The Warburg effect in mycobacterial granulomas is dependent on the recruitment and activation of macrophages by interferon-γ

Abstract

Granulomas are the hallmark of mycobacterial disease. Here, we demonstrate that both the cell recruitment and the increased glucose consumption in granulomatous infiltrates during Mycobacterium avium infection are highly dependent on interferon-γ (IFN-γ). Mycobacterium avium-infected mice lacking IFN-γ signalling failed to developed significant inflammatory infiltrations and lacked the characteristic uptake of the glucose analogue fluorine-18-fluorodeoxyglucose (FDG). To assess the role of macrophages in glucose uptake we infected mice with a selective impairment of IFN-γ signalling in the macrophage lineage (MIIG mice). Although only a partial reduction of the granulomatous areas was observed in infected MIIG mice, the insensitivity of macrophages to IFN-γ reduced the accumulation of FDG. In vivo, ex vivo and in vitro assays showed that macrophage activated by IFN-γ displayed increased rates of glucose uptake and in vitro studies showed also that they had increased lactate production and increased expression of key glycolytic enzymes. Overall, our results show that the activation of macrophages by IFN-γ is responsible for the Warburg effect observed in organs infected with M. avium.

Keywords: glycolysis; infection; interferon-γ; macrophages; mycobacteria.

Figure 1

Mycobacterium avium infected mice show an interferon-γ (IFN-γ)-dependent Warburg effect in the liver and spleen. 18-Fluorodeoxyglucose (FDG) positron emission tomography stack of coronal and sagittal section images (nose in the top, tail in the bottom) of non-infected mice (left) or B6 (second from the left), IFNγ-deficient (third from the left), and macrophages insensitive to interferon-γ (MIIG) (right) mice infected for 75 days with 10⁶ CFU of M. avium ATCC 25291.

Figure 2

Quantification of immune cell infiltrates in Mycobacterium avium-infected mice. Seventy days after M. avium infection, spleen and liver cells from non-infected mice (white bars) or B6 (solid black bars) and macrophages insensitive to interferon-γ (MIIG) (solid square bars) infected mice were stained with specific antibodies and analysed by flow cytometry. Each bar represents the mean ± SD from four to five animals per group. *P < 0·05, **P < 0·01.

Figure 3

Enhanced glycolytic activity in liver and splenic macrophages of Mycobacterium avium-infected mice. In vivo glucose uptake of spleen and liver immune cell populations of non-infected mice (white bars) or B6 (solid black bars) and macrophages insensitive to interferon-γ (MIIG) (solid square bars) infected mice was measured using a fluorescent d-glucose analogue (2-NBDG). As a control sample for in vivo experiments, mice that did not receive 2-NBDG were analysed (dashed line). Mean fluorescence intensity (MFI) of 2-NBDG in each cell population is indicated. Mean ± SD were obtained from four to five mice in each condition. *P < 0·05, **P < 0·01.

Figure 4

Mycobacterium avium-infected macrophages exposed to interferon-γ (IFN-γ) display increased rate of glucose uptake and aerobic glycolysis. Bone marrow-derived macrophages were infected with M. avium ATCC 25291 and/or treated with IFN-γ (100 U/ml). The transcription profile of key glycolytic enzymes was evaluated after 24 hr (a). Levels of intracellular lactate dehydrogenase (LDH) activity (b) and extracellular lactate (c) were measured during the 3 days following infection and/or treatment. The levels of glucose uptake (2-NBDG uptake, mean fluorescence intensity) were quantified by flow cytometry during the 3 days following infection and/or treatment (d). Means ± SD are from three independent experiments. *P < 0·05, **P < 0·01, ***P < 0·001.