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* Immunity and Infection Research Center, Vancouver Hospital and Health Sciences Center, Departments of
Medical Genetics and
Medicine (Division of Infectious Diseases), Faculty of Medicine, and
Department of Microbiology and Immunology, Faculty of Science, University of British Columbia, Vancouver, British Columbia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent apoptosis demonstrated by a
genomic DNA ladder, nuclear fragmentation, and condensation as well as
by TUNEL labeling (4). Keane et al. (5)
demonstrated further that avirulent M. tuberculosis strains
and Mycobacterium bovis bacillus Calmette-Guérin
(BCG)4 cause more
apoptosis than virulent strains, implicating a role for apoptosis in
host defense against M. tuberculosis. In addition, there is evidence that apoptosis occurs in vivo during the course of pulmonary tuberculosis. Placido et al. (6) reported that bronchoalveolar lavage from patients with reactive or disseminated tuberculosis demonstrated a 3-fold increase in apoptotic cells compared with control patients as determined by propidium iodide staining, TUNEL labeling, and tissue transglutaminase expression. Furthermore, morphological changes consistent with apoptosis have been noted in epithelioid cells from granuloma of tuberculous patients (7).
Apoptosis during M. tuberculosis infection has been shown to
be an innate mechanism of host defense. Apoptosis, but not necrosis,
was shown to reduce M. tuberculosis CFUs recovered during in
vitro infection of monocyte-derived macrophages (MDMs)
(8). Induction of apoptosis in Mycobacterium
avium-infected macrophages has been demonstrated to be the cause
of intracellular growth restriction induced by picolinic acid
(9). Fas ligand (FasL), TNF-, and ATP-induced apoptosis
all caused a reduction in M. tuberculosis strains H37Ra and
H37Rv recovered from infected macrophages in vitro (8, 10).
However, it has been demonstrated that Fas-FasL interactions and ATP-induced apoptosis are not responsible for the intracellular death of M. tuberculosis. CD4-CD8- cytotoxic T cells mediated Fas-FasL interactions but did not cause the death of intracellular mycobacteria (11). Rather CD8+ T cells can cause the death of intracellular M. tuberculosis and other pathogens by a direct granule-dependent mechanism involving the release of the effector protein granuloysin (12). The effect of ATP-induced killing of the intracellular M. tuberculosis is not due to apoptosis but rather has been attributed to enhanced phagosome-lysosome fusion and acidification of the Mycobacterium-containing phagosome (13, 14, 15).
Proapoptotic and antiapoptotic members of the Bcl-2 family have been shown to be differentially regulated by mycobacterial infection. Bcl-2 family members are homologues of the prototypical antiapoptotic protein Bcl-2 (16). The antiapoptotic family member bfl-1, which encodes the protein A1, is up-regulated in macrophages in response to infection with M. bovis BCG (17). The antiapoptotic gene Bcl-xL has also been shown to be up-regulated during M. tuberculosis infection with a coordinate down-regulation of Bcl-2 (18). In neutrophils, M. tuberculosis infection results in an increase in proapoptotic family member Bax and down-regulation of antiapoptotic family member Bcl-xL, and these events were shown to be dependent on the generation of reactive oxygen species (19). In an animal model of primary murine tuberculosis, Bcl-2 was up-regulated while Bax was down-regulated, resulting in a net decrease in apoptosis in the host (20).
Mcl-1 is an antiapoptotic member of the Bcl-2 family. Prosurvival members of the Bcl-2 family may prevent apoptosis by maintaining the integrity of the mitochondrial membrane, thereby preventing release of cytochrome c, activation of caspases. and DNA degradative enzymes (21, 22). The Bcl-2 homologue, Mcl-1, is present in cells of the hemopoietic lineage (14, 15, 16, 23, 24, 25). In polymorphonuclear cells, antisense oligonucleotides to mcl-1 have been used to reduce Mcl-1 protein expression, resulting in increased apoptosis in response to aging and hypoxia (26).
To study the role of Mcl-1 in M. tuberculosis-infected macrophages, we examined steady-state levels of mcl-1 mRNA and Mcl-1 protein expression in M. tuberculosis-infected THP-1 cells and the association of Mcl-1 levels with bacterial survival.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 and HBSS were obtained from StemCell Technologies (Vancouver, BC, Canada). FCS was purchased from HyClone Laboratories (Logan, UT). Middlebrook 7H9, 7H10, and Middlebrook OADC enrichment were obtained from Difco (Detroit, MI). The Kinyoun staining kit was purchased from BBL Microbiology Systems (Cockeysville, MD). Rabbit polyclonal anti-human Mcl-1 Ab was obtained from Upstate Biotechnology (Lake Placid, NY). Goat polyclonal anti-actin Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary goat anti-rabbit Ab conjugated to HRP was obtained from Bio-Rad (Hercules, CA) and secondary anti-goat Ab conjugated to HRP was purchased from Santa Cruz Biotechnology. TRIzol and Superscript Reverse Transcriptase II were obtained from Life Technologies (Grand Island, NY). The Primer Express program and SYBR green master mix were purchased from Applied Biosystems (Foster City, CA). The pGEMT-Easy vector was obtained from Promega (Madison, WI). Annexin V-PE was purchased from BD PharMingen (San Diego, CA). Actinomycin D was obtained from Calbiochem (La Jolla, CA). LipofectAMINE was purchased from Life Technologies. Phosphorothioate-modified oligonucleotides (s-oligos) were synthesized and purified by Life Technologies.
Isolation and culture of human MDMs
MDMs were isolated as described previously (27). Mononuclear cells were allowed to adhere to cell culture flasks for 45 min at 37°C, 5% CO2, in a humidified atmosphere. Nonadherent cells were removed with three washes with HBSS and medium was replenished. Adherent cells were used the following day for infections.
M. tuberculosis infections
The human promonocytic cell line THP-1 was obtained from the
American Type Culture Collection (TIB-202; Manassas, VA). M.
tuberculosis strains H37Rv and H37Ra were used as virulent and
attenuated type strains, respectively. The human myeloid cell line
THP-1 was used as a model for human macrophage cells in infection
studies because of its similarity to human MDMs and its availability
for use (28, 29). THP-1 cells were cultured in RPMI 1640
supplemented with 10% FCS and 2 mM glutamine and were maintained
between 2 and 10 x 105 cells/ml. M.
tuberculosis was grown to late log phase in Middlebrook 7H9 with
OADC. Aliquots were frozen at -70°C. Representative samples were
thawed and CFUs per ml were enumerated by plating on Middlebrook 7H10
with OADC. Heat-killed bacteria were prepared by heating aliquots at
80°C for 2 h, resulting in a decrease in CFUs recovered of
greater than five logs. Cells were infected as described previously
(29). Briefly, THP-1 cells were seeded in six-well
flat-bottom tissue culture plates and allowed to adhere and
differentiate at 37°C in a humidified, 5% CO2
atmosphere for 18 h in the presence of 20 ng/ml PMA. Cells were
washed and medium was replaced to remove PMA 4 h before the
addition of bacteria. Where indicated, bacteria were opsonin coated by
incubating with 50% fresh serum in RPMI 1640 for 30 min before
addition to culture. Bacteria, opsonin coated or not, were added to
THP-1 cells at infection ratios of 5:1, 20:1, 50:1, or 200:1, resulting
in infection rates as reported in Results. Latex beads (LB)
were used at a ratio of 50:1, resulting in a rate of one to seven beads
per cell in 
90% of cells. Nonopsonin-coated bacteria were added to
MDMs at a ratio of 5:1, resulting in an infection rate of one bacilli
per macrophage. Infection was evaluated by CFU assays. After 4 h
of coincubation of bacteria or LB with cells, cells were washed three
times with HBSS to remove noningested particles and the medium was
replenished.
SDS-PAGE and Western immunoblotting
SDS-PAGE was performed according to the method of Laemmli (30). Western-blotted membranes were developed by ECL as previously described (31).
Quantitative PCR (Q-PCR)
Total RNA was isolated from THP-1 cells using TRIzol according to the manufacturer’s instructions. cDNA was prepared by reverse transcription using Superscript Reverse Transcriptase II according to the manufacturer’s instructions. Q-PCR primers for mcl-1 were designed using the Primer Express program and sequences were as follows: forward, 5'-TGGGTTTGTGGAGTTCTTCCA-3'; and reverse, 5'-ACACCTGCAAAAGCCAGCA-3'. Primers were synthesized by Applied Biosystems. The mcl-1 Q-PCR product was cloned into pGEMT-Easy vector according to the manufacturer’s instructions to compare unknown samples to those with known copy numbers of the mcl-1 PCR product. Q-PCR was conducted on cDNA samples and dilutions of plasmid containing the mcl-1 PCR product in triplicate using the SYBR green master mix. Negative controls included RNA with no reverse transcriptase for DNA contamination and no template for environmental contamination.
Sense and antisense s-oligos
s-Oligos were prepared to the sense and antisense strands of mcl-1 and incorporated into cells as described previously (29). Briefly, s-oligos to human mcl-1 were synthesized and HPLC purified by Life Technologies. s-Oligos were phosphorothioate-modified to prevent intracellular degradation and purified to remove incomplete synthesis products. The 19-mer antisense sequence was 5'-GGGGCTTCCATCTCCTCAA-3' as described previously (26) and the sense sequence was 5'-TTGAGGAGATGGAAGCCCC-3'. To incorporate s-oligos into THP-1 cells, 5 x 106 were resuspended in 500 µl of RPMI 1640 containing 2.5% LipofectAMINE/5 µM s-oligos and incubated on a rotary shaker for 4 h at 37°C before differentiation with PMA for THP-1 cells or before allowing monocytes to adhere for MDMs.
Annexin V-PE binding
Cells were scraped, spun down, and washed twice with 1 ml of cold PBS/106 cells. Cells (1 x 106) were resuspended in annexin V-PE binding buffer (10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2) at 106 cells/ml. An aliquot (100 µl) was removed and 5 µl of annexin V-PE was added. The mixture was vortexed gently and incubated for 15 min at room temperature in the dark. The cells were washed once with binding buffer and fixed in 30% formaldehyde in methanol. The volume was increased to 500 µl with binding buffer for analysis by flow cytometry.
TUNEL assays
TUNEL assays were performed using the Fluorescein-FragEL DNA fragmentation kit (Oncogene Research Products, San Diego, CA) according to the manufacturer’s instructions. Treated cells were cytospun onto slides, stained, and examined by fluorescent microscopy. Cells were considered apoptotic if they were TUNEL positive (green fluorescent nuclear staining). A positive control included incubation of THP-1 cells with actinomycin D (50 ng/ml for 19 h).
Determination of CFUs
Enumeration of CFUs was performed as described previously (29, 32). Bacilli were plated immediately after 4 h of coincubation with cells and washing (time 0) and at 4 and 7 days after infection. Bacilli were released from cells in cold PBS/0.1% Triton X-100, serially diluted in Middlebrook 7H9 with OADC, and 20 µl of three dilutions were plated on Middlebrook 7H10 with OADC in triplicate. CFUs were counted after 14 days of incubation at 37°C and plates were maintained for 21 days to ensure that no additional CFUs appeared.
Statistical analyses
Data presented are expressed as means ± SD. Statistical analyses for Q-PCR, annexin V-PE binding, and TUNEL assays were performed by an unpaired Student’s t test. Comparisons for CFUs were done by ANOVA for each time point. Differences were considered significant at a level of p < 0.05.
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Results |
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Mcl-1 protein levels were determined at several M.
tuberculosis infection ratios and at different times after
infection by Western blotting. Multiplicity of infections (MOIs) for
nonopsonized and opsonized M. tuberculosis strain H37Rv were
titrated to determine the optimal MOI for inducing Mcl-1. Infection
with nonopsonized bacteria at initial ratios of 5:1, 20:1, 50:1, or
200:1 resulted in MOIs of 0.04, 0.44, 0.81, or 1.80 bacteria per
macrophage, respectively, as determined by recovery of CFUs immediately
after infection and washing (time 0). Infection at the same ratios with
opsonized bacteria resulted in MOIs of 0.59, 1.20, 4.21, and 8.56,
respectively. Control cells constitutively expressed low levels of
Mcl-1 and enhanced expression of Mcl-1 was observed in macrophages
infected with either nonopsonized or opsonized M.
tuberculosis (Fig. 1
A).
With nonopsonin-coated bacteria, induction of Mcl-1 protein occurred
with a MOI of as low as 0.44 and was maximal at 0.81 (Fig. 1A, lanes 2–5). With opsonized bacteria,
induction of Mcl-1 was detected at a MOI of 0.59 and was maximal at
1.20 (Fig. 1A, lanes 6–9).
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B). Induction of mcl-1 mRNA levels and protein expression requires infection with virulent strain H37Rv
Expression of mcl-1 mRNA was evaluated in THP-1 cells
that were infected with either H37Rv or H37Ra as well as in cells
exposed to either HK H37Rv or LB. In comparison to control cells, there
was a 9.6-fold increase in mcl-1 mRNA in H37Rv-infected
macrophages (Fig. 2A). In
contrast, no significant increases in mcl-1 gene expression
were observed for cells exposed to any H37Ra bacteria, heat-killed
M. tuberculosis strain H37Rv (HK) bacteria, or LB (Fig. 2A). In THP-1 cells, densitometry of ECL-developed bands
revealed a 4.8-fold increase of Mcl-1 protein, as determined by Western
blotting of whole cell lysates, in H37Rv-infected cells vs uninfected
control cells, whereas steady-state levels of Mcl-1 protein were not
increased by exposure to either H37Ra, HK bacteria, or LB (Fig. 2B). Similarly, in MDMs, there was a 2.5-fold increase of
Mcl-1 protein measured in cells infected with H37Rv vs uninfected
control cells, whereas there was no difference in the amount of Mcl-1
protein detected after exposure of cells to either H37Ra, HK bacteria,
or LB (Fig. 2C).
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To determine whether Mcl-1 expression could be reduced in THP-1
cells and MDMs, phosphorothioate modified antisense and sense
oligonucleotides (s-oligos) were synthesized based upon sequences in
human mcl-1. Incorporation of s-oligos into cells was
verified by measuring the fluorescence of cells treated with
FITC-labeled s-oligos compared with control cells by flow cytometry.
FITC-labeled s-oligos were incorporated into 80–90% of cells in two
independent experiments (data not shown). Treatment with antisense, but
not sense, s-oligos decreased the amount of Mcl-1 protein in whole cell
lysates of noninfected THP-1 cells by >90% and in noninfected MDMs by
>96% (Fig. 3A). Mcl-1
protein was similarly reduced by 90% in whole cell lysates of THP-1
cells and MDMs infected with H37Ra and by 86% or 84%, respectively,
in whole cell lysates of THP-1 cells or MDMs infected with H37Rv (Fig. 3B).
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The contribution of Mcl-1 to THP-1 cell and MDM apoptosis during
M. tuberculosis infection was examined by annexin V-PE
binding to phosphatidylserine on the cell surface. Phosphatidylserine
surface expression, an early marker of cell apoptosis, was assessed by
annexin V-PE binding and flow cytometric analysis at 1 and 4 days after
infection. One day after infection, compared with controls, the
frequencies of annexin V-PE-positive cells were increased in response
to infection with H37Ra but not H37Rv (Fig. 4, A and C). This
effect was not influenced by treatment with mcl-1 antisense.
One day after infection with H37Rv, the percentages of annexin
V-PE-positive THP-1 cells were similar in untreated and sense
s-oligo-treated cells at 12 ± 1% and 15 ± 1%,
respectively. However, the percentage of H37Rv-infected cells that were
positive for annexin V-PE binding increased dramatically to 29 ±
4% in THP-1 cells that were treated with antisense s-oligos to
mcl-1 (Fig. 4A, left graph).
Pretreatment of cells with antisense s-oligos to mcl-1
brought about a similar increase in annexin V-PE binding positive cells
at 4 days postinfection with H37Rv (Fig. 4A, right
graph). M. tuberculosis strain H37Rv-infected cells
treated with sense s-oligos were 28 ± 2% annexin V-PE binding
positive, whereas antisense s-oligo treatment increased this to 53
± 6% positive cells (Fig. 4A, right graph).
Whereas annexin V binding was again higher in H37Ra-infected cells,
this time at 4 days after infection (Fig. 4A, right
graph), as had been observed at 1 day postinfection, this response
was not affected by antisense mcl-1 treatment.
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C,
left and right graphs, respectively), although
infection caused a larger amount of annexin V-PE binding in these
cells. Infection with strain H37Ra caused significantly more annexin
V-PE binding than did infection with strain H37Rv. Furthermore,
antisense oligonucleotides to mcl-1 caused an increase in
annexin V-PE binding positivity in H37Rv-infected cells, but not in
control cells or H37Ra-infected cells at 1 and 4 days after infection
(Fig. 4C).
To ensure that the cause of the annexin V-PE binding to cells was
apoptosis, we evaluated the number of treated THP-1 cells displaying
nuclear fragmentation by TUNEL assay at 1 and 4 days after treatment
and infection (Fig. 4B). One day after infection with H37Rv,
the percentages of TUNEL-positive THP-1 cells were similar in untreated
and sense s-oligo-treated cells at 7 ± 1% and 8 ± 1%,
respectively. However, the percentage of H37Rv-infected cells that were
TUNEL positive increased dramatically to 21 ± 2% in THP-1 cells
that were treated with antisense s-oligos to mcl-1 (Fig. 4B, left graph). Pretreatment of cells with
antisense s-oligos to mcl-1 caused a similar increase in
TUNEL-positive cells 4 days after infection with H37Rv (Fig. 4B, right graph). M. tuberculosis
strain H37Rv-infected cells treated with sense s-oligos were 17 ±
2% annexin V-PE binding positive, whereas antisense s-oligo treatment
increased this to 36 ± 3% positive cells (Fig. 4B,
right graph). Although TUNEL positivity was again higher in
H37Ra-infected cells at 4 days after infection (Fig. 4B,
right graph), as had been observed at 1 day postinfection,
this response was not affected by antisense mcl-1
treatment.
Mcl-1 induction in H37Rv-infected cells did not protect host cells from
apoptosis induced by FasL or by ATP (data not shown). THP-1 cells were
noninfected or infected with H37Rv or H37Ra followed by treatment with
either 20 U/ml FasL or 20 mM ATP to induce apoptosis. Apoptosis was
then measured by annexin V-PE binding to the surface of the cell
membrane. FasL treatment resulted in induction of apoptosis in 72%
of cells after 24 h and this was not significantly different in
noninfected cells, H37Rv-infected cells, or H37Ra-infected cells. ATP
resulted in annexin V-PE binding in >84% of cells after 24 h and
this also was not significantly different in noninfected,
H37Rv-infected, or H37Ra-infected cells.
Attenuation of Mcl-1 expression reduces intracellular survival of M. tuberculosis strain H37Rv
To investigate whether M. tuberculosis strain
H37Rv-induced Mcl-1 expression correlated with the intracellular
survival of bacteria, growth of strains H37Ra and H37Rv was evaluated
in THP-1 cells (Fig. 5A) and
MDMs (Fig. 5B) that were left untreated or treated with
sense or antisense s-oligos to mcl-1. Intracellular growth
of H37Ra was similar whether cells were left untreated or treated with
sense or antisense s-oligos to mcl-1. Growth of strain H37Rv
was similar in untreated cells or cells treated with sense s-oligos to
mcl-1. However, there were dramatic decreases in CFUs
recovered from antisense s-oligo-treated cells at both 4 and 7 days
after infection with H37Rv compared with cells treated with sense
s-oligos (Fig. 5). Recovery of CFUs from antisense-treated THP-1 cells
was 42 ± 3% of CFUs recovered from sense s-oligo-treated,
H37Rv-infected cells at 4 days after infection, and this was reduced
further to only 21 ± 2% at 7 days after infection (Fig. 5A). Recovery of CFUs from antisense-treated MDMs was
56 ± 4% of CFUs recovered from sense s-oligo-treated,
H37Rv-infected cells at 4 days after infection, and this was reduced
further to only 19 ± 2% by 7 days after infection (Fig. 5B).
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Discussion |
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The results presented in Fig. 1A demonstrate that induction
of Mcl-1 by infection with virulent M. tuberculosis strain
H37Rv occurred whether or not bacteria were serum opsonized (Fig. 1A). This suggests that Mcl-1 induction is independent of
the route of entry of the bacilli. It has been reported that at least
seven different receptors including CR1, CR3, CR4, mannose receptor,
scavenger receptors, CD14, and sp-A are involved in the entry of
M. tuberculosis into macrophages (34).
Different receptor pathways may be favored by serum opsonization, but
Mcl-1 induction occurs in the presence or absence of serum
opsonins.
It has been observed that low MOIs for M. tuberculosis cause
less apoptosis than high MOIs and in contrast that production of
TNF- was inversely proportional to MOI (35). Whereas
this may represent a mechanism by which M. tuberculosis
reduces host cell apoptosis and replicates within the host macrophage,
we observed Mcl-1 induction to occur over a wide range of MOIs.
Mcl-1 induction was evident at a MOI as low as 0.44 and increased
further up to a MOI of 8.6, suggesting that induction of this
antiapoptotic mechanism does not require low MOIs (Fig. 1A).
Optimal induction of Mcl-1 occurred at 24 h after infection (Fig. 1B), suggesting that this mechanism is not triggered by
phagocytosis per se, but rather by something elaborated by internalized
bacteria.
In this study, H37Rv infection induced mcl-1 gene expression
(Fig. 2A) and Mcl-1 protein expression (Fig. 2, B
and C), an antiapoptotic gene product, but H37Ra does not.
This is consistent with a model in which host cell apoptosis provides
an antibacterial defense mechanism that is subverted by virulent strain
H37Rv (4, 5, 36). Mcl-1 induction did not occur after
infection with H37Ra or phagocytosis of HK or LB, verifying that the
response is not triggered by phagocytosis (Fig. 2), but rather
infection with viable, virulent M. tuberculosis is required
for induction. Taken together with the finding that maximal induction
of Mcl-1 occurred between 24 and 48 h after infection, these
results suggest that the induction of Mcl-1 is dependent upon a factor
elaborated by viable and virulent M. tuberculosis within the
phagocyte.
Antisense oligonucleotides to mcl-1 reduced the expression
of Mcl-1 protein in control cells (Fig. 3A) and in cells
infected with either M. tuberculosis strain H37Ra or strain
H37Rv (Fig. 3B). Mcl-1 expression in antisense
s-oligo-treated, H37Rv-infected cells was lower than basal expression
in untreated, uninduced control cells (Fig. 3B, lane
5 vs lane 1). The magnitude of the reduction in Mcl-1
expression was similar to that observed previously using the same
antisense oligonucleotide sequence (26) where
mcl-1 antisense treatment resulted in an increase in
apoptosis in polymorphonuclear cells induced by hypoxia while having no
effect on control cells (26). Treatment with antisense
s-oligos to mcl-1 also resulted in an increase in apoptotic
host cells in response to infection with H37Rv (Fig. 4). This suggested
that Mcl-1 induction by virulent bacteria is an antiapoptotic
mechanism. In contrast, for the attenuated strain H37Ra, which did not
induce Mcl-1 expression, antisense treatment did not influence the
level of apoptosis observed (Fig. 4).
Suppression of apoptosis by strain H37Rv may provide a means for
increased intracellular bacterial proliferation. Inhibition of
mcl-1 expression by treatment with antisense s-oligos
resulted in a reduced ability of H37Rv to grow within differentiated
THP-1 cells and MDMs, respectively (Fig. 5). The induction of apoptosis
in antisense-treated, H37Rv-infected THP-1 cells (Fig. 4, A
and B) correlated with reduced recovery of CFUs from
infected cells (Fig. 5A). Similarly, the annexin V-PE
binding of antisense s-oligo-treated, H37Rv-infected MDMs (Fig. 4C) correlated with reduced recovery of CFUs from infected
MDMs (Fig. 5B). This finding is consistent with a growing
appreciation that apoptosis of infected macrophages leads to decreased
survival of M. tuberculosis. For example, Molloy et al.
(8) first described the antimycobacterial effect of host
apoptosis in ATP-treated macrophages compared with the lack of
antimycobacterial activity of necrotic macrophages (hydrogen peroxide
treated). In addition, FasL-induced apoptosis had an
antimycobacterial effect in vitro against M. tuberculosis
strains H37Ra and H37Rv as did TNF--induced apoptosis
(10). Recently, the antimycobacterial activity of
picolinic acid against M. avium was attributed to induction
of host cell apoptosis (9).
Although the increase in apoptosis in antisense oligonucleotide,
H37Rv-infected THP-1 cells and MDMs correlated with a decrease in
intracellular proliferation of this strain, the amount of cellular
apoptosis did not correlate with intracellular growth throughout this
study. For example, H37Ra infection was observed consistently to cause
more host cell apoptosis than strain H37Rv (Fig. 4), yet its growth in
untreated cells or sense oligonucleotide-treated cells was comparable
to that of strain H37Rv. Hence, it may be that there is a threshold
amount of apoptosis required to negatively effect intracellular growth
of H37Rv. Alternatively, there may be mechanisms in addition to
apoptosis that are negatively regulated by Mcl-1 which contribute to
the reduced recovery of H37Rv when they are released from the
inhibitory control of Mcl-1.
Attenuation of host cell apoptosis via induction of the antiapoptotic
mechanisms by intracellular pathogens may provide a means to evade the
host response, allowing increased bacterial proliferation
(37). One example of a mycobacterial antiapoptotic
mechanism is reduced Fas expression during M. tuberculosis
infection, which may provide a means for M. tuberculosis to
limit FasL-mediated apoptosis (10), promoting
intracellular survival. A second example may relate to inhibition of
TNF--induced apoptosis by virulent M. tuberculosis.
Despite the fact that infection of macrophages with strains H37Rv or
H37Ra leads to similar levels of TNF- production, M.
tuberculosis strain H37Ra causes more apoptosis than does
strain H37Rv. This paradox may be explained by the finding that
infection with M. tuberculosis strain H37Rv brings about
greater expression of soluble TNFR 2 (sTNFR2), thereby blocking the
activity of TNF- (38). It has been proposed, therefore,
that expression of sTNFR2 provides strain H37Rv with a means to
decrease host cell apoptosis, thereby increasing its intracellular
survival. Increased expression of sTNFR2 by virulent strain H37Rv, but
not attenuated strain H37Ra is consistent with the observation that
avirulent strains of mycobacteria cause more apoptosis in host
macrophages than virulent strains. Furthermore, the association of more
host cell apoptosis with avirulent strains suggests that macrophage
apoptosis is a host defense mechanism used to prevent proliferation of
internalized bacilli (4, 5, 36). In this study, infection
with M. tuberculosis strain H37Ra promoted significantly
more macrophage apoptosis than was found during infection with strain
H37Rv or in uninfected control cells 1 and 4 days after infection (Fig. 4). This observation correlates with the strain’s inability to induce
Mcl-1 expression and provides an additional mechanism whereby virulent
strains of mycobacteria may cause less apoptosis than avirulent
strains.
Mcl-1 induction may be a direct effect of signaling from M.
tuberculosis. Alternatively, it may be an indirect effect of
M. tuberculosis infection-induced cytokine production or a
combination of both. It has recently been shown that TNF- signaling
in human prostate cancer cells leads to antiapoptotic bcl-2
gene expression and bcl-2 protein expression via NF-
B
(39). At present, although there has not been any direct
evidence presented to link induction of other bcl-2 family members to
TNF- signaling or NF-B activation, it is conceivable that other
family members may be similarly regulated. Induction of another
antiapoptotic Bcl-2 family member, A1 protein encoded by the gene
bfl-1, has recently been reported during infection of murine
macrophages with live or dead M. bovis BCG
(18). A1 induction was associated with the protection of
host macrophages from NO-induced apoptosis. Mcl-1 induction did not
protect H37Rv-infected macrophages from either FasL or ATP-induced
apoptosis. The role of A1 in intracellular survival of BCG was not
assessed (18).
Virulent mycobacteria use multiple, distinct strategies to prevent host
cell apoptosis. Infection with virulent strains has been shown to
prevent apoptosis by minimizing external signals that induce host cell
apoptosis. Two such strategies have been demonstrated thus far
including the decrease in Fas expression, which reduces FasL-induced
apoptosis (10), and the increase in sTNFR expression,
which competes for binding of TNF- with the membrane receptor,
thereby reducing TNF--mediated host cell apoptosis
(38). Infection with virulent M. tuberculosis
also increases expression of antiapoptotic proteins and this effect may
be a direct effect of M. tuberculosis or may be an indirect
effect of M. tuberculosis infection-induced cytokine
production. Antiapoptotic genes induced after infection with virulent
M. tuberculosis include bcl-xL,
bfl-1, and mcl-1, as demonstrated here (17, 18).
In this study, Mcl-1 was induced by infection of THP-1 cells with viable and virulent M. tuberculosis, but not in response to phagocytosis or ingestion of heat-killed or attenuated M. tuberculosis. Induction of this antiapoptotic protein limited the extent of apoptosis in virulent M. tuberculosis-infected macrophages as inhibition of Mcl-1 expression resulted in increased apoptosis of infected cells. This mechanism appears to contribute to the differential amounts of apoptosis observed when comparing virulent and avirulent mycobacterial strains and species. Finally, the induction of antiapoptotic Mcl-1 is important for the intracellular survival and/or proliferation of virulent M. tuberculosis as reduction of Mcl-1 expression by antisense oligonucleotides resulted in decreased CFUs recovered from H37Rv-infected macrophages. An important objective at this stage is to determine the nature of the factor produced during infection with viable and virulent strain H37Rv that leads to Mcl-1 induction. The evidence suggests that this factor must be either actively released or synthesized intracellularly since Mcl-1 induction was not observed in response to internalization of either dead bacilli or attenuated strain H37Ra. In summary, these findings present a novel mechanism by which virulent M. tuberculosis evades apoptosis, an innate, protective host cell response to infection, thereby promoting its intracellular survival and proliferation.
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Acknowledgments |
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Footnotes |
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2 L.M.S., and S.M.H.-W. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. W. Robert McMaster, Jack Bell Research Center, 2660 Oak Street, Vancouver, British Columbia, Canada V6H 3Z6; E-mail address: robm{at}interchange.ubc.ca
4 Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; MDM, monocyte-derived macrophage; FasL, Fas ligand; MOI, multiplicity of infection; HK, heat-killed M. tuberculosis strain H37Rv; LB, latex beads; s-oligo, phosphorothioate-modified oligonucleotide; Q-PCR, quantitative PCR; sTNFR2, soluble TNFR 2.
Received for publication December 12, 2001. Accepted for publication October 25, 2002.
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) or infected with M. tuberculosis
strain H37Rv (
) or strain H37Ra (
). A and
C, Annexin V-PE binding positive THP-1 cells or MDMs
were measured by flow cytometry using untreated and uninfected cells as
a negative control and actinomycin D-treated (0.5 µg/ml for 19
h) THP-1 cells as a positive control. B, TUNEL-positive
THP-1 cells were measured by flow cytometry using untreated and
noninfected THP-1 cells as a negative control and actinomycin D-treated
cells as a positive control. Values represent the means ± SD from
three independent experiments counting 10,000 events per treatment
group in each experiment. *, p < 0.05 for
H37Ra-infected cells untreated or treated with sense s-oligos compared
with H37Rv-infected cells with the same treatments; **,
p < 0.01; ***, p <
0.005 for H37Rv-infected and antisense s-oligo-treated vs
H37Rv-infected and control, untreated, or sense s-oligo-treated cells
and vs H37Ra-infected and antisense s-oligo-treated cells.
, •) or from sense
(
, 
) s-oligo-treated cells that were
infected with either M. tuberculosis strain H37Ra (