Macrophages from elders are more permissive to intracellular multiplication of Mycobacterium tuberculosis

Abstract

The elderly account for a disproportionate share of all tuberculosis cases, and the population ageing may not fully explain this phenomenon. We have performed in vitro infection experiments to investigate whether there is an immunological basis for the apparent susceptibility of elders to tuberculosis. In our infection model, Mycobacterium tuberculosis induces a higher production of interleukin (IL)-6 and reactive oxygen species in macrophages from elders than from younger adults. This response did not prevent, however, an increased multiplication of M. tuberculosis in macrophages from elders as compared with the growth observed within cells from adults. By performing a factorial experiment, we have found that IFN-γ, but not IL-1β, IL-6 or TNF-α, stimulate the macrophages to restrict the multiplication of the bacterium in macrophages from elders. Although monocytes from elders seem to be in a higher level of activation, we present evidences that protein tyrosine phosphorylation response induced by M. tuberculosis is stronger in monocytes from adults than from elders. Using a protein array that detects 71 tyrosine phosphorylated kinases, we identified Pyk2 as the only kinase that displayed a difference of intensity larger than 50 % in adults than in elders. Furthermore, monocytes from elders that were incubated in the presence of tyrosine kinase inhibitors (genistein and PP2) allowed a higher level of bacterial multiplication. These observations may help to explain the susceptibility of elders to tuberculosis. An unexpected result was that both genistein and its negative control, daidzein, abundant soy isoflavones, promoted intracellular mycobacterial growth.

Fig 1

Cytokine production by MDM from aged donors. MDM, 5 × 10⁵, from aged (continuous line) and from young (dashed lines) donors were infected with M. tuberculosis (MOI = 1) in 500 μL of RPMI-1640/10 % autologous serum. Medium of 50 μL was removed after 6, 24, 48 and 72 h. IL-1β, IL-6 and TNF-α in culture supernatants were measured by ELISA. Data represent the median of concentration (nanograms per millilitre), and vertical lines are the 95 % confidence interval. Sample sizes for adults and elders were six and four in time points 6, 48 and 72 h, and 15 and 12 in time point 24 h, respectively. At each time point a Mann–Whitney’s test was performed for comparisons between adults and elders. *P < 0.05 is considered significant

Fig. 2

Phagocytic activity of monocytes from aged donors. Monocytes, 10⁵, from either an elder (continuous line) or an adult (dashed line) were incubated with 5 × 10⁵ BCG-DsRed bacteria for 10 min. Non-infected cells were used as controls. Fluorescence intensity (red) was analysed by flow cytometry, and 10⁴ events were measured. The curves that correspond to control cells have inside the label ‘No infection’. The curves that correspond to infected cells are marked with the label ‘BCG’. For statistical analysis, a cutoff value for defining red fluorescent positivity was set arbitrarily at 10¹ to exclude over 97 % of non-infected cells. This result is representative of five independent experiments

Fig. 3

Protein tyrosine phosphorylation in infected monocytes. Monocytes, 7 × 10⁵, were infected with M. tuberculosis (MOI, 20) for 30 min and 12 μg of total protein were analysed by immunoblot with an anti-phosphotyrosine antibody. Two donors from each age group were studied and M. tuberculosis-infected cells were compared with non-infected cells. Molecular weight markers are indicated on the left, and the sizes of specifically phosphorylated proteins are shown on the right. For loading control, the same blot was probed 2 days after with an anti-β-actin antibody

Fig. 4

Identification of tyrosine phosphorylated kinases. a Cells, 3 × 10⁶, were infected with M. tuberculosis (MOI, 20) for 30 min and 12 μg of total protein were analysed by immunoblot with an anti-phosphotyrosine antibody. b Arrays incubated with lysates from either an adult or an elder (120 μg of total protein). Chemiluminiscence was measured as counts with an image analyser, and data were normalized with dots A5-6, which are positive controls included in the kit. Local background was subtracted from each dot, and differences in dot intensities were determined. Dots were included in duplicates in the kit, and correspond to the following kinases: positive controls, A1, A3, A5 and M11. Negative controls: B1, B3 and L11. A7, Abl1; A9, Ack1; A11, ALK; B5, Axl; B7, Blk; B9, BMX; B11, Btk; C1, Csk; C3, Dtk; C5, EGFR; C7, EphA1; C9, EphA2; C11, EphA3; D1, EphA4; D3, EphA5; D5, EphA6; D7, EphA7; D9, EphA8; D11, EphB1; E1, EphB2; E3, EphB3; E5, EphB4; E7, EphB6; E9, ErbB2; E11, ErbB3; F1, ErbB4; F3, FAK; F5, FER; F7, FGFR1; F9, FGFR2; F11, FGFR2 (α); G1, Fgr; G3; FRK; G5; Fyn; G7, Hck; G9, HGFR; G11, IGF-I R; H1, Insulin R; H3, Itk; H5, JAK1; H7, JAK2; H9, JAK3; H11, LCK; I1, LTK; I3, Lyn; I5, MATK; I7, M-CSFR; I9, MUSK; I11, NGFR; J1, PDGF-α; J3, PDGF-β; J5, Pyk2; J7, RET; J9, RORI; J11, ROR2; K1, ROS; K3, RYK; K5, SCFR; K7, SRMS; K9, SYK; K11, Tec; L1, Tie-1; L3, Tie-2; L5, TNK1; L7, TRKB; L9, TXK; M1, Tyk2; M3, TYRO10; M5, VEGFR2; M7, VEGFR3; M9, ZAP70

Fig. 5

Effect of tyrosine kinase inhibitors on protein tyrosine phosphorylation in infected monocytes. Monocytes, 7 × 10⁵, from an adult were preincubated with protein kinase inhibitors (100 μM genistein or 10 μM PP2) for 1 h before infection with M. tuberculosis (MOI, 20) for 30 min and 12 μg of total protein were analysed by immunoblot with an anti-phosphotyrosine antibody. Molecular weight markers are indicated on the left. For loading control, the same blot was probed 2 days after with an anti-β-actin antibody

Fig. 6

ROS production in activated monocytes. Cells, 10⁵, were activated by M. tuberculosis infection (MOI, 5) or by incubation with the strong activator TPA for 30 min in the presence of luminol. Non-activated cells (‘No infection’) were included as negative controls. Data are the cps + standard deviation, and Student’s t test was performed for comparisons between adults (n = 7) and elders (n = 7). *P < 0.05 is considered significant

Fig. 7

Cellular death in infected monocytes. After cell lysis by sonication, the lysate was diluted one tenths and mixed with a luminogenic substrate that detect a ‘dead-cell protease activity’ (2 volumes diluted lysate: 1 volume substrate). After 30 min, chemiluminiscence was measured in a luminometer as cps. Data are the log of the cps + standard deviation. Student’s t test test was performed for comparisons between adults (n = 5) and elders (n = 4). *P < 0.05 is considered significant