Exploration of extracellular vesicles from Ascaris suum provides evidence of parasite–host cross talk

Figures & data

Figure 1. EV-like structures (range: 80–200 nm) released in culture media by Ascaris suum adult worms after three days incubation, visualized by TEM. Scale bar is indicated in the figure. EVs are purified by ultracentrifugation.

Table 1. The size distribution of particles in extracellular vesicles (EVs) samples based on three readings of each. ES EVs: Excretory/Secretory extracellular vesicles; ABF EVs: Ascaris suum body fluid extracellular vesicles; SEC: Size Exclusion Chromatogaphy; UC: ultracentrifugation. ^aSee Figure 2.

Sample name	Purification method	Number of peaks	Mean size ± SE (nm)	Mean size ± SE (nm) of main population^a	Concentration (particles/ml)
ES EVs fraction 7	SEC	5	224.3 ± 9.3	166.4 ± 16.3	6.47 · 10^10 ± 6.47 · 10^9
ES EVs fraction 8	SEC	3	216.3 ± 3.6	157.6 ± 7.6	2.71 · 10^11 ± 4.42 · 10^9
ES EVs fraction 9	SEC	3	213.2 ± 0.6	186.8 ± 9.3	2.77 · 10^11 ± 1.10 · 10^10
ES EVs fraction 10	SEC	3	198.1 ± 3.6	163.0 ± 3.2	1.37 · 10^11 ± 7.49 · 10^9
ES EVs fraction 11	SEC	3	207.0 ± 1.8	195.7 ± 42.5	6.24 · 10^10 ± 1.93 · 10^9
ES EVs fraction 12	SEC	5	203.2 ± 5.5	166.9 ± 10.7	3.97 · 10^10 ± 5.89 · 10^9
ES EVs	UC	4	257.6 ± 5.6	188.4 ± 23.1	2.84 · 10^13 ± 1.38 · 10^12
ABF EVs fraction 7	SEC	0	-	-	-
ABF EVs fraction 8	SEC	5	218.0 ± 27.0	223.9 ± 14.5	5.16 · 10^10 ± 2.86 · 10^9
ABF EVs fraction 9	SEC	9	198.9 ± 13.4	156.9 ± 3.1	5.24 · 10^10 ± 9.33 · 10^9
ABF EVs fraction 10	SEC	5	193.0 ± 7.3	165.2 ± 17.2	3.14 · 10^10 ± 1.94 · 10^9
ABF EVs fraction 11	SEC	7	157.3 ± 28.4	134.5 ± 11.6	1.61 · 10^10 ± 1.90 · 10^9
ABF EVs fraction 12	SEC	7	145.7 ± 10.2	138.3 ± 29.5	1.37 · 10^10 ± 1.33 · 10^9
ABF EVs	UC	4	177.6 ± 3.5	156.5 ± 10.2	9.03 · 10^10 ± 1.08 · 10^9

Figure 2. Finite track length adjustment (FTLA) Concentration/Size graphs for NTA analysis of particles in A-F) Size Exclusion Chromatography (SEC) fractions 7–12 purified from Ascaris suum Excretory/Secretory (ES) products, G-K) SEC fractions 8–12 purified from A. suum adult body fluid (ABF), L) ultracentrifugation pellet of A. suum ES and M) A. suum ABF.

Figure 3. Venn diagram of miRNAs associated with extracellular vesicles of Ascaris suum adults, L3 and L4 ranked according to average abundance (reads per million). miRNAs shown in red were up-regulated and miRNAs shown in blue were down-regulated. ^aAdult vs. L3, ^bAdult vs. L4.

Figure 4. Venn diagram of miRNAs from Ascaris suum adult Excretory/Secretory (E/S) products associated with extracellular vesicles (EVs) and the EV-depleted supernatant ranked according to average abundance (reads per million). miRNAs shown in blue were down-regulated in A. suum adult ES EVs. None of the common miRNAs were up-regulated.

Figure 5. Venn diagram of miRNAs associated with extracellular vesicles obtained from Ascaris suum adult Excretory/Secretory (ES) products, A. suum adult body fluid (ABF) and A. suum intestine ranked according to average abundance (reads per million). miRNAs shown in red were up-regulated and miRNAs shown in blue were down-regulated. ^aAdult vs. ABF, ^bAdult vs. Intestine.

Table 2. Predicted targets involved in immunity or inflammation of Ascaris suum micro(mi)RNAs in extracellular vesicles (EVs) or supernatant (>100 reads per million) among de-regulated genes in jejunal mucosa of pigs infected with A. suum, relative to uninfected pigs identified by microarray [11]. Abbreviations: A: Adult Excretory/Secretory (ES) EVs; L3: L3 EVs; L4: L4 EVs; ABF: A. suum body fluid; I: Intestine; S: Supernatant.

Target gene	Gene function	Targeted by (miRNA)	miRNA present in (sample)
C1QA	Component of the complement system	asu-miR-1175-5p	L4, ABF, I
CD80	Induces T-cell proliferation and cytokine production.	asu-miR-5361-5p	A, L3, L4, ABF, I, S
		asu-miR-81a	A, L3, L4, ABF, I, S
		asu-miR-81b-3p	A, L4, ABF, I, S
		asu-miR-81c-3p	ABF, I, S
		asu-miR-81c-5p	A, L3, L4
		asu-miR-9-3p	A, L3, L4, ABF, I, S
CD86	Expressed by antigen-presenting cells and facilitates T-cell activation.	asu-let-7-5p	A, L3, L4, ABF, I, S
		asu-miR-5358b-5p	L4, ABF
		asu-miR-5359-5p	A, L3, L4, ABF, I, S
IFNGR1	Encodes the ligand-binding chain (alpha) of the gamma interferon receptor.	asu-miR-71-5p	A, L3, L4, ABF, I, S
SLA-DOB	Encodes MHC class II, involved in T-cell activation.	asu-miR-57-5p	A, L3, L4, ABF, I, S
		asu-lin-4-5p	A, L3, L4, ABF, I, S
TLR7	Member of the Toll-like receptor (TLR) family.	asu-miR-9-5p	A, L3, L4, ABF, I, S
XCR1	Chemokine receptor belonging to the G protein-coupled receptor superfamily.	asu-miR-34-5p	A, L3, ABF, I, S

Figure 6. Venn diagram of proteins associated with Ascaris suum adult Excretory/Secretory (ES) extracellular vesicles (EVs), A. suum adult ES and A. suum adult ES supernatant (SN). A selection of identified proteins are listed (the full list can be found in Supplementary Table 3 (.xls)). Significant in SN, Significant in EV.

Figure 7. Venn diagram of proteins associated with Ascaris suum adult body fluid (ABF) EVs, ABF and ABF supernatant (SN). A selection of identified proteins are listed (the full list can be found in Supplementary Table 4 (.xls)). Significant in SN, Significant in EV.

Figure 8. Venn diagram of proteins associated with Ascaris suum adult Excretory/Secretory (ES) extracellular vesicles (EVs) and A. suum adult body fluid (ABF) EVs. A selection of identified proteins are listed (the full list can be found in Supplementary Table 3 (.xls) and Supplementary Table 4 (.xls)).

van Liempt E, van Vliet SJ, Engering A, et al. Schistosoma mansoni soluble egg antigens are internalized by human dendritic cells through multiple C-type lectins and suppress TLR-induced dendritic cell activation. Mol Immunol. 2007;44:2605–2615.

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