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Licentiate thesis from the Department of Immunology Wenner-Gren Institute, Stockholm University, Sweden Does IgA play a role in protection against pulmonary tuberculosis? Anna Tjärnlund Stockholm 2005 PUBLICATIONS This licentiate thesis is based on the following papers, which are referred to by their Roman numerals in the text: I. Rodríguez A*., A. Tjärnlund*, J. Ivanyi, M. Singh, I. García, A. Williams, P. D. Marsh, M. Troye-Blomberg, and C. Fernández. 2005. Role of IgA in the defense against respiratory infections. IgA deficient mice exhibited increased susceptibility to intransal infection with Mycobacterium bovis BCG. Vaccine 23: 2565-2572. II. Tjärnlund A*., A. Rodríguez*, P-J. Cardona, E. Guirado, J. Ivanyi, M. Singh, M. Troye-Blomberg, and C. Fernández. 2005. Polymeric Ig receptor knockout mice are more susceptible to mycobacteria infection in the respiratory tract. Submitted to Vaccine * These authors contributed equally CONTENTS ABBREVIATIONS SUMMARY INTRODUCTION 1 TUBERCULOSIS 1 MYCOBACTERIAL INFECTIONS Immune evasion IMMUNE RESPONSE TO MYCOBACTERIAL INFECTION 1 2 3 Innate immune responses 3 Adaptive immune responses 4 Granuloma formation 5 Cellular requirements for protective immunity against TB 5 + 5 + CD8 T cells 6 γδ T cells 7 Macrophages 8 Dendritic cells 9 B cells 10 CD4 T cells MUCOSAL IMMUNITY IN THE RESPIRATORY TRACT 11 Specific immune responses 11 Secretory IgA 13 Functions of IgA 14 Fcα receptors 14 IgA deficiency 15 TB: THE DISEASE 17 Diagnosis 17 Tuberculin skin test 18 Microscopy 18 Cultivation 18 Molecular methods 19 Treatment 19 Current vaccine 19 New vaccine candidates 20 ANIMAL MODELS IN TB 21 PRESENT STUDY 23 AIM 23 MATERIALS AND METHODS 24 Antigen and adjuvant 24 Immunizations 24 Intranasal infection with BCG and quantification of bacterial load 24 M. tuberculosis aerosol infection and quantification of bacterial load 25 Antibody production 25 Cytokine-producing cells 25 Cytokine production 26 Quantification of mRNA expression 26 Histology and morphometry 26 Statistical analysis 27 RESULTS AND DISCUSSION 28 Paper I 28 Paper II 29 CONCLUDING REMARKS 33 ACKNOWLEDGEMENTS 34 REFERENCES 35 APPENDIX: Paper I-II ABBREVIATIONS APC Antigen-presenting cell BAL Broncho-alveolar lavage BCG Mycobacterium bovis Bacillus Calmette-Guérin CFU Colony forming unit CR Complement receptor CT Cholera toxin DC-SIGN DC-specific intercellular adhesion molecule-3 grabbing nonintegrin FcR Fc-receptor HIV Human immunodeficiency virus HPRT Hypoxanthine guanine phosphoribosyl transferase Ig Immunoglobulin i.n. Intranasal iNOS Inducible nitric oxide synthase IFN-γ Interferon-gamma IL Interleukin MALT Mucosal-associated lymphoid tissue MDR Multidrug resistant MHC Major histocompatibility complex MR Mannose receptor MyD88 Myeloid differentiation factor 88 NALT Nasal-associated lymphoid tissue NO Nitric oxide pIgA Polymeric immunoglobulin A pIgR Polymeric immunoglobulin receptor PPD Purified protein derivative RANTES Regulated-upon-activation normal T-cell sequence RMI Reactive-nitrogen intermediate SC Secretory component sIgA Secretory immunoglobulin A sIgAD Selective IgA deficiency TAP Transporters associated with antigen processing TB Tuberculosis TCR T-cell receptor Th Helper-T cell TLR Toll-like receptor TNF-α Tumor necrosis factor-alpha TST Tuberculin skin test SUMMARY More than a century after the identification of the tubercle bacillus and the first attempts at vaccination, tuberculosis (TB) still remains one of the world’s most serious infectious diseases. TB is typically a disease of the lung, which serves both as port of entry and as the major site of disease manifestation. The currently used vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is administered parentally and induces a systemic immune response. However, it fails to protect against pulmonary TB, thereby raising the question whether vaccination targeting the mucosal immunity in the lungs could be favourable. The respiratory mucosal surfaces represent the first line of defence against a multitude of pathogens. Secretory IgA (sIgA) in mucosal secretions has an important function by blocking entrance of pathogenic organisms and preventing infections. Yet, another role for IgA in protection against intracellular pathogens has lately been appreciated, when sIgA was demonstrated to neutralize viruses intracellulary. We aimed to investigate the relevance of sIgA in protection against mycobacterial infections using mice deficient for IgA and the polymeric Ig receptor. Mice were immunized intranasally with a mycobacterial antigen which elicited, in wild-type mice, a strong IgA response in mucosal secretions in the respiratory tract. Gene-targeted mice failed to induce the same response and more importantly, were more susceptible to mycobacterial infections in the respiratory tract, as demonstrated by higher bacterial loads in the lungs than wild-type mice. Analysis of immune responses after infection revealed reduced production of proinflammatory, and protective, factors such as IFN-γ and TNF-α in the lungs of deficient mice, which was in concordance with the higher bacterial burden seen in the lungs of these mice. The mechanisms explaining the defective proinflammatory responses in the lungs of deficient mice are not clear but might involve impaired signalling through Fcα receptors, or homologous receptors, which could lead to inadequate activation of pulmonary macrophages. This could subsequently result in suboptimal induction and production of cytokines and chemokines important for attraction and migration of cells to sites of infection in the lungs. Our results demonstrate a role for IgA in protection against mycobacterial infection in the respiratory tract by blocking the entrance of the mycobacterium into the lungs, and/or by modulating the locally induced proinflammatory immune responses. INTRODUCTION TUBERCULOSIS Tuberculosis (TB) remains one of the leading infectious diseases and causes high mortality in humans, resulting in more than 2 million deaths annually (WHO, 2001). The increasing global health burden of TB is due both to the synergistic pathogenesis of coinfection with the human immunodeficiency virus (HIV), as well as to continued dissemination of multidrug-resistant (MDR) Mycobacterium tuberculosis strains (Coker, 2004; Toossi, 2003). Despite this alarming health challenge, the capacity for treating and preventing TB remains limited, and a uniformly effective vaccine is lacking. The M. tuberculosis complex, the cause of TB, is comprised of M. tuberculosis, M. bovis, M. africanum, M. microtti and M. canetti, and although all members can cause TB, M. tuberculosis is the most prevalent. The natural reservoir of M. tuberculosis and M. canetti is limited to humans and that of M. microtti is mainly limited to small rodents (Kremer et al., 1998). In contrast, the host range of M. bovis is very broad and these species can cause disease among a wide range of wild and domestic animals, as well as in humans (Ayele et al., 2004). M. africanum has been isolated from humans and diverse animal species (Thorel, 1980; Alfredsen and Saxegaard, 1992). All members in the complex are slow-growing organisms with generation times ranging from 12 to 24 hours depending on environmental and microbial variables. MYCOBACTERIAL INFECTIONS The causative agent of infectious TB is M. tuberculosis, a rod-shaped obligate aerobic bacillus, which is shielded by a unique wax-rich cell wall, composed of long-chain fatty acids, glycolipids and other components (reviewed in Kaufmann, 2001). This robust cell wall of the bacteria contributes to intracellular survival in host phagocytes. TB can manifest itself at any tissue site, but the lungs represent both the main port of entry and the most important site of disease manifestation. Droplets containing bacilli are expelled from individuals with active pulmonary TB, and subsequently inhaled into the respiratory 1 tract (Riley et al., 1995) and phagocytosed by alveolar macrophages. These cells are considered to be the main cellular host for mycobacteria, and their major role is rapid killing of the invading organism, through the release of toxic reactive oxygen and nitrogen intermediates, or killing by lysosomal enzymes following fusion with the bacterial phagosome. The receptors that have been implicated in the uptake of mycobacteria include mannose receptors (MRs) that recognize mannose residues on mycobacteria (Schlesinger, 1993; Schlesinger et al., 1996), Fc receptors (FcRs) binding opsonised cells, complement receptor (CR) 1, CR3, and CR4, after opsonisation with complement factor C3 (Aderem and Underhill, 1999; Hirsch et al., 1994; Schlesinger et al., 1990), surfactant receptors (Downing et al., 1995), and scavenger receptors (Zimmerli et al., 1996). Once M. tuberculosis has entered the lungs, one of four potential fates might occur (Dannenberg, 1994): 1. The initial host response can be effective in killing and eliminating the bacilli. These individuals will not develop TB at any time point in the future. 2. The bacilli can grow and multiply immediately after infection, thereby causing clinical disease known as primary TB. 3. The bacilli may become dormant and never cause disease, resulting in a latent infection that is manifested only as positive tuberculin skin test (TST). 4. The dormant bacilli can eventually begin to grow with resultant clinical disease known as reactivation TB. Immune evasion Despite the preferred target cell, whose function is the elimination of microbes; M. tuberculosis can remain viable after phagocytosis through different strategies evolved to evade host immune responses. The use of certain CRs may be advantageous for the bacterium, since engagement of these receptors does not induce the release of cytotoxic reactive oxygen intermediates (Wright and Silverstein, 1983). Moreover, it is well known that mycobacteria can evade the normal phagosome-lysosome fusion pathway resulting in 2 persistence of the bacteria within the host cell (Armstrong and Hart, 1975). In this way the bacteria not only remain viable, but bacterial antigens are prevented from presentation to T cells. Mycobacteria appear to increase retention of a macrophage protein, termed the tryptophan-aspartate containing coat (TACO) protein, on the surface of the mycobacterial phagosome, and thereby preventing phagosome-lysosome fusion (Ferrari et al., 1999). In addition, expression of major histocompatibility complex (MHC) II molecules is decreased in M. tuberculosis-infected macrophages (Noss et al., 2000). Another immune evasion mechanism is the secretion of proteins such as superoxide dismutase and catalases by M. tuberculosis, which are antagonistic to reactive oxygen intermediates (Andersen et al., 1991), and inhibition of macrophage apoptosis (Fratazzi et al., 1999). Macrophages infected with M. tuberculosis produce inhibitory cytokines, such as transforming growth factor-β1 and interleukin (IL)-10, which reduce macrophage activation, thereby leading to decreased clearance of bacteria (Barnes et al., 1992; Toossi et al., 1995). IMMUNE RESPONSE TO MYCOBACTERIAL INFECTION Innate immune responses Besides phagocytosis, recognition of M. tuberculosis or mycobacterial products is crucial for an effective host response. Phagocytic cells play an important role in the initiation and direction of the adaptive immunity by presentation of mycobacterial antigens and expression of costimulatory molecules and cytokines. Central to immune defence against microbial pathogens are pattern recognition receptors such as the Toll-like receptors (TLRs). Emerging evidence suggest that TLRs play an important role in the activation of immune cells by pathogens, including M. tuberculosis. TLR2, TLR4, and more recently, TLR1/TLR6 that heterodimerise with TLR2, have been implicated in the recognition of mycobacterial antigens (Bulut et al., 2001; Hajjar et al., 2001). Predominantly, a role for TLR2 in immune recognition of M. tuberculosis has been demonstrated. Mycobacterial products have been demonstrated to induce secretion of tumor necrosis factor-α (TNF-α) and nitric oxide (NO) by macrophages via interaction with TLRs, as well as inducing apoptosis in the host cell (Aliprantis et al., 1999; Brightbill et al., 1999). Infection studies using TLR gene-disrupted mice have, however, provided conflicting data, depending on the experimental settings, for instance the dose of bacteria used. TLR2-/- mice 3 have been demonstrated to be more susceptible to M. tuberculosis infection than wild-type mice, while others have reported a redundant role for TLR2 in this context (Sugawara et al., 2003; Reiling, et al., 2002; Drennan et al., 2004). In addition, outcomes from infection studies with TLR4 deficient mice show disparity (Kamath et al., 2003; Shim et al., 2003; Reiling et al., 2002). Signal transduction by most TLRs, with the exception of TLR3, requires the adapter molecule myeloid differentiation factor 88 (MyD88) (Medzhitov et al., 1998; Adachi et al., 1998; Kawai et al., 1999). MyD88 is an intracellular adaptor protein in the IL-1 receptor/IL-1 receptor associated kinases (IRAK) pathway that links TLR recognition with activation of IRAK and TNF receptor associated factor (TRAF), translocation of NF-κB, and gene transcription (Akira et al., 2003). Mice deficient in MyD88 fail to generate proinflammatory responses when stimulated through TLRs (Adachi et al., 1998; Kawai et al., 1999), and demonstrate high susceptibility to several infectious agents, including mycobacteria (Muraille et al., 2003; Mun et al., 2003; Feng et al., 2003; Fremond et al., 2004). Along with triggering cytokine secretion, TLR activation triggers the maturation of monocyte-derived dendritic cells (DCs), resulting in higher levels of T-cell costimulatory molecules, such as CD80 and CD86, along with antigen-presentation molecules such as MHC II (Hertz et al., 2001; Michelsen et al., 2001; Tsuji et al., 2000). In this way TLRs contribute to the innate immune system by the induction of antimicrobial effector molecules, upon ligation. In addition, recognition of mycobacterial products by TLRs induces secretion of cytokines and upregulation of immunostimulatory molecules, and subsequently modulation of the adaptive immune response. Adaptive immune responses M. tuberculosis is a classic example of a pathogen for which the protective immune response relies on cell mediated immunity. The initial interactions in the lungs is with alveolar macrophages, but after this first encounter DCs and monocyte-derived macrophages, recruited to the site of infection, also take part in the phagocytic process (Henderson et al., 1997; Thurnher et al., 1997). Infected DCs mature and migrate to draining lymph nodes to prime naïve T cells via processed antigens. Inflammation in the lungs provides the signals that direct the effector T lymphocytes back to the site of infection where granulomas are formed. The 4 anatomic affinity of these cells appears to be mainly determined by site-specific integrins, “homing receptors”, on their surface and complementary mucosal tissue-specific receptors, “addressins”, on vascular endothelial cells (Kunkel and Butcher, 2003). In addition, chemokines produced in the local microenvironment promote chemotaxis toward mucosal tissues and regulate integrin expression on mucosal lymphocytes, thereby controlling cell migration (Champbell et al., 2003). Granuloma formation Granuloma formation is the hallmark of M. tuberculosis infection. Granulomas are formed in response to chronic local antigenic stimulation, and can be observed in different infectious diseases, including schistosomiasis, leprosy, and leishmaniasis (Reyes-Flores, 1986; Modlin and Rea, 1988; Palma and Saravia, 1997; Rumbley and Phillips, 1999; Boros, 1999). Structure and composition of granulomas vary depending on the organism. A tuberculous granuloma is observed concomitant with a highly activated cell-mediated immune response, which generally mediates control of mycobacterial numbers in the lungs. The granuloma is composed of many different cells, including macrophages, CD4+-, and CD8+-T cells, and B cells (Gonzales-Juarrero et al., 2001). These cells control the infection by providing a local environment for the cells to interact, leading to an effective immune response where cytokine production, macrophage activation and CD8+ T cell-effector functions lead to killing of the mycobacteria. Granulomas also provide a way of containing the bacilli by walling off and preventing spread of infection. Altogether these actions lead to inhibition of growth, or death of M. tuberculosis. However, the resulting pathology can cause additional problems for the host. Cellular requirements for protective immunity against TB CD4+ T cells CD4+ T cells are generally considered to be among the most important players in the protective immune response against M. tuberculosis. CD4+ T cells recognize peptide antigens from mycobacteria degraded in the phagolysosomal compartments and complexed with MHC class II molecules (Davis and Björkman, 1988). Murine studies with antibody depletion of CD4+ T cells (Muller et al., 1987), adoptive transfer (Orme and Collins, 1984), or the use of 5 gene-deficient mice (Caruso et al., 1999), have demonstrated that the CD4+ T cell subset is required for control of infection. The primary effector function of CD4+ T cells is the production of cytokines; first and foremost interferon-γ (IFN-γ), which is crucial for induction of microbicidal activities by macrophages, but also TNF-α. Production of these cytokines by CD4+ T cells are important for the control of TB (Flynn et al., 1993; Flynn et al., 1995), and studies using IFN-γ gene depleted mice demonstrate that these mice are highly susceptible to virulent M. tuberculosis, with defective macrophage activation and uncontrolled bacilli growth (Cooper et al., 1993). Humans defective in genes for IFN-γ or the IFN-γ receptor are prone to serious mycobacterial infections, including M. tuberculosis (Ottenhof et al., 1998). TNF-α plays a key role in the granuloma formation (Kindler et al., 1989; Senaldi et al., 1996), induces macrophage activation, and has immunoregulatory properties (Orme et al., 1999; Tsenova et al., 1999). In mice, TNF-α is also important for containment of latent infection in granulomas (Mohan et al., 2001). Although IFN-γ production by CD4+ T cells is a very important effector function, these cells probably have other roles in controlling M. tuberculosis infection. In MHC class II-/- or CD4-/mice, levels of IFN-γ were severely diminished early in infection, but returned to wild-type levels later on (Tascon et al., 1998; Caruso, et al., 1999). Nevertheless, the gene-deficient mice were not rescued by this later IFN-γ production and succumbed to the infection. Both CD4+ T cell clones and mycobacterial-antigen-expanded CD4+ T cells have been shown to exhibit cytolytic effector function against mycobacterial-antigen pulsed- or mycobacteriainfected-macrophages (Orme et al., 1992). CD8+ T cells A role for CD8+ T cells in mycobacterial infection has also been described despite the residence of the bacteria within phagosomes (reviewed in Smith and Dockrell, 2000). Mycobacterial antigen-specific CD8+ T cells are restricted by either MHC class I or CD1 molecules. CD1 molecules are nonpolymorphic molecules that present lipids or glycolipids to T cells, as opposed to peptide epitopes presented by MHC molecules (reviewed in Porcelli and Modlin, 1999). Mice genetically disrupted in the genes for β2-microglobulin or transporter of antigen processing (TAP), and therefore deficient in MHC class I and nonclassical MHC class Ib molecules and CD8+ T cells, displayed increased susceptibility to M. 6 tuberculosis infection compared to wild type mice (Flynn et al., 1992; Behar et al., 1999; Sousa et al., 1999). The mechanism by which mycobacterial proteins gain access to the MHC class I molecules is not clear. Mycobacteria-induced pores or breaks in the phagosomal membrane have been proposed as mechanisms (Myrvik et al., 1984; Mazzaccaro et al., 1996; Teitelbaum et al., 1999). The bacteria could use this as a way of gaining access to cytosolic nutrients and introducing toxic molecules into the cytoplasm. Additionally, mycobacterial antigens would be allowed to enter the cytoplasm of infected cells and the MHC class I pathway. Two principal effector functions for CD8+ T cells have been suggested: lysis of infected cells and production of cytokines, explicitly IFN-γ, although the relative contributions of these functions are not established. CD1- and MHC class I-restricted CD8+ T cells, specific for mycobacterial antigens, have been shown to induce lysis of infected human DCs and macrophages, resulting in reduced intracellular bacterial numbers (Stenger et al., 1997; Cho et al., 2000). This was dependent on perforin, which was required for pore formation (Stenger et al., 1997), while granulysin was responsible for killing the intracellular bacteria (Stenger, et al., 1998). Moreover, antigen-specific CD8+ T cells can target and kill infected macrophages, and also induce growth inhibition of M. tuberculosis, through apoptotic mechanisms (Oddo et al., 1998). The role of IFN-γ in mycobacterial infections, on the other hand, is considered to be activation of macrophages. CD8+ T cells from lungs of infected mice have been shown to be primed for IFN-γ production although this production appeared to be limited in the lungs (Serbina and Flynn, 1999). γδ T cells T cells that express the γδ T-cell receptor (TCR) also participate in the immune response against M. tuberculosis (Kaufmann, 1996), and are believed to play a role in the early immune response. Data from animal studies suggest that γδ T cells play a significant role in the host response to TB in mice (Izzo and North, 1992). In addition, studies using γδ TCR knockout mice indicate that γδ T cells may be involved in regulation of granuloma formation, which is critical for control of mycobacteria (D´Souza et al., 1997). Dieli et al., have demonstrated an early accumulation of γδ T cells in the lungs of BCG-infected mice, reaching a peak 3 weeks 7 before αβ T cells, as well as regulatory role for γδ T cells in the induction of CD8+ T cells in the lungs (Dieli et al., 2003). These results indicate that γδ T cells might be important for control of mycobacterial infection in the period between innate and adaptive immunity. Human γδ T cells, predominantly the Vγ9/Vδ2 TCR subset, have also been demonstrated to respond to M. tuberculosis antigens (Kabelitz et al., 1991), and monocytes infected with live M. tuberculosis were particularly effective in expanding this subset of γδ T cells (Havlir et al., 1991). The effector functions of γδ T cells in the immune response to M. tuberculosis appear to be both cytokine secretion and cytotoxicity. Studies with M. tuberculosis antigen-activated γδ T cell clones or primary cells demonstrated IFN-γ production, but also TNF-α production in response to phosphate antigens (reviewed in Boom, 1999). Likewise, cytotoxicity mediated by γδ T cells has been confirmed and was dependent upon activation through the TCR (Munk et al., 1990; Dieli et al., 2003). M. tuberculosis reactive γδ T cells from the peripheral blood of TST positive subjects were cytotoxic for monocytes pulsed with mycobacterial antigens. Macrophages Macrophages are regarded as the phagocytic cell that initially ingest M. tuberculosis, and provide an important cellular niche during infection. Upon infection, macrophages have been shown to secrete proinflammatory cytokines such as TNF-α, IL-1, and IL-6, suggesting a role for these cytokines in the recruitment of cells to the site of infection (Giacomini et al., 2001). Furthermore, the secretion of TNF-α may aid in the activation of macrophages to produce reactive oxygen and nitrogen intermediates, and help granuloma formation (Roach et al., 2002; Flynn et al., 1995). The significance of these toxic nitrogen oxides in host defence against M. tuberculosis has been well documented, both in vitro and in vivo, particularly in the murine system (MacMicking et al., 1997; Shiloh and Nathan, 2000). In the mouse, reactive nitrogen intermediates (RMI) play a protective role in both the acute and chronic persistent infection (MacMicking et al., 1997; Flynn et al., 1998). More important, accumulating evidence supports a role for these reactive molecules in host defence in human TB (Nicholson et al., 1996; Wang et al., 1998), although this still remains controversial. 8 The mechanisms by which NO and other RMI may affect antimicrobial activity could be through modification of bacterial DNA, proteins and lipids (reviewed in Knowledge et al., 2001). NO can also deaminate, as well as directly damage bacterial DNA. RMI has also the potential to disrupt signalling pathways, and NO has been demonstrated to induce apoptosis. Dendritic cells While DCs do not display effective anti-microbial activity upon mycobacterial encounters, their secretion of cytokines and expression of co-stimulatory molecules help in modulating the adaptive immune response, supporting a helper T cell (Th) 1 biased T-cell response. It was recently shown that DCs, but not macrophages, infected with M. tuberculosis were capable of driving Th 1 polarization of naïve CD4+ T cells (Hickman et al., 2002). Activation of human monocyte-derived DCs with the 19-kDa mycobacterial lipoprotein results in the preferential secretion of IL-12, a key player in host defence against M. tuberculosis (Thoma-Uszynski et al., 2000). An additional property of DCs contributing to their effectiveness in initiating immune responses is their ability to migrate from peripheral tissues to secondary lymphoid tissues after acquiring antigens. Naïve T cells are thereby activated via antigen presentingand costimulatory-molecules in the presence of polarizing cytokines such as IL-12. DCs and macrophages appear to have different roles during infection with mycobacteria. Macrophages but not DCs, for instance, have the ability to kill intracellular M. tuberculosis (Bodnar et al., 2001). The different intracellular behaviour of M. tuberculosis in macrophages and DCs may reflect differences in the receptors involved in bacterial uptake in the two cell types. DCs have lectin-surface receptors such as the recently identified DC-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) (Geijtenbeek et al., 2000), that are not expressed on most macrophages and that facilitate antigen uptake (Engering et al., 2002). Whereas CR3 and MR are also expressed by human DCs, they seem to be neglected by the tubercle bacillus (Tailleux et al., 2003). Ligation of DC-SIGN with the M. tuberculosisderived lipoarabinomanan induces IL-10 secretion by DCs, and thereby suppresses their functions (Geijtenbeek et al., 2003). 9 B cells While an essential role for T cells in the control of M. tuberculosis is well established, the role of B cells is less well understood. Infection studies with B cell-deficient mice have revealed conflicting data. It has been reported that B-cell-deficient mice had an increase in viable bacilli compared to the wild-type mice (Vordermeier et al., 1996). Other studies demonstrated that B cells were recruited to the lungs of mice infected with M. tuberculosis and contributed to granuloma formation, yet mice that lack B cells are able to control the growth of bacteria in the lungs (Johnson et al., 1997; Bosio et al., 2000). The absence of B cells resulted in reduced recruitment of neutrophils, macrophages, and CD8+ T to the lungs, suggesting that B cells may influence the cellular composition at this site. An additional role for B cells as antigenpresenting cells (APCs) has also been suggested (Vordermeier et al., 1996). 10 MUCOSAL IMMUNITY IN THE RESPIRATORY TRACT Mucosal surfaces lining the respiratory-, gastrointestinal-, and urogenital tracts are the major sites of entry for pathogens. These mucosal surfaces thereby provide the first line of defence against entrance of various bacteria and viruses. Protection of mucosal membranes against colonization, possible entry and invasion by microbes is provided by a combination of nonspecific and specific mechanisms. Production of mucos is part of the non-specific mechanisms which acts as a physical barrier by containing substances such as lysozyme, lactoferrin, collectin and defensins. Ciliary action can force microbes out of the respiratory tract. The movement of microbes by the ciliae decreases the time available for adherence by pathogens to the epithelium. Tight junctions between neighbouring epithelial cells lining the mucosal membranes also act as a physical barrier against penetration. Specific immune responses Mucosal surfaces contain specialized mucosa-associated lymphoid tissues (MALT) necessary for antigen sampling and induction of mucosal immune responses. The immune system in the upper and lower respiratory tract can be divided into three parts (Davis, 2000): 1. an epithelial compartment at the surface of the epithelium and the underlying connective tissue that contains immunocompetent cells 2. the MALT, subdivided according to anatomical location: the nose-associated lymphoid tissue (NALT), larynx-associated lymphoid tissue (LALT), and the bronchus-associated lymphoid tissue (BALT) 3. lymph nodes draining the respiratory system Mucosal antigen-specific immune responses are elicited in the MALT, where foreign material from epithelial surfaces can be sampled and transported by microfold (M) cells, and subsequently taken up by underlying DCs and macrophages (reviewed in Kiyono and Fukuyama, 2004). The follicles of the MALT contain all immunocompetent cells, i. e. T cells, B cells and APCs that are required for initiation of an immune response. The MALT, as well as local and regional draining lymph nodes, thereby constitutes the inductive site for 11 generation of mucosal immunity. The common mucosal immune system connects these inductive sites with effector sites where antigen-specific lymphocytes perform their effector functions after extravasation from peripheral blood, directed by the local profile of vascular adhesion molecules and chemokines (Fig. 1). Dendritic cells in the lymphoid tissue capture antigens, process and present them to lymphocytes in the context of MHC molecules. After antigen-induced priming, proliferation and partial differentiation in the MALT, lymphoid memory and effector cells migrate to regional lymph nodes where further differentiation can take place (Brandtzaeg et al., 1999). The lymphocytes thereafter pass into the peripheral blood circulation whereby extravasation at mucosal effector sites occurs. These primed cells express adhesion molecules or “homing receptors” specific for corresponding determinants on endothelial cells in mucosal and exocrine glandular tissues (Butcher and Picker, 1996). Figure 1. The common mucosal immune system. (Modified from Nature Reviews Immunology 2004). 12 Secretory IgA IgA is the predominant immunoglobulin (Ig) isotype induced at mucosal sites (Brandtzaeg, 1989), where it is believed to mediate defense mechanisms (Lamm, 1997; Mazanec et al., 1993). IgA monomers are polymerized through the J chain, which is added just before secretion of IgA by plasma cells (Johansen et al, 2000). IgA-producing plasma cells migrate to the basolateral surface of mucosal epithelial cells, where secreted IgA is transported to the luminal side by the polymeric Ig receptor (pIgR), expressed at the basolateral side of epithelial cells lining the mucosal surfaces (Mostov, 1994) (Fig. 2). Translocation of IgA involves enzymatical cleavage of the pIgR whereby the extracellular part of the molecule, the secretory component (SC), in complex with polymeric IgA (pIgA) forms the secretory IgA (sIgA) that is released into the luminal secretions (Norderhaug et al., 1999). pIgR-mediated transcytosis does not require the presence of a ligand, resulting in a continuous release of the SC into external secretions. In human exocrine fluids, 30 to 60% of SC is normally in a free form (Brandtzaeg, 1973). The constitutive expression of pIgR in epithelial cells can be further upregulated by certain cytokines, such as IFN-γ (Sollid et al., 1987; Loman et al., 1997; Youngman et al., 1994), and TNF-α (Kvale et al., 1988). Figure 2. Transport of mucosal IgA. (A) IgA translocation through the epithelium. (B) Structure of sIgA. 13 Functions of IgA IgA is thought to be the most important Ig for lung defence by shielding the mucosal surfaces from penetration by microorganisms and foreign antigens, as well as neutralizing bacterial products such as enzymes and toxins (Lamm, 1997; Mazanec et al., 1993). Other mechanisms include its ability to agglutinate microbes and interfere with bacterial motility by interacting with their flagella. Immune complexes of IgA and encountered antigens can be transported across epithelial cells from basal to apical surfaces in vitro (Kaetzel et al., 1991), and in vivo (Robinson et al., 2001). Foreign substances that have breached the mucosal surface could thereby be eliminated from the body by IgA-mediated transport back through the epithelium. Additionally, IgA appears to be able to interact with viral antigens during transcytosis and interfere with viral synthesis and/or assembly, thereby neutralizing viruses intracellularly (Mazanec et al., 1992). In vitro studies have demonstrated evidence for such intraepithelial cell neutralization against Sendai and measles viruses (Fujioka et al., 1998; Yan et al., 2002), influenza virus (Mazanec et al., 1995), rotavirus (Burns et al., 1996; Feng et al., 2002), and recently against HIV (Huang et al., 2005). Whereas the role of sIgA is established in mucosal immunology, the function of serum IgA antibodies is mostly unknown. Serum IgA is considered to be a “discrete housekeeper” because IgA-immune complexes can be removed by the phagocytic system with little or no resulting inflammation. Fcα Recptors IgA has traditionally been viewed as a non-inflammatory antibody. The inability of sIgA to fix complement efficiently or to act as an opsonin is an advantage in secretions, where induction of an inflammatory reaction would likely affect the integrity of the mucosal surface (Kerr, 1990). However, characterization of FcRs for IgA (FcαRs) has challenged the paradigm of IgA as a non-inflammatory or even anti-inflammatory immunoglobulin. The human FcαR (FcαRI, CD89) is expressed on eosinophils (Monteiro, et al., 1990), neutrophils (Honorio-Franca et al., 2001), monocytes (Patry et al., 1996), macrophages subsets, Kupffer cells, and DCs (Geissmann et al., 2001). Whereas interaction of IgA with the pIgR is noninflammatory, CD89 mediates a broad spectrum of pro-inflammatory and immunomodulatory 14 effects (Morton et al., 1996). A second line of defence at the interface of mucosal and systemic immunity, provided by FcαR-serum IgA interactions on Kupffer cells has been proposed (van Egmond et al., 2000). Under physiological conditions, sIgA inhibits invasion of pathogens into the mucosal surface, as a first line of defence, without activating inflammatory responses. Under pathological conditions the pathogens can invade the portal circulation due to disruption of the mucosal barrier, where they subsequently will be exposed to serum IgA. Concomitantly produced inflammatory cytokines induce FcαRI expression on Kupffer cells, and FcαRI-positive Kupffer cells can thereby phagocytose the pathogens that have entered the circulation. Despite extensive studies on FcαRs in man, there is scarce knowledge about the structure and function of FcαRs in mice. A CD89 homologue in rats was recently identified (Maruoka et al., 2004), but no mouse homologue has yet been found. A common Fcα/µR was newly characterized on human and mouse B cells and macrophages, and on different tissues like liver, spleen, and intestine (Shibuya et al., 2000). Using the human FcαR probe, transcripts of two cDNAs, PIR-A and PIR-B, isolated from a mouse splenic library, were detected (Kubagawa et al., 1997). The PIR-A and PIR-B genes were fund to be expressed on B cells and cells from the myeloid lineage. IgA deficiency Selective IgA deficiency (SIgAD), using 0.05 g/l as the upper limit for diagnosis in adults, is the most common form of primary immunodeficiency in the western world and affects approximately 1/600 individuals (reviewed in Hammarström et al., 2000). The variability in the prevalence in different ethnic groups is striking, 1/18000 in Japanese and 1/4000 in Chinese, suggesting a genetic implication for the disorder. Although sIgA has a clear biological role, SIgAD is a heterogeneous condition with symptoms ranging from none to recurrent respiratory or gastrointestinal diseases, atopy, asthma, inflammatory or autoimmune disorders such as systemic lupus erythematosus, rheumatoid arthritis, and pernicious anaemia (Burks and Steele, 1986; Burrows and Cooper, 1997; Cunningham-Rundles, 2001), although most individuals remain without symptoms. IgA deficiency can be associated with IgG subclass deficiency (Oxelius et al., 1981), making it, in some cases, difficult to distinguish between the cause and the symptoms. The fact that most IgA-deficient individuals are healthy 15 may be partly explained by a compensatory increase in IgM-bearing B cells and increased secretory IgM in mucosal fluids (Brandtzaeg and Nilssen, 1995; Norhagen et al., 1989). However, secretory IgM does not completely replace sIgA functionally, particularly not in the upper respiratory tract (Brandtzaeg et al., 1987). 16 TB: THE DISEASE TB is primarily a pulmonary infectious disease. Following infection with M. tuberculosis there is an early transient influx of granulocytes, but the hallmark of mycobacterial infections is the development of granulomatous lesions (Rook and Bloom, 1994). Although granuloma formation provides a mean of containing infection, granulomas may displace and destroy adjacent tissues. Initially well-formed granulomas may gradually develop central caseation which may lead to extensive fibrosis or cavity formation in the lungs. TB can involve any organ system in the body. While pulmonary TB is the most common clinical manifestation, extrapulmonary TB is also an important clinical problem. The bacilli can spread from the initial site of infection in the lung through the lymphatics or blood to other parts of the body an cause extrapulmonary TB of the pleura, lymphatics, bone, genitorurinary system, meninges, peritoneum, or skin. Before the HIV pandemic, and in studies involving immunocompetent adults, it was observed that extrapulmonary TB constituted about 10-20% of all cases with TB (Fanning, 1999; Weir and Thornton, 1985). In HIVpositive patients, extrapulmonary TB accounts for more than 50% of all cases of TB (Theuer et al., 1990). The most common extrapulmonary sites in HIV-positive individuals are the lymph nodes. Disseminated, or miliary, TB refers to involvement of two or more organs simultaneously, and can occur during primary infection or after reactivation of a latent infection, as well as after reinfection. Diagnosis Early confirmation of the diagnosis of TB is a challenging problem. The established methods have limitations of speed, sensitivity and specificity. In general, it is more difficult to diagnose extrapulmonary TB than pulmonary TB, since this often requires invasive procedures to obtain diagnostic specimens for histological or bacteriological confirmation. 17 Tuberculin skin test The TST is currently the only widely used method for identifying infection with M. tuberculosis in persons who do not have TB. This test is based on the fact that infection with M. tuberculosis produces a delayed-type hypersensitivity reaction to certain mycobacterial components in the extracts of culture filtrates called “tuberculins”. The test (Mantoux method) is administered by intradermal injection of tuberculin purified protein derivative (PPD), which produces a wheal of the skin. The visible induration (in mm) is measured 48 and 72 hours after injection. There are however concerns regarding this test. Several factors may contribute to false-negative results such as age, poor nutrition and general health, overwhelming acute illness, or immunosuppression, such as medications or HIV infection (American Thoracic Society, 2000). In addition, false-positive results can occur in individuals who have been infected with other mycobacteria, including vaccination with BCG. Because of its low sensitivity, TST can not be used to rule out the possibility of active TB. Microscopy The detection of acid-fast bacilli in stained smears examined microscopically is the first bacteriologic evidence of the presence of mycobacteria in clinical specimens. Acid-fast staining procedure depends on the ability of mycobacteria to retain dye when treated with mineral acid or an acid-alcohol solution. Smear examination is rapid, inexpensive, technically simple, and highly specific for acid-fast bacilli, such as M. tuberculosis. Additionally, it gives a quantitative estimation of the number of bacilli being excreted. Identification of smear positive patients is of major importance because only smear positive pulmonary TB patients are regarded as highly infectious to others. However, smear microscopy can not discriminate between M. tuberculosis and other mycobacteria, and in addition, lacks sensitivity since 5000 to 10000 bacteria/ml is needed for a positive result (American Thoracic Society, 2000). Cultivation Mycobacterial culture is the ultimate proof of mycobacterial infection and is often used as the reference method due to its high sensitivity and specificity (Schirm et al., 1995; Walker, 2001). As few as 10 bacteria/ml of material is necessary for detection using this method (American Thoracic Society, 2000). Also, the cultivation of the etiological agent has been 18 essential for species identification, drug susceptibility testing and monitoring the response to therapy. Nevertheless, the slow growth rate of M. tuberculosis and most other mycobacterial pathogens complicates the use of cultivation as diagnostic technique. Molecular methods The use of nucleic acid amplification for the diagnosis of TB is rapidly evolving. These technologies allow for the amplification of specific target sequences of nucleic acids that can be detected through the use of a nucleic acid probe, and both RNA and DNA amplification systems are commercially available (Cohen et al., 1998). Treatment Treatment against TB requires a combination of different drugs. Isoniazid, rifampicin, pyrazinamide and streptomycin constitute the first line of TB drugs to be used. A combination of three drugs is required to avoid the development of MDR TB, and must be taken for a long period of time, usually 6 months or more. The WHO declared TB as a global emergency in 1993. One reason for this extraordinary declaration was an epidemic of MDR TB in the USA in the late 1980s and early 1990s, predominantly in HIV infected prisoners and homeless (Centers for Disease Control and Prevention, 1991). The management of MDR TB is a challenging problem, given that treatment is less effective, more toxic and much more expensive compared to treatment of patients with drug susceptible TB. In the last few years the fluoroquinolone group of drugs has been added to the chemotherapy to TB, and has been used as a part of regimens to treat patients with MDR TB. Current vaccine The current vaccine against TB, the attenuated M. bovis bacillus Calmette-Guérin (BCG), was developed by the French scientists Calmette and Guérin in the first decade of the 20th century. It has been used for more than 70 years and has been given to more people than any other vaccine. Although it can prevent disseminated and meningeal TB in young childen, its efficacy against the most prevalent disease form, pulmonary TB in adults, has been strongly 19 questioned. Data concerning the protective efficacy of BCG in adults range from 0% in South India to 80% in the UK (Fine, 1995). The reason for this variation in efficacy might depend on several factors, including variation in BCG strains, vaccination dose, vaccination protocols, or inappropriate handling of vaccine (Hess and Kaufmann, 1999). Additionally, studies from animal models suggest that prior exposure to live environmental mycobacteria primes the host immune system against mycobacterial antigens shared with BCG, and recall of this immune response upon vaccination results in accelerated clearance of BCG and therefore decreased protection against TB (Kamala et al., 1996; Brandt et al., 2002). Moreover, the hypothesis that the protection by BCG vaccination wanes over time has been brought up (Sterne et al., 1998). Another factor underlying the failure of BCG could be the route of vaccination. BCG is currently administered intradermally which might not be optimal for inducing protective immunity in the respiratory tract. Vaccination at the mucosal site has been believed to be superior to vaccination at other sites for eliciting protective immune responses against mucosal infectious diseases (Davis, 2001). Falero-Diaz and colleagues reported that intranasal (i.n.) vaccination with BCG conferred potent protection against airway M. tuberculosis challenge (Falero-Diaz et al., 2000). Another study demonstrated that a single i.n. BCG vaccination is superior to the subcutaneous route for protection against pulmonary TB in mice (Chen et al., 2004). In addition, i.n. vaccination offers desirable vaccination advantages, such as ease, feasibility, and the ability to trigger to trigger both mucosal and systemic immune activation (Davis, 2001). New vaccine candidates Over the past several years there has been an intensive effort to develop a new vaccine against TB, and the TB vaccines under development can be divided into two categories: preexposure or postexposure vaccines. Preexposure vaccines prevent infection and subsequent disease and should be given to uninfected persons. Postexposure vaccines aim to prevent or reduce progression to disease, and would be given to persons already infected with M. tuberculosis. To date, a multitude of vaccine candidates have been tested in animal models. The increasing knowledge of the tubercle proteins and development of techniques to help identify the most immunogenic antigens, have generated numerous subunit vaccine candidates. Another area in which there has been substantial interest and progress is the DNA vaccines, and several 20 mycobacterial antigens have been targeted in this manner (Huygen, 1998). Whole bacterial vaccines have the advantage of a built-in adjuvanticity, as well as containing both protein- and non-protein antigens. This strategy includes live attenuated bacteria, as well as engineering and overexpression of distinct antigens to improve the immunogenicity. ANIMAL MODELS IN TB Experimental animal models of TB are central to vaccine development. As for many infectious diseases, there is no ideal model for TB. The most common models of M. tuberculosis infection are the mouse and the guinea pig. In neither of these species does the disease perfectly match that seen in human, however many aspects of immunity are the same. Nevertheless, the importance of being careful when extrapolating results from animal experiments to human TB must be emphasised. The mouse is the most widely used species and provides many advantages (reviewed in Kaufmann, 2003). The mouse genome has been completely sequenced, and besides mice being relatively low in cost, there is a wealth of information on their immune system and the techniques and reagents for mechanistic studies are abundant. Additionally, the availability of genetically targeted mice makes it possible for in vivo studies of the relevance of particular cells and molecules. A large number of gene knockout and knock-in mice, both constitutive and conditional have been generated. However, the mouse is relatively resistant to M. tuberculosis, and does not develop the severe pathology seen in some human patients. The guinea pig is generally considered even more susceptible to M. tuberculosis than humans and therefore provides a very sensitive model for testing the efficacy of novel vaccine candidates. Moreover, granulomatous lesions in guinea pigs are very similar to those in human TB patients. Finally, the group 1 CD1 molecules, responsible for presentation of mycobacterial glycolipids to CD1-restricted T cells, are present in humans and guinea pigs but absent in mice (Ulrichs and Kaufmann, 2002; Schaible and Kaufmann, 2000). The non-human primate model is considered the closest match for human disease in terms of pathology. The immune response in this species is very similar to those in humans and most reagents for human cells and molecules can be applied to primate studies. Experiments in 21 non-human primates should be limited to critical experiments, and for final validations directly preceding clinical trials of vaccines and therapeutic agents in humans. 22 PRESENT STUDY AIM The upper respiratory tract is the port of entrance for the mycobacterium, and the first encounter between the host and the pathogen is taking place in the lung. This raises the hypothesis that protective immunity against mycobacterial infections should include a local respiratory mucosal immunity in the lungs, in addition to a cell-mediated immune response. We therefore aimed to investigate the importance of IgA, the major Ig isotype present at mucosal sites, in the respiratory mucosal immunity against infection with intracellular mycobacteria. The specific objectives were: To evaluate the relevance of IgA in the respiratory tract in protection against mycobacterial infection using IgA-deficient mice (paper I). To study the role of actively secreted IgA in protection against mycobacterial infection in the respiratory tract using pIgR-deficient mice (paper II). 23 MATERIALS AND METHODS Antigen and adjuvant The endotoxin-free recombinant PstS-1 protein was used as antigen for immunizations. This 38 kDa lipoprotein is a putative phosphate-transport receptor, and a known B-, and T-cell stimulant that has been suggested as a potential immunodiagnostic reagent (Wilkinson et al., 1997). As adjuvant, for targeting the mucosal immune system, cholera toxin (CT) was used. CT is a strong mucosal adjuvant that markedly increases antigen presentation by DCs, macrophages and B cells (reviewed in Holmgren, 2005). It acts by up-regulation of MHC-, and costimulatory molecules as well as chemokine receptors on APCs, and by increasing the permeability of the epithelium leading to enhanced uptake of co-administered antigen. Immunizations IgA-deficient mice and wild-type non-targeted littermate (IgA+/+) mice (Harriman et al., 1999), provided by Dr. I Mbawuike (Baylor College of Medicine, Houston, USA), were used in Paper I. pIgR-deficient mice (Shimada et al., 1999), obtained from Dr. M. Nanno (Yakult Central Institute for Microbiological Research, Tokyo, Japan), and wild-type C57BL/6 mice were used in Paper II. All mice were kept and bred in the Animal House in the Arrhenius Laboratories at Stockholm University, Sweden. For investigating the induced mucosal immune responses, groups of mice were immunized i.n. using the PstS-1 protein (10 µg/dose) formulated with CT (1 µg/dose). Each group of mice received three immunizations separated by three-week intervals. I.n. immunizations were performed on mice anaesthetized with isofluorane and the antigen (30 µl) was applied to the external nares, 15 µl in each nostril, using a micropipette. Intranasal infection with BCG and quantification of bacterial load Mice were inoculated with 106 colony-forming units (CFU) of live BCG i.n., in 30 µl PBS, under anesthesia with isofluorane, two weeks after the last immunization with PstS-1. At weeks 1 or 4 after infection, mice were sacrificed and the numbers of viable bacteria in the 24 lungs and broncho-alveolar lavage (BAL) were determined. Serial dilutions of the lung homogenates and BAL samples were plated on Middlebrook 7H11 agar plates. The numbers of CFU were determined after 2-4 weeks of incubation at 37 °C. M. tuberculosis aerosol infection and quantification of bacterial load All animals were kept under controlled conditions in a BL High Security Facility. Mice were placed in the exposure chamber of an airborne infection apparatus. The nebulizer compartment was filled with 7 ml of the bacillar suspension at a previously calculated concentration to provide an approximate uptake of 20 viable bacilli within the lungs. The numbers of viable bacteria in the left lung homogenates on weeks 3 and 8 after infection were followed by plating serial dilutions on Middlebrook 7H11 agar plates and counting bacterial colonies after 21 days incubation at 37 ºC. Antibody production Total Ig and specific antibody levels in serum, BAL and saliva were analyzed by ELISA. ELISA plates were coated with either the PstS-1 protein (2 µg/ml), or unlabeled anti-mouse Ig (1 µg/ml). Samples were applied to the wells in serial dilutions starting from 1:200 for serum, 1:4 for saliva, and 1:50 for BAL. Secondary antibodies, alkaline-phosphatase labelled goat anti-mouse Ig isotypes, were thereafter added. Finally, a colourless substrate for the enzyme, p-nitrophenyl phosphate, was added which will be cleaved and generated into a coloured reaction product. Optical density was read in a multiscan plate reader at 405 nm. Background level was defined as OD obtained using PBS-Tween 0.05% (vol/vol) instead of samples. ODdilution curves were plotted (after background value correction) and titres were defined as the inverse log10 of the lowest dilution giving OD of 0.1 for BAL and serum, and an OD of 0.2 for saliva samples. Cytokine-producing cells The numbers of IFN-γ-, and TNF-α-producing cells in the lungs were determined by ELISPOT. PVDF plates were coated with anti-IFN-γ antibodies (15 µg/ml), or anti-TNF-α antibodies (5 µg/ml). Cell suspensions obtained from the lungs of individual mice were added 25 to the wells and incubated in the presence of the PstS-1 protein or PPD (10 µg/ml), to induce cytokine production. After 12 h (TNF-α), or 36 h (IFN-γ) of incubation at 37°C in a humidified 5% CO2 incubator, biotinylated anti-IFN-γ antibodies (2 µg/ml), or anti-TNF-α antibodies (0.5 µg/ml) were added to the wells. Streptavidin conjugated to alkaline phosphatase at a 1:1000 dilution was thereafter added. Individual cytokine-producing cells were visualized by addition of the BCIP/NBT substrate solution and enumerated in a computerized ELISPOT counter. Cytokine production Cell suspensions obtained from lungs of individual mice, 4 weeks after i.n. infection with BCG, were cultured (2x105 cells/well) with BCG (1:2 ratio) as stimulation. Supernatants were collected after 48 h and production of IFN-γ and TNF-α was assayed by ELISA. Quantification of mRNA expression Levels of IFN-γ-, regulated upon activation normal T-cell sequence (RANTES)-, inducible nitric oxide synthase (iNOS)-, and TNF-α-mRNA expression in the lungs of M. tuberculosisinfected mice were analyzed by real-time PCR. In short, total RNA from the middle right lobe of the lungs of aerosol-infected mice was extracted with a commercial phenol-chloroform method, RNAzol, and reverse transcribed using a Superscript RT kit to obtain cDNA. The quantitative analysis was performed using a LightCycler™ System. Hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA expression was analyzed for every target sample to normalize for efficiency in cDNA synthesis and RNA loading. A ratio based on the HPRT mRNA expression was obtained for each sample. Histology and morphometry Histopathological analysis of the lungs of M. tuberculosis-infected mice were performed. Briefly, two right lung lobes from each mouse were fixed in buffered formalin and subsequently embedded in paraffin. Every sample was stained with hematoxilin-eosin. For histometry, 5 µm thick sections from each specimen were stained with hematoxilin-eosin and photographed at 6x using a Stereoscopic Zoom SMZ800 microscope and a Coolpix 990 26 digital camera. Sections of 8 lung lobes were studied in each case. A sequence of appropriate software programs was used to determine the area of each single lesion and the total tissue area on photomicrographs. Statistical analysis Experimental groups were compared by ANOVA followed by Tukey posttest. Differences in bacterial loads between wild-type and gene-targeted mice were compared by Mann-Whitney U-test. The level of significance was set at p<0.05. 27 RESULTS AND DISCUSSION Paper I In paper I we investigated the role of IgA in the protection against i.n. challenge with BCG using IgA-deficient mice. The mucosal immune system provides the first line of defence against entrance of a multitude of ingested and inhaled microorganisms, and the most characteristic component of mucosal immunity is sIgA. Induction of IgA responses in the respiratory tract could provide a protective function against diseases caused by respiratory infections, among them TB. We therefore wanted to investigate the relevance of IgA in the respiratory mucosal immunity against mycobacterial infection, after i.n. immunization with the mycobacterial antigen PstS-1 formulated with the mucosal adjuvant CT. It is currently well established that protective immunity against the intracellular pathogen M. tuberculosis requires a cell-mediated immune response (Schaible et al., 1999; Orme et al., 1993). The role of B cells and antibodies in protection against mycobacterial infections has been less extensively studied, and the few studies performed, using B-cell deficient mice, reveal contradictory results with reports of increased viable bacterial counts (Vordermeier et al., 1996), reduced granuloma size but similar bacterial growth (Boise et al., 2000), as well as no effect at all (Turner et al., 2001). The role of antibodies in protection against intracellular bacterial infections has been re-evaluated during the last years, and reports demonstrating antibody-mediated protection in this context have been published (Edelson and Unanue, 2001; Winslow et al., 2000; Hellwig et al., 2001; Glatman-Freedman, 2003). In concordance with this, our results demonstrated that IgA-deficient mice, immunized with PstS-1 in combination with CT, were more susceptible to BCG infection compared to immunized wild-type mice, as shown by higher bacterial load in the lungs and BAL. This result suggests a protective role for IgA in protection against infection with mycobacteria in the respiratory tract. To address the mechanisms underlying the increased susceptibility to BCG infection displayed by the IgA-deficient mice, we analyzed the induced local immune responses, after i.n. immunization with PstS-1 formulated with CT, in IgA-knockout and wild-type mice. Whereas no IgA was detected in either saliva or BAL from IgA-/- mice, they displayed higher levels of both antigen-specific and total IgM compared to IgA+/+ mice. Moreover, IgAdeficient mice displayed an impaired T cell response, as revealed by significantly reduced TNF-α- and IFN-γ-production in the lungs, when compared to wild-type mice. 28 Previous studies have demonstrated impaired T cell responses in both IgA-deficient and Bcell-deficient mice (Arulanandam et al., 2001; Zhang et al., 2002; Vordermeier et al., 1996), but the mechanistic details behind have not been clarified. One possibility is involvement of signalling through Fcα receptors. The human FcαR (CD89) has been well characterized whereas no murine homologue has yet been identified. Transcripts of two cDNAs, PIR-A and PIR-B, have been isolated from a mouse splenic library using the human FcαR probe, and their expression was restricted to B cells and myeloid cells, including granulocytes, macrophages, and mast cells (Kubagawa et al., 1997). A common Fcα/µR, both human and murine, has been identified and is expressed on B cells and macrophages (Shibuya et al., 2000; Sakamoto et al., 2001), and moreover, activated mouse T cells express FcαRs (Sandor et al., 1992). In vitro studies have demonstrated that monomeric and polymeric IgA stimulated TNF-α production and apoptosis in mouse macrophage cell lines (Reljic et al., 2004). It is therefore possible that absence of IgA could lead to reduced or inadequate activation of macrophages at mucosal sites. All together, our results suggest a role for IgA in the protection against mycobacterial infection in the respiratory tract, by blocking the entrance of the bacilli into the lungs and/or by modulating the locally induced proinflammatory immune responses. Paper II In addition to investigating a possible role of IgA in protection against mycobacterial infection in the respiratory tract, we also addressed the role of actively secreted antibodies, specifically sIgA. For this purpose we used pIgR-deficient mice. Although B-cell deficient mice have previously been used in infection studies with mycobacteria (Vordermeier et al., 1996; Boise et al., 2000; Turner et al., 2001), mice lacking actively secreted antibodies have never been used. Generation of pIgR-knockout mice present a unique opportunity to explore the relative contribution of locally secretory antibodies versus systemic immunity in the protection against mycobacteria, as has been done previously in virus-infection studies (Asashi et al., 2002). 29 Initially, the immunological status regarding antibodies, as well as the induced immune responses upon i.n. immunization with PstS-1 in combination with CT in the pIgR-/- mice was assessed. Interestingly, although no antigen-specific IgA was detected in saliva from the knockout mice, IgA levels in BAL were comparable between the two groups. This finding indicates differences between the upper and lower respiratory tract concerning the mechanisms of IgA transport to mucosal secretions. The possibility of transport mechanisms other than through the pIgR can not be ruled out, nor can leakage through the epithelial membrane, which has been previously reported (Johansen et al., 1999), and their respective contributions may be different in the upper and lower respiratory tract. Our finding indicates that transport of IgA in the upper respiratory tract is to a larger extent pIgR mediated than in the lower respiratory tract, where contributions from other transport mechanisms or leakage may have a greater impact. Protection studies against i.n. infection with BCG revealed that pIgR-/- mice were more susceptible to mycobacterial infection than wild-type mice, with significantly higher bacterial burden in the lungs. Although these mice had similar levels of IgA antibodies in the BAL after i.n. immunization as wild-type mice, their higher bacterial load in the lungs after BCG infection could indicate that the SC plays a role for the protective capacity of sIgA. The SC has been associated with several functions, such as stabilization and protection of dimeric/polymeric IgA and, through its carbohydrate residues, ensuring appropriate localization of the complex to the mucos (Phalipon et al., 2002). Moreover, BCG infected pIgR-/- mice displayed a considerable reduction in TNF-α and IFN-γ production by lung mononuclear cells. Since these cytokines are central for protective immunity against mycobacterial infections (Flynn et al., 1995; Cooper et al., 1993), this impaired mycobacterium-induced proinflammatory response most likely contributed to the higher bacterial load in the lungs of these mice. We next addressed the role of sIgA in protection against natural infection with virulent M. tuberculosis. Viable bacterial counts in the lungs clearly demonstrated that pIgR-knockout mice were more susceptible than wild-type mice at the early phase of infection. At the late phase of infection no differences between the two groups could be seen. The higher susceptibility to M. tuberculosis infection was associated with significantly reduced expression of important protective factors in the lungs. Expression of the proinflammatory cytokines IFN-γ and TNF-α were substantially reduced, which corroborate with the findings 30 from the BCG-infected pIgR-deficient mice. Moreover, expression of iNOS was also reduced. Production of reactive nitrogen intermediates is an important defence mechanism against microbial pathogens exerted by macrophages. A reduced level of iNOS in the lungs of infected pIgR-/- mice is therefore in line with the higher bacterial load in the lungs of these mice. Moreover, reduced levels of RANTES expression was also seen in the lungs of infected knockout mice. This C-C chemokine is involved in attracting monocytes and lymphocytes to sites of infection, as well as promoting Th1 type of responses (Taub et al., 1996; Dairaghi et al., 1998; Chensue et al., 1999). These findings indicate that pIgR-/- mice display lower expression of factors important for protective immunity against TB and proper granuloma formation. Indeed, histological analysis of granulomas in the lungs demonstrated a decreased infiltration of macrophages and lymphoytes, but increased numbers of neutrophils in the pIgR-knockout mice compared to wild-type mice. In addition, a higher degree of necrosis and karyorrhexis was evident in the granulomas in the knockout mice. This qualitative histopathological difference was detected at the early phase of infection, whereas no major differences in granuloma composition where seen during the late phase. In this context it is interesting to note that human SC has been reported to bind and inactivate IL-8 (Marshall et al., 2001), a chemokine important for the attraction of neutrophils. Although no rodent IL-8 homologue has been identified, a murine IL-8 receptor homologue exists (Cerretti et al., 1993), and mice deficient for this receptor display impaired neutrophil responses and reduced leukocyte migration (Lee et al., 1995; Becker et al., 2000; Hang et al., 2000). It is therefore tempting to speculate that the ligand for this receptor homologue could in a similar fashion bind to SC as human IL-8, whereby mucosal secretions from pIgR-/- mice would contain higher levels of IL-8 compared to wild-type mice, resulting in increased chemotaxis of neutrophils in the knockout mice. These findings suggest that mycobacteria-infected pIgR-/- mice display a delayed immune response, due to reduced expression of RANTES and consequently impaired attraction of macrophages and lymphocytes to the site of infection, as well as lower proinflammatory response. As a consequence, pIgR-deficient mice exhibit higher bacterial growth in the lungs, which in turn triggers an increased granulomatous infiltration in order to control the bacilli. At the later phase of infection pIgR-/- mice have managed to control the bacterial growth and display comparable viable bacterial counts in the lungs as wild-type mice. 31 Collectively, our results imply a role for sIgA in modulation of mycobacteria-induced proinflammatory immune responses and subsequently for protection against mycobacterial infections. 32 CONCLUDING REMARKS The importance of B cells and antibodies in protection against the intracellular pathogen M. tuberculosis remains controversial, although a protective capacity of antibodies has lately been reconsidered. Mucosal surfaces in the respiratory tract are constantly challenged by massive amounts of inhaled microorganisms and antigens, and in many cases the main protective effector function against mucosal infections is the production of pathogen-specific local sIgA responses, achieved solely via activation of the mucosal immune system. Based on the fact that TB is transmitted via the respiratory tract, and IgA in mucosal secretions is the most characteristic component of mucosal immunity, we investigated the relevance of IgA in protection against mycobacterial infection in the respiratory tract. Our results demonstrate that IgA antibodies play a role in the protection against mycobacterial infection in the respiratory tract, by blocking the entrance of the bacillus into the lungs and/or by modulating the locally induced proinflammatory immune responses. The mechanisms explaining the defective proinflammatory responses in the lungs of IgA- and pIgR-deficient mice might involve impaired signalling through the FcαR, or a homologous receptor, which may lead to inadequate activation of pulmonary macrophages. Insufficient activation of macrophages could consequently result in suboptimal production of factors important for attraction or migration of immune cells to the lungs. In a long perspective, it is important to consider the advantages of efficiently targeting vaccine antigens to appropriate components within the systemic and mucosal immune compartments. As TB is primarily an infection of the respiratory tract, a successful vaccine against TB should stimulate appropriate immunological responses to counteract the tubercle bacillus in the lungs. 33 ACKNOWLEDGEMENTS I wish to express my warm gratitude to all who have contributed to these studies and in particular my supervisor Carmen Fernández and co-supervisor Marita Troye-Blomberg. Many thanks to my co-author and dear friend Ariane Rodríguez Munoz for all the good and bad times we have shared, and for our fruitful collaboration. I sincerely thank everyone that has helped me in the lab and especially Margareta “Maggan” Hagstedt, Ann Sjölund, John Arko-Mensah, Esther Julian, Karin Lindroth and Monika Hansson. Nora Bachmayer, Yvonne Sundström, and Lisa Israelsson, thanks for your friendship, support and all the fun we have together! Thanks to everyone at the Department of Immunology for creating a nice and friendly working atmosphere. Finally I would like to express my deepest and warmest gratitude to my family and all my friends. Mamma, pappa, Karin and mormor, thanks for all your love, support and patience with me during the last 30 years. Simon, thanks for all the fun and good football games. 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