Mycobacterium tuberculosis: Immune evasion, latency and reactivation

Immunobiology 217 (2012) 363–374 Contents lists available at ScienceDirect Immunobiology journal homepage: www.elsevier.de/imbio Review Mycobacterium tuberculosis: Immune evasion, latency and reactivation Antima Gupta a,† , Akshay Kaul a,† , Anthony G. Tsolaki b , Uday Kishore b , Sanjib Bhakta a,∗ a b Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London, London WC1E 7HX, UK Centre for Infection, Immunity and Disease Mechanisms, Biosciences, School of Health Sciences and Social Care, Brunel University, Uxbridge, London UB8 3PH, UK a r t i c l e i n f o Article history: Received 14 January 2011 Received in revised form 16 June 2011 Accepted 5 July 2011 Keywords: Immune evasion Immune response Latency Mycobacterium tuberculosis Reactivation a b s t r a c t One-third of the global human population harbours Mycobacterium tuberculosis in dormant form. This dormant or latent infection presents a major challenge for global efforts to eradicate tuberculosis, because it is a vast reservoir of potential reactivation and transmission. This article explains how the pathogen evades the host immune response to establish a latent infection, and how it emerges from a state of latency to cause reactivation disease. This review highlights the key factors responsible for immune evasion and reactivation. It concludes by identifying interesting candidates for drug or vaccine development, as well as identifying unresolved questions for the future research. © 2011 Elsevier GmbH. All rights reserved. Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunology of latent tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Entry, recognition and phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evasion of the immune system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arrest of phagosome–lysosome fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resistance against reactive nitrogen intermediates and nitric oxides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interference with antigen presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CD8+ T cells, NK cells and the complement membrane attack complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The granuloma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disease reactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations: Ab, antibody; Ag, antigen; Ahp, alkyl hydroperoxide; APC, antigen presenting cell; BCG, Bacillus Calmette-Guérin; CIITA, class II transactivator; CLIP, class II-associated invariant chain peptides; CR, complement receptors; DCs, dendritic cells; EEA, early endosome antigen; ER, endoplasmic reticulum; GTP, guanosine triphosphate; HIV, human immunodeficiency virus; HLA-DM, human leukocyte antigen-DM; IL, interleukin; IFN, interferon; Ig, immunoglobulin; LAM, lipoarabinomannan; ManLAM, mannosylated lipoarabinomannan; MAPK, mitogenactivated protein kinase; MR, mannose receptors; MHC, major histocompatibility complex; NET, neutrophil extracellular trap; NK, natural killer; NO, nitric oxide; NOS, nitric oxide synthase; PI-3P, phosphatydilinositol-3-phosphate; RNA, ribonucleic acid; RNI, reactive nitrogen intermediates; ROI, reactive oxygen intermediate; SNARE, soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors; TACO, tryptophan-aspartate-rich coat protein; TB, tuberculosis; TLR, toll-like receptor; TNF, tumor necrosis factor; MBL, mannose-binding lectin; VPS, vaculor protein sorting; WHO, World Health Organization. ∗ Corresponding author. Tel.: +44 20 7631 6355. E-mail address: s.bhakta@bbk.ac.uk (S. Bhakta). † Both authors contributed equally to this work. 0171-2985/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2011.07.008 363 364 364 366 367 367 368 370 370 371 372 372 372 Introduction Tuberculosis (TB) is once more an alarming disease. It claimed 1.8 million lives in 2009 (WHO 2010). Recent estimates suggest a third of the world’s population harbours Mycobacterium tuberculosis, the causative agent of TB in difficult-to-diagnose latent form and shows little or no clinical symptoms. A significant risk of reactivation exists in immune-compromised host including HIV+ individuals, for whom TB has become one of the leading causes of death (Tufariello et al. 2003). The bacteria have begun to show extensive resistance to most anti-TB drugs (Zvi et al. 2008). A comprehensive vaccine for TB remains elusive. The Bacillus Calmette-Guérin (BCG), the most common vaccine, protects children. However the efficacy of BCG is variable across the world and is generally ineffective against adult pulmonary 364 A. Gupta et al. / Immunobiology 217 (2012) 363–374 TB which is the most prevalent form of the disease. (Sierra 2006). Patients with latent TB infection constitute the largest reservoir for its potential transmission but diagnosing and studying latent TB infection in humans has proved difficult, and good animal models that mimic latent TB in humans have been hard to develop. A recent review discussed advances in the detection of latency in humans based on immunological markers, such as immune cell activation and cytokine production (Lin and Flynn 2010). Much remains unknown, however, about the transition from the initial control of acute infection by the host immune system to the establishment of a long, persistent latent presence in the body by the bacteria. A crucial factor in this transition is the pathogen’s ability to evade its complete elimination by the immune system. Immunology of latent tuberculosis Pulmonary TB is by far the most common form of tuberculosis, accounting for 86% of all new and relapsed cases worldwide, based on WHO data. It is also the most extensively investigated in the literature, and therefore forms the major part of this review. Entry, recognition and phagocytosis In lung infections, M. tuberculosis is typically inhaled into the body through the mouth or nose, passes through the airways and reaches the alveolar space in the lungs. Here, the bacteria interact with dendritic cells (DCs), alveolar macrophages and pulmonary epithelial cells. They are capable of invading all three cell types, but their favoured hosts are alveolar macrophages and other mononuclear phagocytes (Bermudez and Goodman 1996). M. tuberculosis gains entry into alveolar macrophages through receptor-mediated phagocytosis, a normal feature of the innate immune system. There are two main routes through which these cells recognise the bacteria. One is where bacterial cell surface molecules or secreted proteins can activate complement proteins present in the alveolar space, which are then recognised by complement receptors (CRs) on macrophages (Schlesinger 1993). The other is where alveolar macrophages can recognise bacterial mannose residues [particularly mannose-capped lipoarabinomannan (LAM)] directly through binding with macrophage mannose receptors (MR) (Fenton et al. 2005). The majority of studies currently indicate that phagocytosis through CRs is the route most favoured by M. tuberculosis (Schlesinger 1993). Whilst CR-mediated phagocytosis triggers the minimal superoxide production (Russell 2005), MR-mediated phagocytosis directs the bacilli to a phagosomal compartment that has limited ability to fuse with lysosomes (Kang et al. 2005). However, MR-mediated phagocytosis into alveolar macrophages may be a more important route in the early stages of primary infection, because alveolar macrophages exhibit high MR activity, and serum opsonins (which might bind to CRs) are scarce or absent in the alveolar environment (Stokes et al. 2004). On the other hand, the invasion of mycobacteria inside dendritic cells occurs through a C-type lectin receptor DC-SIGN (dendritic-cellspecific intercellular adhesion molecule-3-grabbing non-integrin) (Appelmelk et al. 2008). The receptor binds to high mannosecapped LAM (ManLam) structure and ligand proteins such as DnaK, Cpn60.1, glyceraldehydes-3 phosphate dehydrogenase (GAPDH) and lipoprotein lprG (Carroll et al. 2010). Alveolar macrophages are attractive targets for M. tuberculosis because they are adapted to the task of removing small airborne particles through phagocytosis, and do not induce strong inflammatory responses. Their ability to produce anti-microbial chemicals such as nitric oxide (NO) and reactive oxygen intermediates (ROI) is blunted (Fenton et al. 2005). Innate immune response: The encounter of alveolar macrophages with M. tuberculosis in the alveolar space initiates innate immunity. The principal effector mechanisms of innate immunity are neutrophils, tissue macrophages (derived from blood-borne monocytes), natural killer (NK) cells and the complement system. Neutrophils are known to play an important role in host defence against tuberculosis (Eruslanov et al. 2005). These blood-borne leukocytes are believed to be recruited through the secretion of IL8 by infected alveolar macrophages (Sawant and McMurray 2007). They are typically the first recruited cells to arrive on the scene. Work on murine models suggests neutrophils play a regulatory, non-phagocytic role against M. tuberculosis (Pedrosa et al. 2000). Their ability to kill the bacteria is debated (Eruslanov et al. 2005). They are believed to play a bigger role in facilitating the adaptive immune response through cytokine signaling. Neutrophils typically act against microbes through phagocytosis and intracellular killing action. However, a recent paper describes a new mechanism through which neutrophils may act against microbes: neutrophil extracellular traps (NETs) (Ramos-Kichik et al. 2009). These NETs are chromatin-based structures studded with granule proteins that bind and kill bacteria. NETs act successfully against many Gram-positive and Gram-negative bacteria as well as fungi. M. tuberculosis, although liable to bind to NETs, also finds a way to evade their killing action (Ramos-Kichik et al. 2009). Blood monocytes are less abundant than neutrophils in circulating blood, but are a critical part of the innate immune response to TB. These leukocytes are recruited through chemokine signals produced by infected alveolar macrophages, and migrate rapidly across the blood vessels to the site of infection. Within the tissue, they differentiate into macrophages with the ability to ingest and kill the bacteria. The interaction between macrophages and T cells (and in particular, the activation of macrophages by IFN-␥ secreted by T cells) is considered central in the elimination of M. tuberculosis (Boom et al. 2003). T-cell activated macrophages are believed to be the dominant effector cells against M. tuberculosis (Flynn and Chan 2001). NK cells are large granular lymphocytes found in circulating blood. These cells recognise and destroy infected host cells. They also activate the effector functions of macrophages by producing IFN-␥ (Abbas and Lichtman 2006). Recent in vitro studies show that NK cells can lyse M. tuberculosis-infected macrophages (Vankayalapati and Barnes 2009). They also facilitate an adaptive immune response by producing IFN-␥, and by stimulating macrophages to produce the cytokines IL-12, IL-15 and IL-18. These in turn induce expansion of CD8+ T cells (Vankayalapati and Barnes 2009). Their in vivo role is however not yet well understood. NK cells are recruited in response to lipid and glycolipid antigen presented on CD1 molecules on the infected cells and produce IFN-␥ (Mendelson et al. 2005). The complement system is a central humoral wing of innate immunity against pathogens. It has three functions, (a) opsonisation of microbes, which facilitates their ingestion by macrophages, (b) osmotic lysis of microbial cells through the formation of the complement membrane attack complex (MAC) and (c) recruitment of leukocytes by chemokine signaling. Recently, M. bovis BCG (which has 99.9% gene sequence identity with M. tuberculosis) has been shown to activate all three pathways of complement – the classical pathway (through binding via complement protein C1q), the lectin pathway (through binding of mannose-binding lectin or L-ficolin to the bacterial cell surface) and the alternative pathway (through the deposition of C3b on the bacterial cell surface) (Carroll et al. 2009). There is also evidence to suggest C3 binding to whole M. tuberculosis through both classical and alternative pathways (Ferguson et al. 2004). The source of complement proteins (C1q, C4b, C3b and C3bi) in the first instance is probably secretion by pneumocytes A. Gupta et al. / Immunobiology 217 (2012) 363–374 and alveolar macrophages (Cole et al. 1983; Strunk et al. 1988). However, as some bacteria disseminate into the bloodstream (e.g. through infecting the epithelial cells), serum complement (particularly C3bi) could be activated as well. Complement activation through increased ligation of C3b does appear to enhance phagocytic uptake by alveolar macrophages, as well as recruitment of the inflammatory response – both of which may be argued to paradoxically work in the long-term favour of the bacteria (Ferguson et al. 2004; Flynn and Chan 2005). Specific proteins of the complement system as well as their proteolytic forms that are bound to M. tuberculosis may affect the subsequent fate of the organism. However, it has also been shown that M. bovis BCG binds Factor H, a key regulator of complement (Carroll et al. 2009). If the same turns out to be true of M. tuberculosis, this would suggest it follows a strategy of selectively up-regulating complement opsonisation to facilitate entry into macrophages, whilst somehow down-regulating (through binding with Factor H) the effector functions of complement such as the formation of the membrane attack complex. Adaptive immune response: In most cases, innate immunity is insufficient on its own to control M. tuberculosis infection. As antigen concentration increases, the adaptive immune response is activated and is crucial in controlling the primary infection. Frequently, adaptive immunity controls but does not eliminate the infection (for reasons reviewed below), and thus, on-going adaptive immunity remains important in preventing reactivation of infection at a later stage (Boom et al. 2003). The adaptive immune response consists of humoral immunity (mediated by B lymphocytes) and cell-mediated immunity (mediated by T lymphocytes). Both types of adaptive response are typically activated in the peripheral lymphoid organs. Here, naïve T and B cells regularly circulate searching for antigen processed and presented on cell-surface MHC molecules by professional antigenpresenting cells (APCs). B cells can recognise and present antigen directly to T cells. For some time, the dominant view in the literature had been that the adaptive immune response against M. tuberculosis relies exclusively on the cell-mediated immunity through CD4+ T cells, but more recent work has shed new light on the role of CD8+ and other T cell subsets, as well as B cells. An unusual feature of M. tuberculosis is that it takes much longer time after infection for the adaptive immune response to be triggered in humans and mice, compared with other pathogens such as Salmonella, Listeria, Haemophilus or Plasmodium (King et al. 2003; Malaguarnera and Musumeci 2002; Zenewicz and Shen 2007). Various explanations have been proposed for this. This could be a result of (a) the slow growth of the bacteria, thus, it takes time for adequate antigen concentration to build up in the lungs (and hence, in the peripheral lymph organs), (b) delay in trafficking antigen from the infected sites to peripheral lymph organs, (c) interference by the bacteria in the pathways for processing and display of antigen to naïve T or B lymphocytes, and/or (d) delay in the recruitment and migration of lymphocytes from the peripheral lymph organs to the infected site (Wolf et al. 2008). A recent study examined Ag85B-specific CD4+ T cells in vivo in mice (Wolf et al. 2008). It found that the delay in the initiation of the adaptive immunity in response to M. tuberculosis infection was directly linked to a delay in the activation of CD4+ T cells. These cells were activated first in the local lung-draining mediastinal lymph nodes. Increasing bacterial numbers in the lungs did not accelerate the T cell response, suggesting that slow bacterial growth is not a causal factor in the delayed immune response. Nor was the immune response accelerated when additional DCs were mobilised in the lymph nodes and the lungs using a pro-inflammatory agent like lipopolysaccharide. Furthermore, the migration rate of T cells from the lymphoid organs to the infected site did not appear abnormal. This suggests that the bacteria are either located in a cellular compartment which impedes the 365 transport of antigen to the peripheral lymphoid organs, or the bacteria actively impede the presentation of antigen to T cells in the lymph nodes. The delayed adaptive immune response could be significant in establishing latency by giving the bacteria time to establish sufficient numbers to evade complete elimination. Activation and recruitment of T lymphocytes: It is becoming increasingly clear that the human T cell response to M. tuberculosis involves CD4+ , CD8+ and ␥␦ T cells. The contributions of each type at different stages of infection are still being ascertained, but it is likely that a diverse T cell repertoire that is able to recognise many different types of bacterial epitopes (proteins as well as lipids) may enhance the efficiency of the adaptive immune response against M. tuberculosis (Boom et al. 2003). For instance, MHC Class I and II molecules are able to display peptide antigens to T cells, whereas CD1 molecules (found mainly on antigen-presenting cells) can display lipid antigens, including mycolic acids. Finally, ␥␦ T cells can recognise non-proteinaceous antigen containing phosphate without the need for any presentation molecules. CD4+ T cells: For quite some time, CD4+ T cells were thought to be the central adaptive immune defence against TB. This is because, unlike CD8+ T cells, they recognise antigens presented only on MHC Class II molecules. These MHC Class II molecules have easy access to vacuoles, phagosomes and other endocytic vesicles, but (typically) not the cytoplasm. Since M. tuberculosis is known to reside in DC phagosomes and macrophage, it follows that its peptide antigens are more likely be displayed on MHC Class II molecules and hence trigger a CD4+ T cell response. The T cell response to M. tuberculosis is activated in the local lung-draining lymph node, where circulating naïve T cells recognise antigens through different receptors like toll like receptors (TLRs), NOD-like receptors and C-type lectins presented by infected DCs (Mendelson et al. 2005). Upon M. tuberculosis recognition, pro-inflammatory cytokines such as IL-6, IL-21, IL-1␤, IL-12p40 and anti-inflammatory TGF-␤ are expressed (Torrado and Cooper 2010). IL-12p40 is the subunit component for both IL-12 and IL-23 cytokines. Further, IL-12 and IL-23 induces the development of TH 1 and TH 17 cells respectively (Torrado and Cooper 2010). Therefore, the balance between TH 1 and TH 17 response depends upon the levels of specific cytokines produced by mycobacterial infected cells. Furthermore, TH 17 cells produce IL-17, IL-21 and IL-22 as their signature cytokines which are the key inducers of inflammation and protective immune response (Korn et al. 2007; Liang et al. 2006). It is suggested that IL-17 is essential for initial granuloma formation by promoting early neutrophil recruitment in TB infection (Torrado and Cooper 2010). During mycobacterial high dose infection, the ␥␦ T cells amplify the production of IL-17 which stimulates the differentiation of TH 1 responses by promoting release of IL-12 (Torrado and Cooper 2010). Therefore, further investigations are required to understand the balancing mechanism of TH 1 and TH 17 response related to the dose dependent TB infection. As discussed above, TGF-␤ helps the differentiation of TH 17 cells; however it also acts in the differentiation of regulatory T (Treg) cells. In a recent study it has been found that during active tuberculosis Tregs inhibit protective TH 1 responses, but not the proinflammatory TH 17 responses which facilitate the mycobacterial replication within macrophages (Marin et al. 2010). A number of T cell antigens specific for latent TB have been identified, including the proteins Rv2003, Rv1733c, Rv3407, Rv2005c and Rv0140 (Schuck et al. 2009). These induce stronger T-cell responses in patients with latent TB as compared to those with evident infection. Once activated, T cells undergo clonal expansion, differentiate into CD4+ effector cells and migrate out of the lymph nodes to the sites of infection, guided by chemokine gradients. At the infection site, these T cells recognise bacterial antigen on macrophages and DCs, and release IFN-␥ to activate the microbicidal mechanisms within infected macrophages. Moreover, the genetically 366 A. Gupta et al. / Immunobiology 217 (2012) 363–374 inherited human IFN-␥ receptor deficiencies and susceptibility to mycobacterial infection highlights the protective role of IFN-␥ in tubercular infection (Newport et al. 1996). Another cytokine IL18 from macrophages and DCs has recently been explored for its protective immunity against tuberculosis (Schneider et al. 2010). M. tuberculosis activated CD4+ T cells also release TNF-␣ (which is thought to play a role in the formation and maintenance of granulomas) and can trigger cell lysis in infected macrophages or kill intracellular bacteria (using perforin and granzymes) (Canaday et al. 2001). However, it is not clear if cell lysis/apoptosis of infected macrophages is detrimental to intracellular M. tuberculosis (Tufariello et al. 2003). Additionally, CD4+ T cells secrete cytokines such as IL-2 which aid for CD8+ and ␥␦ T cells (Boom et al. 2003). The critical role of CD4+ T cells in the control of TB is best shown by data on HIV+ individuals, whose CD4+ T cell populations are severely depleted (Sester et al. 2010). These patients show a marked increase in susceptibility to primary TB infection, re-infection as well as reactivation of latent infection. This is corroborated by studies on knockout mice with deleted genes for CD4+ or MHC Class II molecules, showing that the loss of CD4+ cells correlates with increased susceptibility to M. tuberculosis (Caruso et al. 1999). An important factor that can determine the outcome of infection is the balance between the TH 1 and TH 2 subsets of CD4+ T lymphocytes. The former activates macrophages by secreting IFN-␥, whilst the latter plays a key role in limiting damage from an excessive immune response by inhibiting macrophage activation through the secretion of cytokines such as IL-4, IL-10 and IL-13 (Abbas and Lichtman 2006). There is evidence to suggest that M. tuberculosis is able to inhibit the production of TH 1 cells, inducing a dominant TH 2 response which helps it to evade killing by macrophages (Zhang et al. 1995). However, the relative significance of this balance in the outcome of infection is still unclear. CD8+ T cells: The exact role of CD8+ T cells in defence against TB is not yet clearly understood. Evidence from in vivo work on mice, bovine and primate models suggest critical roles for CD8+ T cells in immunity against TB (Chen et al. 2009). Studies from murine models indicate that CD8+ T cells are primed in the mediastinal lymph nodes within 2 weeks of aerosol infection, and can be found in the lungs after 2 weeks following infection (Chen et al. 2009). The lungs and granulomas in non-human primate models contain equal proportions of CD4+ and CD8+ T cells (Chen et al. 2009). Significantly, people with latent TB have high concentrations of mycobacteriaspecific CD8+ T cells (Randhawa 1990; Tufariello et al. 2003). CD8+ T cells can recognise peptide antigens presented on MHC Class I molecules, which are formed in the cytoplasm of all nucleated cells. They are also able to recognise lipid antigens presented by CD1 molecules. However, how M. tuberculosis antigens make their way from the macrophage phagosome into the cytoplasm for processing and display on MHC Class I molecules remains unresolved. One intriguing mechanism involves apoptosis of infected macrophages with antigens being carried within apoptotic vesicles, which are then ingested into the cytoplasm of bystander APCs such as DCs (Kaufmann and Flynn 2005). CD8+ T cells can potentially have a number of functions. They can produce cytokines such as IFN-␥ and TNF-␣ to activate macrophages, kill infected host cells or kill intracellular bacteria through their cytotoxic mechanisms. These functions have all been reported in vitro models on murine or bovine TB infection, but have not yet been clearly described in human. Evidence from the mouse model suggests that the cytotoxic functions of CD8+ T cells are at its peak in early infection and then decline to very low levels, whereas cytokine-producing functions are also at peak early but remain high throughout the latent stage of infection (Kaufmann and Flynn 2005). Activation and Recruitment of B lymphocytes: Historically, B cells were not considered to offer any protection against M. tuberculosis (Kumararatne 1997). It is striking that BCG, the main vaccine against TB, confers protection against paediatric TB meningitis, but is not effective against adult pulmonary TB. A large volume of literature on antibody-mediated action against M. tuberculosis is contradictory and inconsistent (Glatman-Freedman 2006). However, studies using monoclonal antibodies to M. tuberculosis antigens namely the heparin-binding hemaglutinin adhesion (HBHA), MPB83 and the 16 kDa acrystallin, in vitro and in mice have shown significant protective effects leading to greater survival, enhanced granuloma formation, reduced lung pathogen concentrations in early stages of infection and growth inhibition (Chambers et al. 2004; Pethe et al. 2001; Williams et al. 2004). The mechanisms through which these particular antibodies exert their effects are unknown, but are thought to include interference with bacterial adhesion to host cells, promotion of phagosome–lysosome fusion in macrophages, neutralization of bacterial toxins, and complement activation. Evasion of the immune system In the majority of the cases, the bacteria are able to evade immune attacks to establish a latent infection. How do they do this? Of the evasion mechanisms listed in Table 1, the ones most welldescribed in the literature are the arrest of phagosome-lysosome fusion, resistance against reactive nitrogen intermediates (RNI) and NO and interference with MHC Class II antigen-presentation. Table 1 Effector mechanisms of innate and adaptive immunity, and their evasion by M. tuberculosis. Innate Immunity Cell type Effector mechanism(s) Evasion mechanism(s) Neutrophils Degranulation Production of ROIs NETs Phagocytosis Production of RNIs and NO Cytolysis of infected cells/intracellular bacteria Activation of macrophages through IFN-␥ Membrane attack complex, opsonisation and phagocytosis by macrophages Cytolysis of infected cells/intracellular bacteria Activation of macrophages through IFN-␥ and TNF-␣ Cytolysis of infected cells/intracellular bacteria Activation of macrophages through IFN-␥ Opsonisation and phagocytosis by macrophages Unknown Genetic resistance (mechanism unknown) Unknown Arrest of phagosome-lysosome fusion Genetic resistance (mechanism unknown) Unknown Unknown Unknown–possibly through binding Factor H Macrophages NK cells Complement system CD4+ T cells Adaptive immunity CD8+ T cells B cells Interference with MHC Class II antigen presentation Interference with MHC Class II antigen presentation Unknown Unknown No evasion mechanism known. M. tuberculosis appears susceptible to Ab-opsonised phagocytosis Abbreviations: Ab, antibody; IFN, interferon; MHC, major histocompatibility complex; NET, neutrophil extracellular trap; NO, nitric oxide; RNI, reactive nitrogen intermediate; ROI, reactive oxygen intermediate; TNF, tumor necrosis factor. A. Gupta et al. / Immunobiology 217 (2012) 363–374 Arrest of phagosome–lysosome fusion Following phagocytosis by macrophages, bacteria reside in plasma-membrane-derived intracellular vesicles called phagosomes. These phagosomes then mature by acquiring low pH, degradative hydrolases and reactive oxygen/nitrogen intermediates, and transiently fuse with lysosomes to form phagolysosomes. This exposes the bacteria to the action of hydrolytic enzymes such as hydrolases, proteases, super oxide dismutase and lysozymes secreted by the lysosomes, which kills the bacteria. This is the principal mechanism through which phagocytes kill ingested microbes (Russell et al. 2009). The maturation and fusion process is complex, but its details are becoming increasingly clearer. Shortly after phagocytosis, the bacteria-containing phagosome acquires a GTPase called Rab5 (either from the cytoplasm or from the plasma membrane). Rab5 then recruits a protein called VPS34, which generates PI(3)Pphosphatidylinositol 3-phosphate – on the cytosolic face of the phagosome. PI(3)P acts as a ligand for EEA1 (early autosomal antigen-1), which also complexes with Rab5 to tether and fuse with early (sorting) endosomes. EEA1 acquisition is believed to be important in the recruitment of another GTPase called Rab7, which facilitates the late fusion with lysosomes. Fusion of phagosomes with early and late endosomes is facilitated by supercoiled membrane-bound proteins called SNAREs (soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors). SNAREs are found on both the transport as well as the target vesicles, and help to ensure correct matching of phagosome cargo with its destination (Vieira et al. 2002). M. tuberculosis evades death by arresting the maturation of phagosomes and preventing their fusion with lysosomes. A number of theories have been put forward to explain how this happens (Fig. 1). It was initially believed that the bacteria produce ammonia, using its urease enzyme, to block the maturation process. However, M bovis BCG in which the urease gene has been blocked continue to inhibit phagosome–lysosome fusion (Sendide et al. 2004). Recent evidence suggests that M. tuberculosis-containing phagosomes retain the early endosomal marker Rab5 far longer than normal, and fail to recruit Rab 7, which in turn prevents fusion with lysosomes. Significantly, the phagosome appears to retain markers derived from plasma membrane, which allow acquisition of nutrients like iron by M. tuberculosis (Vergne et al. 2003a, 2004). Mycobacterial modulation of phagosomal maturation appears to be mediated by the cell wall components. There is considerable evidence that mannosylated lipoarabinomannan (ManLAM) inhibits VPS34 and acquisition of EEA1 (Fratti et al. 2001). A study found that phagosomes containing ManLAM coated beads were defective in recruiting SNARE protein syntaxin 6 and less efficient at accumulating cathepsin D, a lysosomal hydrolase, from the Trans-Golgi Network (TGN) (Fratti et al. 2003). This suggests than ManLAM disrupts the association of the bacteria-containing phagosome with TGN products. The study also found that ManLAM disrupts PI-3K signaling, thereby inhibiting the acquisition of EEA1. ManLAM appears to be incorporated in macrophage membrane rafts via its glycosylphosphatidylinositol (GPI) anchor, after being shed from the cell wall of the bacteria. It is unclear whether this originally happens at the cell or phagosomal membrane level, but it appears to be critical for the blocking action of the glycolipid (Welin et al. 2008). It has also been reported that ManLAM does not inhibit phagosomal maturation completely, suggesting that either all bacteria do not evade the killing mechanism, or there must be other mycobacterial products supplementing the ManLAM action (Welin et al. 2008). A few studies have demonstrated that ManLAM inhibits PI3P and EEA1 acquisition (and thus phagosome–lysosome fusion) by inhibiting Ca2+ signaling (Kusner and Barton 2001; Malik et al. 367 2000; Vergne et al. 2003b). There is evidence that this is achieved through the down-regulation of calmodulin-dependent signal transduction (Malik et al. 2001) and the inhibition of sphingosine kinase (Malik et al. 2003), which prevents the conversion of macrophage sphingosine to sphingosine-1 phosphate (S-1 P) and arrests the S-1P dependent increase in Ca2+ concentration. Significantly, modulation of Ca2+ signaling appears to require live, viable bacteria, which may explain why killed bacteria cannot inhibit phagosome–lysosome fusion. However, the precise links between reduced Ca2+ concentration and EEA1 acquisition are unclear, and there are some contradictions in the literature as to the need for elevated Ca2+ concentration in order to promote phagosome–lysosome fusion (Zimmerli et al. 1996). A recent study showed that the activation of p38 MAPK (mitogen-activated protein kinase) is associated with the exclusion of EEA1 from endocytic membranes, and that its inhibition increases the recruitment of EEA1 and the maturation of phagosomes (Welin et al. 2008). It was hypothesised that ManLAM might be responsible for activating p38 MAPK, but this has been shown recently not to be the case (Welin et al. 2008). The bacterial factor responsible for activating MAPK therefore remains unknown. It has also been suggested that the bacteria may prevent phagosomal maturation by retaining coronin-1 (also known as TACO, for Tryptophan-Aspartate-rich Coat protein) on the phagosomal membrane. However, this seems to be a feature associated with clumped rather than dispersed bacilli, and direct evidence of TACO retention by M. tuberculosis-containing phagosomes in human macrophages remains to be demonstrated (Flynn and Chan 2003; Schuller et al. 2001). Interestingly, a recent study reported that pulmonary surfactant protein D (SP-D), a surfactant associated hydrophilic protein belonging to collection family, binds to and masks M. tuberculosis ManLAM. This appears to prevent ManLAM from binding to the macrophage’s mannose receptors (MRs), and reduces intracellular survival of ingested bacteria by enhancing phagosome–lysosome fusion (Ferguson et al. 2006). In contrast, surfactant protein A (SPA) enhances the phagocytosis by upregulating the macrophage MRs (Beharka et al. 2002). SP-A also binds to M. tuberculosis 60 kDa cell wall protein via the carbohydrate recognition domain although it may not be clear what the major effect of this binding is (Pasula et al. 1997). Resistance against reactive nitrogen intermediates and nitric oxides The mycobacteria are not able to survive in macrophages activated with IFN-␥ produced by T cells (Flynn and Chan 2003). The production of toxic RNI through NO synthase 2 (NOS2) dependent pathway triggered by IFN-␥ is another anti-microbial killing mechanism available to macrophages. This has been shown to be essential for TB control in mice (Ferguson et al. 2006). Significantly, NOS2 is expressed in murine lung granulomas throughout acute as well as latent phases of infection, and inhibition of this enzyme leads to reactivation disease (Flynn and Chan 2003). A critical role for RNIs in the control of human TB remains to be demonstrated (Kaufmann 2001; Lin and Flynn 2010; Yang et al. 2009). It has however been shown that human alveolar macrophages infected with M. tuberculosis produce NO, and that levels of NO are negatively correlated with levels of intracellular bacterial growth (Rich et al. 1997). Increased expression of NOS2 has also been reported in the lungs of patients with active pulmonary TB, and alterations in the NOS2A gene in humans have been associated with increased susceptibility to TB (Lin and Flynn 2010). However, recent immunohistochemical studies have demonstrated low levels of NOS2 expression and NO production in human macrophages (Carsillo et al. 2009). 368 A. Gupta et al. / Immunobiology 217 (2012) 363–374 Fig. 1. The arrest of phagosome–lysosome fusion by M. tuberculosis. Following phagocytosis, the bacteria are internalised into vesicles called phagosomes. These phagosomes then need to find, tether against and merge with toxin-carrying lysosomes for the bacteria to be killed. The matching, tethering and fusion of phagosomes and lysosomes is mediated by a number of enzymes and “marker” molecules that need to be recruited to phagosomal membranes. M. tuberculosis arrests phagosome–lysosome fusion by interfering with the recruitment of these molecules. Abbreviations: EEA1, early endosome antigen 1; ManLAM, mannosylated lipoarabinomannan; Rab, proteins from Ras superfamily of small GTPases; SNARE, soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors; PI-3P, phosphatydilinositol-3-phosphate; VPS, vaculor protein sorting. The cytokines TNF-␣, IL-1␤ and IFN-␥, all of which are produced by TH 1 lymphocytes, are believed to activate the inducible form of the enzyme NO synthase within macrophage phagosomes. This enzyme catalyses the production of NO from arginine (Abbas and Lichtman 2006; Yang et al. 2009). NO is one of several RNIs – it can react with superoxide anion (O2 − ) to form peroxynitrite (ONOO− ), a powerful oxidant. These products (NO and related RNIs) can then attack bacterial DNA, proteins and lipids. NO can also cause enzyme dysfunction in bacteria through its effects on protein accessories such as iron–sulphur groups, heme groups and amines (Yang et al. 2009). In the M. tuberculosis genome ahpC gene encodes alkyl hydroperoxide reductase subunit C (AhpC). This M. tuberculosis enzyme has been shown to protect Salmonella typhimurium and mammalian cells from RNI toxicity (Chan and Flynn 2004). AhpC catalyses the breakdown of ONOO− by forming an antioxidant complex with peroxidase and peroxinitrite reductase activity, in conjunction with dihydrolipoamide dehydrogenase (Lpd), dihydrolipoamide succinyltransferase (SucB) and thioredoxin-like AhpD (Bryk et al. 2002). Another M. tuberculosis gene, msrA, codes for methionine sulfoxide reductase (MsrA) which converts methionine sulfoxide, a product of a reaction between ONOO− and methionine residues of proteins, to methionine (St John et al. 2001). This is believed to be an enzymatic basis for RNI resistance in M. tuberculosis (Fig. 2). In addition to ahpC and msrA, two other genes (nox1 and noxR3) have been implicated in RNI resistance, although their mechanism of action remains unclear. Intriguingly, recent microarray analyses have shown that RNI regulates M. tuberculosis gene expression in vitro. This has been corroborated using a murine model in vivo. The pattern of gene expression appears to be similar for both nitrosative as well as oxidative stress, suggesting that resistance against both might be mediated through a common signal transduction pathway (Schuller et al. 2001). Interference with antigen presentation Antigen presentation is a crucial part of activating killing mechanisms in both innate and adaptive immunity. There are three known routes of antigen presentation. One is seen when M. tuberculosis peptide antigens get processed and presented on MHC Class II molecules by APCs to CD4+ T lymphocytes, which induce the killing of either infected cell and/or intracellular bacteria on their own by secreting IFN-␥ or TNF-␣. The second is seen when M. tuberculosis peptide antigens are processed and displayed on MHC Class I molecules to cytolytic CD8+ T cells, which then kill the infected cells and/or their intracellular bacteria by secreting toxic granules. The third is when M. tuberculosis lipid or glycolipid antigens (particularly mycolic acids and ManLAM) are recognised on CD1 molecules A. Gupta et al. / Immunobiology 217 (2012) 363–374 369 Fig. 2. Synthesis, regulation and anti-mycobacterial action of NO and its evasion by M. tuberculosis. Cytokines TNF-␣ and IL-1␤, and IFN-␥, secreted by APCs and T cells respectively, activate the inducible form of NO Synthase (iNOS) within macrophage phagosomes. iNOS catalyses the conversion of arginine to NO, which reacts with superoxide anion (O2 − ) to form peroxynitrite (ONOO− ), a powerful oxidant. This causes damage to bacterial DNA and kills M. tuberculosis within phagosomes. Cytokines released by TH 1 cells activate iNOS, whilst those from TH 2 cells inhibit it. M. tuberculosis has evolved mechanisms to resist nitrosative stress by using reductases to break down oxidizing agents such as peroxynitrite. by CD8+ T cells or NK cells, which then kill the infected cell through the release of cytotoxic granules. Since M. tuberculosis is able to survive these cytolytic attacks in latent infection, the pathogen is likely to have evolved a method to inhibit antigen presentation, and thus evade killing mechanisms. It has been shown in murine and in vitro human macrophage models that (a) infection with M. tuberculosis reduces the expression of MHC Class II molecules on APCs, and diminishes antigen presentation to T lymphocytes, and (b) the bacteria appear to inhibit the IFN-␥ induced up-regulation of MHC Class-II expression in infected APCs. The degree of inhibition is much higher with viable compared to heat-killed bacteria, indicating a mechanism that relies at least partially on metabolic activity rather than simply a constitutive element of the bacteria. The expression of MHC Class I molecules does not appear to be affected by infection (Hmama et al. 1998). The antigen processing and presentation pathways are distinct for MHC Class I and Class II molecules. For Class I presentation, the phagocytosed antigen proteins are delivered in an endoplasmic reticulum (ER)-independent, but a cathepsin-S-dependent manner and the process is called cross presentation (Rock and Shen 2005). For Class II presentation, the phagocytosed antigens must be degraded within phagosomes or phagolysosomes into peptide fragments, which are then secreted together with HLA-DM (human leukocyte antigen-DM) in an endosomal vesicle. At the same time, MHC Class II molecules are synthesised in the ER, carrying with them an attached protein called the class II-associated invariant chain peptides (CLIP) which binds into the peptide-binding cleft and stabilises the molecule. This molecule is then translocated towards the cell membrane in an exocytic vesicle, which fuses with the endosomal vesicle containing processed peptides. The HLADM molecule removes the CLIP, and enables a peptide fragment to be loaded on to the molecule. If this occurs, the molecule is once again stabilised and can be delivered to the cell surface. If not, it is degraded by proteases in the endosome (Abbas and Lichtman 2006). Inhibition of antigen presentation could occur on one or more steps of this MHC Class II pathway. Table 2 considers the possibilities and the evidence from the literature to support or refute each of them (Fig. 3). For MHC Class I presentation, M. tuberculosis enters into the cytosol of the infected cells through different possible routes where mycobacterial proteins undergo proteolysis and then transfer to ER by the transporter associated with antigen processing (TAP), The one potential pathway involves the translocation of mycobacteria to cytosol through a transporter referred as dislocon in the phagosomal membrane (Baena and Porcelli 2009). It is also possible that some membrane disrupting proteins of mycobacteria like ESAT-6 and CFP-10 are responsible for the leakage into cytosol (Baena and Porcelli 2009). An alternative pathway is the vacuolar model where peptides are generated within phagosomes and loaded on MHC I molecules directly (Baena and Porcelli 2009). There is another pathway known as the detour pathway which exists for MHC Class I presentation of M. tuberculosis antigens, and it depends upon the uptake by apoptotic cells (Baena and Porcelli 2009). M. tuberculosis clearly uses multiple pathways to disrupt antigen-presentation on MHC Class II molecules. First, it is possible that its ability to inhibit phagosome–lysosome fusion also impairs antigen processing. Second, although the evidence is conflicting, it appears likely that M. tuberculosis is able to inhibit the expression of MHC Class II molecules (particularly upon IFN-␥ activation) by down-regulating the expression of CIITA (class II transactivator). Third, this is consistent with the ability of the bacteria to interfere with intracellular processes for co-localising MHC Class II molecule and peptide antigen during peptide loading. It has been argued that the mechanisms described above fall into two categories: those that have an immediate effect on the cell’s ability to display antigen (i.e., between 1 and 10 h post-infection), and those that act after 10 h (Chang et al. 2005). The first set includes disruption to antigen presentation on MHC molecules already available, and so involve either inhibition of antigen processing, MHC Class II-endosome co-location, or peptide loading. The second set 370 A. Gupta et al. / Immunobiology 217 (2012) 363–374 Table 2 Antigen presentation pathway for MHC Class II and its inhibition by M. tuberculosis. Pathway step Possible interference Evidence 1 Antigen processing Inhibition of proteolysis within phagosome 2 MHC Class II molecule formation Inhibition of gene expression (i.e., mRNA synthesis) for MHC Class II, including the normal up-regulation on activation with IFN-␥ Macrophages cultured with M. tuberculosis LAM fail to display antigen from whole viruses, but are able to display synthesised epitopes (Moreno et al. 1988) M. tuberculosis does not affect gene expression, synthesis or steady state levels of class II molecules (Hmama et al. 1998), but this is contradicted by another study (Noss et al. 2000), which finds that M. tuberculosis 19-kDA lipoprotein inhibits MHC Class-II expression. Further, it has been demonstrated (Wojciechowski et al. 1999) that M. bovis BCG down-regulates the mRNA expression of a regulatory protein called CIITA, which down-regulates the expression of MHC Class II in murine macrophages. This result has been confirmed this result for human macrophages (Pai et al. 2003) M. tuberculosis infection appears to have minimal effect on assembly or processing of Class II molecules through the Golgi apparatus (Hmama et al. 1998) The intersection of vesicles containing MHC Class II molecules with late endosomal structures containing antigen is impaired in infected cells (Hmama et al. 1998) Once the MHC Class II molecule reaches the endosomal compartment, either the removal of the CLIP or the loading of the peptide is blocked (Hmama et al. 1998) No evidence to show that this part of the pathway is affected by M. tuberculosis infection Disruption to assembly of MHC Class II complex, or their transport to endosomal vesicles Prevention of co-localisation of MHC Class II with processed antigen 3 MHC Class II–peptide co-location in late endosomes 4 Peptide loading Inhibition of peptide loading on to MHC Class II molecules in endosome 5 Endosome transport to cell membrane Inhibition of endosomal transport of loaded peptide to cell membrane Abbreviations: CLIP, class II-associated invariant chain peptides; LAM, lipoarabinomannan; RNA, ribonucleic acid. includes processes required for the continued supply of new MHC Class II molecules, of which the most important is the regulation of gene expression for MHC Class II synthesis. The argument is that it is beneficial for the bacteria to maintain several evasion pathways that act over different time scales, because this ensures continual inhibition of antigen presentation, evasion of the immune surveillance and the establishment of latent infection. A few mycobacterial factors implicated in interference with antigen presentation have been identified. (1) ManLAM attenuates IFN-␥-induced expression of MHC-Class II molecules by a human macrophage cell line in vitro (Chan et al. 1991), (2) a M. tuberculosis 25-kDa glycolipoprotein has been implicated in the suppression of MHC Class II molecules (Wadee et al. 1995); and (3) a M. tuberculosis 19-kDa lipoprotein inhibits IFN-␥ - induced MHC Class II presentation through a toll-like receptor (TLR)-2-dependent pathway (Noss et al. 2001). It also induces maturation of DCs through TLR2 shortly after primary infection, but in the latent stage, it might be responsible for down-regulating MHC II expression and priming of T cells (Flynn and Chan 2003). The ManLAM mediates the inhibition of phagosome–lysosome fusion as well as disrupts antigen presentation to lymphocytes. CD8+ T cells, NK cells and the complement membrane attack complex It is evident that M. tuberculosis is able to evade the effector functions of macrophages and CD4+ T cells in order to establish a latent infection. Thus, the interaction between these two cell types is the mainstay of the human immune response to TB. However, the involvement of CD8+ T cells, NK cells and the complement system in anti-TB defense is nevertheless interesting and important. NK and CD8+ T cells are clearly recruited against M. tuberculosis. There is no evidence that (unlike MHC Class II) antigen presentation on macrophage MHC Class I or CD1 molecules is inhibited by the bacteria. It is also clear from in vitro studies that both NK and CD8+ T cells are capable of killing the bacteria. As to the complement system, there is evidence now to show that M. tuberculosis activates all three pathways of complement, and that opsonin C3b binds to the bacteria, which ought to lead to cytolysis through the formation of a membrane attack complex. Why is the bacteria then not eliminated by one or all of these three complement pathways? A speculative explanation might be that in fact, CD8+ T cells and NK cells do lyse infected cells. However, the rate of elimination just balances the rate of replication of bacteria, and so a steady latent infection concentration is maintained. As to the complement system, it is possible that complement opsonisation is so effective in inducing phagocytosis by macrophages that the bacteria are never exposed to the late steps of the complement pathway. Alternatively, the bacteria’s ability to bind factor H might be responsible for diminishing the generation of the lytic complex of complement (Carroll et al. 2009). Further research in this area will shed more light on whether these speculations are valid. The granuloma An important question concerning the immunology of latent TB is the location of the bacteria within the host. There is evidence to suggest that most immuno-competent hosts mount a strong immune response that limits the bacteria to the lungs and the local draining lymph node (together called the “Ghon complex”) (Gomez and McKinney 2004). It is therefore generally assumed that the bacteria must latently reside within lung or lymph node granulomas (Bouley et al. 2001). However, the evidence on this is not conclusive. Granulomas are the characteristic feature of human latent pulmonary TB. They are formed when innate and adaptive immune responses are not able to rapidly eliminate the pathogen. As a result, immune cells and cytokines accumulate around infected cells to control the spread of infection. In TB, mature granulomas evolve when undifferentiated mononuclear phagocytes are recruited in response to M. tuberculosis infection, collected around the infected cell(s), andactivated by cytokines such as IFN-␥. Activation is associated with an increase in size and cell organelles, and a “ruffled” cell membrane. The activated macrophages develop elongated nuclei, and link up their cell membranes in zipper-like arrays to form a physical cellular barrier to the further expansion of the pathogen. Human tuberculous granulomas are typically well-organised, with a central core of macrophages surrounded by T and B lymphocytes, DCs, endothelial cells, fibroblasts and granulocytes (Tufariello et al. 2003). The interaction between activated macrophages and A. Gupta et al. / Immunobiology 217 (2012) 363–374 371 Fig. 3. Expression of microbial peptide fragments on MHC Class II molecules. Microbes are first phagocytosed into vesicular compartments called endosomes, which then merge with lysosomes to produce digested peptide fragments. These fragments are then co-located in a vesicle with MHC Class II molecules synthesised in the ER. The peptide fragment is loaded on to the MHC Class II molecule. This stabilises the molecule, and it is then delivered to the cell surface. Abbreviations: ER, endoplasmic reticulum; CLIP, class II-associated invariant chain peptides; HLA-DM, human leukocyte antigen-DM. IFN-␥-secreting lymphocytes (particularly CD4+ T cells) is considered to be critical in controlling the infection. Additionally, TNF-␣ is involved in the formation and maintenance of granuloma integrity through its effects on expression of adhesion molecules and chemokines (Lin and Flynn 2010). Studies show that many asymptomatic humans harbour virulent bacteria in their tuberculous granulomas (Bouley et al. 2001; Tufariello et al. 2003). Whilst being responsible for controlling the infection, it is possible that the granulomas also paradoxically offer a niche for long-term survival of the bacteria. This can happen because macrophage-activating T lymphocytes are typically organised around the periphery of the granulomas, whilst the infected cells are located in the centre. Activated macrophages and giant multinucleated cells form a wall which, whilst preventing the bacteria from escaping out, also appear not to let more T lymphocytes in (Tufariello et al. 2003). This allows the bacteria to survive in a latent state within infected but inactivated cells. It has been suggested that, there are probably two populations of bacteria in the granulomas – those killed in infected but activated macrophages, and those that remain alive in non-activated macrophages. Disease reactivation Reactivation disease is difficult to distinguish clinically from re-infection. It occurs when latent bacteria from old, scarred granulomatous lesions are reactivated into an active, virulent state. Reactivation disease is most frequently triggered when the host immune response weakens or is suppressed – probably the stark case is that of HIV+ individuals, who are low in CD4+ T counts and face 10% risk per year of reactivation disease. The co-incidence of TB with HIV is now well documented (Shen et al. 2004). Studies with mice suggest that the immune response associated with the latent phase of infection may differ from those in the primary infection, with a more significant role for CD8+ T cells and a lesser role for CD4+ T cells (Flynn and Chan 2001). However, this is not completely consistent with studies in primates and humans (Shen et al. 2004). In addition, a large number of mycobacterial factors play leading roles either in immune evasion or in enabling reactivation. A group of proteins called the resuscitation promoting factors (coded by rpf genes) appear to be important in re-activation. It has been shown that deletion of rpf genes renders the bacteria unable 372 A. Gupta et al. / Immunobiology 217 (2012) 363–374 to reactivate even after immunosupression of the host (Biketov et al. 2007; Russell-Goldman et al. 2008). M. tuberculosis has five Rpf factors (Rpf A to E), and can survive multiple mutations across the underlying genes (Hett and Rubin 2008). Although they do not appear to be necessary for general viability, rpf genes are believed to play a crucial role in inducing mycobacteria out of a dormant (and hence possibly a latent) state. The Rpf proteins are believed to act by hydrolyzing peptidoglycan, possibly acting in conjunction with another (unknown) protein. The basic idea is that the bacteria “shut down and close tight” in response to environmental stress, with very heavy cross-linking between peptidoglycan strands causing a thickening and decreased permeability of the cell wall. When the stress agent is removed, rpf genes are activated, and these produce Rpf proteins that snip the tight peptidoglycan strands and enable the bacteria to re-enter a growth phase (Hett and Rubin 2008). Each of these could potentially be a drug target, and finding novel methods to block the persistence factor(s) would force the bacteria out of the dormant stage and help prevent latent infection. The dormancy (DosR) regulon of three-gene operon including dosR (Rv3133c) has been found to be crucial for rapid resumption of the growth once M. tuberculosis exits an anaerobic or NO-induced non-respiring state and essential for M. tuberculosis to transit between respiring and non-respiring conditions without loss of viability (Leistikow et al. 2010). Reactivation of latent tuberculosis infection with necrosis and death of mice was observed in TNF deficient mice (Botha and Ryffel 2003). Recently, a monkey model of co-infected with latent TB and mucosal simian immunodeficiency virus was developed to explore the factors associated with reactivation of latent TB infection in immunodeficient humans (Diedrich et al. 2010). Immunosupressed animals with low peripheral CD4T cells show higher reactivation of TB (Diedrich et al. 2010). The results of the study suggest that initial T cell depletion strongly influence the outcomes of HIV-M. tuberculosis co-infection and disease progression (Diedrich et al. 2010). Conclusions and perspectives Having reviewed the principal aspects of immune evasion, dormancy and reactivation in M. tuberculosis, it is tempting to ask: (1) what are the major gaps in our knowledge in this field, (2) what unresolved questions should future researchers focus on as a matter of priority?, and (3) what are the implications of the current state of knowledge for identifying targets for drug and vaccine design? On the aspect of the Immunology of TB infection, the following unresolved questions arise: (1) How is the initial entry of the pathogen into alveolar macrophages mediated and does the route it takes make a difference to bacterial survival within the macrophage? Answering this question could help us design strategies to interfere with these interactions, thus deviating the bacteria to take alternative routes that are likely to inhibit its intracellular survival within macrophages; (2) Evidence based on the cases of extra-pulmonary tuberculosis do suggest that the bacilli can seed, remain latent and cause pathology in other organs such as bone and meninges. Does it establish a latent infection everywhere or only in specific niches like lung granulomas? Is the pathogen sequestered in unknown extra-pulmonary locations? Answering this question would add information about simulations of the latent phase that currently rely principally on granulomatous niches; (3) What roles do B lymphocytes, NK cells, complement proteins and CD8+ T cells play in defence in vivo against the pathogen? Can these immune components be recruited to kill the bacteria and/or the infected macrophages? Answering this question would help design therapies to boost individuals’ innate immunity and prevent the establishment of latent infection; (4) granulomas appear to control the infection but also protect the bacilli by limiting access to T lymphocytes. Is it possible to modulate the structure of the granuloma so that T lymphocytes are given better access to infected macrophages in order to completely eliminate the primary infection? Understanding the precise role of TNF-␣ would be a crucial step forward in this goal. Acknowledgements The authors of this manuscript acknowledge the supports from the Medical Research Council, UK for a New Investigators Research Grant (G0801956) to SB, a Collaboration Grant to TB Drug Discovery-UK (www.tbd-uk.org.uk) and Brunel University for BRIEF awards and provisional of infrastructure funding to AT and UK. 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