Recombinant MPT83 Derived from Mycobacterium tuberculosis Induces Cytokine Production and Upregulates the Function of Mouse Macrophages through TLR2

Infection with Mycobacterium tuberculosis is a significant cause of mortality worldwide, and the risk for mortality increases when persons are infected with antibiotic-resistant strains of M. tuberculosis and/or are coinfected with HIV. Host resistance to M. tuberculosis infection depends on both innate and adaptive immune responses (1, 2).

During the early stages of infection with M. tuberculosis, macrophages serve as the main effector cells, and they phagocytose the bacilli, which limits bacterial survival and proliferation (3, 4). Macrophages also possess several antimicrobial mechanisms that control the intracellular replication of bacilli (5, 6).

TLRs on APCs recognize microbial molecules and pathogen-associated molecular patterns and induce innate immune responses (e.g., cytokine production) and modulate adaptive immune responses (1). TLR2 mediates cellular responses to diverse molecular structures. In the context of M. tuberculosis infection, the recognition of M. tuberculosis cell wall components by TLR2 results in the production of proinflammatory molecules (7–9), the induction of apoptosis (10), and the formation of macrophage giant cells (11); these effects contribute to both host protection and immunopathology. Polymorphisms in the human TLR2 gene are associated with enhanced susceptibility to leprosy and tuberculosis (12–14). In mice, TLR2 deficiency increases susceptibility to M. tuberculosis infection (15, 16). Several mycobacterial ligands for TLR2 have been identified, including lipomannan (17), lipoarabinomannan (18, 19), and the lipoproteins LpqH (19 kDa; Rv3763) (20–22), LprG (Rv1411c) (23), LprA (24), MPB83 (25), and a 38-kDa lipoprotein (26).

The binding of ligands to TLRs triggers signal transduction that results in proinflammatory responses that are mediated by the activation of MAPKs and transcription factors, such as NF-κB and IFN regulatory factors (27). MAPKs and transcription factors are important for many cellular functions, ranging from proliferation to differentiation to apoptosis (28). Three types of MAPKs exist in mammalian cells—ERK, JNK, and p38—and they can be activated independently and simultaneously. p38 and ERK are of critical importance for the release of proinflammatory cytokines, such as TNF-α and IL-1 (29, 30).

IFN-γ is produced by activated T cells and is a central regulator of the immune response to M. tuberculosis (31, 32). IFN-γ upregulates the expression of MHC class II (MHC-II) mRNA and protein and, thereby, regulates Ag processing by macrophages (33). In mice, the activation of macrophages by IFN-γ stimulates the production of NO, which results in the death of M. tuberculosis bacilli (34, 35). Mice that are deficient in IFN-γ or the IFN-γR and are infected with M. tuberculosis develop a fatal disease associated with widespread dissemination of M. tuberculosis (36, 37); similarly, in humans, genetic mutations that result in alterations in IFN-γ signaling are associated with bacterial dissemination after bacillus Calmette-Guérin vaccination and increased susceptibility to M. tuberculosis infection (38, 39). The survival of M. tuberculosis may be associated with an inhibition of IFN-γ–dependent responses. Prolonged (>18 h) infection of macrophages with M. tuberculosis or exposure to lipoarabinomannan, 19-kDa lipoprotein, LprG, or LprA inhibits the IFN-γ–induced expression of certain genes (20–24). M. tuberculosis also inhibits macrophage expression of CIITA and genes that are regulated by CIITA, including MHC-II, H2-M, and invariant chain (40). In addition, M. tuberculosis inhibits the expression of other accessory proteins that are required for Ag presentation (40, 41), which might then reduce IFN-γ production by CD4⁺ T cells.

MPT83 is a lipoylated and glycosylated M. tuberculosis lipoprotein that is attached to the cell surface and has several exposed epitopes (42). MPT83 also is a sero-dominant Ag in cattle infected with Mycobacterium bovis, and it is recognized by T cells from infected cattle but not by T cells from cattle vaccinated with bacillus Calmette-Guérin (43, 44). Previously, we found that vaccines against M. bovis that combined Ag85B and MPT64 with MPT83 produced a larger protective immune response than did vaccines containing individual components (45, 46). In the current study, we focused on the effects of recombinant MPT83 (rMPT83) on macrophages. Our results showed that rMPT83 is devoid of any posttranslational modification and is able to induce the production of TNF-α, IL-6, and IL-12 p40 by macrophages. We also demonstrated a direct interaction between rMPT83 and TLR2 with concomitant rMPT83-mediated enhancement of IFN-γ–induced Ag presentation and MHC-II expression.

C57BL/6 mice were purchased from Vital River Company (Beijing, China). TLR2^−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). MyD88^−/− mice were obtained from Osaka University (Osaka, Japan). All mice were housed in the Animal Center of Peking University, maintained in individual cages, and fed commercial mouse chow and water. All experimental procedures followed institutional guidelines for the ethical treatment of animals and were approved by the institutional ethical committee. A mouse macrophage cell line (RAW264.7) and a human embryonic kidney cell line (HEK293) were originally purchased from the American Type Culture Collection (Manassas, VA). HEK293 cells were stably transfected with an empty enhanced GFP (eGFP) vector (BD Biosciences, Clontech, CA), an eGFP-TLR2 plasmid, or an eGFP-TLR4 plasmid to obtain HEK293-Vector, HEK293-TLR2, and HEK293-TLR4 cell lines, respectively. All of the plasmids were constructed in our laboratory (State Key Laboratory of Protein and Plant Gene Research). Cells were cultured in DMEM (HyClone Laboratories, Logan, UT) supplemented with 10% FBS (Life Technologies BRL, Gaithersburg, MD), penicillin (100 U/ml), and streptomycin (100 μg/ml) and maintained at 37°C in a humidified incubator (5% CO₂).

The gene encoding MPT83 was amplified using PCR based on the genomic DNA sequence of M. tuberculosis H37Rv. The PCR product then was cloned using the prokaryotic expression plasmid pET22b⁽⁺⁾ (Novagen, Darmstadt, Germany), as described previously (47). The recombinant proteins were purified by affinity chromatography using Ni-NTA (Qiagen, Chatsworth, CA) for testing of their ability to induce cytokine production by macrophages.

Cytokine ELISA MAX Set Deluxe kits (BioLegend, San Diego, CA) were used to measure cytokine levels, following the manufacturer’s instructions, in supernatants from ligand-treated RAW264.7 cells and TLR2- or TLR4-transfected HEK293 cells and from supernatants collected from an MHC-II Ag-processing assay. In experiments designed to block TLR signaling, RAW264.7 cells were pretreated for 30 min at 37°C with a mouse Ab against TLR2 (30 μg/ml) or TLR4 (30 μg/ml) or a mouse IgG isotype-matched control Ab (30 μg/ml). Additional experiments involved pretreating RAW264.7 cells for 30 min at 37°C with inhibitors to p38 (SB203580; 30 μM), ERK (PD98059; 50 μM), or JNK (JNK inhibitor II; 40 μM). The RAW264.7 cells were then incubated with rMPT83 for 4, 16, or 24 h at 37°C. Anti-TLR2 and anti-TLR4 were purchased from Imgenex (San Diego, CA), the IgG isotype control Ab was from Santa Cruz Biotechnology (Santa Cruz, CA), and the inhibitors were from Sigma-Aldrich (St. Louis, MO).

RAW246.7 cells were treated for various times with rMPT83 (0 or 5 μg/ml) or Pam₃CSK₄ (10 μg/ml), a TLR1/2 agonist. After treatment, cell culture supernatants were removed, and the cells were rinsed twice with PBS. Five hundred microliters/well of TRIzol (Invitrogen, Carlsbad, CA) was added to the plates. The cells were cultured at room temperature for 5 min and mixed thoroughly two times with chloroform:isoamyl alcohol (1:1) to extract RNA. The cells were washed with 75% ethanol and dissolved in diethypyrocarbonate-treated, double-distilled water. First-strand cDNA was synthesized by reverse transcription using total RNA Transcript II reverse transcriptase (TransGen Biotech Company, Beijing, China). The target genes were amplified using conventional methods and the appropriate cDNA templates, with upstream and downstream primers. RNA levels of the analyzed genes were normalized to the amount of β-actin or GAPDH present in each sample. All primers were synthesized by Sangon (Shanghai, China), and their sequences were as follows: β-actin, sense: 5′-TGCTGTCCCTGTATGCCTCT-3′, antisense: 5′-GGTCTTTACGGATGTCAACG-3′; TNF-α, sense: 5′-GGCGGTGCCTATGTCTCA-3′, antisense: 5′-GGCAGCCTTGTCCCTTGA-3′; IL-6, sense: 5′-TGCCTTCTTGGGACTGAT-3′, antisense: 5′-CTGGCTTTGTCTTTCTTGTT-3′; IL-12p40, sense: 5′-CAGAAGCTAACCATCTCCTGGTTTG-3′, antisense: 5′-TCCGGAGTAATTTGGTGCTTCACAC-3′; and CIITA, sense: 5′-AGACGTGTTCTGCTCATC-3′, antisense: 5′-AGTTCTCAAAGTAGTGCCTCAT-3′.

RAW264.7 cells were stimulated for various times and treated with cell lysis buffer supplemented with proteinase inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN). The cells were centrifuged at 4°C, 12,000 × g, for 30 min, and protein concentrations were measured using the Bradford protein-quantification assay. Cell pellets were processed using the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL), following the manufacturer’s instructions, to separate the cytoplasmic and nuclear fractions. Equal amounts of protein were separated by SDS-PAGE and then transferred electrophoretically to polyvinyldifluoride membranes (Millipore, Bedford, MA). After blocking with 5% nonfat milk in PBS ([pH 7.4], 0.05% (v/v) Tween-20), the membranes were incubated overnight at 4°C with primary Abs, including rabbit anti-ERK2, rabbit anti-p38 (Santa Cruz Biotechnology), mouse anti–phospho-ERK1/2, rabbit anti–phospho-p38, rabbit anti–stress-activated protein kinase/JNK, rabbit anti–phospho–stress-activated protein kinase/JNK, rabbit anti-Stat1,rabbit anti–phospho-Stat1 (Y701), and rabbit anti–phospho-Stat1 (S727) (Cell Signaling Technology, Beverly, MA), according to the manufacturer’s instructions. After a wash with PBS, the membranes were incubated for 1–2 h at 37°C with the appropriate HRP-conjugated anti-mouse IgG or anti-rabbit IgG secondary Ab (1:5000) (Santa Cruz Biotechnology). The peroxidase-positive bands were detected using an ECL reaction solution (Vigorous, Beijing, China) and visualized by exposure to x-ray film (XAR5; Kodak, Rochester, NY).

An HEK293-TLR2 stable cell line, an HEK293-TLR4 stable cell line, and an HEK293-Vector stable cell line were grown overnight on coverslips and then incubated for 30 min at 37°C with rMPT83 (5 μg/ml) in Hank’s buffer. The cells were washed with PBS and fixed for 15 min using 4% paraformaldehyde. Then the cells were stained by sequential 1-h incubations with mouse anti-MPT83 IgG (mAb purified by our laboratory [State Key Laboratory of Protein and Plant Gene Research]) and Cy3-conjugated rabbit anti-mouse IgG (Santa Cruz Biotechnology). Between each staining step, the cells were washed three times with PBS. After staining, the cells were mounted on slides using Mowiol solution (Sigma-Aldrich) and observed using a 63× oil objective on an Axiovert 200M microscope (Carl Zeiss, Munich, Germany). For flow cytometry, the cells were collected from the slides after staining, and 10,000 total events per sample were analyzed using a BD FACScan calibrator (BD Biosciences, San Jose, CA).

HEK293-Vector, HEK293-TLR2, and HEK293-TLR4 stable cell lines were washed twice with PBS and lysed with cell lysis buffer. After centrifugation, the supernatant from the cell lysates was incubated with rMPT83 immobilized on Ni-NTA beads at 4°C on a rotor. The beads were washed and boiled in 5× Laemmli buffer for 10 min. Proteins were separated on 10% SDS-PAGE and then transferred electrophoretically to polyvinyldifluoride membranes. The membranes were incubated with anti-GFP, followed by incubation with HRP-conjugated anti-rabbit IgG secondary Ab (1:5000; Santa Cruz Biotechnology). Immunoreactive bands were detected using an ECL reaction solution and visualized by exposure to x-ray film.

RAW264.7 cells (2 × 10⁶/well) were seeded in six-well culture plates with glass coverslips; treated with rMPT83 (5 μg/ml) for 0, 15, or 30 min; and washed twice with PBS. The cells were then fixed for 5 min on ice, using prechilled methanol. To reduce background staining, the cells were incubated for 30 min in PBS containing 5% BSA before incubation with rabbit anti-mouse NF-κB. After a 1.5-h incubation, the cells were washed, FITC-conjugated goat anti-rabbit IgG was added, and the cells were incubated for 1 h at room temperature. The cells were washed, and the coverslips were turned upside down onto a solution of DAPI (100 ng/ml) with Mowiol, using nail polish to delineate the edge of the fixed cover slips. NF-κB localization was observed using a Zeiss LSM 710 confocal microscope equipped with a 63×, 1.4-NA, oil-immersion objective (Carl Zeiss).

Cells were incubated in 24-well plates (2 × 10⁵ cells/well) with rMPT83 (0 or 5 μg/ml) and IFN-γ (0 or 20 ng/ml) (PeproTech, London, U.K.) for 12, 24, or 48 h. Cells were harvested and washed with prechilled PBS. The cells were incubated on ice for 30 min with a 1:100 dilution of Fc Block (BD Pharmingen, San Diego, CA) in PBS with 1% BSA, and they were then incubated on ice for 1 h in the dark with PE-CY5–conjugated anti-MHC-II (eBioscience), PE-conjugated anti–IFN-γR β-chain, or an appropriate isotype-matched negative control Ab (BioLegend). The cells were fixed with 1% paraformaldehyde and analyzed using a FACScan calibrator with 10,000 total events/sample, as described previously (48).

To obtain a large number of rMPT83-specific CD4⁺ T cells for an Ag-processing assay, C57BL/6 mice received three immunizations over a 3-wk period using an MPT83 DNA vaccine. The vaccination consisted of a mixture of plasmid pJW4303-MPT83 (100 μg) and adjuvant pJW4303-IL12 (50 μg), and it was administered by i.m. injection into the quadriceps muscle. The DNA used in the vaccination was purified using the Mega plasmid DNA kit (Qiagen) and diluted in saline to a final concentration of 1 mg/ml. Three weeks after the final immunization, the mice were euthanized by cervical dislocation. Splenic lymphocytes were isolated using lymphocyte separation medium (Dakewe Biotech, Shenzhen, China), following the manufacturer’s instructions. After cell counting, the cell concentration was adjusted to 2 × 10⁶ cells/ml with RPMI 1640 containing 10% FBS, and the cells were seeded in 96-well cell culture plates and cultured at 37°C in 5% CO₂.

TLR2^−/− or C57BL/6 mice were killed by cervical dislocation and immersed in 75% ethanol for 5 min. The peritoneal cavities were flushed with 5 ml RPMI 1640 medium without FBS. After washing, 5 × 10⁵ cells were plated on sterile glass coverslips in 12-well tissue culture plates in 1 ml RPMI 1640 containing 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin and incubated overnight at 37°C with 5% CO₂. Nonadherent cells were removed by washing twice with PBS and were treated with medium alone, rMPT83 (5 μg/ml), or other ligands, as described in the 15Results.

RAW264.7 cells or mouse peritoneal macrophages (1 × 10⁵ cells/well) were incubated in 96-well plates with rMPT83 (0 or 5 μg/ml) for 24 h at 37°C. The cells were then washed with PBS and fixed for 20 min in 0.5% paraformaldehyde at 37°C. The rMPT83 Ag-specific CD4⁺ T cells that were isolated from immunized mice were added at a concentration of 5 × 10⁵ cells/well, and the plates were cultured for 48 h at 37°C. Supernatants were harvested, and the concentration of IL-2 in each supernatant sample was measured using ELISA, as described above.

Results were calculated as the mean ± SD of triplicate experiments. Statistical testing was conducted using a one-way ANOVA, followed by Tukey test, using SigmaStat 3.5 software (Systat Software, Richmond, CA). For all tests, p < 0.05 was considered statistically significant.

RAW264.7 cells were treated with various concentrations of rMPT83 for 4, 16, or 24 h, the culture supernatants were collected, and cytokine levels were measured by ELISA. The results showed that rMPT83 significantly increased, in a dose-dependent manner, the production of TNF-α, IL-6, and IL-12p40 (p < 0.001; Fig. 1A). rMPT83 produced a similar increase in the relative expression of mRNA for TNF-α, IL-6, and IL-12p40 (Fig. 1B).The effect was specific to rMPT83, because both proteinase K and anti-rMPT83 (but not the isotype control Ab) abrogated the rMPT83-induced increase in TNF-α, IL-6, and IL-12p40 (p < 0.05; Fig. 1A). Polymyxin B inhibited the LPS-induced increase in TNF-α, IL-6, and IL-12p40 production but did not affect the rMPT83-induced increase in TNF-α, IL-6, and IL-12p40 production (data not shown), indicating that the effects of rMPT83 were not due to inadvertent contamination with LPS.

RAW264.7 cells were incubated with or without anti-TLR2, anti-TLR4, or isotype control Ab for 30 min before stimulation with rMPT83. Supernatants were then harvested, and levels of TNF-α, IL-6, and IL-12p40 were measured using ELISA. Anti-TLR2 significantly blocked the rMPT83-induced increase in the production of TNF-α (p < 0.001; ∼50% inhibition at 30 μg/ml) and the production of IL-6 and IL-12p40 (p < 0.001; ∼90% inhibition at 30 μg/ml), whereas neither anti-TLR4 nor the isotype control Ab altered the rMPT83-induced increase in the production of TNF-α, IL-6, or IL-12p40 (Fig. 1C). Furthermore, rMPT83-induced cytokine production was significantly lower in macrophages from TLR2^−/− mice than in macrophages from wild-type mice (p < 0.05 or p < 0.01), indicating that the effects of rMPT83 were dependent on TLR2 (Fig. 1D).

HEK293 cells lack TLRs, yet retain downstream TLR-signaling components. HEK293 cells transfected to express specific TLRs secrete IL-8 in response to TLR signaling. To test whether rMPT83 can signal through TLR2, HEK293 cells were transfected with mouse TLR2 (HEK293-TLR2), TLR4 (HEK293-TLR4), or a control vector (HEK293-Vector) and incubated for 24 h with various concentrations of rMPT83. Levels of IL-8 in supernatants were measured by ELISA. rMPT83 induced, in a dose-dependent manner, the production of IL-8 in HEK293-TLR2 cells but not in HEK293-TLR4 or HEK293-Vector cells (Fig. 2A). Flow cytometry showed that rMPT83 bound to the surface of HEK293-TLR2 cells but not to the surface of HEK293-TLR4 or HEK293-Vector cells (Fig. 2B). The percentage of HEK293-TLR2 cells that bound rMPT83 increased with increasing concentrations of rMPT83 (p < 0.01). Likewise, immunofluorescence microscopy showed strong fluorescence of anti-rMPT83 on the surface of HEK293-TLR2 cells exposed to rMPT83 but little fluorescence on the surface of HEK293-TLR4 cells or HEK293-Vector cells (Fig. 2C). To further ascertain whether rMPT83 physically interacts with the TLR2 molecule, we conducted a pull-down assay using whole-cell extracts from HEK293 cells transfected with mouse TLR2, TLR4, or a control vector. The cell extracts were incubated with rMPT83 immobilized on Ni-NTA beads. Western blots probed with anti-GFP showed that immobilized rMPT83 was able to pull-down TLR2 (Fig. 2D). No band was visible in the vector-control group or the group containing TLR4 (Fig. 2D). These observations suggested that rMPT83 interacts specifically and predominantly with TLR2.

Many cellular responses to external stimuli are dependent on MAPKs; thus, we examined the effect of rMPT83 on MAPK activation. RAW264.7 cells were stimulated with rMPT83 (5 μg/ml), and Western blot analysis was used to examine the phosphorylation of p38, JNK, and ERK1/2 during various time courses. rMPT83 produced a strong phosphorylation of p38, JNK, and ERK1/2 after 10–30 min of stimulation. The peak phosphorylation occurred within 30 min of stimulation with rMPT83 (Fig. 3A). Little phosphorylation was observed in untreated cells. We also examined whether rMPT83 activated the p38, JNK, and ERK1/2 pathways through TLR2 or TLR4 signaling. Levels of phosphorylated p38, JNK, and ERK1/2 induced by rMPT83 were measured after preincubation of RAW264.7 cells with an Ab against TLR2 or TLR4 or with an isotype-matched control Ab. Immunoblotting was used to measure the phosphorylation state, which is a well-defined indicator of enzymatic activity level. As shown in Fig. 3B, the rMPT83-mediated phosphorylation of p38 was significantly reduced, and the rMPT83-mediated phosphorylation of JNK was attenuated when RAW264.7 cells were pretreated with anti-TLR2 or anti-TLR2 plus anti-TLR4; in contrast, the rMPT83-induced phosphorylation of ERK1/2 was minimally affected by anti-TLR2 or anti-TLR2 plus anti-TLR4. The phosphorylation state of p38, JNK, and ERK1/2 was not affected by pretreatment with anti-TLR4 or the isotype-matched control. Therefore, the phosphorylation of MAPKs in response to rMPT83 is mediated primarily by TLR2, not by TLR4.

RAW264.7 cells were cultured for 30 min with or without SB203580 (an inhibitor of p38 MAPK), PD98059 (an inhibitor of MEK via upstream, activator-dependent phosphorylation), or JNK inhibitor II, and rMPT83-induced production of TNF-α, IL-6, and IL-12p40 was examined using ELISA. TNF-α production was significantly blocked by JNK inhibitor II (p < 0.001; ∼90% inhibition at 5 μg/ml rMPT83) but only partially blocked by SB203580 or PD98059 (Fig. 4A). The production of IL-6 and IL-12p40 was prominently blocked by JNK inhibitor II (p < 0.001; ∼95% inhibition) and attenuated by SB203580 (∼75–80% inhibition) but was not affected by PD98059 (Fig. 4C, 4E). Similar results were obtained when the expression of mRNA for TNF-α, IL-6, and IL-12p40 was examined (Fig. 4B, 4D, 4F).

NF-κB is an important transcription factor that is involved in the induction of proinflammatory cytokines (49–51) and the expression of costimulatory molecules. The promoters of IL-12 p40 (52, 53) and TNF-α (51, 54) contain NF-κB binding sites, and transcription of these genes is dependent essentially on the binding of NF-κB/rel transcription factors to the NF-κB binding sites. Therefore, the effects of rMPT83 on NF-κB activation and translocation were examined. Confocal microscopy demonstrated the localization of NF-κB p65 to the nuclei of rMPT83-treated cells. After 15 min of stimulation with rMPT83, the nuclei were filled with active NF-κB p65 (Fig. 5A), but the level decreased after 1 h of stimulation (data not shown). In unstimulated cells, NF-κB p65 was present primarily in the cytoplasm (Fig. 5A). Western blot analysis also showed that stimulation of RAW264.7 cells with rMPT83 induced the expression of nuclear NF-κB. Nuclear translocation of NF-κB was preceded by degradation of IκBα, the inhibitor of NF-κB (Fig. 5B).

To investigate the effect of rMPT83 on IFN-γ–induced MHC-II expression, RAW264.7 cells were incubated with rMPT83 for 0, 12, 24, or 48 h in the presence of IFN-γ, and flow cytometry was used to examine MHC-II expression. MHC-II was expressed at low levels in RAW264.7 cells cultured in medium alone for 0–48 h (Fig. 6A, 6F). The addition of IFN-γ resulted in a 2.7-fold increase in MHC-II expression (Δ mean fluorescence value [MFV] at 24 h was 53 ± 6 for medium alone and 165 ± 33 with the addition of IFN-γ). When cells were coincubated with rMPT83 and IFN-γ, MHC-II expression was significantly increased after 24 h of culture (p < 0.05). Treatment of cells with rMPT83 increased the expression of MHC-II compared with treatment with medium alone, with a 2.7-fold increase after 48 h of culture (ΔMFV at 48 h was 59 ± 45 for medium alone and 148 ± 16 with the addition of rMPT83). When cells were coincubated with rMPT83 and IFN-γ, there was a 3.5-fold increase in MHC-II expression after 48 h of culture (p < 0.001) (ΔMFV at 48 h was 59 ± 45 for medium alone and 356 ± 73 with the addition of both IFN-γ and rMPT83). MHC-II expression after coincubation with both IFN-γ and rMPT83 increased from 24 h to a maximum after 48 h of culture (Fig. 6A–D).

To determine the role of TLR2 in the enhancing effects of rMPT83 on IFN-γ–induced MHC-II expression in RAW264.7 cells, anti-TLR2 was used in the flow cytometric analysis. Anti-TLR2 significantly blocked the enhancing effects of rMPT83 on IFN-γ–induced MHC-II expression (p < 0.01) (Fig. 6E, 6F). Treatment with anti-TLR4 or isotype control Ab did not block the rMPT83-induced increase in MHC-II expression (data not shown). C57BL/6 or TLR2^−/− macrophages also were incubated with IFN-γ for 24 or 48 h, and flow cytometry was used to examine MHC-II expression. The results showed that rMPT83 remained capable of enhancing IFN-γ–induced expression of MHC-II in macrophages from both types of mice, although the expression of MHC-II at 48 h was significantly lower in TLR2^−/− mice than in C57BL/6 mice (p < 0.05 or 0.01; Fig. 6G), which suggested a role for TLR2 in the enhancing effects of rMPT83 on IFN-γ–induced MHC-II expression.

To determine whether increases in IFN-γ–regulated MHC-II expression affects the ability of mouse macrophages to present rMPT83 to CD4⁺ T cells, rMPT83-specific CD4⁺ T cells were purified from mice immunized with the plasmid pJW4303-MPT83. Specific CD4⁺ T cells express the α/β TCR and produce IL-2 in concentrations that are proportional to the number of specific MHC-II–peptide complexes recognized by the TCR. rMPT83-specific CD4⁺ T cells were incubated for 48 h with RAW264.7 cells that were pretreated with IFN-γ, rMPT83, or rMPT83 plus IFN-γ, and supernatant levels of IL-2 were measured using ELISA. Treatment with IFN-γ or rMPT83 alone enhanced the ability of macrophages to present rMPT83 peptide to CD4⁺ T cells, as reflected by increased IL-2 levels (p < 0.01 or p < 0.05; Fig. 7A). MHC-II presentation of rMPT83 peptide was enhanced even more after treatment with both rMPT83 and IFN-γ (p < 0.001; Fig. 7A). The rMPT83-induced increase in MHC-II presentation of rMPT83 peptide was blocked by anti-TLR2 but not by anti-TLR4 or the isotype control Ab (Fig. 7A). In addition, the ability of macrophages to present rMPT83 peptide to CD4⁺ T cells was enhanced by rMPT83 in macrophages from C57BL/6 mice compared with macrophages from TLR2^−/− mice (Fig. 7B).

Effective host defense against mycobacterial infection requires an innate immune response that facilitates the clearance of mycobacteria by macrophages. Activation of the TLR2-signaling pathway results in the production of inducible NO synthase and the secretion of cytokines, such as TNF-α and IL-6, and these effects enhance the innate immune response and promote the activation of CD4⁺ T cells (1). Our results showed that rMPT83 induced the expression of mRNA and protein for TNF-α, IL-6, and IL-12 p40, and these effects were attenuated or blocked by anti-TLR2, indicating that the effects of rMPT83 on cytokine production are mediated by TLR2. The production of cytokines was lost when rMPT83 was digested with proteinase K, similar to the effects observed with two other mycobacterial lipoglycoproteins, p19 and 38-kDa Ag (9, 10). Furthermore, the production of cytokines, particularly TNF-α, was higher in macrophages from wild-type mice than in macrophages from TLR2^−/− mice. TNF-α is important for granuloma formation and the induction of cytotoxicity against M. tuberculosis bacilli (55, 56). In contrast, IL-12 plays a crucial role in activating the protective Th1 immune response (57–59). Interestingly, the virulence of pathogenic mycobacteria is correlated inversely with the levels of IL-12 and TNF-α (60–63). Our results also showed that rMPT83, IFN-γ, or rMPT83 plus IFN-γ produced significantly higher levels of NO than that produced in medium alone. NO production by macrophages is a major antimicrobial mechanism, and our results indicated that NO production is upregulated by rMPT83 and IFN-γ (Supplemental Fig. 1).

rMPT83 bound more strongly to HEK293-TLR2 cells and stimulated greater IL-8 production in HEK293-TLR2 cells than in HEK293-TLR4 cells or HEK293-Vector cells. These results demonstrated that rMPT83 is a potent agonist for TLR2. The TLR2 ectodomain contains leucine-rich repeats, a motif that is commonly involved in protein–protein interactions (64), and so, the fact that rMPT83 is a potent agonist for TLR2 might have been predicted. The protein moiety of lipoproteins is critical for interaction with TLR2. Immunoprecipitation was used to demonstrate the binding of rMPT83 and TLR2 in the absence of acylation or glycosylation, consistent with previous results showing that the recombinant mycobacterial proteins ESAT6, PPE18, and MPB83 bind to TLR2 (25, 65, 66). The current findings also are consistent with observations that other LprG and LprA lipoproteins trigger the TLR2-signaling pathway, resulting in the secretion of proinflammatory cytokines, such as TNF-α.

TLR2 signaling is dependent on MyD88, IRAK, and TNFR-associated factor 6, and it results in the activation of NF-κB and MAPK (67–69). Our studies indicated that rMPT83-induced activation of the TLR2-signaling pathway was dependent on MyD88 because the production of TNF-α was significantly higher in macrophages from wild-type mice than in macrophages from MyD88^−/− mice (Supplemental Fig. 2). In addition, our results demonstrated that p38, ERK, and JNK are phosphorylated rapidly in response to rMPT83. p38 activity is believed to be of critical importance for the release of TNF-α and IL-1 by LPS-stimulated monocytes (30). The present results showed that the rMPT83-induced production of TNF-α, IL-6, and IL-12 p40 was reduced drastically by JNK inhibitor II and a p38 inhibitor (SB203580), indicating that the p38 and JNK MAPK pathways are critically important in rMPT83-induced production of TNF-α, IL-6, and IL-12 p40.

NF-κB is a dimeric transcription factor formed by the interactions of five Rel family proteins, and it is the primary transcription factor activated in TLR signaling (69). The inhibition of NF-κB activation in dendritic cells decreases the expression of MHC-II and costimulatory molecules and, thereby, decreases the priming of T cells (70). NF-κB binding sites within the MHC-II invariant chain promoter are downregulated by the NF-κB subunit, p50, in promonocytic U937 cells (71). Recent evidence suggested that NF-κB may also play a role in resolving inflammation, because a shift in NF-κB subunits from p50-p65 to p50 homodimers is associated with the resolution of inflammation (69, 72). Our results indicated that rMPT83 reduced the level of IκBα in cytoplasm and increased the expression of NF-κB p65 in the nucleus; these effects may then lead to an increase in MHC-II expression.

IFN-γ is critically important in controlling M. tuberculosis infection. Defects in the IFN-γ–signaling pathway lead to severe mycobacterial infections in both mice and humans (33, 36, 37). Previous studies showed that, in cells that are chronically infected with M. tuberculosis, it inhibits the regulation of certain genes by IFN-γ. The current results demonstrated that after a 24- or 48-h incubation, rMPT83 enhanced the ability of IFN-γ to induce MHC-II expression in RAW264.7 cells. The results further demonstrated that rMPT83 enhanced the ability of macrophages to present rMPT83 peptide to CD4⁺ T cells for recognition, as reflected by an increase in IL-2 production. The effects of rMPT83 on MHC-II expression and IL-2 production were blocked or attenuated by anti-TLR2, which indicated that the effects are mediated by the TLR2-signaling pathway. IL-2 is secreted by Ag-activated CD4⁺ T cells during the primary immune response, and it is believed to enhance memory/effector function by increasing the antigenic sensitivity and expression of effector cytokines in secondary immune responses (73). In M. tuberculosis infection, an enhancement in macrophage MHC-II Ag expression would be an effective immune response strategy because MHC-II–restricted CD4⁺ T cells are central for host defense against M. tuberculosis.

Ting et al. (74) demonstrated that M. tuberculosis inhibits IFN-γ transcriptional responses without altering STAT1 activation. The present findings showed that rMPT83 increased the expression of IFN-γR and did not prevent IFN-γ–induced phosphorylation of Stat1 (Supplemental Fig. 3), suggesting that the enhancing effect of rMPT83 on IFN-γ signaling occurs at a distal site in the signaling pathway, perhaps involving chromatin remodeling. Induction of CIITA mRNA is dependent on changes in chromatin structure, and IFN-γ stimulation results in the remodeling of the CIITA locus (41). Our results indicated that the induction of CIITA mRNA by IFN-γ was increased by rMPT83 (Supplemental Fig. 4), suggesting that rMPT83 exerts distal control of sets of IFN-γ–responsive genes. Transcriptional control of MHC-II expression might be an important mechanism in the regulation of APC function during infection with M. tuberculosis. CIITA is the master transcriptional regulator of MHC-II molecules (41). It is well known that MHC-II molecules have a crucial role in the development and function of the immune system. In addition to the classical function of MHC-II molecules in presenting Ag to CD4⁺ T cells, MHC-II molecules can activate various cellular functions in immune or nonimmune cells when cross-linked by Ab or superantigen (75–78). Previous studies showed that prolonged TLR2 signaling by mycobacterial lipoproteins, such as 19-kDa lipoprotein, inhibits MHC-II expression and Ag processing in macrophages (20, 22). In our studies, prolonged TLR2 signaling by rMPT83 enhanced MHC-II expression and Ag presentation by macrophages. Several explanations exist for the discrepant findings with 19-kDa protein and rMPT83. The potential explanation is that we and other investigators described increased vaccine efficacy in a murine M. tuberculosis challenge model when rMPT83 was used as the immunogenic Ag (43, 45–48), whereas other investigators described decreased vaccine efficacy in a murine M. tuberculosis challenge model when 19-kDa lipoprotein, expressed in Mycobacterium vaccae, was used as the immunogenic Ag (79). rMPT83 is an immunodominant Ag that is recognized by T cells because it enhances the expression of the gene encoding CIITA, which upregulates the transcription of MHC2TA and the expression of MHC-II molecules and plays a major role in host protection. The 19-kDa lipoprotein may block CD4⁺ T cell activation by decreasing peptide–MHC-II expression and inhibiting the IFN-γ–induced chromatin remodeling of MHC2TA. CCAATT/enhancer-binding protein-β and -δ are induced by TLR2 signaling and bind to CIITA promoters, which contributes to the inhibition of CIITA and results in decreased MHC-II molecule expression and inhibition of Ag presentation (22, 41, 80); together, these effects may promote the survival of M. tuberculosis within macrophages.

In conclusion, our results demonstrated that rMPT83 is a potent TLR2 agonist, induces innate immunity (e.g., cytokine production), and upregulates APC function in mouse macrophages through the TLR2-signaling pathway.

The authors have no financial conflicts of interest.