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Infection and Immunity, August 2003, p. 4487-4497, Vol. 71, No. 8
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.8.4487-4497.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Mycobacterium tuberculosis 19-Kilodalton Lipoprotein Inhibits Gamma Interferon-Regulated HLA-DR and Fc{gamma}R1 on Human Macrophages through Toll-Like Receptor 2

Adam J. Gehring,1,2 Roxana E. Rojas,2 David H. Canaday,2 David L. Lakey,3 Clifford V. Harding,1 and W. Henry Boom2,4*

Department of Pathology,1 Division of Infectious Disease,2 Tuberculosis Research Unit, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio,4 Division of Infectious Disease, Center for Pulmonary and Infectious Disease Control, University of Texas Health Center at Tyler, Tyler, Texas3

Received 18 March 2003/ Accepted 15 May 2003


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ABSTRACT
 
Mycobacterium tuberculosis survives in macrophages in the face of acquired CD4+ T-cell immunity, which controls but does not eliminate the organism. Gamma interferon (IFN-{gamma}) has a central role in host defenses against M. tuberculosis by activating macrophages and regulating major histocompatibility complex class II (MHC-II) antigen (Ag) processing. M. tuberculosis interferes with IFN-{gamma} receptor (IFN-{gamma}R) signaling in macrophages, but the molecules responsible for this inhibition are poorly defined. This study determined that the 19-kDa lipoprotein from M. tuberculosis inhibits IFN-{gamma}-regulated HLA-DR protein and mRNA expression in human macrophages. Inhibition of HLA-DR expression was associated with decreased processing and presentation of soluble protein Ags and M. tuberculosis bacilli to MHC-II-restricted T cells. Inhibition of HLA-DR required prolonged exposure to 19-kDa lipoprotein and was blocked with a monoclonal antibody specific for Toll-like receptor 2 (TLR-2). The 19-kDa lipoprotein also inhibited IFN-{gamma}-induced expression of Fc{gamma}RI. Thus, M. tuberculosis, through 19-kDa lipoprotein activation of TLR-2, inhibits IFN-{gamma}R signaling in human macrophages, resulting in decreased MHC-II Ag processing and recognition by MHC-II-restricted CD4 T cells. These findings provide a mechanism for M. tuberculosis persistence in macrophages.


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INTRODUCTION
 
Mycobacterium tuberculosis infection remains a serious health problem throughout the world. In most healthy persons, acquired immunity, mediated by T cells, controls but does not eradicate M. tuberculosis infection, resulting in persistent bacilli within macrophages. One-third of the world's population is thought to harbor such persistent bacilli and thus at risk for reactivation disease when immunity fails. The mechanisms used by M. tuberculosis to establish and maintain persistent infection in human macrophages are not understood. Major histocompatibility complex type II (MHC-II)-restricted CD4 T cells play a key role in protective immunity to M. tuberculosis during primary infection as well as in containing persistent bacilli (18, 24, 25, 31, 33). Depletion of CD4 T cells results in reactivation tuberculosis in both mice and humans (26, 38).

Gamma interferon (IFN-{gamma}) produced by activated T cells is a central regulator of the immune response to M. tuberculosis (8, 42). IFN-{gamma} activates antimicrobial mechanisms of macrophages and regulates MHC-II antigen (Ag) processing by up-regulating MHC-II mRNA and protein expression (5). The role of IFN-{gamma} in M. tuberculosis infection differs between mice and humans. In mice, IFN-{gamma} activation of macrophages stimulates production of nitric oxide (NO), resulting in killing of M. tuberculosis bacilli (9, 37). Direct activation of human macrophages by IFN-{gamma} does not result in increased killing of intracellular bacilli (12, 35). Furthermore, the role of NO in controlling M. tuberculosis in human macrophages remains controversial (4). Thus, the primary role of IFN-{gamma} in human immunity to M. tuberculosis may lie in its ability to up-regulate MHC-II Ag processing for CD4 T cells. Inhibition of IFN-{gamma}-regulated processing of mycobacterial Ags for CD4 T cells provides a mechanism for M. tuberculosis to escape detection and persist within macrophages. Infection of macrophages with mycobacteria or exposure to mycobacterial constituents can inhibit IFN-{gamma} signaling (15, 41). However, the molecular ligands and mechanism(s) responsible for inhibition of IFN-{gamma} signaling pathways by M. tuberculosis in macrophages are not understood.

M. tuberculosis activates macrophages via Toll-like receptors (TLRs). M. tuberculosis contains pathogen-associated molecular patterns, recognized by TLRs, that result in production of proinflammatory cytokines (tumor necrosis factor alpha, interleukin-1 [IL-1], IL-12, and IL-18) (14, 21). Mycobacteria contain well-defined ligands for TLR-2 and may also have ligands for TLR-4 (22). The 19-kDa lipoprotein of M. tuberculosis (lqpH/Rv3763) is a TLR-2 ligand (6, 10). Earlier studies in our laboratory determined that this 19-kDa lipoprotein inhibited MHC-II Ag processing in murine bone marrow macrophages (29, 30).

The present study was undertaken to determine the effect of TLR-2 signaling by the 19-kDa lipoprotein on IFN-{gamma}-regulated responses in human macrophages. Prolonged exposure to 19-kDa lipoprotein decreased IFN-{gamma}-regulated expression of HLA-DR protein and mRNA and was not associated with macrophage apoptosis. Inhibition of IFN-{gamma}-mediated expression of HLA-DR by 19-kDa lipoprotein resulted in decreased processing and presentation of soluble protein Ags and M. tuberculosis bacilli to MHC-II-restricted CD4 T cells. The 19-kDa lipoprotein also decreased expression of the IFN-{gamma}-regulated protein Fc{gamma}RI. Blocking of TLR-2 on macrophages prevented 19-kDa lipoprotein-mediated inhibition of HLA-DR Ag processing and presentation. Thus, prolonged signaling through TLR-2 by the 19-kDa lipoprotein of M. tuberculosis blocked IFN-{gamma} activation of human macrophages.


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MATERIALS AND METHODS
 
Cells and media. Unless otherwise specified, cells were cultured at 37°C in a 5% CO2 atmosphere. THP-1 cells (American Type Culture Collection [ATCC]) were maintained in RPMI 1640 (BioWhittaker, Walkersville, Md.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, Utah), 50 µM 2-mercaptoethanol, 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES buffer, nonessential amino acids, 100 U of penicillin per ml, and 100 µg of streptomycin per ml (BioWhittaker). The T-cell hybridomas DB1 and 1T1A were maintained in Dulbecco's modified Eagle's medium (DMEM) (BioWhittaker) supplemented as indicated above (complete DMEM). Infection medium was DMEM supplemented with 10% non-heat-inactivated FBS without antibiotics.

Abs and reagents. The HLA-DR specific antibody (Ab) (clone TÜ 36, immunoglobulin G2b [IgG2b]), the Fc{gamma}R1-specific Ab (clone 10.1, IgG1), and isotype-matched control Abs (mouse IgG2b and IgG1) used for flow cytometry were obtained from Caltag Laboratories (Burlingame, Calif.). The L243 Ab used for HLA-DR blocking was purified from L243 B-cell hybridoma (ATCC) supernatant by using a protein G column. The 2392 anti-TLR-2 blocking Ab was graciously provided by Paul Godowski, Genentech Inc. (San Francisco, Calif.). Recombinant Ag85B of M. tuberculosis was produced as described previously (19).

M. tuberculosis H37Ra and purification of the 19-kDa lipoprotein. M. tuberculosis H37Ra (ATCC) was grown to log phase in Middlebrook 7H9 medium (Difco, Detroit, Mich.) with albumin, dextrose, and catalase enrichments (Difco), harvested, and frozen at -80°C in RPMI 1640-10% FBS-6% glycerol. The bacterial titer was determined by counting CFU on 7H10 agar plates (Difco). To remove large clumps from bacterial suspensions prior to THP-1 infection, aliquots were thawed, vortexed, and allowed to settle for 5 min. M. tuberculosis was taken from the top of the tube, washed once in infection medium, and pelleted at 7,500 x g for 8 min. M. tuberculosis bacilli were resuspended, passed four to five times through a 26-gauge needle, and sonicated for 30 s. Bacterial dilutions for infection were calculated based on the titer of the frozen stock.

The 19-kDa lipoprotein was purified from M. tuberculosis H37Ra with slight modification of previously described methods (30). Instead of lysate, M. tuberculosis cell wall, obtained by passage of bacteria through a French press followed by centrifugation, was used for detergent extraction with 20% Triton X-114 (Sigma, St. Louis, Mo.). The detergent layer was further fractionated by electroelution with a Prep-cell chamber (Bio-Rad, Hercules, Calif.). The fractions collected were assayed by Western blotting, and those containing highly purified 19-kDa lipoprotein were pooled and reextracted with Triton X-114. The Triton X-114 layer was then precipitated by overnight incubation at -20°C with acetone. Recovered 19-kDa lipoprotein was resuspended in 90% dimethyl sulfoxide-10% HEPES (5 mM) and frozen at -70°C. The 19-kDa lipoprotein used for experiments was diluted to 50 µg/ml in phosphate-buffered saline and frozen at -70°C in small aliquots to avoid freeze-thawing. Preparations of 19-kDa lipoprotein used in experiments were tested for lipopolysaccharide contamination by Limulus amebocyte lysate assay (BioWhittaker) and contained less than 20 pg of endotoxin per ml at the concentrations of 19-kDa lipoprotein used in experiments.

Animals. HLA-DR1 transgenic mice were provided by Dennis Zaller (Merck, Whitehouse Station, N.J.). The mice, B10.M, are transgenic for the human HLA-DR1 molecule on an H-2f mouse MHC background (36). Mice were bred and colonies were maintained at the Animal Resource Center at Case Western Reserve University.

Flow cytometry. THP-1 cells were incubated with phorbol myristate acetate (10 ng/ml) in 24-well tissue culture plates. After 24 h, cells were washed and treated with or without 19-kDa lipoprotein (200 ng/ml) and with or without IFN-{gamma} (100 U/ml; Endogen) for 6 to 72 h. To recover cells, wells were treated with trypsin-Versene for 10 min, washed with RPMI containing 10% heat-inactivated FBS, and centrifuged at 400 x g. THP-1 cells were incubated with 5% bovine serum albumin in PBS on ice for 10 min and then with phycoerythrin-conjugated monoclonal Ab (MAb) to human HLA-DR or Fc{gamma}RI (Caltag Laboratories) or isotype-matched control Ab (Caltag Laboratories) for 30 min on ice. Cells were fixed with 1% paraformaldehyde and analyzed with a FACScan flow cytometer (Becton Dickinson), using CellQuest software (Becton Dickinson). Ten thousand events were recorded. Changes in mean fluorescence values ({Delta}MFV) were calculated by subtracting the mean fluorescence value with isotype-matched Ab-stained cells from the mean fluorescence value with anti-HLA-DR or -Fc{gamma}R1 Ab.

RNA preparation and quantitative reverse transcription-PCR (RT-PCR). Total RNA was extracted from THP-1 cells with Trizol (Life Technologies) according to the manufacturer's instructions. cDNA was synthesized from total RNA by using specific primers and Moloney murine leukemia virus reverse transcriptase (Life Technologies). The sequence of the reverse transcription primer for HLA-DR{alpha} was 5'-TCGATGAAACAGA-3'. The primer sequence for 18S rRNA (R18), used as internal control, was 5'-GACGGTATCTGATC-3'. cDNA from 20 ng of total RNA was used per PCR. Real-time PCR was performed with the TaqMan sequence detection system (Applied Biosystems). TaqMan primer and probe design was performed with the Primer Express software. The sequences of primers and probes are as follows: for HLA-DR{alpha}, forward primer 5'-GACAAAGCCAACCTGGAAATCA-3', reverse primer 5'-GGACGTTGGGCTCTCTCAGTT-3', and probe 6FAM-5'-CAACTATACTCCGATCACCAATGTACCTCCAGAG-3'-TAMRA; for R18, forward primer 5'-CGCCGCTAGAGGTGAAATTC-3', reverse primer 5'-CATTCTTGGCAAATGCTTTCG-3', and probe 6FAM-5'-ACCGGCGCAAGACGGACCAGA-3'-TAMRA. Samples were quantified by using relative standard curves in each amplification. DNA standards were prepared by PCR amplification of random-primed cDNAs with target-specific primers and cloning into Escherichia coli with the pCR4-TOPO 3,957-bp vector (Invitrogen, Carlsbad, Calif.). All results were normalized with respect to the internal control and expressed as number of copies of HLA-DR{alpha} per 1010 copies of R18.

Generation and characterization of HLA-DR-restricted T-cell hybridomas. HLA-DR1 transgenic mice were immunized with 25 µg of recombinant Ag85B or tetanus toxoid (TT) in complete Freund's adjuvant (Gibco-Life Technologies, Grand Island, N.Y.). Inguinal and popliteal lymph nodes were harvested after 7 days and restimulated in vitro with 10 µg of the immunizing protein per ml. On day 4, 10 U of murine IL-2 (R&D Systems, Minneapolis, Minn.) per ml was added to the culture and left for 24 h before fusion. On day 5, restimulated T cells were fused with the BW5147 CD4-transfected cell line by using polyethylene glycol 1500 (Boehringer Mannheim, Indianapolis, Ind.). Successfully fused cells were selected by using complete DMEM supplemented with hypoxanthine-aminopeterin-thymidine (Gibco-Life Technologies).

T-cell hybridomas were screened for a response to Ag85B or TT presented by THP-1 cells (105 cells/well). T-cell hybridomas DB1 (Ag85B specific) and 1T1A (TT specific) were selected for further study. The peptide specificity of DB1 was determined by using overlapping 16-amino acid synthetic peptides based on the sequence of Ag85B. THP-1 cells (1.5 x 105) and DB1 cells (1 x 105) were incubated with 1 µM peptide in 96-well flat-bottom plates (Falcon/Becton Dickinson, Franklin Lakes, N.J.) for 20 to 24 h.

MHC-II restriction for DB1 and 1T1A cells was determined by incubating T hybridoma cells (105) with Ag-pulsed THP-1 cells (10 µg of TT per ml for 1T1A cells or infection at a multiplicity of infection [MOI] [M. tuberculosis to cells] of 5:1 for DB1 cells) in the presence of 10 µg of L243 HLA-DR blocking Ab or isotype-matched control (IgG2a) per ml for 20 to 24 h. Following incubations, culture supernatants were collected for CTLL-2 assay.

Ag processing and presentation assays. THP-1 cells were incubated in 96-well flat-bottom plates (1.5 x 105 cells/well) with 10 ng of phorbol myristate acetate (Sigma) per ml in infection medium for 24 h to promote adherence to plates. Cells were washed once with DMEM and incubated with 100 U of recombinant human IFN-{gamma} (Endogen, Woburn, Mass.) per ml with or without 19-kDa lipoprotein (200 ng/ml unless otherwise indicated) for 24 h. Following 24 h of incubation all medium was removed from the cells prior to Ag exposure. The cells then were pulsed with Ag85B (0.1 to 50 µg/ml), M. tuberculosis (MOI of 0.1 to 50), or TT (0.1 to 50 µg/ml) for 6 h and fixed in 1% paraformaldehyde, followed by incubation with DB1or 1T1A T hybridoma cells (105 cells/well). Supernatants were harvested after 20 to 24 h, and the amount of IL-2 produced by T hybridoma cells was measured with CTLL-2 cells.

For experiments blocking TLR-2, adherent THP-1 cells (1.5 x 105/well) were treated with 20 µg of anti-TLR-2 MAb (MAb 2392) or isotypic control Ab (IgG1) per ml for 30 min at room temperature. Following incubation with Abs, equal volumes of IFN-{gamma} (200 U/ml) alone or IFN-{gamma} plus 19-kDa lipoprotein (200 ng/ml) were added and cells were incubated. After 20 h, medium was removed and THP-1 cells were pulsed with 20 µg of TT per ml for 6 h. Following the TT pulse, THP-1 cells were fixed and 1T1A T hybridoma cells were added to each well and incubated for 20 to 24 h. The culture supernatant was collected and assayed for IL-2 with CTLL-2 cells.

CTLL-2 assay. Supernatants from experiments with T hybridoma cells were assessed for IL-2 by bioassay using the IL-2-dependent CTLL-2 cell line. Briefly, 5 x 103 CTLL-2 cells in 50 µl of complete DMEM were incubated with 100 µl of culture supernatant for 16 to 20 h. Proliferation of CTLL-2 cells in each well was measured by a colorimetric assay with Alamar blue (15 µl; Trek Diagnostics, Westlake, Ohio) and a Bio-Rad 550 microplate reader. IL-2 production was expressed as optical density at 550 nm minus optical density at 595 nm. Unless otherwise noted, all results of T hybridoma cell experiments represent the mean response for triplicate wells ± standard deviation (SD).

TUNEL assay. Apoptosis was measured by DNA fragmentation by using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay (Roche Diagnostics, Mannheim, Germany). Adhered THP-1 cells (106/ml) were treated for 24 h with or without 19-kDa lipoprotein (200 to 500 ng/ml). Apoptosis was measured by flow cytometry after TUNEL labeling according to the manufacturer's instructions. DNase I served as a positive control. THP-1 cells were incubated in medium for 24 h and then fixed, permeabilized, and incubated with 200 U of DNase I per ml for 10 min at room temperature. Following treatment with DNase I, DNA strand breaks were labeled by using the TUNEL assay. Cells were considered apoptotic if their fluorescence was higher than that observed in medium alone.


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RESULTS
 
The 19-kDa lipoprotein of M. tuberculosis inhibits HLA-DR expression induced by IFN-{gamma}. To investigate the effect of the 19-kDa lipoprotein of M. tuberculosis on IFN-{gamma}-regulated MHC-II Ag processing and presentation by human macrophages, expression of HLA-DR on THP-1 cells was measured. HLA-DR was expressed at low levels on THP-1 cells cultured in medium alone ({Delta}MFV at 24, 48, and 72 h = 105 ± 4, 144 ± 9, and 178 ± 42, respectively; n = 3) (Fig. 1A). The addition of IFN-{gamma} (100 U/ml) resulted in a greater-than-10-fold increase in HLA-DR ({Delta}MFV at 24 h = 1,223 ± 115; n = 3), with maximum expression at 72 h ({Delta}MFV at 72 h = 1,605 ± 107; n = 3) (Fig. 1). When THP-1 cells were coincubated with 19-kDa lipoprotein (200 ng/ml) and IFN-{gamma} for 6 to 72 h, the up-regulation of HLA-DR expression by IFN-{gamma} was inhibited (Fig. 1B to F). HLA-DR expression induced by IFN-{gamma} was decreased after 24 h of coincubation with 19-kDa lipoprotein and continued to decline at 48 and 72 h. The degree of inhibition of HLA-DR expression ranged from 33 to 75%, depending on the length of coincubation, with maximum inhibition at 72 h ({Delta}MFV without 19-kDa lipoprotein = 1,605 ± 107; {Delta}MFV with19-kDa lipoprotein = 400 ± 134 [P = 0.01; n = 3]). Treatment of THP-1 cells with 19-kDa lipoprotein alone decreased constitutive surface HLA-DR slightly compared to treatment with medium alone (to 58 ± 14, 44 ± 26, and 74 ± 83 at 24, 48, and 72 h, respectively; n = 3). The biological significance of this minimal change in baseline values is unclear, since THP-1 cells, in the absence of IFN-{gamma}, are unable to process and present Ag to T cells. These experiments were performed with highly purified 19-kDa lipoprotein obtained by electroelution from Triton X-114-extracted material of the M. tuberculosis cell wall. In preliminary experiments with THP-1 cells, negative control electroelution fractions that lacked the 19-kDa lipoprotein did not affect HLA-DR expression or THP-1 viability, consistent with our previous studies with murine macrophages (30).



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  		FIG. 1. The 19-kDa lipoprotein inhibits MHC-II surface expression on THP-1 cells. (A to E) Histogram representation (MFV) of HLA-DR expression on THP-1 cells at baseline (no treatment) (A) or after treatment with IFN-{gamma} in the presence or absence of the 19-kDa lipoprotein for 6 h (B), 24 h (C), 48 h (D), or 72 h (E). Cells were labeled with isotype control MAb or anti-HLA-DR MAb after treatment with IFN-{gamma} or IFN-{gamma} plus19-kDa lipoprotein. The results are representative of those from three experiments. (F) {Delta}MFVs ({Delta}MFV = MFV of cells labeled with anti-HLA-DR MAb - MFV of cells labeled with isotypic control MAb), assessed by flow cytometry, of cells treated with IFN-{gamma} alone or IFN-{gamma} plus19-kDa lipoprotein. {Delta}MFV was assessed between 6 and 72 h of continuous treatment; maximum inhibition (75%) was observed at 72 h. Values are means ± SDs from three experiments.

Inhibition of IFN-{gamma}-induced expression of HLA-DR by the 19-kDa lipoprotein correlates with decreased HLA-DR mRNA. The HLA-DR{alpha} mRNA level was measured by TaqMan quantitative RT-PCR under the same conditions in which surface HLA-DR expression was assessed by flow cytometry. Figure 2 shows changes of HLA-DR mRNA copy number in THP-1 cells over time after treatment with or without 19-kDa lipoprotein and with or without IFN-{gamma}. In the absence of IFN-{gamma}, baseline HLA-DR{alpha} mRNA levels increased slightly over 48 h of culture and the 19-kDa lipoprotein had no appreciable effect (Fig. 2A). IFN-{gamma} induced up-regulation of HLA-DR mRNA after 6 h of treatment, with peak inducible expression at 24 h (HLA-DR copy number/1010 R18 copies at 24 h: untreated = 8.6 x 104 ± 3 x 104, IFN-{gamma} treated = 1.48 x 108 ± 5 x 107 [P < 0.01; n = 3]). The 19-kDa lipoprotein inhibited induction of HLA-DR mRNA by IFN-{gamma} after 24 h of coincubation (Fig. 2B, 50% inhibition), with maximal inhibition detected after 48 h (90% inhibition) (HLA-DR copy number/1010 R18 copies at 48 h: IFN-{gamma} only = 9.6 ± 2.6 x 107, IFN-{gamma} plus 19-kDa lipoprotein = 9.5 x 106 ± 2 x 105 [P < 0.05; n = 3]). Inhibition of mRNA correlated with inhibition of HLA-DR surface expression. Thus, inhibition of IFN-{gamma}-regulated HLA-DR mRNA synthesis may represent a major mechanism for decreased HLA-DR surface expression in macrophages treated with 19-kDa lipoprotein.



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  		FIG. 2. The 19-kDa lipoprotein inhibits HLA-DR{alpha} mRNA. RNA was extracted from THP-1 cells after culture for the indicated times. Reverse transcription and TaqMan PCR for HLA-DR{alpha} and the control gene R18 was performed. The copy number for the target sequence (HLA-DR{alpha}) was normalized to 1010 copies of R18 in each sample [(HLA-DR{alpha} copies in given sample/R18 copies in given sample) x 1010]. (A) HLA-DR{alpha} mRNA copies were calculated for THP-1cells cultured in medium alone or cultured with 19-kDa lipoprotein. (B) HLA-DR{alpha} mRNA copies were calculated for THP-1 cells treated with IFN-{gamma} or with IFN-{gamma} plus 19-kDa lipoprotein. Twenty-four hours of exposure to the 19-kDa lipoprotein decreased inducible HLA-DR{alpha} mRNA synthesis by 50%. Values are means ± SDs from triplicate samples. The results shown are from a single experiment representative of three.

DB1 and 1T1A T hybridoma cells recognize Ag85B and TT presented by HLA-DR. To determine if decreases in IFN-{gamma}-regulated HLA-DR expression affected the ability of human macrophages to process and present Ag to CD4 T cells, T-cell hybridomas were generated by immunizing HLA-DR1 transgenic mice with either the major M. tuberculosis Ag, Ag85B, or a model Ag, TT, and selected for recognition of peptide-HLA-DR1 complexes on human macrophages. T-cell hybridomas are immortalized murine T cells that express alpha/beta T-cell receptor and produce IL-2 in proportion to the number of specific HLA-DR-peptide complexes that their T-cell receptors recognize.

DB1 cells recognized soluble Ag85B processed and presented by THP-1 cells (HLA-DR1/DR2) (Fig. 3A). To confirm MHC-II restriction, an anti-HLA-DR Ab (L243) was used to block HLA-DR-restricted presentation to DB1 cells. THP-1 cells were infected with M. tuberculosis and then incubated with DB1 cells with or without L243 or isotype-matched control Ab. DB1 cells recognized the Ag85B epitope processed from whole M. tuberculosis, and L243 blocked DB1 recognition of M. tuberculosis-infected THP-1 cells (Fig. 3B). The peptide specificity of DB1 cells was determined with a panel of overlapping 16-amino acid peptides from Ag85B. DB1 cells recognized peptide PAFEWYYQSGLSIVMP, corresponding to Ag85B96-111.



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  		FIG. 3. DB1 and 1T1A T-cell hybridomas respond to Ag and are restricted to HLA-DR presentation. (A) Response of DB1 T hybridoma cells to M. tuberculosis Ag85B. THP-1 cells (1.5 x 105) treated with 100 U of IFN-{gamma} per ml were incubated with Ag85B at the indicated concentrations for 6 h and then fixed with 1% paraformaldehyde. DB1 cells (105) were added to fixed THP-1 cells. (B) IFN-{gamma}-treated THP-1 cells were infected with M. tuberculosis (MTB) (MOI of 5) in the presence of 10 µg of L243 MAb or isotype control MAb per ml for 2 h prior to addition of DB1 cells. (C) 1T1A cells recognize TT processed by THP-1 cells. THP-1 cells were treated with IFN-{gamma}, pulsed with increasing concentrations of TT, and fixed. 1T1A cells (105) were added to fixed THP-1 cells. (D) IFN-{gamma}-treated THP-1 cells were incubated with TT (10 µg/ml) in the presence of 10 µg of L243 MAb per ml for 1 h prior to addition of 1T1A cells. For all experimental conditions, culture supernatants were harvested 20 to 24 h after coincubation of T hybridoma and THP-1 cells. IL-2 production by DB1and 1T1A cells was measured by CTLL assay. OD550, optical density at 550 nm.

Similar experiments were performed to characterize the 1T1A TT-specific, HLA-DR restricted T hybridoma. 1T1A cells recognized TT processed and presented by THP-1 cells, and L243 blocked presentation of TT to 1T1A (Fig. 3 C and D). The antigenic specificity and restriction to HLA-DR displayed by the DB1 and 1T1A T-cell hybridomas made them suitable for studying MHC-II processing and presentation of M. tuberculosis Ag85B or TT by human macrophages.

The 19-kDa lipoprotein inhibits HLA-DR-restricted Ag processing and presentation by human macrophages. Preliminary experiments determined that decreased HLA-DR expression was associated with decreased presentation of peptide Ag85B96-111 to DB1 cells (data not shown). These studies were extended to determine if inhibition of IFN-{gamma}-induced HLA-DR protein expression by the 19-kDa lipoprotein was associated with changes in Ag processing of soluble Ags for MHC-II-restricted T cells. THP-1 cells were pretreated with IFN-{gamma} with or without 19-kDa lipoprotein for 24 to 48 h, followed by an Ag pulse and then fixation of the Ag-presenting THP-1 cells. HLA-DR Ag processing and presentation of Ag85B was measured with DB1 cells, and that of TT was measured with 1T1A cells. THP-1 cells treated with IFN-{gamma} alone processed and presented epitopes recognized by DB1 and 1T1A cells efficiently at Ag concentrations of 10 µg/ml and above for Ag85B and 3 µg/ml and above for TT (Fig. 4 A and B). Treatment with 19-kDa lipoprotein (200 ng/ml) for 24 h markedly reduced HLA-DR Ag processing of Ag85B by IFN-{gamma}-treated THP-1 cells at all Ag concentrations, with maximum inhibition at 30 and 50 µg of Ag85B per ml (Fig. 4A). Processing of TT for HLA-DR presentation to 1T1A cells also was inhibited by the 19-kDa lipoprotein, with the most pronounced inhibition at TT concentrations of 3 to 30 µg/ml (Fig. 4B). Mock extracted buffer or control electroelution fractions not containing the 19-kDa lipoprotein had no effect on HLA-DR Ag processing and presentation (data not shown). The prolonged half-life of peptide-HLA-DR complexes on macrophages explains why inhibition of Ag processing was observed more rapidly than decreases in cell surface HLA-DR expression. MHC-II Ag processing relies on de novo HLA-DR synthesis, which, based on the mRNA results (Fig. 2B), was inhibited by 50% after 24 h of exposure to the 19-kDa lipoprotein.



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  		FIG. 4. The 19-kDa lipoprotein inhibits processing and presentation of Ag85B, TT, and M. tuberculosis. THP-1 cells were treated with IFN-{gamma} with or without 19-kDa lipoprotein for 24 h and then pulsed with Ag85B (A), TT (B), or M. tuberculosis (C) for 6 h. THP-1 cells were fixed and incubated with DB1 (A and C) or 1T1A (B) cells for 20 to 24 h. Culture supernatants were collected, and the IL-2 produced was measured by CTLL assay. Each panel shown is representative of four experiments. OD550, optical density at 550 nm.

To determine if the 19-kDa lipoprotein inhibited processing of Ag85B from whole M. tuberculosis, THP-1 cells were treated with IFN-{gamma} with or without 19-kDa lipoprotein for 24 h and then infected with M. tuberculosis. Uptake of M. tuberculosis by THP-1 cells was not affected by 19-kDa lipoprotein treatment as measured by flow cytometry of THP-1 cells infected with fluorescently labeled M. tuberculosis. HLA-DR processing and presentation of Ag85B originating from phagocytosed live M. tuberculosis bacilli was inhibited threefold by the 19-kDa lipoprotein (Fig. 4C). Thus, the 19-kDa lipoprotein inhibited IFN-{gamma}-regulated processing and presentation of soluble and particulate Ag by human macrophages.

Inhibition of HLA-DR processing by the 19-kDa lipoprotein is concentration dependent, requires prolonged exposure, and cannot be rescued by IFN-{gamma}. Concentrations of 19-kDa lipoprotein required for inhibition were measured by using MHC-II Ag processing and presentation of Ag85B from M. tuberculosis bacilli (MOI, 5:1). THP-1 cells were treated with IFN-{gamma} and increasing concentrations of 19-kDa lipoprotein (0 to 500 ng/ml) for 24 h. Maximal inhibition of HLA-DR Ag processing was measured at concentrations of 200 ng of 19-kDa lipoprotein per ml and above (Fig. 5A).



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  		FIG. 5. Inhibition of IFN-{gamma}-regulated HLA-DR Ag processing by the 19-kDa lipoprotein is concentration dependent, requires prolonged exposure, and cannot be reversed by IFN-{gamma}. (A) THP-1 cells were treated with IFN-{gamma} and increasing concentrations of 19-kDa lipoprotein for 24 h. THP-1 cells were then incubated with or without M. tuberculosis (Mtb) (MOI of 1) for 4 h and fixed, followed by addition of DB1 cells. (B) All THP-1 cells were treated with IFN-{gamma} 24 h prior to Ag pulse, and 19-kDa lipoprotein was added at the indicated times prior to Ag. Following incubations with IFN-{gamma} and 19-kDa lipoprotein, all additions were removed and the THP-1 cells were infected with M. tuberculosis (MOI of 5) for 6 h and fixed, followed by addition of DB1 cells. (C) THP-1 cells were treated with different concentrations of IFN-{gamma} (0 to 1,000 U/ml) with or without 19-kDa lipoprotein (200 ng/ml) for 24 h. THP-1 cells then were pulsed with TT (20 µg/ml) for 6 h and fixed. 1T1A cells were added to the fixed THP-1 cells. For all experimental conditions, culture supernatants were harvested at 20 to 24 h after coincubation of T hybridoma and THP-1 cells. IL-2 production by DB1 and 1T1A cells was measured by CTLL assay. OD550, optical density at 550 nm.

To determine the length of exposure required for inhibition, all THP-1 cells were treated with IFN-{gamma} 24 h prior to an Ag pulse, and 19-kDa lipoprotein was added at 8, 12, 16, 20, and 24 h before pulsing with Ag. For example, cells treated with 19-kDa lipoprotein for 8 h received 16 h of IFN-{gamma} before addition of the 19-kDa lipoprotein. After 24 h (16 h of treatment with IFN-{gamma} and 8 h of treatment with IFN-{gamma} plus 19-kDa lipoprotein), cells were washed, pulsed with Ag (M. tuberculosis MOI of 5), and fixed before addition of T hybridoma cells. Minimal inhibition was observed after 8 h of exposure to 19-kDa lipoprotein, and this progressed to greater than 50% inhibition by 24 h (Fig. 5B). In addition, 1- to 2-h pulses with 19-kDa lipoprotein followed by chase periods of up to 24 h did not inhibit processing and presentation by HLA-DR, indicating that continuous exposure to lipoprotein was required (data not shown).

To analyze the interaction between IFN-{gamma} and the 19-kDa lipoprotein, THP-1 cells were treated with a single concentration of 19-kDa lipoprotein in the presence of increasing concentrations (0 to 1,000 U/ml) of IFN-{gamma} (Fig. 5C). Inhibition of HLA-DR Ag processing and presentation by the 19-kDa lipoprotein was most pronounced at lower concentrations of IFN-{gamma} (10 to 100 U/ml). High concentrations of IFN-{gamma} did not fully rescue HLA-DR Ag processing, suggesting that inhibition of IFN-{gamma} signaling of macrophages by the 19-kDa lipoprotein was not competitive.

The 19-kDa lipoprotein inhibits IFN-{gamma}-regulated Fc{gamma}RI expression on human macrophages. Expression of Fc{gamma}RI is highly regulated by IFN-{gamma} and is a marker of macrophage activation. Fc{gamma}RI expression, measured by flow cytometry, increased almost fourfold following 24 h of IFN-{gamma} treatment (Fig. 6). Addition of 19-kDa lipoprotein during stimulation of THP-1 cells by IFN-{gamma} resulted in a 40% decrease in Fc{gamma}RI expression after 24 h (Fig. 6B) ({Delta}MFV of constitutive expression = 39.95, {Delta}MFV after 24 h with IFN-{gamma} = 149.76, and {Delta}MFV after 24 h with IFN-{gamma} and 19-kDa lipoprotein = 89.63). Expression of IFN-{gamma} receptor (IFN-{gamma}R) molecules was not affected by the 19-kDa lipoprotein during the 24 h incubation (data not shown). These results indicate that inhibition of IFN-{gamma} responses was not due to modulation of IFN-{gamma}R and that treatment with the 19-kDa lipoprotein interfered with IFN-{gamma}R signaling in macrophages.



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  		FIG. 6. The 19-kDa lipoprotein inhibits IFN-{gamma}-induced surface expression of Fc{gamma}RI. (A) Histogram representation (MFV) of THP-1 cells left untreated, treated with IFN-{gamma}, or treated with IFN-{gamma} and 19-kDa lipoprotein for 24 h and stained with anti-Fc{gamma}RI MAb or isotype control MAb. (B) {Delta}MFVs ({Delta}MFV = MFV of cells labeled with anti-Fc{gamma}R1 MAb - MFV of cells labeled with isotype-matched control Ab) for the results in panel A. Results are representative of those from three experiments.

Inhibition of IFN-{gamma}-regulated HLA-DR Ag processing by the 19-kDa lipoprotein is dependent on TLR-2. The 19-kDa lipoprotein is a known ligand for TLR-2. To determine the role of TLR-2 in 19-kDa lipoprotein-mediated inhibition of IFN-{gamma}R signaling, an anti-TLR-2 blocking MAb (MAb 2392) was used in Ag processing assays. Treatment with isotype-matched control Ab or 2392 did not affect processing of TT for 1T1A cells in the absence of the 19-kDa lipoprotein (data not shown). THP-1 cells treated with 19-kDa lipoprotein in the presence of 10 µg of isotypic control Ab per ml were inhibited 64% in their ability to process and present TT to 1T1A cells. Inhibition by the 19-kDa lipoprotein was reversed in the presence of anti-TLR-2 MAb 2392 during incubation of THP-1 cells with IFN-{gamma} plus the 19-kDa lipoprotein (Fig. 7). This finding implicates TLR-2 as the receptor responsible for mediating 19-kDa lipoprotein inhibition of IFN-{gamma}-regulated HLA-DR Ag processing and expression of FcR{gamma}I in human macrophages.



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  		FIG. 7. Blocking TLR-2 on THP-1 cells prevents 19-kDa lipoprotein-mediated inhibition of HLA-DR Ag processing. THP-1 cells were incubated in the presence of 20 µg of anti-TLR-2 MAb 2392 or isotype-matched MAb (IgG1) per ml for 30 min prior to addition of IFN-{gamma} or IFN-{gamma} plus19-kDa lipoprotein. THP-1 cells were incubated for 20 h, medium was removed, and the cells were pulsed with TT for 6 h before fixation. 1T1A cells were added to the fixed THP-1 cells and left for 20 to 24 h, and IL-2 production was measured by CTLL assay. OD550, optical density at 550 nm.

The 19-kDa lipoprotein does not induce apoptosis in THP-1 cells. To determine if native 19-kDa lipoprotein induced apoptosis, THP-1 cells were treated with different concentrations of lipoprotein for 24 h. At concentrations of up to 500 ng/ml, a concentration 2.5-fold higher than that required for maximum inhibition of Ag processing, the 19-kDa lipoprotein did not induce significant apoptosis compared to medium alone, as shown in a representative experiment in Fig. 8. Apoptosis was measured by TUNEL assay; cells treated with DNase I served as a positive control. In three experiments, levels of apoptosis were 0.6% ± 0.2% for medium alone and 0.65% ± 0.12% for 19-kDa lipoprotein-treated THP-1 cells. Addition of IFN-{gamma} (100 U/ml) had no effect on the levels of apoptosis for cells treated with medium alone or with 19-kDa lipoprotein (data not shown). Thus, apoptosis was not the mechanism for 19-kDa lipoprotein-mediated inhibition of IFN-{gamma}-regulated HLA-DR expression and function.



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  		FIG. 8. The 19-kDa lipoprotein does not induce apoptosis in THP-1 cells. Apoptosis was measured in THP-1 cells (106 cells/ml) either cultured in medium alone or treated with 19-kDa lipoprotein (500 ng/ml) for 24 h. After the 24-h incubation, cells were fixed in 2% paraformaldehyde and DNA fragmentation was measured by TUNEL assay. DNase I served as a positive control.


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DISCUSSION
 
A hallmark of M. tuberculosis is its ability to infect, survive in, and persist in human macrophages. Acquired immunity, mediated primarily by MHC-II-restricted IFN-{gamma}-producing CD4+ T cells, controls M. tuberculosis infection but fails to eradicate the organism. When acquired immunity fails because of aging, malnutrition, or human immunodeficiency virus type 1 infection, persistent macrophage-bound M. tuberculosis bacilli emerge to cause reactivation tuberculosis. The mechanism(s) used by M. tuberculosis to persist for many years in macrophages in the face of highly developed and active acquired T-cell responses, reflected in strongly positive tuberculin skin test reactivity, is poorly understood. Earlier studies established that M. tuberculosis can interfere with IFN-{gamma}-mediated activation and IFN-{gamma}R signaling in human macrophages (41). However, the molecules of M. tuberculosis that are responsible for interference with IFN-{gamma} signaling have not been characterized.

In earlier studies, we established that the 19-kDa lipoprotein of M. tuberculosis, a pathogen-associated molecular pattern, inhibits MHC-II Ag processing in murine bone marrow macrophages and that this inhibition was dependent on TLR-2 (30). This study aimed to determine the role of the 19-kDa lipoprotein in activation of human macrophages by IFN-{gamma}. The 19-kDa lipoprotein of M. tuberculosis was found to decrease IFN-{gamma}-regulated processing and presentation by HLA-DR of soluble Ag85B, TT, and whole M. tuberculosis bacilli by human macrophages. The 19-kDa lipoprotein also inhibited up-regulation of Fc{gamma}RI, a molecule regulated primarily through IFN-{gamma} in macrophages. Inhibition of IFN-{gamma}R signaling was dependent on TLR-2, required prolonged exposure to the 19-kDa lipoprotein, and was not associated with apoptosis. Thus, the present studies establish for the first time that the M. tuberculosis 19-kDa lipoprotein inhibits IFN-{gamma} signaling in human macrophages through TLR-2.

The 19-kDa lipoprotein is part of a large family of molecules sharing a common structural motif consisting of an triacyl head group attached to an amino-terminal cysteine (47). The 19-kDa lipoprotein of M. tuberculosis is one of a number of lipoproteins made by the organism, and alteration of its core structure by deacylation abolishes its immunogenic properties (28, 30). Lipoproteins activate macrophages via TLR-2 during the acute phase of infections, resulting in a signaling cascade that leads to production of proinflammatory cytokines and inducible NO synthase and costimulatory molecule expression (6, 16, 23). The findings of this study suggest two phases to TLR-2-mediated responses when the 19-kDa lipoprotein of M. tuberculosis is used as stimulus: an acute activating phase occurring within minutes of exposure and a delayed deactivation phase resulting from prolonged exposure (>16 h) to 19-kDa lipoprotein. Prolonged activation of TLR-2 results in inhibition of signaling through IFN-{gamma}R.

Other studies have reported inhibition of host immune responses associated with the M. tuberculosis 19-kDa lipoprotein. Human macrophage proinflammatory cytokine production was decreased by 19-kDa lipoprotein-transformed Mycobacterium smegmatis compared to wild-type M. smegmatis after a 24-h infection (34). Others have described a decrease or loss in vaccine efficacy in a murine M. tuberculosis challenge model when 19-kDa lipoprotein expressed in Mycobacterium vaccae was used as the immunogen (1, 46). Increased virulence was observed when the gene for the 19-kDa lipoprotein was transformed into a strain of M. tuberculosis naturally defective in 19-kDa lipoprotein expression (20).

How prolonged activation through TLR-2 by the 19-kDa lipoprotein results in inhibition of IFN-{gamma}-mediated responses remains to be determined. Expression of IFN-{gamma}R protein and mRNA was not affected by 24 h of treatment with the 19-kDa lipoprotein (data not shown). Supernatant transfer experiments, to determine if 19-kDa lipoprotein-mediated inhibition occurred through secretion of inhibitory cytokines (e.g., IL-10) or other mediators, did not reveal evidence for a soluble inhibitor (data not shown). IL-10 can inhibit human MHC-II traffic to the plasma membrane. However, this mechanism is not associated with decreases in MHC-II mRNA as observed in our studies (17). Furthermore, the 19-kDa lipoprotein inhibited MHC-II Ag processing in macrophages from IL-10 knockout mice (E. Noss, personal communication).

The 19-kDa lipoprotein may activate a negative feedback mechanism or novel signaling pathway via TLR-2, leading to a block in IFN-{gamma}R signaling. Production of suppressors of cytokine signaling, which negatively regulate Janus kinase (JAK)/STAT activation, represents one possible mechanism (13, 27, 39). Other TLR ligands such as CpG DNA and lipopolysaccharide can induce expression of suppressors of cytokine signaling (11, 40). However, Ting et al. demonstrated that M. tuberculosis inhibits IFN-{gamma} transcriptional responses without altering STAT1 activation (41). Thus, alternative mechanisms may exist for M. tuberculosis to inhibit signaling through IFN-{gamma}R.

Signaling through TLR-2 has been described to cause apoptosis of human macrophages (2, 3). In our experiments with native 19-kDa lipoprotein from M. tuberculosis, we have not found evidence for apoptosis as a mechanism to explain the inhibition of IFN-{gamma}-regulated HLA-DR expression and function. We have addressed this possibility formally by performing TUNEL assays on THP-1 cells treated with 19-kDa lipoprotein. Furthermore, we have not observed a loss in THP-1 viability or adherence following 19-kDa lipoprotein treatment compared to control macrophages. In addition, 19-kDa lipoprotein-treated macrophages demonstrate slightly increased phagocytosis of M. tuberculosis, indicating intact cellular function. It is possible that distinct structural features of TLR-2 ligands determine the outcome of signaling through TLR-2.

The complexities of TLR signaling pathways are being elucidated, with most studies focusing on MyD88-dependent and -independent signaling pathways for TLR-4. TLR-2 signaling also is dependent on MyD88, IRAK, and TRAF-6, resulting in NF-{kappa}B and mitogen-activated protein kinase activation (44, 45). However, ligand specificity for TLR-2 may depend on forming heterodimers with other TLRs (TLR-1 and TLR-6), which may affect the signaling cascade (7, 32). To date, no intersections between the proximal TLR and IFN-{gamma}R signaling pathways have been described. Most studies of TLR signaling focus on the effects of short-term activation. However, our studies depend on prolonged stimulation through TLR-2 for inhibition of IFN-{gamma}, which may result in a different TLR signaling cascade.

M. tuberculosis has evolved a number of mechanisms to invade and persist within macrophages. Macrophages are key antimicrobial effector cells as well as professional antigen-presenting cells for MHC-II-restricted CD4 T cells, the primary T-cell subset in protective immunity to M. tuberculosis. Mechanisms used by M. tuberculosis to resist host immunity will differ depending on the stage of infection. Modulation of the phagosome and resistance to microbicidal mechanisms of macrophages are critical for initial survival before acquired immune responses become operative. Production of inhibitory cytokines such as IL-10 and transforming growth factor ß are most pronounced during active tuberculosis when the organism has overwhelmed host defenses. Inhibition of MHC-II Ag processing by M. tuberculosis would be particularly important during the persistent phase of M. tuberculosis infection, where the organism survives within macrophages in the face of primed M. tuberculosis-specific IFN-{gamma}-secreting CD4 memory T cells. By blocking the ability of one of the key cytokines in M. tuberculosis infection, IFN-{gamma}, the 19-kDa lipoprotein may block CD4 T-cell activation by decreasing peptide-MHC-II expression and thus promote survival of M. tuberculosis within macrophages. To obviate acquired immunity, M. tuberculosis uses one of the primary receptors on phagocytes for recognition of bacterial pathogens, namely, TLR-2. The specificity of this mechanism for M. tuberculosis lies in the unique ability of M. tuberculosis to survive acute activation of macrophages through TLRs. In this setting, the mechanism of prolonged stimulation by TLR-2 ligands such as the 19-kDa lipoprotein can become operative. The presence of TLR-2 in phagosomes brings the receptor responsible for inhibition in close proximity to the bacilli, allowing for prolonged stimulation of TLR-2 (43). Thus, M. tuberculosis takes advantage of an innate host defense mechanism to interfere with its recognition by MHC-II-restricted CD4+ T cells, thereby allowing the organism to persist within the host.


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ACKNOWLEDGMENTS
 
We thank Dennis Zaller for generously providing the HLA-DR1 transgenic mice used to make the DR1-restricted T-cell hybridoma. Rish Pai and Marilyn Convery purified the 19-kDa lipoprotein used in all experiments. Andrew Kanost contributed expertise in developing the TaqMan RT-PCR assay for HLA-DR{alpha}. Erika Noss provided guidance during early experiments with human macrophages.

This work was supported by National Institutes of Health grants AI27243 and HL55967 and contract AI95383 to the Tuberculosis Research Unit (to W.H.B.) and by grants AI34343, AI35726, and AI47255 (to C.V.H. and the CFAR at Case Western Reserve University).

Roxana E. Rojas and Adam J. Gehring contributed equally to the experimental data presented in this study, and Clifford V. Harding shares senior authorship with W. Henry Boom.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Infectious Disease, BRB 1031, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4984. Phone: (216) 368-4844. Fax: (216) 368-2034. E-mail: WHB{at}cwru.edu. Back

Editor: S. H. E. Kaufmann


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Infection and Immunity, August 2003, p. 4487-4497, Vol. 71, No. 8
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.8.4487-4497.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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