Lymphatic filariasis (LF) is a socio-economically devastating mosquito-borne Neglected Tropical Disease caused by parasitic filarial nematodes. The interaction between the parasite and host, both mosquito and human, during infection, development and persistence is dynamic and delicately balanced. Manipulation of this interface to the detriment of the parasite is a promising potential avenue to develop disease therapies but is prevented by our very limited understanding of the host-parasite relationship. Exosomes are bioactive small vesicles (30–120 nm) secreted by a wide range of cell types and involved in a wide range of physiological processes. Here, we report the identification and partial characterization of exosome-like vesicles (ELVs) released from the infective L3 stage of the human filarial parasite Brugia malayi. Exosome-like vesicles were isolated from parasites in culture media and electron microscopy and nanoparticle tracking analysis were used to confirm that vesicles produced by juvenile B. malayi are exosome-like based on size and morphology. We show that loss of parasite viability correlates with a time-dependent decay in vesicle size specificity and rate of release. The protein cargo of these vesicles is shown to include common exosomal protein markers and putative effector proteins. These Brugia-derived vesicles contain small RNA species that include microRNAs with host homology, suggesting a potential role in host manipulation. Confocal microscopy shows J774A.1, a murine macrophage cell line, internalize purified ELVs, and we demonstrate that these ELVs effectively stimulate a classically activated macrophage phenotype in J774A.1. To our knowledge, this is the first report of exosome-like vesicle release by a human parasitic nematode and our data suggest a novel mechanism by which human parasitic nematodes may actively direct the host responses to infection. Further interrogation of the makeup and function of these bioactive vesicles could seed new therapeutic strategies and unearth stage-specific diagnostic biomarkers.

Lymphatic filariasis is caused by parasitic nematodes that invade and occupy the host lymphatic system. The extent of lymphatic filariasis is staggering, with over 120 million people infected in 73 endemic countries and an estimated 40 million people suffering from a range of disfiguring and debilitating clinical manifestations of this disease. The mechanisms by which these medically important parasites navigate the host immune response to establish infection are not yet fully understood. In this study, we identify exosome-like vesicles (ELVs) that are abundantly released from infective stage L3 Brugia malayi, an etiological agent of human lymphatic filariasis. We show that these vesicles have a narrow size distribution and morphology consistent with classical exosomes, and that they contain common exosomal protein markers, putative effector proteins, as well as small regulatory RNAs. We show that ELVs are enriched with microRNAs that are perfectly conserved between parasite and host, suggesting a potentially novel mechanism by which filarial worms can actively manipulate host gene expression. We demonstrate that parasite ELVs are internalized by macrophages and elicit a classically activated phenotype in these host cells. The discovery of exosome-like vesicle release by human nematode parasites newly enlightens the roadmap to understanding the pathology of LF and related helminthiases. These vesicles also present promising new targets for intervention and diagnostics.

Funding: This work was supported by National Institute of Food and Agriculture Award 1001501 to MJK. URL: www.csrees.usda.gov . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2015 Zamanian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Here we show that larval and adult stage B. malayi secrete prodigious quantities of extracellular vesicles in vitro whose size and morphology are consistent with exosomes. These exosome-like extracellular vesicles (ELVs) contain small RNA species, including specific miRNA and are enriched in miRNA that are identical to host miRNAs with known immunomodulatory roles [ 31 – 34 ]. The protein cargo of the vesicles is relatively scant but includes bioactive proteins, proteins with putative RNA binding properties and proteins commonly associated with exosomes [ 35 ]. The parasite ELVs are internalized by host macrophages and elicit a classically activated phenotype in these cells. The demonstration that filarial nematodes secrete exosomal RNA and proteins that potentially function at the host-parasite interface is significant. Defining this parasite effector toolkit exposes an array of new molecules that may be exploited in novel LF control strategies.

Exosomes are a subtype of extracellular vesicle categorized by size (30–120 nm diameter) and defined by a particular biogenic pathway [ 23 ]; exosomes are formed by inward budding of vesicles in the late endosomal pathway to create multivesicular endosomes that fuse with the plasma membrane to effect release [ 24 , 25 ]. Originally thought to be a means of cellular waste disposal, exosomes are now considered highly bioactive extracellular vesicles that facilitate cell-to-cell communication and are the focus of renewed investigation. The cargo of exosomes is complex and variable, containing bioactive proteins, functional mRNA, miRNA and other small non-coding RNA species [ 18 , 26 ], likely reflecting both source and target environments. Fusion of the exosome to a target cell delivers this heterogeneous bioactive cargo and selectively alters the biology of the target tissue [ 19 , 21 , 26 , 27 ]; the isolation of exosomes from circulatory systems and an array of biofluids suggests effector sites can be far from the point of release. Parasites are known to release exosome-like vesicles [ 27 – 30 ] and it is compelling to hypothesize that bioactive molecules secreted by parasitic nematodes, packaged in exosomes, function as cell-to-cell effectors in the host-parasite interaction. Indeed recently, extracellular vesicles secreted by the gastrointestinal nematode Heligmosomoides polygyrus, containing proteins and small RNA species, have been shown to alter gene expression in host cells and suppress innate immune responses in mice [ 26 ].

The parasitic filarial nematodes Wuchereria bancrofti, Brugia malayi and B. timori are etiological agents of Lymphatic filariasis (LF), a chronic and debilitating disease infecting over 120 million people in 73 endemic countries [ 1 ]. Adult parasites reside in the lymphatic vasculature of infected individuals and release larvae called microfilariae, which are taken up by vector mosquitoes during the blood meal. Parasites rapidly develop within the mosquito, molting twice to the infective L3 stage [ 2 , 3 ] before transmission to the definitive host during a subsequent blood meal. Following penetration of the vertebrate host via the puncture wound left by the mosquito, L3 stage parasites migrate to the lymphatics and undergo further growth and development, molting to the L4 stage and again to adulthood. The longevity of patent infection is remarkable; adults live for at least 8–10 years by general consensus. The ability of larval stages to successfully invade the host, and for adult worms to maintain infection for such an extended period of time, suggest filarial worms have developed strategies to both facilitate the establishment of infection and evade or manipulate the host immune response. Although the immunomodulatory capabilities of infecting larval and adult stage filarial worms have been well documented and reviewed [ 4 – 8 ], the parasite effector molecules responsible for manipulating host biology and their mechanisms of release have been difficult to define. Actively secreted proteins have historically been considered the principal candidates and several secreted proteins have been identified with demonstrable bioactivity at the host-parasite interface [ 9 – 12 ]. Adding to these, the characterization of parasitic nematode secretomes has revealed a complex array of potential proteinaceous effectors [ 13 – 16 ]. Other types of effector, including molecules expressed on the parasite surface may have a role [ 17 ] and the emergence of small noncoding RNAs as cell-to-cell agents of genetic regulation [ 18 – 22 ] hint at exciting alternative mechanisms.

Results and Discussion

Infective-stage B. malayi release exosome-like vesicles In order to ascertain whether exosomes are released by B. malayi, extracellular vesicles were isolated from parasites incubated in culture media using a filtration and ultracentrifugation protocol. We focused our initial discovery efforts on larval and adult stage parasites. L3, adult male, and adult female B. malayi were incubated in vitro for 24 hour periods under standard culture conditions, and purified vesicle preparations were evaluated with electron microscopy (EM). Infectious stage L3 parasites in culture release abundant 50–120 nm microvesicles consistent with the classical “deflated ball” morphology of mammalian and non-mammalian exosomes reported in the literature [36] (Fig 1A & 1B). We refer to these as exosome-like vesicles (ELVs) throughout this manuscript, in recognition that they cannot be unequivocally designated as exosomes, rather than another class of extracellular vesicles, because their biogenesis has not been determined. Preparations from adult stage B. malayi were more heterogenous and dilute, not allowing for the definitive categorization of putative exosome-like vesicles (Fig 1C). This, despite the fact a much higher mass of total parasite tissue was used for adult preparations as compared to larval preparations. These data suggest ELV release to be a predominantly larval phenomenon in B. malayi, a working hypothesis supported by analysis of RNA associated with the vesicles. We therefore chose to focus our subsequent experiments on L3 stage parasites. A compelling overall hypothesis for the function of B. malayi ELVs is that they mediate the secretion and trafficking to host cells of effector molecules that facilitate parasitism and the observation that ELV secretion occurs primarily in those parasite stages that infect the host and establish parasitemia is consistent with this narrative. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pntd.0004069.g001

Time course profile of exosome-like vesicle release from infectious stage B. malayi To more accurately resolve the dynamics of ELV release in L3 B. malayi, we used a nanoparticle tracking analysis (NTA) system to measure vesicle output in a 72 hr in vitro time course. Media was collected from 300 worms after three successive 24 hr incubation periods, vesicles were purified by ultracentrifugation as before and individual vesicle preparations were analyzed via NanoSight LM10 as shown in Fig 2 (sample recording: S1 Video). Day 1 (0–24 hr in culture) preparations reveal a prolific ELV release rate (> 9,000 ELVs/parasite/min) with a very narrow size distribution centered at ∼90 nm. Day 2 (24–48 hr in culture) preparations show an essentially equivalent rate of release, but a stark broadening of the size distribution. Day 3 (48–72 hr in culture) preparations are associated with significantly lower levels of release (<4,000 ELVs/parasite/min) and an even wider multimodal size distribution. These data suggest an overall time-dependent decay in vesicle rate of release and size specificity, which correlates to decreased L3 viability in vitro. The release of considerable quantities of precisely-sized ELVs in viable worms (Days 1–2) is followed by the release of smaller quantities of a broader size range of particles that potentially include larger membrane vesicles and apoptotic blebs (Days 2–3). This suggests an active and regulated mechanism of ELV release in healthy and viable L3 stage parasites, as opposed to a passive mode of noisy cellular deterioration. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pntd.0004069.g002

The protein cargo of Brugia exosome-like vesicles The protein content of B. malayi ELVs was determined using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC/MS/MS). A total of 32 proteins each containing at least two unique peptides were identified using MASCOT (Table 1). Specific proteins identified within the pellet included characteristic markers of exosomes including Hsp70, elongation factor-1α, elongation factor-2, actin, and Rab-1. In addition, over 80% of the proteins identified are orthologous to proteins identified in mammalian exosome proteomes, strongly suggesting that these vesicles are exosome-like in nature and supporting our ELV designation here. Interestingly, this set of vesicle-specific proteins is entirely distinct from the proteins previously identified in pre- and post-molt L3 secretions [37]. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Annotation of Brugia ELV proteome. https://doi.org/10.1371/journal.pntd.0004069.t001 UniProt-GOA and quickGO were used to sort proteins into functional groups based on assigned gene ontology (GO) terms [38, 39], as shown in Fig 3. Based on GO annotations, 20% of the proteins identified are involved in binding of bioactive molecules including nucleic acids and other proteins, 16% function in the transport of various ions and proteins and 14% are ribosomal proteins. In addition, a large fraction of proteins identified (21%) appear to be involved in various metabolic processes including hydrolase and transferase activities while the remaining 29% comprises proteins with translational, cytoskeletal and other functions. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pntd.0004069.g003 Included in the list of Brugia ELV proteins are potential effector molecules. Bm-CPL-1 is a cathepsin L-like cysteine protease robustly expressed across the B. malayi life cycle [40]. Upregulation of Bm-cpl-1 expression coincides with transition between life cycle stages and an important role in the modulation of parasite molting has been confirmed [41–43]. This is the first demonstration that B. malayi secretes CPL-1 although other cathepsin-like cysteine proteases have been identified in the B. malayi secretome [14, 37] and a cathepsin L-like molecule is secreted by intra-mammalian stage Haemonchus contortus [44]. The exogenous function of exosomal Bm-CPL-1 is not clear but evidence points to some manipulation of the host-parasite interface. In a previous study, we suppressed Bm-cpl-1 expression using in vivo RNAi during the mosquito life stages [42]. Loss-of-function reduced prevalence of infection in mosquitoes by nearly 40%, suggesting Bm-CPL-1 is important for establishing or maintaining parasitemia. In flatworms, an immunomodulatory role for secreted cathepsin L-like proteases is better established [45]; in Fasciola infection cathepsin L contributes to the permissive polarized Th2 > Th1 host response. The proteomic profiles of parasitic helminth exosomes are broad in range; for example, over 350 proteins were identified in the putative exosomes secreted by Heligmosomoides polygyrus [26] whilst 45 and 79 proteins were identified in exosome-like vesicles from Echinostoma caproni and Fasciola hepatica, respectively [46]. The B. malayi L3 stage profile identified here is relatively scant but consistent with this broad distribution. It may be that this is a stage-specific observation and ELV secreted by other B. malayi life stages display a more complex and abundant protein cargo tailored to distinct functional demands. Reflecting the small RNA component of these ELVs (see later sections), it may also be that larval stage Brugia ELVs are primarily vehicles for protected RNA secretion. Replication of the experiments conducted here might add depth to the MS data set and identify further ELV-associated proteins.

Brugia ELVs are internalized by host macrophages Macrophages are critical mediators of the early immune response to invasive Brugia parasites [8]. To test the hypothesis that secreted Brugia ELVs interact with host macrophages, we used fluorescent lipophilic dyes to visualize the interaction between J774A.1 murine macrophages and ELVs. This cell line was chosen because it is commercially available, can be cultured readily and because it recapitulates the biology of primary macrophages and dendritic cells [57]. ELVs were labeled with PKH67, a green fluorescent dye, and incubated with J774A.1 labeled with PKH26, a red fluorescent dye. Confocal microscopy revealed efficient internalization of the ELVs by this macrophage cell line (Fig 8). Internalization was observed diffusely throughout the cell cytoplasm with focus around membrane-rich puncta associated with the surface of the macrophages (Fig 8B). This pattern of internalization is consistent with other studies describing a phagocytic route of vesicle internalization [58, 59]. Macrophages were counterstained with DAPI to determine the efficiency of cell labeling and ELV uptake. PKH26-labeling of J774A.1 was very efficient and all cells were visualized although intensity of labeling was variable (Fig 8D). Approximately 40–50% of macrophages internalized labeled ELVs to some degree (Fig 8E) with approximately 10% of macrophages internalizing ELVs at markedly higher levels (Fig 8E). There was no correlation between strong PKH 26-labelling of macrophages and vesicle uptake indicating internalization is not a factor of receptiveness to labeling. PPT PowerPoint slide

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larger image TIFF original image Download: 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). https://doi.org/10.1371/journal.pntd.0004069.g008