Figure 6.

Regulation of cytokine secretion by mitoROS relies on the presence of NOX2 (gp91phox). (A) Microscopic analysis of mitoROS in BMDMs from wild-type (wt) or NOX2-deficient (gp91phox^-/-) mice. Cells were either left untreated (Control) or infected with swollen A. fumigatus conidia for 2 h, 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) The abundance of mitoROS in BMDMs was determined by image analysis of MitoSOX-stained BMDMs with ImageJ software. Means of CTCF for each condition were compared. (C) MitoSOX CTCF was measured in untreated (Control) gp91phox^-/- BMDMs and those exposed to swollen conidia for 2 h. A relative CTCF was calculated in relation to an average of CTCF measured in untreated (Control) gp91phox^-/- BMDMs. (D, E) Evaluation of cytokine secretion by infected NOX2-deficient (gp91phox^-/-) BMDMs. Macrophages were infected with swollen conidia and supernatants were collected after incubation overnight. Levels of TNF-α and IL-1β in supernatants were analyzed by ELISA. No cytokines were detected in the supernatant from BMDMs (gp91phox^-/-) treated rotenone, but not treated with swollen conidia (not shown). (F) Schematic of the regulation of antifungal responses in macrophages through NOX2 complex and RET-associated mitoROS production. 1. Following infection with swollen conidia, gp91phox is recruited to the phagosomal membrane. 2. MitoROS production is increased due to RET. Activation of gp91phox is necessary for the regulation on cytokine secretion via RET-associated mitoROS. Data are from two 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), the Mann Whitney U Test (C), the t-test (D, E): ** indicates p<0.01.