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Abstract
Apoptosis is an important host defense mechanism against mycobacterial infection. Recent reports suggest that links between apoptosis and endoplasmic reticulum (ER) stress are critical for the regulation of mycobacterial survival; however, the exact regulatory mechanisms are not well known. In this study, we isolated 20 Mycobacterium tuberculosis (Mtb) clinical strains from Korean patients and examined ER stress-mediated apoptosis in Mtb-infected macrophages. Most Mtb strains increased the rates of apoptosis and production of ER stress-sensing molecules in mouse macrophages, similar to Mtb H37Rv infection. Moreover, the intracellular survival of Mtb clinical isolates in macrophages was similar to that of H37Rv. Our data suggest that infection with Mtb downregulated MCP-1 and MCPIP. The regulation of MCPIP may decrease ROS production, leading to a reduction in ER stress-mediated apoptosis.
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Figure 1.
Induced apoptosis and endoplasmic reticulum (ER) stress activation by clinical isolates of Mycobacterium tuberculosis (Mtb). Raw264.7 cells were infected with H37Rv and 20 clinical isolates of Mtb (multiplicity of infection; MOI=1) for 3 h, and incubated for an additional 24 h. (A) The percentage of sub-G1 cells was measured using flow cytometry for the detection of apoptosis. Stimulation by 500 nM staurosporine (STS) for 6 h was used as a positive control. (B) Necrosis was assessed by assaying lactate dehydrogenase (LDH) release from culture supernatants. (C) Western blotting was performed using antibodies against C/EBP homologous protein (CHOP), phospho-eukaryotic initiation factor (eIF2)α, binding immunoglobulin protein (Bip), caspase-3, and β-actin. As a positive control, cells were treated with 500 ng/ ml tunicamycin (TM) for 6 h. The densitometry values for each protein were normalized to the β-actin level, and compared to cells infected with H37Rv. ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001.†Indicates statistically significant differences, compared to cells infected with the clinical isolate, IN1302.†p < 0.05; ††p < 0.01; and †††p < 0.001. The data are representative of at least three independent experiments. Bars represent the means ± SD.
Figure 2.
Intracellular survival of Mtb isolates in Raw264.7 cells. Colony-forming unit (CFU) assays were performed to measure the intracellular survival of H37Rv and 20 clinical isolates of Mtb in Raw264.7 cells. Statistical differences in Mtb survival, compared to H37Rv, were calculated using a one-way ANOVA with Bonferroni's multiple comparison test. ∗ p < 0.05; ∗∗ p < 0.01; and ∗∗∗ p < 0.001. N≥3. Bars represent the means ± SD.
Figure 3.
The production of proinflammatory cytokines by Raw264.7 cells infected with clinical isolates of Mtb. Raw264.7 cells were infected with H37Rv and clinical isolates of Mtb at a ratio of 1:1. Twenty-four hours after infection, supernatants were collected and subjected to ELISAs for tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP-1), and interleukin (IL)-6. As a positive control, cells were treated with 500 ng/ml lipopolysaccharide (LPS) for 24 h. ∗Indicates significant differences compared to cells infected with H37Rv. ∗ p < 0.05; ∗∗ p < 0.01; and ∗∗∗ p < 0.001. † Indicates significant differences compared to cells infected with the clinical isolate, IN1302. † p <0.05 and †† p < 0.01. N≥3. Bars represent the means ± SD.
Figure 4.
The expression of MCP-induced protein (MCPIP) through the mitogen-activated protein kinase pathway during Mtb infection. Raw264.7 cells were infected with clinical Mtb isolates for 30 or 60 min, and then subjected to Western blot analyses targeting (A) phospho-c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38, or (B) MCPIP. Treatment with 500 ng/ml LPS for 1 h was used as a positive control. The densitometry values for each protein were normalized to the β-actin level. ∗Indicates significant differences compared to cells infected with H37Rv. ∗∗p < 0.01. †Indicates significant differences compared to cells infected with the clinical isolate, IN1302. † p < 0.05 and †† p < 0.01. The data are representative of at least three independent experiments. Bars represent the means ± SD.
Figure 5.
ROS synthesis in Raw264.7 cells infected with 1 of 20 clinical isolates of Mtb. Raw264.7 cells were incubated for 3 h with H37Rv or clinical Mtb isolates, and then cultured for 24 h in DMEM. For the analysis of intracellular ROS production, cells were incubated with dihydroethidium (20 μM) for 30 min and then analyzed by flow cytometry. H2O2 (10 mM, 30 min) was used as a positive control. Statistical differences between the Mtb- and H37Rv-infected cells were examined using a one-way ANOVA with Bonferroni's multiple comparison test. † p < 0.05. N≥3. Bars represent the means ± SD.
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