Mycobacterium tuberculosis-induced expression of granulocyte-macrophage colony stimulating factor is mediated by PI3-K/MEK1/p38 MAPK signaling pathway

Abstract

Members of the colony stimulating factor cytokine family play important roles in macrophage activation and recruitment to inflammatory lesions. Among them, granulocyte-macrophage colony stimulating factor (GM-CSF) is known to be associated with immune response to mycobacterial infection. However, the mechanism through which Mycobacterium tuberculosis (MTB) affects the expression of GM-CSF is poorly understood. Using PMA-differentiated THP-1 cells, we found that MTB infection increased GM-CSF mRNA expression in a dosedependent manner. Induction of GM-CSF mRNA expression peaked 6 h after infection, declining gradually thereafter and returning to its basal levels at 72 h. Secretion of GM-CSF protein was also elevated by MTB infection. The increase in mRNA expression and protein secretion of GM-CSF caused by MTB was inhibited in cells treated with inhibitors of p38 MAPK, mitogen-activated protein kinase kinase (MEK-1), and PI3-K. These results suggest that up-regulation of GM-CSF by MTB is mediated via the PI3-K/MEK1/p38 MAPK-associated signaling pathway.

Fig. 1.. mRNA expression of GM-CSF was affected by MTB. THP-1 cells were treated with PMA (100 nM) for 48 h and were incubated in the presence of MTB for the indicated times (0, 1.5, 3, 6, 9, 12, 24 h). cDNA were prepared from total RNA of infected cells, and was subjected to PCR to amplify (A) chemokines (CKβ8, CKβ8-1, MCP-1, MIP-1α), (B) DC markers (HLA-DR, DC-SIGN, DEC205, CCR7), and (C) colony stimulating factors (M-CSF, G-CSF, GM-CSF). The PCR products were resolved by 1.8% agarose gel. GAPDH was used as an internal control.

Fig. 2.. MTB induces the increased expression and secretion of GM-CSF. (A) Differentiated THP-1 cells were infected with the indicated concentrations (0, 1, 2, 5, 10, 20, 40 or 50 MOI) of MTB for 6 h. Total RNA was extracted and cDNA was prepared. PCR analysis was performed using GM-CSF-specific primers. The PCR products were resolved by 1.8% agarose gel (upper panel), to detect GM-CSF. GAPDH was used as an internal control. Densitometric analysis was performed (lower panel). Data are expressed as mean ± SD, and are presented as the expression levels of GM-CSF mRNA relative to GAPDH mRNA (The expression level of GM-CSF relative to GAPDH in the absence of mycobacterial infection was set to 1.0). The data represent results from three independent experiments (*P ＜ 0.05 relative to uninfected control). (B) THP-1 cells were differentiated for 48 h and were incubated in the presence of MTB for the indicated times (0, 1.5, 3, 6, 9, 12, 18, 24, 48 or 72 h). Next, semi-quantitative RT-PCR was carried out, as above. Densitometric analysis was performed (lower panel). Data are expressed as mean ± SD, and are presented as the expression levels of GM-CSF mRNA relative to GAPDH mRNA. (The level of GM-CSF relative to GAPDH without infection by MTB was set to 1.0). The data represent results from five independent experiments (*P ＜ 0.05, **P ＜ 0.01 relative to uninfected control). (C) Differentiated THP-1 cells were incubated in the absence or presence of MTB for 24 h, and secretion of GM-CSF was measured by ELISA using cell culture supernatants. Data are expressed as the mean ± SD of three independent experiments and are presented as pg/ml.

Fig. 3.. MTB-induced expression of GM-CSF is mediated by PI3-K, MEK1, and p38 MAPK. (A) PMA-treated THP-1 cells were pre-incubated with the inhibitors SB202190 (20 μM), PD98059 (50 μM), Ro-31-8425 (50 nM), Ly294002 (10 μM), U73122 (50 ng/ml), SP600125 (10 μM) for 45 min, followed by mycobacterial infection (10 MOI) for 4 h. cDNA was prepared from total RNA extracted from treated cells. PCR analysis was performed using GM-CSF-specific primers. PCR products were analyzed by 1.8% agarose gel (upper panel) to detect GM-CSF expression. GAPDH was used as an internal control. Densitometric analysis was performed (lower panel). Data are expressed as the mean ± SD, and are presented as the expression levels of GM-CSF mRNA relative to GAPDH mRNA (The level of GM-CSF relative to GAPDH in the absence of mycobacterial infection was set to 1.0). Data are the results from three independent experiments (**P ＜ 0.01 relative to MTB infection alone). (B) Differentiated THP-1 cells were pre-treated with the indicated concentrations of SB202190, PD98059, Ly294002, U73122 or SP600125 for 45 min, followed by MTB infection (10 MOI). Supernatants were harvested 24 h after infection and secretion of GM-CSF was measured by ELISA. Data are presented as mean ± SD of three independent experiments performed in duplicate (**P ＜ 0.01 relative to MTB alone). (C) Differentiated THP-1 cells were treated for 45 min with the indicated concentration of SB202190, PD98059 or Ly294002. Subsequently, MTB was added at 10 MOI. Supernatants were harvested after 24 h, and the protein contents of GM-CSF in culture supernatants were determined by ELISA. Data shown are the mean ± SD of three independent experiments performed in duplicate (**P ＜ 0.01 or ***P ＜ 0.001 relative to MTB alone).