Fig. 9 | Nature Communications

Fig. 9

From: Peritoneal tissue-resident macrophages are metabolically poised to engage microbes using tissue-niche fuels

Fig. 9

The mitochondrial electron transport chain is required for antimicrobial function in peritoneal tissue-resident macrophages. a Quantification of cytokines from peritoneal tissue-resident macrophage (pTRMØ) supernatant 24 h after Saccharomyces cerevisiae (S. cerevisiae) (50 μg/ml) ±dimethylmalonate (DMM,10 mM) or ±atpenin A5 (1 μM). The far-right panel shows IL-1β after 1 h post-stimulation with adenosine triphosphate (ATP, 3 mM), 23 h after S. cerevisiae. Data (n = 4 separate wells per group) represent two independent experiments. For TNF, IL-10 and KC, data were analysed by one-way ANOVA with Dunnett’s post-tests; for IL-1β, data were analysed by two-way ANOVA with Sidak’s post-tests vs vehicle control or vs no ATP as indicated (interaction p < 0.0001, ATP p < 0.0001, complex-II p < 0.0001). Error bars = mean ± SEM. b Relative expression of Il1b RNA from samples in a. c Representative photographs of pTRMØ. In c, d, zymosan-AlexaFluor488 (5 μg/ml) are green, mitochondria labelled with Mitotracker Red CMX-Ros (25 nM) are red, whereas the nuclei are labelled blue with 6-diamidino-2-Phenylindole (DAPI,125 ng/ml). The yellow box denotes mitochondria surrounding zymosan. Zymosan cores can be seen with non-specific Mitotracker Red fluorescence. d A characteristic z-stack showing mitochondria surrounding the phagolysosome. The yellow numbers denote distance from coverslip. e Quantification of the number of Mitotracker Red positive pixels expressed as percentage of total (termed mitochondrial localisation %) at different radial distances from zymosan. Data shown are 53 zymosan particles from five images. Data were analysed by a one-way ANOVA (p < 0.0001) with a linear regression post-test shown. f Box and whisker plots showing quantification of size for >800 mitochondrial units per group taken from five images of pTRMØ in the presence or absence of zymosan-AlexaFluor488. g Representative pictures (left) of microbial plates with S. cerevisiae colonies. Data shown are from S.cerevisiae cultured ± pTRMØ with atpenin A5 (1 μM) or control for 4½ h. The graph on the right shows quantification of the average microbial killing. Data are from three experiments (n = 13 per group) and were analysed by two-way ANOVA (interaction + experimental repeat p = 0.26, atpenin A5 p = 0.10). h Representative pictures (left) and quantification of data (right) as in e, but with antimycin A (1 μM) or control. Data are from four experiments (n = 18 per group) and were analysed by two-way ANOVA (interaction + experimental repeat p = 0.10, antimycin A p < 0.0001). Whiskers e–h show 10–90% of the range. White scale bars are 10 μm

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