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Originally published online as doi:10.1189/jlb.0505250 on November 7, 2005

Published online before print November 7, 2005
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(Journal of Leukocyte Biology. 2006;79:80-86.)
© 2006 by Society for Leukocyte Biology

Virulent clinical isolates of Mycobacterium tuberculosis grow rapidly and induce cellular necrosis but minimal apoptosis in murine macrophages

Jae Seuk Park, Marcela Henao Tamayo, Mercedes Gonzalez-Juarrero, Ian M. Orme1 and Diane J. Ordway

Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins

1 Correspondence: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523. E-mail: Ian.Orme{at}colostate.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we investigated the ability of four clinical isolates of Mycobacterium tuberculosis representing a range of virulence for their capacity to grow in bone marrow-derived macrophages. The rate of growth of each of the isolates in macrophages reflected their known virulence, but the most virulent isolates strongly induced production of the cytokine tumor necrosis factor {alpha}. A key difference, however, was the degree of cell cytotoxicity observed with the more virulent strains after several days in culture. Staining of cell monolayers for DNA fragmentation indicative of apoptosis showed that this was minimal and only evident to any degree in macrophages infected with the most virulent strains. In contrast, electron microscopy revealed damage of macrophages consistent with cell necrosis. These results suggest that rapid intracellular growth rate and induction of necrotic cell death within host macrophages are virulence factors of M. tuberculosis in the early stages of bacterial infection. They further imply that infected cell apoptosis, regarded as a defense mechanism or cross-priming mechanism, plays a minimal role.

Key Words: tuberculosis • virulence factors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tuberculosis remains the most frequent cause of death as a result of infectious disease throughout the world [1 2 3 ]. Approximately 8 million cases are reported each year with approximately 2 million deaths [4 ]. As a result of genetic analysis, new families of Mycobacterium tuberculosis have been recognized, including the Beijing/W strain [5 6 7 8 ]. This organism, as well as many other recent isolates, appears to be of high virulence based on studies in animal models [9 ].

It remains unclear exactly why M. tuberculosis is virulent. Various suggestions have been put forward, including the ability to divide (relatively) rapidly in the infected host, its intracellular niche, and the ability to enter dormancy and its possession of a complex cell wall, which is impervious to attack and contains noxious lipids [10 11 12 13 14 15 16 17 18 19 ]. More recently, it has been demonstrated that the lack of virulence in attenuated strains such as the bacillus Calmette-Guerin (BCG) vaccine, reflects the loss of genetic material encoding cytotoxic materials including culture filtrate protein-10 (CFP-10) and early secretory antigenic target-6 (ESAT-6) [20 21 22 ]. Clearly, a better identification of M. tuberculosis virulence factors is essential to understanding the pathogenesis of tuberculosis and may reveal salient components of the host defense.

Macrophages are the host cells for M. tuberculosis and represent the initial innate defense against this organism [23 ]. After uptake, the bacilli are to be found in phagosomes, where they survive initially by preventing host mechanisms such as phagosomal acidification and lysosome fusion [17 , 24 ]. Once the bacilli begin to grow, the initial host macrophage is probably destroyed, and other macrophages and incoming monocytes become infected. Destruction of the initial host cell may be cytotoxic or apoptotic, and the latter has been proposed as a defense mechanism to slow the dissemination of the disease process [25 26 27 28 29 ]. Soon thereafter acquired immunity is initiated, containing the site of infection by a granulomatous inflammatory mechanism [30 ].

In the current study, we used a mouse macrophage model to examine early events soon after infection, and clinical isolates of M. tuberculosis representing a range of virulence, measured as their growth in the lungs of mice after aerosol exposure [31 ]. Those of higher virulence grew faster in macrophages, but all isolates induced similar amounts of host cytokines, and each could be inhibited by the addition of interferon-{gamma} (IFN-{gamma}) to cultures. Where the more virulent strains differed, however, was in their ability to cause host cell death. Given the popularity of the idea that apoptosis underlies much of this, we stained for DNA fragmentation but found only a small percentage of cells undergoing this process in macrophages infected with these isolates. Instead, examination of cells by electron microscopy suggested that macrophages infected with the more virulent strains were being destroyed by necrosis. Accordingly, these findings question the hypothesis that apoptosis is a major defense mechanism used by infected macrophages in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Eight- to 10-week old-specific pathogen-free C57BL/6 female mice were purchased from the Jackson Laboratories (Bar Harbor, ME). All mice were housed in a Biosafety Level III animal facility and given mouse chow and water ad libitum.

Isolation of bone marrow-derived macrophages (BMM)
BMM were obtained from C57BL/6 mice. Briefly, mice were killed by cervical dislocation, and the femur and tibia were aseptically removed. Bone marrow was flushed out of the bones by using ice-cold complete cell culture medium (cCM) consisting of Dulbecco’s modified Eagle’s medium containing 10 mM HEPES, 2 mM L-glutamine, 100 U penicillin per ml, 100 µg streptomycin per ml, and nonessential amino acids with 10% L-929 fibroblast-conditioned medium and 10% heat-inactivated fetal calf serum. Bone marrow cells were plated at 2 x 106 cells/well in 24-well tissue-culture plates in 1 ml cCM and incubated at 37°C in 5% CO2. After 24 h, 1 ml cCM was added, and nonadherent cells were removed after another 24 h by replacing the medium with fresh cCM. Cells were incubated for another 6 days with one change of medium on Day 5. cCM was replaced with incomplete cCM (lacking antibiotics and L-929-conditioned medium) on Day 7 and used for infection on Day 8. The number of macrophages at the time of infection was ~2 x 105 cells/well.

Bacteria
Virulent M. tuberculosis strain H37Rv (TMC 107, Trudeau Institute, Saranac Lake, NY) and clinical strains obtained from patients with active tuberculosis (CSU93, CSU22, CSU15, and CSU21) were grown from a low passage number seed on glycerol alanine salts medium or Proskauer-Beck liquid media to mid-log phase, aliquoted, and stored at –70°C. Before inoculation, the aliquots of mycobacteria were resuspended in incomplete medium, and bacterial clumps were dispersed by passing through a 26-gauge needle five times. For each experiment, the adequacy of dispersion was checked by acid-fast staining of bacterial suspension. The number of viable bacilli in the suspension was measured by serially diluting the bacterial suspensions in saline and plating onto Middlebrook 7H11 agar plates. The colonies, which appeared on the plates 3–4 weeks of incubation at 37°C, were counted to determine the number of viable bacilli per milliliter.

Experimental infection of BMM and bacterial enumeration
Macrophages were infected with mycobacteria, and 6 h later, the monolayers were washed to remove extracellular bacilli. In some studies, recombinant murine IFN-{gamma} (BD PharMingen, San Diego, CA) was added at a dose of 100 U every other day. At each time-point, supernatants were collected and frozen at –70°C. The numbers of intracellular mycobacteria were measured using acid-fast staining and by plating. For the latter, 1 ml double-distilled H2O containing 0.05% Tween 80 was added to monolayers and incubated for 10 min to lyse macrophages. After passing through a 26-gauge needle five times, the lysates were serially diluted and plated onto Middlebrook 7H11 agar plates.

Deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay
The presence of apopotic cells in macrophage cultures was analyzed by the TUNEL assay with the MEBSTAIN apoptosis kit (Medical and Biological Laboratories Co., Japan). The protocol provided by the manufacturer was modified to be used for immunohistochemistry. In brief, BMM were placed in wells in chamber slides (Sigma Chemical Co., St. Louis, MO), infected as described previously, and fixed for 15 min with a solution containing 4% paraformaldehyde. Therafter, the slides were washed twice with phosphate-buffered saline (PBS), and 50 µl terminal deoxynucleotidyl transferase (TdT) buffer II was added and incubated for 10 min at room temperature. The TdT reaction was performed in the presence of biotin-dUTP for 1 h at 37°C. After washing three times with distilled water, the slides were incubated with blocking solution for 10 min at room temperature. The blocking solution was removed, and 200 µl streptavidin:horseradish peroxidase (Serotec Inc., Raleigh, NC) was incubated for 1 h at 37°C. Following three washes with PBS, the reaction was developed using airway epithelial cells (BioGenex, San Ramon, CA) as substrate. For counterstaining, Meyer’s hematoxylin was used and mounted using crystal/mount (Biomedia Corp., Foster City, CA). As a positive control for apoptosis, camptothecin was used at a concentration of 20 µm/ml (this molecule inhibits the nuclear enzyme DNA topoisomerase type I).

Cellular viability assays
Cell death was assayed by trypan blue exclusion and by Naphthol blue-black cell viability-staining methods [32 ].

Measurement of host cytokines
A cytometric bead array kit (CBA; BD Biosciences, San Jose, CA) was used to measure inflammatory cytokines. BMM were incubated with the different strains of M. tuberculosis at a multiplicity of infection (MOI) of 1:5 at 4 h and supernatants were collected and then frozen back at –80°C on Days 2, 4, and 6. After thawing the supernatants, the CBA mouse inflammatory cytokine assay procedure was performed according to kit instructions. Assays were completed with duplicate samples, and results are expressed as a mean of two experiments. Values are represented by the mean cytokine pg/ml minus the noninfected media control. The beads were analyzed on a Becton Dickinson (San Jose, CA) FACSCalibur flow cytometer.

Transmission electron microscopic analysis
For transmission electron microscopy, BMM were infected with M. tuberculosis at a MOI of 5 for 6 h, followed by extensive washing of extracellular bacteria and further incubation for 6 days. Infected cells were prepared for electron microscopy by first adding fixative consisting of 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer with 5 mM CaCl2 and MgCl2 and incubating the cells for 1 h at room temperature and then overnight at 4°C. After washing cells with sodium cacodylate buffer, the cells were fixed with 1% osmium tetroxide, resuspended in warm agar, and solidified at 4°C. Sections were cut and stained with uranyl acetate, dehydrated using acetone, and embedded in resin. The sections were analyzed with a JEOL 2000EXII transmission electron microscope (Peabody, MA).

Statistics
The data are presented as mean ± SD of three independent experiments. Statistical analysis of the data was carried out with Student’s t-test. Differences were considered significant at a P level of <0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Growth of M. tuberculosis in mouse BMM
BMM were infected in vitro with M. tuberculosis isolates representing a range of virulence. The growth of each isolate was determined by acid-fast staining (Fig. 1A ) or by the more accurate method of plating to measure colony-forming units (CFU; Fig. 1B ). Using both methods, it was observed that the more virulent isolates, CSU22 and CSU93, grew much better in the BMM cells.



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  		Figure 1. Growth of M. tuberculosis strains in macrophages cultured from C57BL/6 mice. BMM were infected with M. tuberculosis strains at a MOI of 1, and the numbers of intracellular bacteria were determined using the acid-fast staining method [acid-fast bacilli (AFB); A] or the bacterial colony count method (CFU; B) immediately after infection or at 3 or 6 days after infection. Values shown are the means ± SD from three independent experiments. Growth of the CSU22 strain was significantly higher than the other isolates (*, P<0.05).

 
Production of host effector molecules by M. tuberculosis-infected mouse BMM
Upon infection, macrophages secrete a variety of cytokines and other molecules. To determine if this differed between clinical isolates, the concentrations of tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-6, and IL-10 in culture supernatants, as well as the chemokine monocyte chemoattractant protein-1 (MCP-1), were determined over time as illustrated in Figure 2 . TNF was rapidly produced within 4 h after infection with the two virulent isolates. It is interesting that the highest production of IL-6 and IL-10 was observed in cultures of H37Rv and with much slower kinetics. This was also seen in terms of MCP-1, although production of this chemokine to the two virulent strains was also observed.



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  		Figure 2. Cytokine and chemokine production induced by different isolates. Supernatants were analyzed by CBA using flow cytometry. Values are the means ± SD from two experiments.

 
As further evidence that the macrophages remained functional despite infection, their capacity to be activated by IFN-{gamma} was then determined. As shown in Figure 3 , preactivation of macrophages with this cytokine reduced the numbers of bacilli per cell and concomitantly reduced cell death.



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  		Figure 3. Intracellular growth of M. tuberculosis (A) and cytotoxicity (B) of macrophages infected with M. tuberculosis were inhibited by IFN-{gamma}. BMM were infected with M. tuberculosis strains (MOI of 5) and incubated with or without IFN-{gamma} treatment (100 U/ml). Numbers of intracellular bacteria were determined by acid-fast staining (A), and the percentages of dead cells were determined by trypan blue dye exclusion (B) after 6 days of infection. Values are the means ± SD from three independent experiments. *, Statistically significant decreases (P<0.01).

 
Evidence for minimal apoptosis in infected macrophage cultures
To determine if the observed cell death seen in infected macrophages was a result of apoptosis, a TUNEL apoptosis kit was used to assay DNA fragmentation. The results of this study are shown in Figure 4 , and Figure 4A shows control uninfected-cultured macrophages in which no apoptosis was observed. Figure 4B shows the development of apoptosis in macrophages infected with the five isolates at a MOI of 1:5 after 2, 4, or 6 days of infection. No apoptotic cells were seen in the macrophage monolayers infected with the control strain H37Rv. Apoptosis was detected in monolayers with all four clinical isolates, but it was far more evident in cells infected with the two more virulent isolates CSU22 and CSU93. Having said that, the percentages of cells identified by the TUNEL method were low. CSU22 had the highest levels of apoptotic cells, but this was also associated with cell aggregation, as shown in Figure 5 . This latter figure also shows cells treated with the apoptosis inducer camptothecin, used here as a positive control.



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  		Figure 4. Staining of macrophage monolayers for DNA fragmentation by the TUNEL method on Day 6 of the culture period. Arrows indicate cells staining positive. The data are representative of two separate experiments.

 


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  		Figure 5. Higher power field of macrophage monolayers showing cell aggregation and positive staining for apoptosis in infected cells. Cultures were analyzed on Day 6, after initial infection with a MOI of 1:5. A positive control apoptosis was induced using camptothecin. Original magnification, 100x.

 
Cell death was associated with cellular necrosis
Given the observation that measurable cell death did not correlate with the degree of apoptosis that could be measured, we examined infected macrophages under electron microscopy. As shown in Figure 6 , macrophages infected with the low virulence CSU21 strain showed intact cell walls and organelles, and bacilli were clearly encapsulated within individual phagosomal membranes. In contrast, macrophages infected with CSU22 were necrotic, with cell swelling, lysis of cytoplasmic membranes, and release of intracellular constituents including the bacilli. It further appeared that the phagosomal membranes had become disorganized so that multiple bacilli occupied each vesicle.



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  		Figure 6. Electron micrographs of murine macrophages infected by M. tuberculosis strain CSU21 (A) and CSU22 (B–D). Transmission electron microscopy was performed on M. tuberculosis-infected BMM (MOI of 5) after incubating for 6 days. Macrophages infected with M. tuberculosis strain CSU21 showed intact morphology (A), and each bacterium was located in separate vacuoles (arrow). Macrophages infected with M. tuberculosis strain CSU22 show necrotic features, including swelling of organelles and cell membrane (B, C) leading to cell lysis, allowing the release of bacterial particles (D). Note that multiple bacteria can be seen located in phagosomal vacuoles (C). Original magnification, 4000x.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study show that clinical isolates of M. tuberculosis, which are virulent in mice infected by aerosol exposure [31 ], also grow more readily in cultures of BMM. The infected cells remained functional in the sense that they could secrete large amounts of the cytokine TNF-{alpha}, but those infected with the more virulent strains also exhibited increasing cell death (up to 40% by Day 6 in the case of CSU22). This could be reversed by prior activation of macrophages with the cytokine IFN-{gamma}. Staining of monolayers for DNA fragmentation indicating apoptosis revealed that only the virulent isolates induced this to any extent and that this extent was quite low. Instead, observation of cells by electron microscopy suggested cell death by necrosis, which of course is a primary marker of the disease in humans [33 ].

A better propensity to initially grow in such cells would presumably result in the bacillus being able to produce more secreted products. Amongst these, it is now becoming clear that genes present in the RD1 region of the bacterial genome encode for the proteins CFP-10 and ESAT-6, which have recently been implicated as having cytopathic qualities [22 ]. We have recently shown that BCG, which has lost the RD1 region, fails to induce necrosis in the lungs, even in IFN-{gamma} gene-disrupted mice [34 ]. Thus, it is possible that better production of these molecules by faster growing virulent clinical isolates underlies the cell damage and death we observed in the present study.

The macrophage monolayer system appears to be a reasonable way to evaluate this further in vitro [35 ]. Differences in virulence have been demonstrated in a variety of models, including Mycobacterium avium isolates [36 ] and the laboratory strains M. tuberculosis H37Rv and H37Ra [12 , 37 ]. In this regard, it has recently been suggested by several laboratories that macrophages infected by M. tuberculosis are triggered to enter a state of apoptosis and that this represents a protective mechanism to slow bacterial growth and dissemination [25 26 27 28 29 ]. This is an attractive idea, but it is inconsistent with the progressive growth of the infection seen in the lungs in vivo [31 ]. The results presented here are much more consistent with bacterial virulence correlating with cell cytotoxicity. Indeed, other studies also suggest this [38 , 39 ]. Apoptosis has been suggested a prerequisite to macrophage or dendritic cell cross-priming [40 ], but we would argue that cell death by necrosis could also serve this purpose equally well. The observation above that H37Rv induced no detectable apoptosis is not surprising to us, as we routinely use 8- to 10-day-infected monolayers as part of our tuberculosis drug-screening program [35 ].

It is interesting to note that other studies have linked virulence of M. tuberculosis to a gene locus-designed sst1 [41 ]. In contrast to our results, studies using the Erdman strain (which has equivalence to H37Rv in terms of mouse virulence) induced apoptosis in macrophages expressing the resistance allele of sst1, including those from C57BL/6 as used above, and necrosis in macrophages from animals with the susceptibility allele. We did not test Erdman in the current experiments, but our data suggest the reverse.

In fact, it is not widely understood or appreciated that necrosis is a major element of the mouse model, but it develops relatively slowly and is contained to some degree by a vigorous fibrotic response [30 , 42 ]. Much more rapid necrosis is seen in the guinea pig model, but it remains unclear if the bacteria present in the lungs are directly responsible for this [43 , 44 ]. Clearly, this is an area that should be investigated further, especially as it is this necrotic process that is ultimately fatal in tuberculosis.

In longer term cultures overgrown with CSU22, we observed cording of the bacilli, especially when released by dead cells late in the culture period. Cord factor has been chemically identified as trehalose 6,6-dimycolate (TDM) [45 ] and has long been considered to be a virulence factor of M. tuberculosis [46 , 47 ]. Removal of TDM by petroleum ether stripping of the bacterium cell wall caused decreased bacterial survival in murine macrophages, but there was no loss of bacterial growth in broth culture [48 ]. Conversely, greater production and/or shedding of TDM by the more virulent isolates may well cause damage to host membranes and may have been responsible for the apparent disorganization of the phagosomes, which usually contain only a single bacillus, as we observed here by electron microscopy. Together, these data support the hypothesis that necrosis of cells by virulent isolates of M. tuberculosis is a major factor contributing to the pathogenesis of the disease process.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grants AI-40488 and AI-44072.

Received May 9, 2005; revised July 14, 2005; accepted August 4, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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