Mycobacterium bovis Induces Endoplasmic Reticulum Stress Mediated-Apoptosis by Activating IRF3 in a Murine Macrophage Cell Line

Abstract

Mycobacterium bovis (M. bovis) is highly adapted to macrophages and has developed multiple mechanisms to resist intracellular assaults. However, the host cells in turn deploy a multipronged defense mechanism to control bacterial infection. Endoplasmic reticulum (ER) stress-mediated apoptosis is one such primary defense mechanism. However, the role of interferon regulatory factor 3 (IRF3) between ER stress and apoptosis during M. bovis infection is unknown. Here, we demonstrate that M. bovis effectively induced apoptosis in murine macrophages. Caspase-12, caspase-9, and caspase-3 were activated over a 48 h infection period. The splicing of XBP-1 mRNA and the level of phosphorylation of eIF2α, indicators of ER stress, significantly increased at early time points after M. bovis infection. The expansion of the ER compartment, a morphological hallmark of ER stress, was observed at 6 h. Pre-treatment of Raw 264.7 cells with 4-PBA (an ER stress-inhibitor) reduced the activation of the ER stress indicators, caspase activation and its downstream poly (ADP-ribose) polymerase (PARP) cleavage, phosphorylation of TBK1 and IRF3 and cytoplasmic co-localization of STING and TBK1. M. bovis infection led to the interaction of activated IRF3 and cytoplasmic Bax leading to mitochondrial damage. Role of IRF3 in apoptosis was further confirmed by blocking this molecule with BX-795 that showed significant reduction expression of caspase-8 and caspase-3. Intracellular survival of M. bovis increased in response to 4-PBA and BX-795. These findings indicate that STING-TBK1-IRF3 pathway mediates a crosstalk between ER stress and apoptosis during M. bovis infection, which can effectively control intracellular bacteria.

Keywords: ER stress; IRF3; M. bovis; apoptosis; mycobacterium.

Figure 1

M. bovis infection induces apoptosis and caspase activation. (A) Raw 264.7 cells were screened for induction of apoptosis using Annexin V/PI staining after 24 h infection with M. bovis at an MOI of 10. Tunicamycin (5 μg/ml) was used as a positive control for apoptosis. 4-PBA, an ER stress inhibitor was treated before M. bovis infection in Raw 264.7 cells. After washing and Annexin V/PI staining, cells were analyzed by flow cytometry. (B) Quantitative analysis of the percentage of apoptotic cells (sum of early and late apoptotic cells) from (A), a statistically significant difference (^*P < 0.05) is observed between 4-PBA pretreated and non-pretreated groups using the two-tailed t-test. (C) Raw 264.7 cells were stimulated with M. bovis for 24 h and total cell lysates were subjected to Western blot for cleaved caspase-3 (p17 specific), caspase-9, and caspase-12. (D) Bands corresponding to each protein were quantified, and the intensities of each protein were normalized to the intensity of tubulin. Data are representative of at least three independent experiments, each performed in triplicate with similar results. The asterisks indicate statistically significant differences compared with untreated cells (^*P < 0.05, ^**P < 0.01).

Figure 2

ER stress is involved in M. bovis-mediated apoptosis. Raw 264.7 cells were infected with M. bovis at an MOI of 10, and then incubated for 0–48 h. (A) TEM analysis of Raw 264.7 cells after M. bovis infection for 6 h. The perinuclear rough ER regions on the images in the left panel are magnified on the right panels. Arrows indicate ER lamellae before and after infection. (B) XBP-1 mRNA splicing was determined by RT-PCR using specific primers that were used to amplify products of unspliced and spliced mRNA. (C) The results represent the ratio of spliced XBP-1 to intact (or unspliced) XBP-1 (XBP-1S/U ratio). (D) Total cell lysates were subjected to Western blot to identify phosphorylation of eIF2α. (E) Bands corresponding to each protein were quantified, and the intensities of each protein were normalized to the intensity of tubulin. Data are representative of at least three independent experiments, each performed in triplicate with similar results. The asterisks indicate significant differences compared with untreated cells (^*P < 0.05, ^**P < 0.01, ^***P < 0.001).

Figure 3

Effect of ER stress mediated apoptosis on intracellular survival of M. bovis. (A–D) 4-PBA was treated before M. bovis infection in Raw 264.7 cells and total cell lysates were subjected to Western blot for phosphorylation of eIF2α, cleaved caspase-12, and caspase-3 (p17 specific) and PARP. Bands corresponding to each protein were quantified, and the intensities of each protein were normalized to the intensity of tubulin. (E) Quantification of intracellular survival of M. bovis in Raw 264.7 cells pretreated for 3 h with tunicamycin (5 μg/ml). Cells were harvested at 6 and 24 h post infection with M. bovis and bacteria number was determined by CFU counting. (F) Quantification of intracellular survival of M. bovis in Raw 264.7 cells pretreated for 3 h with 4-PBA (5 mM). Cells were harvested at 24 h post infection with M. bovis and bacterial numbers were determined. Data are representative of at least three independent experiments, each performed in triplicate with similar results. The asterisks indicate significant differences compared with untreated cells (^*P < 0.05, ^**P < 0.01).

Figure 4

ER stress results in the phosphorylation and nuclear translocation of IRF3. (A–D) BX-795 and 4-PBA were respectively treated before M. bovis infection in Raw 264.7 cells and total cell lysates were subjected to Western blot for phosphorylation of IRF3. Bands corresponding to each protein were quantified, and the intensities of each protein were normalized to the intensity of tubulin. The asterisks indicate significant differences compared with untreated cells (^*P < 0.05, ^**P < 0.01). (E) 4-PBA was treated before M. bovis infection in Raw 264.7 cells. Fixed cells were incubated with anti-IRF3 monoclonal antibody followed by FITC-conjugated goat anti-rabbit antibody as the secondary antibody and visualized by immunofluorescence microscopy. Results are representative of three independent experiments, each performed in triplicate with similar results.

Figure 5

Activation of IRF3 requires the mobilization of STING and TBK1. (A–D) Raw 264.7 cells were infected with M. bovis at an MOI of 10, and then incubated for 0–48 h. Total cell lysates were subjected to Western blot for phosphorylation of TBK1. Bands corresponding to each protein were quantified, and the intensities of each protein were normalized to the intensity of tubulin. (C,D) 4-PBA was treated before M. bovis infection in Raw 264.7 cells and total cell lysates were subjected to Western blot for phosphorylation of TBK1. (E,F) 4-PBA was treated before M. bovis infection in Raw 264.7 cells. Fixed cells were incubated with anti-STING antibody and anti-TBK1 antibody followed by fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat (green) or Alexa Fluor-conjugated goat anti-rabbit (red). Results are representative of at least three independent experiments, each performed in triplicate with similar results. The asterisks indicate significant differences compared with untreated cells (^*P < 0.05, ^**P < 0.01).

Figure 6

IRF3 causes the mitochondrial damage and caspase-8 activation. Raw 264.7 cells were untreated or treated with M. bovis at an MOI of 10 for 24 h. (A) Mitochondrial transmembrane potential (ΔΨm) is measured using JC-1 as the fluorescent marker. The JC-1-aggregate form, indicating normal ΔΨm, appears red and the monomeric form, indicating low ΔΨm (i.e., disrupted mitochondrial membrane), is green by confocal microscopy. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), as a positive control for the mitochondrial damage. Scale bar = 50 μm. (B) Mitochondrial fractions were isolated from whole cell lysates from Raw 264.7 cells. Mitochondrial fractions and supernatant were immunoblotted for the indicated proteins. Left and right parts represent the same membrane. (C) Samples were immunoprecipitated with control IgG or anti-phospho-IRF3. Whole-cell lysate (input) or immunoprecipitations were resolved by SDS-PAGE and immunoblotted with anti-phospho-IRF3 or Bax. (D) BX-795 was treated before M. bovis infection in Raw 264.7 cells and total cell lysates were subjected to Western blot for cleaved caspase-3 (p17 specific) and caspase-8. (E) Bands corresponding to each protein were quantified, and the intensities of each protein were normalized to the intensity of tubulin. (F) Quantification of intracellular survival of M. bovis in Raw 264.7 cells pretreated for 3 h with BX-795 (10 μM). Cells were harvested at 24 h post infection with M. bovis and bacteria number was determined by CFU counting. Data are representative of at least three independent experiments, each performed in triplicate with similar results. The asterisks indicate significant differences compared with untreated cells (^*P < 0.05, ^**P < 0.01, ^***P < 0.001).

Figure 7

Schematic representation of ER stress-mediated apoptosis via the activation of IRF3 following M. bovis stimulation. M. bovis induced ER stress in macrophages triggers the translocation of STING, which facilitates phosphorylation of IRF3 by the downstream phosphorylated TBK1. Subsequently, (i) phosphorylated IRF3 translocates to the nucleus to induce the transcription of putative apoptosis-related factors. (ii) phosphorylated IRF3 associates with Bax and triggers mitochondrial pathway of macrophages apoptosis. (iii) phosphorylated IRF3 activates the downstream caspase-8. All these three pathways finally lead to apoptosis.