Extended Data Fig. 10: FcγR1 mediates mtDNA uptake followed by delivery to TLR9 and endosomal rupture.
a, Treatment and analysis scheme. 8-week-old B6 males were i.p. injected with 2 μg Biotin-Ox-mtDNA and 24 h later, BM, splenic and peritoneal pDC were purified and FC analyzed for intracellular biotin-Ox-mtDNA uptake (n = 3 independent experiments). b, IF analysis of splenic pDC that were incubated with 2 μg Biotin-mtDNA or Biotin-Ox-mtDNA for 1 h and stained for biotin (red) and TLR9 (green). Scale bars, 5 μm and 1 μm (n = 3). c, Relative fluorescence intensities of the biotin and TLR9 signals along the broken white lines in b, demonstrating (Ox-)mtDNA-TLR9 co-localization. d, FC analyses of TLR9hi cells in WT and Tlr9−/− Flt3L-induced BMDC cultures (including pDC, cDC1 and cDC2) 4 h after incubation with Ox-mtDNA (n = 3 independent experiments). e, GSEA pathway analyses of bulk RNA-seq data (n = 3). f, IF analysis of splenic pDC that were incubated with 2 μg biotin-labelled non-oxidized or oxidized mtDNA (red) for 1 h and stained for ruptured endosomes with galectin 8 (green) and TLR9 (cyan) antibodies. Scale bar, 5 μm. g, A working model illustrating how (Ox-) mtDNAs are internalized by pDC and activate TLR9-dependent NF-κB and NLRP3 inflammasome signaling (created with BioRender). Although both mtDNA forms activate TLR9 and access the cytoplasm via ruptured endosomes, only Ox-mtDNA binds NLRP3 and activates the inflammasome to produce IL-1β that potentiates NF-κB activation via IL-1R. Results in (a and d) are mean ± SD. Kruskal–Wallis test (a and d). Two-sided adaptive multilevel splitting Monte Carlo approach with Benjamini-Hochberg procedure (e). Micrographs (b, c and f) are representative of at least three independent experiments.
