Alpha-galactosylceramide as a therapeutic agent for pulmonary Mycobacterium tuberculosis infection

Abstract

Rationale: Invariant natural killer T (iNKT) cells are a unique subset of T cells that recognize lipid antigens presented by CD1d molecules. Recent studies have shown that iNKT cells can protect mice against Mycobacterium tuberculosis (Mtb) infection. We sought to determine whether pharmacological activation of iNKT cells by α-galactosylceramide (α-GalCer) could be used to treat tuberculosis (TB).

Objectives: We hypothesized that α-GalCer, either alone or in combination with isoniazid, could be used to treat pulmonary TB.

Methods: The ability of α-GalCer-activated iNKT cells to suppress Mtb replication was evaluated using an in vitro coculture system. To test its potency in vivo, mice infected with virulent Mtb were treated with α-GalCer alone or in combination with isoniazid.

Measurements and main results: Quantitative colony-forming unit counts were compared for both experimental systems. Our results show that α-GalCer plus isoniazid controls bacterial growth better than α-GalCer or INH alone, and single or multiple α-GalCer administrations prolong the survival of the mice infected via the aerosol route.

Conclusions: Our results demonstrate that α-GalCer administration can improve the outcome of Mtb infection, even when transmitted by the aerosol route. However, a combination of isoniazid and α-GalCer treatment has a synergistic effect on infection control. We conclude that more efficient treatment of TB will be achieved through a combination of classic chemotherapy and modulation of the host immune response.

Figure 1.

α-Galactosylceramide (α-GalCer)-activated splenocytes suppress Mycobacterium tuberculosis (Mtb) replication. (A) Infected macrophages (Mϕ) were cultured alone or with splenocytes. Some cultures included α-GalCer (100 ng/ml), IFN-γ (2.5 U/ml) or both. After 72 hours, the total colony-forming unit (CFU) was measured. Statistical testing was done using a one-way analysis of variance with Tukey posttest comparing all possible pairs. Differences between all columns are statistically significant (*P < 0.05). Bars, mean ± SEM of replicate cultures. (B) Treatment with α-GalCer mediates a dose-dependent increase in IFN-γ production that correlates with control of bacterial replication. Immediately before cell lysis supernatants were sampled to measure IFN-γ by ELISA. Values shown in B are mean ng/ml ± SEM of IFN-γ (open symbols) or CFU ± SEM (solid symbols).

Figure 2.

Treatment with α-galactosylceramide (α-GalCer) (100 μg·kg⁻¹ or 500 μg·kg⁻¹) significantly prolongs the survival of mice infected by the aerosol route. (A) Survival of mice after the administration of α-GalCer (100 μg·kg⁻¹) by the intraperitoneal or intratracheal route (n = 10). Mice were divided in four groups. One group (solid diamonds) received α-GalCer group by intraperitoneal administration on Days −5, −3, and −1. A second group (solid squares) received α-GalCer group by intraperitoneal administration on Days 1, 5, and 9. A third group (closed circles) received α-GalCer group by the intratracheal route on Day 1. Mice were infected with Mycobacterium tuberculosis (Mtb) by low-dose aerosol (LDA) on Day 0. (B) Vehicle (open circles) or α-GalCer group (500 μg·kg⁻¹) (solid circles) was administered intraperitoneally 1 day after LDA infection with Mtb. (C) Survival of mice after the administration of α-GalCer (500 μg·kg⁻¹) by the intraperitoneal route (n = 10). No animals died in the uninfected group treated with α-GalCer. Mice received vehicle (open circles), α-GalCer on Day 1 by intraperitoneal administration (solid circle), or multiple doses of α-GalCer (solid squares) starting on Day 1 and then every 30 days. (D) Mice (solid diamonds) received multiple intraperitoneal administrations starting on Day 30 and then every 30 days. C and D are from the same experiment and the data are separated for clarity. Survival analysis was performed using Kaplan-Meier method and comparisons were done with log-rank test. *P < 0.05, ***P < 0.0005.

Figure 3.

Lung IFN-γ levels and bacterial burden. C57BL/6 mice (n = 5) were infected by the aerosol route and 24 hours after infection α-galactosylceramide (α-GalCer) (500 μg·kg⁻¹) or α-C-GalCer (500 μg·kg⁻¹) or saline were administered intraperitoneally. The lung inoculate was 118 colony-forming units (CFU) (B6). (A) IFN-γ levels were measured in total lung homogenate and the (B) bacterial burden was quantified at 3 weeks post infection. (C) The bacterial burden in the lungs of Mycobacterium tuberculosis (Mtb)-infected C3H mice 3 weeks after α-GalCer (500 μg·kg⁻¹) or saline treatment. A one-way analysis of variance with Bonferroni post test was used for statistical testing. *P < 0.05.

Figure 4.

Combination of α-galactosylceramide (α-GalCer) and INH has an additive effect on the reduction of the mycobacterial burden. C3H mice (n = 6) were inoculated intravenously with Mycobacterium tuberculosis (Mtb) and then treated with α-GalCer alone (Day 1 post infection) or α-Galcer (Day 1 post infection) plus isoniazid (INH) (Day 14 post infection). Five weeks later, the bacterial burden in the lung and spleen were quantified. *P < 0.05 by a one-way analysis of variance. All colony-forming unit (CFU) comparisons between groups were statistically significant in the lung.