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Transcriptional Responses Without Inhibiting Activation of STAT11
Division of Infectious Diseases, University of California, Rosalind Russell Arthritis Research Laboratory and Loewenstein Laboratory for Mycobacterial Research, San Francisco General Hospital, San Francisco, CA 94143
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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activates macrophages to kill diverse intracellular
pathogens, but does not activate human macrophages to kill virulent
Mycobacterium tuberculosis. We tested the hypothesis
that this is due to inhibition of IFN- signaling by M.
tuberculosis and found that M. tuberculosis
infection of human macrophages blocks several responses to IFN-,
including killing of Toxoplasma gondii and induction of
FcRI. The inhibitory effect of M. tuberculosis is
directed at transcription of IFN--responsive genes, but does not
affect proximal steps in the Janus kinase-STAT pathway, as STAT1
tyrosine and serine phosphorylation, dimerization, nuclear
translocation, and DNA binding are intact in M.
tuberculosis-infected cells. In contrast, there is a marked
decrease in IFN--induced association of STAT1 with the
transcriptional coactivators CREB binding protein and p300 in M.
tuberculosis-infected macrophages, indicating that M.
tuberculosis directly or indirectly disrupts this
protein-protein interaction that is essential for transcriptional
responses to IFN-. Gamma-irradiated M. tuberculosis
and isolated cell walls reproduce the effects of live bacteria,
indicating that the bacterial component(s) that initiates inhibition of
IFN- responses is constitutively expressed. Although
lipoarabinomannan has been found to exert effects on macrophages, it
does not account for the inhibitory effects of cell walls. These
results indicate that one mechanism for M. tuberculosis
to evade the human immune response is to inhibit the IFN- signaling
pathway, and that the mechanism of inhibition is distinct from that
reported for Leishmania donovani or CMV, in that it
targets the interaction of STAT1 with the basal transcriptional
apparatus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Macrophages are essential in host defense against many infections. When
macrophages encounter most bacteria, they phagocytose and kill them. In
contrast, macrophages phagocytose, but do not kill, M.
tuberculosis. Indeed, macrophages support the intracellular growth
of M. tuberculosis (reviewed in Ref. 2), and
the observation that M. tuberculosis has evolved ligands for
at least seven distinct macrophage surface receptors suggests that
entry into macrophages provides an advantageous environment for
induction of mycobacterial gene expression and subsequent growth and
spread (3). Although several cytokines and
hormones can modulate the activity of macrophages, the predominant
activator of macrophage microbicidal activity is IFN- (4, 5). IFN- is secreted by lymphocytes in response to Ag
stimulation and has been shown to be a determinant of resistance to
tuberculosis in mice, as demonstrated by the severe, disseminated
tuberculosis seen in IFN- knockout mice (6, 7). Studies
of patients with tuberculosis have demonstrated the presence of IFN-
in pleural fluid (8, 9), lung fluid (10), and
lymph nodes (11), suggesting that a defect in response to
IFN- rather than the absence of its production allows tuberculosis
to progress. In vitro, IFN- activates human macrophages to control
the growth of intracellular pathogens, including Toxoplasma
gondii, Leishmania donovani, Chlamydia
psittaci, and Legionella pneumophila (4, 12, 13, 14), but is unable to activate human macrophages to restrict
or kill virulent M. tuberculosis (12, 15). This
suggests that M. tuberculosis might interfere with cellular
signal transduction pathways that are activated by IFN- and thereby
avoids being killed within macrophages.
The signal transduction pathway initiated by IFN- is becoming
increasingly well characterized. Binding of IFN- to cell surface
receptors results in activation of the tyrosine kinases
JAK13 and JAK2, leading to
phosphorylation of cytoplasmic STAT1. Tyrosine-phosphorylated STAT1
homodimerizes through interaction of the SH2 domain on one molecule
with phosphotyrosine on another and translocates to the nucleus. In the
nucleus, STAT1 homodimers activate transcription of specific genes that
possess -activation sequences (GAS; consensus sequence is
TTNCNNNAA). Human genes that contain GAS include Fc receptor
type I (CD64), guanylate binding protein-2, class II
trans-activator, and indoleamine-2,3-dioxygenase
(16, 17, 18, 19). Although phosphorylation of STAT1 on
Tyr701 is sufficient for dimerization,
nuclear translocation, and activation of transcription, maximal
transcriptional activity of STAT1 also requires phosphorylation on a
single serine (Ser727) (20).
Interaction of STAT1 with the basal transcriptional apparatus is
mediated by interaction with the CREB binding protein (CBP)/p300 family
of transcriptional coactivators, an interaction that may be regulated
in part by phosphorylation of STAT1 at Ser727
(21). Dephosphorylation of STAT1 is one means of
terminating IFN signaling (22), although at least a
portion is ubiquinated and catabolized by proteasomes
(23).
In the present study we tested the hypothesis that M.
tuberculosis blocks IFN--initiated activation of macrophages,
using human macrophages infected with a virulent strain of M.
tuberculosis. We found that M. tuberculosis infection
inhibits IFN--activated microbicidal activity and IFN--induced
gene expression without inhibiting proximal steps in the JAK-STAT
signaling pathway. Rather, M. tuberculosis infection
disrupts the essential interaction of STAT1 with CBP and p300. We also
found that the effect of live bacteria could be reproduced by
gamma-irradiated M. tuberculosis and by crude cell wall
fragments but not by purified lipoarabinomannan.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human monocytes were isolated from buffy coats and cultured for 6 days in 24- or 6-well tissue culture plates (Corning, Corning, NY) as previously described (24) with a minor modification: cells were cultured in RPMI 1640 with 2 mM L-glutamine (Life Technologies, Gaithersburg, MD) and 2.5% autologous serum overnight after plating to improve adherence. Culture medium and nonadherent cells were removed by aspiration after 1 and 5 days of culture, and monolayers were subsequently incubated with fresh culture medium supplemented with 10% autologous serum.
Culture of M. tuberculosis and infection of macrophages
M. tuberculosis (Erdman) was used to infect macrophages as previously described (3). All experiments with M. tuberculosis-infected macrophages were performed with cells 3 days after infection unless stated.
Culture and infection of macrophages with Toxoplasma gondii
T. gondii (RH strain) were obtained from Dr. Kami Kim
(Albert Einstein College of Medicine, New York, NY) and maintained by
passage in human foreskin fibroblasts grown in DMEM with 10%
heat-inactivated FBS and 2 mM L-glutamine (Life
Technologies). Freshly isolated parasites from lysed human foreskin
fibroblast monolayers were resuspended in RPMI 1640 with 2 mM
L-glutamine and 2.5% heat-inactivated FBS and
added to macrophage monolayers at an MOI of 1. After 1 h of
incubation at 37°C, extracellular parasites were removed by washing
the monolayers three times with PBS. Some cells were fixed for
enumeration of toxoplasma at this time corresponding to time zero,
while others were incubated with RPMI 1640 with autologous serum alone
or medium containing IFN- (100 ng/ml; human rIFN-; 3 x
107 U/mg; Genentech, South San Francisco, CA) at
37°C for 8–24 h. Cells were then fixed, and intracellular toxoplasma
were enumerated after staining with 4',6-diamidino-2-phenylindole,
dihydrochoride (DAPI; 0.5 µg/ml; Molecular Probes, Eugene,
OR).
Flow cytometry analysis of FcRI expression
M. tuberculosis-infected and uninfected human
macrophages cultured in six-well plates were either treated or
untreated with IFN- (20 ng/ml) for 24 h at 37°C. Cell
monolayers were washed twice with PBS, chilled in PBS containing 0.5 mM
EDTA on ice, and scraped from the wells. Cells were washed and
resuspended in PBS with 1% human serum and 0.1%
NaN3 at 5 x 106
cells/ml. Aliquots of 5 x 105 cells were
incubated for 45 min on ice with FITC-conjugated anti-FcRI
(anti-CD64; Ancell, Bayport, MN) using the concentrations
recommended by the manufacturer. Cells were then washed three times
with PBS and fixed overnight in 1% paraformaldehyde. Ten thousand
cells were analyzed for FcRI expression on a FACSort flow cytometer
with CellQuest software (Becton Dickinson, Mountain View, CA).
Northern hybridization analysis
Total cellular RNA was isolated (RNeasy, Qiagen, Chatsworth, CA)
from either M. tuberculosis-infected or uninfected
macrophages cultured in six-well plates that were treated with IFN-
(20 ng/ml) at 37°C for the indicated times. RNA was quantitated by UV
absorbance, and equal amounts (10 µg) were fractionated by
electrophoresis, transferred to nylon membranes, and hybridized with a
32P-radiolabeled FcRI cDNA probe
(106 cpm/ml) containing a fragment of human
FcRI cDNA (nucleotides 488–845 of the a1 splicing product). After
washing and exposure to film, blots were stripped for 2 h at
65°C in stripping solution (5 mM Tris-HCl (pH 8.0), 2 mM EDTA, and
0.1x Denhardt’s solution) to remove bound FcRI probe and then
rehybridized with a 32P-radiolabeled GAPDH probe
containing nucleotides 536–899 of the human GAPDH cDNA to verify the
amount of RNA in each lane. Quantitation of signals on Northern blots
was performed on IS-1000 Digital Imaging System (Alpha Innotech, San
Leandro, CA). Both radiolabeled FcRI and GAPDH cDNA probes were
generated by random priming with [-32P]dCTP
(Amersham, Arlington Heights, IL) using a Prime-It RmT Random Primer
Labeling Kit (Stratagene, La Jolla, CA).
Preparation of macrophage cytoplasm and nuclear extracts
Before treatment with IFN-, M.
tuberculosis-infected and uninfected macrophages were preincubated
with fresh culture medium for 2 h at 37°C. The cells were then
treated with IFN- (20 ng/ml) for the indicated times, washed with
ice-cold PBS, scraped into PBS, and pelleted by centrifugation
(500 x g) at 4°C for 10 min. Cell pellets were
resuspended in buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA,
1 mM EGTA, 1 mM sodium orthovanadate, 1 mM PMSF, 20 µg/ml aprotinin,
20 µg/ml antipain, 20 µg/ml leupeptin, and 10 µg/ml pepstatin A)
and incubated on ice for 10 min. Nonidet P-40 was added to a final
concentration of 0.2%, and the cell suspension was passed through a
26-gauge needle to break open cells. After centrifugation (15,000
x g, in a microcentrifuge) at 4°C for 1 min, supernatants
were collected as cytoplasmic extracts, and the pellets (crude nuclei)
were washed with buffer A and then resuspended in buffer C (20 mM HEPES
(pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM sodium
orthovanadate, and the same protease inhibitors as in buffer A). After
incubation at 4°C for 60 min, insoluble materials were pelleted by
centrifugation (15,000 x g) at 4°C for 10 min, and
supernatants were collected as nuclear extracts. Whole cell lysates
were prepared by lysis of cells in RIPA lysis buffer containing
phosphatase and protease inhibitors (50 mM Tris (pH 7.5), 1% Nonidet
P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 2 mM EDTA, 50
mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF, 20 µg/ml
aprotinin, 20 µg/ml antipain, 20 µg/ml leupeptin, and 10 µg/ml
pepstatin) at 4°C for 10 min. The protein concentration was measured
by the method of Bradford (25) using a protein assay kit
and BSA standards (Bio-Rad Laboratories, Hercules, CA).
Immunoblot analysis of STAT1
Proteins were subjected to SDS-PAGE on 8% polyacrylamide,
transferred to nitrocellulose membranes, and blocked with 5% nonfat
milk in Tris-buffered saline-Tween-20 (20 mM Tris (pH 7.6), 0.14 M
NaCl, and 0.1% Tween-20) for 1 h at room temperature. The
membranes were then incubated with a rabbit polyclonal Ab specific for
phosphorylated (at Tyr701) STAT1 (New England
Biolabs, Beverly, MA), a rabbit polyclonal Ab specific for
serine-phosphorylated (at Ser727) STAT1
(provided by Dr. David A. Frank, Dana-Farber Cancer Institute, Boston,
MA) (26), a mAb that recognizes total STAT1 (Transduction
Laboratories, Lexington, KY), or anti-annexin I polyclonal Ab 4800
(27) at a 1/50,000 dilution. After washing with
Tris-buffered saline-Tween-20, blots were incubated with HRP-conjugated
goat anti-rabbit IgG secondary Ab (for anti-phosphorylated
STAT1 and anti-annexin I) or goat anti-mouse (for
anti-total STAT1; Zymed, South San Francisco) for 1 h at room
temperature, and bound Ab was visualized by enhanced chemiluminescence
(Amersham, Arlington Heights, IL). Densitometry was performed by
measuring the density of bands using an IS-1000 Digital Imaging System
(Alpha Innotech).
EMSAs
Cytoplasmic extract (10 µg protein) or nuclear extract (1.5
µg protein) from equal numbers of cells was incubated with
105 cpm of 32P end-labeled
double-stranded oligonucleotide probe as previously described
(28). The phosphorylated probe containing the sequence
GTATTTCCCAGAAAAAGGA found in the promoter region of the FcRI
gene was provided by Drs. Sarah Gaffen and Mark Goldsmith (Gladstone
Institute of Virology and Immunology, San Francisco, CA). DNA-protein
complexes were separated from free probe by electrophoresis through 4%
nondenaturing polyacrylamide gels in 0.25x TBE buffer (22.5 mM
Tris-borate and 0.5 mM EDTA). The gels were dried, and bands were
visualized by autoradiography.
STAT1 pull-down assay
Whole cell extracts of macrophages were prepared as described by Nandan et al. (29). An extended oligonucleotide containing a biotinylated 5' nucleotide, a six-base spacer, and the GAS from the human guanylate binding protein gene (29) was annealed to its complementary strand, and 100 pmol was used to bind dimerized STAT1 in whole cell extracts (200 µg of protein/condition). After allowing the GAS oligonucleotide to bind STAT1 in cell extracts (2 h, 4°C, on a rotator), streptavidin-agarose beads (Ultralink Streptavidin, Pierce, Rockford, IL; 20 µl of a 50% slurry) were added, and incubation was continued for an additional hour at 4°C. After washing three times with lysis buffer containing 100 mM KCl with protease and phosphatase inhibitors, GAS binding proteins were eluted by boiling in Laemmli sample buffer, and bound proteins were analyzed by SDS-PAGE and immunoblotting. Initial experiments established the optimal quantity of GAS oligo and streptavidin-agarose beads needed to bind all the competent STAT1 in macrophage cell extracts and established that STAT1 binding was displaceable with the addition of excess GAS oligonucleotide (not shown). Bound STAT1 was detected with anti-Tyr701-phospho-STAT1 (New England Biolabs), and bound CBP and p300 were detected with an Ab from Zymed that recognizes both proteins. The density of immunoreactive STAT1 and CBP/p300 bands was quantitated using an image analysis system (Alpha Innotech).
Neutralization of TGF-ß
A neutralizing mAb to TGF-ß1, -ß2, and -ß3 was purchased from Genzyme (Cambridge, MA), and latency-associated peptide was purchased from R & D Systems (Minneapolis, MN). Antagonists or carrier were added (at the indicated concentrations) to macrophage culture medium at the same time as addition of M. tuberculosis.
M. tuberculosis components
Gamma-irradiated M. tuberculosis, whole cell lysates,
cytosol, membranes, total lipid extract, cell wall, and
lipoarabinomannan were all derived from M. tuberculosis
H37Rv and were provided by Dr. John Belisle (Colorado State University,
Fort Collins, CO). The monoclonal Ab CS-35 was also provided by Dr.
Belisle. Killed bacteria or isolated components were incubated with
macrophages for 1 h, followed by washing with fresh medium, then
macrophages were incubated for 2 days before stimulation with
IFN-.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induction of T. gondii killing
The observation that human macrophages cannot kill M.
tuberculosis despite IFN- treatment could be due to one of two
possible general mechanisms. One is that M. tuberculosis is
intrinsically resistant to the microbicidal mechanisms of human
macrophages. The other is that M. tuberculosis inhibits
IFN--initiated activation of macrophages by blocking one or more
steps in the IFN- signal transduction pathway. Because IFN- can
activate macrophages to restrict the growth of T. gondii, we
reasoned that if M. tuberculosis blocks IFN- signaling,
M. tuberculosis-infected macrophages should not be able to
respond to IFN- by restricting T. gondii. Shown in Fig. 1
are results that demonstrate that
coinfection of macrophages with M. tuberculosis inhibits the
ability of IFN- to cause macrophages to restrict T. gondii. T.
gondii invaded as efficiently and replicated within M.
tuberculosis-infected macrophages at the same rate as in
uninfected macrophages. Although IFN- activated control macrophages
to restrict T. gondii, coinfection of macrophages with
M. tuberculosis completely abrogated the ability of IFN-
to activate macrophages to kill or restrict growth of T.
gondii. These results support the hypothesis that M.
tuberculosis blocks IFN- signaling in human macrophages.
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induction of FcRI
To further test the hypothesis that M. tuberculosis
inhibits IFN- signaling, we examined another macrophage response:
induction of expression of the type I receptor for the Fc domain of IgG
(FcRI or CD64). Analysis by flow cytometry demonstrated that cell
surface FcRI was expressed constitutively on human macrophages and
that IFN- increased expression of cell surface FcRI on uninfected
and M. tuberculosis-infected macrophages 
3-fold (Fig. 2). In this assay, M.
tuberculosis infection inhibited IFN- induction of FcRI
expression by 50% (range in 16 independent experiments using cells
from different donors was 34–60%) compared with that in uninfected
cells (Fig. 2). The effect of M. tuberculosis was not simply
a consequence of phagocytosis, because macrophages that phagocytosed
serum-opsonized zymosan or latex beads did not exhibit any reduction in
IFN- up-regulation of FcRI (not shown).
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responses is exerted at the level of
transcriptional activation of IFN-responsive genes
The increase in expression of FcRI in response to IFN- is
due to direct transcriptional activation of the FcRI gene
(30). IFN- induced expression of mRNA in both
uninfected and M. tuberculosis-infected macrophages;
however, M. tuberculosis markedly inhibited the increase in
FcRI mRNA in response to IFN- (Fig. 3). Densitometric analysis of the blot
revealed that M. tuberculosis infection resulted in
reduction of IFN--induction of FcRI mRNA transcription, with up
to 60% reduction in cells treated with IFN- for 4–8 h. These
results indicate that M. tuberculosis inhibits IFN-
induction of FcRI cell surface expression by inhibiting activation
of transcription, the end point in the IFN- signaling pathway.
M. tuberculosis inhibition of IFN- transcriptional
activation was exerted on all IFN--responsive genes that we
examined, including all three alternatively spliced transcripts of the
FcRI gene, indoleamine 2,3-dioxygenase (31), and
guanylate binding protein-2 (32) (not shown).
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To further characterize the effect of M. tuberculosis
infection on IFN- signaling, we examined an intermediate step in the
IFN- signal transduction pathway: tyrosine phosphorylation of STAT1.
As shown in Fig. 4A, IFN-
induced tyrosine phosphorylation of STAT1 and -ß in both
uninfected and M. tuberculosis-infected macrophages in a
similar time-dependent manner. Instead of inhibition of STAT1 tyrosine
phosphorylation, we found that STAT1 phosphorylation was greater in
M. tuberculosis-infected macrophages than in uninfected
cells. The ultimate extent of tyrosine phosphorylation of STAT1 and
-ß in M. tuberculosis-infected macrophages was 2.1-fold
higher than that in uninfected cells at 30 min of IFN- treatment, as
quantitated by densitometry. When the same samples were analyzed using
an Ab that detects total STAT1 and -ß, densitometric analysis
revealed that the abundance of STAT1 protein was increased
2-fold in M. tuberculosis-infected macrophages compared with
that in uninfected cells, suggesting that M. tuberculosis
infection results in up-regulation of both STAT1 and STAT1ß
expression in macrophages either by increasing synthesis or by
decreasing degradation of the protein (Fig. 4B). To address
the possibility that M. tuberculosis nonspecifically
increases the abundance of cytoplasmic proteins in macrophages, we
examined the level of another cytosolic protein, annexin I. The annexin
I content was the same in all the samples (Fig. 4C),
indicating that increase in STAT1 abundance does not extend to other
cytoplasmic proteins.
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Because tyrosine-phosphorylated STAT1 must dimerize and
translocate from the cytoplasm to the nucleus to activate
transcription, we investigated the effect of M. tuberculosis
infection on translocation of STAT1 from the cytoplasm to the nucleus.
As shown in Fig. 5,
tyrosine-phosphorylated STAT1 is present in nuclei isolated from either
uninfected or M. tuberculosis-infected macrophages treated
with IFN-, indicating that IFN--induced nuclear translocation of
STAT1 is not inhibited by M. tuberculosis infection.
Immunofluorescence microscopy using Abs to total STAT1 or to
tyrosine-phosphorylated STAT1 confirmed that IFN- induces nuclear
translocation of STAT1 in M. tuberculosis-infected
macrophages (not shown).
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-activated DNA binding activity of STAT1 is not reduced in
M. tuberculosis-infected macrophages
Because M. tuberculosis infection did not inhibit
IFN--induced tyrosine phosphorylation or nuclear translocation of
STAT1, we examined the effect of M. tuberculosis infection
on IFN--activated DNA binding activity of STAT1 by EMSA. Treatment
of macrophages with IFN- induced formation of STAT1 complexes in the
cytoplasmic and nuclear extracts that were competent to bind a
synthetic GAS derived from the human FcRI gene (Fig. 6). M. tuberculosis infection
resulted in enhanced formation of the IFN--activated DNA-protein
complex, reflecting the increased amount of cytoplasmic and nuclear
STAT1 in M. tuberculosis-infected macrophages (Fig. 5
A). These results demonstrate that M.
tuberculosis infection does not affect the ability of STAT1 to
dimerize, translocate to the nucleus, or bind specific DNA target
sequences.
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-induced serine phosphorylation of STAT1
Although phosphorylation of STAT1 on tyrosine 701 is sufficient to
cause dimerization, nuclear translocation, and DNA binding of STAT1,
full transcriptional activity of STAT1 also requires phosphorylation
at serine 727 (20, 33). Using an Ab that specifically
recognizes STAT1 phosphorylated at Ser727
(26), we found that M. tuberculosis-infected
macrophages contain a small amount of serine-phosphorylated STAT1,
and that addition of IFN- stimulated serine phosphorylation of
STAT1 to a similar extent in M. tuberculosis-infected and
uninfected macrophages (Fig. 7).
Therefore, the mechanism of M. tuberculosis inhibition of
IFN- responses is not exerted through reduced phosphorylation or
enhanced dephosphorylation of STAT1
Ser727.
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Transcriptional activation of IFN--responsive genes depends on
association of STAT1 dimers with the transcriptional coactivators CBP
and p300, which link STAT1 to the basal transcriptional apparatus and
RNA polymerase II (21, 34). Initial efforts to examine the
association of CBP and p300 with STAT1 by immunoprecipitation were
unsuccessful, because the anti-STAT1 Abs used recognize the N- and
C-terminal domains of STAT1, which are the same domains that interact
with CBP and p300. Consequently, we used oligoprecipitation with a
GAS-containing oligonucleotide bound to agarose beads to isolate STAT1
dimers and STAT1-associated proteins from M.
tuberculosis-infected and uninfected macrophages. These studies
revealed an IFN-- and time-dependent association of STAT1 with the
GAS oligonucleotide in extracts of infected and uninfected macrophages
(Fig. 8). Although the amount of STAT1
that bound the GAS oligonucleotide in lysates of M.
tuberculosis-infected macrophages was the same as (or slightly
greater than) that in lysates of uninfected macrophages, lower amounts
of CBP/p300 were present in eluates from M.
tuberculosis-infected compared with uninfected macrophages at all
time points examined. Densitometric analysis of the samples harvested
after 30 min of IFN- treatment (when the quantity of oligo-bound
STAT1 was maximal), revealed an apparent 77% reduction in the amount
of CBP/p300 associated with STAT1 (when normalized to the amount of
bound STAT1) in M. tuberculosis-infected macrophages
compared with that in uninfected macrophages. This reduction in
CBP/p300 association with STAT1 closely resembles the extent of
reduction in transcriptional responses to IFN- in M.
tuberculosis-infected macrophages.
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responses is
not blocked by neutralizing TGF-ß
The finding that M. tuberculosis inhibits IFN-
transcriptional responses without affecting known steps in the JAK-STAT
signaling pathway suggested that its mechanism of inhibition resembles
that of TGF-ß (29, 35, 36). Moreover, TGF-ß is
secreted by human monocytes containing an avirulent strain of M.
tuberculosis, H37Ra (37). However, neither an mAb to
TGF-ß nor latency-associated peptide, a protein that binds TGF-ß
with high affinity and prevents receptor binding, restored the ability
of M. tuberculosis-infected macrophages to respond to
IFN- (Fig. 9). To further evaluate the
possibility that TGF-ß mediates the inhibition of IFN- responses,
we assayed TGF-ß in medium of macrophages infected with the Erdman
strain of M. tuberculosis. We found that there was no
biologically active TGF-ß detectable in untreated medium, but we
found that acid activation of the medium revealed up to 120 pg/ml of
activatable TGF-ß. When we used this concentration of active TGF-ß
to treat macrophages, we did not observe any inhibition in IFN-
responses (data not shown). Taken together, these results strongly
indicate that the inhibitory effect of M. tuberculosis on
macrophage responses to IFN- is not mediated by the secretion of
TGF-ß.
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responses
To determine whether inhibition of IFN- responses by M.
tuberculosis is due to a bacterial component induced after
infection of macrophages or is due to a constitutively expressed
bacterial component, we substituted killed (gamma-irradiated) M.
tuberculosis (H37Rv) for live bacteria before stimulation with
IFN-. As shown in Fig. 10, there was
a dose-related inhibition of IFN--induced expression of FcRI that
closely corresponded to the inhibition observed with live M.
tuberculosis.
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responses indicates that one or more
preformed components of the bacteria are sufficient for initiating a
pathway that results in inhibition of IFN- responses. In a
subsequent experiment, we found that a whole cell lysate of M.
tuberculosis H37Rv also inhibited IFN--induced up-regulation of
cell surface FcRI, indicating that the essential bacterial component
was able to exert its effects even when it was not presented in the
form of whole particulate bacteria (not shown). We therefore surveyed
several subcellular fractions of gamma-irradiated M.
tuberculosis H37Rv. We found that cytosol (up to 100 µg/ml),
crude membranes (up to 50 µg/ml), or total lipids (up to 50 µg/ml)
had no effect on the ability of macrophages to respond to IFN- (data
not shown). In contrast, we found that the cell wall fraction possessed
a potent ability to initiate inhibition of IFN- up-regulation of
FcRI (Fig. 11). As little as 0.5
µg/ml (quantitated by protein assay) of M. tuberculosis
cell wall was capable of maximum inhibition of IFN- up-regulation of
FcRI. Because lipoarabinomannan (LAM) is a major component of the
M. tuberculosis cell wall, we examined the ability of
purified LAM to cause inhibition of IFN- up-regulation of FcRI.
Although LAM exhibited some activity, it did not inhibit the response
to IFN- to the extent observed with unfractionated cell walls, even
at very high concentrations of LAM (Fig. 11B). To determine the
relationship between the concentration of purified LAM that exerted a
partial inhibitory effect and the concentration present in highly
active unfractionated cell walls, we quantitated LAM in cell walls by
immunoblotting, compared with a standard curve constructed using
purified LAM. This revealed that the content of LAM in unfractionated
cell walls did not exceed 350 ng per 0.5 µg of cell wall protein.
Because 25 µg/ml of LAM caused less inhibition of IFN- than 0.5
µg/ml of unfractionated cell wall, we conclude that LAM cannot be the
sole component of the M. tuberculosis cell wall that
initiates the inhibition of IFN- responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activates macrophages to kill other pathogens but cannot activate human
macrophages to kill M. tuberculosis, we tested the
hypothesis that M. tuberculosis survives in macrophages by
inhibiting IFN- signaling. We found that M. tuberculosis
infection of macrophages indeed inhibits macrophage responses to
IFN-, and that inhibition is exerted at the level of transcription
of IFN--responsive genes. At least two other pathogens have been
found to inhibit IFN- signaling. Leishmania donovani
inhibits IFN- responses by inhibiting tyrosine phosphorylation of
STAT1 by JAK1 and JAK2 (38), and human CMV inhibits
responses to IFN- in infected fibroblasts and endothelial cells, by
depleting cells of JAK kinases through degradation by proteosomes
(39). In contrast, infection with M.
tuberculosis does not inhibit STAT1 tyrosine or serine
phosphorylation, dimerization, nuclear translocation, or recognition of
specific DNA sequences. Rather, infection with M.
tuberculosis inhibits IFN- responses by directly or indirectly
disrupting the essential interaction of STAT1 with the
transcriptional coactivators CBP and p300. The underlying mechanism by
which M. tuberculosis infection disrupts the
STAT1-CBP/p300 interaction remains to be elucidated, and one or more
mechanisms could be responsible. First, another transcription factor
could compete with STAT1 for the same binding site(s) on CBP/p300. CBP
and p300 are present in limiting quantities in most, if not all, cells.
A recent study found that activation of Jurkat cells with IFN-
inhibits TNF- activation of transcription of the HIV-1 long terminal
repeat because STAT2 (activated by IFN-) binds the same domain on
p300 as does NF-
B (activated by TNF-). Because Jurkat cells
possess 28,000 molecules of p300 and 150,000 molecules of STAT2
per cell, activation of STAT2 sequestered p300 so that it was
unavailable to NF-B (40). Similarly, STAT1 and
AP-1/ets have been found to inhibit one another’s actions by
competition for a limiting quantity of CBP (34). In view
of these findings, M. tuberculosis might indirectly inhibit
IFN- responses by activating a macrophage signaling pathway that
requires CBP and/or p300 and thereby restricts the availability of
these coactivators for use by STAT1. Alternatively, M.
tuberculosis could directly target the domains of either STAT1 or
CBP that are involved in their protein-protein interaction. The
interaction of STAT1 with CBP is mediated by binding of the
N-terminal domain of STAT1 to Cys/His-rich domain 1 of CBP and
binding of the C-terminal domain of STAT1 to Cys/His-rich domain 3
(21), and M. tuberculosis could target any of
these domains on either protein to disrupt their association. A
precedent for such a mechanism was recently reported; in addition to
the well-established interaction of adenovirus E1A with p300 (41, 42), E1A also directly interacts with the C-terminal domain of
STAT1 and blocks IFN- activated transcription (43).
Another potential target of such a mechanism is the N-Myc interactor
protein (Nmi). Nmi markedly stabilizes the interaction of STAT1 and
CBP and thereby enhances IFN- responses (44).
Therefore, interference with Nmi could yield the decrease in
STAT1-CBP/p300 association that we observed in M.
tuberculosis-infected macrophages. Additional experiments will be
necessary to determine which of these mechanisms may account for the
decreased association of STAT1 and CBP/p300 observed in M.
tuberculosis-infected macrophages.
An alternative, distinct, mechanism for M. tuberculosis
inhibition of IFN- responses is disruption of a pathway that is
distinct from the JAK-STAT pathway but that converges with it to
activate transcription of IFN--responsive genes. Considering the
findings that we report here, a plausible candidate target of such a
pathway is CBP. Although there is not yet any information regarding
post-translational modification of CBP in response to IFN-,
CREB/CBP-dependent transcriptional activation in pituitary cells
requires a CBP activating signal that includes nuclear calcium
signaling and calcium/calmodulin-dependent kinase IV (45).
If an analogous CBP-activating signal is required for IFN-
signaling, it represents an additional potential target of M.
tuberculosis.
M. tuberculosis LAM has been reported to inhibit
mitogen-activated protein kinase (MAPK) activation in human monocytes
by activating the protein phosphatase SHP-1 (Src homology domain
2-containing tyrosine phosphatase-1) (46). Because MAPK
can catalyze the phosphorylation of STAT1
Ser727 (20), it was essential for us
to consider inhibition of MAPK activity as a possible mechanism by
which M. tuberculosis inhibits IFN- responses. However,
our finding that STAT1 is phosphorylated on
Tyr701 and Ser727 to the
same extent and with the same kinetics in uninfected and M.
tuberculosis-infected macrophages makes this effect unlikely to
account for inhibition of IFN- responses. Moreover, we have not
observed any inhibition of phosphorylation of ERK2 at
Thr183 or Tyr185 in
response to IFN- in M. tuberculosis-infected macrophages
(L.-M. Ting and J. D. Ernst, unpublished
observation). An additional possible target of M.
tuberculosis is one or more additional kinases activated by
IFN-, such as the renaturable tyrosine kinases whose activation by
IFN- is sensitive to inhibition by TGFß (29).
Although the effects of M. tuberculosis in our experiments
are not attributable to TGFß, these or similar kinases may be
sensitive to inhibition by other mediators activated by M.
tuberculosis.
Previous studies have demonstrated that Mycobacterium
leprae-infected murine macrophages are refractory to IFN-
induction of microbicidal activity, cytotoxicity for tumor cells,
superoxide anion production, and surface Ia Ag expression (47, 48). In addition, exposure of macrophages to high concentrations
of purified M. tuberculosis LAM results in defective
responses to IFN-, including transcriptional activation,
intracellular microbicidal activity, expression of MHC class II
molecules, and cytotoxicity for tumor cells (49, 50). We
found that these effects are also found in human macrophages infected
in vitro with a virulent strain of M. tuberculosis. Although
M. tuberculosis infection did not completely inhibit
transcriptional activation of FcRI in response to IFN-,
IFN--induced killing of T. gondii was completely
abolished by M. tuberculosis infection in macrophages,
suggesting that the block of IFN--activated signaling is
functionally significant and may at least partially account for the
failure of IFN- to activate macrophages to kill M.
tuberculosis. Because these experiments were performed using high
concentrations of IFN- (20–100 ng/ml; 600-3000 U/ml) it is likely
that a more complete block may occur in vivo, where the local
concentration of IFN- is lower.
In contrast to the findings of the aforementioned studies, we found
that LAM is unlikely to be the sole component of M.
tuberculosis that initiates the inhibition of IFN- responses.
Although the most potent inhibitory activity was found in a cell wall
fraction, the concentration of LAM required for inhibition of IFN-
responses was >100-fold greater than that found in the cell wall
fraction that exerted even greater inhibitory activity. Although this
observation does not completely exclude a role for LAM in initiating a
pathway that results in inhibition of IFN- responses, it suggests
that if LAM is involved at all, it depends on another cell wall
component for its effect. The role for this other hypothetical cell
wall component could be a structural one, in which it orients LAM in a
manner that enables it to interact more productively with macrophages
than it can when it is in a purified (probably micellar) form. The
alternative role for the other hypothetical cell wall component is to
provide an additional signal to macrophages that, when combined with a
signal initiated by LAM, causes potent inhibition of IFN- responses.
Such a mechanism is reminiscent of the need for both lipoteichoic acid
and peptidoglycan of Staphylococcus aureus to initiate
septic shock (51). Further analysis of the precise
components and conformations of the cell wall will be necessary to
definitively identify the molecule(s) responsible for initiating the
inhibition of IFN- responses.
The survival of M. tuberculosis in macrophages and
resistance to the human immune system are likely to involve more than
one mechanism, and identification of these mechanisms will be crucial
to understanding the pathogenesis of this common and serious disease.
The ability of M. tuberculosis to block macrophage responses
to IFN- is likely to be an important trait developed by the bacteria
in response to the development of cell-mediated immunity. Overcoming
this blockade may allow the immune system to better contain and
eradicate M. tuberculosis and may be a valuable adjunct in
developing improved therapies for latent and active tuberculosis.
|
Acknowledgments |
|---|
RI GAS oligonucleotide probe and advice
concerning the EMSA experiments. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Joel D. Ernst, University of California, Box 0868, San Francisco, CA 94143-0868. E-mail address:
3 Abbreviations used in this paper: JAK, Janus kinase; GAS, gamma-activation sequence; CBP, CREB binding protein; FcRI, Fc receptor type I; MOI, multiplicity of infection; DAPI, 4',6-diamidino-2-phenylindole, dihydrochoride; LAM, lipoarabinomannan; Nmi, N-Myc interactor protein; MAPK, mitogen-activated protein kinase.
Received for publication March 19, 1999. Accepted for publication July 14, 1999.
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S. P. Coller, J. M. Mansfield, and D. M. Paulnock Glycosylinositolphosphate Soluble Variant Surface Glycoprotein Inhibits IFN-{gamma}-Induced Nitric Oxide Production Via Reduction in STAT1 Phosphorylation in African Trypanosomiasis J. Immunol., August 1, 2003; 171(3): 1466 - 1472. [Abstract] [Full Text] [PDF]
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R. K. Pai, M. Convery, T. A. Hamilton, W. H. Boom, and C. V. Harding Inhibition of IFN-{gamma}-Induced Class II Transactivator Expression by a 19-kDa Lipoprotein from Mycobacterium tuberculosis: A Potential Mechanism for Immune Evasion J. Immunol., July 1, 2003; 171(1): 175 - 184. [Abstract] [Full Text] [PDF]
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A. Gross, M. Bouaboula, P. Casellas, J.-P. Liautard, and J. Dornand Subversion and Utilization of the Host Cell Cyclic Adenosine 5'-Monophosphate/Protein Kinase A Pathway by Brucella During Macrophage Infection J. Immunol., June 1, 2003; 170(11): 5607 - 5614. [Abstract] [Full Text] [PDF]
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S. Prabhakar, Y. Qiao, Y. Hoshino, M. Weiden, A. Canova, E. Giacomini, E. Coccia, and R. Pine Inhibition of Response to Alpha Interferon by Mycobacterium tuberculosis Infect. Immun., May 1, 2003; 71(5): 2487 - 2497. [Abstract] [Full Text] [PDF]
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P. Kumar, R. R. Amara, V. K. Challu, V. K. Chadda, and V. Satchidanandam The Apa Protein of Mycobacterium tuberculosis Stimulates Gamma Interferon-Secreting CD4+ and CD8+ T Cells from Purified Protein Derivative-Positive Individuals and Affords Protection in a Guinea Pig Model Infect. Immun., April 1, 2003; 71(4): 1929 - 1937. [Abstract] [Full Text]
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R. Condos, B. Raju, A. Canova, B.-Y. Zhao, M. Weiden, W. N. Rom, and R. Pine Recombinant Gamma Interferon Stimulates Signal Transduction and Gene Expression in Alveolar Macrophages In Vitro and in Tuberculosis Patients Infect. Immun., April 1, 2003; 71(4): 2058 - 2064. [Abstract] [Full Text]
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L.-Y. Gao, R. Groger, J. S. Cox, S. M. Beverley, E. H. Lawson, and E. J. Brown Transposon Mutagenesis of Mycobacterium marinum Identifies a Locus Linking Pigmentation and Intracellular Survival Infect. Immun., February 1, 2003; 71(2): 922 - 929. [Abstract] [Full Text] [PDF]
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D. M. Mosser The many faces of macrophage activation J. Leukoc. Biol., February 1, 2003; 73(2): 209 - 212. [Full Text] [PDF]
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A. Jeevan, T. Yoshimura, K. E. Lee, and D. N. McMurray Differential Expression of Gamma Interferon mRNA Induced by Attenuated and Virulent Mycobacterium tuberculosis in Guinea Pig Cells after Mycobacterium bovis BCG Vaccination Infect. Immun., January 1, 2003; 71(1): 354 - 364. [Abstract] [Full Text] [PDF]
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T. Greenwell-Wild, N. Vazquez, D. Sim, M. Schito, D. Chatterjee, J. M. Orenstein, and S. M. Wahl Mycobacterium avium Infection and Modulation of Human Macrophage Gene Expression J. Immunol., December 1, 2002; 169(11): 6286 - 6297. [Abstract] [Full Text] [PDF]
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 M. J. Scott, C. J. Godshall, and W. G. Cheadle Jaks, STATs, Cytokines, and Sepsis Clin. Vaccine Immunol., November 1, 2002; 9(6): 1153 - 1159. [Full Text] [PDF]
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S. K. Roach and J. S. Schorey Differential Regulation of the Mitogen-Activated Protein Kinases by Pathogenic and Nonpathogenic Mycobacteria Infect. Immun., June 1, 2002; 70(6): 3040 - 3052. [Abstract] [Full Text] [PDF]
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S. P. Hickman, J. Chan, and P. Salgame Mycobacterium tuberculosis Induces Differential Cytokine Production from Dendritic Cells and Macrophages with Divergent Effects on Naive T Cell Polarization J. Immunol., May 1, 2002; 168(9): 4636 - 4642. [Abstract] [Full Text] [PDF]
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J. Zerrahn, U. E. Schaible, V. Brinkmann, U. Guhlich, and S. H. E. Kaufmann The IFN-Inducible Golgi- and Endoplasmic Reticulum- Associated 47-kDa GTPase IIGP Is Transiently Expressed During Listeriosis J. Immunol., April 1, 2002; 168(7): 3428 - 3436. [Abstract] [Full Text] [PDF]
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B. Samten, P. Ghosh, A.-K. Yi, S. E. Weis, D. L. Lakey, R. Gonsky, U. Pendurthi, B. Wizel, Y. Zhang, M. Zhang, et al. Reduced Expression of Nuclear Cyclic Adenosine 5'-Monophospate Response Element-Binding Proteins and IFN-{gamma} Promoter Function in Disease Due to an Intracellular Pathogen J. Immunol., April 1, 2002; 168(7): 3520 - 3526. [Abstract] [Full Text] [PDF]
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D. Wagner, F. J. Sangari, S. Kim, M. Petrofsky, and L. E. Bermudez Mycobacterium avium infection of macrophages results in progressive suppression of interleukin-12 production in vitro and in vivo J. Leukoc. Biol., January 1, 2002; 71(1): 80 - 88. [Abstract] [Full Text] [PDF]
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T. D. Joseph and D. C. Look Specific Inhibition of Interferon Signal Transduction Pathways by Adenoviral Infection J. Biol. Chem., December 7, 2001; 276(50): 47136 - 47142. [Abstract] [Full Text] [PDF]
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S. Raman, T. Song, X. Puyang, S. Bardarov, W. R. Jacobs Jr., and R. N. Husson The Alternative Sigma Factor SigH Regulates Major Components of Oxidative and Heat Stress Responses in Mycobacterium tuberculosis J. Bacteriol., October 15, 2001; 183(20): 6119 - 6125. [Abstract] [Full Text] [PDF]
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S. Ehrt, D. Schnappinger, S. Bekiranov, J. Drenkow, S. Shi, T. R. Gingeras, T. Gaasterland, G. Schoolnik, and C. Nathan Reprogramming of the Macrophage Transcriptome in Response to Interferon-{gamma} and Mycobacterium tuberculosis: Signaling Roles of Nitric Oxide Synthase-2 and Phagocyte Oxidase J. Exp. Med., October 15, 2001; 194(8): 1123 - 1140. [Abstract] [Full Text] [PDF]
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E. H. Noss, R. K. Pai, T. J. Sellati, J. D. Radolf, J. Belisle, D. T. Golenbock, W. H. Boom, and C. V. Harding Toll-Like Receptor 2-Dependent Inhibition of Macrophage Class II MHC Expression and Antigen Processing by 19-kDa Lipoprotein of Mycobacterium tuberculosis J. Immunol., July 15, 2001; 167(2): 910 - 918. [Abstract] [Full Text] [PDF]
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J. L. Flynn and J. Chan Tuberculosis: Latency and Reactivation Infect. Immun., July 1, 2001; 69(7): 4195 - 4201. [Full Text] [PDF]
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L.-G. Bekker, S. Freeman, P. J. Murray, B. Ryffel, and G. Kaplan TNF-{{alpha}} Controls Intracellular Mycobacterial Growth by Both Inducible Nitric Oxide Synthase-Dependent and Inducible Nitric Oxide Synthase-Independent Pathways J. Immunol., June 1, 2001; 166(11): 6728 - 6734. [Abstract] [Full Text] [PDF]
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F. A. Post, C. Manca, O. Neyrolles, B. Ryffel, D. B. Young, and G. Kaplan Mycobacterium tuberculosis 19-Kilodalton Lipoprotein Inhibits Mycobacterium smegmatis-Induced Cytokine Production by Human Macrophages In Vitro Infect. Immun., March 1, 2001; 69(3): 1433 - 1439. [Abstract] [Full Text] [PDF]
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H.-J. Ullrich, W. L. Beatty, and D. G. Russell Interaction of Mycobacterium avium-Containing Phagosomes with the Antigen Presentation Pathway J. Immunol., December 1, 2000; 165(11): 6073 - 6080. [Abstract] [Full Text] [PDF]
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S. Thoma-Uszynski, S. Stenger, and R. L. Modlin CTL-Mediated Killing of Intracellular Mycobacterium tuberculosis Is Independent of Target Cell Nuclear Apoptosis J. Immunol., November 15, 2000; 165(10): 5773 - 5779. [Abstract] [Full Text] [PDF]
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P. F. Barnes and B. Wizel Type 1 Cytokines and the Pathogenesis of Tuberculosis Am. J. Respir. Crit. Care Med., June 1, 2000; 161(6): 1773 - 1774. [Full Text]
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