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Promoter Function in Disease Due to an Intracellular Pathogen1
,
,,
Departments of
*
Microbiology and Immunology,
Medicine, and
Biochemistry, and
Center for Pulmonary and Infectious Disease Control, University of Texas Health Center, Tyler, TX 75708;
¶ National Institute of Aging, National Institutes of Health, Baltimore, MD 21224;
||
Crippled Children’s Foundation Research Center, Le Bonheur Children’s Hospital, and Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN 38103;
#
Department of Internal Medicine, University of North Texas Health Sciences Center, Fort Worth, TX 76107;
**
Inflammatory Bowel Disease Research Center, Cedars-Sinai Medical Center, Los Angeles, CA 90048;
Department of Immunology and Microbiology, University of Miami School of Medicine, Miami, FL 33124; and
*
Laboratory of Experimental Immunology, National Cancer Institute at Frederick, Frederick, MD 21702
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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protein
and mRNA expression have been shown to be reduced in tuberculosis
patients, compared with healthy tuberculin reactors. To determine
whether this decrease was associated with reduced activity of the
IFN- promoter, we first studied binding of nuclear proteins to the
radiolabeled proximal IFN- promoter (-71 to -40 bp), using EMSAs
with nuclear extracts of freshly isolated peripheral blood T cells.
Nuclear extracts of T cells from most tuberculosis patients showed
markedly reduced expression of proteins that bind to the proximal
IFN- promoter, compared with findings in nuclear extracts of T cells
from healthy tuberculin reactors. These DNA-binding complexes contained
CREB proteins, based on competitive EMSAs, supershift assays, and
Western blotting with an anti-CREB Ab. Transient transfection of
PBLs with a luciferase reporter construct under the control of the
IFN- promoter revealed reduced IFN- promoter activity in
tuberculosis patients. Transient transfection of Jurkat cells with a
dominant-negative CREB repressor plasmid reduced IFN- promoter
activity. These data suggest that reduced expression of CREB nuclear
proteins in tuberculosis patients results in decreased IFN- promoter
activity and reduced IFN- production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The second reason to study tuberculosis is that it provides an
excellent model to investigate the relationship between the immune
response and clinical manifestations of disease from intracellular
pathogens. Most persons infected with M. tuberculosis are
healthy tuberculin reactors who mount a protective immune response. In
contrast, patients with active tuberculosis have severe disease due to
ineffective immunity, and when PBMC from tuberculosis patients are
cultured with M. tuberculosis, production of IFN- by T
cells is depressed, compared with healthy tuberculin reactors (2, 3). Elucidation of the mechanism for reduced IFN-
production will enhance our understanding of resistance and
susceptibility to disease from intracellular pathogens, as IFN-
production is central to immunity against many of these organisms
(3, 4, 5, 6, 7).
Reduced IFN- production in human tuberculosis is associated with
reduced expression of IFN- mRNA (3, 8, 9).
Transcription of the IFN- gene in activated T cells is controlled by
essential regulatory regions in the distal (-96 to -80 bp) and
proximal (-73 to -48 bp) portions of the IFN- promoter
(10). Binding of specific proteins to these regulatory
regions markedly affects IFN- promoter activity
(10, 11, 12, 13, 14), and the proximal promoter element mediates the
selective expression of IFN- by T cells (12). To test
the hypothesis that reduced IFN- mRNA expression in human
tuberculosis was due to alterations in these DNA-binding proteins, we
evaluated binding to the proximal IFN- promoter in tuberculosis
patients and in healthy tuberculin reactors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood was obtained from 21 healthy tuberculin reactors and from 25 HIV-seronegative patients with culture-proven pulmonary tuberculosis, all of whom had received <4 wk of antituberculosis therapy. Acid-fast stains of sputum were positive in all patients. This work was reviewed and approved by the Institutional Review Boards of the University of Texas Health Center (Tyler, TX), and the University of North Texas Health Science Center (Fort Worth, TX), and informed consent was obtained from all participants.
Preparation of T cells
PBMC were isolated by differential centrifugation over Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, NJ). Freshly isolated PBMC were incubated with magnetic beads conjugated to anti-CD3 (Miltenyi Biotec, Auburn, CA), and a magnetic cell separator was used to positively select CD3+ cells. The purity of CD3+ cells was 98%, as measured by cytofluorometric analysis with an EPICS C flow cytometer (Beckman Coulter, Miami, FL).
Extraction of nuclear proteins
Nuclear proteins were prepared as previously described (11). The cellular pellets of CD3+ cells were suspended in cell lysis buffer containing 50 mM KCl, 0.5% Nonidet P-40, 25 mM HEPES buffer (pH 7.8), 1 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 100 µM DTT (all from Sigma Aldrich, St. Louis, MO), and subsequently incubated for 5 min on ice. After centrifugation at 550 x g, the pellets (nuclei) and supernatants (cytosol) were collected. The nuclei were washed in cell lysis buffer without Nonidet P-40, repelleted at 550 x g for 5 min, and resuspended for 5 min on ice in extraction buffer containing 500 mM KCl, 25 mM HEPES (pH 7.8), 10% glycerol, 1 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 10 µg/m chymostatin (Sigma Aldrich), 10 µg/ml trypsin-chymotrypsin (Sigma Aldrich), and 100 µM DTT. The samples were subsequently frozen and thawed twice, using dry ice and a 37°C water bath, incubated with gentle agitation at 4°C for 25 min, and centrifuged at 18,000 x g for 10 min. The clear supernatants were collected as nuclear protein extracts, and the amount of nuclear proteins obtained were quantified (bicinchoninic acid, Pierce, Rockford, IL), aliquoted, and stored at -70°C.
EMSA
The nuclear protein extracts (5 µg) were incubated with 1 µg of poly(dI-dC) (Amersham Pharmacia Biotech, Piscataway, NJ), 20 mM Tris (pH 7.5), 60 mM KCl, 2 mM EDTA, 0.5 mM DTT, 4% Ficoll, and 8,000 cpm of 32P-labeled oligonucleotide DNA probes in a total volume of 10 µl. The binding reaction was conducted at room temperature for 30 min. In some experiments, 100-fold molar excess of nonlabeled double-stranded oligonucleotides were added as cold competitors, or different Abs were added, and the mixture was incubated on ice for 25 min before adding the labeled DNA probe. Nuclear protein complexes were resolved by electrophoresis on 5% nondenaturing polyacrylamide gels in 0.5 x Tris borate-EDTA buffer at 12 V/cm for 2 h at room temperature. Dried gels were exposed to hyperfilm (Amersham Pharmacia Biotech) at -70°C with intensifying screens.
The double-stranded probes were end-labeled with T4 polynuclear kinase
(New England Biolabs, Beverly, MA) and
[-32P]dATP (DuPont NEN Life Science
Products, Boston, MA), and 
1 ng of labeled DNA was used in a
standard EMSA reaction. The following double-stranded oligonucleotides
were used as labeled probes or cold competitors: IFN- -71 to -40,
5'-AAAACTTGTGAAAATACGTAATCCTCAGGAGA-3' (12);
CREB consensus binding site (wild type),
5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3'; CREB consensus
binding site (mutated), 5'-AGAGATTGCCTGTGGTCAGAGAGCTAG-3'
(11); NF-
B, 5'-AGT TGA GGG GAC TTT CCC AGG C-3'. The
underlined nucleotides on the wild-type probe are essential for binding
to CREB.
For supershift assays, we used anti-CREB1 mAb (Santa Cruz Biotechnology, Santa Cruz, CA), which detects both phosphorylated and nonphosphorylated CREB, anti-junB (Santa Cruz Biotechnology), and mouse IgG (BD PharMingen, San Diego, CA).
Western blotting
Reducing SDS-PAGE (10%) was performed as previously described (15), using 10 µg of nuclear extract in each sample. The gel was then electroblotted in Tris glycine buffer containing 40% methanol onto a nitrocellulose membrane (Trans-blot; Bio-Rad, Hercules, CA). After blocking the membrane with blocking buffer (5% fat-free milk in TBS: 10 mM Tris (pH 8.0), 150 mM NaCl) for 2 h at room temperature, primary Ab was added at 1/2000 dilution (100 ng/ml) in 5% BSA in TBS with 0.05% Tween overnight at 4°C. The membrane was washed three times with TBS/Tween, and incubated with secondary Ab (rabbit anti-mouse IgG conjugated to HRP; Santa Cruz Biotechnology) at 1/8,000 dilution in blocking buffer for 1 h at room temperature. After washing five times in TBS/Tween and once in TBS, the membrane was drained briefly and Ab binding was detected by ECL (Amersham Pharmacia Biotech).
Real-time PCR for quantification of CREB mRNA
Total RNA was extracted from 2.5–5 x 106 PBMC with TRIzol reagent (Life Technologies, Gaithersburg, MD), according to the manufacturer’s instructions, and treated with RQ RNase-free DNase (Promega, Madison, WI) and Rnasin Ribonuclease inhibitor (Promega). The DNase-treated RNA was extracted with phenol/chloroform, and precipitated with sodium acetate and ethanol, using standard methods. Total RNA was reverse transcribed using the OligodT15 primer (Promega) and Omniscript reverse transcriptase (Qiagen, Valencia, CA).
Primers and probes for the CREB1 gene were designed with Primer Express
software (PE Biosystems, Foster City, CA) and synthesized by Operon
Technologies (Alameda, CA). The forward and reverse primers were
5'-GGTTTGTGCTGAGCTCCTTGA and 5'-TGCGGCCCACACATTACTT, respectively.
The probe was 5'-CTTAGGGACAGAATTACCCCAGCCTCTTGA, 5'-fluoroscein
phosphoramidite-labeled and 3'-TAMRA labeled. The Taqman
-actin control reagents (PE Biosystems) were used as the internal
standard.
Real-time PCR assays were performed in a sealed 96-well microtiter
plate (PE Biosystems) on a spectrofluorometric thermal cycler (Applied
Biosystems 7700 Prism, PE Biosystems). An equal quantity of each cDNA
sample was aliquoted into paired wells containing 1x Taqman Universal
PCR master mix (PE Biosystems), 5 pmols of either the CREB1 or
-actin primers, and 1 pmol of the corresponding probe in a total
volume of 25 µl. Standard curves for the CREB1 and -actin genes
were generated using serial 10-fold dilutions of 10 ng/ml human genomic
DNA (PE Biosystems) and the appropriate primers and probes. Each sample
and each standard curve dilution were run in triplicate. Amplification
was performed as follows: 50°C for 2 min, 95°C for 10 min, and 40
cycles of 95°C for 15 s, and 60°C for 1 min. For each sample,
the fluorescent signal was measured and plotted during each 60°C
annealing and extension step. Using the cycle threshold (the number of
PCR cycles required for the fluorescent dye to be detectable), and the
constructed standard curve for each gene, the relative amount of CREB1
and -actin cDNA in each sample was determined. To normalize for the
amount of RNA in each sample, data for each patient were expressed as
the ratio of CREB cDNA to -actin cDNA, expressed in arbitrary
units.
Plasmid constructs
The following plasmids were used: 1) a luciferase reporter
construct under the control of the IFN- proximal promoter,
containing head-to-tail (5' to 3') dimers of the IFN- proximal
promoter, -71 to -44 bp from the transcription start site
(16); 2) a -galactosidase reporter construct driven by
the SV40 promoter (16); 3) KCREB, a pRc/RSV CREB
dominant-negative repressor plasmid containing CREB cDNA with a
mutation in the DNA-binding domain that blocks binding of wild-type
CREB to target DNA (17); 4) a pRc/RSV empty vector
control. The latter two vectors were kindly provided by Dr. R. Goodman
(Vollum Institute for Advanced Biomedical Research, Oregon Health
Sciences University, Portland, OR).
Transient transfection assays
Freshly isolated nonadherent PBMC (2 x
106 ml) were cultured for 20 h in RPMI 1640
(Life Technologies) with 10% FCS (HyClone Laboratories,
Logan, UT) and 1 µg/ml of PHA-L (Sigma Aldrich), as
previously described (18). Cells were then washed and
resuspended in 250 µl of fresh medium at 2 x
107/ml, and electroporated at room temperature
with 40 µg of plasmid DNA, using a Gene Pulser (Bio-Rad), with
settings of 250 V and 975 µF. The cells were then resuspended in
fresh medium, rested for 1 h at 37°C, and stimulated with 50
ng/ml of PMA and 400 nM of ionomycin (both from Sigma Aldrich) for
4 h. Cell lysates were prepared, and luciferase activity was
measured in a luminometer (Zylux, Maryville, TN), and expressed as
relative light units
(RLU),3 using a
luciferase assay kit (Promega). -Galactosidase activity was measured
with a commercially available kit (Promega).
Confluent Jurkat cells were washed and resuspended at 2.5 x
107 cells/ml in 400 µl of buffer containing 300
mM KCl, 10 mM K3PO4, 10 mM
CaCl2, 10 mM MgCl2, 25 mM
HEPES, 5 mM EGTA and 2 mM ATP (Sigma Aldrich), 1 mM reduced
glutathione (Sigma Aldrich), and 30 µg of plasmid DNA for 10
min on ice, then electroporated as described above for PBLs. The cells
were then rested on ice for 10 min, diluted to 2 x
106 cells/ml with fresh RPMI 1640 and 10% FCS,
and cultured for 5 h at 37°C. The cells were then stimulated
with 20 ng/ml PMA and 400 nM ionomycin for 22 h. Luciferase
activity and -galactosidase activity were measured as outlined above
for PBLs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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promoter region in T
cells from tuberculosis patients and healthy tuberculin reactors
We and others have shown that M. tuberculosis-induced
IFN- production and mRNA expression are reduced in tuberculosis
patients, compared with healthy tuberculin reactors (2, 3, 19, 20). Transcription of the IFN- gene depends on binding of
specific nuclear proteins to the IFN- promoter
(10, 11, 12, 13, 14). To determine whether reduced IFN- mRNA
expression in tuberculosis patients was associated with reduced binding
of nuclear proteins to the IFN- promoter, we performed EMSAs using
the radiolabeled proximal IFN- promoter as a probe on nuclear
extracts from freshly isolated CD3+ cells
obtained from PBMC of 21 healthy tuberculin reactors and 15
tuberculosis patients. Fig. 1
shows
representative results for five healthy tuberculin reactors and five
patients. The binding pattern observed was similar for T cells from all
21 healthy tuberculin reactors. However, in T cells from 12 of 15
tuberculosis patients, a low-mobility complex binding to the IFN-
promoter (indicated by the arrow in Fig. 1) was absent
(p < 0.001, Fisher’s exact test), suggesting
that DNA-binding proteins to the IFN- promoter were reduced in
tuberculosis patients. EMSAs with T cells from healthy
tuberculin-negative donors showed DNA binding that was
indistinguishable from that observed for healthy tuberculin reactors
(data not shown). M. tuberculosis-induced IFN- production
by PBMC from healthy tuberculin reactors was 3050 ± 242 pg/ml,
compared with 854 ± 460 pg/ml for tuberculosis patients
(p < 0.001, Student’s t test).
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, nuclear extracts from T cells of healthy tuberculin reactors
were incubated with excess unlabeled proximal IFN- promoter probe,
before performance of EMSA. Excess unlabeled probe completely abrogated
binding of the low-mobility complex binding to the radiolabeled
proximal IFN- promoter (Fig. 2, lanes 1 and 2), demonstrating the specificity of
binding.
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proximal promoter binding complex
with decreased expression in tuberculosis patients
Because CREB proteins have been shown to bind to the proximal
IFN- promoter (11), competitive EMSAs were performed on
nuclear extracts of CD3+ cells obtained from five
healthy tuberculin reactors, using 100-fold molar excess of the
unlabeled wild-type CREB consensus binding site, the mutated CREB
consensus binding site and the NF-B binding site. Fig. 2 shows a
representative result. Binding of the low-mobility complex to the
IFN- promoter was eliminated by excess of the unlabeled wild-type
CREB consensus binding site, but not by the mutant CREB or the NF-B
binding site, suggesting that the protein complex binding to the
IFN- promoter contained CREB protein. As an alternative means to
confirm this finding, nuclear extracts from five healthy tuberculin
reactors were incubated with anti-CREB1 mAb, before addition of the
labeled proximal IFN- promoter probe and performance of EMSA. Fig. 3 shows a representative result.
Anti-CREB1 mAb bound to the nuclear protein complex, resulting in a
supershifted complex of higher m.w. In contrast, control IgG and
anti-junB Abs had no effect. These results confirm that
the IFN- promoter binding complex in T cells from healthy tuberculin
reactors contained CREB proteins.
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The EMSA data above suggest that the capacity of CREB protein to
bind the IFN- promoter was reduced in T cells from tuberculosis
patients, compared with healthy tuberculin reactors. To determine
whether this reduction in DNA binding was due to reduced levels of CREB
protein, we performed Western blotting with the anti-CREB mAb on
nuclear extracts of CD3+ cells from freshly
isolated PBMC of eight tuberculosis patients and nine healthy
tuberculin reactors. Representative results for three tuberculosis
patients and three healthy tuberculin reactors are shown in Fig. 4, demonstrating that CREB protein
expression was markedly decreased in tuberculosis patients. This
reduction in CREB protein was not due to a mutation that changed a
single epitope of CREB, as Western blotting with a polyclonal Ab also
showed reduced CREB protein in nuclear extracts of
CD3+ cells from tuberculosis patients (data not
shown).
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-actin in
CD3+ cells from PBMC of seven tuberculosis
patients and six healthy tuberculin reactors. The ratio of CREB cDNA to
-actin cDNA, expressed in arbitrary units, was similar in
tuberculosis patients and healthy tuberculin reactors (11.6 ± 4.0
vs 10.8 ± 2.1 arbitrary units, p = 0.86, Fig. 5). Therefore, the reduced expression of
CREB protein in tuberculosis patients was not due to reduced CREB mRNA
expression. Pulse-chase experiments with
[35S]methionine and cysteine, in combination
with immunoprecipitation with anti-CREB mAb, revealed that the rate
of degradation of CREB protein was not enhanced in tuberculosis
patients (data not shown).
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B site
To determine whether the decrease in CREB protein production in
tuberculosis patients was specific or was due to a generalized decrease
in transcription factors, we performed EMSAs, using the radiolabeled
consensus NF-B binding site as a probe, on nuclear extracts from
freshly isolated CD3+ cells obtained from PBMC of
four healthy tuberculin reactors and four tuberculosis patients. The
amount of NF-B binding complex was increased in T cells from
tuberculosis patients (Fig. 6A), paralleling the enhanced
NF-B expression reported in monocytes from tuberculosis patients
(21). These findings indicate that T cells from
tuberculosis patients did not have a generalized reduction in
transcription factors. Fig. 6B shows that the dominant
low-mobility complex was specific for binding to the consensus NF-B
site, as it was eliminated by excess of the unlabeled NF-B site, but
not by the proximal IFN- promoter or by the wild-type CREB consensus
binding site.
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promoter activity
To determine whether reduced CREB protein production in
tuberculosis patients was associated with diminished IFN- promoter
activity, we transiently transfected PBLs with an IFN- proximal
promoter luciferase reporter plasmid and a -galactosidase reporter
plasmid. For each sample, luciferase activity was normalized for
-galactosidase activity, which controlled for differential
transfection efficiency between samples. Cells from five tuberculosis
patients stimulated with PMA and ionomycin showed significantly reduced
luciferase activity, compared with cells from five healthy tuberculin
reactors (16,893 ± 3,658 vs 53,148 ± 10,097 RLU,
respectively, p = 0.01, Fig. 7). This indicates that functional
IFN- promoter activity is reduced in tuberculosis patients.
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promoter activity, we
attempted to evaluate the effects of the dominant-negative repressor
KCREB plasmid on IFN- promoter activity in T cells from healthy
tuberculin reactors. However, because these experiments involve
cotransfection of three constructs and transfection efficiency in PBLs
is low, inconsistent results were obtained that were not interpretable.
Therefore, we transfected the Jurkat T cell line with the IFN-
proximal promoter luciferase reporter plasmid, the -galactosidase
reporter plasmid, and either the pRc/RSV KCREB plasmid or the pRc/RSV
empty plasmid. The cells were then stimulated with PMA and ionomycin,
and -galactosidase and luciferase activity were measured. After
normalization for -galactosidase activity, cotransfection of the
KCREB plasmid reduced luciferase expression by 70%, compared with
cotransfection with the empty plasmid (12,208 ± 2,523 vs
38,690 ± 6,085 RLU, respectively, p = 0.04, Fig. 8). This demonstrates that neutralization
of CREB inhibits IFN- promoter function.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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promoter in
patients with disease due to an intracellular pathogen. T cells from
80% of tuberculosis patients showed significantly reduced binding of
low-mobility nuclear proteins to the proximal IFN- promoter,
compared with findings in T cells from healthy tuberculin reactors.
These nuclear proteins specifically bound to the IFN- promoter
region and contained CREB, based on competitive EMSAs with wild-type
and mutant CREB, and supershift assays with an anti-CREB Ab.
Decreased levels of CREB in T cells from tuberculosis patients were
demonstrated by Western blot, and were associated with reduced IFN-
promoter activity. Inhibition of CREB binding to DNA by a
dominant-negative KCREB plasmid reduced IFN- promoter activity in
Jurkat cells, demonstrating that CREB is needed for optimal IFN-
promoter activity. The sum of these findings, in combination with prior
studies showing reduced expression of IFN- mRNA and protein by T
cells from tuberculosis patients (3, 9, 20), strongly
suggest that severe disease due to M. tuberculosis reduces
CREB expression, which in turn contributes to decreased IFN-
promoter activity and IFN- production.
IFN- plays a pivotal role in resistance to intracellular pathogens,
including viruses, bacteria, fungi, and parasites (4, 5, 6, 7, 22). Animals lacking IFN- because of a gene deletion and
patients with defective IFN- receptors are highly susceptible to
mycobacterial disease, including tuberculosis (23, 24, 25, 26). A
growing body of evidence suggests that pathogenic mycobacteria have
developed strategies to minimize the antimicrobial effects of IFN-
by interfering with IFN--mediated signal transduction in macrophages
(27, 28). Our current findings suggest that M.
tuberculosis may also combat the effects of IFN- by reducing
IFN- promoter activity and mRNA expression through down-regulating
CREB transcription factors. Further studies are needed to determine
whether these findings can be generalized to patients with other
infectious diseases.
CREB proteins are essential for T cell function and cytokine
production. Transgenic mice expressing a dominant-negative form of CREB
show defective T cell proliferation, and reduced production of IFN-,
IL-2, and IL-10 (14, 29). CREB also contributes to
activation of the IL-2 promoter in T cells stimulated with CD28 and
mitogen (30). IFN- production is controlled at the
transcriptional level, and the distal and proximal elements
in the IFN- promoter are essential for activation-specific
production of IFN- by T cells (10). CREB proteins bind
to the IFN- proximal element, but the resultant effects on
transcription of the IFN- gene remain controversial. Some
investigators found that CREB proteins inhibited IFN- transcription
(12, 13), whereas others reported that they enhanced
IFN- transcription (11). Because these studies were
performed in Jurkat T cells and in transgenic mice, their applicability
to IFN- production in humans in vivo is uncertain. We found that
healthy tuberculin reactors with protective immunity against
tuberculosis and high M. tuberculosis-induced IFN-
production had normal levels of CREB protein that bound to the proximal
element of the IFN- promoter. In contrast, tuberculosis patients had
diminished CREB protein levels, reduced binding to the IFN-
promoter, diminished IFN- promoter activity, and low IFN-
production. Binding to the IFN- proximal promoter was also reduced
in nuclear extracts of T cells from patients with lepromatous leprosy
and extensive disease due to Mycobacterium
leprae, compared with those from patients with limited
disease and tuberculoid leprosy, although the nature of the binding
complex was not characterized (31). Our current findings
suggest that CREB protein expression in human T cells in vivo
correlates closely with the capacity to produce IFN- in response to
M. tuberculosis.
The mechanism that results in reduced CREB proteins in tuberculosis patients remains uncertain. CREB mRNA expression was not reduced in tuberculosis patients, and the t1/2 of CREB protein was not reduced, as assessed by pulse-chase experiments. Other potential mechanisms include failure of CREB proteins to translocate from the cytoplasm to the nucleus, and defects in translation or posttranslational modifications of CREB protein. It is also possible that the t1/2 of CREB proteins in tuberculosis patients in vivo is reduced, due to characteristics of the cellular milieu that are not mimicked during the pulse-chase experiments performed in vitro. Further studies are needed to clarify this important question.
Transcription factors play a central role in orchestrating the immune
response by controlling cytokine production. A wide variety of
microbial pathogens, including M. tuberculosis, enhance
production of inflammatory cytokines, such as TNF-
, IL-6, and IL-8,
through up-regulation of transcription factors, particularly NF-B
(21, 32, 33, 34, 35). Inhibition of cytokine production by
microorganisms through down-regulation of transcription factors has
rarely been described. HIV infection was reported to inhibit STAT5
expression (36), but this finding was not confirmed
(37). Our current report is consistent with the hypothesis
that severe disease due to M. tuberculosis reduces
expression of CREB protein, which in turn decreases IFN- promoter
activity and IFN- production, weakening host defenses and favoring
development of extensive disease. Alternatively, individuals with
reduced expression of CREB protein and IFN- may have enhanced
susceptibility to progression of tuberculosis infection to disease,
whereas persons with normal CREB protein and IFN- levels may contain
the infection and not develop tuberculosis disease. Distinguishing
these possibilities would require evaluation of patients before
development of tuberculosis, but this is not generally feasible.
However, studies of CREB protein and IFN- expression in animals
infected with M. tuberculosis may provide insight into the
contribution of CREB proteins to IFN- production in vivo. It is
intriguing to speculate that modulation of CREB protein expression may
contribute to IFN- production and to host defenses against
intracellular pathogens other than M. tuberculosis. An
improved understanding of the role of CREB proteins may permit
development of novel strategies to treat and prevent infectious
diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter F. Barnes, Center for Pulmonary and Infectious Disease Control, University of Texas Health Center, 11937 US Highway 271, Tyler, TX 75708-3154. E-mail address: peter.barnes{at}uthct.edu
3 Abbreviation used in this paper: RLU, relative light units.
Received for publication September 20, 2001. Accepted for publication February 6, 2002.
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 X. Pang, P. Vu, T. F. Byrd, S. Ghanny, P. Soteropoulos, G. V. Mukamolova, S. Wu, B. Samten, and S. T. Howard Evidence for complex interactions of stress-associated regulons in an mprAB deletion mutant of Mycobacterium tuberculosis Microbiology, April 1, 2007; 153(4): 1229 - 1242. [Abstract] [Full Text] [PDF]
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A. Colmone, S. Li, and C.-R. Wang Activating Transcription Factor/cAMP Response Element Binding Protein Family Member Regulated Transcription of CD1A J. Immunol., November 15, 2006; 177(10): 7024 - 7032. [Abstract] [Full Text] [PDF]
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B. Samten, S. T. Howard, S. E. Weis, S. Wu, H. Shams, J. C. Townsend, H. Safi, and P. F. Barnes Cyclic AMP Response Element-Binding Protein Positively Regulates Production of IFN-{gamma} by T Cells in Response to a Microbial Pathogen J. Immunol., May 15, 2005; 174(10): 6357 - 6363. [Abstract] [Full Text] [PDF]
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S. Yano, P. Ghosh, H. Kusaba, M. Buchholz, and D. L. Longo Effect of Promoter Methylation on the Regulation of IFN-{gamma} Gene During In Vitro Differentiation of Human Peripheral Blood T Cells into a Th2 Population J. Immunol., September 1, 2003; 171(5): 2510 - 2516. [Abstract] [Full Text] [PDF]
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B. Samten, B. Wizel, H. Shams, S. E. Weis, P. Klucar, S. Wu, R. Vankayalapati, E. K. Thomas, S. Okada, A. M. Krensky, et al. CD40 Ligand Trimer Enhances the Response of CD8+ T Cells to Mycobacterium tuberculosis J. Immunol., March 15, 2003; 170(6): 3180 - 3186. [Abstract] [Full Text] [PDF]
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