Hindawi Publishing Corporation
Clinical and Developmental Immunology
Volume 2011, Article ID 678570, 11 pages
doi:10.1155/2011/678570
Review Article
Modulation of Cell Death by M. tuberculosis as a Strategy for
Pathogen Survival
Markos Abebe,1, 2, 3 Louise Kim,2 Graham Rook,2 Abraham Aseffa,1 Liya Wassie,1, 3
Martha Zewdie,1 Alimuddin Zumla,2 Howard Engers,1 Peter Andersen,3
and T. Mark Doherty3
1 Armauer
Hansen Research Institute, P.O. Box 1005, Addis Ababa, Ethiopia
Centre for Infectious Diseases and International Health, Windeyer Institute of Medical Sciences,
Royal Free and University College Medical School, London WC1T 4JF, UK
3 Department of Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, København S, 2300 Copenhagen, Denmark
2 The
Correspondence should be addressed to T. Mark Doherty, tmd@ssi.dk
Received 17 September 2010; Accepted 27 November 2010
Academic Editor: Nicholas West
Copyright © 2011 Markos Abebe et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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 IL4, [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].
2
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
Clinical and Developmental Immunology
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 TNFα 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
Clinical and Developmental Immunology
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. IL12 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 IL12R 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
3
controlling TB in its latent stage [20] and that the IL-4δ2/IL4 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-γinducible 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 TNFα 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 TNFα 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
4
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 humanderived 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 TNFα 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
Clinical and Developmental Immunology
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 mycobacteriainduced 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, IL4 appears to bias cell death slightly towards necrosis over
apoptosis, and this effect was enhanced by BCG infection
(Table 1).
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
Clinical and Developmental Immunology
5
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.
Fold change over uninfected THP-1 cells
Infected with:
null
BCG
BCG
Erdmann
Erdmann
+ IL-4
− IL-4
+ IL-4
− IL-4
+ IL-4
Apoptosis
Necrosis
TNF-α
TNFR1
TNFR2
Fas
FasL
0.67 ± 0.13
3.38 ± 0.04
3.29 ± 0.08
0.44 ± 0.22
0.37 ± 0.12
0.79 ± 0.19
1.06 ± 0.18
2.68 ± 0.14
1.13 ± 0.30
1.34 ± 0.07
0.51
2.15
0.05
17.43
4.21
0.68
15.59
2.27
4.96
1.38
0.57
18.99
3.00
5.20
1.89
0.66
0.13
<0.1
0.65
0.34
0.96
ND
ND
1.30
1.15
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 TNFα 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
Caspase
8
0.67
4.35
0.61
0.36
0.35
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 TNFα. 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 THP1 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. tuberculosisinfected cells, the opposite was seen, with falling proCaspase 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
6
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 Rv1280cRv1283c as inhibitors of apoptosis and this is associated
with decreased production of cytokines, including TNFα [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 TNFαproduction, 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
Clinical and Developmental Immunology
[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 antiinflammatory) 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-CT1999-10005, 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).
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