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