of CD43 in cell-cell interactions has been heavily debated, as there is contradictory evidence whether the molecule has an adhesive or anti-adhesive role (133,  21  134). The large size of CD43, and its heavily glycosylated and negatively charged extracellular region would suggest a tendency for the molecule to repel cell contact. In agreement with these physical properties, it has been shown that CD43 expression causes decreased cell adhesion and that targeted disruption of the molecule increases cell-cell contact (135-137, 200, 201). Conversely, there is also substantial evidence pointing to a pro-adhesive role for CD43. Although the specific in vivo interactions are unknown, several ligands have been proposed for CD43, including intercellular adhesion molecule-1 (ICAM-1) (202), complement component C1q (203), E-selectin (204, 205), galectin-1 (206), sialoadhesin (207), and major histocompatibility complex class 1 molecules (MHC-1) (141). Additionally, anti-CD43 mAbs are able to inhibit cell interaction, further supporting adhesive functions of CD43 (139, 140, 208). The structural properties discussed earlier, and the experimental evidence for the diverse roles of CD43 suggest that the function of the molecule may depend on the specific interactions in play. Clearly, there is evidence supporting a dual-functionality of CD43 that is integral to regulation of cell interactions.  1.4.4  CD43 & disease  Dysregulation of CD43 expression has been shown to have a potential role in a number of disease processes including graft-versus-host disease (GVHD), WiskottAldrich Syndrome (WAS), Human Immunodeficiency Virus/Acquired Immune  22  Deficiency Syndrome (HIV/AIDS), and Rheumatoid Arthritis. The evidence for the involvement of CD43 in these conditions will be further discussed in this section.  Graft-versus-host Disease GVHD is a syndrome characterized by pathology in several organs including the skin, liver, lungs and gastrointestinal tract. This disease is a common complication of allogenic stem cell transplantation and can be classified as acute or chronic. Acute GVHD is mediated by immunocompetent donor T cells migrating to lymphoid tissues after infusion, where they recognize host alloantigens and become activated upon interaction with host antigen-presenting cells (209). These activated donor T cells display differential expression of the CD43 glycoforms. In GVHD patients, activated donor T cells show increased expression of 135 kDa CD43 (210). In the mouse model of GVHD, it has been shown that both 115 kDa and 135 kDa structures are upregulated on CD4+ T cells in the spleen. Interestingly, on CD8+ cells, CD43 135 kDa was found to be upregulated while CD43 115 kDa was down regulated. Additionally, other activation markers such as IL-2α/β and ICAM-2 were found to be upregulated on these cells, lending further evidence for 135 kDa CD43 being characteristic of activated T cells (211). Moreover, a diverse population of small intestine epithelial T cells called intraepithelial lymphocytes (IEL), which normally express either 115 kDa or 135 kDa CD43, express both CD43 glycoforms in GVHD patients (212). These reports link CD43 to effector-like activities in GVHD that are related to the glycosylation of the molecule.  23  Wiskott-Aldrich Syndrome Wiskott-Aldrich syndrome (WAS) is an inherited immunodeficiency syndrome characterized by thrombocytopenia, eczema, recurrent infections, and increased incidence of autoimmune diseases and malignancies (213, 214). Several studies have found abnormal glycoforms of CD43 exhibiting altered glycosylation patterns in WAS patients (170, 172, 215, 216). Further, circulating T cells in WAS patients were found to express the high MW glycoform of CD43, which is normally only associated with activated T cells (216). The activity of the enzyme responsible for the branching of Oglycans on CD43, C2GnT, was found to be increased in resting T cells, B cells and platelets of WAS patients (216). The abnormal branching on CD43 and increased activity of C2GnT indicates a dysregulation of T cell activation in WAS patients, and it has been proposed that this causes escape of self-reactive T cells into the periphery leading to autoimmunity. In addition, T cells from WAS patients show reduced proliferation in response to immobilized CD43 antibody and produce less IL-2, leading to further immune defects (217). A role for CD43 has also been proposed in eczema seen in WAS patients, as altered T cell binding to glycoproteins in the epidermis has been linked to CD43 and atopic dermatitis and other forms of cutaneous inflammation (218-220).  HIV/AIDS Acquired immune deficiency syndrome (AIDS) is caused by one of two lentiviruses termed human immunodeficiency viruses (HIV-1 and HIV-2), which disable the immune system, thus predisposing patients to an array of opportunistic infections. Auto-antibodies to CD43 have been found in HIV patients’ T cells (221). Furthermore, T  24  cells from these patients appear to be sulphated and hyposialated, which could impair CD43-mediated homotypic aggregation and cause the formation of auto-antibodies, which may accelerate disease progression (222, 223). Such auto-antibodies may affect the viability of T cells in HIV patients as CD43 ligation has been shown to induce apoptosis (191, 192). In addition, one group found that HIV patients that have progressed to AIDS expressed a partially sialated CD43 in the activation-associated form (135 kDa), whereas an asymptomatic HIV patient expressed the 115 kDa glycoform on T cells, consistent with healthy controls suggesting that MW of CD43 increased with disease progression (224, 225).  Rheumatoid Arthritis Rheumatoid arthritis (RA) is an autoimmune disease primarily characterized by chronic inflammation of the joints and surrounding tissue leading to destruction and functional disability, but it can also affect other organs leading to systemic illness. T cells in the synovial joint fluid of RA patients have been shown to have higher than normal levels of the high MW CD43 135 kDa. It has also been demonstrated that adhesion molecules, crucial to inflammation, are expressed in neutrophils of RA patients’ joint tissue, and that the integrin CD11b is upregulated, while L-selectin, CD43 and CD44 are down-regulated (226). Despite this decreased expression of CD43, the interaction of these neutrophils with the endothelium remains intact. CD43 shedding was found to be enhanced by a neutrophil elastase that is present in high quantities in synovial joint fluid of RA patients (167). The significance of these findings to RA pathology remains to be elucidated.  25  1.5  Introduction to the thesis  The information presented in this chapter validates the importance of research into TB pathogenesis. It is quite apparent that novel preventative and therapeutic strategies are required and thus we must broaden our knowledge of the disease processes from both the perspective of host defenses and bacterial evasion of such defenses. The interactions that occur between the pathogen and host are pertinent to our understanding of mycobacterial biology and TB pathogenesis. The initial event in infection, recognition of M. tuberculosis by host Mφ, is a critical step in the establishment of infection and therefore merits thorough investigation. Numerous Mφ receptors have been identified as having a role in the initial association with M. tuberculosis but the differences in the outcome of infection depending on the route of bacterial entry are not fully understood. A study in 2000 by Fratazzi et al. was the first evidence of a potential role for CD43 in mycobacterial infections (75). CD43 was found to be required for efficient binding of mycobacteria by macrophages and CD43-transfected HeLa cells, and addition of soluble human CD43 restored binding of M. avium to splenic macrophages from CD43 deficient mice. The authors suggested that CD43 is important in promoting a stable interaction between mycobacteria and host cells. Although no specific evidence was given for in vivo mycobacterial disease, this was the first description of a possible role for CD43 in an infectious process, and further investigation was indeed warranted. In addition to this important evidence that CD43 may be involved in the host response to mycobacteria, it has been shown to be involved in a number of  26  immunological events and disease processes as previously mentioned. The described roles of CD43 in cell migration, activation, signaling and apoptosis led us to hypothesize that the role of CD43 in mycobacterial pathogenesis may extend beyond initial Mφbacteria interactions and that CD43 may be a significant feature of the innate response to TB infection. We tested our hypotheses by first confirming the findings of Fratazzi, et al. and by extending those findings to additional cell types and mycobacterial strains, as outlined in Chapter 2. 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Expression of Aberrant OGlycans Attached to Leukosialin in Differentiation-deficient HL-60 Cells. Cancer Res 51:2854.  46  225.  Saitoh, O., F. Piller, R. I. Fox, and M. Fukuda. 1991. T-lymphocytic leukemia expresses complex, branched O-linked oligosaccharides on a major sialoglycoprotein, leukosialin. Blood 77:1491.  226.  Alicia Humbría, F. Diaz-Gonzalez, M. R. Campanero, A. G. Arroyo, A. Laffon, R. Gonzalez-Amaro, and F. Sanchez-Madrid. 1994. Expression of L-Selectin, CD43, and CD44 in Synovial Fluid Neutrophils from Patients with Inflammatory Joint Diseases. Arthritis & Rheumatism 37:342.  47  Chapter 2  2.1  CD43 is required for optimal growth inhibition of Mycobacterium tuberculosis in macrophages and in mice1  Summary We explored the role of macrophage (Mφ) CD43, a transmembrane glycoprotein,  in the pathogenesis of M. tuberculosis. Using gene-deleted mice (CD43-/-), we assessed the association of the bacterium with distinct populations of Mφ and found that CD43-/Mφ bound less M. tuberculosis than CD43+/+ Mφ. Increased infective doses did not abrogate this difference. However, reduced association due to the absence of CD43 could be overcome by serum components. Mφ from heterozygote mice, which express 50% of wild type CD43, bound more bacteria than CD43-/- but less than CD43+/+ proving that the gene dose of CD43 correlates with binding of M. tuberculosis. Furthermore, the reduced ability of CD43-/- Mφ to bind bacteria was restricted to mycobacterial species. We also found that the survival and replication of M. tuberculosis within Mφ was significantly enhanced in the absence of CD43, making this the first demonstration that the mechanism of mycobacterial entry influences its subsequent growth. Most importantly, we show here that the absence of CD43 in mice aerogenically infected with M. tuberculosis results in an increased bacterial load during both the acute and chronic stages of infection and more rapid development of granulomas, with greater lung involvement and distinctive cellularity.  1  A version of this chapter has been published as:  Randhawa, A. K., H. J. Ziltener, J. S. Merzaban, and R. W. Stokes. 2005. CD43 is required for optimal growth inhibition of Mycobacterium tuberculosis in macrophages and in mice. Journal of Immunology 175: 1805-1812. 48  2.2  Introduction  Mycobacterium tuberculosis infects over 8 million people and causes nearly 2 million deaths annually, making it the deadliest human pathogen. The World Health Organization estimates that between 2002 and 2020, 1 billion people will become infected with M. tuberculosis and over 36 million people will die of Tuberculosis (TB) if the rise in incidence is not controlled. A critical step in the pathogenesis of M. tuberculosis is the initial interaction of the pathogen with the host macrophage (Mφ). This interaction is mediated by several Mφ receptors in association with ligands on the bacterium including the complement receptors CR1, CR3, and CR4, (1-6), Fcγ receptor (7), mannose/glucan receptors (1, 8), CD14, (9, 10), scavenger receptors (4) and surfactant protein receptors A (11, 12) and D (13). It has also been shown that CD43 (leukosialin; sialophorin) may be important in promoting a stable interaction of mycobacteria with Mφ (14) . CD43 is a negatively charged transmembrane sialoglycoprotein expressed on most hematopoietic cells (15). The function of this molecule has been the subject of debate; it has been shown that CD43 on T cells and B cells acts as a barrier molecule restricting cell-cell contact (16-19) but that it can also have a pro-adhesive quality (2022). Thus, it has been proposed that CD43 may play a dual role in intercellular contact (23, 24). Involvement of CD43 in leukocyte homing and tissue infiltration, possibly due to its adhesive or anti-adhesive properties, has been shown in several studies (19, 25, 26). It has also been demonstrated that CD43 can regulate cell survival (27, 28) and is involved in the apoptosis of T cells and hematopoietic progenitor cells (29-32).  49  Fratazzi et al. first described a role for CD43 in mycobacterial pathogenesis when they found that splenic Mφ from CD43-/- mice could not bind M. tuberculosis or Mycobacterium avium in vitro but that the ability to bind M. avium could be restored by addition of the extracellular region of CD43. They also found that CD43-transfected HeLa cells bound M. avium but not other bacteria, and that CD43 was required for TNFα production by Mφ in response to infection with M. avium (14). In this study, we further explore the role of CD43 in the binding and uptake of M. tuberculosis by Mφ and to determine the role of CD43 in M. tuberculosis pathogenesis using a gene-deleted mouse model that lacks expression of CD43 (33).  50  2.3  Materials and methods  Bacteria M. tuberculosis strain Erdman (TMC #107; ATCC #35801), M. tuberculosis strain H37Rv (TMC #102, ATCC #27294), and M. avium (TMC #724, ATCC #25291) were grown to late log phase in Proskauer & Beck medium supplemented with 0.05% Tween 80. Cultures were stored and tested for viability as previously described (2). Salmonella enterica serovar Typhimurium (S. typhimurium) and Listeria monocytogenes were grown to mid-log phase in tryptic soy broth (Difco) and washed in PBS before use.  Mice Wild-type (WT) control mice (CD43+/+), CD43-/-, and CD43+/- mice backcrossed 7 generations on C57Bl/6 background (33) were housed in a specific pathogen-free animal facility in micro isolator cages.  Experiments were done in  accordance with the standards set by the Canadian Council on Animal Care. For all experiments, mice were age- and sex-matched and controls were littermates.  Macrophage monolayers Resident alveolar, peritoneal, and bone marrow-derived Mφ (AMφ, PMφ and BMMφ, respectively) were obtained from CD43-/- and CD43+/+ mice as previously described (2, 34, 35). Splenic Mφ (SpMφ) were obtained by gently disrupting spleens into single cell suspensions, washing in PBS, and resuspending cells in supplemented RPMI (RPMI 1640 medium with 10% fetal calf serum, 10 mM L-glutamine, and 10 mM  51  sodium pyruvate, all from Life Technologies, Inc., Grand Island, NY) at a concentration of 2x106 cells per mL. Cells were plated in 1 mL aliquots onto 13 mm acid-washed, sterile glass coverslips in 24 well plates and incubated at 37°C/5% CO2 for 24 hours, at which point the non-adherent cells were removed and 1 mL of fresh media added to each well. The cells were incubated for an additional 4 days before use.  Particles for probing macrophage receptors The function of FcγR was examined using sheep red blood cells (SRBC) coated with IgG (EIgG), complement receptors were investigated using SRBC coated with IgM and iC3b (EIgMC’), while lectin-like receptors were probed using zymosan particles, all as previously described (2). Latex beads (Diameter 1.07 µm, Polybead® polystyrene microspheres, Polysciences, Inc., Warrington, PA.) were used to investigate non-specific phagocytosis.  Flow Cytometry AMφ, PMφ, BMMφ and SpMφ from CD43-/- and control mice were isolated as described above and plated onto sterile petri dishes (bacteriologic plastic was used to facilitate subsequent removal of adherent cells). Following incubation at 37°C/5% CO2, non-adherent cells were removed by washing with RPMI. Adherent cells were removed by cooling and scraping, washed with DMEM (Life Technologies) and processed for flow cytometry. Cells were stained with mAb S11-FITC (rat anti-pan-CD43, kindly supplied by Dr. John Kemp, University of Iowa), (36, 37) and/or rat anti-mouse Mφ F4/80 (Caltag Laboratories, Burlingame, CA) at 2mg/mL, or with secondary antibody  52  alone (Streptavidin-cychrome). After staining, cells were washed twice with HBSS (Life Technologies), and analyzed on a FACScan IV flow cytometer (Becton Dickinson, Mountain View, CA).  In vitro assay for binding of particles to macrophages Mφ monolayers on coverslips in 24 well plates were washed twice with binding medium (138 mM NaCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, 0.6 mM CaCl2, 1 mM MgCl2, and 5.5 mM D-glucose) (38). A 500 µL aliquot of binding medium was added to each well, and the cells were acclimatized for 10 min at 37°C/5% CO2. For non-opsonic studies, the particles to be tested were diluted to the desired concentration in binding medium and added to monolayers. In studies of opsonic binding, 1% normal, or 1% heat-inactivated, CD43-/- or CD43+/+ mouse serum was added prior to addition of bacteria. For experiments with mycobacteria or particles, monolayers were infected for 1 hour rocking (Nutator, Becton Dickinson, Mountain View, CA) followed by 2 hours stationary at 37°C/5% CO2, whereas in experiments with S. typhimurium and L. monocytogenes, monolayers were infected for 40 min at 4°C. Monolayers were then washed 3 times, fixed, stained with Kinyoun's Carbol Fuchsin and malachite green for mycobacteria-infected Mφ, or Giemsa for other bacteria and control particles. Binding was quantified microscopically by counting 100 Mφ per coverslip and assessing the percentage of Mφ that bound at least 1 bacterium and the average number of bacteria that were associated with each infected Mφ.  53  In vitro survival and replication of M. tuberculosis following phagocytosis by CD43-/- and CD43+/+ Mφ φ Intracellular growth assays were conducted in BMMφ from CD43-/- and WT CD43+/+ mice. Monolayers were infected with M. tuberculosis Erdman at a multiplicity of infection (MOI) of 20:1 (bacteria:Mφ). Because preliminary studies showed that fewer bacteria were able to infect CD43-/- Mφ, these were also infected at 30:1. Monolayers were infected according to the procedures described above. To eliminate extracellular bacteria from the infected Mφ monolayer, coverslips were washed three times in binding medium after infection and transferred to new 24 well plates containing 1 mL of supplemented RPMI per well. At this time (day 0), and on days 1, 4 and 7 post-infection, coverslips and supernatants were processed to assess the CFU, as previously described (39).  Growth and pathogenesis of M. tuberculosis in mice CD43-/- and CD43+/+ mice were infected with a low dose of M. tuberculosis Erdman (50-100 CFU) using an inhalation exposure chamber (Glas-Col, Terre Haute, IN). On specific days post-infection, 4-6 mice in each group were euthanized, and lungs, livers and spleens were aseptically removed and weighed. A portion of each tissue was removed and fixed in 10% buffered formalin for histopathological examination while the remainder (lungs and spleens only) was homogenized in PBS to assess bacterial loads. Serial dilutions of tissue homogenates were plated on 7H10 agar supplemented with OADC. Plates were incubated at 37oC and CFU counted after 21-25 days. Another group  54  of mice was infected intravenously with M. tuberculosis by injecting 1 x 105 bacteria into the tail vein. Bacterial loads were determined as described above.  Histopathology Livers, spleens, and lungs from mice infected with M. tuberculosis (as described above) were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 µm, and stained with Hematoxylin and Eosin (H&E) for histopathological examination using standard techniques. The population of cells in areas of pulmonary inflammation was quantified by classifying cells as either (i) typical Mφ (including epithelioid cells), (ii) “foamy” Mφ - cells having highly vacuolated cytoplasm, which gave a foamy appearance or (iii) lymphocytes.  The proportion of each cell type in representative areas of  inflammation was determined by counting 100 cells, and the results expressed to the nearest 5% to allow for a degree of variation between areas.  Statistics Data are expressed as mean ± SEM. Student’s t test for independent means was used; p < 0.05 was considered significant unless otherwise noted.  55  2.4  CD43-/- Mφ φ of different origins bind M. tuberculosis less readily than CD43+/+ Mφ φ at various multiplicities of infection  We first determined if CD43 affected the non-opsonic binding of M. tuberculosis by using SpMφ, PMφ, BMMφ AMφ from CD43-/- and WT CD43+/+ mice. SpMφ, PMφ and BMMφ were infected at an MOI of 20:1 while AMφ were infected at a higher MOI of 500:1 as it has been shown that these cells do not bind M. tuberculosis very well in vitro (35). After a 3 hour infection, we found that SpMφ and PMφ bound bacteria more efficiently than did BMMφ and AMφ, as assessed by both the percentage of the Mφ population infected (data not shown) and the number of bacteria binding to individual infected Mφ (Figure 2.1A). All CD43-/- Mφ phenotypes bound significantly less bacteria than WT CD43+/+ Mφ (p<0.01); CD43+/+ SpMφ, PMφ, BMMφ and AMφ bound, respectively, 117%, 31%, 162% and 55% more bacilli than did CD43-/- derived Mφ (Figure 2.1A). We chose to use BMMφ as a model in subsequent studies as they can be obtained in large numbers and represent a recently differentiated Mφ, such as those that may be found entering sites of infection during the course of pathogenesis of M. tuberculosis. Infection of BMMφ with increasing numbers of M. tuberculosis demonstrated that reduced association of the bacteria with CD43-/- Mφ was consistent over a wide range of MOI. At ratios of 20:1, 40:1, 60:1, 100:1 and 200:1 (Figure 2.1B) bacteria:Mφ, CD43-/- Mφ associated with significantly less bacteria than did CD43+/+ Mφ (p<0.05). The reduction in non-opsonic binding of M. tuberculosis by CD43-/- BMMφ is  56  manifested not only as a reduction in the number of bacteria binding to the Mφ population, but also a reduction in the percentage of Mφ binding at least one bacillus (data not shown).  2.5  Opsonization of bacteria overcomes the impaired ability of CD43-/- Mφ φ to bind M. tuberculosis  CD43+/+ and CD43-/- BMMφ were infected with M. tuberculosis in the absence or presence of 1% normal- or heat inactivated (HI) (56°C for 30 min) mouse serum at a MOI of 40:1 (Figure 2.2). For these experiments, only serum autologous to the Mφ source was used. In the unopsonized control 27.8 ± 1.4% of CD43+/+ Mφ had associated with bacteria while only 9.3 ± 1.3% of CD43-/- Mφ were infected (p<0.001). In the presence of serum opsonins, a higher percentage of both Mφ populations bound bacteria and there was no significant difference between them (51.0 ± 1.6% of CD43+/+ and 47.3 ± 4.8% of CD43-/- Mφ bound bacteria). However, when monolayers were infected with M. tuberculosis in the presence of HI serum, the difference in binding of bacteria to the two Mφ populations was restored (32.5 ± 2.5% of CD43+/+ Mφ; 20.8 ± 1.9% of CD43-/Mφ associated with bacteria, p<0.01). Interestingly, the level of binding observed with CD43-/- Mφ in the presence of HI serum was higher than that seen in the absence of any serum.  57  2.6  CD43 is involved in binding other mycobacteria but its absence does not abrogate binding of S. typhimurium or L. monocytogenes by BMMφ φ  In order to determine if the reduced ability of CD43-/- Mφ to bind M. tuberculosis strain Erdman was unique, we infected BMMφ from CD43-/- and CD43+/+ mice with the virulent strain, H37Rv, with the opportunistic pathogen M. avium and with representative Gram-positive (L. monocytogenes) or –negative (S. typhimurium) intracellular bacteria (Figure 2.3). For M. tuberculosis and M. avium, BMMφ were infected at MOIs of 40:1 and 20:1, respectively, for 3 hours. For non-mycobacteria, monolayers were infected at a MOI of 10 bacteria per Mφ for 40 min at 4ºC. Less CD43/- Mφ were able to bind the mycobacterial species than could CD43+/+ Mφ. However, there was no difference between CD43-/- and CD43+/+ Mφ in their ability to bind the other bacteria.  2.7  CD43 deficiency does not affect Mφ φ non-specific uptake or phagocytosis via FcγγR and complement receptors, but does enhance binding via lectin-like receptors  To investigate whether CD43 deficiency affects the function of other Mφ receptors, we measured binding of several different control particles that are commonly used to study receptor-ligand interactions of phagocytes (Figure 2.4). EIgG were used to investigate the function of FcγR and EIgMC’ were used to examine complement receptors. Zymosan, a yeast cell wall preparation containing polysaccharides, was used  58  to probe for lectin-like receptors while latex beads were used to examine non-specific interactions. When assessing the actual number of particles associated with Mφ, there was no difference in the ability of the CD43-/- Mφ to phagocytose EIgG, EIgMC’, or latex spheres when compared to the CD43+/+ controls (Figure 2.4). However, CD43-/Mφ bound significantly more zymosan particles than WT Mφ (20.4 ± 0.8 vs. 14.13 ± 1.1, respectively, p<0.01).  2.8  The level of CD43 surface expression differs between Mφ φ phenotypes  CD43 cell surface expression was monitored using mAb S11 (37). The epitope recognized by anti-CD43 mAb S11 is not affected by changes in CD43 glycosylation (40), thus mAb S11 cell surface binding reflects the level of CD43 protein expression. As expected, all Mφ populations from CD43-/- mice did not express CD43 above background. Although all cell types from CD43+/+ mice expressed low levels of CD43 compared to T cells (41), levels on BMMφ were higher than those on SpMφ, PMφ, or AMφ (Figure 2.5). The mean fluorescence intensity for AMφ was 20% of that seen in BMMφ. In addition, a much lower percentage of the AMφ population expressed CD43.  2.9  CD43 gene dose correlates to the ability of BMMφ φ to bind M. tuberculosis  To ascertain whether the amount of CD43 expressed by Mφ affects their ability to bind M. tuberculosis, we compared the binding of the bacterium to Mφ that were heterozygous (+/-) for the CD43 gene (and express 50% less CD43 than do CD43+/+)  59  (33) with CD43-/- and CD43+/+ Mφ (Figure 2.6). We found that the CD43+/- Mφ population associated less with bacteria than did the CD43+/+ Mφ (19.0 ± 2.5% and 37.3 ± 2.11%, respectively). In contrast, only 11.8 ± 1.6% CD43-/- Mφ had associated bacilli (Figure 2.6A). When the actual numbers of bacteria per Mφ were assessed, CD43 gene dose again correlated with the amount of associated bacteria, as CD43+/+ cells bound 2.07 ± 0.24, while CD43+/- bound 1.08 ± 0.24, and CD43-/- bound 0.56 ±0.11 bacilli per Mφ (Figure 2.6B).  2.10  The survival and replication of M. tuberculosis within CD43-/- Mφ φ is enhanced  BMMφ from CD43+/+ and CD43-/- mice were infected with M. tuberculosis and the subsequent growth of the bacteria was measured by determining CFU over 7 days. As shown in Table 1, at a MOI of 20:1, CD43+/+ Mφ phagocytosed more bacteria than CD43-/- Mφ on day 0 (p<0.001) but by day 7 there were comparable amounts in the two populations. However, when CD43-/- Mφ were infected at a MOI of 30:1, the same amount of bacteria were taken up as in CD43+/+ Mφ at 20:1 on day 0 and by day 7 postinfection there were twice as many bacteria in the CD43-/- population (p<0.001). Moreover, the doubling times of M. tuberculosis in CD43-/- Mφ were significantly less than in wild type Mφ, where it took 27.69 ± 0.26 hours for one doubling compared to 24.04 ± 0.18 and 24.12 ±0.39 hours in CD43-/- Mφ infected at 20:1 and 30:1 respectively (p<0.001).  60  2.11  CD43 deficient mice have a reduced ability to control M. tuberculosis growth during the acute and chronic phases of infection following aerosol inhalation of bacteria  To determine the role of CD43 on the in vivo growth and pathogenesis of M. tuberculosis, we infected CD43-/- and CD43+/+ mice aerogenically with a low dose of the bacterium (50-100 bacilli). There appeared to be no differences in bacterial load in either the lung or spleen during the first two weeks of infection (Figure 2.7). However, by day 28 there was a significantly higher bacterial load in both the lungs and spleens of mice lacking CD43. After this initial peak, there was a period of host control of growth in the CD43-/- mice resulting in the bacterial load being reduced to levels similar to those in the control mice. Subsequently, bacterial loads remained relatively constant in CD43+/+ mice whereas in CD43-/- mice, bacterial loads steadily increased until the termination of the experiment. There were no differences in the survival of mice (data not shown). A comparable experiment following infection over a shorter time period (84 days) gave similar results (data not shown). To determine whether the greater bacterial load in the spleens of CD43-/- mice during the acute phase of infection (up to day 28) was due to a greater susceptibility of splenic Mφ in CD43-/- mice or to greater seeding of the spleen with bacteria from the lung, we infected CD43-/- and CD43+/+ mice with M. tuberculosis via the intravenous route. This ensured equal numbers of bacteria were deposited into the spleens and lungs of both mouse strains. No differences were seen in the growth rate of M. tuberculosis over 6 weeks (Figure 2.8).  61  2.12  Organ pathology is exacerbated in CD43 deficient mice  Histopathologic assessment of organs from M.tuberculosis-infected mice revealed that pathology in CD43-/-mice was more severe and developed more rapidly than in CD43+/+ mice. By day 1 post-infection, lymphoid hyperplasia was evident in the spleens of CD43-/- mice and by day 56 these mice displayed multifocal granulomatous inflammation affecting nearly 50% of the lung (Table II). This level of pathology did not appear in the CD43+/+ control mice until day 85. By the final experimental time point, CD43-/- mice had granulomatous inflammation affecting more than 50% of the lung sections, severe lymphoid hyperplasia in the spleen, and vascular, perivascular, and interstitial infiltrates of lymphocytes and neutrophils in the liver. At various time points CD43-/- mice also showed an increased number of foamy Mφ in lung sections. Although granulomas in CD43+/+ mice also contained some foamy Mφ, these were only seen during the chronic stages of infection and decreased in numbers towards the end of the experiment whereas in CD43-/- mice foamy Mφ were present from day 56 onwards and in greater numbers (Figure 2.9). Overall, granuloma formation in CD43-/- mice occurred more rapidly, more extensively, affected a greater proportion of the lung, and included more foamy macrophages.  62  2.13  Discussion  While recent studies have shown that the interaction of M. tuberculosis with Mφ does not necessarily result in uptake of the bacteria, certain Mφ populations can and do ingest them (2, 35, 38).  Understanding how M. tuberculosis enters, survives and  establishes an infection in these Mφ populations is crucial to comprehending the pathogenesis of mycobacterial infections. Recently, Fratazzi et al. described a role for CD43 in mycobacteria-Mφ interactions (14). Their results suggested that CD43 may play a role in promoting a stable interaction of mycobacteria with receptors on host cells and that this interaction regulated TNF-α production by the Mφ. To advance our understanding of this interaction, we further characterized the role of CD43 in mycobacterial infections by analyzing the association of M. tuberculosis with different Mφ phenotypes, by using other bacterial species and control particles, by studying the relationship between CD43 expression and mycobacterial binding, and by monitoring the growth of M. tuberculosis in Mφ monolayers and in CD43 knockout mice. We confirm that CD43 is involved in the ability of Mφ to bind and engulf mycobacteria, as Mφ from CD43 knockout mice were less able to phagocytose M. tuberculosis. Our studies extend this observation and show that this reduction of M. tuberculosis binding depends upon various factors. Firstly, Mφ of distinct origins varied in their ability to bind M. tuberculosis and showed different levels of reduction in binding when CD43 was absent. While the previous study of CD43-TB interactions focused on SpMφ (14) we show here that BMMφ, PMφ, and AMφ also had an impaired  63  association with M. tuberculosis in the absence of CD43. Although AMφ are the cell that first encounters M. tuberculosis in vivo, new mononuclear phagocytes arrive at the site of infection during the course of the disease, where they may differentiate and encounter bacteria. BMMφ are an acceptable model for these elicited Mφ and are commonly used in studies of M. tuberculosis. We therefore used these cells as a model for our studies. Within a single population of Mφ (BMMφ), the level of expression of CD43 directly correlated with binding of M. tuberculosis (Figure 2.6). However, flow cytometry of different Mφ populations showed that expression of CD43 did not directly correlate with binding of M. tuberculosis. AMφ expressed the lowest level of CD43 and bound M. tuberculosis poorly. However, BMMφ bound M. tuberculosis at levels lower than did SpMφ and PMφ, yet expressed the highest levels of CD43 (Figure 2.5). It is possible that the expression of CD43 on the surface of Mφ may not reflect its functional state, as has been seen with other Mφ receptors (2). Alternatively, CD43 may act in conjunction with other Mφ receptors to mediate uptake of M. tuberculosis. Thus, variation in expression of these other receptors would explain differences in binding capacity of the various Mφ populations. This contention is supported by the observation that binding of mycobacteria to CD43-/- Mφ can be restored by the addition of the extracellular portion of CD43 (14). That CD43 is critical for optimal association of M. tuberculosis with these secondary receptors is shown by the direct correlation of binding with CD43 expression within a single Mφ population. Further evidence that CD43 was not the only surface moiety involved in binding M. tuberculosis was that as the MOI was increased, both CD43-/- and WT CD43+/+ Mφ bound higher numbers of bacteria. However, at no point did CD43-/- BMMφ bind the 64  same number of bacteria as did CD43+/+ Mφ and it was calculated that CD43 was accountable for up to 40-50% of M. tuberculosis binding by Mφ. We show that a heat-labile component of serum can overcome the reduction in binding due to the absence of CD43. It has been previously demonstrated that the extracellular mucin region of CD43 is present in, and can be isolated from, plasma (42) and that this molecule can increase binding of mycobacteria by CD43-/- Mφ (14). Therefore it appears, in agreement with previous findings, that soluble CD43 present in serum may potentiate mycobacteria-Mφ interactions. It is also very likely that complement is responsible for at least some of the enhanced binding of the bacterium in the presence of serum, as it has been implicated in facilitating uptake of mycobacteria by Mφ (1, 3, 5, 6) and heat inactivated serum lacks the capacity for complement activation (43). However, other heat-resistant opsonins may contribute to enhanced binding. Even in the presence of heat-inactivated serum, both CD43-/- and CD43+/+ Mφ showed increased binding of M. tuberculosis compared to non-opsonic binding. This suggests that some component of serum that is heat stable can also mediate binding to Mφ. Thus, our studies show that CD43 mediated binding of M. tuberculosis depends upon the Mφ phenotype, the number of infecting bacteria, the presence of serum opsonins and the amount of CD43 expression. Binding studies with control particles demonstrated that the absence of CD43 does not affect non-specific phagocytosis by Mφ, or the function of complement receptors and FcγR. Interestingly, CD43-/- Mφ had an increased affinity for zymosan. This could be due to the removal of factors impeding the interaction of zymosan binding, such as the large negative charge of CD43 sialic acid residues or steric hindrance created  65  by the large size of CD43. The effect of CD43 on bacterial binding to Mφ also had a level of specificity, as representative gram negative and positive bacteria did not require the presence of CD43 to bind to Mφ. However, three strains of mycobacteria all required the presence of CD43 for optimal binding. This supports the contention that CD43 binds specifically to a mycobacterial moiety. The intracellular growth of M. tuberculosis was significantly enhanced in CD43/- Mφ even though the bacteria are less readily phagocytosed by the Mφ. This increased growth rate was independent of the number of bacteria initially ingested. The higher rate of growth could be due to the fact that CD43-/- Mφ have an impaired ability to initiate TNF-α production (14), which is known to be involved in controlling intracellular growth of M. tuberculosis (44-47). Other cytokines have also been shown to be involved in stimulating the release of the chemokines RANTES and macrophage inflammatory protein-1 (48), which could also affect the intracellular growth of M. tuberculosis. Additionally, CD43-/- Mφ could be selectively phagocytosing the more virulent bacteria within the innoculum, or uptake of the bacterium in the absence of CD43 may lead to altered phagosome maturation, which is known to be associated with the survival of intracellular mycobacteria (49-53). Moreover, the induction of killing mechanisms by Mφ may differ in CD43-/- and +/+ cells. For example, it has been demonstrated that CD43 is involved in apoptotic signaling pathways that would affect the fate of mycobacteria-infected Mφ (27, 29-31). CD43 appears to have a significant role in controlling the growth of M. tuberculosis in the murine host. When infected via aerosol, CD43-/- mice had increased bacterial loads in the lung and spleen during the acute phase of infection up to day 28.  66  This increased growth of M. tuberculosis in CD43-/- mice may be attributed to the enhanced growth in CD43-/- Mφ we demonstrated in vitro. Alternatively, it may be due to differences in the type and/or number of cells recruited to sites of infection. In H&E stained tissue samples, lymphoid hyperplasia was seen in the spleens of CD43-/- mice as early as day 1 post-infection and there were more granulomas in the lungs by day 28 compared to CD43+/+ controls. The difference in bacterial growth during the acute phase of infection in CD43+/+ and CD43-/- mice was more pronounced in the spleens. As this effect was not seen in mice infected intravenously with M. tuberculosis, we can conclude that this is not just because of enhanced bacterial growth in CD43-/- SpMφ but is more likely due to increased dissemination from the lungs of CD43-/- mice. Following the development of the adaptive immune response (around day 28) the mice were able to control the infection for a period of time. However, following this period the CD43-/- mice also failed to control bacterial growth during the chronic stage of infection. It is possible this difference can also be ascribed to the increased susceptibility of CD43-/- Mφ. However, the adaptive immune response was able to reduce the bacterial load in CD43-/- mice between days 28 and 56. This suggested that CD43-/- Mφ were capable of being activated to kill intracellular M. tuberculosis just as effectively as CD43+/+ Mφ during this stage of the infection. During the chronic stage of the infection, an effective immune response must be maintained with the corresponding maintenance of granulomata to contain the bacteria. We have shown that during this late stage of infection, bacterial growth is not controlled in CD43-/- mice and histological findings show that normal granuloma formation is impaired in these mice and may account for increased bacterial loads. Other published roles of CD43, including  67  involvement in T cell activation and differentiation, and the recruitment of lymphocytes to sites of infection (19, 25, 26) could also explain the inadequacies in the immune response against M. tuberculosis. It is critical to note that the ability of CD43-/- mice to control infection with M. tuberculosis depended upon the route of infection. While significant differences were seen between CD43-/- and CD43+/+ mice infected aerogenically with M. tuberculosis, there was no significant difference when mice were infected via an intravenous injection with the same bacterium. It has been previously shown that M. tuberculosis may have increased virulence when administered aerogenically as opposed to intravenously (54) and that the pathogenesis of the organism is affected by route of delivery (55). This emphasizes the importance of utilizing experimental infection procedures that most closely mimic natural exposure in order to obtain results that are most physiologically relevant. In summary, this study establishes that CD43 is involved in the binding, uptake and subsequent growth of M. tuberculosis in murine Mφ and in vivo. These results support the theory that CD43 has a dual function in cell-cell interactions (23, 24, 56) and that the nature of particles interacting with CD43 can dictate its function. Additional studies are necessary to determine the biology of M. tuberculosis-CD43 interactions, to identify potential mycobacterial ligands for CD43, and to understand mechanisms of cell recruitment in CD43 knockout mice.  68  Table 2.1  Mφ φ type (MOI)  DAY 0  DAY 1  DAY 4  DAY 7  Doubling Time (h)  CD43+/+ (20:1)  4.76 x104 (7.85 x102)  4.01 x104 (9.35 x102)  1.63 x105 (2.85 x103)  3.19 x106 (5.46 x104)  27.69 (0.256)  CD43-/(20:1)  *2.58 x104 (4.42 x102)  *2.31 x104 (7.38 x102)  1.68 x105 (3.85 x103)  3.31 x106 (4.91 x104)  *24.04 (0.177)  CD43-/(30:1)  4.81 x104 (8.08 x102)  3.96 x104 (9.30 x102)  *2.20 x105 (4.25 x103)  *6.13 x106 (9.59 x104)  *24.12 (0.387)  Intracellular survival and replication of M. tuberculosis is enhanced in CD43-/- BMMφ φ  CD43-/- and CD43+/+ control BMMφ were incubated with M. tuberculosis Erdman at 20:1 bacteria:Mφ and CD43-/- Mφ were also infected at 30:1. Average CFU/mL and doubling times are shown for day 0 and days 1, 4 and 7 post-infection. Results are expressed as mean ± SEM for 3 independent experiments, each with 3 coverslips, plated in duplicate at each time point.  69  Infiltrating cell types (%) Mouse Type  Granuloma formation  Area of Lung affected (%)  Normal Mφ φ  Foamy Mφ φ  Lymphocytes  Day 56  CD43+/+ CD43-/-  Single area Multifocal  <25% ~50%  50 20  0 30  50 50  Day 85  CD43+/+ CD43-/-  Single area Multifocal  <25% ~50%  25 0  25 50  50 50  Day 127  CD43+/+ CD43-/-  Multifocal Multifocal  <25% ~50%  20 0  30 25  50 75  Day 168  CD43+/+ CD43-/-  Multifocal Multifocal  ~50% >50%  25 0  25 25  50 75  Day 210  CD43+/+ CD43-/-  Multifocal Multifocal  ~50% >50%  25 0  5 25  70 75  Time Postinfection  Table 2.2  Granuloma formation in CD43-/- mice is more severe and has altered morphology  At the indicated times post-infection H&E-stained sections of lung from CD43-/- and CD43+/+ mice infected with M. tuberculosis were evaluated for the number and type of granulomas present (multifocal = numerous granulomas throughout the lung), the amount of the section affected (%), and the dominant cell types present in the granulomatous region (%), assessed as described in Materials & Methods. Organ sections are from the same mice for which bacterial loads were assessed in Figure 2.7.  70  Figure 2.1  M. tuberculosis has a reduced ability to associate with CD43 deficient Mφ φ  A) SpMφ, PMφ and BMMφ from CD43-/- and CD43+/+ mice were infected with M. tuberculosis Erdman at 20:1 bacteria:Mφ, while AMφ were infected at 500:1. The average number of bacteria per infected Mφ was assessed microscopically for 100 randomly chosen Mφ. B) CD43-/- and CD43+/+ BMMφ were infected with M. tuberculosis Erdman at MOIs of 20:1, 40:1, 60:1, 100:1 and 200:1 and associated bacteria were quantified as above. For both A) and B) The mean ± SEM from 2 independent experiments, each with 3 coverslips, is shown. All CD43-/- values are statistically less than WT values (p<0.05).  71  Figure 2.2  Heat-labile serum opsonins overcome the reduced binding of M. tuberculosis to CD43-/- Mφ φ  BMMφ from CD43+/+ and CD43-/- mice were infected with M. tuberculosis Erdman in the absence (Serum-free) or presence of 1% normal (+ Serum) or heat-inactivated (+ HISerum) mouse serum at a MOI of 40:1. The percentage of Mφ binding at least 1 bacillus is shown. Values represent the mean ± SEM from 3 independent experiments, each with 3 coverslips. ** = p <0.001 and * = p <0.01 when compared to CD43+/+ control.  72  Figure 2.3  CD43 is involved in Mφ φ binding of other mycobacterial species but not other intracellular bacteria  CD43-/- and CD43+/+ BMMφ were infected with Mycobacterium tuberculosis H37Rv (H37Rv) or Mycobacterium avium (M. av) at MOI of 40:1 and 20:1, respectively, for 3 hours, or with Salmonella enterica serovar Typhimurium (S. ty) or Listeria monocytogenes (L. mo) at 10:1 for 40 min. The mean ± SEM percent of Mφ binding at least 1 bacillus is shown for 3 independent experiments, each with 3 coverslips. * = p <0.01 when compared to control.  73  Figure 2.4  The absence of CD43 on Μφ does not affect phagocytosis via complement receptors, FcγγR, or non-specific uptake, but enhances uptake of zymosan  CD43-/- and CD43+/+ BMMφ were incubated with test particles at the following MOI: EIgG (50:1), EIgMC’ (50:1), latex spheres (25:1), or zymosan (25:1). After 3h, the association of particles was assessed microscopically. The average number of particles bound per Mφ is shown. Results are expressed as the mean ± SEM of 3 independent experiments, each with 3 coverslips. * = p<0.01 when compared to control.  74  Figure 2.5  Surface expression of CD43 varies on different Mφ φ phenotypes  CD43-/- and CD43+/+ SpMφ, PMφ, BMMφ and AMφ were stained with MAb S11 (antipan-CD43). The mean fluorescence intensity ± SEM from 2 independent experiments is shown. Numbers above the bars represent the percentage of the Mφ population that expressed CD43. The dotted line represents background levels of anti-CD43 mAb binding  75  Figure 2.6  M. tuberculosis binding to BMMφ φ is dependent on CD43 gene dose  BMMφ from WT (+/+), CD43 knockout (-/-) and CD43 heterozygous (+/-) mice were incubated with M. tuberculosis Erdman at a ratio of 40 bacteria per Mφ. A) The percentage of Mφ binding at least 1 bacillus is shown. B) The average number of bound bacteria per infected Mφ is shown. Figures represent the mean ± SEM from 2 experiments, each with 3 coverslips * = p <0.001 when compared to CD43+/+, † = p <0.05 when compared to CD43+/-.  76  Figure 2.7  CD43 is necessary for the control of M. tuberculosis growth during both the acute and chronic phase of infection in mice  CD43-/- (open circles) and CD43+/+ (filled circles) mice were infected aerogenically with a low-dose of M. tuberculosis. The bacterial loads in the A) Lung and B) Spleen are shown as the mean CFU/Organ x 104 ± SEM for 4-6 mice per experimental group at each time point. * = p <0.05, ** = p <0.01,when compared to control.  77  Figure 2.8  CD43 deficient mice infected intravenously with M. tuberculosis do not show impaired control of bacterial growth during the acute phase of infection  CD43-/- (open circles) and CD43+/+ (filled circles) mice were infected intravenously with M. tuberculosis. The bacterial loads in the A) Lung and B) Spleen are shown as the mean CFU/Organ x 103 ± SEM for 5 mice per group at each time point. No significant difference was found between the experimental groups.  78  Figure 2.9  Lung pathology is exacerbated in CD43 deficient mice infected with M. tuberculosis  Representative granuloma from A.) CD43+/+ and B.) CD43-/- mice 210 days postinfection with M. tuberculosis via aerosol exposure, 200X magnification. 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Immunologic Research 20:89 - 99.  85  Chapter 3  3.1  CD43 controls intracellular growth of Mycobacterium tuberculosis through the induction of TNF-α α-mediated apoptosis2  Summary Establishment of Tuberculosis infection begins with the successful entry and  survival of the pathogen within macrophages. We previously showed that macrophage CD43 is required for optimal uptake and growth inhibition of Mycobacterium tuberculosis both in vitro and in vivo. Here, we explore the mechanisms by which CD43 restricts mycobacterial growth in murine macrophages. We found that although M. tuberculosis grows more readily in resting CD43-/- macrophages, when these cells were primed with IFN-gamma the bacterial growth rate was similar to that seen in CD43+/+ cells. To discern the mechanisms by which M. tuberculosis exhibits enhanced growth within resting CD43-/- macrophages, we assessed the induction of inflammatory mediators in response to infection. We found that the absence of CD43 resulted in reduced production of nitric oxide, TNF-alpha, IL-12 and IL-6 by M. tuberculosisinfected macrophages. We also found that infected resting, but not activated CD43-/cells showed decreased levels of apoptosis and increased levels of necrosis. Addition of TNF-alpha to CD43-/- macrophages restored control of M. tuberculosis growth and induction of apoptosis to CD43+/+ levels. We thereby propose that CD43 is involved in the inflammatory response to M. tuberculosis and can regulate apoptosis through the induction of pro-inflammatory mediators to control intracellular growth of the organism.  2  This chapter forms part of a manuscript in preparation.  Randhawa, A.K., H.J. Ziltener, and R.W. Stokes. CD43 controls intracellular growth of Mycobacterium tuberculosis through the induction of TNF-α-mediated apoptosis. 86  3.2  Introduction  Despite treatment and prevention efforts, Mycobacterium tuberculosis remains the most-common cause of infection-related mortality worldwide (1, 2). Each year, nearly 2 million deaths occur due to Tuberculosis and over 8 million people are newly infected with the causative bacterium, M. tuberculosis (3). A central feature of the pathogenesis of this organism it the ability to infect and persist within the macrophage (Mφ). Following inhalation of the bacteria, Mφ serve as the first line of host immune defense against infection and act as the primary host cell of M. tuberculosis. For these reasons, it is crucial that we fully understand the events that occur during the initial encounter between the Mφ and the bacterium, and the dynamics of this relationship during the intracellular survival of M. tuberculosis. Several receptors on phagocytic cells have been shown to be involved in the initial detection of M. tuberculosis, including the complement receptors, the mannose receptor, surfactant protein A receptor, scavenger receptors, dendritic cell-specific intercellular  adhesion  molecule-3  (ICAM-3)-grabbing  non-integrin  (DC-SIGN),  mannose-binding lectin, dectin-1, and Toll-like receptor 2 (4-14). Recent studies have also shown that CD43 is involved in the binding and uptake of M. tuberculosis by Mφ (15, 16). CD43 is a transmembrane glycoprotein that is abundantly expressed on lymphohemopoietic cells (17). It appears that CD43 may have a dual function in cell-cell interactions as it has been shown to act as a barrier molecule that can restrict contact between cells (18-20), but has also displayed a role in adhesion (21, 22). In addition, we have shown that the presence of CD43 on Mφ enhances binding  87  of M. tuberculosis, whereas it inhibits binding of zymosan (a mannan-rich yeast cell wall product) (16). It is clear that this molecule has a number of physiological functions as it has also been shown to play a role in the activation of T cells (23-26), monocytes (27), and neutrophils (28), leukocyte homing and tissue infiltration (29, 30) and regulation of apoptosis (31-37). In bacterial infections, it has been shown that CD43 on Mφ is involved in the binding of M. avium and M. tuberculosis in a dose dependent manner, but does not appear to associate with Shigella flexneri, Salmonella typhimurium or Listeria monocytogenes (15, 16). Fratazzi et al. also showed that re-addition of the extracellular portion of the molecule can restore binding of M. avium to CD43-/- Mφ. As with its role in cell-cell interactions, it appears that CD43 may play a dual role in mycobacterial infections as it was not only found to be involved in initial binding of the bacteria, but also plays a role in the subsequent growth within Mφ, as M. tuberculosis grew more readily in CD43-/- BMMφ (16). The enhanced growth of M. tuberculosis due to absence of CD43 was also observed in vivo; CD43-/- mice infected aerogenically with M. tuberculosis displayed increased bacterial loads in lungs and spleens during both the acute and chronic stages of infection and also had altered granuloma formation (16). In this study, we further explored the role of CD43 in mycobacterial pathogenesis. To discern the mechanisms by which CD43 controls intracellular growth of M. tuberculosis, we compared the growth of M. tuberculosis within resting and IFNgamma (IFN-γ) activated CD43-/- and +/+ Mφ and also examined killing mechanisms employed by Mφ in response to M. tuberculosis.  88  3.3  Materials and methods  Bacteria M. tuberculosis strain Erdman (Trudeau Mycobacterial Collection #107; ATCC #35801), was grown to late log phase in Proskauer & Beck medium supplemented with 0.05% Tween 80 under static culture conditions with intermittent agitation (every 2 days over a 2-3 week period). Cultures were stored at -80°C and assessed for colony forming units (CFU) as previously described (6).  Mice Wild type (CD43+/+) and CD43 knockout (CD43-/-) mice backcrossed at least 10 generations on C57Bl/6 background (38) were housed in a specific pathogen-free animal facility in micro isolator cages. Mice were age- and sex-matched, and wherever possible, wild type controls were littermates from CD43 heterozygote (+/-) matings. All experiments were carried out in accordance with the standards set by the Canadian Council on Animal Care.  Culture of bone marrow-derived Mφ φ Bone marrow-derived macrophages (BMMφ) were isolated from CD43+/+ and CD43-/- mice as previously described (6, 39). Mice were euthanized and dissected to remove femurs, tibiae, and humeri. The ends of the bones were cut and using a 25-gauge needle, the marrow was flushed out with supplemented RPMI (RPMI 1640 medium with 10% fetal calf serum, 10mM L-glutamine, and 10mM sodium pyruvate, all from GIBCO  89  Burlington, ON). Bone marrow washes were pooled and red blood cells were lysed using 0.17M ammonium chloride (NH4Cl, pH 7.2). After washing, cells were resuspended in supplemented RPMI and incubated in tissue culture treated flasks (Becton Dickinson (BD) Labware, Franklin Lakes NJ) for 3 hours at 37ºC, 5% CO2 to deplete non-stem cells by adherence. Non-adherent cells were then washed, resuspended in bone-marrow media (supplemented RPMI plus 10% L-cell conditioned media, prepared as described previously (39)), counted, plated in 24-well plates (BD), and allowed to differentiate for 7 days at 37ºC, 5% CO2. Media was replenished on day 5. Wherever indicated, Mφ were primed for 16 hours with 100 U/mL murine recombinant IFN-γ (R&D Systems Inc., Minneapolis MN) prior to infection. IFN-γ was supplemented every 72 hours during in vitro growth assays.  Intracellular growth of M. tuberculosis The growth of M. tuberculosis within Mφ monolayers was evaluated by infecting BMMφ and determining bacterial CFU counts over a 7 day period, as previously described (16, 40). Briefly, BMMφ were cultured on 13 mm acid-washed sterile glass coverslips in 24 well plates for 7 days, at which point media was replaced with 0.5 mL supplemented RPMI +/- 100 U/mL recombinant IFN-γ, and left overnight. Frozen M. tuberculosis stocks were thawed, pelleted, and resuspended in phagocytosis medium (138 mM NaCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, 0.6 mM CaCl2, 1 mM MgCl2, and 5.5 mM D-glucose, pH 7.4). Bacteria were passed through a 25-gauge needle 10 times to disperse the bacterial clumps characteristic of mycobacteria, and diluted to the required concentration using phagocytosis medium. The overlay was removed from  90  BMMφ monolayers and replaced with M. tuberculosis in phagocytosis medium at multiplicities of infection (MOI) of 20:1 or 30:1 (bacteria:Mφ). Infected Mφ were incubated for 1 hour rocking (Nutator, Becton Dickinson, Mountain View, CA) followed by 2 hours stationary at 37°C/5% CO2. Following infection, monolayers were washed twice with phagocytosis medium and coverslips were transferred to new 24 well plates containing 1 mL supplemented RPMI alone or with 100 U/mL IFN-γ. To determine bacterial counts, well contents were sonicated (VC50T 50W probe sonicator, Sonics & Materials, Danbury, CT) for 10 seconds to lyse Mφ and break up clumps of bacteria, serially diluted in PBS + 0.1% Tween 80, and plated in duplicate on 7H10 agar (Difco) supplemented with 0.5% glycerol and 10% oleic acid-albumin-dextrose complex (OADC). OADC was prepared as described previously (41). Plates were incubated in sealed bags for 18-21 days at 37ºC, after which CFU were enumerated. To allow for direct comparison of findings, intracellular growth assays were completed in conjunction with cytokine, nitric oxide, cytotoxicity, and apoptosis assays.  Cytokine detection by enzyme-linked immunosorbant assay (ELISA) The production of TNF-α, IL-12, IL-6, and IL-10 by BMMφ was measured using OptEIA™ ELISA Sets (BD) according to manufacturer’s instructions. In short, 96 well plates were coated with capture antibody, and incubated overnight at 4°C. Plates were washed, blocked, and experimental samples and standards were added. After 2 hours incubation at room temperature, plates were washed and subsequently, detection antibodies and substrates were applied. Absorbance readings (450nm, reference 570nm) were determined using a microplate reader (Bio-Rad Benchmark, Mississauga ON)  91  Measurement of nitric oxide production by Mφ φ The production of nitric oxide (NO) by BMMφ was measured using the Griess Assay, as previously described (39). Briefly, at various time points 100 µL aliquots of culture supernatant were removed from uninfected or M. tuberculosis-infected Mφ monolayers. Serial dilutions of NaNO2 in supplemented RPMI were used to obtain a standard curve. Samples and standards were mixed with 50 µL Griess Reagent 1 (1% w/v sulphanilamide in 2.5% phosphoric acid (H3PO4)) and 50 µL Griess Reagent 2 (0.1% w/v naphthylethylenediamine dihydrochloride in 2.5% H3PO4) for 10 minutes after which absorbance was read (550nm, reference 655nm).  Lactate dehydrogenase assay Levels of cellular necrosis were assessed by measuring the release of lactate dehydrogenase (LDH), a stable cytosolic enzyme, from BMMφ monolayers using the CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison WI). 50 µL of culture supernatant was used for this enzymatic assay, which results in the conversion of a tetrazolium salt into a red formazan product, measurable by OD. The amount of product is proportional to the number of lysed cells in the sample. Background levels were determined by the amount of LDH released from untreated control Mφ, whereas detergent-lysed uninfected Mφ served as a positive control for maximum amount of LDH released. The relative percentage of cytotoxicity in experimental samples was calculated by: [release of LDH from infected cells (OD490)/maximum LDH release (OD490)] x 100.  92  Quantification of histone-associated DNA fragments Apoptosis levels were compared using the Cell Death Detection ELISAPLUS kit (Roche) according to manufacturer’s instructions. Briefly, CD43+/+ and CD43-/- BMMφ were infected as described above. After 4 days of incubation, plates were centrifuged to pellet contents including any cells that may have lifted off the monolayer, and incubated with lysis buffer. Cell lysate was centrifuged to pellet intact nuclei and the cytoplasmic fraction (supernatant) samples retained for ELISA. Sandwich ELISA was performed by placing samples into a streptavidin-coated 96 well plate and incubating with anti-histonebiotin and anti-DNA-peroxidase. Subsequently, unbound antibodies were washed and the amount of peroxidase retained was determined by absorbance (OD405), with ABTS as the substrate. 5 µM staurosporine (STS, Sigma-Aldrich, Oakville ON) was used as a positive control for induction of apoptosis by incubating with uninfected Mφ for 4 hours.  Measurement of Caspase-3 activity Caspase-3 activity was measured using the colorimetric Caspase-3 substrate AcDEVD-pNA (Calbiochem, San Diego CA). CD43+/+ and CD43-/- BMMφ were infected with M. tuberculosis as described above. On day 4 of infection, 24 well plates were centrifuged to pellet any cells that may have lifted off the monolayer and the overlaying medium was removed. STS was used to induce apoptosis in positive controls as described above. Cells were then incubated with 50 µL lysis buffer (10 mM HEPES, pH 7.4, 50 mM NaCl, 2 mM MgCl2, 5 mM EGTA, 0.2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 10 µg/mL aprotinin, 2 µg/mL leupeptin, and 1 mM PMSF, all from Sigma-Aldrich) for 10 min at 4°C. Lysates were  93  centrifuged at high speed for 10 min to pellet cellular debris, the supernatant transferred to microfuge tubes and kept at 4°C. 100 µg of protein lysate, as quantified using the Bradford Assay, was incubated at 37°C for 1 hour with reaction buffer (50 mM HEPES, pH 7.4, 20% glycerol, 2 mM EDTA, and 10 mM DTT added immediately before use) and 1 mM Caspase-3 substrate. A standard curve was produced by diluting a stock solution of pNA (Sigma-Aldrich) from 200 mM to 25 mM and absorbance measured by determining OD at 405nm.  TNF-α α neutralization/reconstitution For TNF-α neutralizing/reconstitutions assays, 100 ng/mL recombinant TNF-α or 10 ng/mL anti-TNF-α antibody (both from R&D Systems) were added along with bacteria at the time of infection and supplemented every 48 hours. In vitro growth assays and apoptosis ELISA were performed as described above.  Statistical analysis Data are expressed as mean ± SEM. Student’s t test for independent means was used; p < 0.05 was considered significant unless otherwise noted.  94  3.4  IFN-γγ activation abrogates the enhanced growth of M. tuberculosis in CD43-/- Mφ φ  In previous studies, we found that M. tuberculosis grows more readily in CD43-/BMMφ (16). In those experiments, the cells used were representative of resident tissue Mφ. To determine whether cells representing T-cell-activated Mφ require CD43 to control the intracellular growth of M. tuberculosis, we compared the growth of the bacterium in wild type (CD43+/+) and knockout (CD43-/-) Mφ with or without prior IFN-γ stimulation. Because previous studies (16) have shown that Mφ deficient in CD43 bind mycobacteria less efficiently than wild-type Mφ, we infected CD43-/- BMMφ at MOIs of 20:1 and 30:1, in addition to the 20:1 MOI used for CD43+/+ cells in order to ensure equal uptake of the bacteria by both populations as well as equal MOIs. When resting (not stimulated with IFN-γ) BMMφ (Figure 3.1A-B) were infected at 20:1, CD43-/- Mφ phagocytosed approximately 40% less mycobacteria than CD43+/+ Mφ on day 0 (Figure 3.1A), as expected. Adding 30:1 bacteria per Mφ to CD43-/- cells resulted in uptake comparable to CD43+/+ at 20:1 (Figure 3.1A), but resulted in significantly greater bacterial load after 7 days (Figure 3.1B, p<0.0001), in agreement with previous findings (16). In assessing growth within IFN-γ-stimulated Mφ (Figure 3.1C-D), both CD43+/+ and CD43-/- Mφ showed reduced bacterial loads by day 7 compared to resting Mφ. Bacterial uptake remained significantly less when CD43 was not expressed (Figure 3.1C, p=0.002) but there were no statistical differences in the growth rate of the bacteria in CD43+/+ and -/- IFN-γ activated Mφ, as illustrated by bacterial load after 7 days (Figure 3.1D). Thus, it appears that although CD43 is required 95  for restricting the growth of M. tuberculosis in resting BMMφ, activation of the cells with IFN-γ overcomes the difference in growth due to lack of CD43. This is illustrated by both the bacterial load (Figure 3.1) and also by the doubling time of the bacteria (Table 3.1). In untreated Mφ, M. tuberculosis grows significantly faster when CD43 is not expressed (doubling time of ~24 hours) compared to within wild-type Mφ (doubling time of ~28 hours). However, in IFN-γ treated Mφ, there is no significant difference in the growth rate of the bacteria regardless of CD43 expression. As expected, the growth rate of M. tuberculosis within IFN-γ activated Mφ is less than within resting Mφ, irrespective of whether CD43 is expressed or not.  3.5  CD43 is required for greatest induction of pro-inflammatory mediators by M. tuberculosis-infected Mφ φ  In order to explore the mechanisms by which CD43 restricts intracellular growth of M. tuberculosis, we compared the induction of various Mφ defense mechanisms that are often employed in the response to this bacterium. Firstly, we examined the production of various cytokines by CD43+/+ and CD43-/- Mφ after infection with M. tuberculosis (Figure 3.2). It has been shown that IFN-γ is essential for protective immunity against Tuberculosis infection in the mammalian host, but several studies have also shown that IFN-γ alone is insufficient for full protection (42-46). TNF-α has also been shown to play an important role in restricting mycobacterial pathogenesis, particularly in regulating the formation of granulomas and regulation of Th1 cytokine expression (45, 47-51). In addition, the IL-12 family of cytokines including IL-12 itself  96  and IL-23, which shares the IL-12p40 subunit, have been shown to be induced by M. tuberculosis and have proven important in restricting mycobacterial growth in mice likely due to their role in inducing IFN-γ, and activation of antigen-specific lymphocytes (52-55). In comparing the production of TNF-α, and IL-12p40, we discovered that CD43/- Mφ produced significantly less of both cytokines than did CD43+/+ cells (Figure 3.2AB). These results were similar irrespective of pre-treatment of Mφ with IFN-γ before infection with M. tuberculosis. Furthermore, it was noted that the amount of bacteria added to CD43-/- Mφ (20:1 or 30:1 bacteria per Mφ) did not affect the resultant cytokine production. IL-6, commonly regarded as an indicator of general inflammation, may also play a role in the acute response to M. tuberculosis (56). We found that CD43-/- Mφ were also deficient in production of this cytokine regardless of IFN-γ activation and again, that MOI was not a factor in cytokine production (Figure 3.2C). We also examined the production of IL-10, which has been shown to play an anti-inflammatory role in response to M. tuberculosis (57-59). Neither CD43+/+ nor CD43-/- Mφ produced large amounts of IL-10 in response to M. tuberculosis and, because some levels were below the detection limit of the assay (50pg/mL), statistical comparison was not possible (Figure 3.2D). Production of reactive nitrogen intermediates (RNI) such as nitric oxide is a wellknown antimicrobial mechanism employed by Mφ (60), and has been shown to be important in host defense against M. tuberculosis (61-63). We thus compared the production of NO by M. tuberculosis-infected CD43+/+ and CD43-/- BMMφ. Figure 3.3 shows the amount of RNI (measured as nitrite (NO2-) – a stable end product of NO  97  production) produced by CD43+/+ and CD43-/- IFN-γ-activated BMMφ in response to infection with M. tuberculosis. It should be noted that in our studies, resting Mφ that were not stimulated with IFN-γ did not produce detectable amounts of RNI (data not shown). We found that throughout the course of infection, CD43 deficient Mφ produced significantly less RNI than CD43+/+ Mφ at both MOI tested (Figure 3.3). As observed with cytokine production, the amount of bacteria added was unrelated to the amount of RNI produced by CD43-/- Mφ. In both cell types, the production of RNI seemed to reach maximum levels by approximately day 4 of the experiment.  3.6  Enhanced growth of M. tuberculosis in CD43-/- Mφ φ is associated with increased levels of necrosis but decreased induction of apoptotic pathways  TNF-α and NO have been linked to the induction of apoptosis (64-66) and CD43 has recently been shown to also play a role in regulating cell death (31-37). Due to this documented involvement of CD43 in regulating apoptosis, and the apparent role we observed for CD43 in the production of TNF-α and NO, we inquired whether the absence of CD43 on BMMφ affected the level and/or type of cell death induced in response to M. tuberculosis infection. To compare overall levels of cytotoxicity in wild-type and CD43-/- Mφ, we measured the release of lactate dehydrogenase (LDH), a stable cytosolic enzyme released upon cell lysis. In uninfected cell cultures, we did not notice any measurable differences in levels of cell death between CD43+/+ and CD43-/- Mφ over 4 days (Figure 3.4). However, when infected with M. tuberculosis, we found that the amount of cytotoxicity  98  was higher in CD43-/- Mφ, as measured by the amount of LDH released into the medium. This was true for both MOI tested but only in resting Mφ (Figure 3.4A). In contrast, there were no measurable differences in the amount of cytotoxicity induced by M. tuberculosis in CD43-/- and CD43+/+ Mφ that were pre-stimulated with IFN-γ (Figure 3.4B). Next, we measured apoptosis levels using a colorimetric enzyme assay that allows for quantification of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) after induction of apoptosis (Figure 3.5). Staurosporine (STS), a potent inducer of apoptosis, produced similar results in both CD43-/- and CD43+/+ Mφ, at various doses regardless of IFNγ stimulation. When infected with M. tuberculosis, resting CD43+/+ Mφ displayed a significantly greater concentration of histone-associated DNA fragments than did CD43-/- Mφ (Figure 3.5A). When Mφ were stimulated with IFN-γ prior to infection, this difference was no longer present (Figure 3.5B). Thus, it appears that CD43 plays a role in the induction of apoptosis in resting BMMφ infected with M. tuberculosis. Apoptosis can occur via several distinct pathways. Some of the most wellcharacterized pathways for the induction of apoptosis are the Caspase-mediated pathways, although the involvement of Caspases in M. tuberculosis infection has been debated (67, 68). To determine the mechanism of apoptosis in this model, we assessed the level of activity of Caspase-3, a downstream component in the Caspase pathway. We found that Caspase-3 was indeed active in staurosporine-induced and M. tuberculosisinfected samples (data not shown). Furthermore, in agreement with our preceding apoptosis assay, we found that resting CD43-/- Mφ had significantly less Caspase-3  99  activity than did CD43+/+ Mφ (Figure 3.6A) but that IFN-γ treatment partially restored Caspase-3 levels in CD43-/- Mφ to levels not significantly different from CD43+/+ Mφ (Figure 3.6B).  3.7  Enhanced growth of M. tuberculosis and dysregulation of apoptosis in CD43deficient Mφ φ is partially regulated by induction of TNF-α α  As previously stated, TNF-α has been shown to be crucial in restricting the growth of M. tuberculosis and is also implicated in the initiation of apoptosis. Since CD43-/- Mφ displayed increased susceptibility to intracellular M. tuberculosis growth and showed lower levels of M. tuberculosis-induced apoptosis, we hypothesized that this could be due to the observed deficiency in TNF-α production. To investigate this further, we infected CD43+/+ and CD43-/- Mφ with M. tuberculosis in the presence of either recombinant TNF-α (rTNF-α) or an anti-TNF-α monoclonal antibody (anti-TNF-α) and assessed both intracellular growth (Figure 3.7) and induction of apoptosis (Figure 3.8). Over a 7 day period, treatment of CD43+/+ Mφ with anti-TNF-α resulted in enhanced survival and growth of M. tuberculosis to levels significantly higher (p=0.02) than in control CD43+/+ Mφ (Figure 3.7A) reaching levels comparable (p=0.17) to untreated CD43-/- Mφ (Figure 3.7B). This difference was reflected in the levels of apoptosis in these Mφ, with anti-TNF-α treatment reducing apoptosis in CD43+/+ Mφ to levels significantly less than in untreated cells (p=0.007) and not significantly different (p=0.39) than untreated CD43-/- Mφ (Figure 3.8). In contrast, anti-TNF-α had no effect on CD43-/- Mφ (Figures 3.7 & 3.8).  100  Addition of rTNF-α to CD43-/- Mφ (Figure 3.7B) significantly reduced the survival and growth of M. tuberculosis (p=0.003) to levels comparable to CD43+/+ Mφ (p=0.19, Figure 3.7A). Again, this difference was mirrored by an increase in apoptosis in CD43-/- Mφ treated with rTNF-α to levels significantly higher (p=0.0003) than in untreated CD43-/- Mφ (Figure 3.8B) and not significantly different (p=0.64) than in CD43+/+ control Mφ (Figure 3.8A). Overall, the results demonstrated that Mφ CD43 is critical for control of intracellular M. tuberculosis through the action of TNF-α mediated apoptosis.  101  3.8  Discussion  Understanding how the antimicrobial activities of host Mφ can affect the intracellular growth of Mycobacterium tuberculosis is of great importance to our knowledge of this devastating pathogen, and could be key to developing novel therapeutic or preventative measures. It has been shown that Mφ CD43 plays a role in the uptake of M. tuberculosis (15, 16) and previous work in our lab has shown that this molecule is also involved in restricting the growth of the bacterium in Mφ and in mice (16). In this study, we have described the mechanisms by which CD43 controls intracellular growth of M. tuberculosis. We confirmed our previous finding that, in resting Mφ, M. tuberculosis grows more readily in the absence of CD43, but also showed that this enhanced growth is abrogated when Mφ are stimulated with IFN-γ. These results correlate with our in vivo data demonstrating that CD43-/- mice were more permissive to bacterial growth during the acute phase of infection (first 3-4 weeks following infection) when only resident tissue Mφ and other innate immune mechanisms would be present. However, following the induction of adaptive immunity, when IFN-γ activated Mφ are thought to become involved, growth of bacteria was controlled (16). Thus, it appears that the defense mechanisms employed by Mφ differ depending on the activation state of the cells. Moreover, our findings do not support the common supposition that resting Mφ cannot control the growth of M. tuberculosis. We have shown that resting Mφ can control mycobacterial growth to some extent, and that this is dependent upon the presence of CD43.  102  The differences seen in the growth of the bacterium in resting versus IFN-γ activated Mφ are likely due to differences in the killing mechanisms employed by these cells, of which not all will require the involvement of CD43. It is well established that Mφ have several ways of resisting intracellular growth of M. tuberculosis, many of which are circumvented by the bacterium (45). Some of these antimycobacterial effector functions include the induction of apoptosis and the production of reactive oxygen and reactive nitrogen intermediates (ROI/RNI), which are enhanced by cytokines such as IFN-γ and TNF-α. We examined the function of CD43 in these processes and found a role for it in the M. tuberculosis-induced production of RNI, production of pro-inflammatory cytokines TNF-α, IL-12, and IL-6, and regulation of apoptosis. However, the activity of these mediators and the associated involvement of CD43 seems to be more pivotal in resting Mφ than in IFN-γ activated Mφ with regards to control of M. tuberculosis growth. Again, this is possibly due to the type of antimycobacterial responses employed by resting versus activated Mφ. For example, it has been shown that resting Mφ do not produce significant levels of ROI/RNI in response to M. tuberculosis (39) so these mechanisms are seemingly irrelevant in these cells but may be critical in IFN-γ activated Mφ. Our results suggest that processes such as the production of protective cytokines and induction of apoptosis are likely more fundamental in the response of resting Mφ to M. tuberculosis. TNF-α has been shown to play a central role in not only host defense against mycobacteria, but in the initiation of apoptotic pathways. Several studies have linked these findings and shown that TNF-α mediated apoptosis may be a fundamental host  103  defense mechanism in mycobacterial infections (59, 69-74) and our data support this assertion, at least for resting Mφ. CD43-/- Mφ were deficient in TNF-α production and showed lower levels of Caspase-3 mediated apoptosis. When TNF-α was added to CD43-/- Mφ, apoptosis levels increased and growth of M. tuberculosis was controlled to levels seen in CD43-expressing cells. Moreover, the neutralization of TNF-α in CD43+/+ cell cultures resulted in decreased levels of apoptosis and increased growth of M. tuberculosis. These results clearly indicate that TNF-α mediated apoptosis is key to controlling the intracellular growth of M. tuberculosis and that CD43 plays a regulatory role in this process. As mentioned previously, apoptosis is emerging as a vital host defense mechanism in controlling M. tuberculosis infection; a number of studies have shown that virulent mycobacteria can inhibit the induction of apoptosis and in doing so, thrive within Mφ (74-77). Circumventing apoptotic pathways allows the pathogen to evade bactericidal effector functions (72, 78) and avoid T cell priming (79-81), leading to a favorable environment for persistence. Recent studies have begun to elucidate mycobacterial gene products that may be involved in averting apoptosis (77, 81). The secA2 gene, which encodes a component of a virulence-associated protein secretion pathway, has been found to be required for the inhibition of apoptosis in M. tuberculosisinfected Mφ (81). Previous studies investigating the growth of a ∆secA2 gene-deletion mutant of M. tuberculosis uncovered results very similar to ours, but examined parameters from the perspective of the bacterium instead of the host (82). The ∆secA2 mutant was defective for growth within Mφ in vitro and elicited greater amounts of RNI, TNF-α, and IL-6. These findings support the contention that these inflammatory  104  mediators, in conjunction with Mφ apoptosis, are important antimycobacterial effectors. In addition, the aforementioned study found that the ∆secA2 mutant showed decreased growth in mice during early stages of an aerosol infection and that the enhanced growth within Mφ was abrogated with IFN-γ stimulation, similar to our results. Thus, it appears that apoptosis-related defense mechanisms may be of greater significance in resident tissue Mφ than in activated cells. In parallel, the lack of a clear role for CD43 in the IFN-γ activated Mφ may be due to the presence of additional host defense mechanisms such as RNI/ROI that compensate for the loss of TNF-α mediated apoptosis in resting CD43-/- Mφ. Indeed, both RNI and ROI are known to induce apoptosis, so it is possible that the similar levels of apoptosis in IFN-γ activated Mφ from CD43+/+ and CD43-/- Mφ may be mediated by these mechanisms. Furthermore, the observation that CD43-/- IFN-γ activated Mφ had comparable levels of cytokine inhibition as resting Mφ, yet restricted mycobacterial growth better, suggests that these cytokines have no role in IFN-γ mediated killing. Our results in this study, taken together with our previous findings, indicate a multifunctional role for CD43 in the growth and pathogenesis of M. tuberculosis. We have shown that CD43 is involved in the binding and uptake of M. tuberculosis and other mycobacteria (16), and that CD43 is necessary for optimal control of intracellular replication of M. tuberculosis by resting Mφ. We have also shown that CD43 is involved in the induction of RNI by IFN-γ activated Mφ infected with M. tuberculosis. Additionally, we have demonstrated a regulatory role for CD43 in the production of proinflammatory cytokines including TNF-α, which mediates apoptosis in M.  105  tuberculosis-infected Mφ and is known to be important for granuloma formation in M. tuberculosis-infected mice (47). Together, these results suggest that CD43 may act as either a pattern recognition receptor or perhaps a co-receptor to facilitate the phagocytosis of M. tuberculosis and regulate downstream effector functions. We are currently undertaking studies to further explore the mechanisms of this phenomenon including comparing signaling capacities of CD43-/- and +/+ Mφ in response to mycobacteria. Further inquiry is also warranted to analyze other antimycobacterial functions of Mφ that may be affected by CD43 such as phagolysosome fusion, and production of reactive oxygen species, chemokines, or other cytokines important in the host response to M. tuberculosis.  106  Table 3.1  CD43+/+ CD43 -/CD43 -/-  MOI 20:1 20:1 30:1  IFN-γγ -  DT ±SEM (h) 27.71 ±0.15 *24.82 ±0.20 *24.07 ±0.19  CD43+/+ CD43 -/CD43 -/-  20:1 20:1 30:1  + + +  77.27 ±6.72 87.91 ±3.73 83.01 ±5.97  M. tuberculosis grows more readily in resting but not IFN-γγ activated CD43-/- Mφ φ than in CD43+/+ Mφ φ.  Bone marrow-derived Mφ from CD43 knockout (CD43-/-) or control (CD43+/+) mice were infected with M. tuberculosis at 20:1 or 30:1 (MOI) with or without overnight stimulation with 100U/mL IFN-γ. The doubling time of the bacteria was calculated by comparing bacterial loads over 7 days. Results are shown as doubling time (DT) in hours ±SEM for 3 independent experiments, each with 3 replicates plated in duplicate. * Indicates p<0.05 when compared to CD43+/+ group.  107  Figure 3.1  IFN-γγ stimulation overcomes enhanced growth of M. tuberculosis in CD43-/- Mφ φ.  CD43-/- and +/+ Mφ were infected with M. tuberculosis at 20:1 (CD43+/+ black bar, CD43-/- white bar) or 30:1 (CD43-/- grey bar) for 3 hours. Monolayers were washed to remove unbound bacteria and infection was monitored for 7 days. Bacterial loads (CFU/mL) are shown ±SEM for 3 independent experiments, each with 3 replicates, plated in duplicate. (**) indicates statistical significance (p<0.01) compared to CD43+/+ control. (A) and (B) show bacterial loads in resting Mφ, while in (C) and (D), Mφ were primed overnight with 100 U/mL IFN-γ before infection. NOTE: y-axis scales are not equivalent.  108  Figure 3.2  CD43-/- Mφ φ are deficient in pro-inflammatory cytokine production in response to M. tuberculosis infection.  Resting or IFN-γ activated BMMφ were infected with M. tuberculosis for 3 hours. Control CD43+/+ Mφ were infected at 20:1 (black bars), while CD43-/- Mφ were infected at both 20:1 (white bars) and 30:1 (grey bars) to equalize bacterial uptake. After infection, the monolayers were washed to remove unbound bacteria and re-incubated for 24 hours, at which point 100 µL of culture supernatant was removed for cytokine detection by ELISA as described in the text. For (A) TNF-α and (B) IL-12p40 results shown are the mean ±SEM for 5 independent experiments, each with 3 replicates, whereas for (C) IL-6 and (D) IL-10 results are the mean ±SEM for 3 independent experiments, each with 3 replicates. Asterisks indicate significant difference compared to CD43+/+ control where (*) indicates p<0.05 and (**) p<0.001.  109  Figure 3.3  IFN-γγ activated CD43-/- Mφ φ infected with M. tuberculosis produce less RNI than do CD43+/+ Mφ φ  IFN-γ activated BMMφ were infected with M. tuberculosis for 3 hours, after which unassociated bacteria were removed by washing, and cells were re-incubated for 7 days. At the onset of infection, and on days 1, 4, and 7 post-infection, 100 µL of culture supernatant was removed from each well to measure RNI production using the Griess Assay as outlined in the text. Control CD43+/+ Mφ were infected at 20:1 bacteria per Mφ (filled circles), while knockout CD43-/- Mφ were infected at MOIs of 20:1 (open squares) and 30:1 (shaded triangles). Results are expressed as the mean ±SEM for 3 independent experiments, each with 3 replicates. Where error bars are not visible, SEM was not beyond the graphic symbol. At days 1, 4, and 7, all CD43-/- values are significantly lower than CD43+/+ controls (p<0.001).  110  Figure 3.4  M. tuberculosis infection causes greater cytoxicity in CD43-/- Mφ φ than CD43+/+ control Mφ φ  Resting (A) and IFN-γ activated (B) CD43+/+ (black bars) and CD43-/- (white bars) BMMφ were left uninfected (CTRL) or infected with M. tuberculosis (MTB) at 20:1 and 30:1 bacteria per Mφ as described. After 4 days of infection, levels of necrosis were assessed by measuring the release of lactate dehydrogenase (LDH) into the culture supernatant. Cytotoxicity was measured as a percentage compared to a fully lysed positive control (100% cytotoxicity). Results are expressed as the mean ±SEM % cytotoxicity for 3 independent experiments, assayed in duplicate. (*) indicates p<0.01 compared to CD43+/+ control at same MOI, whereas (†) indicates a statistical difference of p=0.0003 between CD43-/- 30:1 and CD43+/+ 20:1. Dotted line indicates background levels in media alone.  111  Figure 3.5  CD43 is required for optimal induction of apoptosis in M. tuberculosis-infected Mφ φ  Apoptosis was measured in resting (A) and IFN-γ activated (B) CD43+/+ (black bars) and CD43-/- (white bars) BMMφ after 4 days of infection with M. tuberculosis by determining the concentration of histone-associated DNA fragments as outlined in Materials & Methods. Uninfected cells (CTRL) served as a negative control while the apoptosis inducer staurosporine (STS) was used as a positive control. Results are expressed as the mean ± SEM OD for 3 independent experiments assayed in duplicate. (**) indicates p<0.01 compared to CD43+/+ control at same MOI, whereas (†) indicates a statistical difference of p=0.007 between CD43-/- 30:1 and CD43+/+ 20:1.  112  Figure 3.6  Absence of CD43 results in decreased activity of Caspase-3 in M. tuberculosis-infected Mφ φ  Caspase-3 activity was measured by an enzymatic assay using samples from resting (A) and IFN-γ activated (B) CD43+/+ (black bars) and CD43-/- (white bars) BMMφ after 4 days of infection with M. tuberculosis. Results are shown as mean ±SEM enzyme activity for 3 independent experiments assayed in duplicate. (**) indicates p<0.01 compared to CD43+/+ control at same MOI, whereas (†) indicates statistical difference between CD43-/- 30:1 and CD43+/+ 20:1 (p<0.01).  113  Figure 3.7  Enhanced growth of M. tuberculosis in CD43-/- Mφ φ is due to deficient TNF-α α production  Growth of M. tuberculosis was measured in CD43+/+ (A) and -/- (B) BMMφ to compare the effect of 10 ng/mL anti-TNF-α antibodies (white bars) or 100 ng/mL recombinant TNF-α (rTNF-α, grey bars). Reagents were added along with bacteria at the time of infection and supplemented every 48 hours. Bacterial loads after 7 days of infection are shown as mean CFU/mL ±SEM for 3 independent experiments, each with 3 replicates plated in duplicate. (*) indicates p<0.05 compared to control within the same group, whereas (†) indicates statistical difference of p<0.001 for the CD43-/- group (B) compared to CD43+/+ control (A).  114  Figure 3.8  M. tuberculosis-induced apoptosis in CD43-/- Mφ φ is TNF-α α dependent  Apoptosis was measured in CD43+/+ (A) and -/- (B) M. tuberculosis-infected BMMφ to compare the effect of 10ng/mL anti-TNF-α antibodies (white bars) or 100ng/mL recombinant TNF-α (rTNF-α, grey bars). Reagents were added along with bacteria at the time of infection and supplemented every 48 hours. Apoptosis was compared by measuring histone-associated DNA fragments after 4 days of infection as in Figure 3.5. Results are expressed as mean ±SEM OD for 3 independent experiments (**) indicates p<0.01 compared to control within the same group, whereas (†) indicates p<0.01 for the CD43-/- group (B) compared to the CD43+/+ control (A).  115  3.9  Literature cited  1.  Kochi, A. 1991. 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Bean, C. Demangel, M. P. France, H. Briscoe, and W. J. Britton. 2002. TNF Regulates Chemokine Induction Essential for Cell Recruitment, Granuloma Formation, and Clearance of Mycobacterial Infection. The Journal of Immunology 168:4620  50.  North, R. J., and Y. J. Jung. 2004. Immunity to tuberculosis. Annual Review of Immunology 22:599.  51.  Zganiacz, A., M. Santosuosso, J. Wang, T. Yang, L. Chen, M. Anzulovic, S. Alexander, B. Gicquel, Y. Wan, J. Bramson, M. Inman, and Z. Xing. 2004. TNFα is a critical negative regulator of type 1 immune activation during intracellular bacterial infection. J. Clin. Invest. 113:401.  52.  Flynn, J. L., M. M. Goldstein, K. J. Triebold, J. Sypek, S. Wolf, and B. R. Bloom. 1995. IL-12 increases resistance of BALB/c mice to Mycobacterium tuberculosis infection. J Immunol 155:2515.  53.  Ladel, C. H., G. Szalay, D. Riedel, and S. H. Kaufmann. 1997. Interleukin-12 secretion by Mycobacterium tuberculosis-infected macrophages. Infect. Immun. 65:1936.  54.  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Olivier, P. Gros, L. F. Barrera, and L. F. Garcia. 1999. TNF-α and IL-10 Modulate the Induction of Apoptosis by Virulent Mycobacterium tuberculosis in Murine Macrophages. J Immunol 162:6122.  60.  Fang, F. C. 1997. Mechanisms of Nitric Oxide-related Antimicrobial Activity. J. Clin. Invest. 99:2818.  61.  Chan, J., Y. Xing, R. S. Magliozzo, and B. R. Bloom. 1992. Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages. J. Exp. Med. 175:1111.  62.  MacMicking, J. D., R. J. North, R. LaCourse, J. S. Mudgett, S. K. Shah, and C. F. Nathan. 1997. Identification of nitric oxide synthase as a protective locus against tuberculosis. PNAS 94:5243.  63.  Chan, J., and J. L. Flynn. 1999. Nitric oxide in Mycobacterium tuberculosis infection. In Nitric Oxide and Infection. F. C. Fang, ed. Plenum, New York, p. 281.  64.  Nagata, S. 1997. Apoptosis by Death Factor. Cell 88:355.  65.  Gaur, U., and B. B. Aggarwal. 2003. Regulation of proliferation, survival and apoptosis by members of the TNF superfamily. Biochemical Pharmacology 66:1403.  66.  Li, C.-Q., and G. N. Wogan. 2005. Nitric oxide as a modulator of apoptosis. Cancer Letters 226:1.  67.  Lee, J., H. G. Remold, M. H. Ieong, and H. Kornfeld. 2006. Macrophage Apoptosis in Response to High Intracellular Burden of Mycobacterium tuberculosis Is Mediated by a Novel Caspase-Independent Pathway. J Immunol 176:4267.  121  68.  O'Sullivan, M. P., S. O'Leary, D. M. Kelly, and J. Keane. 2007. A CaspaseIndependent Pathway Mediates Macrophage Cell Death in Response to Mycobacterium tuberculosis Infection. Infect. Immun. 75:1984.  69.  Basu, S., S. K. Pathak, A. Banerjee, S. Pathak, A. Bhattacharyya, Z. Yang, S. Talarico, M. Kundu, and J. Basu. 2007. Execution of Macrophage Apoptosis by PE_PGRS33 of Mycobacterium tuberculosis Is Mediated by Toll-like Receptor 2dependent Release of Tumor Necrosis Factor-{alpha}. J. Biol. Chem. 282:1039.  70.  Spira, A., J. D. Carroll, G. Liu, Z. Aziz, V. Shah, H. Kornfeld, and J. Keane. 2003. Apoptosis Genes in Human Alveolar Macrophages Infected with Virulent or Attenuated Mycobacterium tuberculosis: A Pivotal Role for Tumor Necrosis Factor. Am. J. Respir. Cell Mol. Biol. 29:545.  71.  Riendeau, C. J., and H. Kornfeld. 2003. THP-1 Cell Apoptosis in Response to Mycobacterial Infection. Infect. Immun. 71:254.  72.  Keane, J., B. Shurtleff, and H. Kornfeld. 2002. TNF-dependent BALB/c murine macrophage apoptosis following Mycobacterium tuberculosis infection inhibits bacillary growth in an IFN-γ independent manner. Tuberculosis 82:55.  73.  Fratazzi, C., R. Arbeit, C. Carini, M. Balcewicz-Sablinska, J. Keane, H. Kornfeld, and H. Remold. 1999. Macrophage apoptosis in mycobacterial infections. J Leukoc Biol 66:763.  74.  Balcewicz-Sablinska, M. K., J. Keane, H. Kornfeld, and H. G. Remold. 1998. Pathogenic Mycobacterium tuberculosis Evades Apoptosis of Host Macrophages by Release of TNF-R2, Resulting in Inactivation of TNF-α. J Immunol 161:2636.  75.  Keane, J., H. G. Remold, and H. Kornfeld. 2000. Virulent Mycobacterium tuberculosis Strains Evade Apoptosis of Infected Alveolar Macrophages. J Immunol 164:2016.  76.  Sly, L. M., S. M. Hingley-Wilson, N. E. Reiner, and W. R. McMaster. 2003. Survival of Mycobacterium tuberculosis in Host Macrophages Involves Resistance to Apoptosis Dependent upon Induction of Antiapoptotic Bcl-2 Family Member Mcl-1. J Immunol 170:430.  77.  Velmurugan, K., B. Chen, J. L. Miller, S. Azogue, S. Gurses, T. Hsu, M. Glickman, W. R. Jacobs, S. A. Porcelli, and V. Briken. 2007. Mycobacterium tuberculosis nuoG Is a Virulence Gene That Inhibits Apoptosis of Infected Host Cells. PLoS Pathogens 3:e110.  78.  Fratazzi, C., R. D. Arbeit, C. Carini, and H. G. Remold. 1997. Programmed cell death of Mycobacterium avium serovar 4-infected human macrophages prevents the mycobacteria from spreading and induces mycobacterial growth inhibition by freshly added, uninfected macrophages. J Immunol 158:4320.  122  79.  Schaible, U. E., F. Winau, P. A. Sieling, K. Fischer, H. L. Collins, K. Hagens, R. L. Modlin, V. Brinkmann, and S. H. E. Kaufmann. 2003. Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis. Nat Med 9:1039.  80.  Winau, F., S. H. E. Kaufmann, and U. E. Schaible. 2004. Apoptosis paves the detour path for CD8 T cell activation against intracellular bacteria. Cell Microbiol 6:599.  81.  Hinchey, J., S. Lee, B. Y. Jeon, R. J. Basaraba, M. M. Venkataswamy, B. Chen, J. Chan, M. Braunstein, I. M. Orme, S. C. Derrick, S. L. Morris, W. R. Jacobs, Jr., and S. A. Porcelli. 2007. Enhanced priming of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis. J. Clin. Invest. 117:2279.  82.  Kurtz, S., K. P. McKinnon, M. S. Runge, J. P. Y. Ting, and M. Braunstein. 2006. The SecA2 Secretion Factor of Mycobacterium tuberculosis Promotes Growth in Macrophages and Inhibits the Host Immune Response. Infect. Immun. 74:6855.  123  Chapter 4  4.1  Discussion & future directions  Discussion  Tuberculosis remains a serious threat to global health. With 1/3 of the population infected, over 8 million active cases, and the emergence of multiple- and extensivelydrug-resistant strains of M. tuberculosis, it is clear that improved vaccines and therapeutics are imperative (1). Several factors including its ease of transmission, complex biological structure, efficient evasion of host immune responses, and treatment complexities make M. tuberculosis a successful pathogen. These characteristics have also posed a number of challenges for clinicians and researchers seeking novel approaches to combat TB disease. There are several approaches to studying infectious organisms in efforts to understand pathogenesis including examining bacterial virulence mechanisms and/or host defense strategies.  In these studies, we focused on the initial interaction of M.  tuberculosis with host cells and explored how the dynamics of this interaction can affect the subsequent progression of infection. Mφ, the primary host cells for M. tuberculosis, serve as the first line of defense against infection and can dictate the subsequent innate and adaptive responses, and ultimately the outcome of infection. Optimally, Mφ eliminate intracellular mycobacteria using a number of defense mechanisms. However, M. tuberculosis has successfully evolved mechanisms to evade such defenses and survive and replicate in these cells  124  As mentioned in the introductory chapter, there are several Mφ surface components that have been shown to interact with M. tuberculosis but none of these have been shown to affect the successive control of bacterial growth. CD43 is the first and only receptor whose presence has been shown to affect the survival of M. tuberculosis. This evidence indicates that there is clearly a functional link between the binding of M. tuberculosis by CD43-mediated processes and the subsequent events. This supposition is further supported by our findings that CD43 regulates the production of inflammatory mediators and the induction of apoptosis in response to M. tuberculosis. Apoptosis, or programmed cell death, plays an important role in immunity to pathogens so it is not surprising that many intracellular pathogens exhibit anti-apoptotic mechanisms (2-4). Although some studies have shown that M. tuberculosis may induce host cell apoptosis under  certain conditions (5-8), there is considerable evidence  suggesting that virulent strains of the bacterium actually inhibit apoptosis (9-12). Moreover, it has recently been shown that the differential susceptibility of various mouse strains to M. tuberculosis infection is linked to the induction of necrosis versus apoptosis in infected Mφ (13). In that study, it was shown that cells that became apoptotic upon infection were more resistant to bacterial growth, while cells with a necrotic phenotype were extremely susceptible to the intracellular growth of M. tuberculosis or Listeria monocytogenes (13). This hypothesis that M. tuberculosis evades apoptosis to allow for increased replication and spread within the host is supported by our findings reported here. Our results also suggest that the controlled growth of M. tuberculosis in Mφ via induction of apoptosis is mediated mainly by TNF-α. Consistent with other published  125  studies (14), we found that neutralization of TNF-α resulted in lower levels of apoptotic cell death and enhanced bacterial survival. The significant role for TNF-α in host defense against Tuberculosis infection has been established by several studies using neutralizing antibodies, over expression of TNF receptors, or knockout mice (14-17) but the mechanism by which TNF-α mediates host defense is not fully understood. Our data suggest that TNF-mediated apoptosis is an important process employed by host cells to restrict mycobacterial growth. Furthermore, this may be of significance in the development of granulomas in M. tuberculosis-infected individuals as not only has TNFα been shown to be important in the formation of granulomas, but the CD43-/- mice showed inadequate containment of mycobacterial infection, likely due to their deficiency in TNF-α production. This remains to be elucidated, as other factors may be responsible for the abnormal organ pathology seen in CD43-/- mice infected with M. tuberculosis. For instance, CD43 has been shown to be important in activation of T cells and in lymphocyte homing (18, 19). The preliminary study by Fratazzi et. al implied a potential role for CD43 in the recognition of mycobacteria. This evidence, along with the published roles for CD43 in cell activation, migration and signaling events led us to hypothesize that it may have a significant function in M. tuberculosis infection. We confirmed this hypothesis by showing that CD43 plays a number of roles including the initial binding and uptake of the bacterium, and the production of inflammatory mediators and antimycobacterial effector functions of Mφ. We contend that CD43 is required for the optimal control of M. tuberculosis infection, especially during early stages of infection, and suggest it is a critical component of the innate immune response to Tuberculosis.  126  4.2  Future directions  Several avenues for future research in the area of CD43 interactions with M. tuberculosis exist that were beyond the scope of the studies presented in this manuscript. We have successfully established that CD43 is an important host cell molecule that interacts with the mycobacterial surface. The nature of this interaction is yet to be established and the question of whether CD43 acts as a pattern recognition receptor or as a co-receptor will needs to be resolved in future studies. Perhaps there are other molecules that cooperate with CD43 in binding to the bacterial surface and the characterization of these will aid our understanding of this interaction. To date, no definitive ligands have been identified on the bacterium that may bind to cell surface receptors, but evidence by other researchers in our lab indicates that the outer layer of the cell wall envelope of M. tuberculosis modulates the interaction of the bacterium with Mφ (20). Identification of specific ligands and the mechanisms of ligandreceptor interactions will be central to our understanding of mycobacterial pathogenesis as it is becoming clear that such interactions can determine the induction of innate immune mechanisms and the subsequent outcome of infection. Our results in the present study point to the involvement of CD43 in cell signaling processes in response to M. tuberculosis. Intra- and intercellular signaling events potentiate effector cell activation and are important components of the immune response against invading pathogens, thereby bridging the innate and adaptive branches of immunity. We have begun comparing gene expression using gene array technology based on the findings of this project. These studies are ongoing and the results will be  127  analyzed using bioinformatics tools that will allow comparison of gene expression by CD43-/- and CD43+/+ Mφ in response to M. tuberculosis infection and may lead to the identification of components of interest for further study. CD43 is not only present in high concentrations on Mφ, but on nearly all hematopoietic cell types (18, 21-23) and the role of CD43 on these cells and their interaction with M. tuberculosis will be of interest for future studies. For example, DCs are vital in the progression of innate to adaptive immunity to many pathogens including M. tuberculosis, and the presence of CD43 on these cells may have a function in this role. Additionally, further inquiry into the adaptive immune response to M. tuberculosis is warranted, given our results that granuloma formation is impaired in CD43 deficient mice. Because CD43 has been associated with regulating the homing of lymphocytes to sites of inflammation, analyzing the composition of granulomas and evaluating the infiltrating cell types may offer insight into how this process develops. In summary, studies that elucidate mechanisms involved in M. tuberculosis infection and the innate immune response of infected individuals will lead to a thorough understanding of the molecular mechanisms of disease and ultimately allow identification of therapeutic targets or vaccine candidates.  128  4.3  Literature cited  1.  WHO. 2004. In Global tuberculosis control: surveillance, planning, financing. World Health Organization, Geneva.  2.  Boya, P., B. Roques, and G. Kroemer. 2001. Viral and bacterial proteins regulating apoptosis at the mitochondrial level EMBO Journal 20:4325-4331.  3.  Dockrell, D. H. 2003. The multiple roles of Fas ligand in the pathogenesis of infectious diseases. Clinical Microbiology and Infection 9:766-779.  4.  Hilleman, M. R. 2004. Strategies and mechanisms for host and pathogen survival in acute and persistent viral infections. PNAS 101:14560-14566.  5.  Rojas, M., L. F. Barrera, G. Puzo, and L. F. Garcia. 1997. Differential induction of apoptosis by virulent Mycobacterium tuberculosis in resistant and susceptible murine macrophages: role of nitric oxide and mycobacterial products. J Immunol 159:1352-1361.  6.  Danelishvili, L., J. McGarvey, Y.-j. Li, and L. E. Bermudez. 2003. Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells. Cellular Microbiology 5:649-660.  7.  Schaible, U. E., F. Winau, P. A. Sieling, K. Fischer, H. L. Collins, K. Hagens, R. L. Modlin, V. Brinkmann, and S. H. E. Kaufmann. 2003. Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis. Nat Med 9:1039-1046.  8.  Derrick, S. C., and S. L. Morris. 2007. The ESAT6 protein of Mycobacterium tuberculosis induces apoptosis of macrophages by activating caspase expression. Cellular Microbiology 9:1547-1555.  9.  Sly, L. M., S. M. Hingley-Wilson, N. E. Reiner, and W. R. McMaster. 2003. Survival of Mycobacterium tuberculosis in Host Macrophages Involves Resistance to Apoptosis Dependent upon Induction of Antiapoptotic Bcl-2 Family Member Mcl-1. J Immunol 170:430-437.  10.  Keane, J., H. G. Remold, and H. Kornfeld. 2000. Virulent Mycobacterium tuberculosis Strains Evade Apoptosis of Infected Alveolar Macrophages. J Immunol 164:2016-2020.  11.  Velmurugan, K., B. Chen, J. L. Miller, S. Azogue, S. Gurses, T. Hsu, M. Glickman, W. R. Jacobs, S. A. Porcelli, and V. Briken. 2007. Mycobacterium  129  tuberculosis nuoG Is a Virulence Gene That Inhibits Apoptosis of Infected Host Cells. PLoS Pathogens 3:e110. 12.  Balcewicz-Sablinska, M. K., J. Keane, H. Kornfeld, and H. G. Remold. 1998. Pathogenic Mycobacterium tuberculosis Evades Apoptosis of Host Macrophages by Release of TNF-R2, Resulting in Inactivation of TNF-{alpha}. J Immunol 161:2636-2641.  13.  Pan, H., B.-S. Yan, M. Rojas, Y. V. Shebzukhov, H. Zhou, L. Kobzik, D. E. Higgins, M. J. Daly, B. R. Bloom, and I. Kramnik. 2005. Ipr1 gene mediates innate immunity to tuberculosis. Nature 434:767-772.  14.  Keane, J., B. Shurtleff, and H. Kornfeld. 2002. TNF-dependent BALB/c murine macrophage apoptosis following Mycobacterium tuberculosis infection inhibits bacillary growth in an IFN-γ independent manner. Tuberculosis 82:55-61.  15.  Byrd, T. F. 1997. Tumor Necrosis Factor-α (TNFα) Promotes Growth of Virulent Mycobacterium tuberculosis in Human Monocytes. The Journal of Clinical Investigation 99:2518 - 2529.  16.  Gardam, M. A., E. C. Keystone, R. Menzies, S. Manners, E. Skamene, R. Long, and D. C. Vinh. 2003. Anti-tumor necrosis factor agents and tuberculosis risk: mechanisms of action and clinical management. The Lancet Infectious Diseases 3:148 - 155.  17.  Garica, I., Y. Miyazaki, G. Marchal, W. Lesslauer, and P. Vassalli. 1997. High sensitivity of transgenic mice expressing soluble TNFR1 fusion protein to mycobacterial infections: synergistic action of TNF and IFN-γ in the differentiation of protective granulomas. Eur. J. Immunol. 27:3182-3190.  18.  Ostberg, J. R., R. K. Barth, and J. G. Frelinger. 1998. The Roman god Janus: a paradigm for the function of CD43. Immunol. Today 19:546 - 550.  19.  Rosenstein, Y., A. Santana, and G. Pedraza-Alva. 1999. CD43, a Molecule with Multiple Functions. Immunologic Research 20:89 - 99.  20.  Stokes, R. W., R. Norris-Jones, D. E. Brooks, T. J. Beveridge, D. Doxsee, and L. M. Thorson. 2004. The Glycan-Rich Outer Layer of the Cell Wall of Mycobacterium tuberculosis Acts as an Antiphagocytic Capsule Limiting the Association of the Bacterium with Macrophages. Infect. Immun. 72:5676-5686.  21.  Borche, L., F. Lozano, R. Vilella, and J. Vives. 1987. CD43 monoclonal antibodies recognize the large sialoglycoprotein of human leukocytes. Eur. J. Immunol. 17:1523-1526.  22.  Carlsson, S. R., and M. Fukuda. 1986. Isolation and characterization of leukosialin, a major sialoglycoprotein on human leukocytes. J. Biol. Chem. 261:12779-12786. 130  23.  Fernandez-Rodriguez, J., C. X. Andersson, S. Laos, D. Baeckström, A. Sikut, R. Sikut, and G. C. Hansson. 2002. The Leukocyte Antigen CD43 Is Expressed in Different Cell Lines of Nonhematopeietic Origin. Tumor Biology 23:193-201.  131  

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