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The Journal of Immunology, 2007, 178: 5192-5199.
Copyright © 2007 by The American Association of Immunologists, Inc.

Disparity in IL-12 Release in Dendritic Cells and Macrophages in Response to Mycobacterium tuberculosis Is Due to Use of Distinct TLRs1

Luca Pompei2,*, Sihyug Jang2,*, Beata Zamlynny*, Sharada Ravikumar{dagger}, Amanda McBride*, Somia Perdow Hickman{ddagger} and Padmini Salgame3,*

* Department of Medicine, University of Medicine and Dentistry of New Jersey, Newark, NJ 07101; {dagger} Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140; and {ddagger} Department of Medicine, University of Pennsylvania, Philadelphia, PA 19410


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The control of IL-12 production from dendritic cells (DCs) and macrophages in response to Mycobacterium tuberculosis (Mtb) is not well understood. The objective of this study was to pursue the mechanism underlying our previous report that in response to Mtb infection, DCs release abundant IL-12, whereas secretion is limited in macrophages. An initial comparison of IL-12p35 and IL-12p40 gene induction showed that p35 transcription is similar in murine bone marrow-derived DCs and macrophages, but a rapid and enhanced IL-12p40 transcription occurs only in DCs. Consistent with the p40 gene transcription profile, Mtb-induced remodeling at nucleosome 1 of the p40 promoter also occurs rapidly and extensively in DCs in comparison to macrophages. Removal of IL-10 or addition of IFN{gamma} enhances macrophage IL-12 release to Mtb, but without affecting the kinetics of remodeling at the macrophage p40 promoter. Furthermore, we show that Mtb-induced remodeling at the p40 promoter and IL-12 release in DCs is TLR9 dependent, and in contrast, TLR2 dependent, in macrophages. Data are also presented to demonstrate that a TLR9 agonist induces quantitatively more extensive remodeling at the IL-12p40 promoter and larger IL-12 release in comparison to a TLR2 agonist. Collectively, these findings suggest that DCs and macrophages handle Mtb differently resulting in only DCs being able to engage the more efficient TLR9 pathway for IL-12 gene induction. Our results also imply that TLR2 signaling is not a good inducer of IL-12, supporting the increasingly strong paradigm that TLR2 favors Th2 responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The production of IL-12 is essential for the development of host Th1 immunity and resistance against intracellular pathogens (1). The bioactive IL-12p70 is a heterodimeric protein consisting of covalently linked p40 and p35 subunits, both of which are regulated independently (2). Substantial progress has been made toward understanding the regulation of IL-12p40 transcription. In the proximal region of the IL-12p40 promoter, several transcriptional regulatory elements have been identified, of which the sites binding C/EBP (3) and Rel and AP1 family members contribute significantly to gene induction (4). Deletion or mutation of these sites results in significantly reduced IL-12p40 production. Additionally, a binding site for the IFN consensus sequence-binding protein and the NFAT complex was identified to be important for promoter activity in response to LPS and IFN-{gamma} (5). The genomic DNA in the nucleus complexes with nucleosomes to form a higher order dynamic structure: the chromatin (6). Thus, a foremost control point in regulating the expression of inducible genes is reorganization of the higher order chromatin structure to reveal the DNA binding sites to the transcriptional machinery. Indeed, the endogenous p40 promoter is assembled in four tightly positioned nucleosomes: a positioned nucleosome upstream of the start site spanning the promoter region and three positioned nucleosomes further upstream. Upon activation with LPS, nucleosome 1 encompassing the region from nucleotide –197 to nucleotide –20, which includes the critical sites for Rel and C/EBP binding, was shown to be rapidly and selectively remodeled in a TLR-dependent and c-Rel and IFN-{gamma}-independent manner (7, 8).

Mortality and morbidity caused by the pulmonary pathogen Mycobacterium tuberculosis (Mtb)4 remain alarmingly high. Although much is known about the immunology of tuberculosis, the precise nature of the protective immune mechanisms against the tubercle bacillus has not been completely defined. Nevertheless, existing evidence suggests that IL-12-initiated cellular Th1-mediated immunity plays a critical role in host defense against Mtb (9). Previously, we reported that dendritic cells (DCs), but not macrophages, rapidly synthesized IL-12 in response to Mtb (10), and therefore, the goal of the present study was to understand the molecular basis for the differential IL-12 induction from these two cell types. In this study, we present evidence that the interaction of Mtb with TLRs on DCs and macrophages is distinct and this results in differences in modifications on the chromatin environment at the IL-12p40 locus and subsequent IL-12 release.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and reagents

C57BL/6 mice and the IL-10 knockout (KO) mice were purchased from The Jackson Laboratory. TLR2KO (11), MyD88KO (12), and TLR9KO (13) mice were developed by S. Akira (Osaka University, Osaka, Japan) and were obtained via H. Shen (University of Pennsylvania, Philadelphia, PA), S. Leibovich (University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School, Newark, NJ), A. Sher (National Institutes of Health, Bethesda, MD), respectively. The TLR2/9 double KO (DKO) mice (14) were generated by A. Bafica and A. Sher (National Institutes of Health, Bethesda, MD). TLR2–/– mice were bred at our animal facility at the UMDNJ-New Jersey Medical School. Bones were provided to us from the MyD88KO, TLR9KO, and TLR2/9DKO mice. IL-12 promoter plasmid was a gift of Dr. X. Ma (Weill Medical College, Cornell University, New York, NY). All animal studies were approved by the Institutional Review Board. The virulent M. tuberculosis Erdman strain (Trudeau Institute, Saranac Lake, NY) was prepared as described previously (10). The irradiated H37Rv strain was obtained from the Colorado State University (Fort Collins, CO) under the National Institutes of Health, National Institute of Allergy and Infectious Diseases contract NO1-AI-40091, "Tuberculosis Vaccine Testing and Research Materials Contract." The GM-CSF-secreting cell line was a gift of Dr. B. Stockinger (National Institute of Medical Research, London, U.K.). CpG 1826 and Pam3Csk4 were obtained from InvivoGen.

Macrophage and DC preparation, RNA purification, cDNA synthesis, and real-time PCR

Bone marrow-derived macrophages and DCs were prepared as described previously (10, 15). Total RNA was purified using TRIzol (Invitrogen Life Technologies), precipitated in isopropyl alcohol, and dissolved in diethyl pyrocarbonate water. Superscript II and oligo(dT) (Invitrogen Life Technologies) were used for cDNA synthesis. Transcripts were quantified by real-time PCR on an Mx3000p thermal cycler (Stratagene) using the fluorophore SYBR Green. mRNA abundance of each gene of interest was normalized to the amount of beta-actin.

Detection of activation of IL-12p40 reporter construct and endogenous IL-12p40

The IL-12p40 promoter-YFP reporter construct was generated by cloning the 3.3-kb PstI fragment of the human IL-12p40 promoter (pXP2; Ref. 16) into the XhoI cloning site of the pmaxFP-Yellow-PRL vector (Amaxa Biosystems). Transient transfection of DCs and macrophages was performed with the Nucleofector and the Amaxa Human Dendritic Cell Nucleofector kit I (Amaxa Biosystems) according to the manufacturer’s protocol with modifications. DCs and macrophages were resuspended in 100 µl of human DC nucleofector solution and electroporated at room temperature with 7 µg of the IL-12p40 promoter-YFP reporter construct using the U-02 program. Immediately after transfection, the cells were transferred to a 60-mm plate. One hour after transfection, the cells were stimulated with 300 µg/ml irradiated Mtb for 24 h. To evaluate electroporation efficiency, both cell types were also transfected with a CMV promoter-driven GFP-expression vector. Flow cytometric analysis showed that 40–50% of the electroporated DCs and macrophages were GFP+ (data not shown).

For intracytoplasmic staining of endogenous IL-12p40, DCs and macrophages were stimulated with 300 µg/ml irradiated Mtb for 24 h and then treated with 2 µM monensin for 3 h, harvested, washed in PBS, and fixed in 4% paraformaldehyde at room temperature. After 15 min, the cells were washed once with PBS and once with permeabilization buffer (0.1% saponin, 1% FBS in PBS) and incubated with 2 µg/ml PE-labeled IL-12p40/70 reactive Ab (BD Pharmingen) in 100 µl of permeabilization buffer for 30 min on ice in the dark. After two saponin solution washings and a subsequent PBS washing, the cells were resuspended in FACS buffer.

The labeled cells for endogenous IL-12p40 and transfected cells with the IL-12p40 promoter-YFP reporter construct were analyzed by flow cytometry in a FACSCalibur instrument (BD Biosciences) equipped with the CellQuest software. YFP expression in cells indicates activation of the exogenous IL-12p40 reporter construct and PE staining indicates activation of the endogenous IL-12p40 promoter.

Detection of NF-{kappa}B translocation by Western blotting

Cytoplasmic and nuclear extracts were prepared from DCs and macrophages that were exposed for 4 h to Mtb. Cytosol and nuclei-containing cell extracts were separated by incubating cells in 200 µl of hypotonic buffer (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 5 mM DTT, 5 mM PMSF, 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 1% Nonidet P-40) for 15 min on ice. To separate the cytosol-containing fraction form the nuclei-containing pellet, lysates were centrifuged at 12,000 x g for 30 s at 4°C in a microfuge, and the supernatant obtained was designated the cytosolic fraction. The pellet was resuspended in 100 µl of hypertonic buffer (20 mM HEPES (pH 7.9); 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM DTT; 1 mM PMSF; leupeptin (5 mg/ml) and aprotinin (5 mg/ml)). The tubes were vigorously rocked on a shaking platform at 4°C for 15 min and then recentrifuged at 12,000 x g for 30 s at 4°C, and the supernatant was nuclear extract, which was aliquoted and stored at –80°C for further use. Twenty-five micrograms of cytoplasmic and nuclear extract from each sample was resolved on 12% SDS-PAGE and transferred to nitrocellulose membrane (Amersham Biosciences). The blots were probed sequentially with anti-p65 and anti-c-Rel Abs (Santa Cruz Biotechnology). The membranes were washed and reacted with HRP-conjugated donkey anti-rabbit IgG Ab (Amersham Biosciences). Blots were developed using the ECL Plus system (Amersham Biosciences).

Quantifying remodeling by chromatin accessibility (ChART)

DCs and macrophages were plated at a density of 2 x 106 cells/plate and left untreated or treated with irradiated H37Rv strain (300 µg/ml). At the indicated time point, cells were scraped from the plate and washed with ice-cold PBS containing spermine (0.15 mM) and spermidine (0.50 mM). Cells were then lysed in 1 ml of lysis buffer (10 mM NaCl, 10 mM Tris (pH 7.4), 3 mM MgCl2, 0.5% Nonidet P-40, 0.15 mM spermine, 0.5 mM spermidine) for 10 min, washed in ice-cold PBS supplemented with spermine and spermidine. Chromatin was digested with MseI (50 U) or ApoI (20 U) at 37°C for 45 min and then left overnight in the presence of 60 µg/sample of proteinase K. Digested DNA was purified by phenol/chloroform extraction and ethanol precipitation, dissolved in diethyl pyrocarbonate water, and diluted at the concentration of 5 ng/µl. Ten nanograms of template was used for real-time PCR in a total volume of 25 µl/well using the hot-start ampliTaq gold polymerase (Applied Biosystems) and SYBR Green technology. The remodeling at the IL-12p40 and TNF promoters was measured using primer set 1 and 3 respectively each spanning the restriction sites for MseI (p40) or ApoI (TNF) as described in Fig. 4A. Results were normalized to the cycle threshold (Ct) values obtained using primer set 2, which does not span the MseI or ApoI sites. {Delta}{Delta}Ct values were used to calculate the percentage of remodeling using the following formula: percentage of remodeling was calculated as ({Delta}{Delta}Ct(untreated) – {Delta}{Delta}Ct(exp))/{Delta}{Delta}Ct(untreated)) x 100; {Delta}{Delta}Ct = 2(Ct calibrator – Ct exp)/2(Ct calibrator – Ct exp), where the nominator refers to the Ct values of the locus of interest and the denominator to the Ct values of the normalizer.


Figure 4
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  		FIGURE 4. Mtb induces differential nucleosome 1 remodeling at the IL-12p40 promoter in macrophages and DCs. A, Schematic representation of IL-12p40 promoter indicating the position of the primers and the restriction enzymes used to determine chromatin remodeling by ChART-PCR. To determine IL-12p40 promoter remodeling, genomic DNA was amplified with primer set 1 that spans the remodeling region, and with primer set 2 that spans a region outside of nucleosome 1 and which is not remodeled following stimulation. The primer set 2 was used to normalize for input DNA. The Ct values obtained for both sets of primers and the calibrator (unstimulated cells) were used to calculate and plot the data as the percent remodeling. TNF promoter remodeling was quantitated using primer sets 3 and 2. B, Quantitation of remodeling at the IL-12p40 promoter in WT and MyD88 KO DCs and macrophages in response to Mtb. C, Remodeling at the TNF promoter in WT cells in response to Mtb. For all experiments, data are presented as mean ± SD of at least three independent experiments.

 
The following are the sequences of the primers used in the ChART-PCR assay: set 1 (IL-12p40 promoter): forward 5'-TAGATGCACTCAGGGAGGCAA-3', reverse 5'-TGGAAACCCAAAGTAGAAACTGACT-3'; set 2 (normalizer): forward 5'-CTTTGGGTTTCCATCAGAAAGTTC-3', reverse 5'-CCTGGTCTGATGTGCCTGG-3'; set 3 (TNF promoter): forward 5'-GTCTGAGTTGATGTACCGCAGT-3', reverse 5'-AGGCA GAGCAGCTTGAGAGTT-3'.

For every experiment, a control sample was also included, where cells were treated with Mtb but not subjected to MseI or ApoI digestion. The DNA from these cells was cut with EcoRI and then amplified with primer sets 1 and 2 or 3 and 2. In all cases, the Ct value for these control samples was similar to the untreated samples digested with MseI or ApoI.

Cytokine ELISA

The presence of cytokines in the supernatants was determined by sandwich ELISA using the following Ab pairs from BD Pharmingen: C15.6 and C17.8 (biotinylated) for IL-12p40; JES5-2A5 and JES5-16E3 (biotinylated) for IL-10; G281-2626 and MP6-XT3 (biotinylated) for TNF-{alpha} and 9A5 and C17.8 (biotinylated) for IL-12p70.

Statistics

For statistical analysis of samples, paired and unpaired Student’s t tests and two-way ANOVA were performed (PRISM version 3.0; GraphPad) as appropriate; values of p < 0.05 were considered significant. * signifies p < 0.05; **, p < 0.005; ***, p < 0.001.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Disparity in IL-12 secretion and IL-12p40 gene transcription in DCs and macrophages in response to Mtb

We first compared IL-12p40 release in response to irradiated Mtb by DCs and macrophages derived from wild-type (WT) and MyD88-deficient mice. Consistent with our previous observation with live Mtb Erdman, irradiated Mtb also induced substantial IL-12p40 and IL-12p70 release from WT DCs and a significantly reduced amount of the same in macrophages (Fig. 1, A and B) (10). In addition, here we also show that IL-12p70 (Fig. 1A) and IL-12p40 (Fig. 1B) release is completely abrogated in the absence of MyD88 in both DCs and macrophages. A significant and equivalent induction of TNF was observed in MyD88-sufficient macrophages and DC cultures, underscoring a disparity that is specific to IL-12 (Fig. 1C). Together, these initial cytokine analyses confirm and extend our previous observations and those of others (14), and set the stage for elucidating the molecular mechanism behind the difference in IL-12 secretion from macrophages and DCs in response to Mtb. Unless specified, for most studies described here, we have used irradiated Mtb, because this allowed us to perform a large portion of the molecular work such as the Western blot and remodeling experiments outside of the biosafety level 3 facilities. However, where appropriate, we also provide data with live Mtb Erdman.


Figure 1
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  		FIGURE 1. Unequal IL-12 release by DCs and macrophages in response to Mtb. A total of 5 x 105 each of macrophages and DCs derived from the bone marrow of WT C57BL/6 (WT) and MyD88-deficient (KO) mice were treated with 300 µg/ml irradiated Mtb for 24 h. Levels of IL-12p70 (A), p40 (B), and TNF (C) release in the supernatants was determined by ELISA (20 ). Data are shown as mean ± SD of three independent experiments.

 
To begin to address the mechanism for unequal IL-12 production from DCs and macrophages, we compared by real-time RT-PCR the kinetics of IL-12p40 mRNA induction in DCs and macrophages in response to Mtb. IL-12p40 gene transcription occurred earlier and to significantly higher extent in DCs as compared with macrophages receiving the same stimulus (Fig. 2A). Interestingly, expression of IL-12p35 mRNA was similar between macrophages and DCs (Fig. 2B) indicating that impaired IL-12 release by macrophages in response to Mtb is at least in part due to impaired IL-12p40 gene transcription. The TNF mRNA expression pattern paralleled the protein profile and was similar in DCs and macrophages (Fig. 2C). There was no detectable mRNA induction for IL-12p35, IL-12p40, and TNF in response to Mtb in both DCs and macrophages deficient in MyD88.


Figure 2
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  		FIGURE 2. Impaired IL-12 release by macrophages in response to Mtb is due to impaired IL-12p40 gene transcription. Bone marrow-derived macrophages and DCs were treated with irradiated Mtb for the indicated time points and the mRNA abundance for IL-12p40 (A), IL-12p35 (B), and TNF (C) was evaluated by RT-PCR. The relative amount of the gene of interest was determined as the "{delta} Ct ({Delta}Ct)" relative to the message of the housekeeping gene beta-actin. The change in relative message levels was calculated as the "{delta}-{delta}-Ct ({Delta}{Delta}Ct)". Results are expressed as relative quantity to untreated cells and are the mean ± SD of three independent experiments. At all time points tested, the difference in IL-12p40 expression between WT DCs and macrophages was significant (p < 0.05).

 
DCs and macrophages exhibit equivalent activation of transcription factors

We next investigated whether IL-12p40 gene transcription in macrophages and DCs in response to Mtb was due to a differential accumulation and/or activation of transcription factors required for IL-12 induction. Specifically, Western blotting analysis was performed to assess whether nuclear translocation of c-Rel and p65 was impaired in macrophages. We found that both c-Rel and p65 were expressed in the nuclear extracts of stimulated macrophages and DCs (Fig. 3A), indicating that the nuclear translocation of IL-12p40 promoter-related transcription factors are not defective in macrophages.


Figure 3
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  		FIGURE 3. DCs and macrophages exhibit equivalent activation of transcription factors. A, Cytoplasmic (CE) and nuclear extracts (NE) were prepared following Mtb stimulation. The extracts were normalized for protein concentrations, and 25 µg of protein separated by SDS-PAGE, and analyzed by Western blotting using Abs reactive against p65 and c-Rel. Data presented are representative of three individual experiments. B, A total of 5 x 106 DCs and macrophages were transiently transfected with IL-12p40 promoter-YFP reporter construct. One hour after transfection, cells were stimulated with 300 µg/ml irradiated Mtb for 24 h. The induction of YFP expression as a readout of exogenous IL-12p40 promoter was analyzed by flow cytometry in a FACSCalibur instrument. For intracytoplasmic staining of endogenous IL-12p40, DCs and macrophages were stimulated with 300 µg/ml irradiated Mtb for 24 h and fixed and permeabilized cells were reacted with PE-labeled IL-12p40/70 Ab and analyzed by flow cytometry in a FACSCalibur instrument (BD Biosciences) equipped with the CellQuest software. Results are shown as the mean percentage of YFP+ (level of exogenous promoter activation) and PE+ cells (level of endogenous promoter activation) ± SD of four independent experiments.

 
Because remodeling of the positioned nucleosome 1 in the p40 locus is essential for gene transcription, we hypothesized that IL-12p40 promoter-related transcription factors accumulate in the nucleus of macrophages but cannot access the endogenous p40 promoter locus due to a deficiency in remodeling. To address this, we comparatively examined activation of the endogenous IL-12p40 promoter by IL-12p40/p70 intracytoplasmic staining and activation of the exogenous IL-12p40 promoter-YFP reporter plasmid where YFP expression is the read out for the activation of the promoter. Activation of the endogenous IL-12p40 promoter resulted in 30 ± 2.5% of DCs expressing IL-12, while only 6 ± 0.8% of macrophages produce this cytokine following Mtb stimulation (Fig. 3B). In contrast, the percentage of YFP+ cells was similar in DCs and macrophages (28 ± 1.8 and 28 ± 1.6%, respectively; Fig. 3B) indicating that Mtb induces equivalent activation of the transcription factors involved in IL-12 transcription and that the defect in macrophages may involve a deficiency in p40 remodeling.

Mtb induces differential nucleosome remodeling at the IL-12p40 promoter in macrophages and DCs

Chromatin accessibility (ChART)-PCR is a recently developed quantitative assay to evaluate nucleosome remodeling (17). The ChART has been used previously to quantitatively evaluate nucleosome 1 remodeling at the IL-12p40 promoter (18). We next performed ChART-PCR on genomic DNA obtained from macrophages and DCs before and after Mtb stimulation to determine the status of nucleosome 1 remodeling. The details of the assay, including primer design, normalization of input DNA and the calculation for percent remodeling are described in Materials and Methods. Briefly, in unstimulated cells, the chromatin is condensed and is not accessible to restriction enzyme digestion at the positioned nucleosome resulting in the ability of this region to be amplified with the appropriate primers. If stimulation leads to remodeling of nucleosome 1, then the restriction enzyme has access to the promoter, DNA is cleaved and little or no PCR product is produced following amplification (Fig. 4A). As shown in Fig. 4B, a significant difference in the kinetics and extent of remodeling was present between DCs and macrophages at all time points tested. For example, as early as 60 min following stimulation, DCs had substantially remodeled the p40 promoter in comparison to macrophages, and this effect was still apparent after 180 min of stimulation. Moreover, maximal remodeling in macrophages never attained the levels detected in DCs because remodeling in both cell types plateaued at 360 min following stimulation. These findings indicate that for significant gene transcription to occur, a threshold level of remodeling needs to take place. The interaction of Mtb with macrophages initiates signaling that does not reach this required level, and gene transcription is minimal. In agreement with the equivalent TNF secretion profile of DCs and macrophages following Mtb activation, the NF-{kappa}B binding site in the TNF promoter was protected equally in the two cell types (Fig. 4C). Assessment of remodeling following Mtb stimulation in MyD88 KO DCs and macrophages showed that in the absence of TLR signaling, the p40 promoter remains inaccessible in both cell types (Fig. 4B).

Remodeling at the macrophage p40 promoter is not altered under conditions that enhance IL-12 release from macrophages

Previously, we had reported that Mtb stimulation of macrophages in the presence of IFN-{gamma} significantly enhanced its ability to release IL-12, and this was associated with a concomitant decrease in IL-10 secretion (10). We reasoned that perhaps IL-10 was hampering remodeling at the p40 promoter in Mtb-stimulated macrophages. To test this, we examined remodeling in WT macrophages in the presence and absence of IFN-{gamma} and in macrophages derived from IL-10KO mice. As expected, addition of IFN-{gamma} to macrophages during Mtb stimulation significantly enhanced IL-12p40 production (Fig. 5A) and abrogated IL-10 release (Fig. 5B). In addition, there was high IL-12p40 release from the IL-10KO macrophages as compared with WT cells (Fig. 5A). However, the increased IL-12p40 release was not associated with enhanced remodeling in these cells (Fig. 5C). Thus, conditions that promoted IL-12 release from macrophages did not appear to operate at the level of p40 promoter remodeling. In fact, IL-10 signaling has been reported to inhibit events that are required for RNA polymerase II recruitment to the p40 promoter (19), a step downstream of remodeling. It is conceivable that in macrophages, remodeling is perhaps intrinsically slow in response to Mtb, which offers IL-10 a window of opportunity to inhibit IL-12p40 gene transcription by functioning downstream of p40 locus decondensation and nucleosome 1 remodeling.


Figure 5
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  		FIGURE 5. Lack of IL-10 or the addition of IFN-{gamma} does not enhance nucleosome 1 remodeling at the IL-12p40 promoter in macrophages. Mtb was added to WT macrophages that had been either untreated or pretreated for 1 h with IFN-{gamma} (100 U/ml) and to IL-10–/– macrophages. IL-12p40 (A) and IL-10 (B) release was detected in the supernatants of the stimulated cells by ELISA. Remodeling at the IL-12p40 promoter (C) was quantitated at 180 min following stimulation by methodology described in Fig. 4A.

 
The differential modulation at the p40 promoter of DCs and macrophages is due to the use of different TLRs for IL-12 production

Why is the macrophage p40 promoter remodeling kinetics slow? Albrecht et al. (18) have reported that the extent of remodeling at the IL-12p40 promoter and subsequent IL-12 gene induction can vary between different TLR agonists. Given also that IL-12p40 promoter remodeling is MyD88 dependent in both DCs and macrophages, we thought that a likely possibility for the disparity in remodeling kinetics and subsequent IL-12 release in DCs and macrophages could be that the two cell types use different TLRs for IL-12 gene induction.

To test this possibility, we examined IL-12 release from DCs and macrophages derived from mice singly deficient in either TLR2 or TLR9, or double-deficient in TLR2 and TLR9 (TLR2/9DKO). Consistent with our previous observation (20), lack of TLR2 did not abrogate IL-12p70 (Fig. 6A) or IL-12p40 (Fig. 6B) release from DCs to Mtb stimulation. In contrast to what was observed with DCs, macrophage IL-12p70 and IL-12p40 release was predominantly TLR2 dependent. IL-12p70 and IL-12p40 release from TLR9KO DCs was significantly abrogated while remaining intact in TLR9KO macrophages. No IL-12 was detected in either TLR2/9 DKO macrophages or DCs.


Figure 6
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  		FIGURE 6. Production of IL-12 from DCs and macrophages is dependent on activation of different TLRs. DCs and macrophages were derived from the bone marrow of WT C57BL/6 mice, TLR2KO, TLR9KO, and TLR2/9DKO mice on the same background. A total of 5 x 106 cells were treated with 300 µg/ml irradiated Mtb for 24 h and supernatants harvested and analyzed for the presence of IL-12p70 (A) and IL-12p40 (B) by ELISA. For all experiments, data are presented as mean ± SD of at least three independent experiments.

 
Next, we examined whether the differential dependency on TLR for cytokine production by DCs and macrophages could be recapitulated when live Mtb Erdman was used. In this experiment, TLR usage for IL-12p70 production in macrophages was determined in the presence of IFN-{gamma}, because we have reported previously that macrophages release very low levels of IL-12p70 in response to Mtb infection, unless cocultured with IFN-{gamma}. IL-12p70 release from DCs in response to live Mtb Erdman was substantially reduced in the absence of TLR9, and a similar reduction was not observed in the absence of TLR2. However, IL-12p70 release was drastically reduced in the combined absence of TLR2 and TLR9 (Fig. 7A). As expected, IFN-{gamma}-treated WT macrophages had elevated IL-12p70 release to Mtb Erdman, but this was completely abrogated in the absence of TLR2 and not in the absence of TLR9 (Fig. 7A). In the TLR2/9 DKO, Mtb Erdman did not induce IL-12p70 from either DCs or macrophages (Fig. 7A). In addition, we also examined p40 gene transcription in DCs and macrophages following infection with Mtb Erdman in the presence and absence of IFN-{gamma}.


Figure 7
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  		FIGURE 7. Different TLRs are used by DCs and macrophages for IL-12 production to Mtb Erdman. A, DCs and macrophages (the latter was pretreated with 100 U/ml IFN-{gamma}) were infected with 1 multiplicity of infection (MOI) of Mtb Erdman. Supernatant was harvested at 24 h and tested for IL-12p70 by ELISA. B, DCs and macrophages were infected with 1 MOI of Mtb Erdman in the presence and absence of IFN-{gamma}. Cells were harvested at 4 h for total RNA preparation and then analyzed for IL-12p40 expression by realtime RT-PCR. The significance values are comparing each KO strain to the WT counterpart.

 
We found that IL-12p40 transcription in response to Mtb Erdman in TLR2KO and TLR9KO DCs and macrophages paralleled the protein induction pattern (Fig. 7B). Furthermore, in the presence of IFN-{gamma}, the IL-12p40 transcription was enhanced in macrophages but still in a TLR2-dependent manner, whereas the expression was independent of TLR2 in DCs. Interestingly, IFN-{gamma}-treated DCs expressed significant IL-12p40 mRNA in response to Mtb, despite the absence of TLR9 (Fig. 7B).

Collectively, these data indicate that in Mtb infection DCs predominantly use TLR9 signaling for the production of high quantities of IL-12. However, in the absence of TLR9, IL-12 production in DCs is mediated via TLR2 signaling to release a small amount of protein, which is significantly enhanced in the presence of IFN-{gamma}. In contrast, Mtb-infected macrophages use only TLR2 signaling that results in only low levels of IL-12 release. Although, these data are in most part consistent with a previous study (14) that showed macrophage and DC cytokine production to be largely dependent on TLR2 and TLR9, they however diverge to demonstrate that a clear distinction exists in TLR usage by macrophages and DCs to Mtb.

To determine whether differences in remodeling was the operative mechanism in controlling the TLR9-mediated high IL-12p70 release in DCs and TLR2-mediated low IL-12 release in macrophages, IL-12p40 remodeling was quantitated following Mtb stimulation in WT, TLR2KO, TLR9KO, and TLR2/9 DKO DCs and macrophages (Fig. 8). IL-12p40 remodeling in WT and TLR2KO DCs increased steadily and with similar kinetics as stimulation with Mtb progressed. TLR9 KO DCs, however, not only demonstrated significant impairment in their ability to remodel this locus, but also displayed kinetics that mirrored levels expressed by WT macrophages. In the absence of TLR2/9, negligible remodeling occurred in DKO DCs. A comparison of WT, TLR2KO, TLR9KO, and TLR2/9 DKO macrophages showed that in the absence of TLR2 signaling, remodeling was significantly affected while remaining unchanged in the absence of TLR9. Flow cytometric analysis using anti-TLR9 Abs showed equivalent intracellular TLR9 expression in the two cell types, confirming that lack of TLR9 signaling from macrophages was not just due to the absence of TLR9 (Fig. 8B).


Figure 8
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  		FIGURE 8. A, IL-12p40 promoter remodeling in DCs and macrophages is regulated by TLR2 and TLR9, respectively. DCs and macrophages were derived from WT, TLR2KO, TLR9KO, and TLR2/9DKO mice and were stimulated with irradiated Mtb. Cells were harvested at 180 min following stimulation and remodeling at the p40 promoter was quantitated as described in Fig. 4A. The significance values are comparing each KO strain to the WT counterpart. B, Equivalent TLR9 expression by DCs and macrophages. Intracellular TLR9 expression in bone marrow-derived macrophages and DCs was evaluated by flow cytometry. Briefly, 1 x 106 cells were blocked in 10% BSA for 30 min. Cells were fixed and permeabilized (reagents from the FoxP3 staining buffer set; eBioscience). The fixed and permeabilized cells were then reacted with 0.2 µg of avidin for 10 min (this prevented background staining), followed by anti-TLR9 Ab (clone 5G5, biotinylated Ab from HyCult) or isotype control for 30 min in permeabilization buffer. Cells were then reacted with PerCP-conjugated streptavidin (BD Pharmingen) for 30 min and washed before acquisition on FACS. Gray histogram represents isotype control and the black histogram represents anti-TLR9 Ab. The histograms represent one of two individual experiments.

 
These data demonstrate that macrophages predominantly use TLR2 for remodeling in response to Mtb, while DCs use TLR9 signaling. Furthermore, the data suggest that there is a hierarchy of use for TLR-mediated induction of IL-12, where engagement of the TLR9 but not TLR2 pathway correlates with strong p40 nucleosome remodeling and maximal production of IL-12. To investigate this further, DCs were stimulated with a TLR9 or TLR2 agonist (CpG and Pam3CSK4, respectively), and remodeling at the p40 promoter (Fig. 9B) and release of IL-12 was examined (Fig. 9A). CpG induced a faster and more extensive remodeling event, while Pam3CSK4 induced significantly less. As predicted, the remodeling deficiency in response to Pam3CSK4 was reflected by low IL-12 secretion, while CpG cultures that remodeled extensively expressed copious amounts of IL-12. Thus, TLR2 and TLR9 signaling induces unequal changes at the p40 promoter and demonstrates that an intrinsic difference exists in the pathways engaged by these TLRs.


Figure 9
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  		FIGURE 9. A, DCs were stimulated with varying concentrations of CpG and Pam3CSK4, and release of IL-12p40 was measured by ELISA. B, DCs were stimulated with 1 µM of CpG and Pam3CSK4 and remodeling at the p40 promoter was examined in response to the two ligands.

 
Together, these data implicate that the comparative efficiency of DCs relative to macrophages in IL-12 release in response to Mtb is due to differential TLR engagement or differential signaling after engagement resulting in activation of the robust TLR9-mediated pathway in DCs and the slow TLR2-mediated pathway in macrophages for p40 promoter remodeling.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Results of this study clarify the mechanism regulating the disparity in TLR-mediated IL-12 release in macrophages and DCs in response to Mtb infection. We provide experimental evidence that the low IL-12 release from macrophages is due to their use of TLR2-mediated signaling for IL-12 gene induction, which leads to slow remodeling and modest gene transcription. DCs, in contrast, are able to engage the robust TLR9-mediated signaling pathway for IL-12 gene induction. Interestingly, we found that IL-12p35 gene transcription, unlike IL-12p40, was not dissimilarly regulated in DCs and macrophages. Because no assays are available for determining IL-12p35 protein expression, it is possible that p35 expression could still be dissimilar in the two cell types due to posttranscriptional regulation of the gene. We are therefore open to the option that the low IL-12p70 release from macrophages could not only be due to low p40 expression, but also due to low p35 expression. That p35 and p40 gene induction can be differently regulated in response to the same stimulus has been reported (21). For instance, phagocytosis of apoptotic cells by macrophages was shown to induce a novel transcription factor GC-BP that selectively bound to the GC dinucleotide of the IL-12p35 promoter to repress p35 and not p40 gene transcription. Instead, posttranscriptional mechanisms were suggested to be operative in the down-regulation of IL-12p40 expression (21).

Evaluation of the contribution of TLRs to host resistance has revealed that mice deficient in TLR2 (22), TLR4 (22, 23), or TLR6 (24) are capable of resisting acute Mtb infection. In contrast, mice lacking the TLR adaptor molecule MyD88 are highly susceptible to infection (25, 26), indicating that TLRs are essential for generating host immune defenses against Mtb, and perhaps in vivo Mtb can engage several different TLRs. A recent study showed that TLR9-deficient mice were moderately susceptible to Mtb infection, but mice lacking both TLR2 and TLR9 molecules were highly susceptible. Although, both synergistic and redundant requirements for cytokine regulation by TLR2 and TLR9 was observed, overall, it appeared that TLR9 regulated Th1 response and cooperated with TLR2 to mediate optimal host resistance against Mtb infection (14). Based on our findings, we would suggest that the cooperativity is at the level of distinct requirement for TLR2 and TLR9 for IL-12 gene induction in macrophages and DCs.

An unanswered question is why Mtb does not use the TLR9 pathway in macrophages for IL-12p40 gene induction? TLR2 ligands are recognized predominantly at the cell surface, but there is evidence that they are also recognized in the phagosome (27). In contrast, there is substantial evidence that CpG DNA interacts with TLR9 in intracellular compartments (28) and that endosomal maturation is necessary for initiation of signal transduction (29, 30). Furthermore, when complexed with cationic lipids, CpG DNA can be manipulated to undergo endosomal trafficking and activate signaling pathways in cells that normally do not respond to the ligand (31). Considering that there is spatial segregation and regulation of TLR2 and TLR9 signaling, our findings would suggest that Mtb is probably targeted to distinct intracytoplasmic compartments in DCs and macrophages. This differential targeting could result in the two cell types differentially engaging the TLR signaling pathway. The likelihood that differential handling of Mtb by DCs and macrophages and consequently differential TLR engagement can occur in the two cell types is supported by a recent study which shows that there exists a fine discrimination in the recognition of individual species of phosphatidyl-myo-inositol mannosides (PIMs) from Mtb by the two pattern recognition receptors: mannose receptor (MR) and DC-SIGN (32). MR engages higher-order PIMS, while DC-SIGN engages both higher- and lower-order PIMs, along with lipoarabinomannan. Together with the fact that MR is preferentially expressed by macrophages (33) and DC-SIGN is highly associated with DCs, it is possible that the distinct interaction of Mtb with these two innate cells results in differences in the subsequent intracellular fate of Mtb. Consistent with this idea, lipoteichoic acid engages the TLR2 pathway only in the presence of the phagocytic receptor CD36 (34), and by the recent characterization of plasmacytoid DC-specific surface receptor Siglec-H that has the potential to deliver pathogens to intracellular TLR9 (35).

Trafficking of Mtb within macrophages has been extensively studied and from independent lines of investigations (reviewed in Ref. 36), it is now well-recognized that Mtb persists within macrophage phagosomes by interfering with membrane trafficking and phagolysosome biogenesis, and IFN-{gamma} effectively opposes the survival of Mtb in its modified phagosomal niche by promoting phagosome acidification and phagolysosomal fusion (37, 38). However, much less is known regarding Mtb trafficking within DCs. Clearly, future studies are needed that will compare and contrast uptake, intracellular trafficking of Mtb, and accessibility of Mtb DNA by TLR9 in DCs and macrophages.

Another question raised by our findings is the following: what downstream molecules are activated by TLR9 stimulation that induce rapid IL-12p40 promoter remodeling and how do they diverge from TLR2-activated molecules? Common signaling pathways are activated from all TLRs, but specific pathways can also be triggered by individual TLRs through association with specific cytoplasmic adapter molecules (39). Our studies show that MyD88 controls both the TLR2- and TLR9-mediated IL-12 response in macrophages and DCs, respectively. There is an expanding literature identifying various negative regulators that intercept at different steps of the TLR/MyD88-signaling pathway to temper TLR-mediated responses (40). However, it is unlikely that these negative regulators specifically inhibit TLR2 and not TLR9 signaling. A possibility though is inhibition of TLR2 signaling by other innate receptors. For example, TLR signaling is moderated in DCs following engagement of DC-SIGN by mycobacterial glycolipid lipoarabinomannan (41).

Alternative possibilities include TLR2- and TLR9-mediated activation of distinct downstream signaling molecules. For example, activation of DCs with the TLR2 agonist Pam-3-cys-induced activation of ERK, which negatively regulated IL-12p70 induction by phosphorylating and stabilizing the transcription factor c-Fos. In contrast, the TLR4 agonist LPS induced activation of p38 and JNK1/2, whose activity was essential for IL-12p70 induction by the agonist (42). The PI3K pathway has also been implicated in negatively regulating IL-12 (43, 44, 45). Furthermore, TLR-mediated cytokine production is attenuated by triggering receptor expressed on myeloid cells (TREM)-2 in bone-marrow derived macrophages (46), and TREM-2 signals via the adaptor molecule DAP-12, which is known to activate PI3K (47). Whether ERK and PI3K negatively regulate IL-12 gene induction by affecting remodeling at the p40 promoter remains to be determined, and whether TREM functions vary in DCs and macrophages also needs further scrutiny. Nonetheless, our data show that during Mtb infection, macrophages use the TLR2-signaling pathway for IL-12 production, a pathway that has been extensively recorded as a poor inducer of IL-12 and one that favors Th2 responses.

There is growing evidence that the nature of the NF-{kappa}B dimers and the ability of the dimers to recruit appropriate coactivator complexes to direct the hyperacetylation of promoters and its subsequent remodeling can affect the activation of genes. Distinct NF-{kappa}B dimers exist and specific dimers can interact with histone acetyltransferases (48, 49) to activate gene transcription and others can interact with histone deacetylases to negatively regulate gene expression (49, 50). One could speculate that TLR9 and TLR2 activate distinct NF-{kappa}B dimers to result in differential remodeling at the p40 promoter.

In summary, the studies described here provide new insight into IL-12 regulation in response to intact Mtb in two important innate immune cells: DCs and macrophages. Below, we propose a model of Mtb interaction with macrophages and DCs and the pathway leading to IL-12 release. Mtb interaction with macrophages and DCs leads to the generation of different sets of TLR ligands or alternatively the ligands are generated in different intracytoplasmic compartments. This differential handling of Mtb results in subsequent activation of either TLR2 signaling in macrophages or both TLR9 and TLR2 signaling in DCs. The TLR9 pathway activated in DCs leads to faster remodeling at the p40 promoter and robust release of IL-12. In contrast, the TLR2 pathway activated by Mtb in macrophages results in slower remodeling of the p40 promoter and reduced IL-12 release. Robust TLR2-dependent IL-12 production from macrophages is seen only under conditions where IL-10 levels are lowered, for example, with the addition of IFN-{gamma}, but without enhancing remodeling. We speculate that because of the slow kinetics of remodeling, IL-10 has a chance to block IL-12 gene transcription at a step downstream of remodeling. The biological significance of the finding is underscored by the fact that DCs are the priming APCs and therefore need to rapidly secrete IL-12 for initiating Th1 immunity. DCs are not long-lived and their IL-12 secretion is thus terminated quickly. In contrast, because Mtb reside and replicate within macrophages, the regulated expression of IL-12 from macrophages may be part of a developmental program to prevent excessive Th1 activation in the lung.


   Acknowledgments
 
We thank S. Leibovich for the MyD88KO mice, Drs. A. Sher and A. Bafica for the TLR9KO and the TLR2/9 DKO mice, Dr. B. Stockinger for the GMCSF-secreting cell line, Dr. J. Belisle for supply of irradiated M. tuberculosis through the Tuberculosis Research Materials and Vaccine Testing Contract, Colorado State University.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
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 National Institutes of Health Grant AI055377. Back

2 L.P. and S.J. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Padmini Salgame, Department of Medicine, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Medical Science Building, Room A-902, Newark, NJ 07101. E-mail address: salgampa{at}umdnj.edu Back

4 Abbreviations used in this paper: Mtb, Mycobacterium tuberculosis; DC, dendritic cell; KO, knockout; DKO, double KO; ChART, chromatin accessibility; Ct, cycle threshold; WT, wild type; PIM, phosphatidyl-myo-inositol mannoside; MR, mannose receptor; TREM, triggering receptor expressed on myeloid cells. Back

Received for publication October 10, 2006. Accepted for publication January 24, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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