Figure 5
Damaged mitochondria in Ft-infected lungs represent a source of DAMPs that elicit TH1-type proinflammatory cytokines. (a) Electron micrograph of mitochondria (arrows) within a white blood cell isolated from sham-inoculated lung. Mitochondria have predominantly lamellar cristae. N, nucleus. (b) Electron micrograph of white blood cell isolated from infected lung tissue showing mitochondria (arrows) with abnormal, round/dilated cristae, some forming protrusions at the mitochondrial surface, and swollen size. RBC, red blood cell. (c) Superoxide levels in the mitochondria within lung cells from uninfected (Sham) and day 6 infected mice were determined by staining isolated cells with MitoSOX Red and were quantified by flow cytometry (left panel) as % cells positive for mitochondrial ROS (MtROS) (right panel). (d) The percentage of lung cells from uninfected (Sham) and Ft-infected mice whose mitochondria exhibit dissipated membrane potentials at various time points p.i. were quantified by flow cytometry using JC-1 staining. (e) Quantitative PCR analysis for the presence of mtDNA in the cytosol of lung cells from uninfected (Sham) and Ft-infected mice was performed as described in Materials and methods. (f) Mitochondria isolated from uninfected (Sham) and Ft-infected lungs at day 6 p.i. were evaluated for their proinflammatory capacity. Wild-type C57BL/6 BMDMs (2.5×105 cells/well) were incubated with various amounts of mitochondria for 24 h. Supernatants were collected and assayed for the presence of TNF by ELISA (see also Supplementary Figure S2). Data are presented as the mean±S.E.M. from two to three independent experiments (n=6 mice per group or 12–18 mice total). *P<0.05, **P<0.01, and ***P<0.001. All results shown were subjected to one-way analysis of variance (ANOVA) with Bonferroni’s post-test.
