Journal: MedComm

Article Title: Inflammasome Activation Differences Underpin Different Mycobacterium tuberculosis Infection Outcomes

doi: 10.1002/mco2.70486

Figure Lengend Snippet: Production of NLRP3 inflammasome pathway proteins and differential activation of mitochondrial stress in macrophages during Mtb HN878 or CDC1551 infection. (A) Representative Western blot image of proteins isolated from HN878‐ or CDC1551‐infected THP‐1 showing levels of NLRP3, ASC, IL1β, and Caspase 1 at 4, 8, 24, and 48 hpi. Uninfected cells (0, C) were used as controls. Tubulin and Actin were used to normalize samples. The IL1β panel shows both precursor and mature forms of the protein. The caspase 1 panel shows both uncleaved and cleaved forms of the protein. MW‐molecular weight standard in kDa. (B) Fluorescence measurement of caspase activity in rBMDMs infected with CDC1551 or HN878 for 24 or 48 h. (C) Representative images of uninfected or Mtb‐infected THP‐1 macrophages after staining with MitoTracker (green) and Mitosox (red). The scale bar is 50 µm. (D) Quantifications of mitochondrial mass by MitoTracker staining. (E) Quantifications of mitochondrial ROS production by MitoSOX staining. Fluorescence intensities of MitoTracker (mitochondrial mass) and MitoSOX (mitochondrial ROS) in hu‐MΦ infected with HN878 or CDC1551 or no infection (uninfected) were measured. Experiments were repeated twice with n = 3–5 samples, and statistical analyses were performed using one‐way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001. (F–Q) Apoptosis and necrosis of Mtb HN878‐ or CDC1551‐infected macrophages. Percentages of apoptotic (F, G) or necrotic (H, I) rBMDMs (F, H) and THP‐1 macrophages (G, I) infected with CDC1551 or HN878 at an MOI of 1 or 5 were determined at 24 or 48 h postinfection. Uninfected (UI) cells were included as controls. (J–Q) Representative images (J, L, N, P) and quantifications (K, M, O, Q) of apoptotic (J–M) and necrotic (N–Q) rBMDMs (J, K, N, O) and THP‐1 (L, M, P, Q) using immunocytochemistry. Cells were enumerated manually from the enlarged microscopic images of each field. Experiments were repeated thrice with n = 3 samples, and statistical analyses were performed using one‐way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < .001.

Article Snippet: Generation of mitochondrial reactive oxygen species (ROS), especially superoxide, in hu‐MΦ macrophages during mycobacterial infection was detected using stains MitoTracker (Cell Signaling Technology) and MitoSox (Thermo Fisher Scientific) following manufacturers’ instructions.

Techniques: Activation Assay, Infection, Western Blot, Isolation, Molecular Weight, Fluorescence, Activity Assay, Staining, Immunocytochemistry