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The Journal of Infectious Diseases logoLink to The Journal of Infectious Diseases
. 2018 Feb 8;217(11):1821–1831. doi: 10.1093/infdis/jiy079

Elevated Cyclic AMP Inhibits Mycobacterium tuberculosis-Stimulated T-cell IFN-γ Secretion Through Type I Protein Kinase A

Yoon-tae Chung 1, Virginia Pasquinelli 2, Javier O Jurado 3, Xisheng Wang 1,1, Na Yi 1, Peter F Barnes 1, Veronica E Garcia 3, Buka Samten 1,✉
PMCID: PMC5946837  PMID: 29438524

Abstract

Cyclic adenosine monophosphate (cAMP) is critical in immune regulation, and its role in tuberculosis infection remains unclear. We determined the levels of cAMP in peripheral blood mononuclear cells (PBMC) from tuberculosis patients and the mechanisms for cAMP suppression of IFN-γ production. PBMC from tuberculosis patients contained significantly elevated cAMP than latent tuberculosis infected subjects (LTBI), with an inverse correlation with IFN-γ production. Consistent with this, the expression of cAMP response element binding protein (CREB), activating transcription factor (ATF)-2 and c-Jun were reduced in tuberculosis patients compared with LTBI. PKA type I specific cAMP analogs inhibited Mtb-stimulated IFN-g production by PBMC through suppression of Mtb-induced IFN-γ promoter binding activities of CREB, ATF-2, and c-Jun and also miR155, the target miRNA of these transcription factors. Neutralizing both IL-10 and TGF-β1 or supplementation of IL-12 restored cAMP-suppressed IFN-g production. We conclude that increased cAMP inhibits IFN-g production through PKA type I pathway in tuberculosis infection.

Keywords: cyclic adenosine monophosphate, cytokine, transcription factor, tuberculosis, human

Levels of cAMP in PBMC of tuberculosis patients was higher and remained elevated upon antigens of Mycobacterium tuberculosis stimulation, and increased cAMP inhibited IFN-γ in a PKA type I dependent manner by targeting transcription factors that control the IFN-γ gene.

Tuberculosis caused by Mycobacterium tuberculosis infection remains a major cause of human death. Although interferon-gamma (IFN-γ) produced by T cells protects against tuberculosis infection [1, 2], it is reduced in tuberculosis patients compared with that of latent-tuberculosis–infected individuals [3]. Furthermore, patients with the most advanced tuberculosis disease show the greatest reduction in IFN-γ secretion by peripheral blood mononuclear cells (PBMC) stimulated with M. tuberculosis [4].

Cyclic adenosine monophosphate (cAMP) plays a critical role in the regulation of a variety of cells, including immune cells, as a second messenger in response to stimulation. Cellular cAMP is produced through hydrolysis of adenosine triphosphate (ATP) by adenylate cyclase, a plasma membrane protein, and increased intracellular cAMP activates protein kinase A (PKA). PKA is a multiprotein complex that consists of 2 catalytic (C) subunits and 2 regulatory (R) subunits, and association of the R subunits with the C subunits keeps the C subunits inactive in the cytoplasm. Binding of cAMP molecules to the R subunits leads to conformational changes, and this leads to the release of the C subunits. The free C subunits then enter the nucleus and activate transcription factors, such as cAMP-responsive element binding proteins (CREB), to induce cAMP-responsive gene expression [5]. Increased intracellular cAMP inhibits T-cell responses, both as an effector and a mediator of regulatory T cells [6, 7]. Elevated intracellular cAMP is one of the mechanisms for the pathology of whooping cough and several other microbial infections [8, 9]. Indeed, cAMP produced by M. tuberculosis directly intoxicates macrophage [10] and reduces the expression of cathelicidin [11] in animal models. However, the status and effect of immune cell cAMP on immunity in tuberculosis patients are unclear. To examine the role of cAMP in reduced IFN-γ production in tuberculosis patients, we studied cAMP levels in PBMC from patients and latent-tuberculosis–infected donors, and explored the potential mechanisms of cAMP suppression of IFN-γ secretion. Our results demonstrated that cAMP levels in PBMC from tuberculosis patients were increased compared with that from latent-tuberculosis–infected donors in a reverse correlation with reduced M. tuberculosis-stimulated IFN-γ production. Increased intracellular cAMP inhibited M. tuberculosis-induced T-cell IFN-γ production by inhibiting the transcription factors that regulate IFN-γ gene transcription through PKA type I.

MATERIALS AND METHODS

Study Subjects and Cell Culture Conditions

Blood was obtained from 40 QuantiFERON-TB gold (QFN) positive healthy subjects (latent-tuberculosis–infected individuals) and 14 pulmonary tuberculosis patients, following the protocols approved by the institutional review boards of The University of Texas Health Science Center at Tyler and The Hospital Muniz, Buenos Aires, Argentina. Latent-tuberculosis–infected donors were enrolled based on positive QFN results with no clinical and radiological evidence of active tuberculosis. Tuberculosis patients were diagnosed based on clinical symptoms, chest X-ray examinations, and confirmed by positive acid-fast bacillus and M. tuberculosis culture in the sputa. All the patients were seronegative for human immunodeficiency virus, did not have a medical history of immunosuppressive conditions, and received less than 2 weeks of antituberculosis therapy. All the patients and latent-tuberculosis–infected donors provided written informed consent for the samples collection and subsequent analysis. PBMC were obtained from peripheral blood by differential centrifugation over Ficoll-Paque. The cells were cultured at 2 × 106/mL in RPMI-1640 with 10% heat-inactivated pooled human AB serum, as described previously [12].

cAMP Analogs

The cAMP analogs used were: N6,2′-O-dibutyryladenosine-3′,5′-cAMP sodium, a cell-permeable cAMP analog that activates cAMP-dependent protein kinase A (referred to as db-cAMP), forskolin (both from Sigma-Aldrich), Rp-adenosine-3′,5′-cyclic adenosine monophosphorothioate (Rp-cAMP), N6-benzoyladenosine-3′,5′-cAMP (6-Benz-cAMP), 8-hexylaminoadenosine-3′,5′-cAMP (8-HA-cAMP), 8-piperidino-adenosine-3′,5′-cAMP (8-PIP-cAMP), and 8-pCPT-2′-O-Me-3′,5′-cAMP (8-pCPT-cAMP), all from Biolog Life Science Institute.

Determination of Intracellular cAMP Concentrations

PBMC were cultured at 2 × 105 cells in 200-µL volume in a 96-well plate with or without M. tuberculosis for 48 hours. After centrifugation at 1500 g for 3 minutes, the culture supernatants were stored at −80ºC for cytokine assessment and the cells were lysed by incubation with 200 µL of cell lysis buffer (50 mM acetate buffer, pH 5.8, containing 0.25% dodecyltrimethylammonium bromide) for 10 minutes with gentle shaking at room temperature. The cAMP content in 10 µL of cell lysate with appropriate dilution was determined by cAMP Biotrak Enzymeimmuoassay System (GE Healthcare-Biosciences, Uppsala, Sweden), following the manufacturer’s instructions.

Measurement of Cytokine Concentrations

Cell-free culture supernatants from the cAMP determination experiments were evaluated for IFN-γ levels by enzyme-linked immunosorbent assay (ELISA), using mouse anti-human IFN-γ mAb (clone NIB42) as capture and biotinylated mouse anti-human IFN-γ mAb (clone 4S.B3) as detection antibodies and serially diluted known concentrations of recombinant IFN-γ as standard, all from BD PharMingen (San Diego, CA).

Measurement of IFN-γ mRNA and miR155 by Real-Time PCR

IFN-γ mRNA and miR155 levels in the total RNA of PBMC incubated with or without M. tuberculosis were determined by quantitative polymerase chain reaction (qPCR) using the primer and probe sets (Thermo Fisher Scientific) following the protocols of the manufacturer and as described [13]. 18S rRNA and miRU6 were used as internal controls.

Electrophoretic Mobility Shift Assay

Nuclear protein extracts of PBMC incubated at different conditions were prepared, and electrophoretic mobility shift assays (EMSAs) were performed using [γ−32P]dATP-labled IFN-γ proximal promoter (−71 to −40 bp) as a probe, as previously described [13, 14].

Western Blotting

Expression of the transcription factors in protein extracts of PBMC was determined by western blotting, as previously described [13, 15], with anti-CREB mAb. The blot was then stripped and reblotted with antibodies to activating transcription factor (ATF)-2, c-Jun, and β-actin (Santa Cruz Biotechnology).

Statistical Analysis

Paired and unpaired Student t tests were used, as appropriate. P values of .05 or less were considered statistically significant.

RESULTS

Increased Intracellular cAMP in PBMC from Tuberculosis Patients Correlates With Reduced M. tuberculosis-stimulated IFN-γ Production and Reduced Expression of Transcription Factors that Regulate IFN-γ Gene Expression

Peripheral blood T cells from tuberculosis patients have a reduced capacity to produce IFN-γ upon stimulation with M. tuberculosis compared with T cells from latent-tuberculosis–infected subjects [3, 16]. Because cAMP is a second signal that affects immune cell functions, we determined whether increased cAMP levels could contribute to the reduced IFN-γ production. We measured cAMP and IFN-γ levels in the cell lysates and culture supernatants of PBMC, respectively, from 7 latent-tuberculosis–infected donors and 15 tuberculosis patients. Overall, cAMP levels were higher in PBMC from tuberculosis patients at baseline level cultured in medium alone (1469 ± 122 fM/10 μL of cell lysate) compared to the cells from latent-tuberculosis–infected donors (1039 ± 227.9 fM/10 μL of cell lysate) and remained significantly elevated upon M. tuberculosis stimulation (1182 ± 93.1 fM/10 μL of cell lysate). However, cAMP levels in PBMC from latent-tuberculosis–infected donors were reduced (499 ± 90.3 fM/10 μL of cell lysate) significantly in response to M. tuberculosis stimulation compared to that in tuberculosis patients (Figure 1A; P = .0002). In contrast, PBMC from tuberculosis patients produced significantly less IFN-γ (16656 ± 282.3 pg/mL, P = .004) than PBMC from latent-tuberculosis–infected donors (39712 ± 284.9 pg/mL) upon M. tuberculosis stimulation, consistent with previous reports [16, 17]. Analysis of the correlation between intracellular cAMP levels and IFN-γ production by PBMC from tuberculosis patients showed a negative correlation (Figure 1B). These results suggest that elevated cAMP in PBMC from tuberculosis patients may contribute to reduced IFN-γ production. We have previously shown that reduced M. tuberculosis-stimulated IFN-γ production by PBMC from tuberculosis patients is due to decreased expression of the transcription factors CREB, ATF-2, and c-Jun [13, 14]. Therefore, we determined the total protein expression levels of these transcription factors. Our results demonstrated that the expression of these 3 transcription factors was reduced, both in the unstimulated and M. tuberculosis-stimulated PBMC from tuberculosis patients compared to PBMC from latent-tuberculosis–infected subjects (Figure 2A). Although the patients varied in the expression of these transcription factors (TB1–3 in Figure 2A), the expression of these transcription factors was significantly reduced in tuberculosis patients after normalizing for β-actin expression (Figure 2A and 2B).

Figure 1.

Figure 1.

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Increased cyclic adenosine monophosphate (cAMP) in peripheral blood mononuclear cells (PBMC) from tuberculosis patients inversely correlated with interferon-gamma (IFN-γ) production by Mycobacterium tuberculosis-stimulated PBMC. A, cAMP concentrations in PBMC from tuberculosis patients and latent-tuberculosis–infected donors. PBMC from 7 latent-tuberculosis–infected donors (QFN+) and 15 tuberculosis patients (TB) were cultured in the absence (Medium) or presence of 2 µg/mL heat-killed M. tuberculosis Erdman (M. tb). After 48 hours, cAMP concentrations in 10 µL of cellular lysates were measured by the Enzymeimmunoassay System kit. Mean values and standard errors are shown. B, Correlation analysis of M. tuberculosis-stimulated IFN-γ production and the respective cellular cAMP levels of PBMC from tuberculosis patients after 48 hours’ stimulation.

Figure 2.

Figure 2.

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Reduced expression of cAMP response element binding protein (CREB), activating transcription factor-2 (ATF-2), and c-Jun transcription factors in peripheral blood mononuclear cells (PBMC) from tuberculosis patients. A, PBMC from tuberculosis (TB) patients and latent-tuberculosis–infected (LTBI) donors were cultured without (1) or with (2) heat-killed Mycobacterium tuberculosis (M. tb) Erdman for 48 hours. The expression of the indicated transcription factors in 30 µg protein extracts of PBMC was determined by western blotting, by serially blotting with different antibodies after stripping of the membrane. One representative result is shown. B, Densities of the western blot bands of indicated transcription factors in PMBC from 9 latent-tuberculosis–infected donors and 10 tuberculosis patients, treated as in (A), were determined by Gel Doc System using Quantity One software (BioRad) and normalized by their respective densities of β-actin bands. The means and standard errors of the densitometry analysis are shown. * P < .05 and ** P < .005 compared to that from latent-tuberculosis–infected donors.

cAMP Downregulates M. tuberculosis-Induced IFN-γ Production

To directly evaluate the effects of cAMP on antigen-induced IFN-γ production, PBMC from 12 latent-tuberculosis–infected donors were cultured with heat-killed M. tuberculosis for 48 hours, in the absence or presence of db-cAMP or forskolin, which stimulate cellular cAMP through membrane-associated adenylate cyclase. The compounds reduced M. tuberculosis-stimulated IFN-γ protein production by more than 60% and 90%, respectively (Figure 3A). IFN-γ mRNA levels were also reduced 80%–90% by db-cAMP and forskolin (Figure 3B), indicating that elevation of intracellular cAMP either by extrinsic (db-cAMP) or intrinsic (forskolin) mechanisms markedly suppressed M. tuberculosis-stimulated IFN-γ production at the transcriptional level. To determine if neutralizing cAMP would affect IFN-γ production and to further confirm that the effect of db-cAMP was mediated through binding to PKA, we used Rp-cAMP, which prevents the activation of the catalytic subunit of PKA by competitive binding to the regulatory subunit [18]. The presence of db-cAMP inhibited IFN-γ production by M. tuberculosis-stimulated PBMC from 7 latent-tuberculosis–infected donors. Pretreatment of PBMC with increasing concentrations of Rp-cAMP restored cAMP-suppressed IFN-γ production in a dose-dependent manner with the highest tested dose restoring IFN-γ production to levels similar to that of the cells stimulated with M. tuberculosis alone (Figure 3C). These results indicate that cAMP inhibits M. tuberculosis-stimulated IFN-γ production by PBMC.

Figure 3.

Figure 3.

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Elevation of cellular cyclic adenosine monophosphate (cAMP) by direct supplementation of cAMP or by cAMP inducer forskolin reduced Mycobacterium tuberculosis (M. tb)-stimulated expression of interferon-gamma (IFN-γ), and this was reversed by cAMP inhibitor. A, Peripheral blood mononuclear cells (PBMC) from 12 latent-tuberculosis–infected donors were cultured at 2 × 105 in 200 µL in a 96-well plate. Cell samples were incubated with indicated concentrations of N6,2′-O-dibutyryladenosine-3′,5′-cAMP sodium (db-cAMP) or forskolin for 1 hour before further incubation with 2 µg/mL heat-killed M. tuberculosis Erdman. Samples were untreated (Medium) or incubated with M. tuberculosis alone (None) as controls. After 48 hours, IFN-γ levels in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). * P = .0004 and ** P ≤ .0001, compared to M. tuberculosis-stimulated cells (None). B, PBMC from 6 latent-tuberculosis–infected donors were cultured, as in (A) and IFN-γ mRNA levels were determined by quantitative polymerase chain reaction (qPCR) using 18s rRNA as internal control. The expression levels of IFN-γ mRNA were expressed as fold change over that of PBMC with medium only. * P < .05, compared to M. tuberculosis-stimulated cells. C, PBMC from 7 latent-tuberculosis–infected donors were cultured in medium alone or with cAMP antagonist, Rp-adenosine-3′,5′-cyclic adenosine monophosphorothioate (Rp-cAMP), at the indicated concentrations for 1 hour before addition of db-cAMP. After 1 hour, cells were cultured in medium alone or stimulated with heat-killed M. tuberculosis for 48 hours, and IFN-γ levels in the cell culture supernatants were measured by ELISA. * P ≤ .05, compared to PBMC treated with M. tuberculosis and db-cAMP. IFN-γ levels in M. tuberculosis-stimulated cells (None) and cells stimulated with M. tuberculosis in the presence of both db-cAMP and 500 µM Rp-cAMP were not significantly different (P = .14). Means and standard errors are shown.

cAMP Inhibits M. tuberculosis-induced IFN-γ Production Through PKA Type I

Intracellular cAMP mediates its downstream effects either by acting through PKA or through a family of guanosine nucleotide exchange factors called Epacs (exchange proteins activated directly by cAMP) [19]. To determine whether cAMP reduces antigen-stimulated T-cell IFN-γ production through the PKA or Epac pathway, we treated PBMC with cAMP analogs that were specific for activation of PKA (6-Benz-cAMP) or Epac (8-pCPT-cAMP) [20]. 6-Benz-cAMP reduced IFN-γ levels in a dose-dependent manner that was comparable to that seen with db-cAMP treatment, whereas 8-pCPT-cAMP had no effect (data not shown). These results indicate that PKA rather than the Epac pathway is used by cAMP for its inhibition of IFN-γ.

PKA is a holoenzyme that has 2 major isoforms, PKA type I (PKA RI) and PKA type II (PKA RII). Each isoform consists of 2 regulatory (R) and 2 catalytic (C) subunits. The R subunits inhibits the C subunits by direct interaction. The R subunit has 2 cAMP binding sites, referred to as sites A and B. The binding of the R subunits with cAMP causes conformational changes of the R subunit that lead to release and activation of the C subunits [21, 22]. To identify the PKA isoform that mediates the inhibitory effects of cAMP, we used 8-HA-cAMP, which binds only to the B site of PKA RI, and 8-PIP-cAMP, which binds to the A site of PKA RI and the B site of PKA RII. Although these analogs are relatively specific for PKA RI and PKA RII, respectively, pairing them with 6-Benz-cAMP, which binds to site A of both PKA RI and PKA RII, allows binding to both sites of the R subunits, thus having more potent effects. We applied 6-Benz-cAMP and 8-HA-cAMP to selectively activate both A and B sites of PKA RI and 6-Benz-cAMP and 8-PIP-cAMP to target A and B sites of PKA RII, a strategy applied by others to identify the role of PKA isoforms in immune cell signaling [21].

The PKA RI-specific 8-HA cAMP reduced M. tuberculosis-induced IFN-γ production by approximately 60%, and the combination of 8-HA-cAMP and 6-Benz-cAMP decreased IFN-γ by 80%–85%, which is comparable to the reduced level obtained with db-cAMP (Figure 4) [21]. In contrast, activation of PKA RII with 8-PIP-cAMP did not affect IFN-γ production, and the combination of 8-PIP-cAMP and 6-Benz-cAMP reduced IFN-γ production by up to 50% (P < .05, compared to 8-HA-cAMP and100 µM 6-Benz-cAMP; Figure 4). Together, these results suggest that cAMP inhibits M. tuberculosis-induced IFN-γ production by PBMC predominantly through PKA RI.

Figure 4.

Figure 4.

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Cyclic adenosine monophosphate (cAMP) inhibited Mycobacterium tuberculosis (M. tb)-induced interferon-gamma (IFN-γ) production primarily through protein kinase A (PKA) RI. Peripheral blood mononuclear cells (PBMC) from 6 latent-tuberculosis–infected donors were cultured with medium alone or with heat-killed M. tuberculosis in the presence or absence of db-cAMP (0 µM) or 8-HA-cAMP (100 µM) or 8-PIP-cAMP (250 µM), or combination of 6-Benz-cAMP (at 50 or 100 µM) with either 8-HA-cAMP or 8-PIP-cAMP. IFN-γ levels in 48-hour culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Means and SE are shown. Values for cells treated with M. tuberculosis and db-cAMP versus cells treated with M. tuberculosis and 8-HA-cAMP combined with 50 or 100 µM 6-Benz-cAMP were not significantly different (P = .6 and .36, respectively). Differences between cells treated with M. tuberculosis with db-cAMP versus M. tuberculosis with 8-PIP-cAMP together with 50 or 100 µM 6-Benz-cAMP were of borderline significance (P = .03 and .06, respectively). ** P < .05, compared to the cells treated with M. tuberculosis in the presence of 8-HA-cAMP with 6-Benz-cAMP. Table 1 shows the respective PKA binding sites of the different cAMP analogs and the combination of analogs [21]. Abbreviations: db-cAMP, N6,2′-O-dibutyryladenosine-3′,5′-cAMP sodium; 6-Bnz, N6-benzoyladenosine-3′,5′-cAMP; 8-HA, 8-hexylaminoadenosine-3′,5′-cAMP; 8-PIP, 8-piperidino-adenosine-3′,5′-cAMP.

Table 1.

Cyclic Adenosine Monophosphate (cAMP) Analogs With Their Protein Kinase A (PKA) Targets

Treatment PKA Binding Sites
8-HA-cAMP RI B
8-PIP-cAMP RI A/RII B
6-Benz/8-HA-cAMP RI A + B
6-Benz/8-PIP-cAMP RII A + B
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Abbreviations: 6-Benz-cAMP, N6-benzoyladenosine-3′,5′-cAMP; 8-HA-cAMP, 8-hexylaminoadenosine-3′,5′-cAMP; 8-PIP-cAMP, 8-piperidino-adenosine-3′,5′-cAMP.

cAMP Reduces Binding of Transcription Factors to the IFN-γ Proximal Promoter Through PKA RI and Reduces Expression of miR155

To investigate the mechanisms by which cAMP inhibits IFN-γ production through PKA RI, we studied the effects of cAMP and cAMP analogs on DNA binding activities and expressions of CREB, ATF-2, and c-Jun transcription factors, which mediate selective expression of IFN-γ gene in T cells through the IFN-γ proximal promoter [14, 23]. EMSAs with the IFN-γ proximal promoter as a probe showed that stimulation of PBMC with M. tuberculosis increased binding of 2 low-mobility complexes to the IFN-γ proximal promoter and these complexes were reduced by db-cAMP or by PKA RI-selective cAMP analogs 6-Benz-cAMP and 8-HA-cAMP (Figure 5A). In contrast, PKA RII-specific 6-Benz-cAMP and 8-PIP-cAMP had minimal effect. To determine whether the reduction in DNA-binding activities is due to changes in protein expression, we performed western blotting for total protein expression of these transcription factors on nuclear extracts of M. tuberculosis-stimulated PBMC from 4 latent-tuberculosis–infected donors, with or without db-cAMP and cAMP analogs. Either db-cAMP or the PKA RI-specific combination of 6-Benz-cAMP and 8-HA-cAMP markedly reduced the expression of CREB, ATF-2, and c-Jun in PBMC stimulated with M. tuberculosis compared with that in PBMC with M. tuberculosis only (Figure 5B). The PKA RII-specific combination of 6-Benz-cAMP and 8-PIP-cAMP did not affect the expression of these transcription factors (Figure 5B). To further confirm the functional significance of suppressed CREB/ATF transcription factors in response to cAMP, we evaluated the expression of miRNA in PBMC stimulated with M. tuberculosis in the presence of db-cAMP. These transcription factors have been shown to regulate miR155 in a bacterial infection [24]. Furthermore, miR155 is critical in protective immunity against tuberculosis infection [25, 26] and has been shown to be reduced in tuberculosis patients [27]. Compared to the cells cultured with medium alone, M. tuberculosis stimulation induced elevated expression of miR155 in a time-dependent manner, and the presence of db-cAMP suppressed the expression of miR155 significantly (Figure 5C), confirming that cAMP leads to deficient CREB/ATF function. We conclude that cAMP acts through PKA RI to downregulate the expression of the transcription factors that regulate IFN-γ and mR155, which are critical molecules in protective immunity against tuberculosis infection.

Figure 5.

Figure 5.

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Effects of cyclic adenosine monophosphate (cAMP) and cAMP analogs on transcription factors that bind to the interferon-gamma (IFN-γ) proximal promoter. A, Peripheral blood mononuclear cells (PBMC) from 5 latent-tuberculosis–infected donors were cultured with medium or 2 µg/mL heat-killed Mycobacterium tuberculosis (M. tb) (None), or M. tuberculosis with 40 µM cAMP or M. tuberculosis with 100 µM 8-HA-cAMP and 50 µM 6-Benz-cAMP, or M. tuberculosis with 100 µM 8-PIP and 50 µM 6-Benz-cAMP. After 48 hours, nuclear protein extracts of PBMC were prepared, and electrophoretic mobility shift assay was performed on the nuclear protein extracts with a radiolabeled IFN-γ proximal promoter as probe. The arrows indicate complexes that bind to the IFN-γ proximal promoter. A representative result is shown. B, PBMC from 5 latent-tuberculosis–infected donors were treated as in (A) for 48 hours. Total cell protein extracts of PBMC were prepared and expression of the indicated proteins were determined by western blotting with different antibodies after stripping. A representative result is shown. The numbers indicate the β-actin–normalized densities of the bands of the transcription factors in arbitrary units. C, PBMC from 7 latent-tuberculosis–infected donors were cultured with medium or M. tuberculosis or M. tuberculosis plus cAMP for 24 and 48 hours. The expression of miR155 in total RNA of PBMC was determined by quantitative polymerase chain reaction after normalization for U6 miRNA. Levels of expression is shown as fold change over cells with medium only. Mean values and SE are shown. Abbreviations: db-cAMP, N6,2′-O-dibutyryladenosine-3′,5′-cAMP sodium; 6-Bnz, N6-benzoyladenosine-3′,5′-cAMP; HA, 8-hexylaminoadenosine-3′,5′-cAMP; PIP, 8-piperidino-adenosine-3′,5′-cAMP; CREB, cAMP response element binding protein; ATF-2, activating transcription factor.

Neutralizing Both IL-10 and TGF-β1 or Supplementation of IL-12 Restores cAMP-Induced Inhibition of IFN-γ Production

Interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-β1) play significant roles in suppression of M. tuberculosis-stimulated IFN-γ production by PBMC, and elevated production of these cytokines is associated with active tuberculosis infection [28]. To examine the significance of these cytokines in cAMP-mediated suppression of M. tuberculosis-stimulated IFN-γ production, we measured IL-10 production by PBMC stimulated with M. tuberculosis in the presence of db-cAMP and tested the effects of neutralizing both IL-10 and TGF-β1 on cAMP-mediated suppression of IFN-γ production. Neutralizing these cytokines individually restored IFN-γ production partially, and neutralizing both IL-10 and TGF-β1simultaneously almost completely reversed db-cAMP-mediated inhibition of IFN-γ production (Figure 6A). These results are consistent with the increased IL-10 production by PBMC from latent-tuberculosis–infected donors stimulated with M. tuberculosis in the presence of db-cAMP (data not shown) and a predicted increase in TGF-β1 levels (though we did not succeed in demonstrating consistently elevated TGF-β1 production in the same setting). IL-10 and TGF-β1 have been shown to inhibit IL-12 production by antigen presenting cells (APC) [29–31], and IL-12 is required for T-cell IFN-γ production in response to antigenic stimulation. Increased cAMP inhibits IL-12 production by APC in other infection [32]. Consistent with this, IL-12 added to the cultures of PBMC stimulated with M. tuberculosis restored db-cAMP-suppressed IFN-γ production in a dose-dependent manner (Figure 6B). This suggests that T cells are responsive to IL-12 signaling despite elevated intracellular db-cAMP and that elevated cAMP may suppress IFN-γ production, probably through affecting the cytokine production profile of APC. Thus, we conclude that increased cAMP may lead to reduced T-cell IFN-γ production by elevated IL-10 and TGF-β1 production at least in part by suppression of IL-12 production.

Figure 6.

Figure 6.

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Neutralization of both interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-β1) or supplementation of IL-12 restores cyclic adenosine monophosphate (cAMP) suppression of interferon-gamma (IFN-γ) production. A, Peripheral blood mononuclear cells (PBMC) from 3 latent-tuberculosis–infected donors were incubated with medium or Mycobacterium tuberculosis (M. tb) (None) or M. tuberculosis with N6,2′-O-dibutyryladenosine-3′,5′-cAMP sodium (db-cAMP), or anti-IL-10 with db-cAMP and M. tuberculosis or anti-TGF-β1 with db-cAMP and M. tuberculosis, or both anti-IL-10 and anti-TGFβ1 with db-cAMP and M. tuberculosis for 48 hours. IFN-γ production in the culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). Mean and SE of 3 experiments are shown. * P < .05, ** P < .005, and *** P < .001 compared to the cells treated with M. tuberculosis in the presence of db-cAMP. B, PBMC from 3 latent-tuberculosis–infected donors were cultured in medium alone, or with M. tuberculosis, or M. tuberculosis with 50 µM db-cAMP or indicated concentrations of IL-12 with cAMP and M. tuberculosis. IFN-γ production in the 48-hour culture supernatants was measured by ELISA. Mean and SE of 3 experiments are shown. * P < .05 compared to the cells treated with M. tuberculosis in the presence of db-cAMP.

DISCUSSION

Previous studies demonstrated that M. tuberculosis-stimulated IFN-γ production by PBMC from tuberculosis patients is reduced compared to that from latent-tuberculosis–infected donors and this reduction in IFN-γ production correlates with disease severity [3, 4]. However, the mechanisms of reduced IFN-γ production remain undetermined. This study provides evidence that increased cAMP in PBMC from tuberculosis patients potentially contributes to the reduction in IFN-γ production. In fact, increased cAMP correlated with reduced M. tuberculosis-stimulated IFN-γ production in tuberculosis patients, and the addition of synthetic cAMP analogs or cAMP-inducing agent inhibited IFN-γ production at both transcript and protein levels. This is probably due to reduced DNA binding activities and the expression of CREB, ATF-2, and c-Jun transcription factors in PBMC from tuberculosis patients, consistent with our previous findings [13–15]. Reduced expression and functional deficiency of these transcription factors have been confirmed with reduced expression of miR155, a target microRNA of CREB/ATF transcription factors, in PBMC from latent-tuberculosis–infected donors stimulated with M. tuberculosis in the presence of db-cAMP, providing a potential mechanism for reduced miR155 expression in patients with active tuberculosis [27]. A combination of PKA type I, but not type II, specific cAMP analogs inhibited M. tuberculosis-stimulated IFN-γ production. Neutralization of both IL-10 and TGF-β1 or supplementation of IL-12 restored cAMP-suppressed IFN-γ production by PBMC. Thus, the present findings provide evidence for the potential roles of elevated cellular cAMP in suppression of Th1 immune responses through the reduced expression of the transcription factors that regulate IFN-γ expression in tuberculosis infection. Thus, our results suggest that elevated intracellular cAMP levels in PBMC from tuberculosis patients inhibited the expression of these transcription factors and their targets genes, such as IFN-γ and miR155. This study also provides an experimental system for future detailed studies to delineate the molecular mechanism of this regulatory pathway in tuberculosis infection. Consistent with this line of argument, previous studies have demonstrated that M. tuberculosis initiates signaling pathways to generate bacterial cAMP in response to environmental cues, which leads to elevated secretion of cAMP into the infected macrophages, thereby intoxicating macrophages and regulating secretion of inflammatory cytokines during live infection [10, 11, 33]. Although we cannot directly examine the significance of this mechanism in human studies, it is possible that APC in the lungs of active tuberculosis patients may be intoxicated by M. tuberculosis-derived cAMP after infection and these intoxicated macrophages may even enter the circulation.

Elevation of cAMP in T cells inhibits T-cell function by several mechanisms, including targeting PKA RI to the TCR-CD3 complex during T-cell activation [34], and activation of PKA RI by cAMP for suppression of T-cell proliferation [35]. Moreover, increased expression of PKA RI enhances sensitivity of T cells to cAMP-mediated suppression of T cells [36]. Though we have not explored the potential roles of this mechanism, our data are consistent with PKA-dependent inhibition of T-cell activation and cytokine production by cAMP. However, the effect of cAMP on APC may play a major role in this system, as neutralization of IL-10 and TGF-β1, produced predominantly by APC in this experimental condition, restored cAMP-suppressed IFN-γ production. Furthermore, we found out that T cells are responsive to IL-12 in the presence of db-cAMP. This contradicts previous studies, which showed that increased cAMP in T cells induced by vasoactive intestinal peptide inhibits Th1 immune responses through suppression of IL-12 signaling [37]. Therefore, the current study favors the role of cAMP in inhibition of T-cell IFN-γ production by suppression of IL-12 through elevated IL-10 and TGF-β1 production by APC in tuberculosis infection. Interestingly, it was shown that M. tuberculosis intoxicates infected macrophages through increased intracellular cAMP [10]. The studies from other bacterial infections also support the significance of this mechanism. Bordetella pertussis, a gram-negative bacteria, induces elevated intracellular cAMP through its adenylate cyclase toxin and inhibits IL-12 by suppression of IL-12 p35 and increased IL-10 [32, 38]. Others have also shown that cAMP/PKA stimulates production of TGF-β and IL-10 [31, 39, 40], and thereby inhibits IL-12 production [41]. However, further delineation of this mechanism requires studies to determine the cell-specific cAMP levels and their effects on functions of APC and T cells in live tuberculosis infection.

The mechanisms causing reduced expression and decreased DNA-binding activities of CREB, ATF-2, and c-Jun transcription factors in PBMC from tuberculosis patients remain unclear. Based on the literature, we propose that elevated intracellular cAMP activates PKA and this may lead to constitutive activation of these transcription factors, thus resulting in their degradation by cellular protein degradation mechanism. However, this requires further studies with a focus on the effect of APC cytokines on the mRNA expression and the protein half-life of these transcription factors.

Interestingly, cAMP has been shown to be one of the effector molecules of regulatory T cells (Tregs) for suppression of effector memory T cells by direct injection of cAMP into T cells [42]. Although we have not explored the potential significance of this mechanism in this study, chronic elevation of cAMP in tuberculosis patients may contribute to the development of Tregs and therefore inhibition of host Th1 immune responses, as elevated Tregs are evident in tuberculosis patients [43].

Abnormalities in the cAMP-PKA axis are associated with several other diseases with abnormal T-cell immune responses, such as AIDS [44], systemic lupus erythematosus [45], and sepsis [46]. Thus, these together with our findings suggest that abnormal activation of cAMP/PKA pathway may play a critical role in development of both infectious and noninfectious diseases associated with Th1 immune responses. In conclusion, our findings provide a potential role for elevated intracellular cAMP in reduced Th1 immune responses and targeting this pathway may provide a novel strategy for host-directed therapy to boost the protective immunity, especially in those patients infected with drug-resistant M. tuberculosis.

Acknowledgment. We thank Dr. Amy Tvinnereim for critical reading of the manuscript and helpful discussions.

Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Financial support. This work was supported in part by the funds from the National Institutes of Health (grant number 1R56AI116864) and the University of Texas Health Science Center at Tyler, Texas.

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