Fig. 5: The effect of EV depletion on intracellular LPS sensing.

a, Mice were injected with DMSO or the EV inhibitor (2.5 μg/g GW4869) on days 1 and 2, and plasma numbers of EVs were measured by NTA on day 3 (n=6). b,c, LPS levels in the cytosol of splenic myeloid cells from Casp11−/− mice pretreated with DMSO or GW4869 as described in (a) and injected i.p. with FITC-LPS on day 3 for 5 h as assessed by the LAL (b) and HEK-Blue TLR4 reporter cell (c) assays (n=7). d,e, ImageStream analysis of intracellular LPS in splenic myeloid cells isolated from Casp11−/− mice pretreated with DMSO or GW4869 as in (a) and injected with FITC-LPS on day 3 and stained with anti-CD45 and anti-FITC antibodies (d) and the quantification of intracellular FITC intensity (e). f, TEM of peritoneal lavage cells isolated from Casp11−/− mice pretreated with DMSO or GW4869 on days 1 and 2 and injected i.p. with FITC-LPS on day 3 and stained with gold-conjugated anti-FITC antibody. Arrows indicate cytosolic localization of LPS. g–i, GSDMD and caspase-11 in the liver and spleen (g; n=3) and IL-18 and IL-1β in the plasma (h,i; n=8) of WT mice pretreated with DMSO or GW4869 as in (a) and injected i.p. with LPS on day 3 for 8 h. (j–l) GSDMD, caspase-11 and β-actin in the liver and spleen (j; n=3) and IL-18 and IL-1β in the plasma (k,l; n=8) of WT mice pretreated with DMSO or GW4869 as in (a) and injected i.p. on day 3 with LPS or LPS followed by LPS-EVs 2 h later. Combined data from two or three independent experiments (a–c,h,i,k,l) or a representative experiment of two (d,e,f) or three (g,j) are shown. Each circle represents a mouse (a,b,c,h,i,k,l) or cell (e) and the horizontal lines represent the mean. Each lane represents a mouse (g,j). P values were determined by unpaired two-tailed t-test (a,b,c,e,h,i) or one-way ANOVA with Dunnett’s post-test (k,l). Scale bar: 200 nm (f). BF, brightfield.