Clearance of infection with Mycobacterium bovis BCG in mice is enhanced by treatment with S28463 (R-848), and its efficiency depends on expression of wild-type Nramp1 (resistance allele)

Abstract

The mouse bcg host resistance gene is known to control the activation of host macrophages for killing of intracellular parasites like Leishmania donovani as well as intracellular bacteria, including Mycobacterium bovis BCG and Salmonella enterica serovar Typhimurium. The Nramp1 gene has been mapped to this locus and affects the efficiency of macrophage activation. It has been shown that imidazoquinoline compounds, including S28463, are able to improve the clearance of a number of intracellular pathogens such as herpes simplex virus 2, human papillomavirus, and Leishmania. The goal of this study was to determine whether S28463 is efficient against infection with another intracellular pathogen, M. bovis BCG, and to determine the molecular basis underlying this effect. To achieve this, B10A.Nramp1(r) and B10A.Nramp1(-/-) mice were infected with M. bovis BCG and treated with S28463. The bacterial content in the spleen from these mice was assayed by a colony-forming assay. In addition, in vitro experiments were performed using bone marrow-derived macrophage cell lines from these mice. These cells were treated with S28463 and/or gamma interferon (IFN-gamma), and nitric oxide (NO) production was measured. Our study was able to show that S28463 acts in synergy with IFN-gamma to increase the production of NO in vitro. We were also able to demonstrate that mice that carried the resistant allele of the Nramp1 gene and were infected with M. bovis BCG responded to treatment with S28463, resulting in a decreased bacterial load after 2 weeks of treatment. Mice that do not express the Nramp1 gene responded only to a very large dose of S28463, and the response was not as efficient as that observed in mice carrying a wild-type Nramp1 allele. Our data provide evidence for the potential of S28463 as an immunomodulator that may be helpful in designing efficient strategies to improve host defense against mycobacterial infection.

FIG. 1

Effect of S28463 on NO production. B10A.Nramp1^r and B10A.Nramp1^−/− macrophages were plated on 24-well plates (10⁶ cells/well) and allowed to adhere for 2 to 4 h. The cells were then treated for 24 h with S28463 (25 ng/ml) with or without IFN-γ (10 U/ml). Each stimulus was done in quadruplicate. The amount of NO₂⁻ produced per amount of total protein was determined by using the Griess reagent. Data are presented as the mean + standard deviation (SD) of two independent experiments. There was a significant difference in NO production between B10A.Nramp1^r and B10A.Nramp1^−/− macrophages following S28463 treatment alone (P < 0.001) or following treatment with IFN-γ and S28463 (P < 0.001).

FIG. 2

Bacterial load of B10A.Nramp1^r and F₁ B10A.Nramp1^r × B10A.Nramp1^−/− mice treated with S28463. B10A.Nramp1^r (A) or F1 B10A.Nramp1^r × B10A.Nramp1^−/− mice (B) were infected intravenously with 5 × 10⁵ M. bovis BCG and injected intraperitoneally every 2 days with S28463 (2 μg/mouse). Once the mice were sacrificed, the spleen was removed, homogenized, plated at various dilutions, and incubated for 2 weeks. The colonies were then counted, which enabled the determination of the total amount of bacteria. Data are presented as the median from three and two independent experiments for panels A and B, respectively. Differences in CFU between PBS-treated and S28463-treated mice were significant (P < 0.001) in both panel A and panel B.

FIG. 3

Bacterial load of B10A.Nramp1^−/− mice treated with various doses of S28463. B10A.Nramp1^−/− mice were infected intravenously with 10⁵ M. bovis BCG and injected intraperitoneally every 2 days with 2 μg (panel A), 8 μg (panel B), 16 μg (panel C), or 50 μg (panel D) of S28463. The mice were sacrificed and the spleens were removed. The spleens were homogenized, and various dilutions were plated and left to grow for 2 weeks. The colonies were then counted, and the total amount of bacteria was determined. Results from four different experiments are shown. Filled symbols, PBS; open symbols, S28463. No significant decrease in CFU could be detected for any of the doses tested (P > 0.05 in all cases).

FIG. 4

Effect of high doses of S28463 on bacterial load in B10A.Nramp1^−/− mice. (A) B10A.Nramp1^−/− mice were infected intravenously with 10⁵ M. bovis BCG and injected intraperitoneally every 2 days with S28463 (500 μg/mouse). Once the mice were sacrificed, the spleen was removed, homogenized, plated at various dilutions, and incubated for 2 weeks. The colonies were then counted, which enabled the determination of the total amount of bacteria. Data are presented as the median from three independent experiments. S28463 treatment led to a significant reduction in CFU (P = 0.034). (B) Splenic ratios were calculated from the PBS- and S28463-treated mice by dividing the weight of the spleen by the total body weight of the mouse. Data are presented as the median from three independent experiments. Treatment with 500 μg of S28463 led to a significant increase in the splenic ratio of B10A.Nramp1^−/− (P < 0.001).