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Cyclic AMP Response Element-Binding Protein Positively Regulates Production of IFN- by T Cells in Response to a Microbial Pathogen ¹

Buka Samten^*,, Susan T. Howard^*,, Steven E. Weis, Shiping Wu^*,, Homayoun Shams^*,, James C. Townsend^*, Hassan Safi^*, and Peter F. Barnes^2,,

^* Center for Pulmonary and Infectious Disease Control, Departments of Microbiology and Immunology and Medicine, University of Texas Health Center, Tyler, TX 75708; and Department of Internal Medicine, University of North Texas Health Sciences Center, Fort Worth, TX 76107

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
IFN- is essential for resistance to many intracellular pathogens, including Mycobacterium tuberculosis. Transcription of the IFN- gene in activated T cells is controlled by the proximal promoter element (–73 to –48 bp). CREB binds to the IFN- proximal promoter, and binding is enhanced by phosphorylation of CREB. Studies in human T cell lines and in transgenic mice have yielded conflicting results about whether CREB is a positive or a negative regulator of IFN- transcription. To determine the role of CREB in mediating IFN- production in response to a microbial pathogen, we evaluated the peripheral blood T cell response to M. tuberculosis in healthy tuberculin reactors. EMSAs, chromatin immunoprecipitation, and Western blotting demonstrated that stimulation of PBMC with M. tuberculosis induced phosphorylation and enhanced binding of CREB to the IFN- proximal promoter. Neutralization of CREB with intracellular Abs or down-regulation of CREB levels with small interfering RNA decreased M. tuberculosis-induced production of IFN- and IFN- mRNA expression. In addition, M. tuberculosis-stimulated T cells from tuberculosis patients, who have ineffective immunity, showed diminished IFN- production, reduced amounts of CREB binding to the IFN- proximal promoter, and absence of phosphorylated CREB. These findings demonstrate that CREB positively regulates IFN- production by human T cells that respond to M. tuberculosis.

	Introduction

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Interferon- plays a pivotal role in resistance to intracellular pathogens, including viruses, bacteria, fungi, and parasites (1, 2, 3, 4, 5), and subjects with defects in IFN- receptor expression or IFN- production have markedly increased susceptibility to disease from these organisms (6, 7). Transcription of the IFN- gene in activated T cells is controlled by regulatory regions in the distal (–96 to –80 bp) and proximal (–73 to –48 bp) portions of the IFN- promoter (8). Binding of specific proteins to these regulatory regions markedly affects IFN- promoter activity (8, 9, 10, 11, 12), and the proximal promoter element mediates the selective expression of IFN- by T cells (10). CREB binds to the IFN- proximal promoter element, and binding is enhanced by phosphorylation of CREB (13, 14). However, studies of Jurkat T cells and transgenic mice have yielded contradictory results, showing that CREB enhances or inhibits IFN- transcription in different experimental systems (9, 10, 11).

To investigate the role of CREB in the human T cell response to microbial pathogens, we evaluated production of IFN- by PBMC in response to Mycobacterium tuberculosis, an intracellular bacterium that infects an estimated 2 billion subjects worldwide (15). The clinical manifestations of M. tuberculosis infection correlate with the capacity of PBMC to produce IFN- in response to mycobacterial Ags. Most infected subjects are healthy tuberculin reactors who mount a protective immune response. In contrast, patients with active tuberculosis have severe disease due to ineffective immunity, and M. tuberculosis-induced production of IFN- is depressed, compared with findings in healthy tuberculin reactors (16, 17). We recently found that the expression of CREB is reduced in unstimulated T cells from tuberculosis patients compared with those from healthy tuberculin reactors (18), suggesting that CREB positively regulates IFN- production. To definitively determine the effect of CREB on IFN- production, we measured IFN- mRNA and protein levels after intracellular delivery of anti-CREB Abs or CREB small interfering RNA (siRNA) ³ to M. tuberculosis-stimulated PBMC. In addition, we used chromatin immunoprecipitation to determine whether CREB binds to the IFN- proximal promoter in M. tuberculosis-stimulated T cells, and studied CREB expression and phosphorylation in PBL during stimulation with mycobacterial Ags.

	Materials and Methods

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Mycobacterial Ags

We used heat-killed M. tuberculosis Erdman (provided by Dr. P. Brennan, Colorado State University, Fort Collins, CO).

Cell isolation

Blood was obtained from 21 healthy tuberculin reactors and 14 HIV-seronegative patients with culture-proven pulmonary tuberculosis, all of whom had received <4 wk of therapy. Acid-fast stains of sputum were positive in all patients, all of whom had far-advanced disease. This work was approved by the institutional review boards of University of Texas Health Center at Tyler and University of North Texas Health Science Center of Fort Worth.

PBMC were obtained by differential centrifugation over Ficoll-Paque, and CD3⁺ T cells were obtained by positive selection with magnetic beads conjugated to anti-CD3 (Miltenyi Biotec) or by negative selection, using the human Pan T Cell Isolation Kit (Miltenyi Biotec). The purity of CD3⁺ cells was 98%, as measured by flow cytometry (FACSCalibur; BD Biosciences). In some experiments, adherent cells were prepared by culturing 10⁷ PBMC/well in 1 ml of RPMI 1640 with 10% human serum in 2-ml wells at 37°C in 5% CO₂ for 1 h and washing extensively with warm RPMI 1640 to remove nonadherent cells. Adherent cells were collected with a rubber policeman, washed, and counted.

EMSA

Nuclear proteins and total cell protein extracts were prepared as previously described (18, 19), quantified (bicinchoninic acid; Pierce), aliquoted, and stored at –70°C. EMSAs were performed with nuclear protein extracts or total cell protein extracts and [-³²P]dATP-labeled oligonucleotide DNA probes, as previously described (18). The DNA probes contained the IFN- –71 to –40 bp proximal promoter element, the wild-type and mutated CREB consensus binding sites, and the NF-B binding site, as previously reported (18). In some experiments, a 100-fold molar excess of unlabeled oligonucleotides was added as cold competitors, or different Abs were added, and the mixture was incubated on ice for 25 min before adding the labeled probe. Nuclear protein complexes were resolved by electrophoresis on 5% nondenaturing polyacrylamide gels, and autoradiography was performed using standard methods (18). For supershift assays, we used anti-CREB mAb and control IgG (both from Santa Cruz Biotechnology).

Immunoprecipitation

Total cell protein extracts of activated PBMC (100 µg) were precleared by incubation with 10 µl of protein G-Sepharose beads at 4°C for 1 h with rotation. After centrifugation at 1800 x g for 3 min, the supernatant was collected and incubated with 2 µg/ml anti-CREB mAb for 2 h. Fifteen microliters of protein G-Sepharose beads were added and incubated overnight at 4°C with rotation. The beads were washed with 1% Nonidet P-40 cell lysis buffer three times. Supernatants were removed, SDS-PAGE loading buffer was added, and the samples were boiled for 5 min.

Western blotting

SDS-PAGE and Western blotting were performed as previously described (18) with anti-phosphorylated CREB. The blot was then stripped and reblotted with anti-CREB polyclonal Ab. In some experiments, Western blotting was also performed with Abs to activating transcription factor-2, c-Jun, and -actin. All Abs were obtained from Santa Cruz Biotechnology.

Chromatin immunoprecipitation

The procedure described by Farnham et al. was used (http:mcardle.oncology.wisc.edu/farnham/protocols/chips.html). Briefly, formaldehyde was added to the cells (final concentration, 1%, v/v) to cross-link DNA to protein. After 10 min, cross-linking was stopped by adding glycine (final concentration, 125 mM/l). After 5 min, the cells were collected, washed with PBS, and lysed in cell lysis buffer (5 mM PIPES (pH 8.0), 85 mM KCl, and 0.5% Nonidet P-40 containing protease inhibitors (1 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml aprotinin)) and incubated on ice for 5 min. The nuclei were collected and lysed in nuclei lysis buffer (50 mM Tris-HCl (pH 8.1), containing protease inhibitors). Lysed nuclei were sonicated with a microtip sonicator on ice to yield DNA fragments of 600 bp, based on conditions optimized by pilot experiments. Samples were then diluted 1/10 with immunoprecipitation dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), and 167 mM NaCl) containing protease inhibitors. Staphylococcus A cells (10 µl; Calbiochem) were blocked with BSA and salmon sperm DNA; used to preclear nuclear lysates, which were then immunoprecipitated with 1 µg of anti-CREB mAb or an isotype control anti-Bcl2; and incubated overnight at 4°C with tumbling. Staphylococcus A (10 µl), blocked with BSA and salmon sperm DNA, was then added and incubated for 15 min at 4°C with tumbling. The immunoprecipitate was pelleted by centrifugation at 1800 x g for 3 min. The pellet was washed twice with 1.4 ml of dialysis buffer and four times with immunoprecipitation wash buffer (100 mM Tris-HCl (pH 8.0), 500 mM LiCl, 1% Nonidet P-40, and 1% deoxycholic acid). For each wash, samples were incubated on a rotating platform for 3 min, then centrifuged at 1800 x g for 3 min. Complexes containing Ab, DNA, and protein were eluted twice with 150 µl of elution buffer (50 mM NaHCO₃ with 1% SDS) by vortexing for 15 min at room temperature and then centrifuging at 1800 x g for 3 min. The samples were treated with RNase A, and the cross-links were reversed by heating to 67°C for 4 h. After treatment with proteinase K, DNA was extracted by phenol/chloroform/isoamyl alcohol and ethanol precipitation. DNA pellets were dissolved in 30 µl of water, and 5 µl was used as template for PCR amplification, using primers for the proximal promoter of IFN- (5'-AATGCCACAAAACCTTAGTTATTAA-3' and 5'-ACTTAACTGATCTTTCTCTTCTAAT-3'). PCR conditions and the amount of template were optimized to ensure that amplification was in the linear range. PCR products were resolved on a 1% agarose gel and stained with ethidium bromide.

Introduction of intracellular Ab by scrape-loading

The neutralizing rabbit anti-CREB IgG Ab, W39 (20) (gift from Dr. M. Montminy, The Salk Institute for Biological Studies, La Jolla, CA), and control IgG were affinity-purified from rabbit immune and nonimmune serum, respectively, using protein A columns (Amersham Biosciences). Serum was adjusted to the composition of the binding buffer (20 mM sodium phosphate, pH 7.0) and filtered through a 0.45-µm pore size filter. Columns were washed with 10 column volumes of binding buffer. After applying the serum, the column was washed again with 10 column volumes of binding buffer. IgG was eluted with 2 column volumes of elution buffer (0.1 M citric acid, pH 3.0), and the pH was neutralized with 1 M Tris-HCl, pH 9.0. Eluted IgG was dialyzed against PBS and concentrated using Amicon ultracentrifugal filters (Millipore). The purity of the IgG was confirmed by SDS-PAGE.

Scrape loading of positively selected CD3⁺ T cells with W39 and control rabbit IgG were performed as previously described (21). Briefly, petri dishes were coated with 20 µg/ml poly-L-lysine in PBS for 2 h at 37°C, then blocked with 2% (w/v) BSA in PBS for 1 h at room temperature. Positively selected CD3⁺ T cells were washed and resuspended at 5 x 10⁶ cells/ml in RPMI 1640, and 1 ml was plated on poly-L-lysine-coated petri dishes for 30 min at 37°C. The supernatant was removed, and fresh RPMI 1640 and different amounts of anti-CREB or control IgG were added. After 5 min, a rubber policeman was used to remove the CD3⁺ cells, which were incubated at 37°C in 5% CO₂ for 1 h. Cells were then washed and resuspended in RPMI 1640 with 10% human serum, and 10⁵ CD3 cells were added to 2 x 10⁴ autologous adherent cells in 200-µl flat-bottom wells, and cultured with heat-killed M. tuberculosis for 72 h.

Intracellular staining to detect anti-CREB and IFN-

Intracellular anti-CREB Ab, W39 (rabbit IgG), control IgG, and IFN- in CD3⁺ T cells were detected by intracellular staining using reagents from BD Pharmingen as previously described (19). Briefly, monensin (2 µM) was added to cells 6 h before staining. Cells were then collected, washed, and incubated in permeabilization buffer for 30 min on ice. The permeabilized cells were washed and stained with FITC-goat anti-rabbit IgG and/or PE-labeled mouse anti-human IFN-. After gating on lymphocytes, we measured the percentages of rabbit IgG⁺ and IFN-⁺ cells by flow cytometry. To determine whether W39 was introduced into the nucleus, permeabilized cells were stained with FITC-goat anti-rabbit IgG to detect W39, and with Hoechst 33342 (Molecular Probes) to stain nuclei. A cytocentrifuge preparation of the stained cells was analyzed by confocal microscopy.

Generation of CREB and GFP siRNA

Positively selected CD3⁺ cells were stimulated with PMA (50 ng/ml) and ionomycin (1 µM) for 2 h, and total RNA was isolated with RNA-Bee (Tel-Test). After DNase treatment, the RNA was reverse transcribed to cDNA, using oligo(dT) (Promega) and Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies). CREB cDNA was amplified by PCR with the primers 5'-CGCGGAGTGTTGGTGAG-3' and 5'-CATCAGTGGTCTGTGCATACTG-3'. The product was confirmed as CREB DNA by sequencing and was then reamplified with internal T7 primer-tagged CREB primers (5'-GAATAATACGACTCACTATAGGGAGACCCACCGTAACTCTAGTACAG-3' and 5'-GAATAATACGACTCACTATAGGGAGATCTGATTTGTGGCAGTAAAGG-3', T7 primer sites underlined) to yield a 830-bp product. CREB RNA was then generated by in vitro transcription with T7 RNA polymerase using the Dicer siRNA generation kit (Gene Therapy Systems). The RNA was digested with Dicer at 37°C for 2 h. The resulting siRNA was purified by spin columns. SiRNA purity and size were assessed by electrophoresis on 3% agarose gels. As outlined above for CREB siRNA, GFP siRNA was generated using a GFP expression plasmid as PCR template for GFP DNA and T7-tagged primers for the GFP gene.

Effect of CREB siRNA on expression of CREB and production of IFN- in response to M. tuberculosis

To enhance entry of siRNA into positively selected CD3⁺ T cells, they were stimulated with 1 µg/ml PHA-leukoagglutinin in RPMI 1640 with 10% human serum for 20 h. The cells were then washed and resuspended in RPMI 1640 with 10% human serum (2 x 10⁶/ml) and plated at 10⁶ cells/well in 2-ml wells. The GFP expression plasmid and GFP siRNA were used to optimize transfection conditions to yield maximal suppression of GFP expression with siRNA. Differing amounts of CREB siRNA and 7 µl of transfection reagent (both provided with the Dicer kit) were incubated with PHA-stimulated CD3⁺ cells for 48 h at 37°C in 5% CO₂. The cells were collected, washed, and resuspended in RPMI 1640 with 10% human serum, and 10⁵ CD3⁺ cells were added to 2 x 10⁴ autologous adherent cells in 200-µl flat-bottom wells. Heat-killed M. tuberculosis (1 µg/ml) was added to some wells, and cells were cultured at 37°C in 5% CO₂. After 72 h, culture supernatants were collected for measurement of IFN- by ELISA, using paired Abs (BD Pharmingen). The lower limit of detection was 5 pg/ml. The cells were collected, washed, and used for Western blotting to detect CREB, activating transcription factor-2, and c-Jun.

Effect of CREB siRNA on IFN- mRNA expression in response to M. tuberculosis

Aliquots of 3–4 x 10⁶ negatively selected CD3⁺ cells were resuspended in 100 µl of human T cell nucleofection solution and washed with PBS containing 0.5% BSA, and nucleofection was performed with different concentrations of siRNA using a Nucleofector and protocol U-14 for unstimulated human T cells (Amaxa). Prewarmed RPMI 1640 (0.5 ml) with 10% human serum was then added to the cells, which were transferred to a prewarmed 2-ml well containing 1.5 ml of RPMI 1640 and 10% human serum, and incubated at 37°C. Forty-eight hours postnucleofection, the CD3⁺ cells were washed and counted, and 2 x 10⁶ cells were added to 4 x 10⁵ autologous macrophages in 2-ml wells. Heat-killed M. tuberculosis (1 µg/ml) was added to some wells.

After an additional 48 h, total RNA was extracted with TRIzol LS reagent (Invitrogen Life Technologies). After DNase treatment, total RNA was quantified by OD₂₆₀, and RNA integrity was determined by agarose gel electrophoresis. Two hundred and fifty nanograms of total RNA from each sample was reverse transcribed using random hexamers and Moloney murine leukemia virus reverse transcriptase. Primers and probes for human 18S rRNA and IFN- were designed, and real-time PCR was performed to quantify cDNA expression using a spectrofluorometric thermal cycler (7700 PRISM; Applied Biosystems), as previously described (18). Standard curves for 18S rRNA and IFN- cDNA were generated using serial 10-fold dilutions of RNA from PBMC stimulated with PMA (50 ng/ml) plus ionomycin (1 µg/ml) for 8 h. Results were expressed as the ratio of IFN- cDNA/18S rRNA, in arbitrary units.

Statistical analysis

The paired or unpaired Student’s t test was used to compare data in two groups, as appropriate. A value of p < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CREB binds to the IFN- proximal promoter in T cells exposed to M. tuberculosis

Stimulation of peripheral blood T cells with M. tuberculosis Ags induces secretion of IFN- (16, 17), but the contribution of CREB to IFN- production is undefined. Most studies have demonstrated that CREB binds to the IFN- proximal promoter during activation of human Th1 cells or Jurkat T cells by PMA and ionomycin (8, 22). To determine whether such binding occurs in T cells exposed to microbial Ags, we stimulated PBMC from five healthy tuberculin reactors with M. tuberculosis and performed EMSAs with the radiolabeled IFN- proximal promoter. A representative result (Fig. 1A) shows that M. tuberculosis induced binding of two low mobility complexes, A and B, to the IFN- proximal promoter, with maximal binding at 48 h. In competitive EMSAs, binding of both complexes was abrogated by unlabeled IFN- proximal promoter or wild-type CREB consensus binding site, but not by the mutated CREB consensus binding site or the NF-B consensus binding site (Fig. 1B). Anti-CREB inhibited binding of complex B to the IFN- proximal promoter (Fig. 1C).

View larger version (53K): [in this window] [in a new window]   FIGURE 1. Effect of M. tuberculosis stimulation of PBMC on CREB binding to the IFN- proximal promoter. A, Complexes binding to the IFN- proximal promoter. PBMC from a healthy tuberculin reactor were cultured with medium for 48 h (lane 1) or with 10 µg/ml heat-killed M. tuberculosis for 48 h (lane 2) or 72 h (lane 3). EMSAs were performed on nuclear extracts with a radiolabeled IFN- proximal promoter probe. The arrows show complexes A and B that bind to the IFN- promoter. Results are representative of those obtained for PBMC from five subjects. B, Effect of oligonucleotide competitors on complexes binding to the IFN- proximal promoter. EMSAs were performed, as described in A. Before adding labeled probe, some nuclear extracts were incubated with a 100-fold molar excess of unlabeled proximal IFN- promoter, the consensus NF-B binding site, or the wild-type (wt) or mutated (mt) CREB consensus binding site. The arrows show complexes A and B that bind to the IFN- promoter. Results are representative of six independent experiments. C, Effect of anti-CREB on the complexes binding to the IFN- proximal promoter. EMSAs were performed as described in A. Before adding labeled probe, some nuclear extracts were incubated with anti-CREB mAb or control IgG. The arrow shows that anti-CREB inhibits binding of complex B, but not complex A. Results are representative of eight independent experiments.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Chromatin immunoprecipitation to evaluate binding of CREB to the IFN- proximal promoter in T cells activated by M. tuberculosis or through the TCR. PBMC from a healthy tuberculin reactor were cultured in the presence of medium alone (lane 1) or with heat-killed M. tuberculosis (10 µg/ml) for 24 h (lane 2) or 48 h (lane 3), and immunoprecipitation was performed with anti-CREB, followed by PCR amplification with primers specific for the IFN- proximal promoter to yield the predicted 189-bp product. Positively selected CD3+ T cells were cultured in 5 ml wells containing plate-bound anti-CD3 (5 µg/ml; clone OKT3; Ortho Biotech) and anti-CD28 (5 µg/ml; clone CD28.2; BD Biosciences) for 2 h. Chromatin immunoprecipitation was performed with anti-CREB, as outlined above (lane 4). Positive controls were PCR amplification of input chromatin before immunoprecipitation (lane 6) and of chromosomal DNA (lane 8). Negative controls were performance of immunoprecipitation with anti-Bcl2 (lane 5) and PCR amplification of a sample containing no DNA (lane 7). M, m.w. markers. Results are representative of five independent experiments.

CREB is constitutively expressed in T cells, but phosphorylated CREB has enhanced binding to the IFN- promoter and stimulates recruitment of the coactivator CREB-binding protein, which associates with RNA polymerase II and up-regulates gene transcription (13, 14). To determine whether M. tuberculosis elicited phosphorylation of CREB, we used Western blotting with Abs specific for serine 133-phosphorylated CREB. Stimulation of PBMC with M. tuberculosis resulted in CREB phosphorylation by 48–72 h (Fig. 3A). Activation of CD3⁺ T cells with anti-CD3 and anti-CD28 or with PMA and ionomycin also induced phosphorylation of CREB (Fig. 3B and data not shown, respectively).

View larger version (49K): [in this window] [in a new window]   FIGURE 3. Phosphorylation of CREB during T cell activation by M. tuberculosis (A) or through the TCR (B). A, PBMC from a healthy tuberculin reactor were cultured with medium alone (lane 1) or stimulated with heat-killed M. tuberculosis (10 µg/ml) for 48 h (lane 2) or 72 h (lane 3). Total cell extracts were immunoprecipitated with anti-CREB mAb, then subjected to Western blotting with anti-phosphorylated CREB. The blot was then stripped and reblotted with anti-CREB polyclonal Ab. Results are representative of five independent experiments. B, CD3+ T cells were positively selected from PBMC of a healthy tuberculin reactor and cultured in 2-ml wells containing plate-bound anti-CD3 and anti-CD28, as outlined in Fig. 2, for varying periods. Total cell extracts were subjected to Western blotting with anti-phosphorylated CREB. This Ab also recognizes phosphorylated activating transcription factor-1, which is visible in B, but not in A, because immunoprecipitation was performed with anti-CREB in A before Western blotting. The blot was then stripped and reblotted with anti-CREB polyclonal Ab. Results are representative of three independent experiments.

Intracellular delivery of anti-CREB Ab reduces M. tuberculosis-induced IFN- production

To determine the effect of CREB on IFN- production, we used W39, a polyclonal Ab that neutralizes CREB (20). To introduce the Ab into cells, we used scrape-loading, which involves firmly adhering cells to poly-L-lysine-coated plates, followed by scraping to physically remove cells. This procedure results in transient perforations of the cell lipid membrane, allowing macromolecules from the medium to enter the cell before the membrane reseals. This method has been used to deliver intracellular Abs to the transcription factor activator protein-1 (21). The percentage of cells containing intracellular Ab was measured by immunolabeling with FITC goat anti-rabbit IgG, followed by cytofluorometric analysis. Pilot experiments showed that 35–40% of the cells contained Ab for 72 h. Fig. 4A shows a result representative of five independent experiments. Trypan blue exclusion showed cell viability of >95%. To determine whether W39 could enter the nucleus and neutralize transcription factors, cells were stained with FITC goat anti-rabbit IgG and with Hoechst stain for the nucleus. Confocal microscopy demonstrated colocalization of the Ab in some nuclei (Fig. 4B).

View larger version (42K): [in this window] [in a new window]   FIGURE 4. Scrape-loading introduces anti-CREB into the nucleus of T cells. A, Purified CD3+ T cells from PBMC of a healthy donor were scrape-loaded with the anti-CREB Ab W39 or no Ab. Seventy-two hours after scrape-loading, cells were permeabilized, stained with FITC goat anti-rabbit IgG, and analyzed by flow cytometry. The insets show the percentage of cells in each quadrant. Results are representative of five independent experiments. B, Nuclear localization of W39 in T cells. Purified CD3+ cells were scrape-loaded with W39 and cultured with autologous macrophages and heat-killed M. tuberculosis (10 µg/ml) for 72 h. Cells were fixed, permeabilized, and analyzed by confocal microscopy (x400). Nuclei were treated with RNase and stained with Hoechst 33342 (blue; left panel). W39+ cells were detected by staining with FITC anti-rabbit IgG (green; middle panel). A merge of both stains shows Ab in some nuclei (right panel).

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Effect of anti-CREB on M. tuberculosis-induced IFN- production. A, IFN- production. CD3+ cells from PBMC of healthy tuberculin reactors were scrape-loaded with control IgG (100 µg/ml) or W39 (50 and 100 µg/ml) and cultured with M. tuberculosis-pulsed macrophages for 72 h. IFN- was measured in supernatants by ELISA. The mean and SE of three experiments are shown. B, Intracellular IFN- expression. CD3+ T cells were scrape-loaded with control IgG or W39 and cultured with M. tuberculosis-pulsed macrophages for 72 h. The cells were stained with FITC anti-rabbit IgG and PE-conjugated anti-IFN-. We gated on IgG+ cells and measured the percentages that were IFN-+. Results are representative of four independent experiments.

CREB siRNA decreases M. tuberculosis-induced IFN- mRNA and protein production

To confirm the results obtained with intracellular anti-CREB, we evaluated the capacity of CREB siRNA to inhibit IFN- production by CD3⁺ T cells exposed to macrophages pulsed with heat-killed M. tuberculosis. CREB and GFP siRNAs, produced as outlined in Materials and Methods, consisted of uniform 19- to 21-bp double-stranded RNA (data not shown). CD3⁺ cells were transfected with a GFP expression plasmid, with or without GFP siRNA, and GFP expression was measured by flow cytometry. In five independent experiments, GFP siRNA reduced GFP expression by 50–75% in a dose-dependent manner (data not shown).

CD3⁺ cells from four healthy tuberculin reactors were incubated with CREB siRNA, GFP siRNA, or no siRNA. T cells were then cultured with autologous macrophages and M. tuberculosis for 72 h. CREB expression, as measured by densitometry, was reduced by 71 ± 13% by 1000 ng of CREB siRNA (p = 0.007; Fig. 6A). In contrast, CREB siRNA did not affect the expression of activating transcription factor-2 and c-Jun, which also bind to the IFN- promoter. Because c-Jun is produced by T cells only upon activating, this indicates that CREB siRNA did not have nonspecific toxic effects or prevent cell activation. To determine the effect of modulating CREB on IFN- production, we measured IFN- concentrations in cell culture supernatants. IFN- levels in supernatants of cells that were not treated with siRNA varied widely from 189 to 2691 pg/ml. IFN- concentrations were unaffected by GFP siRNA, but were reduced by 73 ± 6% by 1000 ng of CREB siRNA (p = 0.04; Fig. 6B).

View larger version (21K): [in this window] [in a new window]   FIGURE 6. Effect of CREB siRNA on the expression of CREB and IFN-. A, Expression of CREB by M. tuberculosis-stimulated T cells. CD3+ cells from PBMC of a healthy tuberculin reactor were treated with transfection reagent and diluent alone (lane 1) or were transfected with 1000 ng of GFP siRNA (lane 2), 500 ng of CREB siRNA (lane 3), or 1000 ng of CREB siRNA (lane 4). CD3+ T cells were then cultured with M. tuberculosis-stimulated macrophages for 72 h. Western blotting was performed on total cell extracts with anti-CREB. The blots were stripped and reprobed with anti-activating transcription factor-2, then with anti-c-Jun, and finally with anti--actin. Results are representative of four independent experiments. B, Effect of CREB siRNA on M. tuberculosis-induced IFN- production. CD3+ cells, treated with no siRNA, GFP siRNA, or 500-1000 ng of CREB siRNA, were cultured with M. tuberculosis-stimulated macrophages as described in A, and IFN- concentrations were measured in supernatants collected after 72 h. The mean and SE for results of experiments with four donors are shown. C, Effect of CREB siRNA on M. tuberculosis–induced IFN- mRNA expression. CREB siRNA, GFP siRNA, or no siRNA was introduced into CD3+ cells of healthy tuberculin reactors by nucleofection. CD3+ cells were then added to autologous macrophages stimulated with heat-killed M. tuberculosis. After 48 h, IFN- mRNA expression was determined by real-time PCR after normalization for 18S rRNA content. The mean and SE for the results of experiments with six donors are shown.

CREB expression is reduced in M. tuberculosis-stimulated T cells from tuberculosis patients

The findings presented above demonstrate that in T cells of healthy tuberculin reactors, CREB is required for Ag-induced IFN- production. To determine whether these findings are relevant to the clinical manifestations of M. tuberculosis infection, we evaluated the expression of CREB in tuberculosis patients, because IFN- production by M. tuberculosis-stimulated PBMC is reduced in these individuals (16, 17). EMSAs with the IFN- proximal promoter as a probe showed that the low mobility complexes binding the IFN- proximal promoter were reduced in M. tuberculosis-stimulated PBMC from tuberculosis patients compared with those from healthy tuberculin reactors (Fig. 7A). Western blotting of nuclear extracts of M. tuberculosis-stimulated PBMC from 14 tuberculosis patients confirmed that CREB was absent in 10 patients and reduced in four patients. In contrast, extracts of PBMC from 14 healthy tuberculin reactors all showed high levels of CREB (Fig. 7B). IFN- concentrations were 3823 ± 211 and 1458 ± 303 pg/ml in supernatants of M. tuberculosis-stimulated PBMC from healthy tuberculin reactors and tuberculosis patients, respectively (p < 0.0001, by Student’s t test). To confirm that the changes observed in PBMC reflected those in T cells, Western blotting was performed on nuclear extracts of CD3⁺ T cells from PBMC of tuberculosis patients and healthy tuberculin reactors cultured with M. tuberculosis or with medium alone for 48–72 h. The expression of CREB was reduced in T cells of tuberculosis patients compared with those of healthy tuberculin reactors (Fig. 7C). Phosphorylation of CREB was also reduced in M. tuberculosis-stimulated PBMC from five tuberculosis patients compared with findings in healthy tuberculin reactors (Fig. 7D). The absence of phosphorylated CREB probably resulted from the lack of CREB substrate.

View larger version (48K): [in this window] [in a new window]   FIGURE 7. CREB expression in M. tuberculosis-stimulated T cells from tuberculosis patients and healthy tuberculin reactors. For all panels, PBMC from a healthy tuberculin reactor (PPD+) and a tuberculosis patient (TB) were cultured with medium for 48 h (lane 1) or with 10 µg/ml heat-killed M. tuberculosis for 48 h (lane 2) or 72 h (lane 3). A, Complexes binding to the IFN- proximal promoter. EMSAs were performed on nuclear extracts of PBMC with a radiolabeled IFN- proximal promoter probe. The arrows show complexes that bind to the IFN- promoter. Similar results were obtained for 14 tuberculosis patients and 14 healthy tuberculin reactors. B, Detection of CREB in PBMC by Western blotting. Western blotting was performed with anti-CREB. The blot was stripped and reblotted with anti--actin. Results are representative of five independent experiments. C, Detection of CREB in CD3+ cells by Western blotting. After PBMC had been cultured with medium or heat-killed M. tuberculosis for 48–72 h, CD3 cells were negatively selected, nuclear extracts were prepared, and Western blotting was performed, as outlined in B. D, Detection of phosphorylated CREB. Total PBMC cell extracts were immunoprecipitated with anti-CREB mAb, then subjected to Western blotting with anti-phosphorylated CREB Ab. The blot was stripped and reblotted with anti-CREB polyclonal Ab. Results are representative of five independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

CREB is a transcriptional regulator of genes that mediate central biologic functions, and mice with deleted CREB genes have marked abnormalities in lung development, neuronal transmission, and development of T cells bearing the TCR (13, 25, 26). CREB also plays a critical role in up-regulating the production of IL-2, because T cells from mice transgenic for a dominant negative form of CREB do not proliferate or produce IL-2 when activated through the TCR (27), and CREB enhances transcription of IL-2 in mitogen-stimulated Jurkat T cells (28). However, the effect of CREB on transcription of IFN- is controversial, as detailed below.

Production of IFN- is pivotal for resistance to M. tuberculosis and a wide variety of other intracellular pathogens (1, 2, 3, 4, 5). IFN- production in response to microbial Ags is controlled by regulation of transcription, and levels of IFN- mRNA parallel those of protein in the immune response to infection (17, 29). The region 40–108 bp upstream of the IFN- gene transcriptional start site is necessary and sufficient for transcription in Jurkat T cells activated by PMA and ionomycin. This region includes proximal (–73 to –48 bp) and distal (–96 to –80 bp) regulatory elements that are highly conserved among humans, rats, and mice, suggesting their essential regulatory role (8). CREB binds to the IFN- proximal promoter, but the resultant effects on transcription of IFN- are controversial. Cotransfection of Jurkat T cells with plasmids encoding CREB and a dimer of the IFN- proximal promoter showed that CREB inhibited IFN- transcription (10, 11). In addition, in transgenic mice expressing luciferase under the control of a dimer of the proximal IFN- promoter, differentiation of precursor T cells to effector T cells was accompanied by reduced CREB expression and enhanced IFN- proximal promoter activity (11). These findings suggest that CREB inhibits transcription of IFN-. However, the effects of CREB on dimeric IFN- promoter constructs in Jurkat T cells and transgenic mice may not reflect events in primary human T cells due to differences between cell lines and primary cells, species-specific factors, or because regulatory effects may differ in plasmid or chromosomal DNA segments, as in the case of some enhancers in the IFN- gene (30).

Some published studies suggest that CREB is a positive regulator of IFN- transcription. For example, transgenic mice that express a dominant negative form of CREB in mature T cells showed defective differentiation of cytokine-secreting effector T cells (12). Chromatin immunoprecipitation also showed increased binding of CREB to the IFN- proximal promoter in human Th1 cells, but not in Th2 cells or precursor T cells (22). Our current findings, using a physiologically relevant experimental system of primary human T cells cultured with M. tuberculosis, provide the most definitive evidence to date that CREB stimulates IFN- production by human T cells responding to microbial Ag.

We previously found reduced CREB levels in freshly isolated peripheral blood T cells from tuberculosis patients compared with normal donors (18). Our current findings confirm and extend these observations, demonstrating that CREB levels in T cells from tuberculosis patients remain low despite activation by M. tuberculosis. Furthermore, exposure to M. tuberculosis markedly enhanced the production of phosphorylated CREB in PBMC from healthy tuberculin reactors, but not in those from tuberculosis patients, probably because baseline CREB levels were extremely low. Phosphorylation of CREB facilitates binding to the IFN- promoter and recruitment of the coactivator CREB-binding protein, up-regulating gene transcription (13, 14). These results suggest that decreased CREB levels in T cells of tuberculosis patients contribute to their reduced capacity to produce IFN- in response to mycobacterial Ags. We are currently investigating the pathogenic mechanisms that result in low CREB expression in tuberculosis.

In the current report we focused on determining the contribution of CREB to IFN- production in response to microbial stimuli and did not address the mechanisms underlying reduced CREB expression in tuberculosis patients. Low CREB levels might predispose patients to more severe disease from M. tuberculosis because of the reduced capacity of T cells to produce IFN-. Alternatively, tuberculosis itself may reduce levels of CREB, which, in turn, decreases IFN- promoter activity and IFN- production, weakening host defenses and favoring the development of extensive disease. To distinguish these possibilities, it will be important to measure the expression of CREB in patients before, during, and after treatment for active tuberculosis. Evaluation of the severity of tuberculosis in CREB-deficient animals will also provide insight into this question. Further studies to understand the underlying mechanisms by which CREB is reduced in tuberculosis may permit the development of immunomodulatory strategies to up-regulate CREB, contributing to the treatment and prevention of disease due to M. tuberculosis and other intracellular pathogens.

	Acknowledgments

 
We are grateful to Dr. Marc Montminy for providing us with the W39 Ab, and to Dr. Zissis Chroneos for assistance with confocal microscopy. We thank Ortho Biotech for provision of anti-CD3 Ab, and Drs. Malini Rajagopalan and Murty Madiraju for helpful discussions.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the National Institutes of Health (AI44935), the Potts Memorial Foundation, the Cain Foundation for Infectious Disease Research, and the Center for Pulmonary and Infectious Disease Control. P.F.B. holds the Margaret E. Byers Cain Chair for Tuberculosis Research.

² Address correspondence and reprint requests to Dr. Peter F. Barnes, Center for Pulmonary and Infectious Disease Control, University of Texas Health Center, 11937 U.S. Highway 271, Tyler, TX 75708-3154. E-mail address: peter.barnes{at}uthct.edu

³ Abbreviations used in this paper used: siRNA, small interfering RNA.

Received for publication October 7, 2004. Accepted for publication February 23, 2005.

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