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Mucosal Immunity in Mycobacterial Infections Anna Tjärnlund Stockholm University All previously published papers were reproduced with permission from the publishers. © Anna Tjärnlund, Stockholm 2007 ISBN 91-7155-388-6 Printed in Sweden by Universitetsservice AB, Stockholm 2007 Distributor: Stockholm University Library 2 Integrity without knowledge is weak and useless, and knowledge without integrity is dangerous and dreadful. Samuel Johnson (1709-1784) English author, critic, & lexicographer 3 4 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, caused by the bacterium Mycobacterium tuberculosis, is typically a disease of the lung, which serves both as the port of entry and as the major site of disease manifestation. The currently used vaccine, BCG, is administered parenterally 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 more favourable. The respiratory mucosal surfaces represent the first line of defense against a multitude of pathogens. Secretory IgA in mucosal secretions has an important function by blocking the entrance of pathogenic organisms and preventing infections. Additionally, a role for IgA in the modulation of immune responses is currently being revealed. In this work, we investigated the relevance of mucosal IgA in the protection against mycobacterial infections using mice deficient for IgA and the polymeric Ig receptor, which is the receptor responsible for mucosal secretions of IgA. Gene-targeted mice were more susceptible to mycobacterial infections in the respiratory tract and displayed reduced production of proinflammatory, and protective, factors such as IFN-γ and TNF-α in the lungs. The mechanisms explaining the defective proinflammatory responses in the lungs of deficient mice might involve impaired signalling through Fcα receptors, or homologous receptors, which could lead to an inadequate activation of pulmonary macrophages. This could subsequently result in suboptimal induction and production of cytokines and chemokines important for the attraction and migration of immune cells to the site of infection. An induction of optimal adaptive immune responses to combat mycobacterial infections requires prompt innate immune activation. Toll-like receptors (TLRs) are vital components of the innate branch of the immune system, ensuring early recognition of invading pathogens. Using TLR-deficient mice we demonstrated an important role for TLR2, and partly TLR4, in the protection against mycobacterial infection in the respiratory tract. TLR2-deficient mice failed to induce proper proinflammatory responses at the site of infection, and macrophages derived from the knockout mice displayed impaired anti-mycobacterial activity. 5 Experimental evidence has concluded that the immune response upon an infection can influence the outcome of succeeding infections with other pathogens. Concurrent infections might additionally interfere with responses to vaccinations and have deleterious effects. We developed an in vitro model to study the effect of a malaria infection on a successive M. tuberculosis infection. Our results demonstrate that a malaria blood-stage infection enhances the innate immune response to a subsequent M. tuberculosis infection with a Th1 prone profile. Reduced infectivity of malaria-exposed dendritic cells implies that a malaria infection could impose relative resistance to ensuing M. tuberculosis infection. However, a prolonged Th1 response may interfere with malaria parasite control. The outcome of this work emphasizes the importance of generating effective immune responses in the local mucosal environment upon respiratory mycobacterial infections. It furthermore puts new light on the immunological interaction between parasites and mycobacteria, which could have implications for future vaccine research. 6 ORIGINAL ARTICLES This doctoral thesis is based on the following papers, which are referred to by their Roman numerals in the text: I. Rodríguez, A*., Tjärnlund, A*., , Ivanyi, J., Singh, M., García, I., Williams, A., Marsh, P. D., Troye-Blomberg, M., and Fernández, C. (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(20): 2565-2572. II. Tjärnlund, A*., Rodríguez, A*., Cardona, P-J., Guirado, E., Ivanyi, J., Singh, M., Troye-Blomberg, M., and Fernández, C. (2006). Polymeric Ig receptor knockout mice are more susceptible to mycobacterial infections in the respiratory tract. Int. Immunol. 18(5):807-816. III. Tjärnlund, A., Guirado, E., Julian, E., Cardona, P-J., and Fernández, C. (2006). Determinant role for TLR signalling in acute mycobacterial infection in the respiratory tract. Microbes Infect. 8(7):1790-1800. IV. Tjärnlund, A., Troye-Blomberg, M., Pawlowski, A. (2007). The effect of malaria parasite-derived materials on dendritic cell susceptibility and response to subsequent Mycobacterium tuberculosis infection. Submitted. * These authors contributed equally 7 8 CONTENTS SUMMARY 5 ORIGINAL ARTICLES 7 ABBREVIATIONS 11 INTRODUCTION 13 TUBERCULOSIS MYCOBACTERIAL INFECTIONS Immune evasion IMMUNE RESPONSE TO MYCOBACTERIAL INFECTIONS Innate immunity Macrophages Dendritic cells Neutrophils Toll-like receptors γδT cells Adaptive immunity Cellular immunity CD4+ T cells CD8+ T cells Humoral immunity Granuloma formation TB: THE DISEASE Diagnosis Tuberculin skin test Microscopy Cultivation Molecular methods Treatment The current BCG vaccine New vaccine candidates ANIMAL MODELS OF TB 13 13 14 15 15 16 17 18 19 20 21 22 22 24 25 25 26 27 27 27 28 28 28 29 30 31 MUCOSAL IMMUNITY IN THE RESPIRATORY TRACT SPECIFIC IMMUNE RESPONSES SECRETORY IgA FUNCTIONS OF IgA IgA RECEPTORS IgA DEFICIENCY 33 33 35 36 37 39 MALARIA PARASITE LIFE CYCLE IMMUNITY TO BLOOD-STAGE MALARIA Adaptive immunity 40 40 41 42 9 Humoral immunity Cellular immunity DCs in malaria 42 43 43 CO-INFECTION BETWEEN TB AND MALARIA ANIMAL STUDIES 45 46 PRESENT STUDY AIMS MATERIALS AND METHODS RESULTS AND DISCUSSION PAPER I PAPER II PAPER III PAPER IV 47 47 48 48 48 51 54 58 CONCLUDING REMARKS 62 ACKNOWLEDGEMENTS 64 REFERENCES 68 10 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 Dendritic cell DC-SIGN DC-specific intercellular adhesion molecule-3 grabbing nonintegrin dIgA Dimeric immunoglobulin A DOTS Directly observed treatment short-course FcαR Fc receptor for immunoglobulin A FcR Fc receptor GPI Glycosylphosphatidylinositol HIV Human immunodeficiency virus Hz Hemozoin Ig Immunoglobulin i.n. Intranasal iNOS Inducible nitric oxide synthase IFN-γ Interferon-gamma IL Interleukin IRAK Interleukin-1 receptor associated kinases i.v. Intravenous M cell Microfold cell MALT Mucosa-associated lymphoid tissue MDR Multidrug-resistant MHC Major histocompatibility complex MR Mannose receptor MyD88 Myeloid differentiation factor 88 NO Nitric oxide 11 pIgA Polymeric immunoglobulin A pIgR Polymeric immunoglobulin receptor PPD Purified protein derivative PfEMP1 Plasmodium falciparum erythrocyte membrane protein 1 PfRBC Plasmodium falciparum-infected red blood cell RANTES Regulated upon activation normal T-cell sequence RBC Red blood cell RNI Reactive nitrogen intermediate SC Secretory component SIgA Secretory immunoglobulin A SIgAD Selective immunoglobulin A deficiency TB Tuberculosis TCR T-cell receptor TGF-β Transforming growth factor-beta Th Helper-T cell TLR Toll-like receptor TNF-α Tumor necrosis factor-alpha TST Tuberculin skin test XDR Extensively drug-resistant 12 INTRODUCTION TUBERCULOSIS Tuberculosis (TB) remains one of the leading infectious diseases and causes high mortality in humans, resulting in almost 2 million deaths annually (WHO, 2007). The increasing global health burden of TB is due both to the synergistic pathogenesis of co-infection with the human immunodeficiency virus (HIV), as well as to the continued dissemination of multidrug-resistant (MDR) Mycobacterium tuberculosis strains (Toossi, 2003; Coker, 2004). 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. microti and M. canetti. 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. microti is mainly limited to small rodents (Kremer et al., 1998). In contrast, the host range of M. bovis is very broad and this 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 various 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, M. tuberculosis, is 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. 13 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 tract (Riley et al., 1995) and phagocytosed by alveolar macrophages. Only particles less than 5 μm in diameter can gain access to the alveoli (Hatch, 1942), where macrophages, resident within the alveolar space, can phagocytose the bacillus. Most of the bacteria engulfed by alveolar macrophages will be eliminated from the body through mucocilliary movements, and a few bacteria will be transported by macrophages into the lung interstitium. Once M. tuberculosis has entered the lungs, one of four potential fates is possible (Dannenberg, 1994): 1. The initial host response can effectively kill and eliminate 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 In spite of the targeting macrophages, whose function is the elimination of microbes, M. tuberculosis can remain viable after phagocytosis due to different strategies evolved to evade host immune responses. The use of non-activating complement receptors (CR) 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 prevent the normal phagosome-lysosome fusion resulting in persistence of the bacteria within the host cell (Armstrong and Hart, 1975). In this 14 way the bacteria not only remain viable, but bacterial antigens are prevented from being presented to T cells. Mycobacteria appear to increase the retention of the tryptophan-aspartate containing coat (TACO) protein on the surface of the mycobacterial phagosome, thereby preventing phagosome-lysosome fusion (Ferrari et al., 1999). Moreover, characterization of mycobacterial phagosomes has revealed the presence of the transferrin receptor (CD71), rab5, and early phagosome Ag 1, all markers of early endosomes, while late endosomal protonATPases are blocked from recruitment to mycobacteria-harbouring phagosomes (SturgillKoszycki et al., 1994; Clemens and Horwitz, 1995; Sturgill-Koszycki et al., 1996). In addition, the expression of major histocompatibility complex (MHC) class II molecules is decreased in M. tuberculosis-infected macrophages (Noss et al., 2000). Other immune evasion mechanisms are 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 the inhibition of macrophage apoptosis (Fratazzi et al., 1999). Macrophages infected with M. tuberculosis produce inhibitory cytokines, such as transforming growth factor (TGF)-β 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 INFECTIONS Innate immunity The initial response to an infection is mediated by components of the innate immunity that serves primarily to restrict the multiplication and dissemination of the pathogens, as well as to initiate the ensuing adaptive response. In addition to macrophages, M. tuberculosis also interacts with epithelial cells in the alveolar space of the lung and is able to invade and replicate in this cell type (Bermudez and Goodman, 1996; Garcia-Perez et al., 2003). However, the role of alveolar epithelium in mycobacterial infections has not been fully elucidated. In addition to forming a physical barrier, alveolar epithelial cells can express adhesion molecules and release cytokines and chemokines, such as IL-8 and monocyte chemotactic protein-1, and thereby modulate the local immune response (Lin et al., 1998). M. tuberculosis infection has also been shown to induce the expression of inducible nitric oxide 15 synthase (iNOS) mRNA by epithelial cells and the production of nitric oxide (NO) (Roy et al., 2004), and, more recently, interferon (IFN)-γ (Sharma et al., 2007). Through the presentation of mycobacterial antigens, and the expression of costimulatory molecules and cytokines, phagocytic cells play an important role in the initiation and direction of the adaptive immunity. Macrophages Macrophages are regarded as phagocytic cells that initially ingest M. tuberculosis. Thus, they provide an important cellular niche during infection. The macrophages are considered to be the main cellular host for mycobacteria, and their major role is the 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, the mannose receptor (MR), that recognizes mannose residues on mycobacteria (Schlesinger, 1993; Schlesinger et al., 1996), Fc receptors (FcRs) binding antibody-coated bacteria, CR1, CR3, and CR4, which bind complement factor C3-opsonized bacilli (Schlesinger et al., 1990; Hirsch et al., 1994; Aderem and Underhill, 1999), surfactant receptors (Downing et al., 1995), and scavenger receptors (Zimmerli et al., 1996). Upon infection, macrophages have been shown to secrete proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, IL-1, and IL-6, which are believed to be important for the recruitment of cells to the site of infection (Giacomini et al., 2001). Furthermore, the secretion of TNF-α may also aid in the activation of macrophages to produce reactive oxygen and nitrogen intermediates, and help granuloma formation (Flynn et al., 1995; Roach et al., 2002). The significance of these toxic nitrogen oxides in the host defense 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 (RNIs) play a protective role in both the acute and chronic persistent infection (MacMicking et al., 1997; Flynn et al., 1998). More importantly, accumulating evidence supports a role for these reactive molecules in the host defense against human TB (Nicholson et al., 1996; Wang et al., 1998), although this still remains controversial. 16 The mechanisms by which NO and other RNIs may affect antimicrobial activity, could be through the modification of bacterial DNA, proteins and lipids (reviewed in Chan et al., 2001). NO can deaminate, as well as directly damage bacterial DNA, and has been demonstrated to induce apoptosis. RNIs also have the potential to disrupt signalling pathways, Dendritic cells While dendritic cells (DC) do not display effective anti-microbial activity upon mycobacterial encounters, their secretion of cytokines and expression of costimulatory molecules help in modulating the adaptive immune response, supporting a helper T cell (Th) 1 biased T-cell response. The major role of DCs during mycobacterial infections appears to be that of an antigen-presenting cell (APC). 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 defense against M. tuberculosis (Thoma-Uszynski et al., 2000). An additional property of DCs that contributes 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-presenting-, and costimulatory-molecules in the presence of polarizing cytokines such as IL-12. DCs and macrophages appear to have different roles during infection with mycobacteria. For instance, activated macrophages, but not DCs, 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 DCspecific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) (Geijtenbeek et al., 2000), that facilitate antigen uptake (Engering et al., 2002) and phagocytosis of mycobacteria by DCs (Geijtenbeek et al., 2003; Tailleux et al., 2003). Although CR3 and MR are also expressed by human DCs, they seem to be less important for the uptake of the tubercle bacillus (Tailleux et al., 2003). Ligation of DC-SIGN with the M. tuberculosis- 17 derived lipoarabinomanan induces IL-10 secretion in DCs, and thereby suppresses their function (Geijtenbeek et al., 2003). Neutrophils It has been suggested that neutrophils participate in the host defense against mycobacterial infections since circulating neutrophils become activated and are recruited to the lungs early in infection. They can be found at the infection nidus at the onset of infection, as well as several days after the initial reponse (Pedrosa et al., 2000; Fulton et al., 2002). In vivo depletion of neutrophils prior to mycobacterial infection enhances bacterial growth in the lungs of infected mice, whereas local treatment with the neutrophil chemoattractant macrophage-inflammatory protein-2 enhances neutrophil recruitment and decreases mycobacterial growth (Appelberg et al., 1995; Fulton et al., 2002). The mechanisms by which neutrophils exert their anti-mycobacterial function are not completely resolved, although several hypotheses have been proposed. These include the secretion of chemokines (Riedel and Kaufmann, 1997; Seiler et al., 2003), the induction of granuloma formation (Seiler et al., 2003), and macrophage uptake of neutrophil-specific molecules such as myeloperoxidase (Hanker and Giammara, 1983), and lactoferrin (Silva et al., 1989). Another mechanism whereby neutrophils indirectly contribute to the killing of mycobacteria was recently demonstrated by Tan et al. Mycobacteria-infected macrophages acquire the contents of neutrophil granules and their anti-microbial molecules by the uptake of apoptotic neutrophil debris, which is trafficked to endosomes and colocalize with the intracellular bacteria (Tan et al., 2006). The involvement of neutrophils in direct killing of mycobacteria has been a matter of controversy. In vitro studies have demonstrated the ability of neutrophils to kill virulent M. tuberculosis (Brown et al., 1987; Jones et al., 1990), although this has been questioned (Denis, 1991; Aston et al., 1998). Kisich et al. showed that neutrophils in human pulmonary lesions contained intracellular M. tuberculosis, thereby demonstrating a phagocytic role for neutrophils in human TB. Human neutrophils were furthermore able to kill virulent M. tuberculosis in vitro (Kisich et al., 2002). Neutrophils are clearly important in the early immunity to bacterial infections. They respond rapidly to chemotactic stimuli released by the bacteria or inflammed epithelium and, thus, arrive early at the site of infection. Besides their anti-mirobial role, neutrophils have been 18 implicated in the modulation of the adaptive immune response by the release of chemoattractants, which recruit other immune cells, such as T cells, monocytes, macrophages and DCs to the site of infection (Kasama et al., 1993; Yang et al., 2000; Scapini et al., 2001). It was recently demonstrated that neutrophils and DCs interact physically through DC-SIGN expressed on DCs and Mac-1 expressed on neutrophils (van Gisbergen et al., 2005; Megiovanni et al., 2006). This interaction enables neutrophils to induce maturation of DCs via TNF-α secretion and a preferential production of IL-12 by the matured DCs (van Gisbergen et al., 2005), which in turn results in an enhanced activation of T cells (Megiovanni et al., 2006). Toll-like receptors Besides phagocytosis, the recognition of M. tuberculosis or mycobacterial products is also crucial for an effective host response. Central to the immune defense against microbial pathogens are pattern recognition receptors, such as the toll-like receptors (TLRs). There are 13 members of the TLR family known today, of which TLR1-10 are found in humans (Ulevitch, 2004). Besides microbial products, TLRs also recognize endogenous ligands, such as heat shock proteins (Ohashi et al., 2000; Vabulas et al., 2002), extracellular matrix breakdown products (Termeer et al., 2002; Guillot et al., 2002), and intracellular contents from necrotic cells (Gallucci et al., 1999; Li et al., 2001). Ligation of TLRs initiates a signal transduction pathway that culminates in the activation of NF-κB and induction of several immuno-related genes, including cytokines and chemokines (Hoffmann et al., 1999; Aderem and Ulevitch, 2000). TLR activation is therefore an important link between innate cellular responses and the subsequent activation of adaptive immune response to microbial pathogens. DCs express the broadest repertoire of TLRs through which they can recognize a plethora of microbial compounds. Upon TLR triggering, immature DCs, apart from cytokine secretion, undergo the process of maturation, resulting in an augmented expression of T-cell costimulatory molecules, such as CD80 and CD86, along with antigen-presentation molecules, such as MHC class II (Tsuji et al., 2000; Hertz et al., 2001; Michelsen et al., 2001). Emerging evidence suggests that TLRs play an important role in the activation of immune cells by pathogens, including M. tuberculosis. TLR2, TLR4, and more recently, TLR1/TLR6 19 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 the immune recognition of M. tuberculosis has been demonstrated. Mycobacterial products have been shown to induce secretion of TNF-α and NO by macrophages via interactions 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 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 (Reiling, et al., 2002; Sugawara et al., 2003; Drennan et al., 2004). In addition, outcomes of infection studies with TLR4-deficient mice show disparity (Reiling et al., 2002; Kamath et al., 2003; Shim et al., 2003). Signal transduction by most TLRs, with the exception of TLR3, requires the adaptor 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 the 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). In this way, TLRs contribute to the innate immune system by the induction of antimicrobial effector molecules, upon ligation. In addition, the recognition of mycobacterial products by TLRs induces secretion of cytokines, chemokines, and upregulation of immunostimulatory molecules, and subsequently the modulation of the adaptive immune response. γδ T cells T cells that express the γδ T-cell receptor (TCR) participate in immunity against M. tuberculosis (Kaufmann, 1996), and are believed to play a role in the early immune response 20 (Izzo and North, 1992). Dieli et al. demonstrated an early accumulation of γδ T cells in the lungs of pulmonary BCG-infected mice, reaching a peak 3 weeks before αβ T cells, where they produce IFN-γ, exert cytotoxic activity against BCG-infected macrophages, and play a regulatory role in the induction of CD8+ T cells in the lungs (Dieli et al., 2003). In addition, studies using γδ TCR knockout mice indicate that γδ T cells may be involved in the regulation of granuloma formation, which is critical for the control of mycobacteria (D’Souza et al., 1997). These results indicate that γδ T cells might be important for the control of mycobacterial infection in the period between innate and adaptive immunity. Human γδ T cells, predominantly the Vγ9/Vδ2 TCR subset, which represents a major peripheral blood T cell subset, have also been demonstrated to respond to M. tuberculosis antigens (Kabelitz et al., 1991), and monocytes infected with live M. tuberculosis were seen to be 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, as well as 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. Adaptive immunity M. tuberculosis is a classic example of a pathogen against which the protective immune response relies on cell-mediated immunity. The initial interaction 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 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, 21 “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). Cellular immunity CD4+ T cells It is well established that CD4+ T cells are of utmost importance for protective immunity against M. tuberculosis. CD4+ T cells recognize peptide antigens from mycobacteria, degraded in the phago-lysosomal 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 gene-deficient mice (Caruso et al., 1999), have demonstrated that CD4+ T cell subsets are required for the control of the infection. The primary effector function of CD4+ T cells is the production of cytokines; first and foremost IFN-γ, which is crucial for the induction of microbicidal activities by macrophages, but also TNF-α. Production of these cytokines by CD4+ T cells is 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 infection, with defective macrophage activation and uncontrolled bacillar growth (Cooper et al., 1993). Furthermore, it is known that 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 in turn plays a key role in the granuloma formation (Kindler et al., 1989; Senaldi et al., 1996). It induces macrophage activation, and has immunoregulatory properties (Orme and Cooper, 1999; Tsenova et al., 1999), and is also important for the containment of latent infection in granulomas, both in mice (Mohan et al., 2001) and humans (Ehlers, 2003). Although IFN-γ production by CD4+ T cells is a very important effector function, CD4+ T cells have most likely other roles in controlling M. tuberculosis infection. In MHC class II-/-or CD4-/-mice, the 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 gene22 deficient mice were not rescued by this later IFN-γ production and succumbed to infection. Both CD4+ T cell clones and mycobacterial-antigen-expanded CD4+ T cells have been shown to exhibit cytolytic effector functions against mycobacterial-antigen pulsed or mycobacteriainfected macrophages (Orme et al., 1992). Active TB is characterized by a profound and prolonged suppression of M. tuberculosisspecific T-cell responses, demonstrated by decreased production of IL-2 and IFN-γ (Toossi et al., 1986; Huygen et al., 1988; Zhang et al., 1994; Torres et al., 1994; Hirsch et al., 1999). Overproduction of immunosuppressive cytokines, such as IL-10 and TGF-β, by mononuclear phagocytes has been implicated in decreased T cell function during TB (Hirsch et al., 1996; Gong et al., 1996; Hirsch et al., 1997; Hirsch et al., 1999). However, while IL-10 and TGF-β levels return to normal after anti-TB treatment, M. tuberculosis-stimulated production of IFNγ remains depressed beyond completion of the treatment (Hirsch et al., 1996; Hirsch et al., 1999). Regulatory-T cells constitute a key component of peripheral tolerance suppressing potentially autoreactive T cells and preventing autoimmune diseases (Sakaguchi et al., 1995; Sakaguchi, 2005). Different subsets of regulatory-T cells have been described, such as IL-10-secreting (Tr1) or TGF-β-secreting regulatory T cells (TH3), and CD4+CD25+ regulatory T cells (O’Garra and Vieira, 2004; Sakaguchi, 2005; Belkaid and Rouse, 2005). The latter cells share common markers with conventional, activated CD4+ T cells, and are now identified by the molecular marker FoxP3 (Sakaguchi, 2005; Fontenot and Rudensky, 2005; Roncador et al., 2005). Involvement of IL-10-secreting regulatory-T cells has been proposed in TB anergic patients (Boussiotis et al., 2000). Recent reports indicate a role for CD4+CD25+ regulatory T cells in the negative modulation of anti-TB immune responses. An increased frequency of CD4+CD25+FoxP3+ regulatory-T cells in the blood and at the site of infection was associated with M. tuberculosis infection (Chen et al., 2007), corroborating a previous study showing enhanced frequency of CD4+CD25+ T cells during active TB and sustained enhancement after completion of anti-TB treatment (Ribeiro-Rodrigues et al., 2006). 23 CD8+ T cells Despite the residence of the bacteria within phagosomes, CD8+ T cells take part in the immunity against mycobacterial infections (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 the transporter of antigen processing, and therefore deficient in MHC class I and non-classical MHC class Ib molecules and, thus, unable to activate CD8+ T cells, display an increased susceptibility to M. tuberculosis infection compared to wild-type mice (Flynn et al., 1992; Behar et al., 1999; Sousa et al., 1999), indicating a protective role for these cells. 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 hence 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 yet 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 is required for pore formation (Stenger et al., 1997), while granulysin was seen to be 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 through the activation of macrophages. CD8+ T cells from lungs of infected mice have been shown to be primed for IFN-γ production, although this production appears to be limited in the lungs (Serbina and Flynn, 1999). 24 Humoral immunity While the essential role for T cells in the control of M. tuberculosis infection is well established, the role of B cells and antibodies is less well understood. Although neglected for a long time, their role has recently been reappreciated. In individuals infected with M tuberculosis, a vigorous humoral response is always present, in addition to the well-defined cellular immune response. Infection studies performed using B cell-deficient mice have revealed conflicting data. One study reported an increase in viable bacilli in B cell-deficient mice compared to wild-type mice (Vordermeier et al., 1996), whereas results from another study suggested that absence of B cells, and hence antibodies, does not affect the outcome of infection with M. tuberculosis (Johnson et al., 1997). Yet another study demonstrated that B cell deficiency results in a reduced recruitment of neutrophils, macrophages, and CD8+ T cells to the lungs upon M. tuberculosis infection (Bosio et al., 2000), suggesting that B cells may influence the cellular composition at this site by regulating chemokines and adhesion molecules. An additional role for B cells as APCs has also been suggested (Vordermeier et al., 1996). Passive immunization with monoclonal antibodies has been reported to protect mice against mycobacterial infection. It was recently shown that IgA antibodies specific for the αcrystallin antigen of M. tuberculosis significantly reduced the number of colony forming units (CFUs) in lungs of infected mice (Williams et al., 2004). These results corroborate other studies, reporting a protective role for monoclonal antibodies and passive immunization in experimental mycobacterial infections (Teitelbaum et al., 1998; Pethe et al., 2001; Chambers et al., 2004; Hamasur et al., 2004). 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 many 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). The structure and composition of granulomas vary depending on the organism. A tuberculous granuloma is observed concomitantly with a highly activated cell-mediated 25 immune response, which generally mediates the 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 cellular interactions, 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 the spread of infection. Altogether, these actions lead to an inhibition of growth, or to the death of M. tuberculosis. However, they also result in inflammatory pathology that contributes to the damage of the host tissue. 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 the infection, granulomas may displace and destroy adjacent tissues. Initially well-formed granulomas may gradually develop central caseation that 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 and cause extrapulmonary TB of the pleura, lymphatics, bone, urogenital 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 TB cases (Weir and Thornton, 1985; Fanning, 1999;). In HIV-positive patients, extrapulmonary TB accounts for more than 50% of all TB cases (Theuer et al., 1990). The most common extrapulmonary sites in HIV-positive individuals are the lymph nodes. Disseminated, or miliary, TB refers to the 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. 26 Diagnosis An early confirmation of the diagnosis of TB is a challenging problem. The established methods have limitations in 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. Tuberculin skin test The TST is currently the only widely used method for identifying latent infection with M. tuberculosis in asymptomatic individuals. 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, also known as the Mantoux method, is administered by intradermal injection of the 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 the TST. 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 cannot be used to rule out the possibility of active TB. Microscopy The microscopal detection of acid-fast bacilli in stained sputum smears is the first bacteriological 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. The identification of smear positive patients is of major importance since only smear positive pulmonary TB patients are 27 regarded as highly infectious to others. However, smear microscopy cannot discriminate between M. tuberculosis and other mycobacteria and, in addition, lacks sensitivity, since 5000-10000 bacteria/ml in the sample are needed for a positive result (American Thoracic Society, 2000). Cultivation Mycobacterial growth in cultures 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). With this method, as few as 10 bacteria/ml within a sample are necessary for bacterial detection (American Thoracic Society, 2000). Also, the cultivation of the etiological agent has been 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 a 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 Although TB can be cured; current treatment is complex and long lasting, involving four drugs for 2 months and two drugs for at least another 4 months. The combination of different drugs is neccessary in order to avoid development of resistant and MDR TB. Isoniazid, rifampicin, pyrazinamide and streptomycin constitute the first line of TB drugs that are used and predominantely target actively growing bacteria through the inhibition of cell wall synthesis, DNA replication and protein synthesis. The long duration for chemotherapy is due 28 to the fact that M. tuberculosis is a slow growing organism and, following the initial killing of the majority of bacteria, persistent bacteria can revert to the state of latency. Through the WHO sponsored, directly observed treatment short-course (DOTS) program, effective TB therapy is now available to around 70% of the world’s population. Yet, the likelihood of DOTS therapy resulting in the eradiction of TB is limited by the large reservoir of latently infected individuals, as well as by delays in diagnosis. The treatment regime is additionally demanding for the patient, labour intensive for health staff and is compromised in settings where health services are poorly accessible. In 1993 the WHO declared TB a global health emergency. One reason for this extraordinary declaration was the increase in MDR TB, which was reaching epidemic proportions. The management of MDR TB is a challenging problem, given that treatment is less effective, more toxic and much more expensive compared to the treatment of patients with drug susceptible TB. In the last few years the fluoroquinolone group of drugs has been added to the chemotherapy of resistant forms of TB, and have been used as a part of regimens to treat patients with MDR TB. During the last year, alarming findings of what has been named extensively drug-resistant (XDR) TB has been reported. XDR TB is caused by strains of M. tuberculosis resistant to virtually all second-line drugs. Inappropriate treatment regimens have probably contributed to the development of XDR TB, which raises the concern for a future epidemic of untreatable TB. The current BCG 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 was first given to humans in 1921 and has now been given to more people than any other vaccine (Fine, 1995a). Although it can prevent disseminated and meningeal TB in young children, its efficacy against the most prevalent form of disease, pulmonary TB in adults, has been strongly questioned. Data concerning the protective efficacy of BCG in adults range from 0% in South India to 80% in the UK (Fine, 1995b). The reason for this variation in 29 efficacy might depend on several factors, including variation in the BCG strains used, vaccination dose, vaccination protocols, or inappropriate handling of the 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 an accelerated clearance of BCG and therefore decreased protection against M. tuberculosis (Kamala et al., 1996; Brandt et al., 2002). Helminth infections have, furthermore, been shown to have an impact on the immune response against mycobacterial infections, leading to a Th2 shift in the immune profile, resulting in a reduced protective efficacy of BCG vaccination (Elias et al., 2005a). Moreover, the hypothesis that the protection by BCG vaccination wanes over time has also been brought up (Sterne et al., 1998). Another factor underlying the failure of BCG-aquired protection 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 the protection against pulmonary TB in mice (Chen et al., 2004). In addition, i.n. vaccination offers desirable advantages, such as ease in administration, feasibility, and the ability to trigger both mucosal and systemic immune activation (Davis, 2001). Lastly, BCG vaccination is also known to stimulate cell-mediated immunity, and BCG immunotherapy has been used in the treatment of bladder cancer resulting in improved survival (Alexandroff et al., 1999). New vaccine candidates Over the past several years there has been an intensive effort to develop a new vaccine against TB. The TB vaccines under development can be divided into two categories: prophylactic (preexposure) or therapeutic (postexposure) vaccines. Prophylactic vaccines prevent infection 30 and subsequent disease and should be given to uninfected persons. Therapeutic vaccines aim to prevent or reduce progression to disease, and would be given to individuals already infected with M. tuberculosis. To date, a number of vaccine candidates have been tested in animal models. The increasing knowledge of the tubercle proteins and the 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 DNA vaccines, and several 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 OF TB Experimental animal models of TB are central to vaccine development. As for many infectious diseases, there is no ideal animal model for TB. The most common models used for M. tuberculosis infection are the mouse and the guinea pig. In neither of these species does the disease perfectly match that seen in humans, however, many aspects of immunity are the same. Nevertheless, caution must be advocated when extrapolating results from animal infection experiments to human TB. The mouse is the most widely used species and provides many advantages (reviewed in Kaufmann, 2003). The mouse genome has been completely sequenced, mice are relatively inexpensive, there is a wealth of information on their immune system, and techniques and reagents for mechanistic studies are abundant. Additionally, the availability of genetically targeted mice makes it possible for in vivo studies to elucidate the relevance of particular cells and molecules. A large number of gene knockout and knockin mice, both constitutive and conditional, has 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 seen in 31 human TB patients. Finally, the group 1 CD1 molecules, responsible for the presentation of mycobacterial glycolipids to CD1-restricted T cells, are present in humans and guinea pigs but absent in mice (Schaible and Kaufmann, 2000; Ulrichs and Kaufmann, 2002). The non-human primate model is considered the closest match for human disease in terms of pathology. The immune response in this model is very similar to that seen in humans, and most reagents for human cells and molecules can be applied to primate studies. Due to ethical reasons, experiments in non-human primates should be limited to critical experiments used for final validations directly preceding clinical trials of vaccines and therapeutic agents in humans. 32 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 defense 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 mucus is part of the non-specific mechanisms that acts as a physical barrier 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 the 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, 2001): 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 nasal-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 be taken up by underlying DCs and macrophages (reviewed in Kiyono and Fukuyama, 2004). M cells are specialized epithelial cells that transcytose particles across 33 epithelial barriers to an intraepithelial lymphoid pocket created by the basolateral surface of the M cell (Neutra et al., 1996). The follicles of the MALT contain all immunocompetent cells, i. e. T cells, B cells and APCs, that are required for the initiation of an immune response. The MALT, as well as local and regional draining lymph nodes, thereby constitute the inductive site for the 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). Figure 1. The common mucosal immune system. (Modified from Nat. Rev. Immunol. (2004) 4:699-710). DCs in the lymphoid tissue capture antigens, process and then 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 34 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). M cells moreover express MHC class II molecules (Allan et al., 1993) and ICAM-1 on their cell surface (Ueki et al., 1995), indicating that they can initiate an immune response. There are reports showing that M cells can be found in the lungs as well as in the gut, and that particulate antigens can be transported through pulmonary M cells (Tenner-Racz et al., 1979). Teitelbaum et al. demonstrated that transcytosis by pulmonary M cells facilitates delivery of M. tuberculosis to the broncho-tracheal lymph nodes early after infection, suggesting a role of M cells in the early development of the local immune response (Teitelbaum et al., 1999). SECRETORY IgA Immunoglobulin (Ig) A is the predominant Ig isotype induced at mucosal sites (Brandtzaeg, 1989), where it is believed to mediate defense mechanisms (Mazanec et al., 1993; Lamm, 1997). Monomers of IgA are polymerized through the J chain, which is added just before the 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 enzymatic cleavage of the pIgR, whereby the extracellular part of the molecule, the secretory component (SC), in complex with dimeric IgA (dIgA), 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; Youngman et al., 1994; Loman et al., 1997), and TNF-α (Kvale et al., 1988). 35 Figure 2. Transport of mucosal IgA. (A) IgA translocation through the epithelium. (B) Structure of SIgA. FUNCTIONS OF IgA IgA is thought to be the most important Ig class for lung defense by protecting the mucosal surfaces from penetration by microorganisms and foreign antigens, as well as by neutralizing bacterial products such as enzymes and toxins (Mazanec et al., 1993; Lamm, 1997). Other mechanisms include the ability of IgA 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 interfered with the mucosal surface can thereby be eliminated from the body by an IgAmediated 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 interactions with Sendai virus (Fujioka et al., 1998), measles virus (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). 36 In humans, two subclasses of IgA, termed IgA1 and IgA2, exist, each being the product of a separate gene, whereas in mice there is only one class of IgA. The major difference between IgA1 and IgA2 resides in the hinge region, the short region between the two Fab arms and the Fc region. IgA1 features an extended hinge due to the insertion of a duplicated stretch of amino acids, which is lacking in IgA2. The longer hinge in IgA1 may have evolved to offer advantages in antigen recognition by allowing higher avidity bivalent interactions with distantly spaced antigens (Boehm et al., 1999; Furtado et al., 2004). In secretions, most of the locally produced IgA is, as described previously, polymeric with a relative increase in the proportion of IgA2 in human. The reason for the predominance of this subclass might be due to the increased vulnerability to proteolytic attack that the extended stretch of amino acids found in IgA1 bring about. Highly specific IgA-cleaving proteases are produced by a number of important bacterial pathogens that are able to colonize mucosal surfaces and invade mucosal tissues (Kilian et al., 1996). For instance, Neisseria meningitides, Haemophilus influenzae, and Streptococcus pneumoniae secrete proteases that specifically cleave the IgA1 hinge region (Senior et al., 1991). IgA2 remains resistant to cleavage since the susceptile hinge region is missing. Another strategy evolved by bacteria to circumvent effector functions by host IgA antibodies is the expression of IgA-binding proteins. These are proteins that bind specifically to IgA and are produced by many strains of group A Streptococcus (Stretococcus pyogenes) and group B Streptococcus, which are major human pathogens. IgA is the second most prevalent antibody in serum after IgG, and serum IgA is predominantely (90%) monomeric IgA1 in humans but mainly dIgA in other animals. IgA RECEPTORS IgA has traditionally been viewed as a non-inflammatory antibody. It is a poor activator of complement and does not activate the classical pathway, although its role in activation of the alternative pathway remains controversial. Only recently, it has become apparent that dIgA or polymeric IgA (pIgA), upon binding to mannan-binding lectin, can induce the activation of the lectin pathway (Roos et al., 2001). The significance of complement activation by IgA in vivo remains unclear. It can be speculated that in situations where antigen is limited, IgA could in fact inhibit complement activation by blocking binding of IgG and IgM, which are more potent activators of complement. The inability of SIgA to fix complement efficiently or 37 to act as an opsonin is an advantage at mucosal sites, where the induction of an inflammatory reaction would likely affect the integrity of the mucosal surface (Kerr, 1990). Whereas the role of SIgA in mucosal immunity is being elucidated, 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. However, characterization of FcRs for IgA (FcαRs) has challenged the paradigm of IgA as a non-inflammatory or even anti-inflammatory Ig. The human FcαR (FcαRI, CD89) is expressed on eosinophils (Monteiro, et al., 1990), neutrophils (HonorioFranca et al., 2001), monocytes (Patry et al., 1996), macrophage subsets, Kupffer cells, and DCs (Geissmann et al., 2001). Whereas the interaction of IgA with the pIgR is noninflammatory, antigen-complexed binding to CD89 mediates a broad spectrum of proinflammatory and immunomodulatory effects depending on the cell type involved (Morton et al., 1996; Monteiro and van de Winkel, 2003). These effects include antibody-dependent cell-mediated cytotoxicity, phagocytosis, release of cytokines, superoxide generation, calcium mobilization, degranulation and antigen presentation. Therefore, a second line of defense 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 the invasion of pathogens into the mucosal surface, as a first line of defense, without activating inflammatory responses. Under pathological conditions, the pathogens can invade the portal circulation due to a disruption of the mucosal barrier, where they will subsequently 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 in 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 (paired Ig receptors A and 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 has furthermore been shown to 38 bind to the intracellular lectin Gal-3 (Mac-2) expressed on several cell types including macrophages (Reljic, et al., 2004a). Gal-3 has been implicated in allergic reactions due to its ability to activate mast cells by crosslinking receptor-bound IgE (Liu et al., 1993). 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 among different ethnic groups is striking (1/18000 in Japanese and 1/4000 in Chinese), which suggests a genetic implication for the disorder. Although SIgA has a fairly clear biological role, SIgAD is a heterogeneous condition with symptoms ranging from none at all to recurrent respiratory or gastrointestinal diseases, atopy, asthma, and inflammatory or autoimmune disorders such as systemic lupus erythematosus, rheumatoid arthritis, and pernicious anaemia (Burks and Steele, 1986; Burrows and Cooper, 1997; CunninghamRundles, 2001). However, most individuals remain asymptomatic. 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 may be partly explained by a compensatory increase in IgM-bearing B cells and increased secretory IgM in mucosal fluids (Norhagen et al., 1989). However, secretory IgM does not completely replace SIgA functionally, particularly not in the upper respiratory tract (Brandtzaeg et al., 1987). 39 MALARIA Malaria is caused by a protozoan parasite of the genus Plasmodium. There are four species affecting humans, Plasmodium falciparum, P. vivax, P. ovale and P. malariae. The disease is associated with a variety of clinical syndromes ranging from asymptomatic to lethal infections involving anaemia, organ failure, pulmonary and cerebral disease. P. ovale and P. malariae are relatively infrequent causes of morbidity, while P. vivax is a common cause of severe illness, especially in Asia and South America, but is rarely fatal. The vast majority of severe malaria cases and deaths are caused by P. falciparum, which is endemic in most of subSaharan Africa, where the WHO estimates that 90% of malaria cases occur. There are over 500 million clinical malaria cases every year, with 1-3 million malaria-associated deaths per year, of which the majority are of children under the age of five (Snow et al., 2005). In addition to these human malaria parasites, there are other Plasmodium species that infect various animals. PARASITE LIFE CYCLE The life cycle of the malaria parasite is complex and involves several developmental stages with asexual reproduction in the human host and sexual reproduction in the Anopheles mosquito vector (reviewed by Stevenson and Riley, 2004) (Fig. 3). An infected female mosquito injects 10-30 sporozoites into the host during a blood meal. These sporozoites are carried by the blood to the liver, where they invade hepatocytes within 30 minutes of a bite. Inside the hepatocytes they undergo a process of asexual replication, which gives rise to schizonts. Up to this point the infection is non-pathogenic and clinically silent. The liver schizonts eventually rupture and thousands of merozoites are released into the blood, where they invade red blood cells (RBC). Each merozoite can divide inside the RBC and develop through different stages, such as ring and trophozoite, into schizontes. As the parasite develops, adherent ligands are expressed on the cell membrane of the infected RBC, one being P. falciparum erythrocyte membrane protein 1 (PfEMP1), which enables the parasitized cell to bind to receptors expressed by endothelial cells lining the blood vessels inside organs, such as the brain, lungs and the placenta. Upon rupture of the infected RBC, more than 30 merozoites are released that can then reinfect new RBCs. This gives rise to a cyclic blood- 40 stage infection, which takes 48-72 hours to complete, depending on the Plasmodium species. The RBC stage of the parasite’s life cycle is responsible for the symptoms and pathology of malaria. A small subset of merozoites differentiates into male and female gametocytes that can be taken up by an Anopheles mosquito feeding on the host. Inside the midgut of the mosquito these mature into gametes. This is the sexual stage of the parasite’s life cycle and upon fertilization a motile zygote is formed, which penetrates the epithelial cell layer of the midgut and forms an oocyst. After 10-24 days, thousands of sporozoites are released from the oocyst, which invade the salivary glands of the mosquito and can be injected into a host upon the mosquito’s blood meal. Figure 3. Schematic view of the malaria parasite life cycle. (Nat. Rev. Immunol. (2001) 1:117-125.) IMMUNITY TO BLOOD-STAGE MALARIA Both innate and adaptive immune responses are critical for establishing clinical immunity and control of parasitemia during an infection with P. falciparum. Innate immune responses are important for the control of initial parasitemia, and play a role not only for non-immune individuals infected for the first time, but also for semi-immune individuals who may be infected with a parasite variant they have never encountered before (Stevenson and Riley, 2004). The initial response to infection usually involves splenic removal of parasitized RBCs. 41 Upon rupture of schizonts, parasite products are released into the circulation and trigger the activation of phagocytes, resulting in a release of pro-inflammatory cytokines that cause fever and mediate other pathological effects. One of these bioactive parasite products is glycosylphosphatidylinosiol (GPI), which acts as a malaria pathogen-associated molecular pattern and toxin. GPI induces the production of several factors implicated in malaria pathogenesis, for example pro-inflammatory cytokines, such as TNF-α, IL-1 and IL-12, as well as iNOS and various adhesion molecules that are expressed on vascular endothelium and recognized by PfEMP1 (Carlson et al., 1992; Schofield and Hackett, 1993; Tachado et al., 1997; Naik et al., 2000). Adaptive immunity In endemic areas where there is a continous exposure to the parasite, adaptive immunity is gradually built up and the severity and incidence of malarial illness decrease with increasing age. Since the parasites have the capacity to vary their antigens, which are major targets for protective antibodies, repeated exposure to the parasite is required for a long-lasting immunity. During the first months of life, infants are protected from malaria, most likely by antibodies transferred from the immune mother. Experimental evidence for species- and stage-specific immunity in malaria demonstrates that adaptive immunity is crucial for the protection against malaria (reviewed in Troye-Blomberg, 1994). Humoral immunity Ig levels in individuals, living in highly endemic areas are strongly elevated and the level of total anti-malarial antibodies increases with age. Antibody-mediated protection against bloodstage malaria is primarily mediated by cytophilic IgG antibodies and can involve different mechanisms (Good and Doolan, 1999). Antibodies can inhibit merozoite invasion of RBCs, prevent sequestration of infected RBC by inhibiting binding to adhesion molecules on the vascular endothelium, neutralize parasite GPI, and thereby inhibit the induction of the inflammatory cascade, prevent parasite binding to the placenta, and lastly, enhance the clearance of infected RBCs by binding to their surface and thus promote phagocytosis and removal of the immune complex in the spleen. 42 It has been reported that levels of total IgE, as well as anti-malarial IgE antibodies, are elevated in malaria patients, in parallel to an elevation of TNF-α (Perlmann et al., 1994). A pathogenic role for IgE has been suggested, whereby crosslinking of IgE receptors on monocytes by IgE-containing immune complexes leads to a local overproduction of TNF-α. This has recently been questioned by studies indicating a protective role for IgE (Bereczky et al., 2004; Dolo et al., 2005). Cellular immunity T cells are essential for both generating, as well as regulating, immunity against blood-stage malaria. The major T cells controlling blood stage infections are CD4+ T cells, of both the Thl and Th2 subsets. Evidence from numerous studies, such as selective depletion of CD4+ T cells in vivo (Weinbaum et al., 1978; Suss et al., 1988; Kumar et al., 1989), and adoptive transfer of CD4+ T cells to immunocompromised mice (Brake et al., 1988; Meding and Langhorne, 1991; Taylor-Robinson et al., 1993; Taylor-Robinson and Phillips, 1993), have revealed the critical role for CD4+ T cells in protective immunity against blood-stage malaria infection. Important for the generation of protective immunity is IL-12 production, which induces a polarized Th1 response with a production of IFN-γ and TNF-α. These cytokines activate phagocytic cells, which are important for controlling the level of parasitaemia. The levels of T cells expressing the γδ TCR are elevated in acute malaria infection, and they are believed to have a protective role by exerting cytotoxicity, as well as by secretion of cytokines, such as IFN-γ (reviewed in Dieli et al., 2001). Human NK cells, which are also activated early during malaria infection (Orago and Facer, 1991), can be activated rapidly by parasites in vitro (Theander et al., 1987), resulting in IFN-γ production (Artavanis-Tsakonas et al., 2003). Since RBCs are devoid of MHC molecules, antigen-specific cytotoxic CD8+ T cells are generally not believed to act against the asexual blood stage. DCs in malaria Early interactions between blood-stage malaria parasites and innate cells are thought to be 43 important for shaping the adaptive immune response to blood-stage malaria. However, the role of DCs in the induction of protection against the blood-stage malaria parasite is not well defined. Studies have shown that P. falciparum-infected red blood cells (PfRBC) can bind myeloid DCs and modulate the maturation and function of DCs. Adhesion of PfRBCs has been shown to inhibit the upregulation of maturation makers, costimulatory molecules and adhesion molecules on DCs upon stimulation with LPS. Moreover, the ability of such DCs to induce T cell responses was significantly reduced (Urban et al., 1999). The receptors on DCs mediating this inhibitory effect, CD36 and CD51, are not only receptors for PfRBCs but are also involved in recognition of apoptotic cells by phagocytes. Ligation of CD36 and/or CD51 with PfRBC or apoptotic cells results in a reduced production of IL-12 and an increased production of IL-10 (Urban et al., 2001). The malarial pigment, hemozoin (Hz), has furthermore been shown to have an immunomodulatory effect on phagocytic cells. Hz is the coordinated polymerized form of heme subunits generated by the parasite during hemoglobin catabolism (Slater et al., 1991). Malaria parasites degrade host hemoglobin for its use as source of amino acids, which is accompanied by the release of free heme. Free heme is oxidatively active and toxic to both the host cell and the parasite, causing parasite death. Since the malaria parasite is unable to catabolyze heme, it protects itself through the crystallization of heme into Hz (Sullivan et al., 1996a). During a Plasmodium infection, Hz is released into the blood through the rupture of PfRBC and is readily taken up, together with PfRBC, by macrophages and monocytes (Schwarzer et al., 1992; Schwarzer et al., 1998). Investigations on DC function in murine Plasmodium infections have yielded contradictory results. In vitro and in vivo studies in murine models have shown that, in contrast to the effect on human DCs, P. chabaudi-infected RBC induced the maturation and activation of murine DCs (Seixas et al., 2001; Perry et al., 2004; Leisewitz et al., 2004). In contrast, other studies have reported a suppressive effect on DCs (Ocana-Morgner et al., 2003; Pouniotis et al., 2004). However, a general trend can be noted where there is an early phase of DC activation during a blood-stage infection, with a condition of low parasitemia, and a late phase of inhibition, when the condition is characterised by high parasitemia. Interestingly, a recent study demonstrated that overstimulation of DCs via TLRs renders them refractory to further activation (Perry et al., 2005). Since malaria is associated with an exponential increase in parasite load, DCs are expected to develop TLR tolerance as infection progresses (Perry et al., 2005). The consequence would be subsequent dysfunction of DCs at later stages of infection. However, whether DC function is impaired during malaria infection still remains to be 44 established. CO-INFECTION BETWEEN TB AND MALARIA The WHO estimates that 90% of the malaria cases occur in sub-Saharan Africa where the highest incidence of TB per capita also exists. Considering the overlapping geographical endemic areas for malaria and TB, co-infection is expected to occur very frequently. Given the chronic character of infections caused by M. tuberculosis and P. falciparum, it is highly plausible that an interaction between one of these microorganisms and the host’s immune system would have an impact on the course of infection and disease caused by the other microorganism. Investigations have established that the immune response induced upon infection with a pathogen may influence the outcome of subsequent infections or vaccinations (Curry et al., 1995; Helmby et al., 1998). For instance, helminth infections have been shown to have an impact on the immune response against mycobacterial infections owing to a Th2 shift in the immune profile (Elias et al., 2005a; Elias et al., 2005b). However, the mutual modulation of host immune responses by M. tuberculosis and P. falciparum in concurrent infections has not been extensively studied. Malaria infection affects the host’s T cells, both qualitatively and quantitatively. Since the containment of a mycobacterial infection and protective immunity against TB depend on an intact cellular immunity, malaria-induced impairment of the immune system could influence susceptibility to, or progression of a M. tuberculosis infection. Clinical evidence on the outcome of infection or disease in individuals with double Plasmodium/M. tuberculosis infection, as compared to those infected with a single pathogen, is scarce in the literature. One indirect piece of evidence comes from a study in Guinea-Bissau that demonstrated a lower risk of death due to malaria in children with a BCG scar (Roth et al., 2005). This supports earlier suggestions that the presence of a BCG vaccination scar may be associated with a general non-specific enhancement of the immune responses to some unrelated infections, and reduced mortality in children (Velema et al., 1991; Kristensen et al., 2000; Roth et al., 2004), although revaccination with BCG did not reduce morbidity from malaria in African children (Rodrigues et al., 2007). A statistically significant association between the presence of antimalarial antibodies and the diagnosis of TB was reported by Adebajo et al., indicating that malaria could affect the progression of the mycobacterial disease (Adebajo et al., 1994). 45 ANIMAL STUDIES The effect of malaria on chronic TB has been investigated in a study utilizing a murine model, showing increased M. tuberculosis loads in the lungs, liver and spleen of co-infected mice (Scott et al., 2004). Another study examined the effect of TB on malaria in M. tuberculosisinfected C57BL/6 mice subsequently infected with Plasmodium yoelii. A protective effect of prior M. tuberculosis infection against P. yoelii was detected and was associated with an enhanced Th1 type of immune response (Page et al., 2005). In contrast, Th2-prone BALB/C mice were not protected against malaria infection by prior M. tuberculosis infection. The results obtained with the C57BL/6, but not BALB/c mice, corroborate previous studies demonstrating protection against malaria in animals infected with mycobacteria (BazazMalik, 1973; Clark et al., 1976; Murphy et al., 1981; Stevenson et al., 1984; Matsumoto et al., 2001). Although animal models serve as a valuable tool for studying immune responses and dissecting underlying mechanisms, caution is advised when extrapolating these results to humans. Animal models of malaria infection suffer from the lack of specificity caused by host restriction of malaria species. P. falciparum, the clinically most important human malaria parasite, does not produce disease in mice. 46 PRESENT STUDY AIMS The upper respiratory tract is the port of entrance for the mycobacterium, and the first encounter between the host and the pathogen takes place in the lung. This raises the hypothesis that protective immunity against mycobacterial infections is highly dependent on a local respiratory mucosal immunity in the lungs, in addition to cell-mediated immune responses. In paper I-II 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. An efficient immune response against a pathogen is critically dependent on rapid detection of the invading organism by the innate immunity and the activation of the subseqent adaptive immune response. Emerging evidence supports the concept that innate immune responses are vital for the host defense against M. tuberculosis. TLRs represent critical pathogenrecognition receptors whose signals lead to the generation of important effector responses, including Th1 responses. We therefore studied the role of TLR signalling in early and late stages of mycobacterial infection in paper III. Considering the overlapping geographical endemic areas for two of the leading infectious causes of morbidity and mortality worldwide, malaria and TB, a high frequency of coinfection can be presumed. Since DCs have a unique capacity to prime naïve T cells, and subsequently direct the downstream immunological events that occur after the initial encounter with the pathogens, they provide a critical link between the innate and adaptive immune responses. In paper IV we developed a model for studying concurrent Plasmodium/M. tuberculosis infection using DCs. We assessed the consequences of a malaria blood-stage infection on a subsequent M. tuberculosis infection of DCs. 47 The specific objectives were: To evaluate the relevance of IgA in the respiratory tract in the protection against mycobacterial infection using IgA-deficient mice (paper I). To study the role of actively secreted IgA in the protection against mycobacterial infection in the respiratory tract using pIgR-deficient mice (paper II). To assess the role of different TLRs in the defense against primary mycobacterial infection using TLR-deficient mice (paper III). To analyse the effect of malaria antigens on DC susceptibility and response to a subsequent M. tuberculosis infection (paper IV). MATERIALS AND METHODS The materials and methods used in this work are described in the individual papers (I-IV). 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 (Harriman et al., 1999). The most characteristic component of mucosal immunity is SIgA. An 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 a mycobacterial antigen. The antigen used for immunization was the endotoxin-free recombinant PstS-1 protein. This 38 kDa lipoprotein is a putative phosphate-transport receptor, and a known B-, and T-cell 48 stimulant that has been suggested as a potential immunodiagnostic reagent (Wilkinson et al., 1997). As an adjuvant, for targeting the mucosal immune system, the 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 et al., 2005). It acts by an upregulation 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 antigens. Although protective immunity against the intracellular pathogen M. tuberculosis relies on efficient cell-mediated immune responses, the function of antibodies is being reappreciated. (Winslow et al., 2000; Edelson and Unanue, 2001; Hellwig et al., 2001; Glatman-Freedman, 2003). In concordance with this, our results demonstrated that IgA-deficient mice, immunized with our formulation, were more susceptible to BCG infection compared to immunized wildtype mice, as shown by a higher bacterial load in the lungs and broncho-alveolar lavage (BAL). This result suggests involvement of IgA in the 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. No IgA was detected in either saliva or BAL from IgA-/- mice. However, they displayed higher levels of both antigen-specific and total IgM compared to IgA+/+ mice. Moreover, IgA-deficient 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. Previous studies have demonstrated impaired T cell responses in both IgA-deficient and Bcell-deficient mice (Vordermeier et al., 1996; Arulanandam et al., 2001; Zhang et al., 2002). However, the mechanistic details behind have not been clarified. One possibility is involvement of signalling through FcαRs. The human FcαR (CD89) has been well characterized, whereas no murine homologue has yet been identified, although a common Fcα/μR, both human and murine, has been identified, which is expressed on B cells and macrophages (Shibuya et al., 2000; Sakamoto et al., 2001). Moreover, activated mouse T cells express FcαRs (Sandor et al., 1992). In vitro studies have demonstrated that monomeric 49 and polymeric IgA, but not IgG or IgM, stimulate TNF-α and NO production, as well as induction of apoptosis in mouse macrophage cell lines (Reljic et al., 2004b). It is therefore possible that absence of IgA could lead to a reduced or inadequate activation of macrophages at mucosal sites, and thereby reduced bacterial clearance. This would furthermore affect subsequent immunological events, since activated macrophages secrete TNF-α and other molecules with chemoattractant properties for immune cells. Reljic et al. (2004a) demonstrated that IgA binds to the intracellular lectin Gal-3, expressed on activated macrophages, indicating a further role for IgA-mediated inhibition of intracellular mycobacteria. Gal-3 has been shown to accumulate only in those phagosomes containing live M. tuberculosis through binding to phosphatidylinositol mannosides, and appears to influence the clearance of infection (Beatty et al., 2002), since bacterial interaction with the phagosomal membrane is required for mycobacterial inhibition of phago-lysosome fusion (de Chastellier and Thilo, 1997). It is possible that the interaction between Gal-3 and its mycobacterial ligand could be further amplified by IgA, which provides additional crosslinking of Gal-3. This may keep the pathogen tightly fixed to Gal-3 and away from other membrane components. In other words, IgA antibodies bound to mycobacteria, or mycobacterial components, may interfere with interactions between opsonised bacteria and the membrane of phagosomes, and thereby prevent the survival of mycobacteria within macrophages. Important in this context is the study by Geissmann et al. showing that human DCs located beneath the epithelium express the IgA Fc receptor CD89, and cosslinking by IgA immunocomplexes results in internalization of the ligand, enhanced expression of the costimulatory molecule CD86, as well as MHC class II molecules, and an increase in the allostimulatory activity by the cells (Geissmann et al., 2001). Another indication of an impaired activation of lung macrophages in the absence of IgA antibodies, comes from studies demonstrating that only lung-derived macrophages from IgAdeficient mice, and not bone marrow-derived macrophages, display an impaired activation phenotype and reduced cytokine production upon stimulation, when compared to wild-type controls (Rodríguez A., personal communication). 50 All together, our results suggest a role for IgA in the protection against mycobacterial infection in the respiratory tract, most likely by modulating the locally induced proinflammatory immune responses through macrophage activation. Another possible mechanism is the opsonisation of bacteria and blocking the entrance of the bacilli into the lungs. PAPER II In addition to investigating a possible role of IgA in the 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 (Shimada et al., 1999). Although B-cell deficient mice have previously been used in infection studies with mycobacteria (Vordermeier et al., 1996; Boiso et al., 2000; Turner et al., 2001), mice lacking actively secreted antibodies have never been used. Generation of pIgR-knockout mice presents a unique opportunity to explore the relative contribution of local secretory antibodies versus systemic immunity in the protection against mycobacteria, as has been done previously in virus-infection studies (Asashi et al., 2002). Initially, the immunological status regarding antibodies, as well as the induced immune responses upon i.n. immunization with the same formulation as in paper I, namely PstS-1 in combination with CT, was assessed in the pIgR-/- mice. Interestingly, although no antigenspecific 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 cannot be ruled out, nor can leakage through the epithelial membrane, which has been previously reported (Johansen et al., 1999). Also, their respective contributions may be different in the upper and lower respiratory tract. Our finding indicates that the 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 51 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 regulation of innate, non-specific, responses to pathogens, mainly due to binding of N-glycans of the SC to bacterial and host factors. One such function is the stabilization and protection of dIgA and pIgA, and although proteolytic degradation is less extensive in the respiratory tract than in the gut lumen, the absence of pIgR, and hence the SC, could imply that the IgA antibodies found in the BAL of pIgR-/- mice are more rapidly degraded and therefore not able to perform their protective function. A further function attributed to the SC is its binding to bacterial components, such as Clostridium difficile toxin A (Dallas and Rolfe, 1998) and fimbriae of enterotoxigenic Escherichia coli (de Oliviera et al., 2001), thereby limiting infection and morbidity. Although no interactions between the SC and mycobacteria, or mycobacterial components, have been reported, the possibility cannot be ruled out. Additonally, it was recently demonstrated, in a mouse model, that SIgA was more protective than pIgA against respiratory infection with Shigella flexneri, due to carbohydratedependent adherence of the SIgA to the mucus lining of the epithelial cell layer (Phalipon et al., 2002). SIgA autoantibodies found in saliva from normal subjects were moreover shown to be polyreactive against pathogenic elements, suggesting an innate function of these antibodies (Quan et al., 1997). This property was recently confirmed for gastrointestinal SIgA in an infection study using Salmonella typhimurium (Wijburg et al., 2006). In conclusion, these properties of the SC suggest that dIgA/pIgA antibodies without the SC, which would be found in pIgR-/- mice, are less protective than SIgA antibodies that are produced in wild-type mice. In this study, we also saw that BCG-infected pIgR-/- mice displayed a considerable reduction in TNF-α and IFN-γ production in lung mononuclear cells. Since these cytokines are central for protective immunity against mycobacterial infections (Cooper et al., 1993; Flynn et al., 1995), this impaired mycobacterium-induced proinflammatory response most likely contributed to the higher bacterial load in the lungs of these mice. We demonstrated in paper I that IgA-deficient mice have a defective proinflammatory immune response, probably due to an impaired activation of macrophages or other IgA receptor-bearing cells, and thereby reduced secretion of cytokines and chemokines. Although the pIgR-knockout mice have IgA antibodies, the lack of pIgR and SC might render these IgA antibodies less functionable, even in the sense of their immunomodulary effect. Alveolar macrophages and DCs extending out in the alveoli might therefore not be optimally activated. Interestingly, a recent study by Favre et 52 al. demonstrated that SIgA works as a weak immunopotentiator in the mucosal environment in the gastrointestinal tract (Favre et al., 2005). SIgA transported from lumen to the subepithelial compartment via M cells stimulated mucosal immune responses via upregulated expression of the costimulatory molecules CD80 and CD86 on DCs. We next addressed the role of SIgA in the 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 was substantially reduced, which corroborates the findings in the BCG-infected pIgR-deficient mice. Moreover, expression of iNOS was also reduced. Production of RNIs is an important defense 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 expression of the regulated upon activation normal T-cell sequence (RANTES) 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 lymphocytes, 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 cytokine 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 murine receptor homologue could, in a similar fashion to human IL-8, bind to SC, whereby mucosal secretions from pIgR-/- mice 53 would contain higher levels of the putative IL-8 homologue compared to wild-type mice, resulting in increased chemotaxis of neutrophils in the knockout mice. Our findings suggest that mycobacteria-infected pIgR-/- mice display a delayed immune response, due to reduced expression of RANTES and TNF-α and consequently impaired attraction of macrophages and lymphocytes to the site of infection, as well as a lower proinflammatory response. As a consequence, pIgR-deficient mice allow more extensive 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, displaying comparable viable bacterial counts in the lungs as the wild-type mice. Collectively, our results imply a role for SIgA in the modulation of mycobacteria-induced proinflammatory immune responses and subsequently a role in the protection against mycobacterial infections. PAPER III Resistance against mycobacterial infection and active TB is dependent on the host’s ability to generate a Th1 type of immune response. Activation of TLRs is an important link between innate cellular responses and the subsequent initiation of adaptive immune defenses against microbial pathogens, including mycobacteria. In paper III we investigated the importance of TLR2 and TLR4 in early and late stage of infection using TLR2-/- mice (Takeuchi et al., 2004), and TLR4-/- mice (Hoshino et al., 1999) with a C57BL/6 background. Studies on the interaction between mycobacterial components and TLRs are extensive as well as numerous, and have established that the 19 kDa lipoprotein and lipid derivatives of mycobacterium mainly interact with TLR2 to induce predominantely proinflammatory responses (Krutzik and Modlin, 2004). Although these in vitro studies are essential for dissecting interactions at a molecular level, the physiological role of TLRs can only be evaluated using whole mycobacteria. The engagement of multiple TLRs, as well as other receptors, may affect the outcome of each single interaction. We therefore initially investigated the susceptibility of TLR-deficient mice to BCG infection. Given that TB is 54 primarily acquired through inhalation of airborne droplets containing the bacterium, we infected the experimental animals via the respiratory tract, in order to resemble a natural infection. Analysis of the bacterial burden in the lungs of infected mice demonstrated an impaired control of bacterial growth in the lungs of TLR2-/- mice at the early stage of infection, whereas no difference between wild-type and knockout mice could be detected at the later stage. The role of TLR4 signalling appears less important in the control of mycobacterial growth, both at early and late stages of infection. In addition, TLR6- and TLR9-deficient mice did not differ from wild-type mice in terms of inhibition of bacillary growth. The fact that an increased susceptibility among TLR2-/- mice was only evident during early stages of infection, suggests that the absence of signalling through TLR2 could be compensated for as the infection proceeded. Other danger signals, such as endogenous factors released upon inflammation-induced tissue pathology, might contribute to evoking an immune response through other TLRs or additional receptors. Heat shock proteins (Ohashi et al., 2000; Vabulas et al., 2002), hyaluronan (Termeer et al., 2002) and fibronectin (Smiley et al., 2001) released from damaged cells have been shown to trigger TLR4 signalling. When using the intravenous (i.v.) route of infection no differences between any of the experimental groups could be seen, indicating the importance of the chosen route of infection. Several studies have addressed the role of TLRs in inducing protective immunity against mycobacterial infection by infecting experimental animals i.v. Since TLR expression on resident, as well as attracted immune cells, may differ in various parts of the body, results from i.v. infections might not be relevant for an airborne bacterium. For instance, human alveolar epithelial cells express both TLR2 and TLR4 (Armstrong et al., 2004). However, considering the possibility of dissemination of a mycobacterial infection, i.v. infection can provide supplementary information. In the light of our results, TLR-dependent effector mechanisms appear to be especially important in the respiratory tract. Indirect support for this comes from studies demonstrating TLR-mediated activation of human bronchial epithelial cells and subsequent secretion of beta defensin-2 (Kagnoff and Eckmann, 1997; Hertz et al., 2003). Pulmonary epithelial cells have additionally been shown to produce IFN-γ in response to M. tuberculosis infection (Sharma et al., 2007). Although no receptor interaction was assessed in that study, this finding implies a role for the alveolar epithelial cells in the innate immunity against mycobacterial infections. Data on the TLR distribution on murine pulmonary epithelial cells is, however, lacking. 55 In addition to BCG, we also used virulent M. tuberculosis as the infectious agent. Our results from aerosol infection of mice were in line with the BCG findings, with the exception that TLR4-/- mice, in addition to TLR2-/- mice, displayed an impaired control of mycobacterial growth at the early stage of infection, which was not seen during BCG infection. We believe the reason for this difference could be due to the different mycobacteria used. Considering that the latter experiment involved the pathogen causing TB, these findings indicate that TLR4 signalling, besides TLR2, is of importance for inducing protective immunity against virulent M. tuberculosis infections. We also included TLR6-/-- and TLR9-/- mice in this experiment, however, no differences between these mouse strains and wild-type mice were detected. Alveolar-resident macrophages within the lung are considered to be the main cellular host for mycobacteria, and the major role for these cells is rapid killing of the invading pathogen. This is due to the release of toxic reactive oxygen and nitrogen intermediates, or to the killing by lysosomal enzymes following fusion of the lysosome with the mycobacteria-containing phagosome. In addition, cytokine production by these cells is essential for the initiation of cellular immune responses. Given the important function of macrophages, and the fact that these cells express TLRs (Punturieri et al., 2004), we evaluated the intracellular growth of mycobacteria in bone marrow-derived macrophages from TLR-deficient mice, as well as their cyokine response. Our results suggest that the higher bacterial load in TLR2-/- mice is a result from impaired activation of macrophages, since they display impaired capacity to control intracellular growth of BCG, in addition to a reduced secretion of TNF-α upon infection with mycobacteria. The absence of TLR4 signalling appears less harmful since TLR4-/macrophages were not as affected as macrophages from the TLR2-/- mice. Phagosome maturation has been demonstrated to be regulated by TLR signalling (Blender and Medzhitov, 2004), which could explain the growth advantage of BCG in TLR2-deficient macrophages. TNF-α produced by activated macrophages is important not only for further activation of macrophages, but also for attracting other immune cells and granuloma formation via induction of chemokines (Algood et al., 2004). We moreover detected completely abolished TNF-α production by MyD88-deficient macrophages, indicating that this signalling pathway is the principal mediator in initiating anti-mycobacterial responses in macrophages. Additional experiments revealed a reduced iNOS mRNA expression in TLR2-deficient 56 macrophages upon BCG infection, giving further evidence for the importance of TLR2 signalling for the induction of effector functions against mycobacteria in macrophages. TLR-deficient mice, particularly TLR2-/-mice, furthermore showed defects in the generation of proinflammatory and Th1-associated cytokine responses when using an antigen restimulation assay. This suggests defective T-cell priming in vivo in these mice, which is supported by a previous study (Heldwein et al., 2003). The absence of T cell activation and expansion following infection could be a consequence of impaired antigen presentation and costimulation, involving macrophages as well as DCs. Although an intrinsic defect in the previously reported TLR2-deficient T cell function could not be ascribed the impaired T cell priming in vivo (Heldwein et al., 2003), the possibility still remains. Not only classical innate cells, but also T cells have been shown to express certain TLRs (Kabelitz, 2007). It is therefore possible that TLRs could act as costimulatory molecules to enhance the proliferation and cytokine production of TCR-stimulated T cells. In contrast to in vitro restimulation assays, ex vivo studies investigating the expression of protective molecules, such as IFN-γ, TNF-α, RANTES and iNOS, in the lungs of M. tuberculosis-infected mice demonstrated no differences between TLR-knockout mice and wild-type controls. Since TLR2- and TLR4-deficient mice displayed higher bacterial burdens in the lungs at the early stage of M. tuberculosis infection, it is possible that an impairment of the studied molecules, that initially might have caused the increased susceptbility to infection, had already been compensated for at the time of assessment. Histological evaluation of the lungs revealed a higher granulomatous response in the lungs of infected TLR-knockout mice at the later stage of infection, most likely a consequence of the initially higher bacterial load that triggered a stronger granulomatous infiltration. This response would then sufficient to control the bacterial growth. Collectively, our results demonstrate a protective role for TLR2, and partly TLR4, in the host defense against mycobacterial infection in the respiratory tract. These receptors are particularly important during the early phase of the infection. A deficiency in mainly TLR2, but also to some extent TLR4, results in an impaired anti-mycobacterial macrophage activity, and a defective proinflammatory response. 57 PAPER IV Malaria, an infectious disease causing around 2 million deaths every year, shares a common geographical distribution with TB. The chronic character of the infections caused by the two pahogens, M. tuberculosis and P. falciparum, implies that the interaction of one of these pathogens with the host’s immune system could have an impact on the course of infection caused by the other. Such mutual interactions have previously been demonstrated for other co-infecting pathogens (Curry et al., 1995; Helmby et al., 1998). We therefore established a model to study the effect of blood-stage malaria on human DC response and the susceptibility of DCs to a succeeding M. tuberculosis infection. Given the unique qualities of DCs as inducers and modulators of immune responses upon interactions with microbial substances, we believe that this model is highly relevant for dissecting the effect of a concurrent M. tuberculosis and P. falciparum infection. In endemic areas, malaria infection is likely to precede a M. tuberculosis infection, since Plasmodium infections are aquired during early childhood, whereas the risk of M. tuberculosis infection increases later in life (Chadha et al., 2003; Balasubramanian et al., 2004; Kolappan et al., 2004). Our initial purpose was therefore to assess the effect a malaria blood-stage infection would have on human DCs, in particular with regard to a succeeding M. tuberculosis infection. P. falciparum parasites (strain F32, mycoplasma free) were cultured (Trager and Jensen, 1976) in human O+ RBC, and schizont- and late trophozoite stage PfRBCs were harvested for co-culturing with human DCs. Alternatively, the malarial pigment Hz was used to represent a malaria blood-stage infection. Hz is the aggregated form of heme polymers, generated after parasite degradation of host hemoglobin, and released in the blood stream upon rupture of parasite-infected RBC. To evaluate the effect of Hz, and not contaminating proteins, membranes or GPI molecules, we used synthetic Hz (Egan et al., 2001). Synthetic Hz (βhematin) is structurally (Slater et al., 1991; Sullivan et al., 1996b) and biologically (Sherry et al., 1995) similar to Hz formed naturally by parasites. These two preparations, PfRBC and Hz, were used as malaria parasite-derived material (MDM) in the study. We initially assessed the susceptibility to M. tuberculosis infection of DCs pre-exposed to either PfRBC or Hz. It has previously been reported that PfRBCs suppress DC function (Urban et al., 1999; Urban et al., 2001), and that Hz inhibits the differentiation of human 58 monocytes into DCs, and reduces their responsiveness to maturation signals (Skorokhod et al., 2004). This suggested that DCs exposed to PfRBC or Hz could be more susceptible to M. tuberculosis infection. However, our results revealed the opposite, since MDM-exposed DCs were infected by M. tuberculosis to a lower extent as compared to non-exposed controls. Light microscopic analysis additionally revealed that the two pathogens, or pathogen-derived substances, could co-exist in the same cell. In order to elucidate the mechanism of the reduced susceptibility to M. tuberculosis infection, we analysed the expression of M. tuberculosis uptake-mediating receptors after pre-exposure of DCs to either PfRBC or Hz. The recently identified C-type lectin DC-SIGN is the major receptor for M. tuberculosis on DCs (Geijtenbeek et al., 2003; Tailleux et al., 2003). When evaluating the cell surface expression of DC-SIGN after co-culturing DCs with Hz, we detected no change in expression as compared to untreated cells. In addition, exposing DCs to PfRBC induced a slight increase in the expression of DC-SIGN. The same results were obtained for MR, another M. tuberculosis receptor expressed on DCs, as well as on macrophages (Schlesinger, 1993). These findings imply that the decreased susceptibility of DCs to M. tuberculosis infection cannot be attributed to changed uptake of bacteria due to an altered receptor expression. It is possible that the reduced uptake of M. tuberculosis by DCs after pre-exposure to malaria antigens is simply a consequence of a diminished phagocytic capacity and not a specific event for M. tuberculosis. To address this question we assessed the phagocytosis of latex beads after treatment of DCs with Hz. There was a clear loss in phagocytic ability by DCs exposed to Hz, which indicates a general loss in phagocytic activity as the cause for the reduced uptake of M. tuberculosis by these DCs. Immature DCs are efficient in antigen uptake and act as sentinels in the periphery, where they capture antigens and then migrate to draining lymph nodes to prime T cells. The process of migration is associated with functional and phenotypical maturation such that, upon arrival in the lymph node the DC has acquired the capacity to effectively stimulate naïve T cells. This is related to upregulation of MCH class II- and costimulatory molecules (Steinman, 1991). The functional consequence of maturation is the DCs’ loss in phagocytic ability. After co-culturing DCs with PfRBC or Hz, we detected an upregulation of MHC class II- and the costimulatory molecules CD86 and CD40, which indicated a maturation of the cells, in line with the finding of their reduced phagocytic ability. 59 Efficient induction of T-cell activation is dependent on the interaction between ligands on T cells with costimulatory molecules expressed on APCs. Considering the previously reported inhibition of PfRBC of DC responses to the maturation stimulus LPS (Urban et al., 1999), we expected that the response of DCs to M. tuberculosis infection would be downmodulated after prior treatment with the malaria antigens. In contrast to the findings by Urban et al., the M. tuberculosis-induced maturation of DCs was not affected by exposure to either PfRBC or Hz prior to M. tuberculosis infection. These discrepant findings might be due to the different stimuli used, and their divergent signalling pathways. M. tuberculosis interacts with various receptors, including different TLRs (Geijtenbeek et al., 2003; Quesniaux et al., 2004; Bafica et al., 2005; Floto et al., 2006), while LPS interacts only with TLR4 (Poltorak et al., 1998). In addition to serving as receptor for PfRBCs and apoptotic cells, CD36 was recently implicated in mediating responses to microbial ligands through TLR2, by binding to diacylglycerides and enhancing signalling thorugh the TLR2/TLR6 heterodimer (Hoebe et al., 2005). In addition, TLR2 agonists are able to block TLR4-induced cytokine production upon concomitant engagement of TLR2 and TLR4 in human DCs (Re and Strominger, 2004). Binding to CD36 by PfRBC could thereby recruit TLR2 into a signalling complex and affect the signalling casade induced by LPS through TLR4 (Stevenson and Urban, 2006). Considering the complexity in the interactions between mycobacteria and DCs, and subsequently the divergent signalling pathways engaged, the inhibitory effect of PfRBC may be overcome upon M. tuberculosis infection. Moreover, the induction of maturation by Hz has been shown to be dependent on TLR9 (Coban et al., 2005). Since human myeloid DCs do not express this receptor (Mazzoni and Segal, 2004), this pathway should not be of importance in our model. Besides TCR-mediated recognition of antigens presented by MHC molecules and interaction with costimulatory molecules expressed on the surface of APCs, activation of T cells is highly dependent on production of cytokines by APCs. Analysis of cytokine production by DCs after M. tuberculosis infection demonstrated an enhanced Th1 type of response after prior treatment to malaria-derived antigens. Both PfRBC- and Hz-treated DCs displayed increased secretion of TNF-α and reduced secretion of IL-10, following M. tuberculosis infection. In addition, pre-exposure to PfRBC also triggered IFN-γ production by M. tuberculosis-infected DCs, while Hz had no effect in this context. As stated earlier, a Th1-biased immune response, with initiation of cell-mediated immunity, is crucial for resistance against mycobacterial infections and development of active TB, with IFN-γ and TNF-α being key components. In 60 light of this, our findings allow for the speculation that malaria infection could impose relative resistance to M. tuberculosis infection, although the ensuing T-cell reponse was not studied in our model. Moreover, dissemination of mycobacterial infections can be achieved by migration of infected DCs, implying that the reduced infectivity of PfRBC- or HZ-exposed DCs could result in a decreased spread of a M. tuberculosis infection. However, although the protective effect by TNF-α is well established, an exaggerated production can result in host pathology. Moreover, IL-10 can reduce the extent of inflammatory-induced injury by its immunouppressive effect (Bogdan et al., 1991; Cunha et al., 1992; Flesch et al., 1994; Murray et al., 1997; Marshall et al., 1997; Balcewicz-Sablinska et al., 1998). It is tempting to speculate that an enhanced innate response to M. tuberculosis infection after prior exposure to MDM, which we found in this study, could have a harmful effect on the control of malaria infection. The initial response to malaria infection is characterised by a Th1 type of immunity that eventually shifts to a predominantly Th2 type, in order to initiate antibody-mediated immune mechanisms important for parasite clearance (Taylor-Robinson and Smith, 1991; Langhorne et al., 2002). A prolonged proinflammatory response could potentially interfere with this immunological shift. Taken together, results of our study demonstrate that malaria-blood stage infection can influence the function of human DCs and have an impact on the outcome and response to a concurrent M. tuberculosis infection. 61 CONCLUDING REMARKS TB remains a major health threat worldwide and is the largest cause of death attributable to a single infectious agent. The causative agent of the disease, M. tuberculosis, has a particular tropism for the lungs, making pulmonary TB the most common and dangerous form of the disease. 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. 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. Based on the fact that TB is transmitted via the respiratory tract, where the infection additionally is manifested, and that 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. We conclude from our studies that IgA antibodies play a role in the protection against mycobacterial infection in the respiratory tract, by modulating the locally induced proinflammatory immune responses and/or by blocking the entrance of the bacillus into the lungs. The mechanisms explaining the defective proinflammatory responses in the lungs of IgA- and pIgR-deficient mice might involve impaired signalling through FcαRs, or homologous receptors, 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, as well in reduced killing of the bacteria inside the macrophages. The SC, being extraordinary abundant at mucosal sites, provide additional innate-like properties that could be of importance for protection of mucosal surfaces. Induction of effective immune responses cannot be achived without prior activation of the innate immunity. Production of cytokines and chemokines by macrophages and DCs is crucial for initiating protective immune reponses against M. tuberculosis, making early recognition of the invading pathogen essential for containment of infection. TLRs are key molecules in bridging innate and adaptive immunity. Since M. tuberculosis infects professional phagocytes that express a diverse array of TLRs, interest is focused on the function and importance of 62 these receptors in combating mycobacterial infections. Our studies demonstrate a protective role for TLR2, and partly TLR4, in anti-mycobacterial immune responses. However, important to consider when evaluating involvement of host factors to immunological responses against infectious agents is the natural route of infection. This is obvious from divergent results using different routes of infection. There is increasing evidence that the immune response against one pathogen can influence the outcome of subsequent infections with other pathogens. Similarly, unrelated infections can modulate vaccine-induced protective responses to other pathogens. In the light of alarming reports on the deleterious interaction of co-infecting HIV and M. tuberculosis with the host, and the now generally accepted harmful conseqences of co-infection beween HIV and malaria, studies of concurrent infections with more than one pathogen should be emphasized. Information from such studies will be of importance for vaccine reseach and drug design. Little is known about the outome of concurrent M. tuberculosis/P. falciparum infections, although malaria and TB occur endemically in overlapping geographical areas. We developed an in vitro model for studying the interaction beween the pathogens causing TB and malaria and human DCs, and our results suggest that a malaria blood-stage infection can affect the reponse to and outome of an ensuing M. tuberculosis infection. These findings encourage further studies on the complex immunological events occurring in individuals simultaneously infected with these two extremely important pathogens. For the future 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. The effect of concurrent infectious agents, or potential infections, on vaccination is moreover an essential question to address. 63 ACKNOWLEDGEMENTS These studies were financially supported by the European Commission specific RTD programs QLK2-CT-199900367 and QLK2-CT-2002-00846, Hjärt- och Lungfonden, the Swedish Agency for Research for Research with Developing Countries (Sida/SAREC) and EU LSHP-CT-2004 503578. I wish to express my warm gratitude to everyone who has contributed in any way in making this thesis a reality and given me support during these intense and fascinating years, especially: My supervisors Marita Troye-Blomberg and Andrzej Pawlowski. Marita, my warmest gratitude for always finding solutions, for believing in me and giving me the trust, strength and opportunity to carry on and finishing my studies. Thanks for your concern for me and my situation and for always having my best interest in mind. Andrzej, my deepest gratitude for dedicating your time and interest in me and my studies, for being a warm, caring, patient, and helpful person, and for making me grow as a researcher and as a person, and for making me believe in myself. Thanks for always being willing to answer questions. And for always knowing the answers! My previous supervisor Carmen Fernández for your commitment and dedication for my work and the results we achieved. Gunilla Källenius for welcoming me to SMI and the TB section and letting me continue my PhD studies there, and for being a kind and caring person. My dear friend, former colleague and co-author Ariane Rodríguez for introducing me into the various techniques and strategies at the department. I am immensely grateful for your friendship and all the support you have, and continue to give me. My cherished friend Nancy Vivar for your help, support, patience, laughs, chats at SMI and MTC. Tanks for being my “ventil” and for your endless patience with me and for your willingness to teach me everything you know. You inspire me to be a better person! 64 Margareta “Maggan” Hagstedt, the foundation on which the department is built on. My warmest gratitude to you for all your help and support these years and especially for your help with our “friends”. I have enjoyed working with you and miss our coffee breaks. My collaborators and co-authors in Barcelona, Pere-Joan Cardona, Evelyn Guiardo, Esther Julian, for excellent collaboration and valuable discussions. Former and present seniors at the department of immunology: Klavs Berzins, Manuchehr Abedi Valugerdi, Eva Severinson, Eva Sverremark-Ekström and Alf Grandien. Thank you for sharing your vast knowledge in the field of immunology and for your support and help with my work. Gelana Yadeta, for making the department run smoothly and for all your help with one or the other thing. Shanie Saghafian Hedengren, I treasure our friendship deeply and I am grateful for all your support. When the going gets tough, the tough gets going! Nora Bachmayer and Yvonne Sundström, thanks for your friendship, support and all the fun we have together! What would I do without you? Hedvig Perlmann, for your positive and friendly character and for being an inspiration in science. I would like to recognize late Peter Perlmann whose generous and dedicated personality has been, and continues to be, an inspiration for researchers in the field of immunology. My old roomies Karin Lindroth, Monika Hansson and Masashi Hayano, thanks for your guidance during my first trembling time at the department, and for being caring and good friends, even after leaving the department. Everyone in the TB section at SMI: Maria Andersson, Solomon Ghebremichael, Melles Haile, Beston Hamasur, Sven Hoffner, Emma Huitric, Lech Ignatowicz, Pontus Juréen, Tuija Koivula, Lisbeth Klintz, Jolanta Mazurek, Alexandra Pennhag, Ramona Peterson, Senia Rosales, Juan Carlos 65 Toro and Jim Werngren. Thank you for welcoming me to SMI and making me feel part of the section. I also want to thank everyone at the Department of Bacteriology, SMI, for providing a friendly atmosphere where I have enjoyed working. A special thanks to Kristina Jonas for all your help. Past and present colleagues at the department of immunology – Petra Amoudruz, John Arko-Mensah, Nancy Awah, Halima Balogun, Ahmed Bolad, Mounira Djerbi, Salah Eldin Farouk, Pablo Giusti, Ben Gyan, Ulrika Holmlund, Elisabet Hugosson, Nnaemeka Iriemenam, Lisa Israelsson, Gun Jönsson, Anna-Karin Larsson, Khosro Masjedi, Jacob Minang, Amre Nasre, Alice Nyakeriga, Jubayer Rahman, Camilla Rydström, Valentina Screpanti, Ylva Sjögren, Ankie Söderlund, Khaleda Qazi Rahman, Manijeh Vafa, NinaMaria Vasconcelos, Stefania Varani. A special thanks to Ann Sjölund for your warm personality and “magic hands” with cells. Francesca Chiodi at MTC, and the students in her group for being kind and helpful whenever I came asking for something or desperately wondered where Nancy was. The staff at the animal house, Eva Nygren and Solveig Sundberg, for taking such good care of my mice and always greeting me with a smile. My wonderful and fantastic friends who keep me balanced in life, especially: Anneli and Mats for being the most wonderful people I have ever met! You make my life brighter and happier. Thanks for your generosity and letting me enjoy Minus, Yoda and Snufs whenever I want. Anna H, for being so stable and making me realize that life is more than work and science, and for help and support whenever I needed it. My Friskis companion and dear friend Pernilla for putting up with all my delays during the years. Maybe a gympa-dinner-movie evening soon? Eva, min allra käraste vän, thank you for always listening to me, being there for me, and making my life happy. 66 Marie, you are my role model in life. Your fantastic strength inspires me and keeps me focused on what is import in life. Little Douglas for being the most adorable boy in the world! Finally, and most of all, I would like to express my deepest and warmest gratitude to my beloved family, my mother Margareta, father Sigge, sister Karin and grandmother Vera. Where in this world would I be without you? What would I be? Thank you for your love and support throughout my life, and especially the last five years. Simon, thanks for joining the Tjärnlund family, for your warm and generous personality and for putting up with all the “dos talk”. 67 REFERENCES Adachi, O., Kawai, T., Takeda, K., Matsumoto, M., Tsutsui, H., Sakagami, M., Nakanishi, K., and Akira, S. (1998). Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9:143-150. Adebajo, A. O., Smith, D. J., Hazleman, B. L., and Wreghilt, T. G. (1994). Seroepidemiological associations between tuberculosis, malaria, hepatitis B, and AIDS in West Africa. J. Med. Virol. 42:366-368. Aderem, A., and Ulevitch, R. J. (2000). Toll-like receptors in the induction of the innate immune response. Nature 406:782-787. Aderem, A., and Underhill, D. M. (1999). Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17:593-623. Akira, S. (2003). Mammalian Toll-like receptors. Curr. Opin. Immunol. 15:5-11. Alexandroff, A. B., Jackson, A. M., O’Donnel, M. A., and James, K. (1999). BCG immunotherapy of bladder cancer: 20 years on. Lancet 353:1689-1694. Alfredsen, S., and Saxegaard, F. (1992). An outbreak of tuberculosis in pigs and cattle caused by Mycobacterium africanum. Vet. Rec. 11:51-53. Algood, H. M., Lin, P. L., Yankura D., Jones, A., Chan, J., and Flynn J. L. (2004). TNF influences chemokine expression of macrophages in vitro and that of CD11+ cells in vivo during Mycobacterium tuberculosis infection. J. Immunol. 172:6846-6857. Aliprantis, A. O., Yang, R. B., Mark, M. R., Suggett, S., Devaux, B., Radolf, J. D., Klimpel, G. R., Godowski, P., and Zychlinsky, A. (1999). Cell activation and apoptosis by bacterial lipoproteins through tolllike recptor-2. Science 285:736-739. Allan, C. H., Mendrick, D. L., and Trier, J. S. (1993). Rat intestinal M cells contain acidic endosomallysosomal compartments and express class II major histocompatibility complex determinants. Gastroenterology 104:698-708. American Thoracic Society. (2000). Diagnosis standards and classification of tuberculosis in adults and children. Am. J. Respir. Crit. Care Med. 161:1376-1395. Andersen, P., Askgaard, D., Ljungvist, L., Bennedsen, J., and Heron, I. (1991). Proteins released from Mycobacterium tuberculosis during growth. Infect. Immun. 59:1905-1910. Appelberg, R., Castro, A. G., Gomes, S., Pedrosa, J., and Silva, M. T. (1995). Susceptibility of beige mice to Mycobacterium avium: role of neutrophils. Infect. Immun. 63:3381-3387. Arulanandam, B. P., Raeder, R. H., Nedrud, J. G., Bucher, D. J., Le, J., and Metzger, D. W. (2001). IgA immunodeficiency leads to inadequate Th cell priming and increased susceptibility to influenza virus infection. J. Immunol. 166:226-231. Armstrong, L., Medford, A. R., Uppington, K. M., Robertson, J., Withereden, I. R., Tetley, T .D., and Millar, A. B. (2004). Expression of functional toll-like receptor-2 and -4 on alveolar epithelial cells. Am. J. Respir. Cell. Mol. Biol. 31:241-245. Armstrong, J. A., and Hart, P. D. (1975). Phagosome-lysosome interactions in cultured macrophages infected with virulent tubercle bacilli. Reversal of the usual nonfusion pattern and observations on bacterial survival. J. Exp. Med. 142:1-16. 68 Artavanis-Tsakonas, K., Eleme, K., McQueen, K. L., Cheng, N. W., Parham, P., Davis, D. M., and Riley, E. M. (2003). Activation of a subset of human NK cells upon contact with Plasmodium falciparum-infected erythrocytes. J. Immunol. 171:5396-5405. Asashi, Y., Yoshikawa, T., Watanabe, I., Iwasaki, T., Hasegawa, H., Sato, Y., Shimada, S., Nanno, M., Matsuoka, Y., Ohwaki, M., Iwakura, Y., Suzuki, Y., Aizawa, C., Sata, T., Kurata, T., and Tamura, Y. (2002). Protection against influenza virus infection in polymeric Ig receptor knockout mice immunized intranasally with adjuvant-combined vaccines. J. Immunol. 168:2930-2938. Aston, C., Rom, W. N., Talbot, A. T., and Reibman, J. (1998). Early inhibition of mycobacterial growth by human alveolar macrophages is not due to nitric oxide. Am. J. Respir. Crit. Care. Med. 157:1943-1950. Ayele, W. Y., Neill, S. D., Zinsstag, J., Weiss, M. G., and Pavlik, I. (2004). Bovine tuberculosis: an old disease but a new threat to Africa. Int. J. Tuberc. Lung Dis. 8:924-937. Bafica, A., Scanga, C. A., Feng, C. G., Leifer, C., Cheever, A., and Sher, A. (2005). TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis. J. Exp. Med. 202:1715-1724. Balasubramanian, R., Garg, R., Santha, T., Gopi, P. G., Subramani, R., Chandrasekaran, V., Thomas, A., Rajeswari, R., Anandakrishnan, S., Perumal, M., Niruparani, C., Sudha, G., Jaggarajamma, K., Frieden, T. R., and Narayanan, P. R. (2004). Gender disparities in tuberculosis: report from a rural DOTS programme in south India. Int. J. Tuberc. Lung Dis. 8:323-332. Balcewicz-Sablinska, M. K., Keane, J., Kornfeld, H., and Remold, H. G. (1998). Pathogenic Mycobacterium tuberculosis evades apoptosis of host macrophages by release of TNF-R2, resulting in inactivation of TNFalpha. J. Immunol. 161:2636-2641. Barnes, P. F., Chatterjee, D., Abrams, J. S., Lu, S., Wang, E., Yamamura, M., Brennan, P. J., and Modlin, R. L. (1992). Cytokine production induced by Mycobacterium tuberculosis lipoarabinomannan. Relationship to chemical structure. J. Immunol. 149:541-547. Bazaz-Malik, G. (1973). Increased resistance to malaria after Mycobacterium tuberculosis infection. Indian. J. Med. Res. 61:1014-1024. Beatty, W. L., Rhoades, E. R., Hsu, D. K., Liu, F. T., and Russell, D. G. (2002). Association of a macrophage galactoside-binding protein with Mycobacterium-containing phagosomes. Cell. Microbiol. 4:167-176. Becker, M. D., O’Rourke, L. M., Blackman, W. S., Planck, S. R., and Rosenbaum, J. T. (2000). Reduced leukocyte migration, but normal rolling and arrest, in interleukin-8 receptor homologue knockout mice. Invest. Ophthalmol. Vis. Sci. 41:1812-1817. Behar, S. M., Dascher, C. C., Grusby, D. G., Wang, C. R., and Brenner, M. B. (1999). Susceptibility of mice deficient in CD1D or TAP1 to infection with Mycobacterium tuberculosis. J. Exp. Med. 189:1973-1980. Belkaid, Y., and Rouse, B. T. (2005). Natural regulatory T cells in infectious disease. Nat. Immunol. 6:353-360. Bereczky, S., Montgomery, S. M., Troye-Blomberg, M., Rooth, I., Shaw, M. A., Farnet, A. (2004). Elevated anti-malarial IgE in asymptomatic individuals is associated with reduced risk for subsequent clinical malaria. Int. J. Parasitol. 34:935-942. Bermudez, L. E., and Goodmann, J. (1996). Mycobacterium tuberculosis invades and replicates within type II alveolar cells. Infect. Immun. 64:1400-1406. Blender, J. M., and Medzhitov, R. (2004). Regulation of phagosome maturation by signals from toll-like receptors. Science 304:1014-1018. Bodnar, K. A., Serbina, N. V., and Flynn, J. L. (2001). Fate of Mycobacterium tuberculosis within murine dendritic cells. Infect. Immun. 69:800-809. 69 Boehm, M. K., Woof, J. M., Kerr, M. A., and Perkins, S. J. (1999). The Fab and Fc fragments of IgA1 exhibit a different arrangement from that in IgG: a study by X-ray and neutron solution scattering and homology modelling. J. Mol. Biol. 286:1421-1447. Bogdan, C., Vodovotz, Y., and Nathan, C. (1991). Macrophage deactivation by interleukin 10. J. Exp. Med. 174:1549-1555. Boom, H. W. (1999). γδ T cells and Mycobacterium tuberculosis. Microbes Infect. 1:187-195. Boros, D. L. (1999). T helper cell populations, cytokine dynamics, and pathology of the schistosome egg granuloma. Microbes Infect. 1:511-516. Bosio, C. M., Gardner, D., and Elkins, K. L. (2000). Infection of B cell-deficient mice with CDC 1551, a clinical isolate of Mycobacterium tuberculosis: delay in dissemination and development of lunh pathology. J. Immunol. 164:6417-6425. Boussiotis, V. A., Tsai, E. Y., Yunis, E. J., Thim, S., Delgado, J. C., Dascher, J. J., Berezovskaya, A., Rousset, D., Reynes, J M., and Goldfeld, A. E. (2000). IL-10-producing T cells suppress immune responses in anergic tuberculosis patients. J. Clin. Invest. 105:1317-1325. Brake, D. A., Long, C. A., and Weidanz, W. P. (1988). Adoptive protection against Plasmodium chabaudi adami malaria in athymic nude mice by a cloned T cell line. J. Immunol. 140:1989-1993. Brandt, L., Cunha, J. F., Olsen, A. W. Chilima, B., Hirsch, P., Appelberg, R., and Andersen, P. (2002). Failure of the Mycobacterium bovis BCG vaccine: some species of environmental mycobacteria block multiplication of BCG and induction of protective immunity to tuberculosis. Infect. Immun. 70:672-678. Brandtzaeg, P. (1973). Structure, synthesis and external transfer of mucosal immunoglobulins. Ann. Immunol. 124:417-438. Brandtzaeg, P. (1989). Overview of the mucosal immune system. Curr. Top. Microbiol. Immunol. 146:13-25. Brandtzaeg, P., Farstad, I. N., Jahnsen, F. L., Johansen, F-E., Nilsen, E. M., and Yamanaka, T. (1999). Integration and segregation of the mucosal immune system. 1. What happens in the microcompartments? Immunol. Today 20:141-151. Brandtzaeg, P., Karlsson, G., Hansson, G., Petruson, B., Björkander, J., and Hanson, L. A. (1987). The clinical condition of IgA-deficient patients is related to the proportion of IgD- and IgM-producing cells in their nasal mucosa. Clin. Exp. Immunol. 67:626-636. Brightbill, H. D., Libraty, D. H., Krutzik, S. R., Yang, R. B., Belisle, J. T., Bleharski, J. R., Maitland, M., Norgard, M. W., Plevy, S. E., Smale, S. T., Brennan, P. J., Bloom, B. R., Godowski, P. J., and Modlin, R. L. (1999). Host defense mechanisms triggered by microbial lipoproteins trough toll-like receptors. Science 285:732-736. Brown, A. E., Holzer, T. J., and Andersen, B. R. (1987). Capacity of human neutrophils to kill Mycobacterium tuberculosis. J. Infect. Dis. 156:985-989. Bulut, Y., Faure, E., Thomas, L., Equils, O., and Arditi, M. (2001). Cooperation of Toll-like receptor 2 and 6 for cellular activation by soluble tuberculosis factor and Borrelia burgdorferi outer surface protein A lipoprotein: role of Toll-interacting protein and IL-1 receptor signaling molecules in Toll-like receptor 2 signaling. J. Immunol. 167:987-994. Burks, A. W. Jr., and Steele, R. W. (1986). Selective IgA deficiency. Ann. Allergy 57:3-13. Burns, J. W., Siadat-Pajouh, M., Krishnaney, A. A., and Greenberg, H. B. (1996). Protective effect of rotavirus VP6-specific IgA monoclonal antibodies that lack neutralizing activity. Science 272:104-107. Burrows, P. D., and Cooper, M. D. (1997). IgA deficiency. Adv. Immunol. 65:245-276. 70 Butcher, E. C., and Picker, L. J. (1996). Lymphocyte homing and homeostasis. Science 272:60-66. Carlson, J., Ekre, H. P., Helmby, H., Gysin, J., Greenwood, B. M, and Wahlgren, M. (1992). Disruption of Plasmodium falciparum erythrocyte rosettes by standard heparin and heparin devoid of anticoagulant activity. Am. J. Trop. Med. Hyg. 46:595-560. Caruso, A. M., Serbina, N., Klein, E., Triebold, K., Bloom, B. R., and Flynn, J. L. (1999). Mice deficient in CD4 T cells have only transiently diminished levels of IFN-γ, yet succumb to tuberculosis. J. Immunol. 162:5407-5416. Cerretti, D. P., Nelson, N., Kozlosky, C. J., Morrissey, P. J., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Dosik, J. K., and Mock, B. A. (1993). The murine homologue of the human interleukin-8 receptor type B maps near the Ity-Lsh-Bcg disease resistance locus. Genomics 18:410-413. Chadha, V. K., Vaidyanathan, P. S., Jagannatha, P. S., Unnikrishnan, K. P., Savanur, S. J., and Mini, P. A. (2003). Annual risk of tuberculous infection in the western zone of India. Int. J. Tuberc. Lung Dis. 7:536-542. Chambers, M. A., Gavier-Widen, D., and Hewinson, R. G. (2004). Antibody bound to the surface antigen MPB83 of Mycobacterium bovis enhances survival against high dose and low dose challenge. FEMS Immunol. Med. Microbiol. 41:93-100. Champbell, D. J., Debes, G. F., Johnston, B., Wilson, E., and Butcher, E. C. (2003). Targeting T cell responses by selective chemokine receptor expression. Semin. Immunol. 15:277-286. Chan, E. D., Chan, J., and Schluger, N. W. (2001). What is the role of nitric oxide in murine and human host defense against tuberculosis? Am. J. Respir. Cell Mol. Biol. 25:606-612. Chen, L., Wang, J., Zganiacz, A., and Xing, Z. (2004). Single intranasal mucosal Mycobacterium bovis BCG vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis. Infect. Immun. 72:238-246. Chen, X., Zhou, B., Li, M., Deng, Q., Wu, X., Le, X., Wu, C., Larmonier, N., Zhang, W., Zhang, H., Wang, H., and Katsanis, E. (2007). CD4(+)CD25(+)FoxP3(+) regulatory T cells suppress Mycobacterium tuberculosis immunity in patients with active disease. Clin. Immunol. 123:50-59. Chensue, S. W., Warmington, K. S., Allenspach, E. J., Lu, B., Gerard, C., Kunkel, S. L., and Lukacs, N. W. (1999). Differential expression and cross-regulatory function of RANTES during mycobacterial (type 1) and schistosomal (type 2) antigen-elicited granulomatous inflammation. J. Immunol. 163:165-173. Cho, S., Mehra, V., Thoma-Uszynski, S., Stenger, S., Serbina, N., Mazzaccaro, R. J., Flynn, J. L., Barnes, P. F., Southwood, S., Celis, E., Bloom, B. R., Modlin, R. L., and Sette, A. (2000). Antimicrobial activity of MHC class I-restricted CD8+ T cells in human tuberculosis. Proc. Natl. Acad. Sci. USA 97:12210-12215. Clark, I. A., Allison, A. C., and Cox, F. E. (1976). Protection of mice against Babesia and Plasmodium with BCG. Nature 259:309-311. Clemens, D. L., and Horwitz, M A. (1995). Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J. Exp. Med. 181:257-270. Coban, C., Ishii, K. J., Kawai, T., Hemmi, H., Sato, S., Uematsu, S., Yamamoto, M., Takeuchi, O., Itagaki, S., Kumar, N., Horii, T., and Akira, S. (2005). Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin. J. Exp. Med. 201:19-25. Cohen, R. A., Muzaffar, S., Schwartz, D., Bashir, S., Luke, S., McGartland, L. P., and Kaul, K. (1998). Diagnosis of pulmonary tuberculosis using PCR assays on sputum collected within 24 hours of hospital admission. Am. J. Respir. Crit. Care Med. 157:156-161. Coker, R. J. (2004). Multidrug-resistant tuberculosis: public health challenges. Trop. Med. Int. Health 9:25-40. 71 Cooper, A. M., Dalton, D. K., Stewart, T. A., Griffin, J. P., Russell, D. G., and Orme, I. M. (1993). Disseminated tuberculosis in interferon gamma gene-disrupted mice. J. Exp. Med. 178:2243-2247. Cunha, F. Q., Moncada, S., and Liew, F. Y. (1992). Interleukin-10 (IL-10) inhibits the induction of nitric oxide synthase by interferon-gamma in murine macrophages. Biochem. Biophys. Res. Commun. 182:1155-1159. Cunningham-Rundles, C. (2001). Physiology of IgA and IgA deficiency. J. Clin. Immunol. 21:303-309. Curry, A. J., Else, K. J., Jones, F., Bancroft, A., Grencis, R. K., and Dunne, D. W. (1995). Evidence that cytokine-mediated immune interactions induced by Schistosoma mansoni alter disease outcome in mice concurrently infected with Trichuris muris. J. Exp. Med. 181:769-774. Dairaghi, D. J., Soo, K. S., Oldham, E. R., Premack, B. A., Kitamura, T., Bacon, K. B., and Schall, T. J. (1998). RANTES-induced T cell activation correlates with CD3 expression. J. Immunol. 160:426-433. Dallas, S. D., and Rolfe, R. D. (1998). Binding of Clostridium difficile toxin A to human milk secretory component. J. Med. Microbiol. 47:879-888. Dannenberg, A. M. Jr. (1994). Roles of cytotoxic delayed-type hypersensitivity and macrophage-activating cell-mediated immunity in the pathogenesis of tuberculosis. Immunobiology 191:461-473. Davis, S. S. (2001). Nasal vaccines. Adv. Drug Deliv. Rev. 51:21-42. Davis, M. M., and Björkman, P. J. (1988). T-cell antigen receptor gene and T-cell recognition. Nature 334:395-402. de Chastellier, C., and Thilo, L. (1997). Phagosome maturation and fusion with lysosomes in relation to surface property and size of the phagocytic particle. Eur. J. Cell. Biol. 74:49-62. Denis, M. (1991). Human neutrophils, activated with cytokines or not, do not kill virulent Mycobacterium tuberculosis. J. Infect. Dis. 163:919-920. de Oliviera, I. R., de Araujo, A. N., Bao, S. N., and Giugliano, L. G. (2001). Binding of lactoferrin and free secretory component to enterotoxigenic Escherichia coli. FEMS Microbiol. Lett. 203:29-33. Dieli, F., Ivanyi, J., Marsh, P., Williams, A., Naylor, I., Sireci, G., Caccamo, N., Di Sano, C., and Salerno, A. (2003). Characterization of lung γδ T cells following intranasal infection with Mycobacterium bovis bacillus Calmette-Guerin. J. Immunol. 170:463-469. Dieli, F., Troye-Blomberg, M., Farouk, S. E., Sirecil, G., and Salerno, A. (2001). Biology of gammadelta T cells in tuberculosis and malaria. Curr. Mol. Med. 1:437-46. Dolo, A., Modiano, D., Maiga, B., Daou, M., Dolo, G., Guindo, H., Ba, M., Maiga, H., Coulibaly, D., Perlmann, H., Troye-Blomberg, M., Toure, Y. T., Coluzzi, M., and Doumbo, O. (2005). Difference in susceptibility to malaria between two sympatric ethnic groups in Mali. Am. J. Trop. Med. Hyg. 72:243-248. Downing, J. F., Pasula, R., Wright, J. R., Twigg III, H. L., and Martin II, W. J. (1995). Surfactant protein A promotes attachment of Mycobacterium tuberculosis to alveolar macrophages during infection with human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 92:4848-4852. Drennan, M. B., Nicolle, D., Quesniaux, V. J., Jacobs, M., Allie, N., Mpagi, J., Fremond, C., Wagner, H., Kirschning, C., and Ryffel, B. (2004). Toll-like receptor 2-deficient mice succumb to Mycobacterium tuberculosis infection. Am. J. Pathol. 164:49-57. D’Souza, C. D., Cooper, A. M., Frank, A. A., Mazzaccaro, R. J., Bloom, B. R., and Orme, I. M. (1997). An anti-inflammatory role for gamma delta T lymphocytes in aquired immunity to Mycobacterium tuberculosis. J. Immunol. 158:1217-1221. Edelson, B. T., and Unanue, E. R. (2001). Intracellular antibody neutralizes Listeria growth. Immunity 14:503512. 72 Egan, T. J., Mavuso, W. W., and Ncokazi, K. K. (2001). The mechanism of beta-hematin formation in acetate solution. Parallels between hemozoin formation and biomineralization processes. Biochemistry 40:204-213. Ehlers, S. (2003). Role of tumour necrosis factor (TNF) in host defence against tuberculosis: implications for immunotherapies targeting TNF. Ann. Rheum. Dis. 62:Suppl 2:ii37-42. Elias, D., Akuffo, H., Pawlowski, A., Haile, M., Schon, T., and Britton, S. (2005a). Schistosoma mansoni infection reduces the protective efficacy of BCG vaccination against virulent Mycobacterium tuberculosis. Vaccine 23:1326-1334. Elias, D., Akuffo, H., Thors, C., Pawlowski, A., and Britton, S. (2005b). Low dose chronic Schistosoma mansoni infection increases susceptibility to Mycobacterium bovis BCG infection in mice. Clin. Exp. Immunol. 139:398-404. Engering, A, Geijtenbeek, T. B. H., van Vliet, S. J., Wijers, M., van Liempt, E., Demaurex, N., Lanzavecchia, A., Fransen, J., Figdor, C. G., Piguet, V., and van Kooyk, Y. (2002). The dendritic cellspecific adhesion receptor DC-SIGN internalizes antigen for presentation to T cells. J. Immunol. 168:2118-2126. Falero-Diaz, G., Challacombe, S., Banerjee, D., Douce, G., Boyd, A., and Ivanyi, J. (2000). Intranasal vaccination of mice against infection with Mycobacterium tuberculosis. Vaccine 18:3223-3229. Fanning, A. (1999). Tuberculosis: 6. Extrapulmonary disease. CMAJ 160:1597-1603. Favre, L., Spertini, F., and Corthésy, B. (2005). Secretory IgA possesses intrinsic modulatory properties stimulating mucosal and systemic immune responses. J. Immunol. 175:2793-2800. Feng, C. G., Scanga, C. A., Collazo-Custodio, C. M., Cheever, A. W., Hieny, S., Caspar, P., and Sher, A. (2003). Mice lacking myeloid differentiation factor 88 display profound defects in host resistance and immune responses to Mycobacterium avium infection not exhibited by Toll-like receptor 2 (TLR2)- and TLR4-deficient animals. J. Immunol. 171:4758-4764. Feng, N., Lawton, J. A., Gilbert, J., Kuklin, N., Vo, P., Prasad, B. V., and Greenberg, H. B. (2002). Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6-specific IgA mAb. J. Clin. Invest. 109:1203-1213. Ferrari, G., Langen, H., Naito, M., and Pieters, J. (1999). A coat protein on phagosomes involved in the intracellular survival of mycobacteria. Cell 97:435-447. Fine, P. E. M. (1995a). Bacille Calmette-Guérin vaccines: a rough guide. Clin. Infect. Dis. 20:11-14. Fine, P. E. M. (1995b). Variation in protection by BCG – implication of and for heterologous immunity. Lancet 346:1339-1345. Flesch, I. E., Hess, J. H., Oswald, I. P., and Kaufmann, S. H. (1994). Growth inhibition of Mycobacterium bovis by IFN-gamma stimulated macrophages: regulation by endogenous tumor necrosis factor-alpha and by IL10. Int. Immunol. 6:693-700. Floto, R. A., MacAry, P. A., Boname, J. M., Mien, T. S., Kampmann, B., Hair, J. R., Huey, O. S., Houben, E. N., Pieters, J., Day, C., Oehlmann, W., Singh, M., Smith, K. G., and Lehner, P. J. (2006). Dendritic cell stimulation by mycobacterial Hsp70 is mediated through CCR5. Science 314:454-458. Flynn, J. L., Chan, J., Triebold, K. J., Dalton, D. K., Stewart, T., and Bloom, B. R. (1993). An essential role for Interferon-γ in resistance to Mycobacterium tuberculosis infection. J. Exp. Med. 178:2249-2254. Flynn, J. L., Goldstein, M. M., Triebold, K. J., Koller, B., and Bloom, B. R. (1992). Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection. Proc. Natl. Acad. Sci. USA 89:12013-12017. 73 Flynn, J. L., Goldstein, M. M., Chan, J., Triebold, K. J., Pfeffer, K., Lowenstain, C. J., Schreiber, R., Mak, T. W., and Bloom, B. R. (1995). Tumor necrosis factor-alpha is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2:561-572. Flynn, J. L., Scanga, C. A., Tanaka, K. E., and Chan, J. (1998). Effects of aminoguanidine on latent murine tuberculosis. J. Immunol. 160:1796-1803. Fontenot, J. D., and Rudensky, A. Y. (2005). A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3. Nat. Immunol. 6:331-337. Fratazzi, C., Arbeit, R. D., Carini, C., Balcewicz-Sablinska, M. K., Keane, J., Kornfeld, H., and Remold, H G. (1999). Macrophage apoptosis in mycobacterial infections. J. Leukoc. Biol. 66:763-764. Fremond, C. M., Yeremeev, V., Nicolle, D. M., Jacobs, M., Quesniaux, V. F., and Ryffel, B. (2004). Fatal Mycobacterium tuberculosis infection despite adaptive immune response in the absence of MyD88. J. Clin. Invest. 114:1790-1799. Fujioka, H., Emancipator, S. N., Aikawa, M., Huang, D. S., Blatnik, F., Karban, T., DeFife, K., and Mazanec, M. B. (1998). Immunocytochemical colocalization of specific immunoglobulin A with sendai virus protein in infected polarized epithelium. J. Exp. Med. 188:1223-1229. Fulton, S. A., Reba, S. M., Martin, T. D., and Boom, W. H. (2002). Neutrophil-mediated mycobacteriocidal immunity in the lung during Mycobacterium bovis BCG infection in C57BL/6 mice. Infect. Immun. 70:53225317. Furtado, P. B., Whitty, P. W., Robertson, A., Eaton, J. T., Almogren, A., Kerr, M. A., Woof, J. M., and Perkins, S. J. (2004). Solution structure determination of monomeric human IgA2 by X-ray and neutron scattering, analytical ultracentrifugation and constrained modelling: a comparison with monomeric human IgA1. J. Mol. Biol. 338:921-941. Gallucci, S., Lolkema, M., and Matzinger, P. (1999). Natural adjuvants: endogenous activators of dendritic cells. Nat. Med. 5:1249-1255. Garcia-Perez, B. E., Mondragen-Flores, R., and Luna-Herrera, J. (2003). Internalization of Mycobacterium tuberculosis by macropinocytosis in non-phagocytic cells. Microb. Pathog. 35:49-55. Geijtenbeek, T. B. H., Torensma, R., van Vliet, S. J., van Duijnhoven, G. C. F., Adema, G. J., van Kooyk, Y., and Figdor, C. G. (2000). Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell 100:575-585. Geijtenbeek, T. B. H., van Vliet, S. J., Koppel, E. A., Sanchez-Hernandez, M., Vandenbroucke-Grauls, C. M., Appelmelk, B., and van Kooyk Y. (2003). Mycobacteria target DC-SIGN to suppress dendritic cell function. J. Exp. Med. 197:7-17. Geissmann, F., Launay, P., Pasquier, B., Lepelletier, Y., Leborgne, M., Lehuen, A., Brousse, N., and Monteiro, R. C. (2001). A subset of human dendritic cells expresses IgA Fc receptor (CD89), which mediates internalization and activation upon cross-linking by IgA complexes. J. Immunol. 166:346-352. Giacomini, E., Iona, E., Ferroni, L., Miettinen, M., Fattorini, L., Orefici, G., Julkunen, I., and Coccia, E. M. (2001). Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response. J. Immunol. 166:7033-7041. Glatman-Freedman, A. (2003). Advances in antibody-mediated immunity against Mycobacterium tuberculosis: implications for a novel vaccine strategy. FEMS Immunol. Med. Microbiol. 39:9-16. Gong, J. H., Zhang, M., Modlin, R. L., Linsley, P. S., Iyer, D., Lin, Y., and Barnes, P. F. (1996). Interleukin10 downregulates Mycobacterium tuberculosis-induced Th1 responses and CTLA-4 expression. Infect. Immun. 64:913-918. 74 Gonzalez-Juarrero, M., Turner, O. C., Turner, J., Marietta, P., Brooks, J. V., and Orme, I. M. (2001). Temporal and spatial arrangement of lymphocytes within lung granulomas induced by aerosol infection with Mycobacterium tuberculosis. Infect. Immun. 69:1722-1728. Good, M. F., and Doolan, D. L. (1999). Immune effector mechanisms in malaria. Curr. Opin. Immunol. 11:412-419. Guillot, L., Balloy, V., McCormack, F. X., Golenbock, D. T., Chignard, M., and Si-Tahar, M. (2002). Cutting edge: the immunostimulatory activity of the lung surfactant protein-A involves Toll-like receptor 4. J. Immunol. 168:5989-5992. Hajjar, A. M., O’Mahony, D. S., Ozinsky, A., Underhill, D. M., Aderem, A., Klebanoff, S. J., and Wilson, C. B. (2001). Cutting edge: functional interactions between toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin. J. Immunol. 166:15-19. Hamasur, B., Haile, M., Pawlowski, A., Schröder, U., Källenius, G., Svenson, S. B. (2004). A mycobacterial lipoarabinomannan specific monoclonal antibody and its F(ab') fragment prolong survival of mice infected with Mycobacterium tuberculosis. Clin. Exp. Immunol. 138:30-38. Hammarström, L., Vorechovsky, I., and Webster, D. (2000). Selective IgA deficiency (SIgAD) and common variable immunodeficiency (CVID). Clin. Exp. Immunol. 120:225-231. Hang, L., Frendeus, B., Godaly, G., and Svanborg, C. (2000). Interleukin-8 receptor knockout mice have subepithelial neutrophil entrapment and renal scarring following acute pyelonephritis. J. Infect. Dis. 182:17381748. Hanker, J. S., and Giammara, B. L. (1983). Neutrophil pseudoplatelets: their discrimination by myeloperoxidase demonstration. Science 220:415-417. Harriman, G. R., Bogue, M., Rogers, P., Finegold, M., Pacheco, S., Bradley, A., Zhang, Y., and Mbawuike, I. N. (1999). Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in expression of other Ig isotypes. J. Immunol. 162:2521-2529. Hatch, T. F. (1942). Behavior of microscopic particles in the air and the respiratory system. Aerobiology, Publ. No. 17. Amer. Assn. Adv. Sci. 102-105. Havlir, D. V., Ellner, J. J., Chervenak, K. A., and Boom, W. H. (1991). Selective expansion of human gamma delta T cells by monocytes infected with live Mycobacterium tuberculosis. J. Clin. Invest. 87:729-733. Heldwein, K. A., Liang, M. D., Andresen, T. K., Thomas, K. E., Marty, A. M., Cuesta, N., Vogel, S. N., and Fenton, M. J. (2003). TLR2 and TLR4 serve disinct roles in the host immune response against Mycobacterium bovis BCG. J. Leukoc. Biol. 74:277-286. Hellwig, S. M., van Spriel, A. B., Schellekens, J. F., Mooi, F. R., and van de Winkel, J. G. (2001). Immunoglobulin A-mediated potection against Bordetella pertussis infection. Infect. Immun. 69:4846-4850. Helmby, H., Kullberg, M., and Troye-Blomberg, M. (1998). Altered immune responses in mice with concomitant Schistosoma mansoni and Plasmodium chabaudi infections. Infect. Immun. 66:5167-5174. Hendersen, R. A., Watkins, S. C., and Flynn, J. L. (1997). Activation of human dendritic cells following infection with Mycobacterium tuberculosis. J. Immunol. 159:635-643. Hertz, C. J., Kiertscher, S. M., Godowski, P. J., Bouis, D. A., Norgard, M. V., Roth, M. D., and Modlin RL. (2001). Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2. J. Immunol. 166:2444-2450. Hertz, C. J., Wu, Q., Porter, E. M., Zhang, Y. J., Weismuller, K. H., Godowski, P. J., Ganz, T., Randell, S. H., and Modlin, R. L. (2003). Activation of Toll-like receptor 2 on human tracheobronchial epithelial cells induces the antimicrobial peptide human beta defensin-2. J. Immunol. 171:6820-6826. 75 Hess, J., and Kaufmann, S. H. (1999). Live antigen carriers as tools for improved antituberculosis vaccines. FEMS Immunol. Med. Microbiol. 23:165-173. Hickman, S. P., Chan, J., and Salgame, P. (2002). Mycobacterium tuberculosis induces differential cytokine production from dendritic cells and macrophages with divergent effects on naïve T cell polarization. J. Immunol. 168:4636-4642. Hirsch, C. S., Ellner, J. J., Blinkhorn, R., and Toossi, Z. (1997). In vitro restoration of T cell responses in tuberculosis and augmentation of monocyte effector function against Mycobacterium tuberculosis by natural inhibitors of transforming growth factor beta. Proc. Natl. Acad. Sci. USA. 94:3926-3931. Hirsch, C. S., Ellner, J. J., Russell, D. G., and Rich, E. A. (1994). Complement receptor-mediated uptake and tumor necrosis factor-alpha-mediated growth inhibition of Mycobacterium tuberculosis by human alveolar macrophages. J. Immunol. 152:743-753. Hirsch, C. S., Hussein, R., Toossi, Z., Dawood, G., Shahid, F., and Ellner, J. J. (1996). Cross-modulation by transforming growth factor beta in human tuberculosis: suppression of antigen-driven blastogenesis and interferon gamma production. Proc. Natl. Acad. Sci. USA. 93:3193-3198. Hirsch, C. S., Toossi, Z., Othieno, C., Johnson, J. L., Schwander, S. K., Robertson, S., Wallis, R. S., Edmonds, K., Okwere, A., Mugerwa, R., Peters, P., and Ellner, J. J. (1999). Depressed T-cell interferongamma responses in pulmonary tuberculosis: analysis of underlying mechanisms and modulation with therapy. J. Infect. Dis. 180:2069-2073. Hoebe, K., Georgel, P., Rutschmann, S., Du, X., Mudd, S., Crozat, K., Sovath, S., Shamel, L., Hartung, T., Zahringer, U., and Beutler, B. (2005). CD36 is a sensor of diacylglycerides. Nature 433:523-527. Hoffmann, J. A., Kafatos, F. C., Janeway, C. A., and Ezekowitz, R. A. (1999). Phylogenetic perspectives in innate immunity. Science 284:1313-1318. Holmgren, J., Adamsson, J., Anjuere, F., Clemens, J., Czerkinsky, C., Eriksson, K., Flach, C. F., GeorgeChandy, A., Harandi, A. M., Lebens, M., Lehner, T., Lindblad, M., Nygren, E,. Raghavan, S., Sanchez, J., Stadford, M., Sun, J. B., Svennerholm, A. M., and Tengvall, S. (2005). Mucosal adjuvants and anti-infection and anti-immunopathology vaccines based on cholera toxin, cholera toxin B subunit and CpG DNA. Immunol. Lett. 97:181-188. Honorio-Franca, A. C., Launay, P., Carneiro-Sampaio, M. M., and Monteiro, R. C. (2001). Colostral neutrophils express Fc alpha receptors (CD89) lacking gamma chain association and mediate noninflammatory properties of secretory IgA. J. Leukoc. Biol. 69:289-296. Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K., and Akira, S. (1999). Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccaride: Evidence for TLR4 as the Lps gene product. J. Immunol. 162:3749-3752. Huang, Y. T., Wright, A., Gao, X., Kulick, L., Yan, H., and Lamm, M. E. (2005). Intraepithelial cell neutralization of HIV-1 replication by IgA. J. Immunol. 174:4828-4835. Huygen, K. (1998). DNA vaccines: application to tuberculosis. Int. J. Tuberc. Lung. Dis. 2:971-978. Huygen, K., Van Vooren, J. P., Turneer, M., Bosmans, R., Dierchx, P., and De Bruyn, J. (1988). Specific lymphoproliferation, gamma interferon production, and serum immunoglobulin G directed against a purified 32 kDa mycobacterial protein antigen (P32) in patients with active tuberculosis. Scand. J. Immunol. 27:187-194. Izzo, A. A., and North, R. J. (1992). Evidence for an α/β T cell-independent mechanism of resistance to mycobacteria. Bacillus-Calmette-Guerin causes progressive infection in severe combined immunodeficient mice, but not in nude mice or in mice depleted o CD4+ and CD8+ T cells. J. Exp. Med. 176:581-586. Johansen, F. E., Pekna, M., Norderhaug, I. N., Haneberg, B., Hietala, M. A., Krajci, P., Betsholtz, C., and Brandtzaeg, P. (1999). Absence of epithelial immunoglobulin A transport, with increased mucosal leakiness, in polymeric immunoglobulin receptor/secretory component-deficient mice. J. Exp. Med. 190:915-922. 76 Johansen, F. E., Braathen, R., and Brandtzaeg, P. (2000). Role of J chain in secretory immunoglobulin formation. Scand. J. Immunol. 52:240-248. Johnson, C. M., Cooper, A. M., Frank, A. A., Bonorino, C. B., Wysoki, L. J., and Orme, I. M. (1997). Mycobacterium tuberculosis aerogenic rechallenge infections in B cell-deficient mice. Tuber. Lung Dis. 78:257261. Jones, G. S., Amiarult, H. J., and Andersen, B. R. (1990). Killing of Mycobacterium tuberculosis by neutrophils: a nonoxidative process. J. Infect. Dis. 162:700-704. Kabelitz, D. (2007). Expression and function of Toll-like receptors in T lymphocytes. Curr. Opin. Immunol. 19:39-45. Kabelitz, D., Bender, A., Prospero, T., Wesselborg, S., Janssen, O., and Pechhold, K. (1991). The primary response of human gamma/delta T cells to Mycobacterium tuberculosis is restricted to V gamma 9-bearing cells. J. Exp. Med. 172:1331-1338. Kaetzel, C. S., Robinson, J. K., Chintalacharuvu, K. R., Vaerman, J-P., and Lamm, M. E. (1991). The polymeric immunoglobulin receptor (secretory component) mediates transport of immune complexes across epithelial cells: a local defense function of IgA. Proc. Natl. Acad. Sci. USA 88:8796-8800. Kagnoff, M. F., and Eckmann, L. (1997). Epithelial cells as sensors for microbial infection. J. Clin. Invest. 100:6-10. Kamala, T., Paramasivan, C. N., Herbert, D., Venkatesan, P., and Prabhakar, R. (1996). Immune response and modulation of immune response induced in the guinea-pigs by Mycobacterium avium complex (MAC) & M. fortuitum complex isolates from different sources in the south Indian BCG trial area. Ind. J. Med. Res. 103:201211. Kamath, A. B., Alt, J., Debbabi, H., and Behar, S. M. (2003). Toll-like receptor 4-defective C3H/HeJ mice are not more susceptible than other C3H substrains to infection with Mycobacterium tuberculosis. Infect. Immun. 71:4112-4118. Kasama, T., Strieter, R. M., Standiford, T. J, Burdick, M. D., and Kunkel, S. L. (1993). Expression and regulation of human neutrophil-derived macrophage inflammatory protein 1 alpha. J. Exp. Med. 178:63-72. Kaufman, S. H. E. (1996). Gamma/delta and other unconventional T lymphocytes: what do they see and what do they do? Proc. Natl. Acad. Sci. USA 93:2272-2279. Kaufmann, S. H. E. (2001). How can immunology contribute to the control of tuberculosis? Nat. Rev. Immunol. 1:20-30. Kaufmann, S. H. E. (2003). Immune response to tuberculosis: experimental animal models. Tuberculosis (Edinb.) 83:107-111. Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S. (1999). Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11:115-122. Kerr, M. A. (1990). The structure and function of human IgA. Biochem. J. 271:285-296. Kilian, M., Reinholdt, J., Lomholt, H., Poulsen, K., and Frandsen, E V. (1996). Biological significance of IgA1 proteases in bacterial colonization and pathogenesis: critical evaluation of experimental evidence. APMIS 104:321-338. Kindler, V., Sappino, A P., Grau, G. E., Piguet, P. F., and Vassalli, P. (1989). The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection. Cell 56:731-740. Kisich, K. O., Higgins, M., Diamond, G, and Heifets, L. (2002). Tumor necrosis factor alpha stimulates killing of Mycobacterium tuberculosis by human neutrophils. Infect. Immun. 70:4591-4599. 77 Kiyono, H., and Fukuyama, S. (2004). NALT-versus Peyer’s patch-mediated mucosal immunity. Nat. Rev. Immunol. 4:699-710. Kolappan, C., Gopi, P. G., Subramani, R., Chadha, V. K., Kumar, P., Prasad, V. V., Appegowda, B. N., Rao, R. S., Sashidharan, R., Ganesan, N., Santha, T., and Narayanan, P. R. (2004). Estimation of annual risk of tuberculosis infection (ARTI) among children aged 1-9 years in the south zone of India. Int. J. Tuberc. Lung Dis. 8:418-423. Kremer, K., van Soolingen, D., van Embden, J., Hughes, S., Inwald, J., and Hewinson, G. (1998). Mycobacterium microti: more widespread than previously thought. J. Clin. Microbiol. 36:2793-2794. Kristensen, I., Aaby, P., and Jensen, H. (2000). Routine vaccinations and child survival: follow up study in Guinea-Bissau, West Africa. B. M. J. 321:1435-1438. Krutzik, S. R., and Modlin, R. L. (2004). The role of Toll-like receptor in combating mycobacteria. Semin. Immunol. 16:35-41. Kubagawa, H., Burrows, P., and Cooper, M. D. (1997). A novel pair of immunoglobulin-like receptors expressed by B cells and myeloid cells. Proc. Natl. Acad. Sci. USA 94:5261-5266. Kumar, S., Good, M. F., Dontfraid, F., Vinetz, J. M., and Miller, L. H. (1989). Interdependence of CD4+ T cells and malarial spleen in immunity to Plasmodium vinckei vinckei. Relevance to vaccine development. J. Immunol. 143:2017-2023. Kunkel, E. J., and Butcher, E. C. (2003). Plasma-cell homing. Nat. Rev. Immunol. 3:822-829. Kvale, D., Lovhaug, D., Sollid, L. M., and Brandtzaeg, P. (1988). Tumor necrosis factor-α up-regulates expression of secretory component, the epithelial receptor for polymeric Ig. J. Immunol. 140:3086-3089. Lamm, M. E. (1997). Interaction of antigens and antibodies at mucosal surfaces. Annu. Rev. Microbiol. 51:311340. Langhorne, J., Quin, S. J., and Sanni, L. A. (2002). Mouse models of blood-stage malaria infections: immune responses and cytokines involved in protection and pathology. Chem. Immunol. 80:204-228. Lee, J., Cacalano, G., Camerato, T., Toy, K., Moore, M. W., and Wood, W. I. (1995). Chemokine binding and activities mediated by the mouse IL-8 receptor. J Immunol. 155:2158-2164. Leisewitz, A. L., Rockett, K. A., Gumede, B., Jones, M., Urban, B., and Kwiatkowski, D. P. (2004). Response of the splenic dendritic cell population to malaria infection. Infect. Immun. 72:4233-4239. Li, M., Carpio, D. F., Zheng, Y., Bruzzo, P., Singh, V., Ouaaz, F., Medshitov, R. M., and Beg, A. A. (2001) An essential role of the NF-kappa B/Toll-like receptor pathway in induction of inflammatory and tissue-repair gene expression by necrotic cells. J. Immunol. 166:7128-7135. Lin, Y., Zhang, M., and Barnes, P. F. (1998). Chemokine production by a human alveolar epithelial cell line in response to Mycobacterium tuberculosis. Infect. Immun. 66:1121-1126. Liu, F. T., Frigeri, L. G., Gritzmacher, C. A., Hsu, D. K., Robertson, M. W., and Zuberi, R. I. (1993). Expression and function of an IgE-binding animal lectin (epsilon BP) in mast cells. Immunopharmacology 26:187-195. Loman, S., Radl, J., Jansen, H. M., Out, T. A., and Lutter, R. (1997). Vectorial transcytosis of dimeric IgA by the Calu-3 human lung epithelial cell line: upregulation by IFN-γ. Am. J. Physiol. Lung Cell. Mol. Physiol. 272:L951-L958. MacMicking, J., Xie, Q-W., and Nathan, C. (1997). Nitric oxide and macrophage function. Annu. Rev. Immunol. 15:323-350. 78 Maruoka, T., Nagata, T., and Kasahara, M. (2004). Identification of the rat IgA Fc receptor encoded in the leukocyte receptor complex. Immunogenetics 55:712-716. Marshall, B. G., Chambers, M. A., Wangoo, A., Shaw, R. J., and Young, D. B. (1997). Production of tumor necrosis factor and nitric oxide by macrophages infected with live and dead mycobacteria and their suppression by an interleukin-10-secreting recombinant. Infect. Immun. 65:1931-1935. Marshall, L. J., Perks, B., Ferkol, T., and Shute, J. K. (2001). IL-8 released constitutively by primary bronchial epithelial cells in culture forms an inactive complex with secretory component. J. Immunol. 167:28162823. Matsumoto, S., Yukitake, H., Kanbara, H., Yamada, H., Kitamura, A., and Yamada, T. (2001). Mycobacterium bovis bacillus calmette-guerin induces protective immunity against infection by Plasmodium yoelii at blood-stage depending on shifting immunity toward Th1 type and inducing protective IgG2a after the parasite infection. Vaccine 19:779-787. Mazanec, M. B., Kaetzel, C. S., Lamm, M. E., Fletcher D., and Nedrud, J. G. (1992). Intracellular neutralization of virus by immunoglobulin A antibodies. Proc. Natl. Acad. Sci. USA 89:6901-6905. Mazanec, M. B., Nedrud, J. G., Kaetzel, C. S., and Lamm, M. E. (1993). A three-tiered view of the role of IgA in mucosal defense. Immunol. Today 14:430-435. Mazanec, M. B., Coudret, C. L., and Fletcher D. R. (1995). Intracellular neutralization of influenza virus by immunoglobulin A anti-hemagglutinin monoclonal antibodies. J. Virol. 69:1339-1343. Mazzaccaro, R. J., Gedde, M., Jensen, E. R., van Santem, H. M., Ploegh, H. L., Rock, K. L., and Bloom, B. R. (1996). Major histocompatibility class I presentation of soluble antigen facilitated by Mycobacterium tuberculosis infection. Proc. Natl. Acad. Sci. USA 93:11786-11791. Mazzoni, A., and Segal, D. M. (2004). Controlling the Toll road to dendritic cell polarization. J. Leukoc. Biol. 75:721-730. Meding, S. J., and Langhorne, J. (1991). CD4+ T cells and B cells are necessary for the transfer of protective immunity to Plasmodium chabaudi chabaudi. Eur. J. Immunol. 21:1433-1438. Medzhitov, R., Preston-Hurlburt, P., Kopp, E., Stadlen, A., Chen, C., Ghosh, S., and Janeway, C. A. Jr. (1998). MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways. Mol. Cell 2:253-258. Megiovanni, A. M., Sanchez, F., Robledo-Sarmiento, M., Morel, C., Gluckman, J. C., and Boudaly, S. (2006). Polymorphonuclear neutrophils deliver activation signals and antigenic molecules to dendritic cells: a new link between leukocytes upstream of T lymphocytes. J. Leukoc. Biol. 79:977-988. Michelsen, K. S., Aicher, A., Mohaupt, M., Hartung, T., Dimmeler, S., Kirschning, C. J., and Schumann, R. R. (2001). The role of Toll-like receptors (TLRs) in bacteria-induced maturation of murine dendritic cells (DCs). Peptidoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2. J. Biol. Chem. 276:25680-25686. Modlin, R. L., and Rea, T. H. (1988). Immunopathology of leprosy granulomas. Springer Semin. Immunopathol. 10:359-374. Mohan, V. P., Scanga, C. A., Yu, K., Scott, H. M., Tanaka, K. E., Tsang, E., Tsai, M. M., Flynn, J. L., and Chan, J. (2001). Effects of tumor necrosis factor alpha on host immune response in chronic persistent tuberculosis: possible role for limiting pathology. Infect. Immun. 69:1847-1855. Monteiro, R. C., Kubagawa, H., and Copper, M. D. (1990). Cellular distribution, regulation, and biochemical nature of an Fc alpha receptor in humans. J. Exp. Med. 171:597-613. Monteiro, R. C., and van de Winkel, J. G. (2003). IgA Fc receptors. Annu. Rev. Immunol. 21:177-204. 79 Morton, H. C., van Egmond, M., and van de Winkel, J. G. (1996). Structure and function of human IgA Fc receptors (FcαR). Crit. Rev. Immunol. 16:423-440. Mostov, K. E. (1994). Transepithelial transport of immunoglobulins. Annu. Rev. Immunol. 12:63-84. Muller, I., Cobbold, S. P., Waldmann, H., and Kaufmann, S. H. (1987). Impaired resistance to Mycobacterium tuberculosis infection after selective in vivo depletion of L3T4+ and Lyt-2+ T cells. Infect. Immun. 55:2037-2041. Mun, H. S., Aosai, F., Norose, K., Chen, M., Piao, L. X., Takeuchi, O., Akira, S., Ishikura, H., and Yano, A. (2003). TLR2 as an essential molecule for protective immunity against Toxoplasma gondii infection. Int. Immunol. 15:1081-1087. Munk, M. E., Gatrill, A. J., and Kaufmann, S. H. (1990). Target cell lysis and IL-2 secretion by gamma/delta T lymphocytes after activation with bacteria. J. Immunol. 145:2434-2439. Muraille, E., De Trez, C., Brait, M., De Baetselier, P., Leo, O., and Carlier, Y. (2003). Genetically resistant mice lacking MyD88-adapter protein display a high susceptibility to Leishmania major infection associated with a polarized Th2 response. J. Immunol. 170:4237-4241. Murphy, J. R. (1981). Host defenses in murine malaria: nonspecific resistance to Plasmodium berghei generated in response to Mycobacterium bovis infection or Corynebacterium parvum stimulation. Infect. Immun. 33:199-211. Murray, P. J., Wang, L., Onufryk, C., Tepper, R. I., and Young, R. A. (1997). T cell-derived IL-10 antagonizes macrophage function in mycobacterial infection. J. Immunol. 158:315-321. Myrvik, Q. N., Leake, E. S., and Wright, M. J. (1984). Disruption of phagosomal membranes of normal alveolar macrophages by the H37Rv strain of Mycobacterium tuberculosis. A correlate of virulence. Am. Rev. Respir. Dis. 129:322-328. Naik, R. S., Branch, O. H, Woods, A. S., Vijaykumar, M., Perkins, D. J., Nahlen, B. L., Lal, A. A., Cotter, R. J., Costello, C. E., Ockenhouse, C. F., Davidson, E. A., and Gowda, D. C. (2000). Glycosylphosphatidylinositol anchors of Plasmodium falciparum: molecular characterization and naturally elicited antibody response that may provide immunity to malaria pathogenesis. J. Exp. Med. 192:1563-1576. Neutra, M. R., Frey, A., and Kraehenbuhl, J. P. (1996). Epithelial M cells: gateways of mucosal infection and immunization. Cell 86:345-348. Nicholson, S., Bonecini-Almeida, M., Silva, J. R. L., Nathan, C., Xie, Q-W., Mumford, R., Weidner, J. R., Calaycay, J., Geng, J., Boechat, N., Linhares, C., Rom, W., and Ho, J. L. (1996). Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis. J. Exp. Med. 184:2293-2302. Norderhaug, I. N., Johansen, F. E., Chjerven, H., and Brandtzaeg, P. (1999). Regulation of the formation and external transport of secretory immunoglobulins. Crit. Rev. Immunol. 19:481-508. Norhagen, G., Engström, P. E., Hammarström, L., Söder, P. O., and Smith, C. I. (1989). Immunoglobulin levels in saliva in individuals with selective IgA deficiency: compensatory IgM secretion and its correlation with HLA and susceptibility to infections. J. Clin. Immunol. 9:279-286. Noss, E. H., Harding, C. V., and Boom, W. H. (2000). Mycobacterium tuberculosis inhibits MHC class II antigen processing in murine bone marrow macrophages. Cell. Immunol. 201:63-74. Ocana-Morgner, C., Mota, M. M., and Rodriguez, A. (2003). Malaria blood stage suppression of liver stage immunity by dendritic cells. J. Exp Med. 197:143-151. Oddo, M., Renno, T., Attinger, A., Bakker, T., MacDonald, H. R., and Meylan, P. R. (1998). Fas ligandinduced apoptosis of infected human macrophages reduces the viability of intracellular Mycobacterium tuberculosis. J. Immunol. 160:5448-5454. 80 O’Garra, A., and Vieira, P. (2004). Regulatory T cells and mechanisms of immune system control Nat. Med. 10:801-805. Ohashi, K., Burkart, V., Flohe, S., and Kolb, H. (2000). Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J. Immunol. 164:558-561. Orago, A., and Facer, C. A. (1991). Cytotoxicity of human natural killer (NK) cell subsets for Plasmodium falciparum erythrocytic schizonts: stimulation by cytokines and inhibition by neomycin. Clin. Exp. Immunol. 86:22-29. Orme, I. M., and Collins, F. M. (1984). Adoptive protection of the Mycobacterium tuberculosis-infected lung. Cell. Immunol. 84:113-120. Orme, I. M., and Cooper, A. M. (1999). Cytokine/chemokine cascades in immunity to tuberculosis. Immunol. Today 20:307-312. Orme, I. M., Miler, E. S., Roberts, A. D., Furney, S. K., Griffin, J. P., Dobos, K. M., Chi, D., Rivoire, B., and Brennan, P. J. (1992). T lymphocytes mediating protection and cellular cytolysis during the course of Mycobacterium tuberculosis infection, Evidence for different kinetics and recognition of a wide spectrum of protein antigens. J. Immunol. 148:189-196. Ottenhof, T. H., Kumararatne, D, and Casanova, J. L. (1998). Novel human immunodeficiencies reveal the essential role of type-1 cytokines in immunity to intracellular bacteria. Immunol. Today 19:491-494. Oxelius, V. A., Laurell, A. B., Lindquist, B., Golebiowska, H., Axelsson, U., Bjorkander, J., and Hanson, L. A. (1981). IgG subclasses in selective IgA deficiency: importance of IgG2-IgA deficiency. N. Engl. J. Med. 304:1476-1477. Page, K. R., Jedlicka, A. E., Fakheri, B., Noland, G. S., Kesavan, A. K., Scott, A. L., Kumar, N., and Manabe, Y. C. (2005). Mycobacterium-induced potentiation of type I immune responses and protection against malaria are host specific. Infect. Immun. 73:8369-8380. Palma, G. I., and Saravia, N. G. (1997). In situ characterization of the human host response to Leishmania panamensis. Am. J. Dermatopathol. 19:585-590. Patry, C., Sibille, Y., Lehuen, A., and Monteiro, R. C. (1996). Identification of Fc alpha receptor (CD89) isoforms generated by alternative splicing that are differentially expressed between blood monocytes and alveolar macrophages. J. Immunol. 156:4442-4448. Pedrosa, J., Saunders, B. M., Appelberg, R., Orme, I. M., Silva, M. T., and Cooper, A. M. (2000). Neutrophils play a protective nonphagocytic role in systemic Mycobacterium tuberculosis infection of mice. Infect. Immun. 68:577-583. Perlmann, H., Helmby, H., Hagstedt, M., Carlson, J., Larsson, P., Troye-Blomberg, M., and Perlmann, P. (1994). IgE elevation and IgE anti-malarial antibodies in Plasmodium falciparum malaria: association of high IgE levels with cerebral malaria. Clin. Exp. Immunol. 97:284-292. Perry, J. A., Rush, A., Wilson, R. J., Olvers, C. S., and Avery, A. C. (2004). Dendritic cells from malariainfected mice are fully functional APC. J. Immunol. 172:475-482. Perry, J. A., Olver, C. S., Burnett, R. C., and Avery, A.C. (2005). Cutting edge: the acquisition of TLR tolerance during malaria infection impacts T cell activation. J. Immunol. 174:5921–5925. Pethe, K., Alonso, S., Biet, F., Delogu, G., Brennan, M. J., Locht, C., and Menozzi, F D. (2001). The heparin-binding haemagglutinin of M. tuberculosis is required for extrapulmonary dissemination. Nature 412:190-194. Phalipon, A., Cardona, A., Kraehenbuhl, J. P., Edelman, L., Sansonetti, P. J., and Corthesy, B. (2002). Secretory component: a new role in secretory IgA-mediated immune exclusion in vivo. Immunity 17:107-115. 81 Poltorak, A., He, X., Sminova, I., Liu, M. Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B., and Beutler, B. (1998). Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085-2088. Porcelli, S. A., and Modlin, R. L. (1999). The CD1 system: antigen-presenting molecules for T cell recognition of lipids and glycolipids. Annu. Rev. Immunol. 17:297-329. Pouniotis, D. S., Proudfoot, O., Bogdanka, V., Apostolopoulos, V., Fifis, T., and Plebanski, M. (2004). Dendritic cells induce immunity and long-lasting protection against blood-stage malaria despite an in vitro parasite-induced maturation defect. Infect. Immun. 72:5331-5339. Punturieri, A., Alviani, R. S., Polak, T., Copper, P., Sonstein, J., and Curtis, J. L. (2004). Specific engagement of TLR4 or TLR3 does not lead to IFN-beta-mediated innate signal amplification and STAT1 phosphorylation in resident murine alveolar macrophages. J. Immunol. 173:1033-1042. Quan, C. P., Berneman, A., Pires, R., Avrameas, S., and Bouvet, J. P. (1997). Natural polyreactive secretory immunoglobulin A autoantibodies as a possible barrier to infection in humans. Infect. Immun. 65:3997-4004. Quesniaux, V., Fremond, C., Jacobs, M., Parida, S., Nicolle, D., Yeremeev, V., Bihl, F., Erard, F., Botha, T., Drennan, M., Soler, M. N., Le Bert, M., Schnyder, B., and Ryffel, B. (2004). Toll-like receptor pathways in the immune responses to mycobacteria. Microbes Infect. 6:946-959. Re, F., and Strominger, J. L. (2004). IL-10 released by concomitant TLR2 stimulation blocks the induction of a subset of Th1 cytokines that are specifically induced by TLR4 or TLR3 in human dendritic cells. J. Immunol. 173:7548-7555. Reiling, N., Holscher, C., Fehrenbach, A., Kroger, S., Kirschning, C. J., Goyert, S., and Ehlers, S. (2002). Cutting edge: Toll-like receptor (TLR)2- and TLR4-mediated pathogen recognition in resistance to airborne infection with Mycobacterium tuberculosis. J. Immunol. 169:3480-3484. Reljic, R., Crawford, C., Challacombe, S., and Ivanyi, J. (2004a). Mouse monoclonal IgA binds to the galectin-3/Mac-2 lectin from mouse macrophage cell lines. Immunol. Lett. 93:51-56. Reljic, R., Crawford, C., Challacombe, S., and Ivanyi, J. (2004b). Mouse IgA inhibits cell growth by stimulating tumor necrosis factor-alpha production and apoptosis of macrophage cell lines. Int. Immunol. 16:607-614. Reyes-Flores, O. (1986). Granulomas induced by living agents. Int. J. Dermatol. 25:158-165. Ribeiro-Rodrigues, R., Resende Co, T., Rojas, R., Toossi, Z., Dietze, R., Boom, W. H., Maciel, E., and Hirsch, C. H. (2006). A role for CD4+CD25+ T cells in regulation of the immune response during human tuberculosis. Clin. Exp. Immunol. 144:25-34. Riedel, D. D., and Kaufmann, S. H. (1997). Chemokine secretion by human polymorphonuclear granulocytes after stimulation with Mycobacterium tuberculosis and lipoarabinomannan. Infect. Immun. 65:4620-4623. Riley, R. L., Mills, C. C., Nyka, W., Weinstock, N., Storey, P. B., Sultan, L. U., Riley, M. C., and Wells, W. F. (1995). Aerial dissemination of pulmonary tuberculosis. A two-year study of contagion in a tuberculosis ward. 1959. Am. J. Epidemiol. 142:3-14. Roach, D. R., Bean, A. G., Demangel, C., France, M. P., Briscoe, H., and Britton, W. J. (2002). TNF regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection. J. Immunol. 168:4620-4627. Robinson, J. K., Blanchard, T. G., Levine, A. D., Emancipator, S. N., and Lamm M. E. (2001). A mucosal IgA-mediated excretory immune system in vivo. J. Immunol. 166:3688-3692. Rodrigues, A., Schellenberg, J. A., Roth, A., Benn, C. S., Aaby, P., and Greenwood, B. (2007). Revaccination with Bacillus Calmette-Guerin (BCG) vaccine does not reduce morbidity from malaria in African children. Trop. Med. Int. Health 12:224-229. 82 Roncador, G., Brown, P. J, Maestre, L., Hue, S., Torrecuadrada, J. L., Ling, K. L., Pratap, S., Toms, C., Fox, B. C., Cerundolo, V., Powrie, F., and Banham, A. H. (2005). Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. Eur. J. Immunol. 35:1681-1691. Rook, G. A. W., and Bloom, B. R. (1994). Mechanisms of pathogenesis in tuberculosis. In: Tuberculosis: Pathogenesis, Protection and Control (Bloom, B. R., Ed.), pp. 485-502. ASM Press, Washington, DC. Roos, A., Bouwman, L. H., van Gijlswijk-Janssen, D. J., Faber-Krol, M. C., Stah, G. L., and Daha, M. R. (2001). Human IgA activates the complement system via the mannan-binding lectin pathway. J. Immunol. 167:2861-2868. Roth, A., Gustafson, P., Nhaga, A., Djana, O., Poulsen, A., Garly, M. L., Jensen, H., Sodemann, M., Rodriguez, A., and Aaby, P. (2005). BCG vaccination scar associated with better childhood survival in GuineaBissau. Int. J. Epidemiol. 34:540-547. Roth, A., Jensen, H., Garly, M. L., Djana, Q., Martins, C. L., Sodemann, M., Rodriguez, A., and Aaby, P. (2004). Low birth weight infants and Calmette-Guerin bacillus vaccination at birth: community study from Guinea-Bissau. Pediatr. Infect. Dis. J. 23:544-550. Roy, S., Sharma, S., Sharma, M., Aggarwal, R., and Bose, M. (2004). Induction of nitric oxide release from the human alveolar epithelial cell line A549: an in vitro correlate of innate immune response to Mycobacterium tuberculosis. Immunology 112:471-480. Rumbley, C. A., and Phillips, S. M. (1999). The schistosome granuloma: an immunoregulatory organelle. Microbes Infect. 1:499-504. Sakaguchi, S. (2005). Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat. Immunol. 6:345-352. Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, N., and Toda, M. (1995). Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151-1164. Sakamoto, N., Shibuya, K., Shimizu, Y., Yotsumoto, K., Miyabayashi, T., Sakano, S., Tsuji, T., Nakayama, E., Nakauchi, H., and Shibuya, A. (2001). A novel Fc receptor for IgA and IgM is expressed on both hematopoietic and non-hematopoietic tissues. Eur. J. Immunol. 31:1310-1316. Sandor, M., Ibraghimov, A., Rosenberg, M. G., Teeraratkul, P., and Lynch, R. G. (1992). Expression of IgA and IgM Fc receptors on murine T lymphocytes. Immunol. Res. 11:169-180. Scapini, P., Laudanna, C., Pinardi, C., Allavena, P., Mantovani, A., Sozzani, S., and Cassatella, M. A. (2001). Neutrophils produce biologically active macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20 and MIP-3beta/CCL19. Eur. J. Immunol. 31:1981-1988. Schaible, U. E., and Kaufmann, S. H. (2000). CD1 and CD1-restricted T cells in infections with intracellular bacteria. Trends. Microbiol. 8:419-425. Schirm, J., Oostendorp, L. A., and Mulder, J. G. (1995). Comparison of amplicor, in-house PCR, and conventional culture for detection of Mycobacterium tuberculosis in clinical samples. J. Clin. Microbiol. 33:3221-3224. Schlesinger, L. S. (1993). Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by mannose receptors in addition to complement receptors. J. Immunol. 150:2920-2930. Schlesinger, L. S., Bellinger-Kawahara, C. G., Payne, N. R., and Horwitz, M. A. (1990). Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3. J. Immunol. 144:2771-2780. 83 Schlesinger, L. S., Kaufmann, T. M., Iyer, S., Hull, S. R., and Marchiando, L. K. (1996). Differences in mannose receptor-mediated uptake of lipoarabinomannan from virulent and attenuated strains of Mycobacterium tuberculosis by human macrophages. J. Immunol. 157:4568-4575. Schofield, L., and Hackett, F. (1993). Signal transduction in host cells by a glycosylphosphatidylinositol toxin of malaria parasites. J. Exp. Med. 177:145-153. Schwarzer, E., Turrini, F., Ulliers, D., Giribaldi, G., Ginsburg, G., and Arese, P. (1992). Impairment of macrophage functions after ingestion of Plasmodium falciparum-infected erythrocytes or isolated malarial pigment. J. Exp. Med. 176:1033-1041. Schwarzer, E., Alessio, M., Ulliers, D., and Arese, P. (1998). Phagocytosis of the malarial pigment, hemozoin, impairs expression of major histocompatibility complex class II antigen, CD54, and CD11c in human monocytes. Infect. Immun. 66:1601-1606. Scott, C. P., Kumar, N., Bishai, W. R., and Manabe, Y. (2004). Short report: Modulation of Mycobacterium tuberculosis infection by Plasmodium in the murine model. Am. J. Trop. Med. Hyg. 70:144-148. Seiler, P., Aichele, P., Bandermann, S., Hauser, A. E., Lu, B., Gerard, N. .P., Gerard, C., Ehlers, S., Mollenkopf, H. J., and Kaufmann, S. H. (2003). Early granuloma formation after aerosol Mycobacterium tuberculosis infection is regulated by neutrophils via CXCR3-signaling chemokines. Eur. J. Immunol. 33:26762686. Seixas, E., Cross, C., Quin, S., and Langhorne, J. (2001). Direct activation of dendritic cells by the malaria parasite, Plasmodium chabaudi chabaudi. Eur. J. Immunol. 31:2970-2978. Senaldi, G., Yin, S., Shaklee, C. L., Piguet, P. F., Mak, T. W., and Ulich, T. R. (1996). Corynebacterium parvum- and Mycobacterium bovis bacillus Calmette-Guerin-induced granuloma formation is inhibited in TNF receptor I (TNF-RI) knockout mice and by treatment with soluble TNF-RI. J. Immunol. 157:5022-5026. Senior, B. W., Loomes, L. M., and Kerr, M. A. (1991). Microbial IgA proteases and virulence. Rev. Med. Microbiol. 2:200-207. Serbina, N. V., and Flynn, J. L. (1999). Early emergence of CD8(+) T cells primed for production of type 1 cytokines in the lungs of Mycobacterium tuberculosis-infected mice. Infect. Immun. 67:3980-3988. Sharma, M., Sharma, S., Roy, S., Varma, S., and Bose, M. (2007). Pulmonary epithelial cells are a source of interferon-gamma in response to Mycobacterium tuberculosis infection. Immunol. Cell Biol. In press. Sherry, B. A., Alava, G., Tracey, K. J., Martiney, J., Cerami, A., and Slater, A. F. (1995). Malaria-specific metabolite hemozoin mediates the release of several potent endogenous pyrogens (TNF, MIP-1 alpha, and MIP1 beta) in vitro, and altered thermoregulation in vivo. J. Inflamm. 45:85-96. Shibuya, A., Sakamoto, N., Shimizu, Y., Shibuya, K., Osawa, M., Hiroyama, T., Eyre, H. J., Sutherland, G. R., Endo, Y., Fujita, T., Miyabayashi, T., Sakano, S., Tsuji, T., Nakayama, E., Phillips, J. H., Lanier, L. L., and Nakauchi, H. (2000). Fc alpha/mu receptor mediates endocytosis of IgM-coated microbes. Nat. Immunol. 1:441-446. Shiloh, M., and Nathan, C. F. (2000). Reactive nitrogen intermediates and the pathogenesis of Salmonella and mycobacteria. Curr. Opin. Microbiol. 3:35-42. Shim, T. S., Turner, O. C., and Orme, I. M. (2003). Toll-like receptor 4 plays no role in susceptibility of mice to Mycobacterium tuberculosis infection. Tuberculosis 83:367-371. Shimada, S., Kawaguchi-Miyashita, M., Kushiro, A., Sato, T., Nanno, M., Sako, T., Matsuoka, Y., Sudo, K., Tagawa, Y., Iwakura, Y., and Ohwaki, M. (1999). Generation of polymeric immunoglobulin receptordeficient mouse with marked reduction of secretory IgA. J. Immunol. 163:5367-5373. Silva, M. T., Silva, M. N., and Appelberg, R. (1989). Neutrophil-macrophage cooperation in the host defence against mycobacterial infections. Microb. Pathog. 6:369-380. 84 Skorohod, O. A., Alessio, M., Mordmuller, B., Arese, P., and Schwarzer, E. (2004). Hemozoin (malarial pigment) inhibits differentiation and maturation of human monocyte-derived dendritic cells: a peroxisome proliferator-activated receptor-gamma-mediated effect. J. Immunol. 173:4066-4074. Slater, A. F., Swiggard, W. J., Orton, B. R., Flitter, W. D., Goldberg, D. G., Cerami, A., and Henderson, G.B. (1991). An iron-carboxylate bond links the heme units of malaria pigment. Proc. Natl. Acad. Sci. USA. 88:325-329. Smiley, S. T., King, J. A., and Hancock, W. W. (2001). Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4. J. Immunol. 167:2887-2894. Smith, S. M., and Dockrell, H. M. (2000). Role of CD8 T cells in mycobacterial infections. Immunol. Cell Biol. 78:325-333. Snow, R. W., Guerra, C. A., Noor, A. M., Myint, H. Y., and Hay, S. I. (2005). The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature 434:214-217. Sollid, L. M., Kvale, D., Brandtzaeg, P., Markussen, G., and Torsby, E. (1987). Interferon-γ enhances expression of secretory component, the epithelial receptor for polymeric immunoglobulins. J. Immunol. 138:4303-4306. Sousa, A. O., Mazzaccaro, R., Russell, D. G., Lee, F. K., Turner, O. C., Hong, S., Van Kaer, L., and Bloom, B. R. (1999). Relative contributions of distinct MHC class I-dependent cell populations in protection to tuberculosis infection in mice. Proc. Natl. Acad. Sci. USA 97:4204-4208. Steinman, R. M. (1991). The dendritic cell system and its role in immunogenicity. Ann. Rev. Immunol. 9:271296. Stenger, S., Hanson, D. A., Teitelbaum, R., Dewan, P., Niazi, K. R., Froelich, C. J., Ganz, T., ThomaUszynski, S., Melian, A., Bogdan, C., Porcelli, S. A., Bloom, B. R., Krensky, A. M., and Modlin, R. L. (1998). An antimicrobial activity of cytotoxic T cells mediated by granulysin. Science 282:121-125. Stenger, S., Mazzaccaro, R., Uyemura, K., Cho, S., Barnes, P., Rosat, J., Sette, A., Brenner, M., Porcelli, S., Bloom, B, and Modlin, R. (1997). Differential effects of cytolytic T cell subsets on intracellular infection. Science 276:1684-1687. Sterne, J. A., Rodrigues, L. C., and Guedes, I. N. (1998). Does the efficacy of BCG decline with time since vaccination? Int. J. Tuber. Lung Dis. 2:200-207. Stevenson, M., Lemieux, S., and Skamene, E. (1984). Genetic control of resistance to murine malaria. J. Cell. Biochem. 24:91-102. Stevenson, M. M., and Riley, E. M. (2004). Innate immunity to malaria. Nat. Rev. Immunol. 4:169-180. Stevenson, M., and Urban, B. C. (2006). Antigen presentation and dendritic cell biology in malaria. Parasite Immunol. 28:5-14. Sturgill-Koszycki, S., Schaible, U. E., and Russell, D. G. (1996). Mycobacterium-containing phagosomes are accessible to early endosomes and reflect a transitional state in normal phagosome biogenesis. EMBO J. 15:6960-6968. Sturgill-Koszycki, S., Schlesinger, P. H., Chakraborty, P., Haddix, P. L., Collins, H. L., Fok, A. K., Allen, R. D., Gluck, S. L., Heuser, J., and Russell, D. G. (1994). Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263:678-681. Sugawara, I., Yamada, H., Li, C., Mizuno, S., Takeuchi, O., and Akira, S. (2003). Mycobacterial infection in TLR2 and TLR6 knockout mice. Microbiol. Immunol. 47:327-336. 85 Sullivan Jr., D. J., Gluzman, I. Y., and Goldberg, D. G. (1996a). Plasmodium hemozoin formation mediated by histidine-rich proteins. Science 271:219-222. Sullivan Jr., D. J., Gluzman, I. Y., Russell, D. G., and Goldberg, D. G. (1996a). On the molecular mechanism of chloroquine's antimalarial action. Proc. Natl. Acad. Sci. USA. 93:11865-11870. Suss, G., Eichmann, K., Kury, E., Linke, A., and Langhorne, J. (1988). Roles of CD4- and CD8-bearing T lymphocytes in the immune response to the erythrocytic stages of Plasmodium chabaudi. Infect. Immun. 56:3081-3088. Tachado, S. D., Gerold, P., Schwaz, R., Novakovic, S., McConville, M., and Schofied, L. (1997). Signal transduction in macrophages by glycosylphosphatidylinositols of Plasmodium, Trypanosoma, and Leishmania: activation of protein tyrosine kinases and protein kinase C by inositolglycan and diacylglycerol moieties. Proc. Natl. Acad Sci. USA. 94:4022-4027. Tascon, R. E., Stavropoulos, E., Lukacs, K. V., and Colston, M. J. (1998). Protection against Mycobacterium tuberculosis infection by CD8 T cells requires production of gamma interferon. Infect. Immun. 66:830-834. Tailleux, L., Schwartz, O., Herrman, J. L., Pivert, E., Jackson, M., Amara, A., Legres, L., Dreher, D., Nicod, L. P., Gluckman, J. C., Lagrange, P. H., Gicquel, B., and Neyrolles, O. (2003). DC-SIGN is the major Mycobacterium tuberculosis receptor on human dendritic cells. J. Exp. Med. 197:121-127. Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T., Takeda, K., and Akira, S. (1999). Differential roles for TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11:443-451. Tan, B. H., Meinken, C., Bastian, M., Bruns, H., Legaspi, A., Ochoa, M. T., Krutzik, S. R., Bloom, B. R., Ganz, T., Modlin, R. L., and Stenger, S. (2006). Macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens. J. Immunol. 177:1864-1871. Taub, D. D., Turcovski-Corrales, S. M., Key, M. L., Longo, D. L., and Murphy, W. J. (1996). Chemokines and T lymphocyte activation: I. Beta chemokines costimulate human T lymphocyte activation in vitro. J. Immunol. 156:2095-2103. Taylor-Robinson, A. W., and Phillips, R. S. (1993). Protective CD4+ T-cell lines raised against Plasmodium chabaudi show characteristics of either Th1 or Th2 cells. Parasite Immunol. 15:301-310. Taylor-Robinson, A. W., Phillips, R. S., Severn, A., Moncada, S., and Liew, F Y. (1993). The role of TH1 and TH2 cells in a rodent malaria infection. Science 260:1931-1934. Taylor-Robinson, A. W., and Smith, E. C. (1991). A role for cytokines in potentiation of malaria vaccines through immunological modulation of blood stage infection. Immunol. Rev. 171:105-123. Teitelbaum, R., Cammer, M., Maitland, M. L., Freitag, N. E., Condeelis, J., and Bloom, B. R. (1999). Mycobacterial infection of macrophages results in membrane-permeable phagosomes. Proc. Natl. Acad. Sci. USA 96:15190-15195. Teitelbaum, R., Glatman-Freedman, A., Chen, B., Robbins, J. B., Unanue, E., Casadevall, A., and Bloom, B. R. (1998). A mAb recognizing a surface antigen of Mycobacterium tuberculosis enhances host survival. Proc. Natl. Acad. Sci. USA. 95:15688-15693. Teitelbaum, R., Schubert, W., Gunther, L., Kress, Y., Macaluso, F., Pollard, J. W., McMurray, F., and Bloom, B. R. (1999). The M cell as a portal of entry to the lung for the bacterial pathogen Mycobacterium tuberculosis. Immunity 10:641-650. Tenner-Racz, K., Racz, P., Myrvik, Q. N., Ockers, J. R., and Geister, R. (1979). Uptake and transport of horseradish peroxidase by lymphoepithelium of the bronchus-associated lymphoid tissue in normal and bacillus Calmette-Guerin-immunized and challenged rabbits. Lab. Invest. 41:106-115. 86 Termeer, C., Benedix, F., Sleeman, J., Fieber, C., Voith, U., Ahrens, T., Miyake, K., Freudenberg, M., Galanos, C., and Simon, J. C. (2002). Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. J. Exp. Med. 195:99-111. Theander, T. G., Pedersen, B. K., Bygbjerg, I. C., Jepsen, S., and Larsen, P. B. (1987). Enhancement of human natural cytotoxicity by Plasmodium falciparum antigen activated lymphocytes. Acta. Trop. 44:415-422. Theuer, C. P., Hopewell, P. C., Elias, D., Schected, G. F., Rutherford, G. W., and Chassion, R. E. (1990). Human immunodefiency virus infection in tuberculosis patients. J. Infect. Dis. 162:8-12. Thorel, M. F. (1980). Isolation of Mycobacterium africanum from monkeys. Tubercle 61:101-104. Thurnher, M., Ramoner, R., Gastl, G., Radmayr, C., Bock, G., Herold, M., Klocker, H., and Bartsch, G. (1997). Bacillus Calmette-Guerin mycobacteria stimulate human blood dendritic cells. Int. J. Cancer 70:128134. Thoma-Uszynski, S., Kiertscher, S. M., Ochoa, M. T., Bouis, D. A., Norgard, M. V., Miyake, K., Godowski, P. J., Roth, M. D., and Modlin, R. L. (2000). Activation of Toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10. J. Immunol. 165:3804-3810. Toossi, Z. (2003). Virological and immunological impact of tuberculosis on human immunodeficiency virus type 1 disease. J. Infect. Dis. 188:1146-1155. Toossi, Z., Gogate, P., Shiratsuchi, H., Young, T., and Ellner, J. J. (1995). Enhanced production of TGF-β by blood monocytes from patients with active tuberculosis and presence of TGF-β in tuberculous granulomatous lung lesions. J. Immunol. 154:465-473. Toossi, Z., Kleinhenz, M. E., and Ellner, J. J. (1986). Defective interleukin 2 production and responsiveness in human pulmonary tuberculosis. J. Exp. Med. 163:1162-1172. Torres, M., Mendez-Sampeiro, P., Jimenez-Zamdio, L., Teran, L., Camarena, A., Quezada, R., Ramos, E., and Sada, E. (1994). Comparison of the immune response against Mycobacterium tuberculosis antigens between a group of patients with active pulmonary tuberculosis and healthy household contacts. Clin. Exp. Immunol. 96:75-78. Trager, W., and Jensen, J. B. (1976). Human malaria parasites in continuous culture. Science 193:673-675. Troye-Blomberg, M., Berzins, K., and Perlmann, P. (1994). T-cell control of immunity to the asexual blood stages of the malaria parasite. Crit. Rev. Immunol. 14:131-155. Tsenova, L., Bergtold, A., Freedman, V. H., Young, R. A., and Kaplan, G. (1999). Tumor necrosis factor alpha is a determinant of pathogenesis and disease progression in mycobacterial infection in the central nervous system. Proc. Natl. Acad. Sci. USA 96:5657-5662. Tsuji, S., Matsumoto, M., Takeuchi, O., Akira, S., Azuma, I., Hayashi, A., Toyoshima, K., and Seya, T. (2000). Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis Calmette-Guerin: involvement of Toll-like receptors. Infect. Immun. 68:6883-6890. Turner, J., Frank, A. A., Brooks, J. V., Gonzalez-Juarrero, M., and Orme, I. M. (2001). The progression of chronic tuberculosis in the mouse does not require the participation of B lymphocytes of interleukin-4. Exp. Gerontol. 36:537-545. Ueki, T., Mizuno, M., Uesu, T., Kiso, T., and Tsuji, T. (1995). Expression of ICAM-I on M cells covering isolated lymphoid follicles of the human colon. Acta. Med. Okayama. 49:145-151. Ulevitch, R. J. (2004). Therapeutics targeting the innate immune system. Nat. Rev. Immunol. 4:512-520. Ulrichs, T., and Kaufmann, S. H. (2002). Mycobacterial persistence and immunity. Front. Biosci. 7:d458d469. 87 Urban, B. C., Ferguson, D. J., Pain, A., Willcox, N., Plebanski, M., Austyn, J. M., and Roberts, D. J. (1999). Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells. Nature 400:7377. Urban, B. C., Willcox, N., and Roberts, D. J. (2001). A role for CD36 in the regulation of dendritic cell function. Proc. Natl. Acad. Sci. USA. 98:8750-8755. Vabulas, R. M., Ahmad-Nejad, P., Ghose, S., Kirschning, C. J., Issels, R. D., and Wagner, H. (2002). HSP70 as endogenous stimulus of the Toll/interleukin-1 receptor signal pathway. J. Biol. Chem. 277:1510715112. Vabulas, R. M., Braedel, S., Hilf, N., Singh-Jasuja, H., Herter, S., Ahmad-Nejad, P., Kirschning, C. J., Da Costa, C., Rammensee, H. G., Wagner, H., and Schild, H. (2002). The endoplasmic reticulum-resident heat shock protein Gp96 activates dendritic cells via the Toll-like receptor 2/4 pathway. J. Biol. Chem. 277:2084720853. van Egmond, M., van Garderen, E., van Spriel, A. B., Damen, C. A., van Emersfoort, E. S., van Zandbergen, G., van Hattum, J., Kuiper, J., and van de Winkel, J. G. (2000). FcalphaRI-positive liver Kupffer cells: reappraisal of the function of immunoglobulin A in immunity. Nat. Med. 6:680-685. van Gisbergen, K. P., Sanchez-Hernandez, M., Geijtenbeek, T. B., and van Kooyk, Y. (2005). Neutrophils mediate immune modulation of dendritic cells through glycosylation-dependent interactions between Mac-1 and DC-SIGN. J. Exp. Med. 201:1281-1292. Velema, J. P., Alihonou, E. M., Gandaho, T., and Hounye, F. H. (1991). Childhood mortality among users and non-users of primary health care in a rural west African community. Int. J. Epidemiol. 20:474-479. Vordermeier, H. M., Venkataprasad, N., Harris, D. P., and Ivanyi, J. (1996). Increase of tuberculosis infection in the organs of B cell-deficient mice. Clin. Exp. Immunol. 106:312-316. Walker, D. (2001). Economic analysis of tuberculosis diagnostic tests in disease control: how can it be modelled and what additional information is needed? Int. J. Tuberc. Lung Dis. 5:1099-1108. Wang, C-H., Liu, C-Y., Lin, H-C., Yu, C-T., Chung, K. F., and Kuo, H. P. (1998). Increased exhaled nitric oxide in active pulmonary tuberculosis due to inducible NO synthase upregulation in alveolar macrophages. Eur. Respir. J. 11:809-815. Weinbaum, F. I., Weintraub, J., Nkrumah, F. K., Evans, C. B., Tigelaar, R. E., and Rosenberg, Y. J. (1978). Immunity to Plasmodium berghei yoelii in mice. II. Specific and nonspecific cellular and humoral responses during the course of infection. J. Immunol. 121:629-636. Weir, M. R., and Thornton, G. F. (1985). Extrapulmonary tuberculosis. Experience from a community hospital and review of the literature. Am. J. Med. 79:467-478. WHO. (2007). Global Tuberculosis Control, Geneva, Switzerland, Report no. WHO/HTM/TB/2007.376. Wijburg, O. L., Uren, T. K., Simpfendorfer, K., Johansen, F. E., Brandtzaeg, P., Strugnell, R. A. (2006). Innate secretory antibodies protect against natural Salmonella typhimurium infection. J. Exp. Med. 203:21-26. Wilkinson, R. J., Haslov, K., Rappuoli, R., Giovannoni, F., Narayanan, P. R., Desai, C. R., Vordermeier, H. M., Paulsen, J., Pasvol, G., Ivanyi, J., and Singh, M. (1997). Evaluation of the recombinant 38-kilodalton antigen of Mycobacterium tuberculosis as a potential immunodiagnostic reagent. J. Clin. Microbiol. 35:553-537. Williams, A., Reljic, R., Naylor, I., Clark, S. O., Falero-Diaz, G., Singh, M., Challacombe, S., Marsh, P. D., and Ivanyi, I. (2004). Passive protection with immunoglobulin A antibodies against tuberculous early infection of the lungs. Immunology 111:328-333. Winslow, G. M., Yager, E., Shilo, K., Volk, E., Reilly, A., and Chu, F. K. (2000). Antibody-mediated elimination of the obligate intracellular bacterial pathogen Ehrlichia chaffeensis during active infection. Infect. Immun. 68:2187-2195. 88 Wright, S. D., and Siverstein, S. C. (1983). Recptors for C3b and C3bi promote phagocytosis but not the release of toxic oxygen from human phagocytes. J. Exp. Med. 158:2016-2023. Yan, H., Lamm, M. E., Bjorling, E., and Huang, Y. T. (2002). Multiple functions of immunoglobulin A in mucosal defense against viruses: an in vitro measles virus model. J. Virol. 76:10972-10979. Yang, D., Chen, Q., Chertov, O., and Oppenheim, J. J. (2000). Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells. J. Leukoc. Biol. 68:9-14. Youngman, K. R., Fiocchi, C., and Kaetzel, C. S. (1994). Inhibition of IFN-γ activity in supernatants from stimulated human intestinal mononuclear cells prevents upregulation of the polymeric Ig receptor in an intestinal epithelial cell line. J. Immunol. 153:675-681. Zhang, M., Gong, J., Iyer, D. V., Jones, B. E., Modlin, R. L., and Barnes, P. F. (1994). T cell cytokine responses in persons with tuberculosis and human immunodeficiency virus infection. J. Clin. Invest. 94:24352442. Zhang, Y., Pacheco, S., Acuna, C. L., Switzer, K. C., Wang, Y., Gilmore, X., Harriman, G. R., and Mbawuike, I. N. (2002). Immunoglobulin A-deficient mice exhibit altered T helper 1-type immune responses but retain mucosal immunity to influenza virus. Immunology 105:286-294. Zimmerli, S., Edwards, S., and Ernst, J. D. (1996). Selective receptor blockade during phagocytosis does not alter the survival and growth of Mycobacterium tuberculosis in human macrophages. Am. J. Respir. Cell. Mol. Biol. 15:760-770. 89