Figure 4.

Sensing fixed swollen A. fumigatus conidia but not zymosan or heat-killed swollen conidia induces mitoROS production via RET in BMDMs. (A) Microscopic analysis of mitoROS in BMDMs. BMDMs were left untreated (Control) or exposed to zymosan particles, heat-killed (HK) or formalin-fixed swollen conidia of A. fumigatus. Cells were stained with mitoSOX, fixed and subjected to imaging. Where indicated, BMDMs were pre-treated with rotenone (shown with yellow background) for 1 h. The scale bars are 5 µm. (B–D) The abundance of mitoROS in BMDMs was determined by image analysis of MitoSOX-stained BMDMs with ImageJ software. A relative corrected total cell fluorescence (CTCF) was calculated in relation to an average of CTCF measured in untreated (Control) BMDMs (B) or in BMDMs exposed to zymosan (C) or formalin-fixed swollen conidia (D). (E) Spectrophotometry analysis of enzyme activities of mitochondrial respiratory chain complex I (CI), complex II (CII), and citrate synthase (CS). Enzymatic activity measured in untreated BMDMs were set as 100% in each replicate. The activity of citrate synthase was measured to assess quality of mitochondria. (F, G) Evaluation of cytokine secretion in stimulated macrophages. After treatment with either vehicle or rotenone, BMDMs were exposed to zymosan particles, heat-killed (HK sw) or formalin-fixed (Fixed sw) swollen conidia of A. fumigatus. Levels of TNF-α and IL-1β in supernatants were analyzed by ELISA. Data are from two (B–D, F, G), or three (E) independent experiments. “n” indicates number of cells used for quantification; bars indicate means and standard errors, red rhomb represent means. Statistical significance was calculated with the Kruskal-Wallis test followed by Tukey post-hoc test (B), Mann Whitney U Test (C, D), or Student’s t-test (E–G): ** indicates p<0.01, and *** indicates p<0.001.