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. 2001 Feb;69(2):800-9.
doi: 10.1128/IAI.69.2.800-809.2001.

Fate of Mycobacterium tuberculosis within murine dendritic cells

K A Bodnar  1 , N V Serbina, J L Flynn
Affiliations

Fate of Mycobacterium tuberculosis within murine dendritic cells

K A Bodnar et al. Infect Immun. 2001 Feb.

Abstract

The interaction of microbes with dendritic cells (DCs) is likely to have an enormous impact on the initiation of the immune response against a pathogen. In this study, we compared the interaction of Mycobacterium tuberculosis with murine bone marrow-derived DCs and macrophages (M phi) in vitro. M. tuberculosis grew equally well within nonactivated DCs and M phi. Activation of DCs and M phi with gamma interferon and lipopolysaccharide inhibited the growth of the intracellular bacteria in a nitric oxide synthase-dependent fashion. However, while this activation enabled M phi to kill the intracellular bacteria, the M. tuberculosis bacilli within activated DCs were not killed. Thus, DCs could restrict the growth of the intracellular mycobacteria but were less efficient than M phi at eliminating the infection. These results may have implications for priming immune responses to M. tuberculosis. In addition, they suggest that DCs may serve as a reservoir for M. tuberculosis in tissues, including the lymph nodes and lungs.

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Figures

FIG. 1
FIG. 1
Cell surface phenotype of DCs infected with M. tuberculosis. Purified bone marrow-derived DCs and Mφ were infected with M. tuberculosis or uninfected and harvested 48 h postinfection. (A) The cells were stained with antibodies as follows: MHC class II (IAb)-FITC conjugated and an isotype control mouse IgG2a; ICAM (CD54)-FITC and B7.1 (CD80)-FITC and an isotype control hamster IgG; B7.2 (CD86)-FITC and an isotype control rat IgG2a; and CD-14 (PE conjugated) and an isotype control rat IgG1(κ). The cells were then fixed and analyzed by FACS analysis. Top row, Mφ; bottom row, DCs; dotted line, IgG control; thin gray line, uninfected cells; thick black line, M. tuberculosis-infected cells. (B) The forward versus side-scatter plots show that both the DCs and the Mφ undergo morphologic changes after infection. A representative experiment of 10 independent experiments is shown.
FIG. 2
FIG. 2
Phagocytic capability is decreased in M. tuberculosis-infected DCs but not macrophages. Purified DCs and Mφ were cultured without (top panel) or with M. tuberculosis (bottom panel). After 72 h, cells were incubated with FITC-labeled latex beads (3 μm) for 2 h at 4 or 37°C as indicated and examined by flow cytometry. Dead cells and free beads were excluded from the gate, and 104 cells were collected within the gate for each sample. The MFI is indicated in each histogram. The bar above the histograph represents the gate. Percent gated: DCs, uninfected, 20% at 4°C and 76% at 37°C; DCs plus M. tuberculosis, 5% at 4°C and 37% at 37°C; Mφ, uninfected, 33% at 4°C and 93% at 37°C; Mφ plus M. tuberculosis, 14% at 4°C and 83% at 37°C. The experiment was repeated once.
FIG. 3
FIG. 3
Immature murine DCs internalize live M. tuberculosis. DCs infected with M. tuberculosis were fixed, embedded, sectioned, and examined by transmission electron microscopy. The arrows indicate M. tuberculosis. The micrograph shown is representative of 25 individual cells observed. Bar = 1 μm.
FIG. 4
FIG. 4
Cytokine secretion in response to M. tuberculosis-infected DCs and Mφ. (A) Supernatants from M. tuberculosis-infected unactivated DCs (●) and Mφ (□) were assayed in duplicate for production of IL-12 (ELISA for IL-12p70) or TNF. (B) Time zero represents cytokine production from the cells prior to infection. Recombinant murine cytokines were used as standards in each assay. Representative experiments of four and two total experiments are shown for IL-12 and TNF-α, respectively. (B) Analysis of gene expression by RNase protection assay. A total of 6 μg of RNA from either uninfected or M. tuberculosis-infected DC (•) and Mφ (□) at various time points was used in the assay, and specific genes of interest were standardized to GAPDH by densitometerization of the autoradiograph. A representative experiment (of two experiments) is shown.
FIG. 5
FIG. 5
Stimulation of T-cell proliferation and cytokine production by APCs. T-cell-enriched splenocytes from C57BL/6 mice were cultured for 3 days with bone marrow-derived DCs or Mφ either infected with M. tuberculosis (MOI = 4) or left uninfected at a 1:50 APC/T-cell ratio. (A) Proliferation was measured by [3H]thymidine incorporation and is represented as the SI. Stimulation with ConA resulted in ∼18,000 cpm, and stimulation with infected DCs resulted in ∼16,000 cpm. DCs, Mφ, or T cells alone incorporated fewer than 2,000 cpm. (B) Supernatants of the cultures described above were collected at the end of the culture period, and the concentration of IFN-γ was measured by sandwich ELISA. A representative experiment (of three experiments) is shown.
FIG. 6
FIG. 6
Activated DCs and Mφ are comparable in the inhibition of M. tuberculosis growth via RNI production. (A) Incorporation of [3H]uracil into M. tuberculosis within DCs and Mφ was assessed in untreated and IFN-γ–LPS-activated DCs and Mφ. The lysates of [3H]uracil-labeled uninfected, M. tuberculosis-infected, or IFN-γ–LPS-activated M. tuberculosis-infected wild-type DCs and Mφ were TCA precipitated, collected on glass filters, and counted in a β-scintillation counter. An inhibitor of NO production, aminoguanidine (AG), was added to the cells prior to infection as indicated. (B) Griess assays were performed with supernatants of M. tuberculosis-infected DCs and Mφ and read at 570 nm. The standard curve was generated using NaNO2. (C) Lysates of [3H]uracil-labeled uninfected, M. tuberculosis-infected, or IFN-γ–LPS-activated M. tuberculosis-infected NOS2−/− DCs and Mφ were TCA precipitated and counted using liquid scintillation. Samples were tested in triplicate, and a representative experiment from three experiments is shown. For statistical analysis for all panels, IFN-γ–LPS or IFN-γ–LPS plus AG samples were compared to untreated cells. ∗, P < 0.02.
FIG. 7
FIG. 7
DCs inhibit the growth of but do not kill intracellular M. tuberculosis. Cell pellet lysates of M. tuberculosis-infected DCs or Mφ (MOI = 1), unactivated or activated with IFN-γ–LPS, were serially diluted in PBS plus 0.05% Tween 80 and plated on 7H10 plates, which were incubated for 18 days at 37°C and 5% CO2. Three wells per condition were assessed at each time point, and the mean intracellular CFU values are reported at various time points after infection. (A) Intracellular CFU in resting DCs (●) and resting Mφ (□). (B) Intracellular CFU in activated DCs (●) and activated macrophages (□). At each time point, <1% of the total CFU was found in the supernatant. In panel A, CFU levels within unactivated DCs and Mφ at each time point revealed no statistical differences (P > 0.5). In panel B, bacterial numbers within DC were not significantly reduced (P = 0.2) compared to intracellular CFU at 4 h (input). Comparison of CFU within Mφ at each time point to the CFU level at 4 h revealed statistically significant reductions over time (∗∗, P < 0.01; ∗, P < 0.02). Error bars show the standard error. A representative experiment (of six experiments) is shown.
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