Primary Lung Dendritic Cell Cultures to Assess Efficacy of Spectinamide-1599 Against Intracellular Mycobacterium tuberculosis

Abstract

There is an urgent need to treat tuberculosis (TB) quickly, effectively and without side effects. Mycobacterium tuberculosis (Mtb), the causative organism of TB, can survive for long periods of time within macrophages and dendritic cells and these intracellular bacilli are difficult to eliminate with current drug regimens. It is well established that Mtb responds differentially to drug treatment depending on its extracellular and intracellular location and replicative state. In this study, we isolated and cultured lung derived dendritic cells to be used as a screening system for drug efficacy against intracellular mycobacteria. Using mono- or combination drug treatments, we studied the action of spectinamide-1599 and pyrazinamide (antibiotics targeting slow-growing bacilli) in killing bacilli located within lung derived dendritic cells. Furthermore, because IFN-γ is an essential cytokine produced in response to Mtb infection and present during TB chemotherapy, we also assessed the efficacy of these drugs in the presence and absence of IFN-γ. Our results demonstrated that monotherapy with either spectinamide-1599 or pyrazinamide can reduce the intracellular bacterial burden by more than 99.9%. Even more impressive is that when TB infected lung derived dendritic cells are treated with spectinamide-1599 and pyrazinamide in combination with IFN-γ a strong synergistic effect was observed, which reduced the intracellular burden below the limit of detection. We concluded that IFN-γ activation of lung derived dendritic cells is essential for synergy between spectinamide-1599 and pyrazinamide.

Keywords: GM-CSF; Mycobacterium-tuberculosis; dendritic cells; interferon; intracellular; lung; pyrazinamide; spectinamides.

FIGURE 1

Characterization of lung derived dendritic cell cultures. (A) Phase contrast microscopy of CD11c positive cells isolated from lungs of Balb/c mice and cultured during 1, 7, and 15 days in the presence of 20 ng/mL GM-CSF. Media was replenished every 48 h. (B) Cell density expressed as total cell number/T75 flask after culturing isolated CD11c positive cells during 0, 10, 20, 30, 40, and 50 days in the presence of GM-CSF, p > 0.05. (C) Cell viability of CD11c cultures expressed as percentage of viable cells after culturing 0, 7, 10, 13, 20, and 28 days in the presence of GM-CSF, p > 0.05. (D) Phenotype of CD11c positive cells after cultured as above for 15 days were stained with fluorescence labeled mAb CD11b/CD11c and analyzed by flow cytometry. The gating strategy used selected cells based on forward and size scatter area (FSC-A/SSC-A) followed by analysis of intensity of fluorescence for CD11c and CD11b expression. The quadrants were set using unstained cells (left dot-plot). Cells stained for CD11c and CD11b are shown in the right dot-plot. Numbers in each quadrant represent the percentage of positive cells. Representative data from more than three experiments in similar conditions.

FIGURE 2

Localization and quantification of intracellular Mtb bacilli in lung derived DCs. Lung derived DC cultures were infected with Mycobacterium tuberculosis (H37Ra strain) as explained in Section “Materials and Methods.” Cells were washed and cultured with cRPMI media (without antibiotics) containing GM-CSF as in Figure 1. Acid fast positive bacilli within cells were visualized using a confocal ZEISS LSM 510 microscope after staining of lung DC cells by the acid fast staining method with Sybr Gold (green). (A) Intracellular H37Ra bacilli in lung dendritic cell cultures after 7 days of infection. (B) Magnification of picture in (A), showing cells containing one, two or many bacilli. (C) Demonstration of intracellular location of bacilli in a confocal imaging Z-stack of cells scanned at 0.2 μm steps. (D) The Mtb bacilli burden in lung dendritic cell homogenates infected for 1, 7, 15, and 30 days (black bars) in similar cultures of lung DCs infected with Mtb as in (A). The Mtb bacilli burden in supernatants from each cell culture is represented in gray bars. The number of colony forming units (CFU) is expressed as log₁₀ CFU. The results suggest that almost all bacteria are located intracellularly, and that there is no significant change in the number of intracellular bacteria over the observation period. The dotted line represents the detection limit of the assay. Representative data from more than three experiments in similar conditions.

FIGURE 3

Cell viability of lung derived DCs after drug treatment. (A) The resazurin colorimetric assay was used to determine the half maximal inhibitory concentration (IC₅₀) for pyrazinamide on lung dendritic cell cultures. (B) Cell viability of lung derived DC cultures after 24 h of incubation with cRPMI media containing GM-CSF and single drug treatment of PZA at 3 mg/mL or 9 mg/mL, spectinamide-1599 (20 μg/mL) or IFN-γ (50 U/mL). Statistical analysis revealed no significant difference among the groups (p > 0.05).

FIGURE 4

In vitro efficacy of spectinamide-1599, PZA, and IFN-γ against intracellular Mtb hosted by lung derived DCs. Lung derived DC cultures were infected with Mtb (H37Ra strain) as explained in the Section “Materials and Methods.” Cells were treated with mono, double or triple combinations (X-axis) of PZA (200 or 400 μg/mL), spectinamide-1599 (10 or 20 μg/mL), murine recombinant IFN-γ (50 U/mL) for 7 days. The cell culture media and drugs were replaced every 48 h. Cell homogenates were plated on agar 7H11 and cultured for 3 weeks at 37°C. The number of colony forming units (CFU) is expressed as log₁₀ CFU/5 × 10⁵ cells (Y-axis). Statistical analysis revealed significant difference (p < 0.05) when untreated samples were compared to mono-therapy treatments and for combinations of PZA versus PZA plus IFN-γ. Representative data from a large experiment and two pilot experiments in similar conditions. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 5

Intracellular uptake of spectinamide-1599 in lung derived DCs and J774A.1 cells. (A) Amount of spectinamide-1599 per cell in lung derived DCs that were either uninfected or infected with Mtb after incubation with 25 or 100 μg/mL spectinamide-1599 for 72 h. (B) Amount of spectinamide-1599 per cell after incubation of J774A.1 cells with 25 or 100 μg/mL spectinamide-1599, spectinomycin or streptomycin for 24 h. (C) Amount of spectinamide-1599 per cell after incubation of J774A.1 cells with 25 or 100 μg/mL spectinamide-1599 for 0.5, 1, 2, 4, 8, and 24 h.