Figure 6. Mitochondrial alterations do not correlate with oxidative burst but are mediated by NO.

Peritoneal macrophages but not J774.16 macrophage-like cells produced ROS upon Cn infection. However pattern of ROS production did not follow the pattern of mitochondrial depolarization suggesting non mitochondrial derived ROS. As a positive control we used rotenone (Rot) which blocks mitochondrial respiratory chain, resulting in accumulation of electrons, hyperpolarization and increase in cellular ROS. A) J774.16 cells were infected for 24 h with either H99 or an acapsular strain (cap59). B) J774.16 were exposed to opsonized H₂O₂ killed Cn or zymosan, resulting in mitochondrial hyperpolarization but no ROS production. C) Peritoneal cells were exposed to opsonized H₂O₂ killed Cn or zymosan resulting in Δφm decrease and abundant ROS. D) Addition of 500 μM of SEITU prevented mitochondrial depolarization in peritoneal macrophages and J774.16 cells (not shown). Total ROS were measured by CellROX® Deep Red Reagent and Δφm was measured by the ratio of Jc-1 red/green fluorescence, using flow cytometry. Numbers represent % of cells within each region. Shaded grey represents non-infected macrophages (No Cn) and each experimental condition is shown by the red line. Each experiment was repeated twice for both J774.16 cells and peritoneal macrophages. Shown are plots of representative experiments.