Fig 1.
All B. malayi intra-mammalian life cycle stages release extracellular vesicles (EV).
(A-D) EVs were isolated from spent culture media then sized and quantified using nanoparticle tracking analysis (NTA). NTA of EV preparations from three individual 24 hr cultures of microfilariae (A), L4 (B), adult male (C) and adult female parasites (D) are shown. Particle size is in nm. (E-F) Electron micrograph of representative adult male and female EV (white arrowheads), scale bar 100 nm.
Fig 2.
B. malayi microfilariae release EV from the excretory/secretory (ES) pore.
Confocal scanning laser micrograph showing anti-Alix immunoreactivity (IR, green) focused at the ES pore (white arrowhead). Alix is frequently found in EV proteomes and considered an EV marker. Anti-Alix IR can be observed extending from the ES pore within a duct-like structure (inset). Worms were counterstained with Hoechst 33342 (nuclei, blue) and phalloidin (muscle, purple). Scale bar 20 μm.
Table 1.
Most abundant proteins in B. malayi adult EV proteome.
Fig 3.
B. malayi EV are internalized by murine macrophages.
(A-C) Confocal scanning laser micrographs showing: (A) murine J774A.1 macrophages stained with Hoechst 33342 (nuclei, blue) and phalloidin (muscle, purple); (B) macrophages following incubation with a no EV control or 1 x 107 PKH67-stained EVs (green) isolated from microfilaria (mf), L3, L4, adult male (AM) and adult female (AF) worms; and (C) an overlay of panels A and B showing EV internalization. All images captured at magnification 63X, all scale bars 10 μm. (D) Imaris 3D reconstruction of confocal micrographs showing labeled EV are internalized and not simply adhered to cell surface. All Imaris images captured at magnification 68X, all scale bars 2 μm. (E) Flow cytometry showing all treated macrophages internalized labeled parasite EVs.
Fig 4.
Murine macrophages internalize parasite-derived EV by phagocytosis.
Imaris 3D reconstructed confocal micrographs of murine J774A.1 macrophages. (A) Control macrophages showing internalization of PKH67-labeled EV (green) isolated from microfilaria (mf), L3, adult male (AM) and adult female (AF) worms in parallel with Fluoresbrite Carboxylate Microspheres (red, phagocytosis tracer). Macrophages are counterstained with Hoechst 33342 (nuclei, blue) and phalloidin (muscle, purple). (B) Macrophages treated with labeled EV (green) and microspheres (red) in the presence of 200 μM Dynasore. Absence of green and red indicates internalization of both EV and tracer are blocked. (C) Macrophages treated with labeled EV (green) and Alexa Fluor 555 conjugated transferrin (tracer, red) in the presence of 30 μM Chlorpromazine. Presence of green and absence of red indicates internalization of tracer is blocked but EV is not. (D) Macrophages treated with labeled EV (green) and Alexa Fluor 555 conjugated cholera toxin b (tracer, red) in the presence of 300 μM Genistein. Presence of green and general absence of red indicates internalization of tracer is generally blocked but EV is not. All Imaris images captured at magnification 68X, all scale bars 2 μm.
Fig 5.
Brugia EV proteome is largely stage- and sex-specific.
(A) UpSetR analysis visualizing intersections between adult male (BmAM), female (BmAF) and previous L3 (BmL3) EV proteome datasets. Datasets participating in intersections are shown as filled (black) circles, the number of unique proteins in that intersection is presented in the histogram above. For example, 59 EV proteins are unique to BmAF but 11 are shared between BmAF and BmAM only. (B) Extended UpSetR analysis to include previously published B. malayi secretomes. Secretome appellation reflects lead authors Hewitson (Hsec)(12), Moreno and Geary (MGsec)(14), and Bennuru (Bsec)(14).
Fig 6.
Brugia EV proteome contains markers of exosome biogenesis.
(A-B) Gene Ontology (GO) analysis of adult male (A) and female (B) EV proteomes summarizing cellular location GO terms, including terms associated with the endosomal pathway. (C-D) GO analysis of adult male (C) and female (D) EV proteomes summarizing molecular function GO terms.
Fig 7.
Ivermectin inhibits EV release from filarial nematodes.
(A) 1 μM IVM reduces EV release by B. malayi in vitro. Microfilaria (mf), L3, adult male (AM) and female (AF) were incubated in RPMI containing IVM or vehicle control. Media was collected after 24 hr and EV isolated and quantified. N = 3 (minimum), mean ± SEM, *P<0.05, **P<0.01, ****P<0.0001. (B) 1 μM IVM reduces EV release in vitro by IVM susceptible Missouri strain (MO) D. immitis L3 but not IVM reduced susceptibility JYD-34 strain. N = 3 (minimum), mean ± SEM, **P<0.01, ns not significant.
Fig 8.
Filarial nematodes release EVs that are relevant at the host parasite interface.
Our model proposes that B. malayi (and D. immitis) release EV from structures including the excretory pore. These EV (magnified) are of a size and morphology consistent with exosomes but lack some canonical vertebrate exosome markers. They do, however, contain immunomodulatory effector proteins and are phagocytosed by vertebrate cells such as macrophages and potentially other relevant cell types (pictured), providing a mechanism by which these nematodes may deliver effector molecules and manipulate the host.