The many faces of host responses to tuberculosis

Tuberculosis remains today one of the top three fatal infectious diseases, together with acquired immune deficiency syndrome (AIDS) and malaria. During the last decade, 90 million new infections occurred, resulting in approximately 30 million deaths. Although there is currently effective chemotherapy, consisting of three specific drugs, this regimen must be continued for a period of at least 6 months, which in many cases, results in problems with compliance. Lack of compliance further impacts on the development of multidrug-resistant strains of the bacterium, which consequently raises the cost of treatment, making the expense of curing tuberculosis prohibitive in many developing countries. Despite the enormous numbers of people infected with this organism, it is estimated that only 10% of affected individuals show evidence of clinical symptoms. Many parameters, notably socio-economic factors, co-infection with human immunodeficiency virus (HIV) and genetic predisposition of the host, influence the susceptibility to disease. Much work has been invested to elucidate the biology of the interaction between Mycobacterium tuberculosis and its host, both in experimental animal models and in clinical studies. Here we review some of the latest developments in the understanding of the immune response required to control this pathogen. It is hoped that further progress in this field will lead to a more rational approach towards the development of an effective vaccine and novel chemotherapeutic agents. The establishment of a successful infection by mycobacteria depends on the initial encounter between the pathogen and the host cell, usually the macrophage. Obviously the surface characteristics of both parties will significantly influence the outcome. Although mycobacteria are Gram positive, their wax-rich cell wall confers on them unique features and thus they are classified as acid-fast bacilli. The abundant cell wall glycolipids, including lipoarabinomannan (LAM) and mycolic acids, are responsible for many immunological peculiarities. Mycobacteria have been proposed to bind to a variety of host cell receptors, including Fc receptors (FcR), complement receptors (CR) (both with or without prior opsonization), the macrophage mannose receptor, surfactant protein receptors and CD14 (reviewed in ref. 1), via a variety of its surface molecules (reviewed in ref. 2). It is proposed that the choice of receptor used to enter the macrophage influences the cellular response. For example, internalization of immunoglobulin G (IgG) -opsonized mycobacteria via the FcR induces the production of reactive oxygen intermediates and permits phagosome–lysosome fusion,3 whereas entry of mycobacteria via CR3 prevents the activation of the respiratory burst4 and results in a phagosome that is arrested in its maturation stage at that of an early endosome.5 The diverse array of receptors that could be utilized by mycobacteria to interact with and to enter host cells, makes it unlikely that there is one ‘preferred route’. Indeed, the organism appears to have become rather adept at abusing both host cell receptors and other host cell molecules in order to maximize internalization. Thus, in a mechanism specific for pathogenic mycobacteria, the bacteria can associate with C2a to form a C3 convertase, resulting in opsonization of the organism with C3bi and uptake by macrophage CR1/CR3.6 However, the vast redundancy of this system is illustrated by experiments demonstrating that CR3-deficient mice infected with M. tuberculosis show no difference in survival, bacterial burden or granuloma formation as compared to wild-type animals.7 Recently, an essential role for cholesterol in the entry of mycobacteria into macrophages has been described. The depletion of plasma membrane cholesterol specifically inhibited mycobacterial uptake. This observation has important implications for the subsequent intracellular events, as cholesterol mediates the phagosomal association of tryptophane aspartate-containing coat protein (TACO), which prevents the maturation of the phagosome to a phagolysosome8. Much work has focused on the interaction of mycobacteria with the Toll-like receptors (TLR). This receptor family was first identified in Drosophila, where they are important for resistance to microbial pathogens.9 Subsequently a large number of Toll homologues have been identified in mammals, and TLR4 in particular has been demonstrated to be critical in responses to Gram-negative bacteria.9,10 Two TLRs, TLR2 and TLR4, have been implicated in the activation of macrophages by mycobacteria. Viable M. tuberculosis could activate an NF-κB reporter gene in Chinese hamster ovary cells transfected with TLR2 and TLR4 via distinct ligands, independent of CD14 expression. Thus, a soluble, heat-stable factor mediates TLR2-dependent activation, while a heat-sensitive cell wall-associated factor induced activation via TLR4. The cell wall component, LAM, isolated from fast-growing mycobacteria, stimulated TLR2- but not TLR4-mediated activation, whereas LAM isolated from M. tuberculosis or M. bovis bacillus Calmette–Guèrin (BCG) failed to activate via either receptor.11 The 19 000 MW lipoprotein of M. tuberculosis induced the production of the T helper type 1 (Th1) cell promoting the cytokine interleukin-12 (IL-12) from primary human monocytes in a TLR2-dependent mechanism and this receptor was sufficient to mediate this effect. The ability of this lipoprotein to activate the promotor for inducible nitric oxide synthase (iNOS), was abolished by the presence of a transfected dominant negative mutant TLR2 receptor.12 This enzyme is responsible for the catalysis of reactive nitrogen intermediates (NO) from l-arginine, which forms one of the primary microbicidal mechanisms of macrophages (see below). TLR2 has also been shown to be recruited specifically to macrophage phagosomes containing yeast, and can trigger tumour necrosis factor (TNF) production in response to Gram-positive, but not Gram-negative, bacteria.13 To date it is unclear whether the TLRs are recruited to the mycobacteria phagosome. However, taken together these data indicate that a variety of bacterial components, including a diverse array from mycobacteria, can activate signalling cascades within the host cell which direct the subsequent development of an immune response (Fig. 1). Stages of the immune response to Mycobacterium tuberculosis. Having gained entry into the macrophage, M. tuberculosis faces the problem of establishing residence inside a primary host effector cell. To this end, mycobacteria have evolved mechanisms to exploit the macrophage as an intracellular niche. One of the major problems is acquisition of essential nutrients in the intracellular environment. The macrophage requires iron as a cofactor in the induction of microbicidal effector mechanisms, while the mycobacteria themselves have an obligate requirement for iron for their intracellular survival. Thus, there is competition between the host and M. tuberculosis for the acquisition of this essential molecule. The host cell acquires iron via the transferrin receptor (TfR), which internalizes extracellular iron bound to transferrin and lactoferrin. This complex is then trafficked to an early endosomal recycling compartment, where the mildly acidic conditions facilitate the release of the iron from the receptor. There are several strategies that M. tuberculosis employs to ensure that its iron supply is not restricted. Primarily, the mycobacterial phagosome is restricted in its maturation state to that of an early endosome which resides in the recycling endosomal pathway. This results in free access to the transferrin receptor with its iron bound to transferrin.14 Additionally mycobacteria have developed specialized iron-binding molecules, siderophores, which have a high affinity for intracellular iron and transfer iron from host proteins to specialized mycobactin molecules in the mycobacterial cell wall.15In vitro studies using gallium, which accumulates intracellularly and disrupts iron acquisition by M. tuberculosis, revealed that treatment of infected macrophages with this compound resulted in the killing of M. tuberculosis both extracellularly and intracellularly.16 In line with this, a mutant strain of M. tuberculosis that was deficient in the synthesis of a subset of siderophores was impaired in its intracellular growth.17 In the clinical situation, there is an increased incidence of tuberculosis among people suffering from dietary iron overload, which is characterized by iron deposition in macrophages and parenchymal cells18. This well illustrates the delicate balance that must exist inside the host cell: too much iron down-regulates microbicidal effector mechanisms and favours the growth of the pathogen, whereas too little iron is inhibitory to the induction of antimicrobial processes. Possible mechanisms for manipulating intracellular iron concentration during M. tuberculosis infection have recently become a focus in our laboratory, and preliminary experiments in mice suggest that the local administration of iron-chelating compounds alters the course of pulmonary tuberculosis (Collins et al. unpublished observations). Recently evidence has been provided that in chronically infected lung tissues, i.e. at late stages of infection, M. tuberculosis obtains carbon from fatty acids. One pathway that is required for this acquisition is the glyoxylate shunt. One enzyme of this pathway, isocitrate lyase, is up-regulated by M. tuberculosis organisms when they are inside macrophages.19 A mutant M. tuberculosis strain with a disruption in the gene encoding isocitrate lyase was attenuated in its ability to sustain a persistent infection in mice, but was dispensable during the acute phase of growth. Furthermore, in vitro infection of macrophages revealed that the expression of isocitrate lyase was prolonged in activated macrophages in comparison to resting cells.19,20 This suggests that the manipulation of the nutritional requirements of M. tuberculosis, coupled with the immune status of the host, dramatically alters the course of infection and could open up potential avenues for therapeutic intervention. As critical as it is for M. tuberculosis to reside inside the macrophage to perpetuate infection, it is more important from the host's perspective to eliminate the pathogen via the activation of microbicidal mechanisms, such as the induction of reactive nitrogen and oxygen intermediates. Experimental M. tuberculosis infection of iNOS knockout mice, reveals a greatly increased susceptibility of these mice, as measured by an increase in bacterial loads, as well as a decreased survival time.21,22 Recently a role for superoxide has also been demonstrated by using mice lacking the cytosolic p47 (phox) gene which is essential for NADPH-dependent production of superoxide radicals. Phox–/– mice showed an increase in bacterial loads during the early infection period with M. tuberculosis, however, once interferon-γ (IFN-γ) -secreting antigen-specific T lymphocytes appeared in the lung, the mutant mice were once again able to control the infection, and bacterial loads stabilized.23 This suggests that early IFN-γ production and the induction of iNOS are not sufficient to control initial M. tuberculosis replication. This is similar to the situation observed in experimental infections with Salmonella typhimurium, where NADPH phagocyte oxidase was required early in infection, whereas iNOS-dependent mechanisms were important later for bacterial clearance.24 Despite experimental models implicating NO production as a critical molecule in the anti-mycobacterial response, in humans its role has been more controversial. However, in vitro experiments have demonstrated the induction of NO in human monocytes,25 as well as in human alveolar and the presence of NO with the growth of M. is from of these is that the production of IFN-γ is critical in the control of M. tuberculosis infection, whether early in infection as a of the activation of immune mechanisms, or by antigen-specific T cells the induction of specific This has been demonstrated in a variety of mice deficient in either IFN-γ or a critical cytokine in the induction of were to with M. there a of with or IFN-γ receptor deficiency are to infections with intracellular including mycobacteria such as M. and M. and from M. bovis Furthermore, as in the experimental with in or the receptor are (reviewed in ref. However, these not from M. tuberculosis The for this is the of these as well as their other bacterial makes it that they are to M. tuberculosis. 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