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by Live Virulent Mycobacterium tuberculosis Is Independent of Mature Mycobacterial Lipoproteins but Dependent on TLR21
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* Department of Medicine, Division of Infectious Diseases, New York University School of Medicine, New York, NY 10016;
Biomedical Sciences Graduate Program, University of California, San Francisco, CA 94143;
Howard Hughes Medical Institute and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461; and
Department of Microbiology, New York University School of Medicine, New York, NY 10016
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Abstract |
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. We have previously shown that live M. tuberculosis inhibits selected macrophage responses to IFN-, and that purified M. tuberculosis 19-kDa lipoprotein inhibits induction of selected IFN--responsive genes through a TLR2-dependent pathway, whereas peptidoglycan inhibits responses to IFN- by a TLR2-independent pathway. To determine the relative contribution of lipoproteins to the inhibition of responses to IFN-, we deleted the M. tuberculosis gene (lspA) that encodes lipoprotein signal peptidase. This revealed that M. tuberculosis lipoprotein processing is indispensable for stimulation of TLR2 reporter cells, but that the lspA mutant inhibits macrophage responses to IFN- to the same extent as wild-type bacteria. Macrophages lacking TLR2 are more resistant to inhibition by either strain of M. tuberculosis, suggesting that nonlipoprotein TLR2 agonists contribute to inhibition. Indeed, we found that phosphatidylinositol mannan from M. tuberculosis inhibits macrophage responses to IFN-. M. tuberculosis inhibition of responses to IFN- requires new protein synthesis, indicating that a late effect of innate immune stimulation is the inhibition of responses to IFN-. These results establish that M. tuberculosis possesses multiple mechanisms of inhibiting responses to IFN-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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(6) and TNF-
production at the site of infection (5). The inability to clear M. tuberculosis despite this immune response suggests that M. tuberculosis may interfere with distal effector events. Defective recognition of infected macrophages by T cells and/or defective responses of infected macrophages to effectors of adaptive immunity may contribute to the ability of M. tuberculosis to persist and progress. One specific mechanism that could permit M. tuberculosis to avoid elimination by the immune response is inhibition of macrophage responses to IFN-, an important effector of immunity to intracellular pathogens. Indeed, whereas IFN- is capable of stimulating macrophages to kill intracellular Toxoplasma, Leishmania, Legionella, and Chlamydia, it is incapable of stimulating macrophages to kill M. tuberculosis in vitro unless IFN- is used to prime macrophages before infection (7, 8, 9, 10, 11, 12, 13, 14, 15, 16). Moreover, experiments in mice have provided evidence that virulent M. tuberculosis evades IFN--dependent mechanisms of immune control in vivo (17).
We and others (15, 16, 18, 19, 20, 21, 22) have reported that M. tuberculosis inhibits human and murine macrophage responses to IFN-. Moreover, we have found that M. tuberculosis uses at least two mechanisms to block responses to IFN-. One is initiated by lipoproteins, acting through TLR2 and MyD88, whereas the other is initiated by mycobacterial peptidoglycan, acting in a TLR2- and MyD88-independent fashion (15). In this study, we examined whether lipoprotein-mediated inhibition is dominant in the context of intact M. tuberculosis. We found that disruption of the M. tuberculosis lipoprotein signal peptidase (lspA) resulted in loss of mature lipoproteins and lipoprotein TLR2 agonist activity, but had no effect on the mutant strain’s ability to inhibit macrophage responses to IFN-. The disruption of TLR2 on macrophages, however, significantly reduced the cells’ sensitivity to inhibition by live M. tuberculosis and M. tuberculosis lysates.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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All M. tuberculosis strains were grown in shaking cultures to midlog phase in Middlebrook 7H9 broth (Difco) supplemented with 0.2% glycerol, 10% oleic acid-dextrose-catalase (OADC)4 and 0.05% Tween 80. M. tuberculosis cultures used for infection of macrophages were grown in the same medium with low-endotoxin albumin-dextrose-catalase (ADC) (Sigma-Aldrich) substituted for OADC. This media contained <1 endotoxin unit/ml. -Irradiated M. tuberculosis H37Rv (Colorado State University, Fort Collins, CO, National Institutes of Health, National Institute of Allergy and Infectious Diseases Contract N01 AI-75320) was prepared as described previously (19). Phosphatidylinositol mannan1 + 2 (PIM; Colorado State University) was stored at 1 mg/ml in DMSO (Sigma-Aldrich) at –80°C. (S)-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH, 3HCl (Pam3CSK4; Calbiochem) and (S)-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-(R)-Cys-(S)-Ser-(S)-Lys4 x 3 CF3COOH (Pam2CSK4; InvivoGen) were stored at 1 mg/ml in endotoxin-tested water (Invitrogen Life Technologies) at –80.
Mice
C57BL/6, IL-6–/–, and TLR2–/– mice were obtained from The Jackson Laboratory and maintained under specific pathogen-free conditions. TLR2–/– mice from The Jackson Laboratory have been backcrossed onto the C57BL/6 background for nine generations. All work with animals was approved by the New York University School of Medicine Institutional Animal Care and Use Committee.
Cell lines
RAW264.7 and HEK-293 cells (American Type Culture Collection (ATCC) were grown in DMEM with 10% heat-inactivated FCS and 2 mM L-glutamine (all obtained from Invitrogen Life Technologies). HEK 293-TLR2 cells were provided by Dr. D. Golenbock (University of Massachusetts, Worcester, MA), and were grown in the same medium with 500 µg/ml Geneticin (Invitrogen Life Technologies). Cells were allowed to adhere overnight before M. tuberculosis treatment or infection. L929 cells (ATCC) were maintained in DMEM with 10% FCS, 2 mM L-glutamine, 100 µM nonessential amino acids, and 55 µM 2-ME (Invitrogen Life Technologies). L cell-conditioned medium was collected from confluent L929 cultures and stored at –20°C.
lspA knockout and complement
A lspA knockout mutant was created with the conditionally replicating mycobacteriophage as described previously (23). The upper (886 bp) and lower (1125 bp) allelic exchange substrates (AES) were PCR amplified (for a complete list of primers see Table I) from H37Rv genomic DNA and ligated into pCR2.1-TOPO (Invitrogen Life Technologies). The AES were sequenced and subcloned into pYUB854. Following transduction of H37Rv and plating on Middlebrook 7H9 agar with ADC and hygromycin (50 µg/ml), six colonies were picked and screened for the absence of lspA transcript by real-time RT-PCR. A PCR fragment encoding the entire lspA open reading frame and the preceeding 470 bp was ligated into pMV306 (24) for complementation of the lspA mutant. Transformed bacteria were plated on Middlebrook 7H9 agar supplemented with ADC and 25 µg/ml kanamycin, and four colonies were picked and analyzed for complementation by real-time RT-PCR.
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Protein extraction was performed according to a published protocol (25). Briefly, 2 ml of bacterial culture at midlog phase was sedimented and washed once with 2 ml of PBS and resuspended in 300 µl of extraction buffer (50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.6% SDS, 10 mM NaPO4, and Roche Complete Protease Inhibitor Cocktail) and added to 0.2 ml of 0.1 mm zirconia/silica beads. The tube was vortexed for 5 min and subsequently sedimented for 2 min at 12,600 x g. The supernatant was removed and the protein concentration determined with the BCA protein assay kit (Pierce). Micrograms (0.2–1.5) were separated on a 10% SDS-PAGE gel, and immunoblotting was performed as Previously described (19). Polyclonal anti-MPT83 was provided by Dr. H. G. Wiker (Gades Institutt, Bergen, Norway) and incubated overnight at 4°C at a dilution of 1/2,000.
Infection of cell monolayers
Bacteria from midlog cultures (A580 
0.5) were sedimented, resuspended in cell culture media, vortexed for 3 min in O-ring tubes containing two 5-mm glass beads (Fisher Scientific), and passed through a 5-µm sterile filter (Millipore). Bacteria were enumerated in a Petroff-Hausser chamber and also serially diluted and plated on Middlebrook 7H10 and 7H9/ADC agar plates.
Bacterial lysates for cell stimulation
Midlog cultures of M. tuberculosis grown in low-endotoxin media were sedimented, washed once with PBS, and resuspended in 1 ml of extraction buffer or PBS with or without protease inhibitors. Bacteria were disrupted in O-ring tubes containing 0.5 ml of 0.1-mm zirconia/silica beads and mechanically disrupted with three 1-min pulses at maximum speed in a BioSpec Products BeadBeater with 3-min intervals on ice. The lysates were sedimented for 1 min at 12,600 x g, the supernatants were transferred, and the efficacy of disruption was assayed as soluble protein concentration (BCA protein assay kit; Pierce) and enumeration of viable bacteria before and after beadbeating. Equivalent amounts of protein (0.8 µg/1 x 106 CFU) were released from all three strains upon bead beating, indicating a comparable efficiency of lysis. Furthermore, a 104-fold difference was seen in the number of viable bacteria before and after beadbeating of H37Rv (our unpublished observations).
Isolation and culture of bone marrow-derived macrophages (BMDM)
Bone marrow cells were isolated as described previously (26), then erythrocytes were lysed using ACK lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 100 µM EDTA (pH 7.4); all obtained from Sigma-Aidrich). Cells were plated in 150 x 25-mm bacterial grade petri dishes (Falcon) at 3–4 x 106/plate, in DMEM with 10% FCS, 20% L929-cell conditioned medium, 1 mM sodium pyruvate, 2 mM L-glutamine, and 100 U/ml penicillin/100 µg/ml streptomycin sulfate (Invitrogen Life Technologies). The cells were incubated at 37°C in 5% CO2 for 3 days, after which the medium was replaced. Adherent cells were harvested between days 6 and 10; the cells were incubated in ice-cold Dulbecco’s PBS (DPBS) containing 5 mM EDTA for 20 min, detached from the plates by vigorous pipetting, then washed and replated at 5 x 105/well in 12-well tissue culture plates in DMEM supplemented with 10% FCS, 10% L929-cell conditioned medium, 1 mM sodium pyruvate, and 2 mM L-glutamine.
Flow cytometry
After infection with live M. tuberculosis or treatment with PIM or Pam3CSK4, RAW264.7 cells were treated with 20 ng/ml recombinant murine IFN- (400–4,000 U/ml; BD Pharmingen). Cells were rinsed with DPBS and incubated for 10 min in DPBS containing 1 mM EDTA. Cells were then scraped, washed, and stained with PE-conjugated anti-mouse I-A/I-E (BD Pharmingen). For experiments with live M. tuberculosis, cells were fixed overnight with 1% paraformaldehyde (Sigma-Aldrich). Cells were analyzed on a FACSCalibur (10,000 total events gated by forward and side scatter; BD Biosciences).
mRNA quantitation
For experiments with Pam3CSK4, total RNA was harvested using Qiagen RNeasy columns. For M. tuberculosis cultures and cells infected with live M. tuberculosis or treated with M. tuberculosis whole cell lysates, total RNA was harvested using TRIzol reagent (Invitrogen Life Technologies). DNA was removed using DNA-free (Ambion). Total RNA was quantitated using RiboGreen (Molecular Probes). Ten nanograms of bacterial or 1 µg of mammalian RNA was reverse transcribed using the Reverse Transcription System (Promega). For mammalian RNA, reverse transcription was primed with random hexamers and oligo(dT). For bacterial RNA, reverse transcription was primed with gene-specific primers (listed in Table I). The cDNA equivalent of 0.2 ng of total bacterial RNA or 10 ng of total mammalian RNA (50 ng for CIITA) was analyzed by quantitative PCR using Platinum SYBR Green qPCR SuperMix UDG (Invitrogen Life Technologies) on an MJ Research Opticon 2 (for primers, see Table I). For quantitation, the relative values were determined by comparing the threshold cycle of each sample to a standard curve consisting of serial dilutions of a positive control cDNA sample.
ELISA
Cell supernatants for ELISA were harvested after the indicated time and stored at –80°. ELISAs were performed with optimized Ab sets specific for human IL-8 (BD Pharmingen) or murine TNF- (eBioscience), used according to the manufacturers’ directions. Samples were diluted 1/10, 1/100 or 1/1000 to allow detection within the range of each assay. Results for experiments with live or lysed M. tuberculosis H37Rv were quantitated using a SpectraMax 340PC384 spectrophotometer, all other results were quantitated using a ELx800UV spectrophotometer (Bio-Tek Instruments).
Inhibition of protein synthesis
Cells were pretreated with cycloheximide (CHX) (Calbiochem) or solvent control (0.02% ethanol; Sigma-Aldrich) for 1 h before addition of M. tuberculosis whole cell lysates. After 8 h, media was removed and stored at –80°C for ELISA and cytotoxicity assays. Cells were treated with IFN- or left untreated for 4 h, and RNA was harvested as described above.
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Results |
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We and others (15, 21) have found that the M. tuberculosis 19-kDa lipoprotein inhibits macrophage responses to IFN- and that this inhibition is dependent on TLR2 and MyD88. Although considerable attention has been focused on the properties of the M. tuberculosis 19-kDa lipoprotein, it is becoming increasingly clear that this lipoprotein is not the sole mediator of M. tuberculosis inhibition of macrophages. Not only is the 19-kDa lipoprotein not required for M. bovis bacille Calmette-Guerin (BCG) (4) inhibition of IFN--dependent Ag processing in infected macrophages, at least one other M. tuberculosis lipoprotein is capable of causing this inhibition (27).
We have also previously found that in addition to native, full-length 19-kDa lipoprotein, a synthetic triacylated hexapeptide containing the N-terminal 6 aa residues of the 19-kDa lipoprotein can also inhibit macrophage responses to IFN- (15). To determine whether treatment with an unrelated lipopeptide could reproduce this inhibition, we examined Pam3CSK4, a triacylated hexapeptide that has no peptide sequence identity with any M. tuberculosis open reading frame, as determined by BLASTP search (28) of all nonredundant GenBank coding sequences of M. tuberculosis. Treatment of RAW264.7 cells with Pam3CSK4 profoundly inhibited IFN- induction of MHC class II surface expression; as little as 1 nM Pam3CSK4 caused >90% inhibition of IFN- induction of surface MHC class II (Fig. 1). IFN- induction of CIITA mRNA was also potently inhibited in Pam3CSK4-treated cells (our unpublished observations), and this inhibition was likely responsible for the defect in MHC class II surface expression. These findings are consistent with previous work (15, 21) showing the inhibitory effects of higher (micromolar) concentrations of synthetic bacterial lipopeptides on responses to IFN-.
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The finding that a synthetic lipoprotein unrelated to the 19-kDa lipoprotein is a potent inhibitor of IFN- suggests that other M. tuberculosis lipoproteins could contribute to inhibition of macrophages via TLR2. Bioinformatic analysis of the M. tuberculosis genome revealed 99 putative lipoproteins, accounting for 2.5% of its proteome (29). Given the number and variety of lipoproteins and the evidence that the TLR2 agonist activity is attributable to the triacylated amino terminus rather than other protein domains, it is unlikely that deletion of any single lipoprotein would render M. tuberculosis unable to inhibit macrophage responses to IFN-. To evaluate the global role of mature lipoproteins in the modulation of macrophage responses to IFN-, we chose to disrupt the lspA gene that encodes prolipoprotein signal peptidase II, the enzyme that cleaves the signal sequence from diacylated prolipoproteins at a site directly preceding the lipidated cysteine residue. The exposure of the primary amine group on the newly exposed N-terminal cysteine then allows for the final acylation and creation of the mature triacylated lipoproteins capable of serving as TLR2 agonists (Fig. 2A).
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To characterize the role of prolipoprotein processing in generating M. tuberculosis TLR2 agonist activity, we examined the ability of wild-type M. tuberculosis H37Rv, 
lspA, and the lspAattB::lspA complemented strain to stimulate HEK293 cells stably transfected to constitutively express human TLR2 (293-TLR2 cells). These cells produce IL-8 in response to IL-1
or lipopeptide TLR2 agonists (our unpublished observations).
Although wild-type H37Rv induced 293-TLR2 cells to produce IL-8 in a dose-dependent manner, the response to lspA bacteria was reduced by >95% compared with that of wild-type H37Rv and the complemented strain (Fig. 4A). HEK293 cells that did not express TLR2 did not produce IL-8 upon treatment with any of the strains of M. tuberculosis tested (our unpublished observations).
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lspA bacteria. Treatment of 293-TLR2 cells with wild-type whole-bacteria lysates induced 25-fold greater amounts of IL-8 than that seen with live bacteria, but lysis of the lspA strain did not correct the defect in activation of 293-TLR2 cells observed with live bacteria: the amount of IL-8 secreted in response to lysates from lspA bacteria was only 2% percent of that seen with lysates from wild-type bacteria (Fig. 4B). This finding indicates that the defect in stimulation of 293-TLR2 cells is not simply due to sequestration or mistrafficking of TLR-2 agonists in the lspA mutant. Furthermore, expression of transfected CD14, a coreceptor for some TLR2 agonists, had only a minor effect on IL-8 secretion in response to lysates from the lspA strain; in transfected cells, there was a >90% decrease in the IL-8 secreted in response to the lspA mutant compared with the wild-type strain (our unpublished observations). Macrophage activation by lipoprotein and nonlipoprotein components in M. tuberculosis
M. tuberculosis has been reported to activate macrophages through TLR2 and TLR4 (31), and through one or more MyD88-independent mechanisms (32). To determine the relative contributions of lipoprotein and nonlipoprotein proinflammatory agonist activities of M. tuberculosis, we infected RAW264.7 cells with live wild-type H37Rv, lspA, and the lspAattB::lspA complement. At 8 h postinfection, the lspA mutant stimulated RAW264.7 cells to secrete 20–30% less TNF- than RAW264.7 cells infected with wild-type bacteria at the same multiplicity of infection (MOI) (Fig. 5A). To compare TNF- secretion in response to wild-type, lspA and complemented strains at different times posttreatment, we treated RAW264.7 cells with whole cell lysates of the three strains for 4, 8, 12, and 24 h. After 8 and 12 h of treatment, significantly less TNF- was secreted by RAW264.7 cells treated with whole cell lysates from the lspA strain than from the wild-type or complemented strain (Fig. 5B). This difference persisted at the 24-h time point, but was no longer statistically significant. The partial response of RAW264.7 cells to the lspA mutant indicates that lipoproteins are responsible for a portion of the proinflammatory stimulation of macrophages in response to infection with live M. tuberculosis. There remains, however, significant lipoprotein-independent stimulation of macrophage TNF- secretion in response to M. tuberculosis.
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To investigate the relative contribution of lipoproteins to M. tuberculosis inhibition of macrophage responses to IFN-, we infected RAW264.7 cells with wild-type H37Rv, lspA, and the lspAattB::lspA complement. This revealed no difference in the ability of the three strains to inhibit the induction of MHC class II surface expression in response to IFN- at a wide range of MOI (Fig. 6). Similarly, we found no difference between wild-type H37Rv and lspA in the inhibition of IFN- induction of CIITA mRNA in infected RAW264.7 cells (our unpublished observations). These results suggest that, although lipoproteins contribute to inhibition of macrophage responses to IFN-, they are dispensable for the inhibition of macrophage responses to IFN- by live M. tuberculosis.
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Having found that expression of mature lipoproteins was not required for M. tuberculosis inhibition of macrophage responses to IFN-, we tested the contribution of TLR2 to the inhibitory effects of wild-type M. tuberculosis and the lspA mutant. If all of TLR2 agonist activity of lspA mutant was lost, as suggested by the 293-TLR2 cells, then we would expect to see no difference in M. tuberculosis inhibition of TLR2+/+ and TLR2–/– macrophages by the lspA mutant and wild-type strains. Infection with either strain of M. tuberculosis resulted in inhibition of IFN- induction of CIITA mRNA in TLR2–/– macrophages, but a significantly higher MOI was required for TLR2–/– compared with TLR2+/+ macrophages (Fig. 7A). To a test a broader range of concentrations of M. tuberculosis, we also assayed the inhibition of IFN- responsiveness of wild-type and TLR2–/– macrophages treated with M. tuberculosis whole cell lysates (high inocula of live M. tuberculosis result in death of macrophages). We found a significant difference in dose-response to M. tuberculosis between the two types of macrophages (Fig. 7B). In contrast, there was relatively little difference in the inhibition caused by the wild-type and lspA mutant strains of M. tuberculosis. These results confirm that, whereas M. tuberculosis lipoprotein-TLR2 interactions contribute to inhibition of macrophage responses to IFN-, they are not essential, and indicate that additional TLR2-dependent and TLR2-independent mechanisms of inhibition exist.
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To examine the difference in sensitivity to TLR2 agonists between murine macrophages and 293-TLR2 cells, we compared the response of RAW264.7 and 293-TLR2 cells to lipopeptides and PIM, a nonlipoprotein TLR2 agonist from M. tuberculosis (33, 34). 293-TLR2 cells responded to stimulation by 2 nM Pam3CSK4, a triacylated lipopeptide that signals via TLR2/TLR1 heterodimers (35), with secretion of IL-8, whereas 10, 1, or 0.1 µg/ml PIM (10 µg/ml PIM is 5.6 µM) induced <1% as much IL-8 as Pam3CSK4 (Fig. 8A). 293-TLR2 cells also secreted significant amounts of IL-8 in response to nanomolar concentrations of Pam2CSK4, the diacylated analog on Pam3CSK4, and macrophage-activating lipopeptide-2, a diacylated lipopeptide that signals via TLR2/TLR6 heterodimers (36) (our unpublished observations). RAW264.7 cells, by contrast, secreted a similar amount of TNF- in response to 2 nM Pam2CSK4, 2 nM Pam3CSK4, and 5 µg/ml PIM (Fig. 8B). In addition, treatment of RAW264.7 cells with as little as 0.5 µg/ml PIM inhibited subsequent IFN- induction of MHC class II surface expression (Fig. 8C). These results indicate that at least one nonlipoprotein TLR2 agonist that is not detected by 293-TLR2 cells is able to inhibit macrophage responses to IFN-, and suggest that PIM and related mycobacterial cell wall glycolipids may be responsible for the effects of the lspA strain on murine macrophages.
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requires new protein synthesis
Much of the work on TLR2 and other innate pattern recognition receptors has focused on their proinflammatory activities, and has examined acute effects of innate immune activation. Because our experiments reveal an inhibitory effect of innate immune activation on responses to IFN-, we investigated the mechanism by which the observed inhibition occurs. We found that 8 h of pretreatment with M. tuberculosis was required for maximal inhibition of responses to IFN- (Fig. 9A). The need for extended pretreatment suggested that inhibition of the response to IFN- requires new protein synthesis. Accordingly, CHX inhibition of macrophage protein synthesis prevented the ability of either wild-type M. tuberculosis or the lspA mutant strain to inhibit IFN- induction of CIITA gene expression (Fig. 9B). These results indicate that although M. tuberculosis stimulation of macrophages via innate immune receptors has an immediate proinflammatory effect, longer treatment with M. tuberculosis induces the expression of one or more cellular proteins that inhibit macrophage responses to IFN-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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, with consequences that include defective killing of M. tuberculosis (15) and class II Ag presentation (20, 21, 37, 38, 39).
Efforts to elucidate the mechanisms used by M. tuberculosis to block macrophage responses to IFN- have revealed that a purified 19-kDa bacterial lipoprotein, acting through TLR2 and MyD88, can initiate signals that disrupt IFN- gene regulation (15, 21, 37). Moreover, we found that peptidoglycan from M. tuberculosis (or a component that copurifies with peptidoglycan) can act in a TLR2- and MyD88-independent manner to block responses to IFN- (15). Although substantial attention has been focused on the ability of the 19-kDa lipoprotein to inhibit responses to IFN- and class II Ag presentation, a 19-kDa lipoprotein-null strain of BCG was as capable as a 19-kDa lipoprotein-replete strain at inhibiting class II Ag presentation by IFN--stimulated macrophages (27). Moreover, other lipoproteins and nonlipoprotein components of M. tuberculosis can exhibit the same effects when examined as isolated components (15, 27). Therefore, we have used genetic modification of M. tuberculosis to determine the relative roles of lipoproteins and nonlipoprotein components in inhibition of macrophage responses to IFN-, in the context of the whole bacteria.
We reasoned that because acylation of a small synthetic lipopeptide was essential for inhibition of macrophage responses to IFN- (15), disruption of prolipoprotein processing would provide valuable information on the relative role of M. tuberculosis lipoproteins for inhibition of responses to IFN-. Therefore, we disrupted bacterial lipoprotein processing by deleting the gene (lspA) that encodes the lipoprotein signal peptidase of M. tuberculosis. We found that the lspA mutant strain lacked 
95% of the TLR2 agonist activity of its parental strain, as assayed by a reporter cell line. Because we found that whole cell lysates of the lspA mutant were also unable to stimulate 293-TLR2 cells, it does not appear that the lack of lipoprotein processing results only in sequestration of lipoproteins or other TLR2 agonists. We also found that the lspA mutant strain induced less TNF- secretion from murine macrophages than the wild-type or complemented strains. This decreased induction of TNF- was most apparent after 8 and 12 h of M. tuberculosis treatment. Extrapolation of the curves from the time course of treatment suggests that >48 h of treatment would be required for the lspA mutant to reach the level of TNF- induction of the wild-type and complemented strains.
We then examined the relative importance and/or potency of lipoproteins in the inhibition of macrophage responses to IFN- in the context of whole, live M. tuberculosis. The lspA strain exhibited no distinguishable difference in the potency or extent of inhibiting IFN- induction of CIITA mRNA or surface MHC class II protein in macrophages, which indicates that lipoproteins are redundant in the context of inhibition by whole M. tuberculosis.
To examine the contribution of TLR2-independent pathways in the inhibition of macrophage responses by whole M. tuberculosis, we compared the M. tuberculosis inhibition of response to IFN- in macrophages from TLR2+/+ and TLR2–/– mice. We found that macrophages lacking TLR2 were significantly less sensitive to inhibition by M. tuberculosis than wild-type macrophages. This difference in sensitivity was seen using both wild-type M. tuberculosis and the lspA mutant strain. This finding was surprising because it suggests that the 293-TLR2 cells do not respond well to nonlipoprotein TLR2 agonists in the lspA mutant.
We compared the responses of macrophages and 293-TLR2 cells to PIM, a glycolipid purified from the cell wall of M. tuberculosis. PIM has been shown to activate CHO cells expressing human TLR2 (33), and PIM activation of murine macrophages requires TLR2 (34). We found, however, that PIM did not activate 293-TLR2 cells, even when the cells were transfected with human CD36. Concentrations of PIM that potently induced TNF- secretion from macrophages did not induce significant IL-8 secretion from 293-TLR2 cells. In addition, concentrations of PIM unable to stimulate 293-TLR2 cells potently inhibited macrophage responses to IFN-. These results suggest that, in the absence of mature lipoproteins, nonlipoprotein TLR2 agonists from M. tuberculosis are able to inhibit macrophage responses to IFN-. In addition to PIM, lipomannan from M. tuberculosis has been reported to act as a nonlipoprotein TLR2 agonist (40, 41). Peptidoglycans from Gram-positive bacteria (42) and BCG (43) have also been shown to exhibit TLR2 agonist activity, although the interpretation of these results is controversial (44). It is likely that, although the 293-TLR2 cells respond to both diacylated and triacylated lipopeptides, they lack one or more coreceptors required to sense nonlipoprotein TLR2 agonists.
Recent work (45) has demonstrated that disruption of lspA in M. tuberculosis results in a loss of virulence in wild-type BALB/c and CBA/J mice. This attenuation may indicate a specific role for lipoproteins in virulence, or it may simply reflect the requirement for one or more of the 99 putative lipoproteins processed by LspA for optimal M. tuberculosis viability in mouse macrophages and tissues. That work did not examine the TLR2 agonist activity of the mutant strain, nor did it examine the ability of lspA-null M. tuberculosis to activate macrophages.
The preponderance of attention to TLR2 and other pattern-recognition receptors has been concentrated on activating signals that result in cytokine secretion and cellular phenotypic changes such as dendritic cell maturation. The finding that a later effect of TLR2 stimulation includes inhibition of IFN- signaling, through induction of one or more proteins, extends the spectrum of activities attributable to innate immune receptors. Although our results indicate that inhibition of IFN- signaling is relieved by inhibition of protein synthesis, we have not yet identified the essential protein(s) that mediates inhibition of responses to IFN-. One obvious candidate was suppressor of cytokine signaling-1 (SOCS1), which can be induced by M. tuberculosis (16), and which inhibits cellular responses to IFN- by blocking the interaction of JAK1 and JAK2 with STAT1 (46). Our previous finding that M. tuberculosis inhibits responses to IFN- without inhibiting activation (18) or function (19) of STAT1 provides strong evidence against a role for SOCS1 in this context. Moreover, recent experiments (38) directly excluded a role for SOCS1 by finding that 19-kDa lipoprotein inhibited IFN--dependent MHC class II Ag presentation in macrophages from SOCS1-deficient mice. In addition, we have previously reported that M. tuberculosis-induced IL-6 can provide "bystander" inhibition of IFN- induction of MHC class II (26). To determine whether induction of IL-6 was sufficient for M. tuberculosis inhibition of macrophage responses to IFN-, we tested the effects of M. tuberculosis treatment on IL-6–/– BMDM. Consistent with our previous observations, both C57BL/6 and IL-6–/– BMDM treated with M. tuberculosis had a defective response to IFN- (our unpublished observations). These results indicate that although M. tuberculosis stimulation of macrophages via innate immune receptors has an immediate proinflammatory effect, a late effect of innate immune receptor stimulation includes induction of one or more proteins that act to inhibit macrophage transcriptional responses to IFN-. M. tuberculosis, by virtue of its ability to survive in nonactivated macrophages, appears to have evolved mechanisms to take advantage of this late effect to enhance its survival in macrophages exposed to an important mediator of adaptive cellular immunity.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by National Institutes of Health Grants R01 AI46097 and K08 AI061105 and a graduate student fellowship from the Western Affiliate of the American Heart Association. 
2 N.B. and E.Z.K. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Joel D. Ernst, Departments of Medicine (Infectious Diseases) and Microbiology, New York University School of Medicine, 550 First Avenue, NBV16S7, New York, NY 10016. E-mail address: joel.ernst{at}med.nyu.edu
4 Abbreviations used in this paper: OADC, oleic acid-dextrose-catalase; ADC, albumin-dextrose-catalase; PIM, phosphatidylinositol mannan1+2; Pam3CSK4, (S)-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH, 3HCl; Pam2CSK4, (S)-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-(R)-Cys-(S)-Ser-(S)-Lys4 x 3 CF3COOH; AES, allelic exchange substrate; BMDM, bone marrow-derived macrophage; CHX, cycloheximide; BCG, Mycobacterium bovis bacille Calmette-Guerin; MOI, multiplicity of infection; SOCS1, suppressor of cytokine signaling-1.
Received for publication July 21, 2005. Accepted for publication December 21, 2005.
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-secreting CD8+ T lymphocytes specific for Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 95: 270-275. , IL-4 and TNF- protein and CD8 T cells producing IL-12p40 mRNA in human lung tuberculous granulomas. Immunology 105: 325-335. [Medline] Interferon activates human macrophages to become tumoricidal and leishmanicidal but enhances replication of macrophage-associated mycobacteria. Infect. Immun. 50: 1-8. -activated bone marrow macrophages and differential susceptibility among strains of Mycobacterium tuberculosis. J. Immunol. 138: 4408-4413. [Abstract]-interferon, interleukin-4, and tumor necrosis factor. Infect. Immun. 58: 2675-2677. Interferon-activated human macrophages and Toxoplasma gondii, Chlamydia psittaci, and Leishmania donovani: antimicrobial role of limiting intracellular iron. Infect. Immun. 59: 4684-4686. as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158: 670-689. . J. Exp. Med. 160: 600-605. -interferon on human monocytes and murine peritoneal macrophages. Immunology 59: 333-338. [Medline] through myeloid differentiation factor 88-dependent and -independent mechanisms. J. Immunol. 172: 6272-6280. and Mycobacterium tuberculosis: signaling roles of nitric oxide synthase-2 and phagocyte oxidase. J. Exp. Med. 194: 1123-1140. transcriptional responses without inhibiting activation of STAT1. J. Immunol. 163: 3898-3906. without inhibiting STAT1 function. J. Immunol. 171: 2042-2049. . J. Immunol. 171: 4750-4757. yet mediates few responses to Mycobacterium tuberculosis. J. Exp. Med. 198: 987-997. interferon-regulated HLA-DR and FcR1 on human macrophages through Toll-like receptor 2. Infect. Immun. 71: 4487-4497. -induced class II transactivator expression by a 19-kDa lipoprotein from Mycobacterium tuberculosis: a potential mechanism for immune evasion. J. Immunol. 171: 175-184. This article has been cited by other articles:
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M. Jeyanathan, J. Mu, K. Kugathasan, X. Zhang, D. Damjanovic, C. Small, M. Divangahi, B. J. Petrof, C. M. Hogaboam, and Z. Xing Airway Delivery of Soluble Mycobacterial Antigens Restores Protective Mucosal Immunity by Single Intramuscular Plasmid DNA Tuberculosis Vaccination: Role of Proinflammatory Signals in the Lung J. Immunol., October 15, 2008; 181(8): 5618 - 5626. [Abstract] [Full Text] [PDF]
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P. A. Sieling, P. J. Hill, K. M. Dobos, K. Brookman, A. M. Kuhlman, M. Fabri, S. R. Krutzik, T. H. Rea, D. G. Heaslip, J. T. Belisle, et al. Conserved Mycobacterial Lipoglycoproteins Activate TLR2 but Also Require Glycosylation for MHC Class II-Restricted T Cell Activation J. Immunol., May 1, 2008; 180(9): 5833 - 5842. [Abstract] [Full Text] [PDF]
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 W. W. Yew and C. C. Leung Update in Tuberculosis 2006 Am. J. Respir. Crit. Care Med., March 15, 2007; 175(6): 541 - 546. [Full Text] [PDF]
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M. Rezwan, T. Grau, A. Tschumi, and P. Sander Lipoprotein synthesis in mycobacteria Microbiology, March 1, 2007; 153(3): 652 - 658. [Abstract] [Full Text] [PDF]
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), 
), or 
) at a range of MOIs, as indicated. Cell supernatants were harvested after 24 h, and secretion of IL-8 into the medium was quantitated by ELISA (mean of duplicate assays). Background level of IL-8 secreted into media of untreated cells (70.9 pg/ml) was subtracted from all values. Results are representative of four independent experiments. Because small changes in MOI led to significant changes in IL-8 secretion, all MOIs were confirmed by quantitative culture. B, 293-TLR2 cells were treated with whole cell lysates from wild-type H37Rv, 
) mice were infected with wild-type H37Rv (solid lines) or the 
), or mock treatment (
) in the presence of CHX or ethanol. Cells were then treated with IFN-