Centre for Molecular Microbiology and Infection, Department of Infectious Diseases and Microbiology, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK1
Author for correspondence: Douglas B. Young. Tel: +44 20 7594 3962. Fax: +44 20 7594 3095. e-mail: d.young{at}ic.ac.uk
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ABSTRACT |
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Keywords: tuberculosis, drug screening, pyrazinamide, luciferase
Abbreviations: BCG, bacille CalmetteGuérin; FCS, fetal calf serum; IFN, interferon-
; r.l.u., relative light units
a These authors contributed equally to the work.
b Present address: The Wellcome Trust, 183 Euston Road, London NW1 2BE, UK.
c Present address: Department of Biology and Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK.
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INTRODUCTION |
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In the conventional approach to drug discovery, compounds are initially optimized for activity against mycobacteria growing in in vitro culture and subsequently assessed for in vivo activity in animal models during preclinical development (Global Alliance for TB Drug Development, 2001 ). A limitation of this approach is that compounds that are particularly active against in vivo phenotypes of mycobacteria may be overlooked in the early discovery phase. The anatomical location and physiological status of mycobacteria within the infected host remains a topic of active research (McKinney, 2000
). The requirement for prolonged chemotherapy to prevent relapse suggests that, in addition to the population of actively dividing mycobacteria present in the lungs of tuberculosis patients, there is a subset of non-dividing organisms that remain relatively resistant to the action of the drugs. This persistent population may include intracellular bacteria held under control by the immune response, as well as oxygen-deprived bacteria sequestered within fibrotic caseous lesions. The aim of the present study was to develop a cell-culture model in which we could reproduce aspects of the complex environment encountered by Mycobacterium tuberculosis during infection, and which could be used for early assessment of the in vivo activity of novel drugs. As a starting point, we wished to establish a culture system that reflected the influence of host immunity on mycobacterial viability.
The effect of the host immune response on mycobacterial growth has been extensively characterized in intact animal models (Orme & Collins, 1994 ). During the initial acute stage of infection in naive mice, mycobacterial numbers increase over a period of several weeks prior to the induction of a mature cell-mediated immune response. This results in a reduction or cessation of bacterial multiplication, leading to a chronic phase of the disease during which bacterial load and mortality depend on the genetic background of the animal and on the initial dose and route of infection. Prior vaccination with BCG a live attenuated variant of Mycobacterium bovis results in accelerated induction of the immune response, reducing the extent of bacterial multiplication in the acute phase, and prolonging survival.
In the first part of this study we have demonstrated that mycobacterial viability in cell cultures prepared from tissues of infected mice is determined by the activity of the cell-mediated immune response. In the second part, we have analysed the effect of this immune-mediated control on the antimycobacterial activity of current drugs, and demonstrated a synergy between pyrazinamide and an active immune response.
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METHODS |
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Infection model and ex vivo culture.
Female C57BL/6 mice (68 weeks old) were infected with M. tuberculosis/pSMT1 (5x105 c.f.u.) or BCG Pasteur/pSMT1 (5x106 c.f.u.) by intravenous injection in the tail. In experiments to monitor the effect of vaccination, groups of mice were vaccinated by subcutaneous injection of 5x105 c.f.u. BCG or saline, and challenged by intravenous injection 810 weeks later. Groups of 35 mice were killed by cervical dislocation, and spleens were removed aseptically and disaggregated by passing through a 100 mm cell strainer (Becton Dickinson, 2360), to produce a single-cell suspension in RPMI culture medium. Samples from individual spleens were serially diluted in PBS and plated out for c.f.u. and r.l.u. measured in triplicate. Splenocyte suspensions from individual mice generally gave luminescence readings differing by less than 20%. Splenocytes with very low luminescence were occasionally found. These were assumed to result from failure of intravenous inoculation and were discarded. The remaining samples were pooled. Red blood cells were lysed using 0·83% (w/v) ammonium chloride (pH 7·6), and splenocytes washed twice with PBS containing 10% (v/v) FCS and once with RPMI culture medium. A sample was stained using 0·4% (w/v) Trypan Blue solution and counted in an Improved Neubauer haemocytometer, from which total cell counts per spleen were calculated. Splenocyte preparations were then resuspended in culture medium at 1x106 cells ml-1, seeded in NUNC 24-well tissue-culture dishes, and incubated at 37 °C with 5% (v/v) CO2 for 28 days. Analysis of the cell composition of splenocyte preparations revealed a predominance of B lymphocytes (5060% of total cells), with 1415% CD4+ T cells and 1112% CD8+ T cells.
Stocks of rifampicin (1 mg ml-1), isoniazid (10 mg ml-1) and pyrazinamide (10 mg ml-1) were prepared in RPMI culture medium and added to splenocyte preparations before distribution into 24-well tissue culture dishes so that the final concentrations were 1, 0·1 and 0·01 µg ml-1 for rifampicin and isoniazid, and 500, 50 and 5 µg ml-1 for pyrazinamide. In some experiments splenocytes from uninfected mice were prepared in the same way and infected in vitro with BCG (1x104 c.f.u. ml-1) either at the start of the incubation period or after 4 days in culture.
To monitor mycobacterial viability in ex vivo cultures, cells were lysed using 0·1% (v/v) Triton X-100, r.l.u. measured and c.f.u. plated out from triplicate wells. Cultures were also examined by light microscopy after staining by the Kinyoun (cold) acid-fast procedure.
Immune manipulation: T cell depletion.
T cell subsets were selectively depleted from ex vivo splenocyte cultures by complement-mediated cell lysis. Splenocyte samples were resuspended at 1x107 cells ml-1 and left for 1 h at 4 °C with a 1/100 dilution of anti-CD4+ and/or anti-CD8+ antibodies (monoclonal antibodies RL172.4 and 31M were kindly provided by Dr Ingrid Muller, Department of Immunology, Imperial College of Science, Technology and Medicine). After washing, rabbit complement (Cedarlane) was added at 1/10 dilution and incubated for 30 min at 37 °C. After two washes with PBS/FCS (10%, v/v) and one wash with RPMI culture medium, cells were resuspended in RPMI culture medium at 1x106 ml-1, and seeded in 24-well dishes as for the ex vivo method. Controls where no antibody was added and controls with no complement added were included in each experiment. A sample of each preparation was retained for flow cytometry analysis; these samples were stained for CD4+ and CD8+ T cell populations (antibodies from Sigma), fixed and analysed using a FACSCalibur Flow Cytometer (Becton Dickinson). Complement-mediated cell lysis typically achieved between 60 and 80% depletion in the total cell number for the targeted T cell subset.
Interferon- assay.
Interferon- (IFN
) was measured in supernatants removed from ex vivo cultures by ELISA (Pharmingen OptEIA kit).
Statistical analysis.
Error bars in figures, and error terms for values reported in tables, show one standard deviation from the mean assessed in at least triplicate samples and experiments were repeated at least once. P values for Table 2 were calculated using a two-tailed paired Students t-test.
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RESULTS |
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The pattern of differential growth in naive and vaccinated splenocytes was dependent on the time at which the mice were killed (Fig. 2). Partial inhibition of mycobacterial growth could be detected in spleens removed from vaccinated mice as early as 1 day after infection, with a stronger inhibition evident by day 7. Two weeks after infection, mycobacterial growth was controlled in both naive and vaccinated splenocytes, although the total bacterial load was higher in the naive cultures. The timecourse for development of mycobacterial growth inhibition matched that of total cell recruitment measured in spleens from the naive and vaccinated mice (Fig. 2d
).
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The timing of pyrazinamide activity in the ex vivo model coincided with the induction of a cell-mediated immune response. To further explore the possible association between these two events, the effect of pyrazinamide was compared in ex vivo splenocytes from naive and BCG-vaccinated mice 11 days after infection with M. tuberculosis (Table 3). This timepoint was chosen to represent the stage of the infection at which a cell-mediated immune response was evident in vaccinated but not naive animals (see Fig. 2
). While pyrazinamide had no effect on mycobacterial viability in splenocytes from naive mice at this timepoint, a clear dose-dependent inhibition was seen in the cultures from vaccinated mice.
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DISCUSSION |
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We have exploited the ex vivo model to study the influence of host immunity on the action of antimycobacterial drugs. These experiments were monitored by assessment of c.f.u. in addition to the rapid luminescence readout used in the immunology experiments. Drugs differ in their effect on luminescence output in short-term assays. Isoniazid triggers a rapid drop in r.l.u., for example, but this is not the case for rifampicin. The reporter construct used in the assay constitutively expresses a high level of the luciferase enzyme and the drop in r.l.u. suggests that isoniazid induces rapid depletion of the reduced FMN cofactor required for luminescence (Meighen, 1991 ), possibly related to changes in the permeability of the mycobacterial cell wall. The continued luminescence of rifampicin-treated cultures indicates availability of a pool of cofactor within the mycobacteria which may persist for some days. Luminescence provides a measure of the amount of luciferase enzyme and the energy balance in the bacterium. This is not directly related to replication, although decreased luminescence may provide an early indication of the fact that replication is unlikely to take place. Therefore, although there is a consistent correlation between r.l.u. and c.f.u. in actively growing cultures (Snewin et al., 1999
), this is not necessarily preserved when mycobacteria are being killed. It is important to note that luminescence provides an immediate measure of the physiological status of the bacteria, while the ability to form colonies is influenced by drug-induced changes that occur during the subsequent weeks in culture.
The most striking observation from the assay of drug activity in the ex vivo model was the change in the effect of pyrazinamide at different times after infection. Pyrazinamide had maximal activity in splenocyte cultures prepared at the time of onset of the antimycobacterial immune response. Pyrazinamide is an important component of current treatment regimens but is unique amongst tuberculosis drugs in being ineffective against bacteria growing in conventional laboratory culture. It does reduce the viability of in vitro cultures exposed to acidic pH (Heifets & Lindholm-Levy, 1992 ), and has some inhibitory effect when administered in the mouse model (Klemens et al., 1996
), although there are conflicting reports of its ability to influence growth of intracellular mycobacteria in macrophage cell cultures (Crowle et al., 1986
; Heifets et al., 2000
). Our experimental observations are consistent with a model in which pyrazinamide acts against a population of mycobacteria exposed to an acidic environment as a result of the action of the host immune response (Mitchison, 1985
). While interference with phagosome maturation is considered the predominant strategy for mycobacterial survival in macrophages (Russell, 2001
), the pyrazinamide-susceptible population may represent some subset of bacteria that are present within acidified phagolysosomes. It is interesting to note that the window of pyrazinamide susceptibility does not extend into the chronic phase of infection in the mouse model. Although there is evidence of a continued cell-mediated immune response at 42 days, our observations indicate that this does not generate a pyrazinamide-susceptible population of bacteria. This suggests that acidic conditions do not provide the environment for persistence, although it does not exclude the possibility that bacteria that survive exposure to acidic conditions subsequently contribute to the persistent pool. This model is of interest in relation to the clinical use of pyrazinamide. Pyrazinamide is included during the initial 2 month phase of therapy and has been shown to reduce the risk of relapse at the end of the standard 6 month regimen (Global Alliance for TB Drug Development, 2001
). This profile is consistent with an early effect of pyrazinamide on the development of persistent organisms rather than a direct activity against an established persistent population.
While the simple ex vivo model described in the present study has been useful in exploring the window of pyrazinamide susceptibility, there is considerable scope for future refinement. Preliminary experiments indicate that a similar approach can be applied to infected lung tissues, for example, and modification of cell-culture techniques may extend the length of the assay period by reducing the lytic effects observed in M. tuberculosis cultures. The limitations of the luciferase readout as a marker for drug activity suggest a potential role for alternative reporter constructs in further optimization of the ex vivo approach. An attractive strategy is to engineer expression of the reporter gene under the control of promoters induced in response to drug action (Wilson et al., 1999 ). This approach has been successfully employed in incorporating the ex vivo assay into a development programme for improved ethambutol derivatives (C. E. Barry, personal communication).
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ACKNOWLEDGEMENTS |
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Received 5 April 2002;
revised 28 June 2002;
accepted 8 July 2002.