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Keywords:

  • Burkholderia pseudomallei;
  • Macrophage;
  • Lymphocyte;
  • C57BL/6;
  • BALB/c

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

The mechanisms involved in the pathogenesis of melioidosis, caused by the intracellular bacterium Burkholderia pseudomallei, are unclear. C57BL/6 mice are resistant to infection, while BALB/c mice are highly susceptible. Previous studies have demonstrated that peritoneal exudate cell preparations enriched for macrophages are capable of effectively eliminating intracellular pathogens. In this study we present evidence showing that interaction of macrophages with lymphocytes is necessary for efficient anti-B. pseudomallei activity.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

Burkholderia pseudomallei is the causal agent of melioidosis, a life-threatening disease which affects humans and animals in tropical and subtropical areas. Infection may manifest as an acute, subacute, or chronic illness. Acute infection is often septicaemic, resulting in death within days of exposure. In contrast, chronic disease can occur asymptomatically and may persist for years [1, 2]. However, the pathogenesis of melioidosis is poorly understood [3].

We have previously demonstrated that BALB/c mice are highly susceptible to B. pseudomallei infection, while C57BL/6 mice are relatively resistant [4, 5]. These mouse strains provide an animal model for the acute and chronic forms of human melioidosis respectively [5]. Peritoneal exudate cells (PEC) from C57BL/6 mice have been shown to be significantly more microbicidal towards B. pseudomallei than PEC from BALB/c mice in ex vivo studies [5]. Adherent PEC cultures, enriched for macrophages, have been used extensively to investigate the role of macrophages in the elimination of intracellular organisms including Salmonella typhimurium[6], Listeria monocytogenes[7], Legionella pneumophila[8], Francisella spp. [9], Mycobacterium leprae[10] and Toxoplasma gondii[11]. The current in vitro study demonstrates that efficient antimicrobial activity of murine PEC towards B. pseudomallei depends on interactions between macrophages and lymphocytes.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

A virulent strain of B. pseudomallei was obtained at autopsy from a fatal case of melioidosis at Townsville General Hospital and cultured on Ashdown agar. C57BL/6 and BALB/c mice were obtained from the University of Queensland Central Animal Breeding House in 1988, and have been single pair sib-mated since that time at the James Cook University Small Animal Breeding Unit. Mice were maintained in a positive pressure environment at 23°C, fed a pelleted protein-enriched diet, and water was provided ad libitum.

Inbred C57BL/6 and BALB/c mice (12–16 weeks) were killed according to NHMRC guidelines and PEC collected by peritoneal lavage. PEC were pooled, washed in DMEM (400×g for 10 min) and resuspended at a concentration of 1.5×106 cells ml−1 in DMEM supplemented with 10% foetal bovine serum (FBS). Cell culture grade tubes (Sarstedt) were seeded with 1 ml of the PEC suspension and incubated for 2 h at 37°C in 5% CO2. Another series of tubes were seeded with 1 ml of the PEC suspension, incubated for 2 h at 37°C in 5% CO2, and non-adherent PEC (NAPEC) were removed by decanting the supernatant into fresh tubes. Tubes containing adherent PEC macrophages (PECM) were rinsed with phosphate buffered saline three times, and fresh DMEM supplemented with 10% FBS was added. Replicate tubes of each type of cell preparation were used to determine viability (trypan blue exclusion) and identify macrophages (non-specific esterase). All cell preparations were infected at a multiplicity of infection (MOI) of 1:12 000 (bacteria:cells) and incubated at 37°C in 5% CO2. Challenge inocula were adjusted based on the percentage of cells (NAPEC) decanted from PEC so that all cultures received the same infection rate. Preliminary experiments using higher (1:20) and lower (1:200 000) infection rates did not show significant differences in antimicrobial activity between preparations (data not shown). At 8, 16 and 24 h following infection, replicate samples were removed and viable bacteria were enumerated by plating serial dilutions on Ashdown agar. Control cultures contained only bacteria in DMEM supplemented with 10% FBS. Bacterial counts were performed in triplicate from two separate tubes at each time point for each cell preparation. Similar results were obtained when the experiment was repeated using a MOI of 1:12 000 and 1:2000. Statistical analyses were performed using two-way ANOVA for microbicidal activity data. Arcsine transformation was performed on percentage viable and esterase-positive data to meet the assumptions of ANOVA.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

Concentrations of PECM and NAPEC, following the decanting procedure were 0.8×106 and 0.7×106 cells ml−1. There were no significant differences in the viability of the various cell fractions. The percentage of viable cells in PECM and NAPEC preparations was 83±5 and 90±4 for C57BL/6 cultures, and 85±6 and 90±5 for BALB/c cultures. The percentages of non-specific esterase-positive cells were 75±15, 89±8, and 21±10 for C57BL/6 PEC, PECM and NAPEC respectively. These values for BALB/c preparations were 63±11, 96±6, and 21±7. Significantly more esterase-positive cells were present in PECM when compared to PEC (P<0.02), and PEC when compared to NAPEC (P<0.001). There were no significant differences between the same cell fractions from BALB/c and C57BL/6 mice.

At the end of the 24-h culture period, PEC cultures from C57BL/6 mice demonstrated significantly more microbicidal activity towards B. pseudomallei than PEC cultures from BALB/c mice (P<0.001) (Fig. 1). PECM and NAPEC cultures from C57BL/6 mice also demonstrated significantly more anti-B. pseudomallei activity than the same cell fractions from BALB/c mice. BALB/c PECM and NAPEC cultures contained 9.5×105±8.9×104 cfu ml−1 and 1.6×105±1.2×104 cfu ml−1, compared to 5.5×105±6.8×104 cfu ml−1 (P<0.01) and 2.5×104±1.5×103 cfu ml−1 (P<0.001) in C57BL/6 cultures.

image

Figure 1. Microbicidal efficiency of PEC cultures from C57BL/6 (hatched bars) and BALB/c (white bars) mice towards B. pseudomallei. PEC were infected at a MOI of 1:12 000 (bacteria:cells) and incubated at 37°C in 5% CO2. At 8, 16 and 24 h, replicate cultures were removed from incubation and viable bacteria were enumerated. Control cultures (black bars) contained only bacteria in DMEM supplemented with 10% FBS. Data represent the mean number of cfu as a percentage of the initial challenge±S.E.M.

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In C57BL/6 cultures, microbicidal activity was significantly lower in PECM (P<0.001) and NAPEC (P<0.001) cultures when compared to PEC cultures (Fig. 2). No cell preparations from BALB/c mice demonstrated significant anti-B. pseudomallei activity at any time point. Similar trends in antimicrobial activity were observed at MOI of 1:2000 and 1:12 000. No significant microbicidal activity was observed at higher (1:20) or lower (1:200 000) infection rates.

image

Figure 2. Microbicidal efficiency of PEC (hatched bars), NAPEC (grey bars) and PECM (white bars) cultures from C57BL/6 mice towards B. pseudomallei. The various cell fractions were infected at a MOI of 1:12 000 (bacteria:cells) and incubated at 37°C in 5% CO2. At 8, 16 and 24 h, replicate cultures were removed from incubation and viable bacteria were enumerated. Control cultures (black bars) contained only bacteria in DMEM supplemented with 10% FBS. Data represent the mean number of cfu as a percentage of the initial challenge±S.E.M.

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4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

C57BL/6 and BALB/c mice have been used extensively for the characterisation of susceptibility to intracellular infection [7, 10, 12–14]. Previous studies have attributed resistance in C57BL/6 mice to efficient production of γ-interferon (IFN-γ) and a strong Th1 response, and susceptibility in BALB/c mice to less efficient production of IFN-γ and a strong Th2 response [12–14]. Macrophages kill intracellular pathogens through antimicrobial processes such as the respiratory burst and nitrogen oxidation [3, 8]. However, interaction of macrophages with other cells of the immune system is necessary for optimal antimicrobial activity [8, 11, 15]. T lymphocytes and natural killer (NK) cells produce cytokines such as IFN-γ which enhance antimicrobial activity in macrophages [15–17]. Currently, two main pathways of cytokine-mediated macrophage activation are recognised: the T cell-independent pathway, involving NK cell-derived IFN-γ acting during the early stages of intracellular infections, and the antigen-sensitised T cell activation of macrophages via the cytokine cascade occurring at later stages of infection [16–18].

Regarding the mechanisms responsible for the acute and chronic clinical presentations of B. pseudomallei infection, it is possible that the balance between Th1/Th2 responses determines the clinical course of infection in humans. In Mycobacterium lepraemurium infection, the balance between Th1/Th2 responses determines the level of host susceptibility to infection [19]. Similarly, in Leishmania major infection, the balance between early T helper cell development determines clinical outcome [20]. The role of early cytokine responses in determining the progression of B. pseudomallei infection is unknown. Considering the role of Th1/Th2 cytokines as determinants of clinical outcome in other intracellular infections, it is possible that early T helper cell activity influences the course of B. pseudomallei infection. Studies investigating cytokine responses to B. pseudomallei infection will be required to assess the role of Th1/Th2 cytokines as determinants of clinical outcome of B. pseudomallei infection.

Previous studies have demonstrated that enriched peritoneal macrophages (PECM) are capable of killing intracellular pathogens [3, 6–10]. Our findings demonstrate that enriched peritoneal macrophages alone are unable to effectively kill B. pseudomallei. The increased antimicrobial capacity of PEC from C57BL/6 mice towards B. pseudomallei depends on the presence of sufficient numbers of both macrophages and lymphocytes. The necessity of macrophage-lymphocyte interactions for efficient anti-B. pseudomallei activity may reflect reliance on cytokine-mediated regulatory pathways.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References
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