Extended Data Fig. 6: Sepsis induces extracellular damaged mitochondria accumulation and TREM2^hi Mac1 cells eliminate cardiomyocyte-derived mitochondria during sepsis, related to Fig. 5.

a, TEM images showing vesicles in hearts of WT mice 3 days post-CLP. b, TEM images showing cardiac interstitial mitochondria vesicles (red arrowheads) of WT mice at steady state (SS) or 3 days post-CLP. Bar graph showing free mitochondria numbers per field of view (FOV). Each symbol represents a FOV. Five fields of view are randomly selected for assay from each animal (n = 3). c, Schematic illustration of the gating strategy for collecting vesicles from hearts of mice. d, Flow cytometric analysis of purified vesicles stained with the mitochondria-selective probes MitoTracker Green and MitoNIR (n = 3 per group). e, Mitochondrial membrane potential in cardiac vesicles and cultured fibroblasts as assessed by MFI levels of MitoNIR in basal conditions and in the presence of depolarizing (FCCP) or hyperpolarizing (Oligomycin) agents (n = 3-4 per group). f, Pseudo-colored representative TEM images of two mononuclear cells (grey) taking up vesicles containing mitochondria (brown) from cardiomyocytes (green) of WT mice 7 days post-CLP. g, The incorporation of cardiomyocyte-derived tdTomato protein in CD45⁺CD11b⁺F4/80⁺CD163⁺RETNLA⁺ macrophages, CD45⁺CD11b⁺F4/80⁺ (non-CD163⁺RETNLA⁺) macrophages and CD45⁺CD11b⁺F4/80^-LY6C⁺ monocytes from αMHC^Cre/+ (control) and αMHC^Cre/+:Rosa26^TdTom (Card^RED) mice at SS and 7 days post-CLP (control mice, n = 4; Card^RED SS, n = 5; Card^RED CLP 7d, n = 8). h, The incorporation of cardiomyocyte-derived mt-Dendra2 protein in CD45⁺CD11b⁺F4/80⁺CD163⁺RETNLA⁺ macrophages, CD45⁺CD11b⁺F4/80⁺(non-CD163⁺RETNLA⁺) macrophages and CD45⁺CD11b⁺F4/80^-LY6C⁺ monocytes from αMHC^Cre/+ (control) and αMHC^Cre/+:mtD2^Flox/Flox (MitoCard) mice at SS and 7 days post-CLP (control mice, n = 4; MitoCard SS, n = 6; MitoCard CLP 7d, n = 6). i, Proportions of TUNEL⁺ and TUNEL⁺cTnI⁺ cells in hearts of WT mice at SS and 3 days post-CLP (n = 6 mice per group). All scale bars are indicated in the images. Data are presented as mean ± SEM (b,e,g,h,i). Two-sided P values were determined by Mann-Whitney U test (b), or by Kruskal–Wallis with Dunn’s multiple comparison test (e,g,h,i). Results represent three (a,b,d,g-i) and two (e,f) independent experiments.

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