Immunomodulatory Effects of Mycobacteria. Toh S.S. 1 and Seah G.T. 2

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1 Immunomodulatory Effects of Mycobacteria Toh S.S. 1 and Seah G.T. 2 Department of Microbiology, Faculty of Medicine, National University of Singapore MD4, 5 Science Drive 2, Singapore ABSTRACT It has been suggested that the variable efficacy of Mycobacterium bovis bacille Calmette- Guérin (BCG) in protection against tuberculosis is attributable to prior immune priming by nontuberculous mycobacteria (NTM) ubiquitously present in the environment. A potential mechanism for this relationship was explored, based on the hypothesis that memory T cells recognising antigens from fast- and slow-growing NTM species differentially affect the functional ability of the lymphocytes to limit growth of intracellular BCG. Memory T cells in peripheral blood of healthy volunteers were stimulated with lysates of M. fortuitum and M. smegmatis (fast-growing NTM) and M. gordonae (slow-growing NTM). The antigen-expanded lymphocytes were co-cultured with autologous monocytes infected with live M. bovis BCG. The infected monocytes were then lysed and surviving bacteria enumerated by radiolabelling. The relative efficacy of BCG growth inhibition by the differentially stimulated lymphocytes was thus determined. The integrated results suggested that differences in the abilities of effector cells primed by the three NTM species to kill intracellular BCG were not statistically significant, although the overall trend suggested that immune recognition of M. fortuitum enhanced growth inhibition of intracellular BCG. More study subjects could be investigated to verify this effect as there are important implications on antigen selection for tuberculosis vaccines. INTRODUCTION Mycobacterium tuberculosis is the causative organism for tuberculosis (TB), a globally significant infectious disease. Cell-mediated immunity plays an important role in the recognition and elimination of this intracellular pathogen. The only TB vaccine in clinical use is an attenuated M. bovis strain called bacille Calmette-Guérin (BCG). This vaccine confers highly variable levels of protective immunity to people living in different parts of the world. The prevailing hypothesis for this phenomenon relates the protective efficacy of the BCG vaccine to prior exposure to free-living, environmental non-tuberculous mycobacteria (NTM). The hypothesis is difficult to prove experimentally because exposure to NTM is ubiquitous and cannot be controlled. The mechanism for the relationship thus remains unclear. This project aims to explore a potential mechanism for this relationship by studying inhibition of intracellular mycobacterial growth by memory T cells following stimulation by different NTM. A study conducted in the Karonga District in Malawi (Fine, 21) showed that the frequency that NTM can be isolated from clinical samples of patients in the area corresponds to relative sensitivity of the community to NTM antigens on skin testing. It was found that regardless of BCG vaccination status, people with high levels of skin-test sensitivity to fast-growing NTM (relative to the tuberculin reaction) had reduced risk of contracting leprosy and tuberculosis, but not people strongly sensitised by slow growing NTM. Mycobacteria which form colonies within seven days are termed fast-growers, whereas those which take longer than seven days are slowgrowers. 1 UROPS student 2 Assistant Professor, Department of Microbiology, NUS 1

2 In a different study, an assay which measures memory immune responses capable of inhibiting the growth of intracellular mycobacteria was described (Worku, 2). The authors found that memory T cells that were expanded with M. tuberculosis whole cell lysates were functionally able to inhibit intracellular growth of M. bovis BCG. (BCG was used here as a surrogate organism for M. tuberculosis.) They showed, by means of appropriate controls, that the effect was not seen following priming with tetanus toxoid, nor with non-activated T cells or mitogen-activated T cells. The two studies above suggested both a hypothesis for and a method of determining the effect of NTM immune priming on host responses to pathogenic mycobacteria. This present project was designed to characterise how expansion of memory T cells recognising antigens from fast- and slow-growing NTM species would affect their functional ability to limit growth of intracellular BCG, in an in vitro macrophage infection model. The hypothesis was that the fast-growers would be more effective than slow-growers in BCG inhibition, as would be predicted from the Karonga study results. MATERIALS AND METHODS Isolation of Mononuclear Cells Fifty millilitres of heparinised blood were collected from four normal healthy individuals who were previously vaccinated with BCG. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation over Ficoll-Paque Plus (Amersham Biosciences). Preparation of mycobacterial lysates M. fortuitum, M. gordonae (both clinical isolates) and M. smegmatis (strain mc 2 155) were cultured in Sauton s broth medium to mid-log phase. The bacteria were dispersed by cup-horn sonication and washed in phosphate-buffered saline (PBS). For lysis of the bacteria, to 2 ml vials half-filled with sterile.1 mm diameter zirconium-silica beads, 1 g of bacteria (wet weight) was added from a suspension ratio of 4 ml of PBS (with a protease inhibitor cocktail) to 3 g cells. The vials were agitated in a Mini Beadbeater (Biospec Products) at a speed of 2, 5 rpm in three cycles of 6 s, with 1 min rest on ice in between (Parish, 1998). The vials were then centrifuged at 13, g for 1 min and the supernatants harvested and filtered through.45 µm syringe filters. The lysates were quantified using the Bio-Rad DC protein assay. Culture of lymphocytes and preparation of effector cells Some of the PBMCs were plated at 5 x 1 5 cells/ml in 24-well tissue culture plates (BD Falcon) in complete medium (Dulbecco s Modified Eagle s Medium with 2% foetal calf serum), to a final volume of 1.2 ml. Bacterial lysates or phytohaemagglutinin (PHA) were added to the cells and incubated for 5 days. In some wells, the PBMCs were left unstimulated (medium-rested control) and in other wells, only PBS was added (PBS control). In preliminary experiments, lymphocyte proliferation assay by tritiated thymidine incorporation was carried out for a range of different bacterial lysate and PHA concentrations to determine the optimal concentrations for equivalent levels of stimulation of PBMCs in the wells. Infection of monocyte cultures with BCG The remaining PBMCs were plated for 48 h to obtain adherent cells. These were counted and re-incubated at 1 5 cells per well in 2 µl of complete medium in a 96-well flat-bottomed tissue culture plate (Nunc) for a further 48 h. M. bovis BCG (Pasteur strain) Sauton s broth cultures were sonicated, washed and counted then resuspended in 2.5 x 1 6 bacterial cells per ml in complete medium with 5 µg/ml of ferric ammonium citrate. The supernatant of the 4-day monocyte culture was replaced with 2 µl of the BCG preparation, at an infection ratio of 5 2

3 BCG to 1 monocyte. This optimal BCG-monocyte infection ratio does not compromise monocyte viability and was determined by counting live cells by trypan blue exclusion in wells infected at a range of different infection ratios. After infection overnight, extracellular BCG were killed by incubation with 2 µl of 1 µg/ml gentamycin solution for 3 h. Enumeration of surviving intracellular BCG The effector cells (lysate-stimulated lymphocytes) were harvested, washed and enumerated by trypan blue exclusion, then added to target cells (BCG-infected macrophages) at an effectortarget ratio of 1:1, for a 72h co-incubation period. The supernatants were aspirated and the macrophages lysed with.25% sodium dodecyl sulphate (SDS). The reagents used for macrophage lysis were titrated to achieve a concentration that would lyse the cells effectively but not compromise viability of the BCG released (data not shown). The bacteria were pulsed with 1 µci of [ 3 H]-uridine and further cultured in Middlebrook 7H9 broth for 72 h, then the amount of tritiated uridine incorporated was determined by liquid scintillation counting. RESULTS Determining appropriate stimulation conditions In PHA-stimulated wells, there was a clear trend of increasing lymphocyte proliferation with increasing concentration of PHA used (Figure 1A). With M. fortuitum (Figure 1B), M. gordonae (Figure 1C) and M. smegmatis (Figure 1D) bacterial lysates, concentrations of 1 µg/ml did not yield counts above background (data not shown). At 2-8 µg/ml (Figure 1) there was significant lymphocyte proliferation, but no significant difference between different concentrations within this range, except for M. smegmatis. Hence, for equivalent levels of lymphocyte stimulation, the final concentrations of each antigen used were: 1 µg/ml PHA, 5 µg/ml M. fortuitum, 5 µg/ml M. gordonae and 7 µg/ml M. smegmatis. Optimisation of BCG-macrophage infection ratio There was no change in the macrophage viability at infection ratios of 1:1 and 5:1, when compared to uninfected macrophages. At an infection ratio of 1:1, there was a significant decline in the macrophage viability when compared to cells infected at 1:1 (p=.5) and 1:5 (p=.3) infection ratios (statistics by t-test for independent samples, data not shown). Hence, the optimal infection ratio was five BCG per macrophage, and this was used in the study. BCG growth inhibition: effects of mycobacterial lysates Figure 2A shows the individual results of each of the four subjects (A D). For two subjects (A and B), the M. fortuitum lysate-stimulated lymphocytes were significantly more inhibitory to BCG growth than medium rested lymphocytes and PHA-stimulated lymphocytes (p <.5 by t- test for independent samples). This observation is consistent in the other two subjects as well, although not statistically significant at the 95% confidence level. Figure 2B shows the results of the four donors, grouped by the antigen used for stimulation of lymphocytes. The data were analysed by the non-parametric Mann-Whitney U test, comparing each group (different antigens) against the others. They suggest that at the 95% confidence level, the antigen-stimulated lymphocytes were not significantly different from the controls in influencing the survival of intracellular BCG. Bacteria in control wells (no effector cells, medium-rested cells and PHA-stimulated cells) showed consistently similar viability in all experiments. 3

4 25 A. PHA B. M. fortuitum Final Concentration of PHA(ug/ml) Final concentration of M. fortuitum lysate(ug/ml) C. M. gordonae D. M. smegmatis Final concentration of M. gordonae lysate(ug/ml) Final concentration of M. smegmatis lysate(ug/ml) Figure 1. Lymphocyte proliferation measured by [ 3 H]-thymidine incorporation in antigenstimulated wells. Mean CPM per well ± 1 SD shown for triplicate wells of cells from one representative subject. Statistics were performed using the Student t-test for independent samples. (*p<.5 when compared to 4 and 8 µg/ml lysates) (CPM = counts per minute) No lymphocytes Medium-rested lymphocytes PHA-stimulated lymphocytes M. fortuitum-stimulated lymphocytes M. gordonae-stimulated lymphocytes M. smegmatis-stimulated lymphocytes * * * 12 A. B A B C D Subjects No lymphocytes Rested lymphocytes PHA M. fortuitum M.gordonae Antigens used for lymphocyte stimulation M. smegmatis Figure 2. Effects of differentially stimulated lymphocytes on BCG survival, results of individual subjects (A) and overall results from four subjects, grouped by antigens used (B). A: Means of triplicates ± 1 SD are shown. Compared to medium-rested lymphocytes from the same subject, the lysates which gave significantly different BCG survival are marked (*p =.3 and **p =.6). B: Median data from four subjects are presented, and error bars indicate the 25 th percentile and the 75 th percentile values for the group. 4

5 DISCUSSION Based on this limited study, whether the NTM are fast- or slow-growers may not determine if immune priming by these environmental species influences the ability of memory T cells to defend against intracellular pathogenic mycobacteria. However, investigating a larger number of study subjects and more different NTM species would be necessary to confirm the trend observed. The assay used in this study could be improved by using BCG expressing green fluorescent protein to improve sensitivity and reduce the background counts encountered with scintillation counting. Dilution plate counts of the bacteria would also help to verify the results obtained. As there is international interest in whether immune priming by environmental NTM affects responses to BCG vaccincation, this work is relevant to the selection of antigens for inclusion in prospective new TB vaccines. There is current debate over whether TB-specific antigens or genus-specific antigens are protective if the latter, then prior exposure to different NTM in different geographical regions may influence the efficacy of the vaccine. REFERENCES Fine, P.E.M., Floyd, S., Standford, J.L., Nkhosa, P., Kasunga, A., Chaguluka, S., Warndorff, D.K., Jenkins, P.A., Yates, M. and Ponnighaus, J.M. (21) Environmental mycobacteria in northern Malawi: implications for the epidemiology of tuberculosis and leprosy. Epidemiol. Infect. 126, Parish, T. and Wheeler, P.R. (1998) Preparation of cell-free extracts from mycobacteria. In: Mycobacteria Protocols. Eds Parish, T. and Stoker, N.G. Methods in Molecular Biology, Humana Press, 11: Worku, S. and Hoft, D.F. (2) In vitro measurement of protective mycobacterial immunity: antigen-specific expansion of T-cells capable of inhibiting intracellular growth of Bacille Calmette-Guérin. Clin. Infect. Dis. 3 (Suppl 3), S

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