Intracellular pathogens escape immune recognition by hiding inside the host cell, enabling them to survive an active immune response. Antibodies and hu-moral effector molecules rarely reach these pathogens. However, intracellular mycobacterial components are proc-essed by the infected host cells and pre-sented on the cell surface through ma-jor histocompatibility complex (MHC) gene products. Recognition of these complexes can prime pathogen-specific naïve T lymphocytes. In turn, activated T cells mediate host protection through activation of bactericidal functions of infected macrophages, transforming the quiet habitat into a hostile environ-ment for the pathogen.
Intracellular bacteria are generally as-sociated with low toxicity for the host cells. Therefore, immune responses are able to control pathogen replication. As the immune responses are usually un-able to fully eliminate the pathogen, it will persist in the host in a state of symp-tom-free infection. This is the start-point for a long-lasting struggle between in-tracellular pathogens and activated im-mune cells.
These main features of intracellular bac-teria could explain the characteristic clinical features of disease due to infec-tion with M. tuberculosis:
(1) Disease is not an inevitable re-sult of infection and latency is the rule,
() TB is characterized by a chronic course and finally,
() The pathogenicity is mainly determined by the immune re-sponses.
Immune responses against M. tu-berculosis
Immune responses against pathogens can be subdivided into two main types of immunity. The first one, innate immu-nity or natural immuimmu-nity, depends on various leukocyte subsets, in particular the cells of the mononuclear phagocyte system (the macrophages), the cells of the dendritic cell (DC) lineage, the granulocytes and the natural killer (NK) cells. The second subtype corresponds to the adaptive immunity that involves lymphocytes. Other cells behave like intermediates between the innate and adaptive immune system, like
uncon-ventional lymphocytes (γδ T cells and NK-T cells). Innate and adaptive immune responses are components of an inte-grated system of host defence, and they cooperate and influence each other. For example, cells from the innate response present antigens to lymphocytes and secrete mediators that will regulate adaptive immune responses. In turn, ac-tivated lymphocytes may turn on effec-tor mechanisms, coordinating some ac-tions of the innate immune responses.
Innate immunity
In order to establish infection, M tuber-culosis has to circumvent the epithe-lial barrier of the respiratory system.
Roughly estimated, only 10 % of the in-haled bacterial load finally reaches the deep lung. Most of the bacilli encounter the upper respiratory epithelium where they will be removed by the mucociliary escalator.
Innate immunity is a fast, non-specific reaction that initiates antimicrobial re-sponses but lacks memory. It is respon-sible for relatively heavy inflammatory responses directly or indirectly toxic to pathogens. The initiation of such re-sponses is mainly based on the capacity of the mononuclear phagocytes and DC to recognize molecules exclusively syn-thesized by bacteria, such as lipopoly-saccharides (LPS), bacterial lipoproteins and lipoteichoic acids. These invariant molecular patterns are recognized by pattern-recognition receptors, like the LPS-receptor (CD14), macrophage man-nose receptor, scavenger receptors and Toll-like receptors (TLR).
Toll-like receptors
TLR are membrane receptors that were first described in Drosophila. These re-ceptors allow the host immune system to quickly detect the presence of for-eign pathogens and consequently gen-erate a signal in order to rapidly mount a vigorous defence. Interactions between microbial products and TLR facilitate the transcription of genes that regulate both innate and adaptive immune responses.
The family of TLR is currently composed of 1 proteins that bind pathogen-asso-ciated molecular patterns (PAMP), some of which are expressed by M. tuberculo-sis. For instance, mycobacterial products are mainly recognized by TLR (19 kD li-poprotein, LAM), TLR4 (LAM, HSP65) and TLR9 (TB DNA) (figure 13) (67).
TLR are expressed by several cell types (e.g. monocyte/macrophages or DC), and in several tissues. The largest diver-sity of TLR mRNAs is found in profession-al phagocytes, suggesting a key role of TLR in innate immunity. TLR-PAMP inter-actions trigger the secretion of chemok-ines/cytokines, the upregulation of cos-timulation molecules (i.e. CD80/CD86) and antigen presenting molecules, and enhance the antigen presenting cell (APC) survival. As TLR are also a prereq-uisite for the induction of clonal anti-gen-specific immune responses, these molecules can be considered as bridges between natural and adaptive immune responses.
The adapter protein MyD88 is one of the major players of the signal transduction following TLR engagement. It has been recently shown that MyD88-/- mice die within 4 weeks after infectious challenge with M. tuberculosis because of excessive
growth of the pathogen, illustrating the importance of the TLR signalling during M. tuberculosis infection(85). MyD88 -/-mice were capable of adaptive immune responses (e.g. normal lymphocytic re-cruitment). However, they were unable to control the infection because of low nitric oxide (NO) synthesis by infected macrophages and low expression of crucial cytokines, such as TNF-α or 1. In this model, low expression of IL-1 is responsible for the Th skewing of the CD4+ T cells, illustrating the role of TLR interactions in the induction of Th1 responses. Based on these observa-tions, the investigators concluded that
in absence of MyD88, and indirectly of TLR-mediated signals, T cell-mediated immunity can only provide a partial pro-tection from TB infection.
Natural killer cells
The natural killer (NK) cells are large granular lymphocytes characterized by surface expression of the CD16 mol-ecule, a receptor for the Fc portion of immunoglobulin (FcRγIII), and/or by the CD56 marker, an adhesin molecule.
These cells are distinct from T cells or B cells but share some T cell functions.
NK cells are able to kill the M. tubercu-losis-infected cells notably in a perforin/
granulysin-dependent manner. How-ever, the precise effector mechanism remains unclear (86, 87). NK cells also regulate the cytotoxic activity of macro-phages against M. tuberculosis (88) and are potent IFN-γ producers, a cytokine crucial for macrophage activation (see below). NK cells are not only key effec-tors in the innate immune response but also regulate adaptive immune respons-es, such as the maturation of cytotoxic CD8+ T cells or the proliferation of γδ T cells during antigenic stimulations with mycobacterial antigens (89). Despite ef-fector functions and early expansion of NK cells after infection with M. tubercu-losis, depletion of NK cells did not sub-stantially impair host resistance (87).
This observation probably illustrates the redundancy with respect to other IFN-γ-producing lymphocytes. However, in situations with weakened adaptive im-mune responses, NK cells can mediate T-cell-independent mechanisms of host resistance to M. tuberculosis. Based on the results in the RAG-/- mouse model, it was hypothesized that NK cells may
Figure13.MainToll-likereceptorsinvolvedinanti-TBimmuneresponsesandtheirmycobacterial ligands. (See text). Abbreviations : HSP65, heat shock protein 65; LAM, lipoarabinomannan; LM, Lipomannan ; PIMs, phosphatidyl-myoinositol mannosidases; STF, soluble tuberculosis factor;
TIRAP, TIR domain-containing adapter protein.
Reproduced from: Doherty TM, Arditi M. Tb, or not tb: That is the question -- does TLR signalling hold the answer? The Journal of Clinical Investigation 004; 114:1699-170
be a major barrier in immunocompro-mised individuals (90). However, this role of substitution by NK cells is not demonstrated in immunocompromised humans (e.g. HIV infection). For exam-ple, even if the percentage of NK cells dramatically increases during acute HIV infection, their proportion significantly diminishes during the chronic phase of the HIV infection, the latter being more strongly correlated with TB onset (91).
Neutrophils
With concentrations from to 5 million cells/ml, neutrophils are the most abun-dant leukocytes in the blood (up to 40-65 % of the white blood cells). As neu-trophils are part of the first line defence against bacterial and fungal infections, neutrophils dysfunction is associated with susceptibility to severe infections caused by these pathogens. Although the role of the neutrophils during M.
tuberculosis infection is not well under-stood and needs further characteriza-tion, it is known that the influx of neu-trophils is the earliest response after invasion of macrophages by mycobac-teria (9, 9). Neutrophils are also found in BAL and pleural spaces during active human TB (94). This rapid neutrophil response contributes to the control of replicating intracellular bacteria by both phagocytic and non-phagocytic func-tions. However, neutrophils are useless against slow or non-replicating bacilli (95, 96). This may explain why studies of neutrophils in a murine model of TB failed to show a dominant protective role of these cells. When exposed to M. tuberculosis or its components, neu-trophils generate potent chemokines (e.g. IL-8, GRO-α, MIP-1α, etc…) that
contribute to the recruitment of ad-ditional inflammatory cells. However, neutrophils have a short half-life, and the presence of pro-inflammatory cy-tokines, such as TNF-α, or the presence of M. tuberculosis itself, accelerates this phenomenon by inducing apoptosis (97, 98). In conclusion, neutrophils may be considered as important players in the first-line defence against mycobacterial invasion: their chemokines initiate the inflammation and recruit more relevant cells. Moreover, neutrophils contribute to the early control of replication of the bacilli. Their role during later phases of infection remains uncertain.
Macrophages and effector mecha-nisms
Macrophages are large phagocytic mononucleated cells. After bacterial in-fection, they play a role as APC, as well as effector cells. M. tuberculosis enters macrophages using receptor-mediated phagocytosis (complement receptors, mannose receptors and scavenger re-ceptors). In contrast to polymorpho-nuclear cells, resting macrophages are long-lived cells with low antimicrobial potential. Therefore resting macrophag-es are a suitable habitat for intracellular bacteria. However, the microbicidal functions of these cells are strongly ac-tivated by the release of IFN-γ from NK cells during early phases and from lym-phocytes during later phases of infec-tion. Once macrophages are activated, they turn into a hostile environment for the bacilli, and ingested M. tuberculosis may be killed in the phago-lysosomal compartment. The toxic components of these vesicles are lysosomal hydrolases, reactive oxygen (ROI) and nitrogen (RNI)
intermediates. The protective role of the RNI against M. tuberculosis was dem-onstrated in several studies (99-101). In contrast, the role of ROI in the control of TB infection is still a matter of debate. It is thought that ROI alone are insufficient to kill M. tuberculosis, and synergistic in-teractions with RNI are likely (10).
M. tuberculosis has elaborated numer-ous strategies to allow it to survive and multiply within activated macrophages.
These include 1) invasion by means of complement receptor, in order to by-pass IL-1 and ROI triggering pathways (10); ) synthesis of LAM and other mycobacterial products that scavenge ROI (104); ) synthesis of superoxide dismutase and catalase to inhibit the respiratory burst that generates ROI; 4) intraphagosomal survival by inhibition of phagolysosome maturation (105); 5) downregulation of TNF-α activity (106).
Macrophages are the main source of IL-1 and of Tumor-Necrosis Factor-alpha
Monocytes/macrophages secrete sev-eral cytokines such as IL-1 and TNF-α, which are important for the granuloma formation (107).
IL-1 is produced early in infection by the components of the innate immunity, including infected macrophages and NK cells. It promotes rapid leucocyte migration and activation, and therefore it contributes significantly to the early phases of granuloma formation (107). IL-1 is a key molecule that links the innate to the adaptive immune response. For instance, IL-1 stimulates the expression of the IL- receptor by effector/memory T cells, allowing them to rapidly expand (108). However, inconsistent results from IL-1 neutralization experiments
prob-ably demonstrate the somewhat redun-dant role of different cytokines (107).
TNF-α belongs to the family of tumor-necrosis factors, also including TNF-β and lymphotoxins α and β. TNF-α seems to be a “leader” in the granuloma for-mation. This is illustrated by M. tubercu-losis infection of TNF-α deficient mice.
These mice form impaired and aberrant granulomas with subsequent severely repressed resistance to mycobacteria.
TNF-α is secreted by different cell types, such as macrophages, NK cells, poly-morphonuclear cells and lymphocytes.
In combination with IL-1, TNF-α is a key cytokine in early leukocyte recruitment to the granuloma. The development of Th1 responses is a positive feedback for its production and further contributes to the dynamic late-phase containment of M. tuberculosis within the granuloma (107). Moreover, TNF-α triggers mac-rophage activation, thereby creating a hostile habitat for M. tuberculosis. In humans, the use of drugs that neutral-ize TFN-α (e.g. infliximab) is associated with a dramatic loss of pathogen con-trol and development of postprimary TB (109). However, conflicting data are emerging about the exact role of inflixi-mab. The effect of infliximab on TB re-activation could be mediated by other mechanisms than the neutralization of TNF-α. For example, treatment of PBMC with infliximab is associated with con-comitant downregulation of important genes required for effective control of M. tuberculosis, such as IFN-γ and IL1-R-β (110). Unfortunately, TNF-α is a dou-ble-edge sword because when abun-dant, it also mediates systemic effects like pyrexia and cachexia. TNF-α is also involved in tissue destruction and the
generation of TB cavities. One hypoth-esis concerns the induction of matrix-metalloproteinase-9 (MMP-9) by TNF-α (111). For instance, MMP-9 is secreted by monocytes upon M. tuberculosis infec-tion and mediates the degradainfec-tion of the extracellular matrix.
Chemoattraction of monocytes: the example of MCP-1
The recruitment of fresh monocytes within the site of infection is important for the establishment and the mainte-nance of the infection within the granu-loma. One of the cytokines involved in the chemoattraction is the “Monocyte Chemoattractant Protein-1” (MCP-1 or CCL). MCP-1 is a powerful chemoat-tractant for monocytes, lymphocytes and NK cells that express its receptor CCR. MCP-1 is significantly produced and released during M. tuberculosis in-fection in humans and is associated with protection in murine models of TB (11).
Promoted by the release of TNF-α, the secretion of MCP-1 also attracts CD4+
and γδ T cells to the site of mycobac-terial infection. Flores-Villanueva et al.
have demonstrated that some genetic variations of the gene encoding MCP-1 may predispose M. tuberculosis-infected individuals to develop clinical disease (11). During human pleural TB, MCP-1 is responsible for about one third of the chemo-tractant activities which attract monocytes to the pleural space (114).
This latter observation allows us to un-derstand how genetic variations con-cerning this chemokine may significant-ly influence the outcome of the disease.
Dendritic cells
DC are professional APC that are crucial for the protection against M. tuberculo-sis infection by both eliciting primary and secondary T cell responses. Of course, it is well described that other cells can also present antigens, such as monocytes/macrophages, B cells and even epithelial/endothelial cells after activation by cytokines. In contrast to DC, the role of such cells in the antigen presentation process is less well char-acterized during the course of TB infec-tion. DC are present in the lung paren-chyma, and their number significantly increases during inflammation (115). In this context, mycobacterial antigens are presented to lymphocytes using both classical (MHC) and non-classical (e.g.
CD1) pathways. The uptake of M. tuber-culosis by DC is mediated by several pro-cedures depending on surface C-type lectins and Fc receptors (116-118). This is followed by phagocytosis and by mac-ropinocytosis. In this process, the spe-cialized C-type lectin DC-SIGN interacts with ManLAM resulting in the specific activation of the DC. In contrast, non-pathogenic environmental mycobac-teria express AraLAM, a LAM devoid of the mannose cuff that does not interact with DC-SIGN. DC-SIGN interactions al-low the pathogen to be targeted to lys-osome-associated membrane protein 1 (LAMP-1)-containing lysosomes. Within DC, LAMP-1+ lysosomes mature to late endosomes/lysosomes and thereby fa-cilitate the antigen processing and pres-entation. Conflicting data have been published with regard to the fate of M.
tuberculosis within the human mono-cyte-derived immature DC.
Maturation and migration of DC after in-ternalization of mycobacterial antigens is important for the determination of the type of immune response. After infec-tion, maturing DC express CCR7, a chem-okine receptor involved in the homing to the regional lymph nodes, where stimulation of T cells occurs. Migration of DC also involves a large network of chemokines, as demonstrated by ex-periments carried out in CCR knockout mice. In these mice, trafficking of DC is considerably reduced and associated with high mortality (119). This process of migration is involved in the dissemina-tion of M. tuberculosis from the primary site of infection to the regional lymph nodes (10). M. tuberculosis is also able to undermine the maturation of DC by: (1) reducing the expression of CD1, thereby diminishing the capacity of those cells to present mycobacterial lipid antigens (see below) (11) ; () inhibiting their expression of maturation markers and their capacity to induce T cell prolifera-tion (1, 1) ; () promoting the induc-tion of IL-10 secreinduc-tion (1).
It is quite possible that not all the mech-anisms of interference with DC matu-ration have already been described.
However, it is clear that such interac-tions between M. tuberculosis and DC depend on the M. tuberculosis strains. As a consequence, the ability to interfere with DC maturation could be a signifi-cant virulence factor (14, 15) (figure 14). As noted by Mendelson et al. (16), host factors also have to be taken into account. For instance, only a very weak proportion of DC was found within both the respiratory tract of rats at birth (17) and the tracheo-bronchial mucosa of infants less than one year of age (18).
Considering the high susceptibility of infants to severe clinical forms of TB and the incomplete maturation state of their few DC at the main site of entry of M.
tuberculosis, it might be hypothesised that the reduced capacity of infants to control M. tuberculosis infection may be due, at least partially, by quantitative and qualitative differences of their res-piratory tract DC compared to adults.
Beside their role in antigen presenta-tion, M. tuberculosis-matured DC secrete Th1-polarizing cytokines, such as IL-1, IL-18 and IL-. These cytokines drive the differentiation of activated T cells into IFN-γ-producing CD4+ and CD8+ T cells.
In addition, there is a balance between the proportion of incompletely matured DC, responsible for reduced protection, and the proportion of fully matured DC that better manage the control of infec-tion. This balance is also drastically influ-enced by both the M. tuberculosis strain characteristics (e.g. LAM ) and the host factors (figure 14).
Other cell types: the example of the alveolar epithelial cells
Other cellular subsets elaborate fast cytokine responses after exposure to M. tuberculosis. Of interest, alveolar epi-thelial cells are able to secrete IL-8, NO and even IFN-γ after M. tuberculosis chal-lenge (15). Moreover, these alveolar epi-thelial cells also express costimulatory molecules (19). Therefore, these cells might contribute at least to early im-mune responses against M. tuberculosis infection, by initiation of an inflamma-tory process, as well as anti-microbial responses.
In addition, their capacity to efficiently present antigens is a feature that may be relevant to their possible contribu-tion in the maintenance of a long-term immune memory. In this case, it is inter-esting to remind that HBHA is a privi-leged partner in the interaction of M.
tuberculosis with alveolar epithelial cells
and that the expression of HBHA is
and that the expression of HBHA is