Immunostimulatory CpG oligodeoxynucleotide confers protection in a murine model of infection with Burkholderia pseudomallei

Abstract

Although CpG oligodeoxynucleotides (CpG ODNs) are known to enhance resistance against infection in a number of animal models, little is known about the CpG-induced protection against acute fatal sepsis such as that associated with the highly virulent bacterium Burkholderia pseudomallei. We previously demonstrated in an in vitro study that immunostimulatory CpG ODN 1826 enhances phagocytosis of B. pseudomallei and induces nitric oxide synthase and nitric oxide production by mouse macrophages. In the present study, CpG ODN 1826 given intramuscularly to BALB/c mice 2 to 10 days prior to B. pseudomallei challenge conferred better than 90% protection. CpG ODN 1826 given 2 days before the bacterial challenge rapidly enhanced the innate immunity of these animals, judging from the elevated serum levels of interleukin-12 (IL-12)p70 and gamma interferon (IFN-gamma) over the baseline values. No bacteremia was detected on day 2 in 85 to 90% of the CpG-treated animals, whereas more than 80% of the untreated animals exhibited heavy bacterial loads. Although marked elevation of IFN-gamma was found consistently in the infected animals 2 days after the bacterial challenge, it was ameliorated by the CpG ODN 1826 pretreatment (P = 0.0002). Taken together, the kinetics of bacteremia and cytokine profiles presented are compatible with the possibility that protection by CpG ODN 1826 against acute fatal septicemic melioidosis in this animal model is associated with a reduction of bacterial load and interference with the potential detrimental effect of the robust production of proinflammatory cytokines associated with B. pseudomallei multiplication.

FIG. 1.

Effect of CpG ODN 1826 pretreatment on bacterial load expressed as CFU (A), IFN-γ levels (B), and TNF-α levels (C). BALB/c mice were injected with either CpG ODN 1826 (○) or PBS (•) on day −2 relative to the time of B. pseudomallei challenge (50 LD₅₀) on day 0. Blood was collected on day 2, and each specimen was used for colony count and cytokine assays. Results from individual mice (n = 15 animals per group) and group means (horizontal line) are shown. Data are representative of three independent experiments. Differences between the CpG and control groups were statistically different for panels A (P = 0.0001) and B (P = 0.0002) by the Mann-Whitney U test.

FIG. 2.

Effects of CpG ODN 1826 pretreatment on IFN-γ (A) and TNF-α (B) serum profiles of B. pseudomallei-infected animals. Animals were treated with CpG ODN 1826 and challenged with B. pseudomallei (50 LD₅₀) as described in the legend to Fig. 1. The animals in each group (n = 15) were bled serially as shown (the numbers of specimens at each time point were not equal since some animals in the control group died before day 15). Differences between groups at each time point were calculated by the Mann-Whitney U test, and those indicated by * were statistically significant (P = 0.0002 and P = 0.0047 for IFN-γ on days 2 and 4, respectively, and P = 0.032, P = 0.0058, and P = 0.0087 for TNF-α on days 7, 9, and 15, respectively). Error bars represent SEM.

FIG.3.

Innate cytokine profiles in the serum of infected BALB/c mice with or without a prior CpG ODN 1826 pretreatment. The animals were injected with 100 μg of CpG ODN 1826 on day −2 (arrow) relative to the challenge on day 0 (arrow) with B. pseudomallei (50 LD₅₀), as described in the legend to Fig. 1. The animals were bled serially on day −5, day −1 (1 day after either CpG ODN 1826 or PBS injection), day 1 (1 day after the B. pseudomallei challenge), day 2, day 6, and day 7. It should be noted that compared with day −5 (**), CpG ODN 1826 induced a significant elevation of both IL-12p70 (A) and IFN-γ (B) cytokines on day −1 (P = 0.0001 for both by the unpaired t test). The CpG ODN treatment was associated with a marked attenuation of the IFN-γ (P = 0.0002) and, to a lesser extent, IL-12p70 (P = 0.0486) production on day 2 (*) following a bacterial challenge. The number at the bottom of each bar indicates the number of specimens available for analysis at each time point. Data are representative results of two independent experiments. Error bars indicate SEM.