Early Activation of iNKT Cells Increased Survival Time of BALB/c Mice in a Murine Model of Melioidosis

Abstract

Melioidosis is an infectious disease caused by Burkholderia pseudomallei. High interferon gamma (IFN-γ) levels in naive mice were reported to mediate protection against B. pseudomallei infection. Invariant natural killer T (iNKT) cells can produce and secrete several cytokines, including IFN-γ. When iNKT cell-knockout (KO) BALB/c mice were infected with B. pseudomallei, their survival time was significantly shorter than wild-type mice. Naive BALB/c mice pretreated intraperitoneally with α-galactosylceramide (α-GalCer), an iNKT cell activator, 24 h before infection demonstrated 62.5% survival at the early stage, with prolonged survival time compared to nonpretreated infected control mice (14 ± 1 days versus 6 ± 1 days, respectively). At 4 h after injection with α-GalCer, treated mice showed significantly higher levels of serum IFN-γ, interleukin-4 (IL-4), IL-10, and IL-12 than control mice. Interestingly, the IFN-γ levels in the α-GalCer-pretreated group were decreased at 4, 24, and 48 h after infection, while they were highly increased in the control group. At 24 h postinfection in the α-GalCer group, bacterial loads were significantly lower in blood (no growth and 1,780.00 ± 51.21, P < 0.0001), spleens (no growth and 34,300 ± 1,106.04, P < 0.0001), and livers (1,550 ± 68.72 and 13,400 ± 1,066.67, P < 0.0001) than in the control group, but not in the lungs (15,300 ± 761.10 and 1,320 ± 41.63, P < 0.0001), and almost all were negative at 48 h postinfection. This study for the first time shows that early activation of iNKT cells by α-GalCer helps clearance of B. pseudomallei and prolongs mouse survival.

Keywords: Burkholderia pseudomallei; Traj18-deficient mice; invariant natural killer T (iNKT) cells; knockout mice; melioidosis; α-galactosylceramide (α-GalCer).

FIG 1

Gating strategy used to identify iNKT cells. Mouse organs and their mononuclear cells (MNCs) were harvested as described in the Materials and Methods. MNCs were stained with monoclonal antibodies (MAbs) to mouse T and iNKT cells and analyzed by flow cytometry. After selection for lymphocytes by forward scatter-side scatter (FSC-SSC), T cells were defined (CD3⁺/CD45⁺) by using anti-mouse CD45 and anti-mouse CD3. Finally, iNKT cells were then further identified (CD3⁺/α-GalCer/CD1d tetramer⁺) by anti-mouse CD3 and α-GalCer/CD1d tetramer (compared with unloaded/CD1d tetramer as a control).

FIG 2

Number of iNKT cells in each organ during B. pseudomallei infection. (A to C) The percentages of iNKT cells in spleens (A), lungs (B), and livers (C) were compared between mice infected with 1,000 CFU (50LD₅₀) of B. pseudomallei (gray bars) and control-infected mice (black bars; 9 mice/group). The numbers of iNKT cells were determined by flow cytometry (Fig. 1). Data are shown as means ± SD. The results are representative of three independent experiments (total of 27 mice/group). Statistically significant differences were evaluated using the unpaired Student’s t test. The asterisks (*) indicate statistical significance (P < 0.05).

FIG 3

Survival curves and IFN-γ concentrations of mice infected with B. pseudomallei. WT BALB/c mice (n = 3) and iNKT-KO mice (n = 3) were infected intraperitoneally with 100 CFU (5LD₅₀) of B. pseudomallei. (A) Survival of mice was observed for 14 days p.i. (x axis), and the percent survival (mean ± SD; y axis) of WT mice (white circles) was compared with that of iNKT-KO mice (black squares). Statistically significant differences were evaluated by Kaplan-Meier analysis and a log-rank test. (B) The amount of serum IFN-γ (x axis) of WT mice (white bars) and iNKT-KO mice (black bars) before and after 4, 24, and 48 h of infection with B. pseudomallei. The results are representative of two independent experiments (total of 6 mice/group). Statistically significant differences were evaluated using the unpaired t test. The asterisks (*) indicate statistical significance (P < 0.05), whereas “ns” indicates not significant (P > 0.05).

FIG 4

Survival of WT BALB/c mice pretreated with α-GalCer or PBS before infection with B. pseudomallei. The survival of BALB/c mice (8 mice/group) in percent (y axis) that received 2 μg of α-GalCer (black squares) or PBS (clear circles) 24 h before infection with 1,000 CFU (50LD₅₀) of B. pseudomallei was observed for 14 days. The results are representative of two independent experiments (total of 16 mice/group). Statistically significant differences were evaluated by using Kaplan-Meier analysis and a log-rank test, and asterisks (****) indicate statistical significance (P < 0.0001).

FIG 5

Cytokine levels and bacterial loads in mice pretreated with α-GalCer before and after B. pseudomallei infection. (A to D) Mice (6 mice/group) were pretreated with α-GalCer (black bars) or with PBS (white bars). The levels of IFN-γ (A), IL-4 (B), IL-10 (C), and IL-12 (D) in sera were measured at different time points. The arrows indicate the time of α-GalCer and B. pseudomallei injections. (E and F) Bacterial counts in blood, spleens, lungs, and livers of α-GalCer-pretreated (black bars) and control (white bars) groups were assessed at 24 h (E) and 48 h (F) after infection. All data are presented as mean ± SE of mice per group per time point and are representative of two independent experiments (total of 12 mice/group). Statistically significant differences were evaluated by using the Mann-Whitney test, and asterisks (**) indicate statistical significance (P < 0.01).