It has been clearly demonstrated that in vitro, virulent M. tuberculosis can favor necrosis over apoptosis in infected macrophages, and this has been suggested as a mechanism for evading the host immune response. We recently reported that an effect consistent with this hypothesis could be observed in cells from the blood of TB patients, and in this paper, we review what is known about evasion strategies employed by M. tuberculosis and in particular consider the possible interaction of the apoptosis-inhibiting effects of M. tuberculosis infection with another factor (IL-4) whose expression is thought to play a role in the failure to control M. tuberculosis infection. It has been noted that IL-4 may exacerbate TNF-α-induced pathology, though the mechanism remains unexplained. Since pathology in TB typically involves inflammatory aggregates around infected cells, where TNF-α plays an important role, we predicted that IL-4 would inhibit the ability of cells to remove M. tuberculosis by apoptosis of infected cells, through the extrinsic pathway, which is activated by TNF-α. Infection of human monocytic cells with mycobacteria in vitro, in the presence of IL-4, appears to promote necrosis over apoptosis in infected cells—a finding consistent with its suggested role as a factor in pathology during M. tuberculosis infection.
1. Introduction
It is generally accepted that tuberculosis (TB) is responsible for 2-3 million deaths and more than 8 million new cases annually [1]. The majority of these occur in developing countries, especially in Sub-Saharan Africa [2], where a substantial proportion of the population (perhaps as much as a third) is thought to be latently infected. Though they are able to control the initial infection, they may later reactivate their disease if they become immunocompromised [3]. Infection with M. tuberculosis is associated with an active inflammatory immune response, characterized by elevated expression of both TNF-α [4–7] and IFN-γ [8–10]. These two cytokines are essential for controlling mycobacterial infections [11–14] but it is clear that in many cases, M. tuberculosis is able to survive this inflammatory process. Indeed, M. tuberculosis depends on the induction of an inflammatory response and the subsequent tissue damage for cavitation and dissemination via pulmonary disease to new hosts. It is probably for this reason that it expresses multiple molecules on its surface to promote inflammatory responses by the host.
It is therefore no surprise that M. tuberculosis has evolved a number of mechanisms by which it interacts with, and modulates, the host’s immune response. In addition to inflammation-promoting molecules [15], M. tuberculosis also expresses surface antigens that can induce IL-10 and IL-4, [16–18] that typically have an anti-inflammatory effect. Elevated expression of IL-4 (a cytokine with pleiotropic activity) has been implicated as a potential virulence factor, both for its anti-inflammatory capacity and apparent ability to promote tissue damage in association with TNF-α [19]. Higher levels of IL-4 expression also correlate with heightened immune responsiveness to ESAT-6, a proxy marker for infection in TB contacts [20–22] and for bacterial load. Finally, the ratio of IL-4 to IFN-γ or the IL-4 antagonistic splice variant, IL-4δ2, appears to be correlated with clinical status and in particular, with TB-related pathology [23–26] rather than of infection.
These studies suggest that IL-4 (alone or together with TNF-α) may play a role in tissue destruction and/or cell death during M. tuberculosis infection. Since cell death (by apoptosis) is a mechanism by which the host can remove infected cells [27, 28] while minimizing cell death and tissue destruction in adjacent, uninfected cells [29], this has obvious relevance for the control of M. tuberculosis infection. Indeed, there is a substantial body of literature suggesting that M. tuberculosis can directly interfere with the apoptosis of infected cells in vitro [30, 31] and that this appears to be directly related to virulence [32, 33]. In contrast, nonvirulent mycobacteria have a much weaker effect and, being dependant on dose, may even promote apoptosis [30].
This question has come under increasing scrutiny in the last few years, and the mechanisms by which M. tuberculosis can inhibit apoptosis are being rapidly identified [34]. However, the relative importance of apoptosis as a virulence mechanism in vivo and interaction of apoptotic mechanisms with the host cytokine response have until recently been largely unexplored and it is only recently that this area has come into focus [35].
2. M. tuberculosis and the Generation of Pathology
M. tuberculosis normally enters the host though the mucosal surfaces—via the lung after inhalation of exhaled droplets containing bacteria or less frequently through the gut after ingestion of bacteria (e.g., in milk from an infected animal). Although some M. tuberculosis-exposed individuals show no signs of infection or T cell memory—having possibly eliminated the pathogen via the innate immune response—the majority of exposed persons display the induction of a rapid inflammatory response. Cytokine and chemokine release triggers the swift accumulation of a variety of immune cells and, with time, the formation of a granuloma, characterized by a relatively small number of infected phagocytes, surrounded by activated monocyte/macrophages and lymphocytes [36]. Traditionally, the granuloma has been thought of as a containment mechanism of the host, but recent work suggests that granulomas are dynamic entities, growing and shrinking as cells are recruited and die [37]. The granuloma may eventually disappear, leaving a small scar or calcification, and the patient’s T cells become responsive to M. tuberculosis-derived antigens. However, if bacterial replication is not successfully controlled, the granuloma can increase in size and cellularity. The end point of this process is necrosis, and the tissue destruction caused by necrosis, can breach the mucosal surface allowing the granuloma contents to leak into the lumen of the lung or allowing M. tuberculosis to escape into the blood vessels of the lung, leading to further dissemination. Destruction of the lumen of the lung—a process referred to as cavitation—gives rise to the prototypic symptom of TB, a persistent cough with blood in the sputum. At this point the patient is infectious, spreading the bacteria by aerosol.
3. Inhibition of Early Host Responses
M. tuberculosis’s ability to persist within the host is directly linked to the fate of the immune cells which phagocytose it. The macrophage/monocyte thus occupies a pivotal place, as the prototypic host cell for M. tuberculosis, and also as the cell responsible for both killing the bacteria directly and priming immune responses by antigen presentation. M. tuberculosis interferes with immune activation at virtually every stage. The processes involved in the pathogen’s interference with vesicle trafficking and intracellular killing have been well described [38]. The processes involved in large-scale tissue destruction and cell death, however, remain to be mapped out.
Tissue destruction is not mediated directly by M. tuberculosi; the bacterium has little or no lytic activity; it is primarily an immunopathological process. Unlike pathogens such as Leishmania spp, which can establish chronic infection by evading the host immune response, M. tuberculosis actively provokes it. The pathogen expresses a number of molecules that bind to the host’s pathogen-associated molecular pattern (PAMP) receptors, such as the Toll-like Receptor (TLR) family [39]. Interestingly, despite M. tuberculosis’s long coevolutionary history with humanity, these molecules are largely conserved, even though most of them do not appear to be essential for the pathogen’s growth or invasive ability [40, 41]. The simplest explanation is that M. tuberculosis depends on the immunopathology that promotes necrosis both for dissemination within the host and for spread to new hosts, but also subverts this response, to allow it to persist in the host. Moreover, the ability of M. tuberculosis to rapidly alter its pattern of gene expression in response to stress [42] suggests that the pathogen may do both: in response to the local microenvironment, it may manipulate immune responses so as to favor apoptosis (reducing inflammation, thus allowing persistent infection) or necrosis (promoting tissue destruction, cavitation, and spread to new hosts).
Inhibition of inflammation at early stages may give M. tuberculosis a breathing space to initiate a productive infection. It has been suggested that invasion of phagocytes which are not yet activated is important for the bacteria’s survival since exposure of macrophages to IFN-γ and/or TNF-α before—but not after—infection decreases the ability of pathogenic mycobacteria to inhibit phagosome maturation and function [43] at least partially by upregulating the production of reactive oxygen and nitrogen derivatives [44–48]. Mannose derivatives on the pathogen’s surface molecules from pathogenic (but not nonpathogenic) mycobacteria inhibit phagocytosis by activated macrophages [49] perhaps targeting the pathogen to cells less prepared to contain it and inhibiting the initiation of inflammatory responses.
It does this in part, by targeting the very mechanisms involved in activating cell-mediated immunity. Though TLR2/4 ligation can initiate the inflammatory cascade in response to mycobacterial infection [50–53], it appears that interference in IFN-γ-signaling via TLR signaling is also a potential virulence mechanism [54]. The 19 kDa lipoprotein of M. tuberculosis appears to be a virulence factor [55] that reduces overall immunity to the bacterium in mice [56]. It is known to bind to TLR1/2 on host cells [57, 58] with resulting inhibition of inflammatory cytokine production (reducing expression of over a third of the IFN-γ-activated genes [59]), and reduced antigen-processing and MHC II expression [59–61]. The virulence factor ESAT-6 has a similar effect, also operating through TLR-2 [62], apparently by modulating TCR signaling pathways downstream of the proximal TCR signaling molecule, ZAP70 [63]. And other factors such as phosphoglycolipids bind to other PAMPs to induce IL-4 and IL-13, apparently contributing to virulence [16, 17, 64], and modulating cytokine expression in concert with other factors [65].
Indeed, M. tuberculosis appears to actively modulate cytokine expression at multiple levels. The mannose derivative lipoarabinomannan (LAM), which is expressed by pathogenic (but not nonpathogenic) mycobacteria, can bind to the DC-SIGN molecule, expressed on the surface of dendritic cells. The binding of LAM to DC-SIGN inhibits maturation and induces dendritic cells to secrete IL-10 [18, 66]. This inhibits antigen presentation, expression of MHC molecules, and expression of costimulatory receptors. Consistent with this, recent studies have found that expression of IL-10 is significantly elevated in TB patients with active disease [67–69]. LAM binding to DC-SIGN also inhibits the production of IL-12 by affected antigen-presenting cells. IL-12 is crucial to immunity to M. tuberculosis, as indicated by the effect of gene polymorphisms on susceptibility to TB, and the extreme susceptibility to mycobacterial disease of individuals with lesions in genes of the IL-12 and IL-12R pathways [70, 71]. Control of IL-12 expression is key to the expansion and activation of IFN-γ—secreting CD4 T cells which are crucial for immunity to TB, as shown by the susceptibility of animals or patients defective in CD4 T cell function or IFN-γ expression or recognition [72–76].
4. Activating and Modulating the Adaptive Immune to M. tuberculosis
Both CD4 and (to a lesser extent) CD8 T cells are thought to be crucial to containing M. tuberculosis infection via IFN-γ production and possibly cytotoxicity [77–79]. As discussed above, M. tuberculosis appears to subvert the host’s immune response, in part by directly countering the activation of T cell—particularly Th1-responses.
Consistent with this, IFN-γ recall responses are generally reduced in patients with advanced TB [80], while IL-4 is elevated [81–83]. The level of IL-4 gene expression appears to correlate with both disease severity in TB patients [81, 82] and risk of subsequent disease in TB-exposed individuals [23, 25]. The IFN-γ/IL-4 ratio increases in most patients during therapy, but decreases in contacts that become ill, suggesting that this state is directly related to the disease [25]. This is supported by reports that increased production of the IL-4 antagonist IL-4δ2 is seen in individuals who are controlling TB in its latent stage [20] and that the IL-4δ2 /IL-4 ratio increases during treatment of TB patients [25] and in those TB patients who respond most rapidly to therapy [84]. Similar observations have also been made in animal models of TB [85, 86]. A poor prognosis in TB is associated with a low IFN-γ/IL-10 ratio just as is seen for IFN-γ/IL-4 [8, 25, 87]. Altering the balance between IFN-γ and IL-4 or IL-10 production and function thus seems to be a second major survival strategy for M. tuberculosis, and the studies above suggest that when this balance is shifted towards IL-4, the result is increased pathology.
Although IL-4 can inhibit the effect of IFN-γ by decreasing the production of IFN-γ response factor-1 (IRF-1), a transcriptional element that enhances expression of IFN-γ-inducible genes such as iNOS [88], high levels of IL-4 are not associated with an absence of inflammatory factors. The proinflammatory cytokine TNF-α is a crucial component for protection against M. tuberculosis, as shown by the rapid reactivation of latent M. tuberculosis infection in people treated with TNF-α receptor antagonists [89, 90] and the susceptibility of TNF-α-deficient animals to M. tuberculosis [5, 7]. Nonetheless, TNF-α mRNA is elevated in TB patients [4] and in TB/HIV-infected patients elevated levels of TNF-α were associated with necrosis [91]. It has been suggested that while it is essential for protection, that in the presence of elevated levels of IL-4, TNF-α appears to promote tissue damage rather than protection [19, 92], possibly by a cooperative effect of transcription [93, 94]. These studies indicate that M. tuberculosis seems to have multiple mechanisms devoted to inhibiting both IFN-γ and TNF-α function and that the pathogen can evade killing by the immune system while still generating the pathology it needs for dissemination—and suggest that IL-4 may play a crucial role.
5. Cytokines, Cell Death, and Pathology
One clue to the mechanisms possibly involved is reports showing that resolving granulomas are rich in apoptotic cells and that inhibition of apoptotic capacity leads to reduced ability to control M. tuberculosis [95]. Granulomas are metabolically active sites, with cells being continually recruited and eliminated [37]. This can occur by several processes—but apoptosis or necrosis feature prominently. It has been suggested that apoptosis is a “silent” method whereby the host can remove infected cells [27, 28] while minimizing cell death in adjacent, uninfected cells, thus decreasing tissue destruction [29]. Antigens from engulfed apoptotic cells are presented, thus enabling cross-priming of the immune response [96]. Modeling studies suggest that TNF-α is one of the strongest factors controlling monocyte recruitment to the granuloma and that TNF-α-driven apoptosis is the strongest negative factor [97]. This is not surprising: TNF-α is a potent inducer of cell death by apoptosis [98]. Necrosis, on the other hand, is associated with the lysis of the infected cell, release of viable M. tuberculosis, and damage to the surrounding tissue [29] and TNF-α is also a major player here [91]. The centre of large unresolved granulomas often becomes necrotic and as mentioned above, this tissue destruction is an essential feature in the spread of M. tuberculosis.
There is now a substantial body of evidence from both in vitro and in vivo studies indicating that virulent M. tuberculosis (but not avirulent mycobacteria) can inhibit apoptosis, and that this may represent an escape mechanism whereby the pathogen can avoid the death of its host cell by apoptosis (and the internalized bacteria along with it as the apoptotic cell is digested) [32, 99–105]. Recent work suggests that M. tuberculosis can actively promote necrosis over apoptosis, consistent with the idea that this is a survival/virulence mechanism for the bacteria [106–109]. Supporting this hypothesis, studies indicate that elevated levels of necrosis are associated with genetic susceptibility to M. tuberculosis in mice [110] or virulence of human-derived clinical isolates [111] and that control of apoptosis via CD43/TNF-α inflammatory responses is important for control of M. tuberculosis [112]. Some of the genes involved such as nuoG have already been identified [113].
6. Interplay between TNF-α, IL-4, and Cell Death In Vivo
We therefore have started to examine the significance of TNF-α-mediated apoptosis in human TB. Recently published data [4] indicates that there is a strong upregulation of genes for factors that promote apoptosis in PBMC from individuals with active disease, including TNF-α and its receptors, Fas and FasL and pro-Caspase 8, when compared to exposed individuals without active disease. This is consistent with an important role for apoptosis in human TB. The fact that expression of these molecules are elevated in those with overt disease and also that the degree of expression of TNF-α correlated with severity of pathology in humans (author’s unpublished data and [91, 114]) suggests that TNF-α is directly involved in the generation of immunopathology. However, it is hard to reconcile inhibition of apoptosis as a mechanism for pathology if expression of apoptotic genes is highest in those with the worst pathology. A possible explanation for this is the observation that while expression of proapoptotic markers was elevated in PBMC from TB patients, when the CD14+ monocytic fraction was examined, the reverse was true [4]. Our conclusion was that monocytes from TB patients—but not monocytes from those infected with M. tuberculosis but asymptomatic, such as individuals with latent TB—were likely less responsive to extrinsic stimuli promoting apoptosis such as TNF-α. Further, we hypothesized that since it was highly unlikely that the majority of CD14+ cells in the blood were infected with M. tuberculosis, this effect was likely modulated by soluble factors. IL-4 is an obvious candidate, given that it is also the most elevated in these patients, it declines as symptoms abate during treatment [25], and its modulation of necrosis induced by TNF-α has been suggested in the past [115, 116]. Increased IL-4 and TNF-α expressions are also apparently associated with severity of pathology in mouse model [117], but interestingly, the only study to look at these factors in granulomas from human disease found no association between IL-4 and necrosis—though as the authors note, they could not distinguish between IL-4 and the IL-4 antagonistic splice variant IL4δ2 [91] which renders this difficult to interpret.
7. Interplay between TNF-α, IL-4, and Cell Death In Vitro
In the current absence of more data from human studies, we have examined this hypothesized interaction in vitro, infecting the human monocytic cell line THP-1 to observe what, if any, effect IL-4 had on the expression of genes that have been shown to be differentially regulated by mycobacterial infection [118–122] particularly those involved in activation of the extrinsic (inflammation-induced) pathway of apoptosis (TNF-α, TNFR1, TNFR2, Fas, FasL, and Caspase 8). Although not a perfect substitute, THP-1 cells have been frequently used as proxies for alveolar macrophages and have been used in many prior studies of mycobacteria-induced apoptosis [123]. We therefore infected these cells with the virulent M. tuberculosis Erdman strain (a clinical isolate) as a prototypical virulent mycobacteria, while the TB vaccine strain BCG Danish 1337 was used as the prototypical avirulent strain. Pilot experiments with the H37Rv and H37Ra virulent and avirulent strains were also done, with similar results to those reported here (data not shown). Bacterial infections were titrated down from a high dose (MOI of 50) down to a low dose (MOI of 5) based on previous publications [124]. We chose the higher dose, based on previous work, which suggested that a higher MOI was needed to induce rapid and detectable apoptosis in vitro [30, 123, 125].
To ensure that the infection protocol used induced significant levels of apoptosis, we infected THP-1 cells in the presence or absence of IL-4 at 10 ng/ml and monitored apoptosis and necrosis with a cell death ELISA kit, optimized to detect both apoptosis and necrosis. As can be seen in Table 1, after 24 hours, infection with BCG led to a major increase in the amount of apoptosis. Interestingly, the level of apoptosis was slightly reduced by IL-4 treatment alone, though this did not significantly decrease the increase of apoptosis induced by BCG. However, supernatants from the same cultures were assayed for total cell death to assess necrosis and this revealed that IL-4 had a significant effect on the balance of cell death. While IL-4 alone did not significantly affect the level of necrosis, in combination with BCG infection, it had a clear pronecrotic effect. Thus, IL-4 appears to bias cell death slightly towards necrosis over apoptosis, and this effect was enhanced by BCG infection (Table 1).
Alteration in cell death by apoptosis or necrosis in THP-1 cells infected with BCG or M. tuberculosis Erdmann (MOI 50) assessed at 24 hours by the Cell Death Detection ELISAPLUS photometric enzyme immunoassay (Roche Diagnostics, Lewes, UK) which measures cell death by both apoptosis and necrosis on fractionated samples. Associated changes in mRNA for the major host genes involved in the activation of the extrinsic pathway of apoptosis were assessed by quantitative Real-Time PCR, using HuPO as a housekeeping gene for normalization. Results shown are relative to untreated cells, of the means of assays from a single experiment (representative of 4) performed in triplicate (ELISA) or quadruplicate (RT-PCR). Values marked in bold text represent a significant increase, those in italics a significant decrease. The ANOVA test (with Dunnett’s multiple comparison posttest for all groups against untreated controls) was used for analyses between groups. In all instances, a P value <.05 was considered significant. A value of “<.1” indicates below the limit of detection, with “ND” indicates that the experiment was not done.
Infected with:
Fold change over uninfected THP-1 cells
Apoptosis
Necrosis
TNF-α
TNFR1
TNFR2
Fas
FasL
Caspase 8
null
+ IL-4
0.67 ± 0.13
0.79 ± 0.19
0.51
0.68
0.57
0.66
0.96
0.67
BCG
− IL-4
3.38 ± 0.04
1.06 ± 0.18
2.15
15.59
18.99
0.13
ND
4.35
BCG
+ IL-4
3.29 ± 0.08
2.68 ± 0.14
0.05
2.27
3.00
<0.1
ND
0.61
Erdmann
− IL-4
0.44 ± 0.22
1.13 ± 0.30
17.43
4.96
5.20
0.65
1.30
0.36
Erdmann
+ IL-4
0.37 ± 0.12
1.34 ± 0.07
4.21
1.38
1.89
0.34
1.15
0.35
The results with M. tuberculosis Erdman were strikingly different from BCG. At 24 hours, M. tuberculosis infection slightly reduced apoptosis and this effect was marginally (but not significantly) augmented by IL-4 (Table 1). But either M. tuberculosis infection or IL-4 treatment led to an increased ratio of cell death by necrosis compared to apoptosis.
This effect required living bacteria, as heat killed bacteria had no significant effect on apoptosis or necrosis after 24, 48, or 72 hours of culture (data not shown). We also looked at the effect of BCG and M. tuberculosis infection (with or without IL-4) after 72 hours of culture and obtained very similar results (data not shown). Finally, we confirmed the ELISA data by FACS analysis for annexin V, which was expressed by 16.28% after 24 hours of culture with BCG, while only 7.26% of control cells were positive. In the presence of IL-4, only 7.06% of BCG-exposed cells were annexin-V positive, confirming the ELISA data. These data are thus consistent with earlier studies suggesting that virulent (but not avirulent) mycobacteria are capable of inhibiting apoptosis, possibly as a defence mechanism against clearance by the host [32, 33]. In addition, the data suggest that IL-4 can also have a mild antiapoptotic effect—though it appears in this in vitro model that this inhibition of apoptosis by IL-4 does not prevent cell death, so much as renders host cells more susceptible to death by necrosis—potentially releasing bacteria which could reinfect adjacent cells, thus further promoting inflammation and immunopathology.
8. Effect of IL-4 and Mycobacterial Infection on Expression of Apoptosis-Modulating Genes
To examine the mechanism behind the IL-4 effect, we examined expression of multiple genes involved in activating pathways of induced cell death. It was clear from the apoptosis data (Table 1) that the processes driving apoptosis had already started by 24 hours. We thus performed the PCR analyses after 24 hours of culture, using quantitative PCR to compare the mRNA expression in infected and uninfected cells with or without IL-4 added to the cultures. As shown in Table 1, mycobacterial infection induced a strong TNF-α response at 24 hours, and strongly activated expression of the genes for the two TNF-α receptors. All of these activating effects were antagonized by IL-4. We also analyzed the supernatants from these cultures and found that in parallel with the induction of TNFR2 mRNA by BCG and M. tuberculosis, there was a significant increase (P<.01) in the amount of soluble TNFR2 protein detectable in culture supernatants 24 hours after infection (data not shown). This increase was identical for BCG and M. tuberculosis and was not inhibited in the presence of IL-4, suggesting that in the presence of IL-4, infected cells continue to shed the TNFR2 receptor at increased levels (compared to uninfected cells), at the same time in which mRNA production is downregulated by IL-4, potentially leading to reduced surface expression and further decreasing the responsiveness of these cells to TNF-α. This is consistent with the picture we drew from patient PBMC [4].
Gene expression for the proapoptotic molecule Fas was not affected by BCG infection, although it was significantly decreased by IL-4. In M. tuberculosis-infected cells, however, Fas expression declined significantly, (Table 1). Since this is likely to render M. tuberculosis-infected cells more resistant to Fas-mediated death, we also assessed expression of FasL in these cells, to gain an idea of what effect they might have on sensitized cells that came into contact with them. However, despite some variability, no significant differences in FasL expression were seen that could be attributed to IL-4 or M. tuberculosis infection (Table 1).
Downstream of both Fas and the TNF-α receptor complexes lies one of the major activating molecules of the extrinsic death pathway, Caspase 8. In BCG-infected THP-1 cells, pro-Caspase 8 transcription increased dramatically and this increase was inhibited by IL-4 consistent with the effects seen on apoptosis. In contrast, in M. tuberculosis-infected cells, the opposite was seen, with falling pro-Caspase 8 expression. IL-4 also reduced pro-Caspase 8 expression by itself, but this effect was not significantly different from that induced by M. tuberculosis infection. To determine if the decrease in Caspase 8 induced by M. tuberculosis infection could be countered by falling levels of apoptosis-antagonising molecules, we also assessed the levels of gene expression for the antiapoptotic molecule FLIPs. Here, however, we found significantly increased expression induced by M. tuberculosis infection (P<.01), suggesting that if anything, the antiapoptotic effect of decreased Caspase 8 would be amplified. Neither IL-4 nor BCG had a significant effect on FLIPs (data not shown).
In total, these data are consistent with prior findings that M. tuberculosis has an apoptosis-blocking effect and indicate that this affects not just the intrinsic pathway but also extrinsic activation of apoptosis mediated through the pro-Caspase 8 molecule, which avirulent mycobacteria do not share. In addition, they suggest that this is potentiated by IL-4, which promotes necrosis instead, supporting a role in the virulence of M. tuberculosis. The data also indicate that this antiapoptotic effect occurs at the gene transcription level and affects multiple gene pathways—though the simple experiments presented here are indicative, not definitive.
9. A Model for M. tuberculosis Pathogenesis
There is a significant body of evidence from both in vitro and in vivo studies indicating that virulent M. tuberculosis can inhibit apoptosis and that this may represent an escape mechanism whereby the pathogen can avoid the death of its host cell—and the internalized bacteria along with it [32, 99–105]. Knock-in studies using the nuoG gene of M. tuberculosis showed that this gene conferred the ability to inhibit apoptosis and increased virulence in mice to avirulent mycobacteria, while its deletion rendered M. tuberculosis less able to inhibit apoptosis of infected THP-1 cells [113]. A number of genes involved in membrane repair and lipid biosynthesis have also been identified [34]. All of these studies indicate that M. tuberculosis actively interferes with the intrinsic pathway of apoptosis in the infected host cell as a means of virulence and that disregulation of the host’s lipid metabolism is a major pathway for generating pathology [126] and promoting necrosis over apoptosis [34].
The picture for inhibition via the extrinsic apoptotic pathway is also rapidly becoming filled in. Knockout studies of the OppA and OppD genes have implicated the peptide transporters encoded by Rv3665c-Rv3662c and Rv1280c-Rv1283c as inhibitors of apoptosis and this is associated with decreased production of cytokines, including TNF-α [127]. Likewise, the hypothetical proteins Rv3654c and Rv3655c appear to interfere with the extrinsic pathway by diminishing the availability of active Caspase 8 through posttranscriptional modification [128]. Inhibition of signaling via members of the TNF receptor superfamily (TNF-α and Fas) has long been suggested as a major factor [32, 129, 130] for modulating pathology and more of the genes apparently involved in this process are being identified [131–133]. Interestingly, these findings are tying identified genotypes (such as nuoG mutants) to the same mechanisms—production of TNF-α and reactive oxygen species—already associated with defence and immunopathology in TB [113, 131, 132].
M. tuberculosis infection is known to induce TNF-α production, but in vivo, infection of host cells does not occur in a vacuum, but in the presence of a variety of immunomodulating factors. We hypothesize that one such factor, IL-4, a cytokine whose expression appears to correlate with a poorer prognosis after M. tuberculosis infection [21, 23–25, 92, 134] when combined with TNF-α, may worsen TB-related pathology, possibly by biasing cell death towards necrosis instead. If this effect is replicated in vivo, (and our data in clinical studies suggest it is [4]) it might help explain why a bias toward IL-4 expression can lead to aggravated pathology in TB [20, 26, 134, 135]. In addition, IL-4 strongly inhibits the expression of the pro-apoptotic molecule TNF-α and its two receptors, which are otherwise increased by mycobacterial infection—an effect which may be exacerbated since mycobacterial infection appears to promote the shedding of the soluble form of the receptors [4] that can act as competitive inhibitors. Inhibiting TNF-α in primate studies appears to promote pathology [136]. All of this supports the hypothesis that control of apoptosis via CD43/TNF-a inflammatory responses is important for control of M. tuberculosis [106, 108, 112]. Finally, IL-4 appears to play a role in the differentiation of M2 (or anti-inflammatory) macrophages, [137–139], which not only promote IL-4 and IL-10 production, but also handle arginine and iron—two important resources for M. tuberculosis— differently from M1 macrophages [140, 141]. We suggest expanding the mechanisms by which M. tuberculosis actively interferes in this process to suggest that the induction of IL-4, which has been linked to virulence, does so via multiple pathways, and at least partially by promoting cell death by necrosis instead of apoptosis. Identifying the mycobacterial factors which drive this process could offer potential new targets for vaccine and drug development and we are thus investigating M. tuberculosis factors that may be involved.
Acknowledgments
The authors would like to acknowledge Mrs Kidist Bobosha for technical assistance. They appreciate AHRI’s administration for the support they provided. Part of the work described here was funded by EU INCO contracts ICA-CT-1999-10005, IC4-2001-10050, EU FP6 contract no. 503367, and the institutes’ core budgets. AHRI is supported by the Governments of Ethiopia, Norway, and Sweden. M. Abebe and L. Kim contributed equally to the work described in this paper. The VACSEL study group also includes University College London, Helen Fletcher (until 2003), University of Zambia School of Medicine, Lusaka, Zambia: Professor Chifumbe Chintu MD, Gina Mulundu MSc, Dr. Peter Mwaba MD.PhD. MRC, Gambia: Professor KPWJ McAdam (until 2003), Patrick Owiafe, Dr. David Warndorff (2001), Dr. Christian Lienhardt (until 2001), Dr. R Brookes, and Dr. Phillip Hill (2001–2006).
BorgdorffM. W.FloydK.BroekmansJ. F.Interventions to reduce tuberculosis mortality and transmission in low- and middle-income countries20028032172272-s2.0-0036210480DyeC.ScheeleS.DolinP.PathaniaV.RaviglioneM. C.Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project1999282677686Úriz AyestaránJ.RepárazJ.CastielloJ.SolaJ.Tuberculosis in patients with HIV infectionTuberculosis en pacientes infectados por el VIH20073021311422-s2.0-34848843264AbebeM.DohertyT. M.WassieL.AseffaA.BoboshaK.DemissieA.ZewdieM.EngersH.AndersenP.KimL.HuggettJ.RookG.YamuahL. K.ZumlaA.FletcherH.ChintuC.MulunduG.MwabaP.McAdamK. P. W. J.OwiafeP.WarndorffD.LienhardtC.BrookesR.HillP.Expression of apoptosis-related genes in an Ethiopian cohort study correlates with tuberculosis clinical status20104012913012-s2.0-7424909934710.1002/eji.200939856FlynnJ. L.GoldsteinM. M.ChanJ.TrieboldK. J.PfefferK.LowensteinC. J.SchreiberR.MakT. W.BloomB. R.Tumor necrosis factor-α is required in the protective immune response against mycobacterium tuberculosis in mice1995265615722-s2.0-0028979703MohanV. P.ScangaC. A.YuK.ScottH. M.TanakaK. E.TsangE.TsaiM. C.FlynnJ. L.ChanJ.Effects of tumor necrosis factor alpha on host immune response in chronic persistent tuberculosis: possible role for limiting pathology2001693184718552-s2.0-003511869810.1128/IAI.69.3.1847-1855.2001OgawaT.UchidaH.KusumotoY.MoriY.YamamuraY.HamadaS.Increase in tumor necrosis factor alpha- and interleukin-6-secreting cells in peripheral blood mononuclear cells from subjects infected with Mycobacterium tuberculosis1991599302130252-s2.0-0025914254HussainR.rabia.hussain@aku.eduTalatN.ShahidF.DawoodG.Longitudinal tracking of cytokines after acute exposure to tuberculosis: association of distinct cytokine patterns with protection and disease development200714121578158610.1128/CVI.00289-07KawamuraI.Protective immunity against Mycobacterium tuberculosis200681116876912-s2.0-39049177243MaJ.ChenT.MandelinJ.CeponisA.MillerN. E.HukkanenM.MaG. F.KonttinenY. T.Regulation of macrophage activation20036011233423462-s2.0-034439351610.1007/s00018-003-3020-0FletcherH. A.Correlates of immune protection from tuberculosis2007733193252-s2.0-3424865597310.2174/156652407780598520Goter-RobinsonC.DerrickS. C.YangA. L.JeonB. Y.MorrisS. L.Protection against an aerogenic Mycobacterium tuberculosis infection in BCG-immunized and DNA-vaccinated mice is associated with early type I cytokine responses20062417352235292-s2.0-3364523745110.1016/j.vaccine.2006.02.005Hervas-StubbsS.MajlessiL.SimsovaM.MorovaJ.RojasM. J.NouzéC.BrodinP.SeboP.LeclercC.High frequency of CD4+ T cells specific for the TB10.4 protein correlates with protection against Mycobacterium tuberculosis infection2006746339634072-s2.0-3364691491410.1128/IAI.02086-05SullivanB. M.JobeO.LazarevicV.VasquezK.BronsonR.GlimcherL. H.KramnikI.Increased susceptibility of mice lacking T-bet to infection with Mycobacterium tuberculosis correlates with increased IL-10 and decreased IFN-γ production20051757459346022-s2.0-25444482624DaoD. N.KremerL.GuérardelY.MolanoA.JacobsW. R.PorcelliS. A.BrikenV.Mycobacterium tuberculosis lipomannan induces apoptosis and interleukin-12 production in macrophages2004724206720742-s2.0-184258868510.1128/IAI.72.4.2067-2074.2004MancaC.ReedM. B.FreemanS.MathemaB.KreiswirthB.BarryC. E.3rdKaplanG.Differential monocyte activation underlies strain-specific Mycobacterium tuberculosis pathogenesis2004729551155142-s2.0-454438041810.1128/IAI.72.9.5511-5514.2004ReedM. B.DomenechP.MancaC.SuH.BarczakA. K.KrelswirthB. H.KaplanG.Barry IIIC. E.clifton_barry@nih.govA glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response20044307004848710.1038/nature02837van KooykY.GeijtenbeekT. B. H.DC-SIGN: escape mechanism for pathogens2003396977092-s2.0-014168829210.1038/nri1182SeahG. T.RookG. A. W.IL-4 influences apoptosis of mycobacterium-reactive lymphocytes in the presence of TNF-α20011673123012372-s2.0-0035421789DemissieA.AbebeM.AseffaA.RookG.FletcherH.ZumlaA.WeldinghK.BrockI.AndersenP.DohertyT. M.ChintuC.MulunduG.MwabaP.McAdamK. P. W. J.OwiafeP.WarndorffD.LienhardtC.BrookesR.HillP.Healthy individuals that control a latent infection with Mycobacterium tuberculosis express high levels of Th1 cytokines and the IL-4 antagonist IL-4δ2200417211693869432-s2.0-2442692860DemissieA.WassieL.AbebeM.AseffaA.RookG.ZumlaA.AndersenP.DohertyT. M.FletcherH.ChintuC.MulunduG.MwabaP.McAdamK. P. W. J.OwiafeP.WarndorffD.LienhardtC.BrookesR.HillP.The 6-kilodalton early secreted antigenic target-responsive, asymptomatic contacts of tuberculosis patients express elevated levels of interleukin-4 and reduced levels of gamma interferon2006745281728222-s2.0-3364637933710.1128/IAI.74.5.2817-2822.2006FletcherH. A.OwiafeP.JeffriesD.HillP.RookG. A. W.ZumlaA.DohertyT. M.BrookesR. H.ChintuC.MulunduG.MwabaP.AndersenP.DemissieA.McAdamK. P. W. J.WarndorffD.LienhardtC.Increased expression of mRNA encoding interleukin (IL)-4 and its splice variant IL-4δ2 in cells from contacts of Mycobacterium tuberculosis, in the absence of in vitro stimulation200411246696732-s2.0-404310835410.1111/j.1365-2567.2004.01922.xOrdwayD. J.CostaL.MartinsM.SilveiraH.AmaralL.ArrozM. J.VenturaF. A.DockrellH. M.Increased interleukin-4 production by CD8 and γδ T cells in health-care workers is associated with the subsequent development of active tuberculosis200419047567662-s2.0-394310604410.1086/422532OrdwayD. J.PintoL.CostaL.MartinsM.LeandroC.ViveirosM.AmaralL.ArrozM. J.VenturaF. A.DockrellH. M.Gamma delta T cell responses associated with the development of tuberculosis in health care workers20054333393502-s2.0-1364425219010.1016/j.femsim.2004.09.005WassieL.DemissieA.AseffaA.AbebeM.YamuahL.TilahunH.PetrosB.RookG.ZumlaA.AndersenP.DohertyT. M.TMD@ssi.dkFetcherH.ChintuC.MulunduG.MwabaP.McAdamK. P.W.J.OwiafeP.WarndorffD.LienhardtC.BrookesR.HillP.Ex vivo cytokine mRNA levels correlate with changing clinical status of Ethiopian TB patients and their contacts over time200831e152210.1371/journal.pone.0001522WuH. P.WuC. L.YuC. C.LiuYU. C.ChuangD. Y.Efficiency of interleukin-4 expression in patients with tuberculosis and nontubercular pneumonia200768108328382-s2.0-3534899962010.1016/j.humimm.2007.07.003GutierrezM. G.MasterS. S.SinghS. B.TaylorG. A.ColomboM. I.DereticV.Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages200411967537662-s2.0-1094425314510.1016/j.cell.2004.11.038KremerL.EstaquierJ.BrandtE.AmeisenJ. C.LochtC.Mycobacterium bovis Bacillus Calmette Guerin infection prevents apoptosis of resting human monocytes1997279245024562-s2.0-003086851510.1002/eji.1830270945BocchinoM.GalatiD.SanduzziA.ColizziV.BrunettiE.MancinoG.Role of mycobacteria-induced monocyte/macrophage apoptosis in the pathogenesis of human tuberculosis2005943753832-s2.0-17144415540RojasM.BarreraL. F.PuzoG.GarciaL. F.Differential induction of apoptosis by virulent Mycobacterium tuberculosis in resistant and susceptible murine macrophages: role of nitric oxide and mycobacterial products19971593135213612-s2.0-0031203033SantucciM. B.AmicosanteM.CicconiR.MontesanoC.CasariniM.GiosuèS.BisettiA.ColizziV.FrazianoM.Mycobacterium tuberculosis-induced apoptosis in monocytes/macrophages: early membrane modifications and intracellular mycobacterial viability20001814150615092-s2.0-003402274710.1086/315371KeaneJ.Balcewicz-SablinskaM. K.RemoldH. G.ChuppG. L.MeekB. B.FentonM. J.KornfeldH.Infection by Mycobacterium tuberculosis promotes human alveolar macrophage apoptosis19976512983042-s2.0-0031015731KeaneJ.RemoldH. G.KornfeldH.Virulent Mycobacterium tuberculosis strains evade apoptosis of infected alveolar macrophages20001644201620202-s2.0-0034651738BeharS. M.sbehar@rics.bwh.harvard.eduDivangahiM.RemoldH. G.hremold@rics.bwh.harvard.eduEvasion of innate immunity by mycobacterium tuberculosis: is death an exit strategy?20108966867410.1038/nrmicro2387BrikenV.MillerJ. L.Living on the edge: inhibition of host cell apoptosis by Mycobacterium tuberculosis2008344154222-s2.0-4864910227410.2217/17460913.3.4.415Gonzalez-JuarreroM.TurnerO. C.TurnerJ.MariettaP.BrooksJ. V.OrmeI. M.Temporal and spatial arrangement of lymphocytes within lung granulomas induced by aerosol infection with Mycobacterium tuberculosis2001693172217282-s2.0-003511212310.1128/IAI.69.3.1722-1728.2001RussellD. G.BarryC. E.FlynnJ. L.Tuberculosis: what we don't know can, and does, hurt us201032859808528562-s2.0-7795235663710.1126/science.1184784RussellD. G.Mycobacterium tuberculosis: here today, and here tomorrow2001285695772-s2.0-003543238310.1038/35085034KorfJ.StoltzA.VerschoorJ.De BaetselierP.GrootenJ.The Mycobacterium tuberculosis cell wall component mycolic acid elicits pathogen-associated host innate immune responses20053538909002-s2.0-1584438234410.1002/eji.200425332BroschR.PymA. S.GordonS. V.ColeS. T.The evolution of mycobacterial pathogenicity: clues from comparative genomics2001994524582-s2.0-003544854510.1016/S0966-842X(01)02131-Xde JongeM. I.BroschR.BrodinP.DemangelC.ColeS. T.Tuberculosis: from genome to vaccine2005445415512-s2.0-2474444216410.1586/14760584.4.4.541BettsJ. C.LukeyP. T.RobbL. C.McAdamR. A.DuncanK.Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling20024337177312-s2.0-003627073910.1046/j.1365-2958.2002.02779.xDereticV.SinghS.MasterS.HarrisJ.RobertsE.KyeiG.DavisA.de HaroS.NaylorJ.LeeH. H.VergneI.Mycobacterium tuberculosis inhibition of phagolysosome biogenesis and autophagy as a host defence mechanism2006857197272-s2.0-3364554267910.1111/j.1462-5822.2006.00705.xAxelrodS.OschkinatH.EndersJ.SchlegelB.BrinkmannV.KaufmannS. H. E.HaasA.SchaibleU. E.Delay of phagosome maturation by a mycobacterial lipid is reversed by nitric oxide2008107153015452-s2.0-4524911397710.1111/j.1462-5822.2008.01147.xDavisA. S.VergneI.MasterS. S.KyeiG. B.ChuaJ.DereticV.Mechanism of inducible nitric oxide synthase exclusion from mycobacterial phagosomes2007312, article e1862-s2.0-4304917221810.1371/journal.ppat.0030186GreentS. J.SchellerL. F.MarlettaM. A.SeguinM. C.KlotzF. W.SlayterM.NelsonB. J.NacyC. A.Nitric oxide: cytokine-regulation of nitric oxide in host resistance to intracellular pathogens1994431-287942-s2.0-002864357810.1016/0165-2478(94)00158-8NathanC.Role of iNOS in human host defense2006312578218742-s2.0-33745605778SchönT.ElmbergerG.NegesseY.Hernandez PandoR.SundqvistT.BrittonS.Local production of nitric oxide in patients with tuberculosis200489113411372-s2.0-4444315943StokesR. W.Norris-JonesR.BrooksD. E.BeveridgeT. J.DoxseeD.ThorsonL. M.The glycan-rich outer layer of the cell wall of Mycobacterium tuberculosis acts as an antiphagocytic capsule limiting the association of the bacterium with macrophages20047210567656862-s2.0-464427647710.1128/IAI.72.10.5676-5686.2004JoE. K.YangC. S.ChoiC. H.HardingC. V.Intracellular signalling cascades regulating innate immune responses to Mycobacteria: branching out from Toll-like receptors200795108710982-s2.0-3404726287910.1111/j.1462-5822.2007.00914.xQuesniauxV.FremondC.JacobsM.ParidaS.NicolleD.YeremeevV.BihlF.ErardF.BothaT.DrennanM.SolerM. N.Le BertM.SchnyderB.RyffelB.Toll-like receptor pathways in the immune responses to mycobacteria20046109469592-s2.0-404317198610.1016/j.micinf.2004.04.016SalgameP.Host innate and Th1 responses and the bacterial factors that control Mycobacterium tuberculosis infection20051743743802-s2.0-2054446278510.1016/j.coi.2005.06.006StengerS.steffen.stenger@mikrobio.med.uni-erlangen.deModlinR. L.rmodlin@medicine.medsch.ucla.eduControl of Mycobacterium tuberculosis through mammalian Toll-like receptors200214445245710.1016/S0952-7915(02)00355-2ReilingN.EhlersS.HölscherC.MyDths and un-TOLLed truths: sensor, instructive and effector immunity to tuberculosis2008116115232-s2.0-3854917408610.1016/j.imlet.2007.11.015Henao-TamayoM.Junqueira-KipnisA. P.OrdwayD.Gonzales-JuarreroM.StewartG. R.YoungD. B.WilkinsonR. J.BasarabaR. J.OrmeI. M.A mutant of Mycobacterium tuberculosis lacking the 19-kDa lipoprotein Rv3763 is highly attenuated in vivo but retains potent vaccinogenic properties20072541715371592-s2.0-3464882888710.1016/j.vaccine.2007.07.042YeremeevV. V.LyadovaI. V.NikonenkoB. V.AptA. S.Abou-ZeidC.InwaldJ.YoungD. B.The 19-kD antigen and protective immunity in a murine model of tuberculosis200012022742792-s2.0-003411138710.1046/j.1365-2249.2000.01212.xSugawaraI.YamadaH.LiC.MizunoS.TakeuchiO.AkiraS.Mycobacterial infection in TLR2 and TLR6 knockout mice20034753273362-s2.0-0037570681TakedaK.TakeuchiO.AkiraS.Recognition of lipopeptides by Toll-like receptors2002864594632-s2.0-003695403710.1179/096805102125001073PaiR. K.PenniniM. E.TobianA. A. R.CanadayD. H.BoomW. H.HardingC. V.Prolonged toll-like receptor signaling by Mycobacterium tuberculosis and its 19-kilodalton lipoprotein inhibits gamma interferon-induced regulation of selected genes in macrophages20047211660366142-s2.0-704422287710.1128/IAI.72.11.6603-6614.2004FortuneS. M.SolacheA.JaegerA.HillP. J.BelisleJ. T.BloomB. R.RubinE. J.ErnstJ. D.Mycobacterium tuberculosis inhibits macrophage responses to IFN-γ through myeloid differentiation factor 88-dependent and -independent mechanisms200417210627262802-s2.0-2442544416NossE. H.PaiR. K.SellatiT. J.RadolfJ. D.BelisleJ.GolenbockD. T.BoomW. H.HardingC. V.Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19-kDa lipoprotein of Mycobacterium tuberculosis200116729109182-s2.0-0035879108PathakS. K.BasuS.BasuK. K.BanerjeeA.PathakS.BhattacharyyaA.KaishoT.KunduM.BasuJ.Direct extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages2007866106182-s2.0-3424901213010.1038/ni1468SamtenB.buka.samten@uthct.eduWangX.BarnesP. F.Mycobacterium tuberculosis ESX-1 system-secreted protein ESAT-6 but not CFP10 inhibits human T-cell immune responses200989supplement 1S74S7610.1016/S1472-9792(09)70017-4TsenovaL.EllisonE.HarbacheuskiR.MoreiraA. L.KurepinaN.ReedM. B.MathemaB.BarryC. E.KaplanG.Virulence of selected Mycobacterium tuberculosis clinical isolates in the rabbit model of meningitis is dependent on phenolic glycolipid produced by the bacilli20051921981062-s2.0-2084443941110.1086/430614SinsimerD.HuetG.MancaC.TsenovaL.KooM. S.KurepinaN.KanaB.MathemaB.MarrasS. A. E.KreiswirthB. N.GuilhotC.KaplanG.The phenolic glycolipid of Mycobacterium tuberculosis differentially modulates the early host cytokine response but does not in itself confer hypervirulence2008767302730362-s2.0-4644911034810.1128/IAI.01663-07AppelmelkB. J.van DieI.van VlietS. J.Vandenbroucke-GraulsC. M. J. E.GeijtenbeekT. B. H.Van KooykY.Cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells20031704163516392-s2.0-0037442109JangS.UzelacA.SalgameP.Distinct chemokine and cytokine gene expression pattern of murine dendritic cells and macrophages in response to Mycobacterium tuberculosis infection2008845126412702-s2.0-5554914681110.1189/jlb.1107742OloboJ. O.GeletuM.DemissieA.EgualeT.HiwotK.AderayeG.BrittonS.Circulating TNF-α, TGF-β, and IL-10 in tuberculosis patients and healthy contacts200153185912-s2.0-003515474910.1046/j.1365-3083.2001.00844.xRedpathS.GhazalP.GascoigneN. R. J.Hijacking and exploitation of IL-10 by intracellular pathogens20019286922-s2.0-003525265610.1016/S0966-842X(00)01919-3de JongR.AltareF.HaagenI. A.ElferinkD. G.De BoerT.Van Breda VriesmanP. J. C.KabelP. J.DraaismaJ. M. T.Van DisselJ. T.KroonF. P.CasanovaJ. L.OttenhoffT. H. M.Severe mycobacterial and Salmonella infections in interleukin-12 receptor-deficient patients19982805368143514382-s2.0-003257729510.1126/science.280.5368.1435TsoH. W.YuL. L.TamC. M.WongH. S.ChiangA. K. S.Associations between IL12B polymorphisms and tuberculosis in the Hong Kong Chinese population200419059139192-s2.0-434471731610.1086/422693CooperA. M.DaltonD. K.StewartT. A.GriffinJ. P.RussellD. G.OrmeI. M.Disseminated tuberculosis in interferon γ gene-disrupted mice19931786224322472-s2.0-002748232710.1084/jem.178.6.2243FlynnJ. L.ChanJ.TrieboldK. J.DaltonD. K.StewartT. A.BloomB. R.An essential role for interferon γ in resistance to Mycobacterium tuberculosis infection19931786224922542-s2.0-002736033310.1084/jem.178.6.2249OttenhoffT. H. M.VerreckF. A. W.HoeveM. A.van de VosseE.Control of human host immunity to mycobacteria2005851-253642-s2.0-1594442176610.1016/j.tube.2004.09.011SaundersB. M.FrankA. A.OrmeI. M.CooperA. M.CD4 is required for the development of a protective granulomatous response to pulmonary tuberculosis20022161-265722-s2.0-003640308610.1016/S0008-8749(02)00510-5ScangaC. A.MohanV. P.YuK.JosephH.TanakaK.ChanJ.jchan@aecom.yu.eduFlynnJ. L.joanne@pitt.eduDepletion of CD4+ T cells causes reactivation of murine persistent tuberculosis despite continued expression of interferon γ and nitric oxide synthase 22000192334735810.1084/jem.192.3.347LazarevicV.NoltD.FlynnJ. L.Long-term control of Mycobacterium tuberculosis infection is mediated by dynamic immune responses20051752110711172-s2.0-23244432880SudD.BigbeeC.FlynnJ. L.KirschnerD. E.Contribution of CD8+ T cells to control of Mycobacterium tuberculosis infection20061767429643142-s2.0-33646025858WoodworthJ. S. M.BeharS. M.Mycobacterium tuberculosis-specific CD8+ T cells and their role in immunity20062643173522-s2.0-33748866849SodhiA.GongJ. H.SilvaC.QianD.BarnesP. F.Clinical correlates of interferon γ production in patients with tuberculosis1997253617620JalapathyK. V.PrabhaC.DasS. D.Correlates of protective immune response in tuberculous pleuritis20044021391452-s2.0-134234519610.1016/S0928-8244(03)00303-1Jiménez-MartínezM. C.LinaresM.BáezR.MontañoL. F.Martínez-CairoS.GorocicaP.ChávezR.ZentenoE.LascurainR.Intracellular expression of interleukin-4 and interferon-γ by a Mycobacterium tuberculosis antigen-stimulated CD4+ CD57+ T-cell subpopulation with memory phenotype in tuberculosis patients200411111001062-s2.0-2024437463610.1111/j.1365-2567.2004.01785.xRobertsT.teri@sun.ac.zaBeyersN.AguirreA.WalzlG.Immunosuppression during active tuberculosis is characterized by decreased interferon-γ production and CD25 expression with elevated forkhead box P3, transforming growth factor-β, and interleukin-4 mRNA levels2007195687087810.1086/511277SiawayaJ. F. D.joe@sun.ac.zaBapelaN. B.RonacherK.BeyersN.Van HeldenP.WalzlG.Differential expression of interleukin-4 (IL-4) and IL-4δ2 mRNA, but not transforming growth factor beta (TGF-β), TGF-βRII, Foxp3, gamma interferon, T-bet, or GATA-3 mRNA, in patients with fast and slow responses to antituberculosis treatment20081581165117010.1128/CVI.00084-08da FonsecaD. M.SilvaC. L.WowkP. F.PaulaM. O.RamosS. G.HornC.MarchalG.BonatoV. L. D.Mycobacterium tuberculosis culture filtrate proteins plus CpG oligodeoxynucleotides confer protection to Mycobacterium bovis BCG-primed mice by inhibiting interleukin-4 secretion20097712531153212-s2.0-7244912610610.1128/IAI.00580-09RhodesS. G.SawyerJ.WhelanA. O.DeanG. S.CoadM.EwerK. J.WaldvogelA. S.ZakherA.CliffordD. J.HewinsonR. G.VordermeierH. M.Is interleukin-4δ3 splice variant expression in bovine tuberculosis a marker of protective immunity?2007756300630132-s2.0-3424988355410.1128/IAI.01932-06SahiratmadjaE.AlisjahbanaB.De BoerT.AdnanI.MayaA.DanusantosoH.NelwanR. H. H.MarzukiS.Van Der MeerJ. W. M.Van CrevelR.Van De VosseE.OttenhoffT. H. M.t.h.m.ottenhoff@lumc.nlDynamic changes in pro- and anti-inflammatory cytokine profiles and gamma interferon receptor signaling integrity correlate with tuberculosis disease activity and response to curative treatment200775282082910.1128/IAI.00602-06MorrisK. R.LutzR. D.BaiX.McGibneyM. T.CookD.OrdwayD.ChanE. D.Suppression of IFNγ + mycobacterial lipoarabinomannan-induced NO by IL-4 is due to decreased IRF-1 expression20098942943032-s2.0-6765062156510.1016/j.tube.2009.03.004AnonTuberculosis associated with blocking agents against tumor necrosis factor-alpha—California, 2002-200320045330683686Gómez-ReinoJ. J.CarmonaL.Rodríguez ValverdeV.MolaE. M.MonteroM. D.Treatment of rheumatoid arthritis with tumor necrosis factor inhibitors may predispose to significant increase in tuberculosis risk: a multicenter active-surveillance report2003488212221272-s2.0-004165331510.1002/art.11137BezuidenhoutJ.RobertsT.MullerL.van HeldenP.WalzlG.Pleural tuberculosis in patients with early HIV infection is associated with increased TNF-alpha expression and necrosis in granulomas2009412-s2.0-6414911153310.1371/journal.pone.0004228e4228SharmaS.BoseM.Role of cytokines in immune response to pulmonary tuberculosis20011932132192-s2.0-0035687859BarksJ. L.McQuillanJ. J.IademarcoM. F.TNF-α and IL-4 synergistically increase vascular cell adhesion molecule-1 expression in cultured vascular smooth muscle cells19971599453245382-s2.0-0031278369IademarcoM. F.BarksJ. L.DeanD. C.Regulation of vascular cell adhesion molecule-1 expression by IL-4 and TNF-α in cultured endothelial cells19959512642712-s2.0-0028861841PatelN. R.ZhuJ.TachadoS. D.ZhangJ.WanZ.SaukkonenJ.KozielH.HIV impairs TNF-α mediated macrophage apoptotic response to Mycobacterium tuberculosis200717910697369802-s2.0-38449106038DivangahiM.DesjardinsD.Nunes-AlvesC.RemoldH. G.BeharS. M.Eicosanoid pathways regulate adaptive immunity to Mycobacterium tuberculosis2010117517582-s2.0-7795430231410.1038/ni.1904MarinoS.simeonem@umich.eduMyersA.FlynnJ. L.KirschnerD. E.kirschne@umich.eduTNF and IL-10 are major factors in modulation of the phagocytic cell environment in lung and lymph node in tuberculosis: a next-generation two-compartmental model2010265458659810.1016/j.jtbi.2010.05.012StengerS.steffen.stenger@mikrobio.med.uni-erlangen.deImmunological control of tuberculosis: role of tumour necrosis factor and more200564supplement 4iv24iv2810.1136/ard.2005.042531KremerL.EstaquierJ.WolowczukI.BietF.AmeisenJ. C.LochtC.Ineffective cellular immune response associated with T-cell apoptosis in susceptible Mycobacterium bovis BCG-infected mice2000687426442732-s2.0-003391784910.1128/IAI.68.7.4264-4273.2000LoeuilletC.MartinonF.PerezC.MunozM.ThomeM.MeylanP. R.Mycobacterium tuberculosis subverts innate immunity to evade specific effectors20061779624562552-s2.0-33750315759OzekiY.KanedaK.FujiwaraN.MorimotoM.OkaS.YanoI.In vivo induction of apoptosis in the thymus by administration of mycobacterial cord factor (trehalose 6,6′-dimycolate)1997655179317992-s2.0-0030900578PlacidoR.MancinoG.AmendolaA.MarianiF.VendettiS.PiacentiniM.SanduzziA.BocchinoM. L.ZembalaM.ColizziV.Apoptosis of human monocytes/macrophages in Mycobacterium tuberculosis infection1997181131382-s2.0-0031038856Ríos-BarreraV. A.Campos-PeñaV.Aguilar-LeónD.Lascurain LedesmaR.Meraz-RíosM. A.MorenoJ.Figueroa-GranadosV.Hernández-PandoR.rhpando@quetzal.innsz.mxMacrophage and T lymphocyte apoptosis during experimental pulmonary tuberculosis: their relationship to mycobacterial virulence200636234535310.1002/eji.200535202RogerP. M.BermudezL. E.Infection of mice with Mycobacterium avium primes CD8+ lymphocytes for apoptosis upon exposure to macrophages20019933783862-s2.0-003501003910.1006/clim.2001.5037WatsonV. E.HillL. L.Owen-SchaubL. B.DavisD. W.McConkeyD. J.JagannathC.HunterR. L.ActorJ. K.Apoptosis in Mycobacterium tuberculosis infection in mice exhibiting varied immunopathology200019022112202-s2.0-0033965118BudakF.UzaslanE. K.CangurS.GoralG.OralH. B.Increased pleural soluble Fas ligand (sFasL) levels in tuberculosis pleurisy and its relation with T-helper type 1 cytokines200818653373432-s2.0-5244912446810.1007/s00408-008-9107-5GanH.LeeJ.RenF.ChenM.KornfeldH.RemoldH. G.Mycobacterium tuberculosis blocks crosslinking of annexin-1 and apoptotic envelope formation on infected macrophages to maintain virulence2008910118911972-s2.0-5254912356910.1038/ni.1654MustafaT.MoggaS. J.MfinangaS. G. M.MørkveO.SvilandL.Significance of Fas and Fas ligand in tuberculous lymphadenitis200511422552622-s2.0-1344431086610.1111/j.1365-2567.2004.02080.xPorcelliS. A.JacobsW. R.Tuberculosis: unsealing the apoptotic envelope2008910110111022-s2.0-5264912023610.1038/ni1008-1101KramnikI.Genetic dissection of host resistance to Mycobacterium tuberculosis: the sst1 locus and the Ipr1 gene20083211231482-s2.0-5114911675110.1007/978-3-540-75203-5_6ParkJ. S.TamayoM. H.Gonzalez-JuarreroM.OrmeI. M.OrdwayD. J.Virulent clinical isolates of Mycobacterium tuberculosis grow rapidly and induce cellular necrosis but minimal apoptosis in murine macrophages200679180862-s2.0-3014443531710.1189/jlb.0505250RandhawaA. K.ZiltenerH. J.StokesR. W.CD43 controls the intracellular growth of Mycobacterium tuberculosis through the induction of TNF-α-mediated apoptosis20081010210521172-s2.0-5144911920110.1111/j.1462-5822.2008.01194.xVelmuruganK.ChenB.MillerJ. L.AzogueS.GursesS.HsuT.GlickmanM.JacobsW. R.PorcelliS. A.BrikenV.Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells.200737, article e1102-s2.0-4154910821310.1371/journal.ppat.0030110HeX. Y.XiaoL.ChenH. B.HaoJ.LiJ.WangY. J.HeK.GaoY.ShiB. Y.T regulatory cells and Th1/Th2 cytokines in peripheral blood from tuberculosis patients20102966436502-s2.0-7795287780210.1007/s10096-010-0908-0DhedaK.BoothH.HuggettJ. F.JohnsonM. A.ZumlaA.RookG. A. W.Lung remodeling in pulmonary tuberculosis20051927120112102-s2.0-2544444480110.1086/444545Hernandez-PandoR.RookG. A. W.The role of TNF-α in T-cell mediated inflammation depends on the Th1/Th2 cytokine balance19948245915952-s2.0-0028168447AguilarL. D.HanekomM.MataD.Gey van PittiusN. C.Van HeldenP. D.WarrenR. M.Hernandez-PandoR.rhdezpando@hotmail.comMycobacterium tuberculosis strains with the Beijing genotype demonstrate variability in virulence associated with transmission201090531932510.1016/j.tube.2010.08.004KausalyaS.SomogyiR.OrlofskyA.PrystowskyM. B.Requirement of A1-α for bacillus Calmette-Guérin-mediated protection of macrophages against nitric oxide-induced apoptosis20011667472147272-s2.0-0035297708KrzyzowskaM.SchollenbergerA.PawłowskiA.HamasurB.WinnickaA.Augustynowicz-KopećE.NiemiałtowskiM.Lipoarabinomannan as a regulator of the monocyte apoptotic response to Mycobacterium bovis BCG danish strain 1331 infection200756289962-s2.0-34447507906MoggaS. J.MustafaT.SvilandL.NilsenR.Increased Bcl-2 and reduced Bax expression in infected macrophages in slowly progressive primary murine Mycobacterium tuberculosis infection20025643833912-s2.0-003637838310.1046/j.1365-3083.2002.01140.xSlyL. M.Hingley-WilsonS. M.ReinerN. E.McMasterW. R.Survival of Mycobacterium tuberculosis in host macrophages involves resistance to apoptosis dependent upon induction of antiapoptotic Bcl-2 family member Mcl-1200317014304372-s2.0-0037218568ZhangJ.JiangR.TakayamaH.TanakaY.Survival of virulent Mycobacterium tuberculosis involves preventing apoptosis induced by Bcl-2 upregulation and release resulting from necrosis in J774 macrophages20054998458522-s2.0-25444486695RiendeauC. J.KornfeldH.THP-1 cell apoptosis in response to Mycobacterial infection20037112542592-s2.0-003721826810.1128/IAI.71.1.254-259.2003LeeJ.RemoldH. G.IeongM. H.KornfeldH.Macrophage apoptosis in response to high intracellular burden of Mycobacterium tuberculosis is mediated by a novel caspase-independent pathway20061767426742742-s2.0-33646027045KlinglerK.Tchou-WongK. M.BrändliO.AstonC.KimR.ChiC.RomW. N.Effects of mycobacteria on regulation of apoptosis in mononuclear phagocytes19976512527252782-s2.0-0030731701KimM. J.WainwrightH. C.LocketzM.BekkerL. G.WaltherG. B.DittrichC.VisserA.WangW.HsuF. F.WiehartU.TsenovaL.KaplanG.RussellD. G.dgr8@cornell.eduCaseation of human tuberculosis granulomas correlates with elevated host lipid metabolism20102725827410.1002/emmm.201000079DasguptaA.SurekaK.MitraD.SahaB.SanyalS.DasA. K.ChakrabartiP.JacksonM.GicquelB.KunduM.BasuJ.joyoti@vsnl.comAn oligopeptide transporter of Mycobacterium tuberculosis regulates cytokine release and apoptosis of infected macrophages201058e1222510.1371/journal.pone.0012225DanelishviliL.YamazakiY.SelkerJ.BermudezL. E.Luiz.Bermudez@oregonstate.eduSecreted Mycobacterium tuberculosis Rv3654c and Rv3655c proteins participate in the suppression of macrophage apoptosis201055e1047410.1371/journal.pone.0010474Balcewicz-SablinskaM. K.KeaneJ.KornfeldH.RemoldH. G.Pathogenic Mycobacterium tuberculosis evades apoptosis of host macrophages by release of TNF-R2, resulting in inactivation of TNF-α19981615263626412-s2.0-0032167519LeeJ.HartmanM.KornfeldH.Macrophage apoptosis in tuberculosis20095011112-s2.0-6274915687310.3349/ymj.2009.50.1.1HincheyJ.LeeS.JeonB. Y.BasarabaR. J.VenkataswamyM. M.ChenB.ChanJ.BraunsteinM.OrmeI. M.DerrickS. C.MorrisS. L.JacobsW. R.PorcelliS. A.Enhanced priming of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis20071178227922882-s2.0-3454761405610.1172/JCI31947MillerJ. L.VelmuruganK.CowanM. J.BrikenV.The type I NADH dehydrogenase of Mycobacterium tuberculosis counters phagosomal NOX2 activity to inhibit TNF-alpha-mediated host cell apoptosis201064e10008642-s2.0-7795395143310.1371/journal.ppat.1000864StadthagenG.JacksonM.CharlesP.BoudouF.BariloneN.HuerreM.ConstantP.LiavA.BottovaI.NigouJ.BrandoT.PuzoG.DafféM.BenjaminP.CoadeS.BuxtonR. S.TasconR. E.RaeA.RobertsonB. D.LowrieD. B.YoungD. B.GicquelB.GriffinR.r.griffin@imperial.ac.ukComparative investigation of the pathogenicity of three Mycobacterium tuberculosis mutants defective in the synthesis of p-hydroxybenzoic acid derivatives2006882245225310.1016/j.micinf.2006.04.008RookG. A. W.Hernandez-PandoR.DhedaK.Teng SeahG.IL-4 in tuberculosis: implications for vaccine design20042594834882-s2.0-544427239210.1016/j.it.2004.06.005Hernandez-PandoR.AguilarD.HernandezM. L. G.OrozcoH.RookG. A. W.Pulmonary tuberculosis in BALB/c mice with non-functional IL-4 genes: changes in the inflammatory effects of TNF-α and in the regulation of fibrosis20043411741832-s2.0-164247316110.1002/eji.200324253LinP. L.MyersA.SmithL.BigbeeC.BigbeeM.FuhrmanC.GrieserH.ChioseaI.VoitenekN. N.CapuanoS. V.KleinE.FlynnJ. L.Tumor necrosis factor neutralization results in disseminated disease in acute and latent Mycobacterium tuberculosis infection with normal granuloma structure in a cynomolgus macaque model20106223403502-s2.0-7574909972710.1002/art.27271HoV. W.SlyL. M.Derivation and characterization of murine alternatively activated (M2) macrophages20095311731852-s2.0-6564909517310.1007/978-1-59745-396-7_12MartinezF. O.SicaA.MantovaniA.LocatiM.Macrophage activation and polarization20081324534612-s2.0-3844910267610.2741/2692RedenteE. F.HigginsD. M.Dwyer-NieldL. D.lori.nield@ucdenver.eduOrmeI. M.Gonzalez-JuarreroM.MalkinsonA. M.Differential polarization of alveolar macrophages and bone marrow-derived monocytes following chemically and pathogen-induced chronic lung inflammation201088115916810.1189/jlb.0609378CornaG.CampanaL.PignattiE.CastiglioniA.TagliaficoE.BosurgiL.CampanellaA.BrunelliS.ManfrediA. A.ApostoliP.SilvestriL.CamaschellaC.Rovere-QueriniP.rovere.patrizia@hsr.itPolarization dictates iron handling by inflammatory and alternatively activated macrophages201095111814182210.3324/haematol.2010.023879EdwardsJ. P.ZhangX.FrauwirthK. A.MosserD. M.Biochemical and functional characterization of three activated macrophage populations2006806129813072-s2.0-3384539821510.1189/jlb.0406249