Fig 1.
MAB-R strains enhanced mtROS in infected murine macrophages compared to the effects of MAB-S strains.
(A) Representative TEM images of BMDMs infected with Mab-R or -S strains at an M.O.I. of 10 for 24 h. As a control, representative images of uninfected cells are shown. The mitochondrial morphology in infected cells and their magnified images are shown (black box region and marked with a red asterisk). Bar indicates 0.5 or 2 μm. (B) To evaluate mtROS (MitoSOX), BMDMs or J774A.1 cells were infected with strains of MAB-R, MAB-S or M. smegmatis (Msm) at an M.O.I. of 10 for 24 h. Additionally, cells were pre-treated with rotenone (Rot; 5 μM) as a positive control (for induction of ROS) for 30 min. The infected cells were stained with MitoSOX and then analysed by flow cytometry (FACSCalibur). (C) Induced mtROS levels in J774A.1 cells infected with live or heat-killed (HK) mycobacteria (M.O.I. of 10) for 24 h were measured by flow cytometry (FACSCalibur). Error bars represent the SD. Statistical significance was determined by ANOVA with Tukey's multiple comparison test (B and C) and two-tailed Student’s t-test (D).
Fig 2.
MAB-R strains led to increased cytosolic oxidized mtDNA in an mtROS-dependent manner in infected murine macrophages.
(A) Cytosolic mtDNA was extracted from the nuclear and cytosolic fractions of J774A.1 cells that were infected with MAB-R, MAB-S, or M. smegmatis (Msm) (M.O.I. of 10) and pre-treated with or without mito-TEMPO (100 μM) for 24 h. Measurement of cytosolic mtDNA expression by qRT-PCR using the mitochondrial D-loop (D-loop-1, -2, and -3), CytB, ND4 and 16S primer sets. Normalization was performed as described in the materials and methods. (B) Representative confocal microscopic images of 8-OHdG induction in infected cells. J774A.1 cells were pre-treated with mito-TEMPO and infected with CFSE-labelled Mab-R or -S (green) at an M.O.I. of 10 for 24 h. Then, immunofluorescence staining using anti-8-oxyhydrodioxy guanosine (8-OHdG), a marker of DNA oxidative damage (red) was performed, and macrophage nuclei were stained with DAPI (blue). All images were captured at 100× magnification. (C) J774A.1 cells were infected with strains of MAB-R, MAB-S or M. smegmatis (Msm) at an M.O.I. of 10 for 24 h with or without mito-TEMPO treatment. Cytosolic DNA was obtained from infected cells, and the levels of 8-OHdG were measured by using an ELISA kit. Error bars represent the SD. Statistical significance was determined by two-tailed Student’s t-test (A and C).
Fig 3.
MAB-R-induced mtROS increased IFN-β and IL-1β production via activation of IRF-3 and the NLRP3 inflammasome.
(A) BMDMs or J774A.1 cells were infected with strains of MAB-R, MAB-S or M. smegmatis (Msm) at an M.O.I. of 10 for 24 h. The supernatants of the infected cells were collected, and IL-1β levels were measured by ELISA. (B) Supernatants from J774A.1 cells infected with live or heat-killed (HK) strains of MAB-R, MAB-S or M. smegmatis (Msm) at an M.O.I. of 10 for 24 h were collected, and the levels of IL-1β were measured by ELISA. (C) J774A.1 cells were pre-treated with or without mito-TEMPO (100 μM) and infected with strains of MAB-R, MAB-S or M. smegmatis (Msm) at an M.O.I. of 10 for 24 h. IL-1β and IFN-β cytokine levels in the supernatants of infected cells were analysed by ELISA. (D) Representative immunoblot images and quantitative bar graphs of NLRP3 (110 kDa), IL-1β (pro form, 35 kDa; active form, 17 kDa), p-IRF3 (44~55 kDa), IRF3 (44~55 kDa) and caspase-1 (Casp1; pro form, 45 kDa; active form, 10 kDa) induced by the indicated mycobacteria (M.O.I. of 10) infection for 4 or 24 h in BMDMs pre-treated with or without mito-TEMPO. The expression levels of IL-1β, NLRP3 and caspase-1 were normalized to that of GAPDH. Error bars represent the SD. Statistical significance was determined by ANOVA with Tukey's multiple comparison test (A) and two-tailed Student’s t-test (C and D).
Fig 4.
MAB-R infection-induced mtROS led to intracellular bacterial growth via enhanced cytosolic escape of the bacteria.
(A) J774A.1 cells were pre-treated with mito-TEMPO (100 μM) and infected with CFSE-labelled (green) Mab-R at an M.O.I. of 10 for 4 or 24 h. Then, infected cells were stained with LAMP -1 (red) and DAPI (blue). Representative images are shown for LAMP-1 (red) and Mab-R (green) co-localization. Bar graph of Pearson's correlation coefficient values for the co-localization of MAB-R and LAMP-1. Twenty randomly selected bacteria were analysed and are representative of two independent experiments. (B) Representative TEM images of Mab-R (M.O.I. of 10 for 24 h) infected BMDMs with or without mito-TEMPO treatment are shown. Red dashed lines indicate the Mab-R-containing phagosomes. (C) Untreated or mito-TEMPO-pre-treated J774A.1 cells infected with Mab-R at an M.O.I. of 10 for different times (2, 4, 8, 14 and 24 h). The infected cells were analysed by AFB staining and observed under a microscope at 100× magnification. The black arrows indicate MAB-R (red-stained bacilli) in methylene blue-stained J774A.1 cells. The bar graph shows the result of intracellular bacterial numbers in each phagosome by randomly counting 15 phagosomes in MAB-R-infected cells. (D) Mito-TEMPO-treated or untreated J774A.1 cells infected with strains of MAB-R, MAB-S or M. smegmatis (Msm) at an M.O.I. of 10 for 4 or 24 h. Cytosolic DNA was extracted using cellular fractionation and the phenol-chloroform-isoamyl alcohol (PCI) method from J774A.1 cells infected with the indicated bacteria. Cytosolic mycobacterial DNA was detected by PCR amplification of the hsp65 (603 bp) gene (non, uninfected cells; +, mycobacterial DNA; −, contains only primers). Error bars represent the SD. Statistical significance was determined by two-tailed Student’s t-test (A and C).
Fig 5.
mtROS led to enhanced intracellular survival of MAB-R strains via IFN-I-dependent cell-to-cell spreading and enhanced cell cytotoxicity.
(A) J774A.1 cells pre-treated with mito-TEMPO (100 μM) and infected with rEGFP_Mma-R or rEGFP_Msm (M. smegmatis) at an M.O.I. of 10 for different times (2, 6 and 24 h). The percentage of EGFP-positive cells was measured by flow cytometry (FACSCalibur). (B) J774A.1 cells were pre-treated with mito-TEMPO and infected with strains of MAB-R, MAB-S or M. smegmatis (Msm) at an M.O.I. of 10 for 24 h. Infected cell lysates were serially diluted and plated onto 7H10 agar plates for CFU assays. (C) Cytotoxicity was quantitated by measuring lactate dehydrogenase (LDH) in the infected cell supernatants. Error bars represent the SD. Statistical significance was determined by two-tailed Student’s t-test (A-C).
Fig 6.
MAB-R infection enhanced IFN-β production via the cGAS−STING axis in infected murine macrophages.
(A) RAW264.7 cells were transfected with siRNAs targeting cGAS or STING or a scramble siRNA (siNT). Forty-eight hours after transfection, protein and transcription levels were measured in total cell lysates by immunoblotting (left panels) and RT-qPCR (right panels), respectively. (B-D) RAW264.7 cells were transfected with cGAS, STING or a scramble siRNA (siNT). Forty-eight hours after transfection, the cells were infected with MAB-R or -S strains (10 M.O.I) and then harvested 24 h after infection. The transcription levels of IFN-β and IL-1β in infected cells were measured via qRT-PCR. Normalization was performed using the β-actin gene (B). IL-1β and IFN-β cytokine levels in the supernatant of infected cells were measured by ELISA (C), and infected cell lysates were serially diluted and plated onto 7H10 agar plates for CFU assays (D). Error bars represent the SD. Statistical significance was determined by ANOVA with Tukey's multiple comparison test (A) two-way ANOVA with multiple comparison test (B-D).
Fig 7.
Crosstalk between IFN-I and IL-1β in MAB-R infection of murine macrophages.
(A) BMDMs were obtained from wild-type (WT) or IFNAR1 KO mice and infected with MAB-R and -S strains at an M.O.I. of 10 for 24 h. Supernatants from the infected cells were collected, and IL-1β and IFN-β cytokine levels were measured by ELISA. (B-C) BMDMs were treated with anti-mouse IgG (10 μg/ml) or anti-mouse IL-1β receptor (10 μg/ml) neutralizing antibodies. Then, the cells were infected with MAB-R and -S stains at an M.O.I. of 10 for 24 h. IL-1β and IFN-β cytokine levels in the infected cell supernatants were measured by ELISA (B), and infected cell lysates were serially diluted and plated onto 7H10 agar plates for CFU assays (C). Error bars represent the SD. Statistical significance was determined by two-tailed Student’s t-test (A-C).
Fig 8.
Schematic diagram showing mitochondrial oxidative stress induced by MAB-R infection of murine macrophages.
MAB-R enters macrophages by phagocytosis and leads to mitochondrial oxidative stress. Our results suggest that MAB-R infection elicits both the i) cGAS−STING−IRF3−IFN-β and ii) NLRP3 inflammasome-IL-1β pathways in murine macrophages: i) Excess formation of mtROS leads to phagosomal rupture-mediated bacterial escape into the cytosol. Cytosolic mitochondrial and mycobacterial DNA induce IFN-I production via the cGAS−STING−IRF3 pathway. ii) Additionally, oxidized mtDNA released into the cytosol leads to activation of the NLRP3 inflammasome. However, inhibition of mtROS by treatment with mito-TEMPO or CsA decreases the levels of mtROS and also leads to decreased IFN-I and IL-1β production. In addition, inhibition of mtROS leads to a reduction in MAB-R replication in phagosomes and inhibition of phagosomal rupture, which ultimately reduces bacterial survival in infected murine macrophages. These two pathways induce opposite results for host defence against MAB-R infection. IL-1β is considered to have antibacterial effects, while IFN-I is recognized to be highly detrimental to bacterial replication.