Phagosomal Processing of Mycobacterium tuberculosis Antigen 85B Is Modulated Independently of Mycobacterial Viability and Phagosome Maturati
http://www.100md.com
感染与免疫杂志 2005年第2期
Department of Pediatrics
Department of Pathology
Division of Infectious Diseases, Case Western Reserve University
University Hospitals of Cleveland, Cleveland, Ohio
ABSTRACT
Control of Mycobacterium tuberculosis infection requires CD4 T-cell responses and major histocompatibility complex class II (MHC-II) processing of M. tuberculosis antigens (Ags). We have previously demonstrated that macrophages process heat-killed (HK) M. tuberculosis more efficiently than live M. tuberculosis. These observations suggested that live M. tuberculosis may inhibit Ag processing by inhibiting phagosome maturation or that HK M. tuberculosis may be less resistant to Ag processing. In the present study we examined the correlation between M. tuberculosis viability and phagosome maturation and efficiency of Ag processing. Since heat treatment could render M. tuberculosis Ags more accessible to proteolysis, M. tuberculosis was additionally killed by antibiotic treatment and radiation. Processing of HK, live, radiation-killed (RadK), or rifampin-killed (RifK) M. tuberculosis in activated murine bone marrow macrophages was examined by using an I-Ab-restricted T-cell hybridoma cell line (BB7) that recognizes an epitope derived from Ag 85B. Macrophages processed HK M. tuberculosis more rapidly and efficiently than they processed live, RadK, or RifK M. tuberculosis. Live, RadK, and RifK M. tuberculosis cells were processed with similar efficiencies for presentation to BB7 T hybridoma cells. Furthermore, phagosomes containing live or RadK M. tuberculosis expressed fewer M. tuberculosis peptide-MHC-II complexes than phagosomes containing HK M. tuberculosis expressed. Since only live M. tuberculosis was able to prevent acidification of the phagosome, our results suggest that regulation of phagosome maturation does not explain the differences in processing of different forms of M. tuberculosis. These findings suggest that the mechanisms used by M. tuberculosis to inhibit phagosomal maturation differ from the mechanisms involved in modulating phagosome Ag processing.
INTRODUCTION
Mycobacterium tuberculosis is an intracellular pathogen that survives inside macrophage phagosomal compartments. CD4 T-cell responses are critical to the control of M. tuberculosis infection in both animals and humans (5, 6, 22, 45) and require processing of mycobacterial antigens (Ags) to generate peptide-major histocompatibility complex class II (MHC-II) complexes. The ability of M. tuberculosis to inhibit MHC-II expression and Ag presentation may contribute to evasion of immune surveillance (16, 19, 23, 25, 28-30, 33, 52). M. tuberculosis components like the 19-kDa lipoprotein have been shown to inhibit MHC-II expression and Ag processing via a mechanism that is dependent on Toll-like receptor 2 but requires hours to days to establish (16, 30). During the initial stages of macrophage infection, live bacteria are processed and presented by MHC-II molecules less efficiently than heat-killed (HK) bacteria are processed and presented (38), suggesting that live M. tuberculosis may inhibit phagosome maturation soon after phagocytosis to inhibit Ag processing and enhance bacterial survival.
After phagocytosis of most bacteria, phagosomes generally fuse with endosomes and eventually lysosomes to form phagolysosomes in a process known as phagosome maturation. In contrast, M. tuberculosis and some other slowly growing mycobacteria (e.g., Mycobacterium bovis and Mycobacterium avium) modify the phagosomal environment to support their survival inside macrophages. These bacteria inhibit phagosome maturation into a phagolysosome by decreasing phagosomal acidification, fusion of phagosomes with lysosomes, and phagosomal acquisition of lysosomal markers and characteristics (3, 4, 8, 15, 26, 31, 44, 53). Thus, phagosomes containing live mycobacteria exclude vesicular H+-ATPase and prevent acidification of this compartment (44). They stain intensely for markers of early endosomes (e.g., transferrin receptor and Rab5) but only weakly for markers of late endosomes, lysosomes, and mature phagolysosomes (e.g., CD63, lysosome-associated membrane proteins 1 and 2, and cathepsin D) (8-10). In murine macrophages, loss of maturation is also associated with phagosome retention of TACO, a coat protein with an unknown function (11).
Processing of M. tuberculosis Ags for presentation by MHC-II molecules is essential to elicit CD4 T-cell responses. Newly synthesized MHC-II molecules are targeted to endocytic compartments (e.g., the MHC-II compartment [MIIC] and class II vesicles [1, 21, 34, 35, 41, 46, 51]), where they bind peptides derived from internalized Ags. The resulting peptide-MHC-II complexes are transported to the cell surface for presentation to CD4 T cells. Particulate Ags (e.g., bacteria) residing in phagosomes are processed in phagosomal compartments to generate peptides. These peptides then bind newly synthesized MHC-II molecules within phagosomes to form peptide-MHC-II complexes, which are transported to the cell surface (36-40).
We recently investigated the role of phagosomes containing HK or live M. tuberculosis in phagosomal processing of M. tuberculosis Ag 85 (38). Flow organellometry and Western blot analysis of phagosomes isolated from gamma interferon (IFN-)-activated bone marrow macrophages (BMMs) demonstrated that phagosomes containing HK M. tuberculosis and phagosomes containing live M. tuberculosis had similar levels of MHC-II, but only phagosomes containing HK M. tuberculosis acquired increasing levels of lysosome-associated membrane protein 1 (an indication of phagosome maturation) with time. Analysis of Ag processing of HK and live M. tuberculosis with an I-Ab-restricted T-cell hybridoma cell line (BB7) that recognizes an epitope derived from M. tuberculosis Ag 85B (30) demonstrated that macrophages processed HK M. tuberculosis more rapidly and efficiently than they processed live M. tuberculosis. Phagosomes containing live M. tuberculosis expressed fewer M. tuberculosis Ag 85B(241-256)-I-Ab complexes than phagosomes containing HK M. tuberculosis expressed. Since live M. tuberculosis may inhibit Ag processing or HK M. tuberculosis may be less resistant to Ag processing (due to the heat treatment), we also killed M. tuberculosis by radiation or antibiotic treatment in the present study. Macrophages processed HK M. tuberculosis more rapidly and efficiently than they processed live, radiation-killed (RadK), or rifampin-killed (RifK) M. tuberculosis. Live, RadK, and RifK M. tuberculosis cells were all similarly processed for presentation to BB7 T hybridoma cells. Phagosomes containing live or RadK M. tuberculosis expressed fewer M. tuberculosis Ag 85B(241-256)-I-Ab complexes than phagosomes containing HK M. tuberculosis expressed, and only live M. tuberculosis was able to prevent phagosomal acidification. These findings suggest that the mechanisms used by M. tuberculosis to inhibit phagosomal maturation differ from the mechanisms involved in modulating phagosome Ag processing.
MATERIALS AND METHODS
Cells and media. B6D2F1/J mice were obtained from Jackson Laboratories (Bar Harbor, Maine). All animal procedures were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University. Incubation in the absence of M. tuberculosis was performed at 37°C in 5% CO2 in standard medium composed of Dulbecco modified Eagle medium (DMEM) (Life Technologies, Grand Island, N.Y.) supplemented with 10% decomplemented fetal calf serum (HyClone, Logan, Utah), 5 x 10–5 M 2-mercaptoethanol, L-arginine HCl (116 mg/liter), L-asparagine (36 mg/liter), NaHCO3 (2 g/liter), sodium pyruvate (1 mM), 10 mM HEPES buffer, and antibiotics. For incubation with M. tuberculosis, standard medium was modified by using nondecomplemented fetal calf serum and no antibiotics. BMM precursors were harvested from femurs of B6D2F1/J mice and cultured for 7 days in six-well plates in standard medium supplemented with 20% LADMAC (43) cell-conditioned medium. Macrophages were then stimulated for 24 h with 1 U of recombinant IFN- (Genzyme, Cambridge, Mass.) per ml. The resulting confluent cultures contained approximately 1.5 x 106 cells/well. The T-cell hybridoma BB7 recognizes M. tuberculosis Ag 85B(241-256) bound to I-Ab (30). The T-cell hybridoma DOBW recognizes OVA(323-339) bound to I-Ad (or to a lesser degree I-Ab) (20).
Bacteria. M. tuberculosis H37Ra (American Type Culture Collection) was grown in Middlebrook 7H9 broth (Difco, Detroit, Mich.) supplemented with 1% glycerol, 0.05% Tween (Sigma, St. Louis, Mo.) (to prevent clumping), and 10% Middlebrook oleic albumin dextrose catalase enrichment (Difco). Bacteria were harvested and frozen at –70°C as described previously (22). Bacterial titers were determined by counting CFU on Middlebrook 7H10 medium (Difco). Bacteria were serially diluted, and 50-μl samples were plated in triplicate. The detection limit for the CFU assay was approximately 200 CFU/ml. M. tuberculosis H37Ra was killed by heating it to 80°C for 30 min to produce HK M. tuberculosis, treated with rifampin (50 μg/ml; Sigma) for 48 h to produce RifK M. tuberculosis, or exposed to 137Cs radiation for 90 min (559 rads/min; total radiation, 5.03 x 104 rads) to produce RadK M. tuberculosis. The viability of M. tuberculosis was assessed by counting CFU and by measuring the release of radioactive 14CO2 from [14C]palmitic acid over a period of 4 days by the BACTEC radiometric method (Becton Dickenson, Franklin Lakes, N.J.). According to the manufacturer, the detection limit for the BACTEC assay is approximately 400 bacteria. The percentage of live M. tuberculosis in an M. tuberculosis culture was determined by using a LIVE/DEAD BacLight bacterial viability kit from Molecular Probes and varied between 65 to 72%. It should be noted that BacLight viability kit results reflect membrane permeability and are not infallible.
Prior to macrophage infection, M. tuberculosis was opsonized in medium containing 10% non-heat-treated fetal calf serum and 1% non-heat-treated normal mouse serum (Sigma) with no antibiotics for 30 min at 37°C and was declumped to remove large M. tuberculosis aggregates. For declumping, M. tuberculosis was pelleted, washed, passaged 10 times through a 26-gauge needle, and sonicated for 1 min in a tabletop ultrasonic cleaning system (Fisher Scientific). The sample was centrifuged at 150 x g for 3 min to remove clumps and generate the infection stock.
To label M. tuberculosis with fluorescein, 109 bacteria were pelleted, resuspended in 1 ml of phosphate-buffered saline (PBS) (pH 9.1), and combined with 25 μl of a 20-mg/ml solution of FLUOS (Boehringer, Mannheim, Germany) in dimethyl sulfoxide for 5 min at room temperature. M. tuberculosis was similarly labeled with AlexaFluor 488 (Molecular Probes, Eugene, Oreg.) in PBS (pH 9.0) for 15 or 30 min at room temperature. All samples were washed twice in medium, opsonized, and declumped as described above. Labeling of live bacteria with FLUOS or AlexaFluor 488 resulted in no significant change in the viability of M. tuberculosis as determined by counting CFU. FLUOS-labeled M. tuberculosis was used to determine the number of bacteria (HK, live, RadK, and RifK M. tuberculosis) internalized per macrophage by flow cytometry (38). AlexaFluor 488-labeled bacteria were used in the LysoTracker colocalization studies described below.
Antibodies and other reagents. Rabbit antiserum specific for the 30-kDa polypeptide of M. tuberculosis Ag 85 was obtained through the Tuberculosis Research Materials and Vaccine Testing Contract at Colorado State University. Y-3P (American Type Culture Collection), a murine immunoglobulin G2a that recognizes the MHC-II molecule I-Ab, was iodinated by the chloramine T method. LysoTracker Red DND-99 was obtained from Molecular Probes.
Ag processing and presentation assays. Macrophages were replated in 96-well flat-bottom plates at a concentration of 1.5 x 105 cells/well prior to stimulation with IFN- (1 U/ml, 24 h). M. tuberculosis was treated and declumped (as described above) and added in 100 μl (final volume). Bacteria were spun onto cells by centrifugation at 900 x g for 10 min at 37°C. Cells were incubated at 37°C for 10 min (providing a total pulse period of 20 min) and washed in ice-cold DMEM to remove extracellular bacteria. Prewarmed medium was added, and cells were incubated at 37°C for various lengths of time. Macrophages were fixed with 1% paraformaldehyde and washed. T hybridoma cells (1 x 105 cells) were added to each well (total volume, 200 μl) and incubated for 24 h. Supernatants (100 μl) were harvested and then assessed for interleukin-2 (IL-2) by using a CTLL-2 proliferation assay, monitored by addition of Alamar blue (Alamar Biosciences, Sacramento, Calif.) as an indicator dye and measured as the difference between absorbance at 550 nm and absorbance at 595 nm after 24 h. Blanks for spectrophotometry were provided by wells containing medium alone (added at the initiation of the CTLL-2 assay) and Alamar blue (added at the same time that it was added for the other wells). All analyses were performed in triplicate.
Percoll density gradient fractionation for T-cell assay. Three six-well plates containing confluent macrophage cultures were activated with 50 U of IFN- per ml for 24 h and used for each fractionation. The plates were processed as described above to achieve a 20-min pulse with M. tuberculosis (multiplicity of infection [MOI], 40), followed by a 10-min chase incubation. Cells were washed, detached by scraping, washed, resuspended in homogenization buffer (0.25 M sucrose, 10 mM HEPES; pH 7.2), and homogenized in a Dounce homogenizer (Kontes Co., Vineland, N.J.) to obtain 80 to 85% lysis (39, 40). Intact cells and nuclei were removed by three consecutive spins at 200 x g for 5 min at 4°C. The supernatant (containing phagosomes) was collected and layered on 9 ml of 27 or 40% Percoll in homogenization buffer and then centrifuged in a Ti50 fixed-angle rotor (Beckman Instruments, Palo Alto, Calif.) at 4°C for 60 min at 36,000 x g. The gradients were manually fractionated from the top into 30 333-μl fractions, which were divided into replicate 10-, 30-, or 50-μl aliquots and frozen at –80°C.
For identification of fractions containing plasma membrane, phagosomes, and MIIC, the following procedure was performed as previously described (38, 39). The plasma membrane was marked prior to homogenization by incubation of macrophages for 60 min at 4°C with 125I-labeled Ab Y-3P. Fractions containing phagosomes were identified by the presence of FLUOS-labeled bacteria with a Spectra Fluor Plus fluorimeter (Tecan, Reading, United Kingdom). Macrophages were incubated with soluble ovalbumin (OVA) (Sigma) at a concentration of 3 mg/ml for 2 h to label MIIC vesicles with OVA(323-339)-I-Ad complexes. Fractions containing OVA(323-339)-I-Ad complexes were identified by the T-cell assay described below.
For T-cell analysis of Percoll gradient fractions, standard medium and T hybridoma cells (BB7 cells for experiments with M. tuberculosis or DOBW cells for experiments with OVA; 105 cells/well) were added to 200 μl (final volume) in 96-well plates. After 24 h, the T-cell responses were assessed as described above. Wells containing only equivalent amounts of Percoll, T cells, and medium were used to generate control supernatants and blanks for the CTLL-2 assay.
Western blotting. Bacterial pellets were resuspended in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer, sonicated for 1.5 min, boiled for 10 min, and spun for 10 min in an Eppendorf centrifuge. Ten micrograms of protein in the supernatant was electrophoresed on SDS—12% polyacrylamide gels, blotted onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore Corporation, Bedford, Mass.), probed with Ag 85-specific rabbit antiserum, and detected with horseradish peroxidase-conjugated anti-rabbit serum by using an enhanced chemiluminescence kit (Amersham).
LysoTracker colocalization studies. Macrophages were replated on coverslips (no. 1.5; Corning, Corning, N.Y.), activated with 1 U of IFN- per ml for 24 h, and then pulsed for 20 min with AlexaFluor 488-labeled HK, live, RadK, or RifK M. tuberculosis (MOI, 1) (labeling of M. tuberculosis is described above). The cells were washed with warmed DMEM to remove extracellular bacteria. Prewarmed medium was added to the cells, and the chase incubation was continued for 20 or 100 min. In the final 5 min of the chase incubation, cells were incubated with medium containing 500 nM LysoTracker Red DND-99 (Molecular Probes) as recommended by the manufacturer. The cells were washed twice in PBS and analyzed with a Leica TCS SP2 AOBS confocal microscope. The viability of the live M. tuberculosis preparations was determined by using a LIVE/DEAD BacLight bacterial viability kit from Molecular Probes.
Statistical analysis. The statistical analysis was done with the two-sample t test by using the MiniTab statistical software (MiniTab Inc., State College, Pa.).
RESULTS
Analysis of M. tuberculosis viability following treatment with the antibiotic rifampin or radiation. Several methods were used to kill M. tuberculosis in order to explore the relationship between M. tuberculosis viability and the function of M. tuberculosis phagosomes. M. tuberculosis aliquots were heat killed, treated with rifampin, or exposed to radiation as described in Materials and Methods. The decreases in viability following these treatments were determined by counting CFU or by measuring the release of radioactive 14CO2 from [14C]palmitic acid over 4 days by the BACTEC radiometric method. Live bacteria were used as positive controls, and the viability of these cells was defined as 100% (Table 1). Treatment of bacteria with heat successfully killed all the bacteria as no CFU or incorporation of [14C]mycolic acid was observed. Treatment of bacteria with rifampin or radiation resulted in no detectable incorporation of [14C]mycolic acid, although a small percentage of bacteria were viable when CFU were analyzed after 14 days (the viability was 1.8% after rifampin treatment and 0.54% after exposure to radiation). Thus, treatment of M. tuberculosis with heat, rifampin (50 μg/ml, 48 h), and radiation (5.03 x 104 rads) successfully killed 98 to 100% of the mycobacteria.
Analysis of processing of live, HK, RifK, and RadK M. tuberculosis. Our previous observation that HK M. tuberculosis was processed more efficiently than live M. tuberculosis suggested that live M. tuberculosis may inhibit Ag processing or that HK M. tuberculosis may be less resistant to Ag processing. In the present study we determined the correlation among M. tuberculosis viability, inhibition of phagosome maturation, and MHC-II Ag processing. Since treatment with heat could structurally alter the bacteria and result in the release of mycobacterial constituents, rendering the cells more accessible to protease degradation, processing of M. tuberculosis killed by other treatments (antibiotics, radiation) was compared to processing of live M. tuberculosis.
Processing of M. tuberculosis in IFN--activated BMMs was studied by using an M. tuberculosis-specific T hybridoma, BB7, which recognizes M. tuberculosis Ag 85B(241-256)-I-Ab complexes (38). BMMs do not express MHC-II and must be activated with IFN- to induce MHC-II expression. On the other hand, IFN- promotes phagosome maturation (2, 24, 26, 42, 47, 48, 50), which potentially obscures the effects of M. tuberculosis. Therefore, BMMs were activated with a very low concentration of IFN- (1 U/ml) that was adequate to induce MHC-II expression but still revealed inhibition of phagosome-lysosome fusion by live M. tuberculosis (Table 2). Activated BMMs were incubated with live, HK, RifK, or RadK M. tuberculosis for 20 min, washed, chased for 20 or 100 min, fixed, and incubated with BB7 T hybridoma cells to assess the presentation of M. tuberculosis Ag 85B(241-256)-I-Ab complexes. Processing of HK M. tuberculosis was initiated rapidly, and M. tuberculosis Ag 85B(241-256)-I-Ab complexes were expressed on the cell surface by 20 min even at a low MOI (Fig. 1A). In contrast, processing of live M. tuberculosis and RifK and RadK M. tuberculosis was inefficient following a 20-min chase. Although processing of live, RifK, and RadK M. tuberculosis resulted in detectable presentation of M. tuberculosis Ag 85B(241-256)-I-Ab complexes after a 100-min chase (Fig. 1B), the magnitude of the T-cell response remained low (especially at a low MOI) compared to the magnitude observed with HK M. tuberculosis. Differences in processing between HK M. tuberculosis and live, RifK, or RadK M. tuberculosis were compared by the two-sample t test for all MOIs for both chase incubations and were found to be significant (P < 0.05) for all MOIs except an MOI of 0.156 at the 20-min chase point, at which the responses were very small. The results with M. tuberculosis killed by incubation with streptomycin or isoniazid were similar to the results with RifK M. tuberculosis (data not shown).
To rule out the possibility that there were variations in uptake of the different M. tuberculosis preparations by macrophages, we assessed the uptake of FLUOS-labeled M. tuberculosis by flow cytometry of infected macrophages as previously described (38). No variation was noted in uptake of the different M. tuberculosis preparations (data not shown). Labeling of M. tuberculosis with fluorescein isothiocyanate did not alter processing of HK, live, RifK, or RadK M. tuberculosis (data not shown). To rule out the possibility of a dominant inhibitory effect of the low proportion of viable M. tuberculosis cells remaining in the RifK and RadK M. tuberculosis preparations, we assessed processing of HK preparations spiked with a small proportion (2 or 5%) of live M. tuberculosis cells. The addition of a small proportion of live M. tuberculosis cells to HK M. tuberculosis preparations had no impact on processing of the HK M. tuberculosis (Fig. 2). Thus, the presence of a small percentage of live bacteria in the RifK and RadK samples did not influence the processing of these samples. These results indicate that processing of M. tuberculosis Ags is more efficient with HK M. tuberculosis than with live, RifK, or RadK M. tuberculosis. Furthermore, the M. tuberculosis Ag processing efficiency did not correlate strictly with M. tuberculosis viability, given the differences among HK, live, RifK, and RadK M. tuberculosis.
Analysis of Ag 85 levels in live, HK, RifK, and RadK M. tuberculosis. We tested whether the different processing efficiencies of live, HK, RifK, and RadK M. tuberculosis correlated with variations in Ag 85 levels in these bacteria. Live, HK, RifK, and RadK M. tuberculosis cells were suspended in 1x SDS-PAGE sample buffer, sonicated, and boiled for 10 min. The supernatants were assessed by SDS-PAGE. Treatment of M. tuberculosis with rifampin or radiation had little effect on the levels of Ag 85, as analyzed by Western blotting with rabbit antiserum specific for the 30-kDa polypeptide of M. tuberculosis Ag 85 (Fig. 3). The levels of Ag 85 in the HK cell lysates were 20% lower than the levels in the live cell lysates. Therefore, the differences in the processing efficiencies of live, HK, RifK, and RadK M. tuberculosis were not a consequence of variations in the levels of Ag 85 in these bacteria.
Comparison of phagosomal processing of live, HK, and RadK M. tuberculosis. We previously standardized fractionation of BMMs infected with M. tuberculosis on 27% Percoll gradients to separate intracellular organelles (38). This technique (described in Materials and Methods) allowed successful separation of fractions containing the plasma membrane from fractions containing the MIIC and M. tuberculosis phagosomes (38). By incubating fractions with BB7 T hybridoma cells, we demonstrated that M. tuberculosis Ag 85B(241-256)-I-Ab complexes were formed within phagosomes that contained M. tuberculosis (38). In the present study, we determined if differences in processing of HK M. tuberculosis versus live or RadK M. tuberculosis by IFN--activated BMMs were reflected in the levels of M. tuberculosis Ag 85B(241-256)-I-Ab complexes expressed in phagosomes containing HK, live, or RadK M. tuberculosis. RifK M. tuberculosis was not used in these experiments since 90 min of irradiation of M. tuberculosis had less potential than 48 h of treatment of M. tuberculosis with rifampin to alter expression of proteins by M. tuberculosis and fewer viable organisms persisted after irradiation (Table 1). We also increased the concentration of IFN- used to activate BMMs in these experiments from 1 to 50 U/ml since BB7 T hybridoma cells responded poorly to subcellular fractions isolated from macrophages activated with only 1 U of IFN- per ml. Even at an IFN- concentration of 50 U/ml, the whole-cell processing of HK, live, RifK, and RadK M. tuberculosis by BMMs was similar to that shown in Fig. 1 for 1 U of IFN- per ml (data not shown).
Macrophages were pulsed with M. tuberculosis preparations for 20 min at an MOI of 40, and this was followed by a 10-min chase incubation. The mean number of FLUOS-labeled bacteria taken up per macrophage was determined by flow cytometry as previously described (38). The level of uptake of HK, live, or RadK M. tuberculosis by macrophages was 13 to 15 bacteria per cell. The cells were washed to remove extracellular bacteria, incubated for an additional 10 min (chase) at 37°C, homogenized, and then fractionated on 27% Percoll gradients (Fig. 4). Fractions containing M. tuberculosis phagosomes, MIIC, and plasma membrane were marked by using criteria described previously (38). M. tuberculosis Ag 85B(241-256)-I-Ab complexes were detected in fractions with BB7 T hybridoma cells. BB7 cells responded only to fractions containing HK M. tuberculosis phagosomes and not to fractions containing live or RadK M. tuberculosis phagosomes (Fig. 4A). BB7 cells also detected M. tuberculosis Ag 85B(241-256)-I-Ab complexes in plasma membrane fractions of macrophages pulsed with HK M. tuberculosis. When 0.5 μM Ag 85B(241-256) peptide was added to all fractions during the T-cell assay, BB7 cells responded equally well to all three sets of phagosomal fractions, confirming that there were equivalent amounts of I-Ab in these fractions (Fig. 4B). These observations demonstrate that phagosomes containing HK M. tuberculosis generate M. tuberculosis Ag 85B(241-256)-I-Ab complexes more rapidly and efficiently than phagosomes containing either live or RadK M. tuberculosis generate these complexes.
Comparison of colocalization of LysoTracker with phagosomes containing HK, live, RadK, or RifK M. tuberculosis. The ability of M. tuberculosis to prevent phagosomal maturation is a property of live bacteria (3). In IFN--activated cells, the ability of live M. tuberculosis to prevent phagosomal maturation can be compromised (24, 26, 50). To examine maturation of M. tuberculosis phagosomes, we examined the colocalization of the acidotropic dye LysoTracker Red DND-99 with phagosomes containing HK, live, RadK, and RifK M. tuberculosis in IFN--activated (1 U/ml, 24 h) BMMs by confocal microscopy. The majority of phagosomes containing HK M. tuberculosis colocalized with the LysoTracker dye (Fig. 5A and Table 2) demonstrating the inability of HK M. tuberculosis to prevent phagosomal maturation. Most phagosomes containing live M. tuberculosis did not colocalize with LysoTracker in IFN--activated cells (Fig. 5B and Table 2). It should be noted that the viability of the live M. tuberculosis preparations as determined by the LIVE/DEAD BacLight bacterial viability kit varied from 65 to 72% and could partially account for the number of live M. tuberculosis phagosomes that did colocalize with the LysoTracker dye (Table 2). Even then, the intensity of colocalization of the LysoTracker dye with live M. tuberculosis phagosomes was substantially less than that seen with HK M. tuberculosis phagosomes (determined visually). The low concentration of IFN- used to activate BMMs did not appear to promote phagosome maturation at these early times, since the percentage of live M. tuberculosis phagosomes colocalizing with the LysoTracker dye remained the same irrespective of whether the cells were activated with IFN- (Table 2). The majority of RadK and RifK M. tuberculosis phagosomes colocalized with LysoTracker Red DND-99 (Fig. 5C and D and Table 2), demonstrating that these dead M. tuberculosis preparations were not able to prevent phagosomal maturation. Live, RadK, and RifK M. tuberculosis differed in the ability to modulate M. tuberculosis phagosome maturation, despite the similar efficiencies of bacterial Ag processing observed with these preparations.
DISCUSSION
M. tuberculosis uses multiple mechanisms to evade host defenses, including inhibition of MHC-II expression and Ag processing and presentation (16-19, 23, 25, 28-30, 32, 33, 52), as well as secretion of inhibitory cytokines (e.g., IL-10, IL-6, and transforming growth factor ) (12). While some inhibitory mechanisms may not be active during very early stages of macrophage infection (but may contribute to the maintenance of chronic infection), other mechanisms, including the ability of M. tuberculosis to inhibit phagosome maturation, may be critical for survival of the bacterium during earlier stages of macrophage infection.
Our previous observation that HK M. tuberculosis was processed more efficiently than live M. tuberculosis was processed (38) suggested that during the early stages of macrophage infection, live M. tuberculosis may inhibit Ag processing to help evade host defenses. To further evaluate the effect of M. tuberculosis viability and phagosome maturation on Ag processing, we analyzed processing of M. tuberculosis killed by treatment with radiation or antibiotics. RadK M. tuberculosis and RifK M. tuberculosis were processed with efficiencies similar to that observed with live M. tuberculosis. We therefore concluded that bacterial viability is not necessary to inhibit processing from the high level observed with HK M. tuberculosis. This implies that inhibition of processing by M. tuberculosis does not require active adaptation of M. tuberculosis to the phagosome environment (e.g., by altered expression of M. tuberculosis molecules) or modification of the phagosome via processes that require active bacterial metabolism (e.g., changes in bacterial gene expression or bacterial protein synthesis). Regardless of whether inhibition of processing involves blockade of processing mechanisms or resistance to these mechanisms, the inhibition appears to be generated by heat-labile, constitutively expressed components of M. tuberculosis without a requirement for bacterial viability. These molecules are inactivated or removed from M. tuberculosis by heat killing and could render M. tuberculosis Ags more accessible to proteolysis. Alternatively, the antigen processing capacity of macrophages might be stimulated by HK bacteria (but not by live, RadK, or RifK bacteria).
Recent studies have focused on the role played by constitutively expressed components of M. tuberculosis (e.g., lipoarabinomannan [LAM] and phosphatidylinositol mannoside [PIM]) in modulating phagosome maturation (13, 14, 49). LAM is a heavily glycosylated phosphatidylinositol produced exclusively by mycobacteria, while PIM is a biosynthetic precursor of LAM. Both LAM and PIM have been shown to traffic within infected macrophages and intercalate into host cell endomembranes (7). LAM blocks a trans-Golgi network-to-phagosome phosphatidylinositol 3-kinase-dependent pathway and interferes with phagosomal acquisition of late endosomal and lysosomal markers (e.g., Rab5 and syntaxin 6) (13, 14). PIM facilitates fusion of phagosomes with early endosomal compartments (49). In addition, the cell wall-anchored lipoprotein LprG has been shown to inhibit MHC-II Ag processing in macrophages (17). It would be interesting to determine whether any constitutively expressed components are potentially missing in HK M. tuberculosis and if they play a role in modulating Ag processing that is independent of their ability to modulate phagosome maturation.
We also evaluated the impact of phagosome maturation on processing of M. tuberculosis Ag 85. IFN- has been shown to promote phagosome maturation in different mycobacterial species (2, 24, 26, 42, 47, 48, 50). Fortunately, at the low concentrations of IFN- used to induce MHC-II expression in our BMMs (1 U/ml, 24 h) and within the time that BMMs were incubated with M. tuberculosis (2 h), IFN- did not increase localization of the LysoTracker dye with live M. tuberculosis phagosomes (Table 2). Via et al. did not note a significant increase in colocalization of the LysoTracker dye with phagosomes containing M. bovis BCG in cells activated with IFN- (500 U, 24 h) at early time points (1 h) (50). Schaible et al. did not observe a drop in the pH of phagosomes containing M. avium in IFN- activated cells within 2 h (42).
While most phagosomes containing dead M. tuberculosis preparations (HK, RadK, and RifK) colocalized with the LysoTracker dye, most phagosomes containing live M. tuberculosis preparations did not (Table 2). The viability of our frozen M. tuberculosis stocks ranged from 65 to 72% and is within the range seen by other investigators (9, 27). The percentage of dead bacteria in our preparations could account for the percentage of live M. tuberculosis phagosomes that did colocalize with the LysoTracker dye (although the intensity of colocalization was considerably less than that seen with HK M. tuberculosis phagosomes). It should be noted that our RadK M. tuberculosis preparation was not metabolically viable, as we did not observe any release of radioactive 14CO2 by the BACTEC radiometric method. Frehel et al. previously observed that radiation-killed M. avium initially showed limited fusion with lysosomes (15). However, the metabolic activity of their preparation was not determined. Although live, RadK, and RifK M. tuberculosis differed in the ability to modulate phagosome maturation, they were all processed with similar efficiencies.
We concluded that M. tuberculosis phagosomal Ag processing is modulated by mechanisms that are independent of bacterial viability and modulation of phagosome maturation. Since all the studies that have been described have been done with an avirulent strain of M. tuberculosis (H37Ra), additional studies are required to determine whether similar observations could be made with the virulent strain H37Rv as well. In addition, we need to evaluate whether heat-labile, constitutively expressed M. tuberculosis components present in live, RadK, and RifK M. tuberculosis but inactivated or absent in HK M. tuberculosis contribute to the inhibition of Ag processing seen with live, RadK, and RifK M. tuberculosis.
ACKNOWLEDGMENTS
This work was supported by ALA grant RG-045-N to L.R., by NIH grants AI35726 and AI34343 to C.V.H., and by NIH grants AI27243 and HL 55967 and contract AI95383 (Tuberculosis Research Unit) to W.H.B.
We thank Martha Torres and Ann Morrisey for help with the BACTEC radiometric method. We thank Claire Doerschuk for permission to use the confocal microscope and Sarah Richer for help with confocal microscopy.
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Department of Pathology
Division of Infectious Diseases, Case Western Reserve University
University Hospitals of Cleveland, Cleveland, Ohio
ABSTRACT
Control of Mycobacterium tuberculosis infection requires CD4 T-cell responses and major histocompatibility complex class II (MHC-II) processing of M. tuberculosis antigens (Ags). We have previously demonstrated that macrophages process heat-killed (HK) M. tuberculosis more efficiently than live M. tuberculosis. These observations suggested that live M. tuberculosis may inhibit Ag processing by inhibiting phagosome maturation or that HK M. tuberculosis may be less resistant to Ag processing. In the present study we examined the correlation between M. tuberculosis viability and phagosome maturation and efficiency of Ag processing. Since heat treatment could render M. tuberculosis Ags more accessible to proteolysis, M. tuberculosis was additionally killed by antibiotic treatment and radiation. Processing of HK, live, radiation-killed (RadK), or rifampin-killed (RifK) M. tuberculosis in activated murine bone marrow macrophages was examined by using an I-Ab-restricted T-cell hybridoma cell line (BB7) that recognizes an epitope derived from Ag 85B. Macrophages processed HK M. tuberculosis more rapidly and efficiently than they processed live, RadK, or RifK M. tuberculosis. Live, RadK, and RifK M. tuberculosis cells were processed with similar efficiencies for presentation to BB7 T hybridoma cells. Furthermore, phagosomes containing live or RadK M. tuberculosis expressed fewer M. tuberculosis peptide-MHC-II complexes than phagosomes containing HK M. tuberculosis expressed. Since only live M. tuberculosis was able to prevent acidification of the phagosome, our results suggest that regulation of phagosome maturation does not explain the differences in processing of different forms of M. tuberculosis. These findings suggest that the mechanisms used by M. tuberculosis to inhibit phagosomal maturation differ from the mechanisms involved in modulating phagosome Ag processing.
INTRODUCTION
Mycobacterium tuberculosis is an intracellular pathogen that survives inside macrophage phagosomal compartments. CD4 T-cell responses are critical to the control of M. tuberculosis infection in both animals and humans (5, 6, 22, 45) and require processing of mycobacterial antigens (Ags) to generate peptide-major histocompatibility complex class II (MHC-II) complexes. The ability of M. tuberculosis to inhibit MHC-II expression and Ag presentation may contribute to evasion of immune surveillance (16, 19, 23, 25, 28-30, 33, 52). M. tuberculosis components like the 19-kDa lipoprotein have been shown to inhibit MHC-II expression and Ag processing via a mechanism that is dependent on Toll-like receptor 2 but requires hours to days to establish (16, 30). During the initial stages of macrophage infection, live bacteria are processed and presented by MHC-II molecules less efficiently than heat-killed (HK) bacteria are processed and presented (38), suggesting that live M. tuberculosis may inhibit phagosome maturation soon after phagocytosis to inhibit Ag processing and enhance bacterial survival.
After phagocytosis of most bacteria, phagosomes generally fuse with endosomes and eventually lysosomes to form phagolysosomes in a process known as phagosome maturation. In contrast, M. tuberculosis and some other slowly growing mycobacteria (e.g., Mycobacterium bovis and Mycobacterium avium) modify the phagosomal environment to support their survival inside macrophages. These bacteria inhibit phagosome maturation into a phagolysosome by decreasing phagosomal acidification, fusion of phagosomes with lysosomes, and phagosomal acquisition of lysosomal markers and characteristics (3, 4, 8, 15, 26, 31, 44, 53). Thus, phagosomes containing live mycobacteria exclude vesicular H+-ATPase and prevent acidification of this compartment (44). They stain intensely for markers of early endosomes (e.g., transferrin receptor and Rab5) but only weakly for markers of late endosomes, lysosomes, and mature phagolysosomes (e.g., CD63, lysosome-associated membrane proteins 1 and 2, and cathepsin D) (8-10). In murine macrophages, loss of maturation is also associated with phagosome retention of TACO, a coat protein with an unknown function (11).
Processing of M. tuberculosis Ags for presentation by MHC-II molecules is essential to elicit CD4 T-cell responses. Newly synthesized MHC-II molecules are targeted to endocytic compartments (e.g., the MHC-II compartment [MIIC] and class II vesicles [1, 21, 34, 35, 41, 46, 51]), where they bind peptides derived from internalized Ags. The resulting peptide-MHC-II complexes are transported to the cell surface for presentation to CD4 T cells. Particulate Ags (e.g., bacteria) residing in phagosomes are processed in phagosomal compartments to generate peptides. These peptides then bind newly synthesized MHC-II molecules within phagosomes to form peptide-MHC-II complexes, which are transported to the cell surface (36-40).
We recently investigated the role of phagosomes containing HK or live M. tuberculosis in phagosomal processing of M. tuberculosis Ag 85 (38). Flow organellometry and Western blot analysis of phagosomes isolated from gamma interferon (IFN-)-activated bone marrow macrophages (BMMs) demonstrated that phagosomes containing HK M. tuberculosis and phagosomes containing live M. tuberculosis had similar levels of MHC-II, but only phagosomes containing HK M. tuberculosis acquired increasing levels of lysosome-associated membrane protein 1 (an indication of phagosome maturation) with time. Analysis of Ag processing of HK and live M. tuberculosis with an I-Ab-restricted T-cell hybridoma cell line (BB7) that recognizes an epitope derived from M. tuberculosis Ag 85B (30) demonstrated that macrophages processed HK M. tuberculosis more rapidly and efficiently than they processed live M. tuberculosis. Phagosomes containing live M. tuberculosis expressed fewer M. tuberculosis Ag 85B(241-256)-I-Ab complexes than phagosomes containing HK M. tuberculosis expressed. Since live M. tuberculosis may inhibit Ag processing or HK M. tuberculosis may be less resistant to Ag processing (due to the heat treatment), we also killed M. tuberculosis by radiation or antibiotic treatment in the present study. Macrophages processed HK M. tuberculosis more rapidly and efficiently than they processed live, radiation-killed (RadK), or rifampin-killed (RifK) M. tuberculosis. Live, RadK, and RifK M. tuberculosis cells were all similarly processed for presentation to BB7 T hybridoma cells. Phagosomes containing live or RadK M. tuberculosis expressed fewer M. tuberculosis Ag 85B(241-256)-I-Ab complexes than phagosomes containing HK M. tuberculosis expressed, and only live M. tuberculosis was able to prevent phagosomal acidification. These findings suggest that the mechanisms used by M. tuberculosis to inhibit phagosomal maturation differ from the mechanisms involved in modulating phagosome Ag processing.
MATERIALS AND METHODS
Cells and media. B6D2F1/J mice were obtained from Jackson Laboratories (Bar Harbor, Maine). All animal procedures were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University. Incubation in the absence of M. tuberculosis was performed at 37°C in 5% CO2 in standard medium composed of Dulbecco modified Eagle medium (DMEM) (Life Technologies, Grand Island, N.Y.) supplemented with 10% decomplemented fetal calf serum (HyClone, Logan, Utah), 5 x 10–5 M 2-mercaptoethanol, L-arginine HCl (116 mg/liter), L-asparagine (36 mg/liter), NaHCO3 (2 g/liter), sodium pyruvate (1 mM), 10 mM HEPES buffer, and antibiotics. For incubation with M. tuberculosis, standard medium was modified by using nondecomplemented fetal calf serum and no antibiotics. BMM precursors were harvested from femurs of B6D2F1/J mice and cultured for 7 days in six-well plates in standard medium supplemented with 20% LADMAC (43) cell-conditioned medium. Macrophages were then stimulated for 24 h with 1 U of recombinant IFN- (Genzyme, Cambridge, Mass.) per ml. The resulting confluent cultures contained approximately 1.5 x 106 cells/well. The T-cell hybridoma BB7 recognizes M. tuberculosis Ag 85B(241-256) bound to I-Ab (30). The T-cell hybridoma DOBW recognizes OVA(323-339) bound to I-Ad (or to a lesser degree I-Ab) (20).
Bacteria. M. tuberculosis H37Ra (American Type Culture Collection) was grown in Middlebrook 7H9 broth (Difco, Detroit, Mich.) supplemented with 1% glycerol, 0.05% Tween (Sigma, St. Louis, Mo.) (to prevent clumping), and 10% Middlebrook oleic albumin dextrose catalase enrichment (Difco). Bacteria were harvested and frozen at –70°C as described previously (22). Bacterial titers were determined by counting CFU on Middlebrook 7H10 medium (Difco). Bacteria were serially diluted, and 50-μl samples were plated in triplicate. The detection limit for the CFU assay was approximately 200 CFU/ml. M. tuberculosis H37Ra was killed by heating it to 80°C for 30 min to produce HK M. tuberculosis, treated with rifampin (50 μg/ml; Sigma) for 48 h to produce RifK M. tuberculosis, or exposed to 137Cs radiation for 90 min (559 rads/min; total radiation, 5.03 x 104 rads) to produce RadK M. tuberculosis. The viability of M. tuberculosis was assessed by counting CFU and by measuring the release of radioactive 14CO2 from [14C]palmitic acid over a period of 4 days by the BACTEC radiometric method (Becton Dickenson, Franklin Lakes, N.J.). According to the manufacturer, the detection limit for the BACTEC assay is approximately 400 bacteria. The percentage of live M. tuberculosis in an M. tuberculosis culture was determined by using a LIVE/DEAD BacLight bacterial viability kit from Molecular Probes and varied between 65 to 72%. It should be noted that BacLight viability kit results reflect membrane permeability and are not infallible.
Prior to macrophage infection, M. tuberculosis was opsonized in medium containing 10% non-heat-treated fetal calf serum and 1% non-heat-treated normal mouse serum (Sigma) with no antibiotics for 30 min at 37°C and was declumped to remove large M. tuberculosis aggregates. For declumping, M. tuberculosis was pelleted, washed, passaged 10 times through a 26-gauge needle, and sonicated for 1 min in a tabletop ultrasonic cleaning system (Fisher Scientific). The sample was centrifuged at 150 x g for 3 min to remove clumps and generate the infection stock.
To label M. tuberculosis with fluorescein, 109 bacteria were pelleted, resuspended in 1 ml of phosphate-buffered saline (PBS) (pH 9.1), and combined with 25 μl of a 20-mg/ml solution of FLUOS (Boehringer, Mannheim, Germany) in dimethyl sulfoxide for 5 min at room temperature. M. tuberculosis was similarly labeled with AlexaFluor 488 (Molecular Probes, Eugene, Oreg.) in PBS (pH 9.0) for 15 or 30 min at room temperature. All samples were washed twice in medium, opsonized, and declumped as described above. Labeling of live bacteria with FLUOS or AlexaFluor 488 resulted in no significant change in the viability of M. tuberculosis as determined by counting CFU. FLUOS-labeled M. tuberculosis was used to determine the number of bacteria (HK, live, RadK, and RifK M. tuberculosis) internalized per macrophage by flow cytometry (38). AlexaFluor 488-labeled bacteria were used in the LysoTracker colocalization studies described below.
Antibodies and other reagents. Rabbit antiserum specific for the 30-kDa polypeptide of M. tuberculosis Ag 85 was obtained through the Tuberculosis Research Materials and Vaccine Testing Contract at Colorado State University. Y-3P (American Type Culture Collection), a murine immunoglobulin G2a that recognizes the MHC-II molecule I-Ab, was iodinated by the chloramine T method. LysoTracker Red DND-99 was obtained from Molecular Probes.
Ag processing and presentation assays. Macrophages were replated in 96-well flat-bottom plates at a concentration of 1.5 x 105 cells/well prior to stimulation with IFN- (1 U/ml, 24 h). M. tuberculosis was treated and declumped (as described above) and added in 100 μl (final volume). Bacteria were spun onto cells by centrifugation at 900 x g for 10 min at 37°C. Cells were incubated at 37°C for 10 min (providing a total pulse period of 20 min) and washed in ice-cold DMEM to remove extracellular bacteria. Prewarmed medium was added, and cells were incubated at 37°C for various lengths of time. Macrophages were fixed with 1% paraformaldehyde and washed. T hybridoma cells (1 x 105 cells) were added to each well (total volume, 200 μl) and incubated for 24 h. Supernatants (100 μl) were harvested and then assessed for interleukin-2 (IL-2) by using a CTLL-2 proliferation assay, monitored by addition of Alamar blue (Alamar Biosciences, Sacramento, Calif.) as an indicator dye and measured as the difference between absorbance at 550 nm and absorbance at 595 nm after 24 h. Blanks for spectrophotometry were provided by wells containing medium alone (added at the initiation of the CTLL-2 assay) and Alamar blue (added at the same time that it was added for the other wells). All analyses were performed in triplicate.
Percoll density gradient fractionation for T-cell assay. Three six-well plates containing confluent macrophage cultures were activated with 50 U of IFN- per ml for 24 h and used for each fractionation. The plates were processed as described above to achieve a 20-min pulse with M. tuberculosis (multiplicity of infection [MOI], 40), followed by a 10-min chase incubation. Cells were washed, detached by scraping, washed, resuspended in homogenization buffer (0.25 M sucrose, 10 mM HEPES; pH 7.2), and homogenized in a Dounce homogenizer (Kontes Co., Vineland, N.J.) to obtain 80 to 85% lysis (39, 40). Intact cells and nuclei were removed by three consecutive spins at 200 x g for 5 min at 4°C. The supernatant (containing phagosomes) was collected and layered on 9 ml of 27 or 40% Percoll in homogenization buffer and then centrifuged in a Ti50 fixed-angle rotor (Beckman Instruments, Palo Alto, Calif.) at 4°C for 60 min at 36,000 x g. The gradients were manually fractionated from the top into 30 333-μl fractions, which were divided into replicate 10-, 30-, or 50-μl aliquots and frozen at –80°C.
For identification of fractions containing plasma membrane, phagosomes, and MIIC, the following procedure was performed as previously described (38, 39). The plasma membrane was marked prior to homogenization by incubation of macrophages for 60 min at 4°C with 125I-labeled Ab Y-3P. Fractions containing phagosomes were identified by the presence of FLUOS-labeled bacteria with a Spectra Fluor Plus fluorimeter (Tecan, Reading, United Kingdom). Macrophages were incubated with soluble ovalbumin (OVA) (Sigma) at a concentration of 3 mg/ml for 2 h to label MIIC vesicles with OVA(323-339)-I-Ad complexes. Fractions containing OVA(323-339)-I-Ad complexes were identified by the T-cell assay described below.
For T-cell analysis of Percoll gradient fractions, standard medium and T hybridoma cells (BB7 cells for experiments with M. tuberculosis or DOBW cells for experiments with OVA; 105 cells/well) were added to 200 μl (final volume) in 96-well plates. After 24 h, the T-cell responses were assessed as described above. Wells containing only equivalent amounts of Percoll, T cells, and medium were used to generate control supernatants and blanks for the CTLL-2 assay.
Western blotting. Bacterial pellets were resuspended in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer, sonicated for 1.5 min, boiled for 10 min, and spun for 10 min in an Eppendorf centrifuge. Ten micrograms of protein in the supernatant was electrophoresed on SDS—12% polyacrylamide gels, blotted onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore Corporation, Bedford, Mass.), probed with Ag 85-specific rabbit antiserum, and detected with horseradish peroxidase-conjugated anti-rabbit serum by using an enhanced chemiluminescence kit (Amersham).
LysoTracker colocalization studies. Macrophages were replated on coverslips (no. 1.5; Corning, Corning, N.Y.), activated with 1 U of IFN- per ml for 24 h, and then pulsed for 20 min with AlexaFluor 488-labeled HK, live, RadK, or RifK M. tuberculosis (MOI, 1) (labeling of M. tuberculosis is described above). The cells were washed with warmed DMEM to remove extracellular bacteria. Prewarmed medium was added to the cells, and the chase incubation was continued for 20 or 100 min. In the final 5 min of the chase incubation, cells were incubated with medium containing 500 nM LysoTracker Red DND-99 (Molecular Probes) as recommended by the manufacturer. The cells were washed twice in PBS and analyzed with a Leica TCS SP2 AOBS confocal microscope. The viability of the live M. tuberculosis preparations was determined by using a LIVE/DEAD BacLight bacterial viability kit from Molecular Probes.
Statistical analysis. The statistical analysis was done with the two-sample t test by using the MiniTab statistical software (MiniTab Inc., State College, Pa.).
RESULTS
Analysis of M. tuberculosis viability following treatment with the antibiotic rifampin or radiation. Several methods were used to kill M. tuberculosis in order to explore the relationship between M. tuberculosis viability and the function of M. tuberculosis phagosomes. M. tuberculosis aliquots were heat killed, treated with rifampin, or exposed to radiation as described in Materials and Methods. The decreases in viability following these treatments were determined by counting CFU or by measuring the release of radioactive 14CO2 from [14C]palmitic acid over 4 days by the BACTEC radiometric method. Live bacteria were used as positive controls, and the viability of these cells was defined as 100% (Table 1). Treatment of bacteria with heat successfully killed all the bacteria as no CFU or incorporation of [14C]mycolic acid was observed. Treatment of bacteria with rifampin or radiation resulted in no detectable incorporation of [14C]mycolic acid, although a small percentage of bacteria were viable when CFU were analyzed after 14 days (the viability was 1.8% after rifampin treatment and 0.54% after exposure to radiation). Thus, treatment of M. tuberculosis with heat, rifampin (50 μg/ml, 48 h), and radiation (5.03 x 104 rads) successfully killed 98 to 100% of the mycobacteria.
Analysis of processing of live, HK, RifK, and RadK M. tuberculosis. Our previous observation that HK M. tuberculosis was processed more efficiently than live M. tuberculosis suggested that live M. tuberculosis may inhibit Ag processing or that HK M. tuberculosis may be less resistant to Ag processing. In the present study we determined the correlation among M. tuberculosis viability, inhibition of phagosome maturation, and MHC-II Ag processing. Since treatment with heat could structurally alter the bacteria and result in the release of mycobacterial constituents, rendering the cells more accessible to protease degradation, processing of M. tuberculosis killed by other treatments (antibiotics, radiation) was compared to processing of live M. tuberculosis.
Processing of M. tuberculosis in IFN--activated BMMs was studied by using an M. tuberculosis-specific T hybridoma, BB7, which recognizes M. tuberculosis Ag 85B(241-256)-I-Ab complexes (38). BMMs do not express MHC-II and must be activated with IFN- to induce MHC-II expression. On the other hand, IFN- promotes phagosome maturation (2, 24, 26, 42, 47, 48, 50), which potentially obscures the effects of M. tuberculosis. Therefore, BMMs were activated with a very low concentration of IFN- (1 U/ml) that was adequate to induce MHC-II expression but still revealed inhibition of phagosome-lysosome fusion by live M. tuberculosis (Table 2). Activated BMMs were incubated with live, HK, RifK, or RadK M. tuberculosis for 20 min, washed, chased for 20 or 100 min, fixed, and incubated with BB7 T hybridoma cells to assess the presentation of M. tuberculosis Ag 85B(241-256)-I-Ab complexes. Processing of HK M. tuberculosis was initiated rapidly, and M. tuberculosis Ag 85B(241-256)-I-Ab complexes were expressed on the cell surface by 20 min even at a low MOI (Fig. 1A). In contrast, processing of live M. tuberculosis and RifK and RadK M. tuberculosis was inefficient following a 20-min chase. Although processing of live, RifK, and RadK M. tuberculosis resulted in detectable presentation of M. tuberculosis Ag 85B(241-256)-I-Ab complexes after a 100-min chase (Fig. 1B), the magnitude of the T-cell response remained low (especially at a low MOI) compared to the magnitude observed with HK M. tuberculosis. Differences in processing between HK M. tuberculosis and live, RifK, or RadK M. tuberculosis were compared by the two-sample t test for all MOIs for both chase incubations and were found to be significant (P < 0.05) for all MOIs except an MOI of 0.156 at the 20-min chase point, at which the responses were very small. The results with M. tuberculosis killed by incubation with streptomycin or isoniazid were similar to the results with RifK M. tuberculosis (data not shown).
To rule out the possibility that there were variations in uptake of the different M. tuberculosis preparations by macrophages, we assessed the uptake of FLUOS-labeled M. tuberculosis by flow cytometry of infected macrophages as previously described (38). No variation was noted in uptake of the different M. tuberculosis preparations (data not shown). Labeling of M. tuberculosis with fluorescein isothiocyanate did not alter processing of HK, live, RifK, or RadK M. tuberculosis (data not shown). To rule out the possibility of a dominant inhibitory effect of the low proportion of viable M. tuberculosis cells remaining in the RifK and RadK M. tuberculosis preparations, we assessed processing of HK preparations spiked with a small proportion (2 or 5%) of live M. tuberculosis cells. The addition of a small proportion of live M. tuberculosis cells to HK M. tuberculosis preparations had no impact on processing of the HK M. tuberculosis (Fig. 2). Thus, the presence of a small percentage of live bacteria in the RifK and RadK samples did not influence the processing of these samples. These results indicate that processing of M. tuberculosis Ags is more efficient with HK M. tuberculosis than with live, RifK, or RadK M. tuberculosis. Furthermore, the M. tuberculosis Ag processing efficiency did not correlate strictly with M. tuberculosis viability, given the differences among HK, live, RifK, and RadK M. tuberculosis.
Analysis of Ag 85 levels in live, HK, RifK, and RadK M. tuberculosis. We tested whether the different processing efficiencies of live, HK, RifK, and RadK M. tuberculosis correlated with variations in Ag 85 levels in these bacteria. Live, HK, RifK, and RadK M. tuberculosis cells were suspended in 1x SDS-PAGE sample buffer, sonicated, and boiled for 10 min. The supernatants were assessed by SDS-PAGE. Treatment of M. tuberculosis with rifampin or radiation had little effect on the levels of Ag 85, as analyzed by Western blotting with rabbit antiserum specific for the 30-kDa polypeptide of M. tuberculosis Ag 85 (Fig. 3). The levels of Ag 85 in the HK cell lysates were 20% lower than the levels in the live cell lysates. Therefore, the differences in the processing efficiencies of live, HK, RifK, and RadK M. tuberculosis were not a consequence of variations in the levels of Ag 85 in these bacteria.
Comparison of phagosomal processing of live, HK, and RadK M. tuberculosis. We previously standardized fractionation of BMMs infected with M. tuberculosis on 27% Percoll gradients to separate intracellular organelles (38). This technique (described in Materials and Methods) allowed successful separation of fractions containing the plasma membrane from fractions containing the MIIC and M. tuberculosis phagosomes (38). By incubating fractions with BB7 T hybridoma cells, we demonstrated that M. tuberculosis Ag 85B(241-256)-I-Ab complexes were formed within phagosomes that contained M. tuberculosis (38). In the present study, we determined if differences in processing of HK M. tuberculosis versus live or RadK M. tuberculosis by IFN--activated BMMs were reflected in the levels of M. tuberculosis Ag 85B(241-256)-I-Ab complexes expressed in phagosomes containing HK, live, or RadK M. tuberculosis. RifK M. tuberculosis was not used in these experiments since 90 min of irradiation of M. tuberculosis had less potential than 48 h of treatment of M. tuberculosis with rifampin to alter expression of proteins by M. tuberculosis and fewer viable organisms persisted after irradiation (Table 1). We also increased the concentration of IFN- used to activate BMMs in these experiments from 1 to 50 U/ml since BB7 T hybridoma cells responded poorly to subcellular fractions isolated from macrophages activated with only 1 U of IFN- per ml. Even at an IFN- concentration of 50 U/ml, the whole-cell processing of HK, live, RifK, and RadK M. tuberculosis by BMMs was similar to that shown in Fig. 1 for 1 U of IFN- per ml (data not shown).
Macrophages were pulsed with M. tuberculosis preparations for 20 min at an MOI of 40, and this was followed by a 10-min chase incubation. The mean number of FLUOS-labeled bacteria taken up per macrophage was determined by flow cytometry as previously described (38). The level of uptake of HK, live, or RadK M. tuberculosis by macrophages was 13 to 15 bacteria per cell. The cells were washed to remove extracellular bacteria, incubated for an additional 10 min (chase) at 37°C, homogenized, and then fractionated on 27% Percoll gradients (Fig. 4). Fractions containing M. tuberculosis phagosomes, MIIC, and plasma membrane were marked by using criteria described previously (38). M. tuberculosis Ag 85B(241-256)-I-Ab complexes were detected in fractions with BB7 T hybridoma cells. BB7 cells responded only to fractions containing HK M. tuberculosis phagosomes and not to fractions containing live or RadK M. tuberculosis phagosomes (Fig. 4A). BB7 cells also detected M. tuberculosis Ag 85B(241-256)-I-Ab complexes in plasma membrane fractions of macrophages pulsed with HK M. tuberculosis. When 0.5 μM Ag 85B(241-256) peptide was added to all fractions during the T-cell assay, BB7 cells responded equally well to all three sets of phagosomal fractions, confirming that there were equivalent amounts of I-Ab in these fractions (Fig. 4B). These observations demonstrate that phagosomes containing HK M. tuberculosis generate M. tuberculosis Ag 85B(241-256)-I-Ab complexes more rapidly and efficiently than phagosomes containing either live or RadK M. tuberculosis generate these complexes.
Comparison of colocalization of LysoTracker with phagosomes containing HK, live, RadK, or RifK M. tuberculosis. The ability of M. tuberculosis to prevent phagosomal maturation is a property of live bacteria (3). In IFN--activated cells, the ability of live M. tuberculosis to prevent phagosomal maturation can be compromised (24, 26, 50). To examine maturation of M. tuberculosis phagosomes, we examined the colocalization of the acidotropic dye LysoTracker Red DND-99 with phagosomes containing HK, live, RadK, and RifK M. tuberculosis in IFN--activated (1 U/ml, 24 h) BMMs by confocal microscopy. The majority of phagosomes containing HK M. tuberculosis colocalized with the LysoTracker dye (Fig. 5A and Table 2) demonstrating the inability of HK M. tuberculosis to prevent phagosomal maturation. Most phagosomes containing live M. tuberculosis did not colocalize with LysoTracker in IFN--activated cells (Fig. 5B and Table 2). It should be noted that the viability of the live M. tuberculosis preparations as determined by the LIVE/DEAD BacLight bacterial viability kit varied from 65 to 72% and could partially account for the number of live M. tuberculosis phagosomes that did colocalize with the LysoTracker dye (Table 2). Even then, the intensity of colocalization of the LysoTracker dye with live M. tuberculosis phagosomes was substantially less than that seen with HK M. tuberculosis phagosomes (determined visually). The low concentration of IFN- used to activate BMMs did not appear to promote phagosome maturation at these early times, since the percentage of live M. tuberculosis phagosomes colocalizing with the LysoTracker dye remained the same irrespective of whether the cells were activated with IFN- (Table 2). The majority of RadK and RifK M. tuberculosis phagosomes colocalized with LysoTracker Red DND-99 (Fig. 5C and D and Table 2), demonstrating that these dead M. tuberculosis preparations were not able to prevent phagosomal maturation. Live, RadK, and RifK M. tuberculosis differed in the ability to modulate M. tuberculosis phagosome maturation, despite the similar efficiencies of bacterial Ag processing observed with these preparations.
DISCUSSION
M. tuberculosis uses multiple mechanisms to evade host defenses, including inhibition of MHC-II expression and Ag processing and presentation (16-19, 23, 25, 28-30, 32, 33, 52), as well as secretion of inhibitory cytokines (e.g., IL-10, IL-6, and transforming growth factor ) (12). While some inhibitory mechanisms may not be active during very early stages of macrophage infection (but may contribute to the maintenance of chronic infection), other mechanisms, including the ability of M. tuberculosis to inhibit phagosome maturation, may be critical for survival of the bacterium during earlier stages of macrophage infection.
Our previous observation that HK M. tuberculosis was processed more efficiently than live M. tuberculosis was processed (38) suggested that during the early stages of macrophage infection, live M. tuberculosis may inhibit Ag processing to help evade host defenses. To further evaluate the effect of M. tuberculosis viability and phagosome maturation on Ag processing, we analyzed processing of M. tuberculosis killed by treatment with radiation or antibiotics. RadK M. tuberculosis and RifK M. tuberculosis were processed with efficiencies similar to that observed with live M. tuberculosis. We therefore concluded that bacterial viability is not necessary to inhibit processing from the high level observed with HK M. tuberculosis. This implies that inhibition of processing by M. tuberculosis does not require active adaptation of M. tuberculosis to the phagosome environment (e.g., by altered expression of M. tuberculosis molecules) or modification of the phagosome via processes that require active bacterial metabolism (e.g., changes in bacterial gene expression or bacterial protein synthesis). Regardless of whether inhibition of processing involves blockade of processing mechanisms or resistance to these mechanisms, the inhibition appears to be generated by heat-labile, constitutively expressed components of M. tuberculosis without a requirement for bacterial viability. These molecules are inactivated or removed from M. tuberculosis by heat killing and could render M. tuberculosis Ags more accessible to proteolysis. Alternatively, the antigen processing capacity of macrophages might be stimulated by HK bacteria (but not by live, RadK, or RifK bacteria).
Recent studies have focused on the role played by constitutively expressed components of M. tuberculosis (e.g., lipoarabinomannan [LAM] and phosphatidylinositol mannoside [PIM]) in modulating phagosome maturation (13, 14, 49). LAM is a heavily glycosylated phosphatidylinositol produced exclusively by mycobacteria, while PIM is a biosynthetic precursor of LAM. Both LAM and PIM have been shown to traffic within infected macrophages and intercalate into host cell endomembranes (7). LAM blocks a trans-Golgi network-to-phagosome phosphatidylinositol 3-kinase-dependent pathway and interferes with phagosomal acquisition of late endosomal and lysosomal markers (e.g., Rab5 and syntaxin 6) (13, 14). PIM facilitates fusion of phagosomes with early endosomal compartments (49). In addition, the cell wall-anchored lipoprotein LprG has been shown to inhibit MHC-II Ag processing in macrophages (17). It would be interesting to determine whether any constitutively expressed components are potentially missing in HK M. tuberculosis and if they play a role in modulating Ag processing that is independent of their ability to modulate phagosome maturation.
We also evaluated the impact of phagosome maturation on processing of M. tuberculosis Ag 85. IFN- has been shown to promote phagosome maturation in different mycobacterial species (2, 24, 26, 42, 47, 48, 50). Fortunately, at the low concentrations of IFN- used to induce MHC-II expression in our BMMs (1 U/ml, 24 h) and within the time that BMMs were incubated with M. tuberculosis (2 h), IFN- did not increase localization of the LysoTracker dye with live M. tuberculosis phagosomes (Table 2). Via et al. did not note a significant increase in colocalization of the LysoTracker dye with phagosomes containing M. bovis BCG in cells activated with IFN- (500 U, 24 h) at early time points (1 h) (50). Schaible et al. did not observe a drop in the pH of phagosomes containing M. avium in IFN- activated cells within 2 h (42).
While most phagosomes containing dead M. tuberculosis preparations (HK, RadK, and RifK) colocalized with the LysoTracker dye, most phagosomes containing live M. tuberculosis preparations did not (Table 2). The viability of our frozen M. tuberculosis stocks ranged from 65 to 72% and is within the range seen by other investigators (9, 27). The percentage of dead bacteria in our preparations could account for the percentage of live M. tuberculosis phagosomes that did colocalize with the LysoTracker dye (although the intensity of colocalization was considerably less than that seen with HK M. tuberculosis phagosomes). It should be noted that our RadK M. tuberculosis preparation was not metabolically viable, as we did not observe any release of radioactive 14CO2 by the BACTEC radiometric method. Frehel et al. previously observed that radiation-killed M. avium initially showed limited fusion with lysosomes (15). However, the metabolic activity of their preparation was not determined. Although live, RadK, and RifK M. tuberculosis differed in the ability to modulate phagosome maturation, they were all processed with similar efficiencies.
We concluded that M. tuberculosis phagosomal Ag processing is modulated by mechanisms that are independent of bacterial viability and modulation of phagosome maturation. Since all the studies that have been described have been done with an avirulent strain of M. tuberculosis (H37Ra), additional studies are required to determine whether similar observations could be made with the virulent strain H37Rv as well. In addition, we need to evaluate whether heat-labile, constitutively expressed M. tuberculosis components present in live, RadK, and RifK M. tuberculosis but inactivated or absent in HK M. tuberculosis contribute to the inhibition of Ag processing seen with live, RadK, and RifK M. tuberculosis.
ACKNOWLEDGMENTS
This work was supported by ALA grant RG-045-N to L.R., by NIH grants AI35726 and AI34343 to C.V.H., and by NIH grants AI27243 and HL 55967 and contract AI95383 (Tuberculosis Research Unit) to W.H.B.
We thank Martha Torres and Ann Morrisey for help with the BACTEC radiometric method. We thank Claire Doerschuk for permission to use the confocal microscope and Sarah Richer for help with confocal microscopy.
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