Fig. 5: Lipid membrane anisotropy alters the composition and function of EVs.
a, Changes in the proportion of raft lipids ((SM + cholesterol)/PC) across temporal stages of COVID-19. n = 16, 25, 19 and 18 independent patient samples for S1, S2, S3 and S4 stages, respectively. P values from two-sided Dunn’s tests are indicated. Statistical significance is indicated using a letter-based representation in which two groups sharing a common letter are not statistically different at P < 0.05. b, Membrane anisotropy (γ) in S2 and S3 EVs measured by incorporation of the fluorescent dye 1,6-diphenyl-1,3,5-hexatriene. n = 4 independent experiments. P value from a two-sided Wilcoxon rank–sum test is indicated. c, Immunoblot analysis of the PS-1 C-terminal fragment (PS-1(CTF))—the catalytic subunit of GS—indicates that PS-1 is enriched in exosomal fractions (EXO) relative to the supernatant (SUP) and plasma. Immunoblot analysis of PS-1(CTF), precursor pulmonary surfactant protein-C (pre-SP-C), mature SP-C, IL-6, caspase-3 and cleaved caspase 3 (activated) in EVs across four stages of COVID-19. Equal amounts of total proteins (30 µg) were loaded in each lane. d, Quantified levels of PS-1(CTF), SP-C, IL-6 and cleaved caspase 3 for the four temporal stages. P values from two-sided Dunn’s tests are shown. c,d, n = 3, 4, 3 and 3 independent patient samples for S1, S2, S3 and S4 stages, respectively. e, Confocal images of the uptake of PKH67-stained EVs (green) into cultured HepG2 cells after 4 h of incubation at 37 °C compared with the negative control in which PBS was added. Cell nuclei were stained with DAPI (blue). Representative images are from three independent experiments. Scale bars, 20 μm. f,g, Top 20 pathways from the Reactome pathway database (f) and KEGG pathway database (g) based on nominally significantly enriched gene sets obtained using gene set enrichment analysis (GSEA). n = 3 independent cultures for each treatment condition. h, Transition from S2 hyperinflammatory to S3 resolution phase is marked by a proportional reduction in EV membrane raft lipids (SM + cholesterol). The resultant decrease in membrane anisotropy alters the localization of PS-1 and other protein cargoes, providing EVs from distinct stages of infection with different biological properties. ECM, extracellular matrix; HC, healthy control individual; HDACs, histone deacetylases; eIFs, eukaryotic initiation factors.
