Isolated Exosomes Displayed Increasing Enrichment in GM3s with Elevating Disease Severity
Lipid analyses were conducted on exosomes isolated from the plasma of the same cohort (n = 75). The purity of isolated exosomes was validated by the enrichment in exosome-specific protein marker ALG-2-interacting protein X (Alix) (Théry et al., 2001) (Figure S5A). In line with previous reports on exosome lipid compositions (Subra et al., 2007), the isolated exosomes displayed selective enrichment of raft-associated lipids, including free cholesterol (Cho) and SMs (Figure S5B). Interestingly, we also noted exosome-specific enrichments in 20:4-BMPs, PUFA-PSs, and several GM3s relative to plasma (Figure S5B), suggesting that these lipids might partake in exosome-specific processes. In corroboration with postulations drawn based on plasma lipid profile changes, we found that GM3s were increasingly enriched in the exosomes of COVID-19 patients of elevating disease severity (Figure 5 ). Several GM3s exhibited greater than 2-fold increases in the exosomes (p < 0.05) of COVID-19 patients relative to healthy controls (Figure 5A). Progressive increases with increasing disease severity were similarly noted for GM3s of varying acyl chain lengths, e.g., very long-chain GM3 d18:0/26:0 (p = 0.0008) and GM3 d18:1/24:1 (p = 0.0015), as well as medium-chain such as GM3 d18:1/16:0 (p = 0.0017) (Figure 5B). BMP38:5(18:1/20:4) was reduced in the exosomes of COVID-19 patients compared to controls (Figure 5A). Increasing endosomal BMP content was previously found to impede intercellular transmission of HIV particles (Chapuy-Regaud et al., 2013). PUFA-PSs such as PS 40:7 and PS 40:5 were specifically increased in the exosomes of severe compared to moderate cases (Figure 5C). While plasma changes in sphingolipids, particularly GM3s, were recapitulated in exosomes, many of the changes in phospholipids (with the exception of PSs) were not observed in isolated exosomes. These observations suggested that plasma changes in glycerophospholipids associated with COVID-19 might be attributed to other components, such as lipoproteins, in the circulation.
Figure 5 Lipid Changes in Exosomes of COVID-19 Patients
(A) Lipid changes in isolated exosomes from plasma of healthy controls (n = 25) and COVID-19 patients (n = 50). Colored dots (blue and red) in volcano plot indicate lipids that were significantly different (p < 0.05) between healthy controls and COVID-19 patients. Blue dots indicate significant lipids with fold change ≥ 2 in COVID-19 patients relative to controls listed in the blue panel, while red dots indicate significant lipids with fold change < 2 in patients relative to controls (listed in red and green panels). Lipids on the right side of vertical line at x = 0 were increased in COVID-19 patients compared to controls (blue and red panels), and lipids on the left side were decreased in COVID-19 patients compared to controls (green panel).
(B) Boxplots illustrate sum of all GM3s (p = 0.0037), and three representatives, GM3 d18:0/26:0 (p = 0.0008), GM3 d18:1/16:0 (p = 0.0017), and GM3 d18:1/24:1 (p = 0.0015), that displayed increasing trends in isolated exosomes from healthy controls to COVID-19 patients of increasing severity. Exosome lipids were expressed in nanomoles of lipids per gram of total protein (nmol/g protein).
(C) Volcano plots illustrate exosome lipids that were significantly different (p < 0.05) in pairwise comparisons indicated on top of each plot. Dots corresponding to significant lipids (p < 0.05) were colored (blue and red), and lipids with fold change ≥ 2 were colored blue and listed in the blue panel accompanying each volcano plot, while lipids with fold change < 2 were colored red.
See also Figure S5.