Tumour necrosis factor receptors and apoptosis of alveolar macrophages during early infection with attenuated and virulent Mycobacterium bovis

Abstract

Apoptosis of macrophages has been reported as an effective host strategy to control the growth of intracellular pathogens, including pathogenic mycobacteria. Tumour necrosis factor-α (TNF-α) plays an important role in the modulation of apoptosis of infected macrophages. It exerts its biological activities via two distinct cell surface receptors, TNFR1 and TNFR2, whose extracellular domain can be released by proteolysis forming soluble TNF receptors (sTNFR1 and sTNFR2). The signalling through TNFR1 initiates the majority of the biological functions of TNF-α, leading to either cell death or survival whereas TNFR2 mediates primarily survival signals. Here, the expression of TNF-α receptors and the apoptosis of alveolar macrophages were investigated during the early phase of infection with attenuated and virulent mycobacteria in mice. A significant increase of apoptosis and high expression of TNFR1 were observed in alveolar macrophages at 3 and 7 days after infection with attenuated Mycobacterium bovis but only on day 7 in infection with the virulent M. bovis. Low surface expression of TNFR1 and increased levels of sTNFR1 on day 3 after infection by the virulent strain were associated with reduced rates of apoptotic macrophages. In addition, a significant reduction in apoptosis of alveolar macrophages was observed in TNFR1(-/-) mice at day 3 after bacillus Calmette-Guérin infection. These results suggest a potential role for TNFR1 in mycobacteria-induced alveolar macrophage apoptosis in vivo. In this scenario, shedding of TNFR1 seems to contribute to the modulation of macrophage apoptosis in a strain-dependent manner.

Keywords: Mycobacterium bovis; apoptosis; macrophage; tumour necrosis factor receptor 1; tumour necrosis factor receptor 2.

Figure 1

Surface tumour necrosis factor receptor (TNFR) expression in J774A.1 cells after Mycobacterium bovis bacillus Calmette–Guérin (BCG) infection. J774A.1 macrophages were either mock-infected or infected with M. bovis BCG at a multiplicity of infection (MOI) of 10 : 1. After 24, 48 and 72 hr, cells were stained with FITC-conjugated anti-mouse TNFR1 (a), or phycoerythrin-conjugated anti-mouse TNFR2 (b) and analysed by flow cytometry. The intensity of TNFRs was expressed as the mean fluorescence intensity (MFI) of at least 10 000 cells per sample. Representative histograms show TNFR1 or TNFR2 surface expression on mock-infected cells (dotted line) and BCG-infected cells (black line) or isotype control (light grey-filled area). The means and standard errors from one representative experiment of at least three is shown. *P < 0.001 versus mock-infected control.

Figure 2

Surface expression of tumour necrosis factor receptor 1 (TNFR1) and TNFR2 on alveolar macrophages after infection with attenuated and virulent Mycobacterium bovis. C57BL/6 mice were intratracheally infected with attenuated (bacillus Calmette–Guérin Moreau) or virulent (ATCC19274) M. bovis. Control mice were inoculated with PBS. After 3 and 7 days, cells were obtained by bronchoalveolar lavage. Alveolar macrophages were identified as CD11b⁻ CD11c^+/high (a) and cell surface expression of TNFR1 and TNFR2 was assessed by FACS, as shown by representative histograms (b). Results of specific staining for TNFR1 (c) and TNFR2 (d) were expressed as the mean channel fluorescence intensity from 10 000 events per sample. Each bar represents mean ± SE of one representative experiment of three with similar results. *P < 0.05 versus control, ^#P < 0.05.

Figure 3

Detection of soluble tumour necrosis factor receptor 1 (sTNFR1) and sTNFR2. Levels of sTNFR1 (a) and sTNFR2 (b) in bronchoalveolar lavage fluid from mice infected with attenuated or virulent Mycobacterium bovis and uninfected control were assessed by ELISA. Each bar represents mean ± SE of one representative experiment of three with similar results. *P < 0.05 versus control, ^#P < 0.05.

Figure 4

Quantification of tumour necrosis factor-α (TNF-α). Levels of TNF-α in the lung from mice infected with attenuated and virulent Mycobacterium bovis strains were measured by ELISA. Each bar represents mean ± SE. Results from one representative experiment of three with similar results is shown *P < 0.05 versus control, ^#P < 0.05.

Figure 5

Apoptosis of alveolar macrophages after infection with attenuated or virulent Mycobacterium bovis. C57BL/6 mice were intratracheally infected with attenuated or virulent M. bovis. After 3 and 7 days of infection, alveolar macrophages were stained with annexin V and 7-aminoactinomycin D for analysis of apoptosis by flow cytometry. Each bar represents mean ± SE. Results from one representative experiment of three with similar results is shown. *P < 0.05 versus control, ^#P < 0.05.

Figure 6

Apoptosis of alveolar macrophages in tumour necrosis factor receptor 1-deficient (TNFR1^−/−) mice. Wild-type C57BL/6 mice or TNFR1^−/− mice were intratracheally infected with Mycobacterium bovis BCG. After 3 days of infection, alveolar macrophages were stained with annexin V and 7-aminoactinomycin D for analysis of apoptosis by flow cytometry. Each bar represents mean ± SE. One representative experiment of two with similar results is shown. *P < 0.05 versus control, ^#P < 0.05.

Figure 7

Number of bacilli in alveolar macrophages infected with attenuated (a) or virulent (b) Mycobacterium bovis at day 3 and day 7 post-infection. Cytocentrifuge slides were stained for acid-fast bacilli using the Ziehl–Neelsen stain and the number of bacilli per macrophage was scored as 1–10 bacilli per macrophage (c,d) and > 10 bacilli per macrophage (e). Results are representative of two different experiments. (c, d, e × 1000).