Mycobacterium abscessus infection leads to enhanced production of type 1 interferon and NLRP3 inflammasome activation in murine macrophages via mitochondrial oxidative stress
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).