Fig 1.
Electron microscopy confirms secretion of exosome-like vesicles in intra-host stages of B. malayi.
TEM images of L3 (A and B) and adult female (C) ELV preparations are shown. L3 vesicles take on a distinct morphology often reported in the literature. Adult isolations are more heterogenous and may require further optimization to achieve uniform vesicle preparation. White arrows show canonical L3 ELVs (B) and putative adult ELVs (C). This provides evidence for the release of exosome-like vesicles in the human-infective L3 stage of the parasite and much of the rest of the work we report is focused on vesicles derived from this larval stage.
Fig 2.
Particle tracking analysis reveals prolific larval Brugia exosome-like vesicle release rate.
Profile of ELVs isolated from culture media incubated with 300 L3 parasites for successive 24 hr incubations. The size distribution of L3-derived ELVs from Day 1 (left), Day 2 (center) and Day 3 (right) incubations are shown (mean ± SD). Calculated vesicle release rates are provided in tabular format. ELV rate of release and size specificity decay in a time-dependent manner in vitro. * re-scaled based on dilution for comparison to 0–24 hour (1:20) dilution.
Table 1.
Annotation of Brugia ELV proteome.
Fig 3.
Protein content of B. malayi exosome-like vesicles.
GO functional annotation of 32 proteins identified in ELVs isolated from B. malayi L3 stage parasites.
Fig 4.
Isolation of Small RNAs from larval and adult B. malayi ELV fractions.
Bioanalyzer data are shown for RNAs isolated from L3, adult male, and adult female Brugia preparations. L3 ELVs contain significant amounts of small RNAs in the 25–200 nt range (25 and 200 nt reference peaks labeled), while adult male and female vesicle preparations yield fewer RNAs. Vesicle fractions were prepared from 300 L3 and 30 adults in 24 hr culture incubations. Despite the much higher total tissue amounts used in adult culture, we detect much higher levels of small RNAs in L3-derived ELVs.
Fig 5.
Discovery and profiling of miRNAs in B. malayi exosome-like vesicles.
(A) Comparative abundance of miRNAs in L3 ELV and tissue-derived samples. miRNA discovery and abundance estimation was carried out using the mirDeep2 pipeline. The 20 miRNAs with highest expression in each sample were retained for comparison and abundance was normalized with respect to total miRNA-mapping reads within each sample. Normalized read count is plotted on a log scale for ELV and tissue miRNAs to provide a relative ordering of fractional abundance. Bma-let-7 only appears in the highly expressed subset, and a number of miRNAs with perfect mature sequence identity to host homologs are highlighted (outer blue circle). (B) Sequence conservation between B. malayi ELV-origin miRNAs and the host ( H. sapiens ) miRNA complement. Reduced heat map showing one-to-one homology between a given B. malayi miRNA and its nearest matching human counterpart in terms of percent identity. Bma-let-7, bma-miR-1, bma-miR-9, bma-miR-92, and bma-miR-100b (white asterisks) share 100% identity with a host miRNA, while bma-miR-34 shows high identity with a host miRNA (21/23 nucleotides). This B. malayi miRNA subset (shown in blue) contains potential modulators of host gene expression. (C) Sequence conservation between B. malayi ELV-origin miRNA seed sites and host ( H. sapiens ) miRNA seed sites. miRNAs sharing perfectly conserved seed sites, defined here as nucleotides 2–8 of the mature miRNA, are marked (blue circles).
Fig 6.
Brugia malayi ELV miRNA sequence homology to nematode and mammalian host miRNAs.
miRNAs from B. malayi, A. suum, C. elegans, H. sapiens, and M. musculus were grouped by seed site sequence identity (nucleotides 2–8) for multiple sequence alignments. Alignments are shown for bma-let-7, bma-miR-9 and bma-miR-993. bma-let-7 is shown as an example of a Brugia ELV miRNA that exhibits both seed site and full length sequence conservation extending to other parasitic and free-living nematodes, as well as to mammalian host species. bma-miR-9 and bma-miR-993 are presented as examples where conserved parasite miRNAs have clear host homologs, yet lack one-to-one C. elegans orthologs. The complete set of alignments can be found in S1 Fig.
Fig 7.
Comparison of the B. malayi ELV miRNA complement to miRNAs secreted by other parasitic nematodes species.
(A & B) Comparison of the 20 most abundant B. malayi ELV miRNAs with the complements of miRNAs found circulating in the serum and plasma of definitive and model mammal hosts burdened with filarial infection (Litomosoides sigmodontis [26], Dirofilaria immitis [48], Loa loa [49], Onchocerca volvulus [48, 50], and Onchocerca ochengi [49]). The D. immitis miRNAs in (A) are restricted to the 20 most abundant miRNAs, and the O. volvulus miRNAs in (B) represent the combination of two non-overlapping sets arising from separate reports. (C) Comparison of the 20 most abundant miRNAs identified in B. malayi ELVs and H. polygyrus exosomes. These analyses reveal sets of common markers and a number of miRNAs unique to each species.
Fig 8.
Brugia exosome-like vesicles (ELVs) are internalized by J774A.1 macrophages.
(A and D) J774A.1 macrophages were labeled with PKH26 (red) and counterstained with DAPI (blue) to visualize nuclei. (B and E) B. malayi L3 stage ELVs were purified from a 24 hr parasite culture and labeled with PKH67 (green). 3 × 105 J774A.1 were co-incubated with approximately 3 × 107 labeled ELVs for 6 hrs at 37°C and washed repeatedly to remove unbound ELVs. Vesicles internalized by macrophages appear diffusely throughout cytoplasm and focused in discrete puncta associated with the cell membrane. (C and F) Merged images showing internalization of parasite ELVs. All images were acquired using a using a Leica TCS SP5 X Confocal/multiphoton microscope system with 20X (A-C) or 60X (D-F) objectives. Scale bars: 10 μm (A-C) and 25 μm (D-F).
Fig 9.
Brugia exosome-like vesicles (ELVs) elicit a classically activated phenotype in J774A.1 macrophages.
(A) J774A.1 (5 × 105) were treated with approximately 4 × 108 purified L3 stage ELVs, live L3 stage parasites (10 worms) or naïve culture media (control) and supernatents collected after 48 hr. The presence of 32 cytokines/chemokines was simultaneously assayed using the Milliplex MAP Mouse Cytokine/Chemokine kit (EDM Millipore) interfaced with a Bio-Plex System (Bio-Rad) utilizing Luminex xMAP technology (Luminex). The quantification of identified cytokines is presented. The cytokine profile generated by ELV treatment is consistent with a classically activated phenotype. (B) Cytokine response to ELV treatment is compared to LPS (200 ng/mL). The close correlation of responses indicates ELV treatment generates a classically activated phenotype. (C) J774A.1 (5 × 105) were treated with high dose LPS (200 ng/mL), low dose LPS (0.003 ng/mL), ELV or naïve culture media (control) for 24 hr, supernatant collected and assayed for G-CSF using a Mouse G-CSF Quantikine ELISA kit (R&D Systems). The absence of response to low dose LPS suggests the classically activated response is not due to LPS-like contamination.