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Release of Small RNA-containing Exosome-like Vesicles from the Human Filarial Parasite Brugia malayi.

Zamanian M, Fraser LM, Agbedanu PN, Harischandra H, Moorhead AR, Day TA, Bartholomay LC, Kimber MJ - PLoS Negl Trop Dis (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Iowa State University, Ames, Iowa, United States of America; Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

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).
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pntd.0004069.g008: 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).

Mentions: 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.


Release of Small RNA-containing Exosome-like Vesicles from the Human Filarial Parasite Brugia malayi.

Zamanian M, Fraser LM, Agbedanu PN, Harischandra H, Moorhead AR, Day TA, Bartholomay LC, Kimber MJ - PLoS Negl Trop Dis (2015)

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).
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Related In: Results  -  Collection

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pntd.0004069.g008: 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).
Mentions: 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.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Iowa State University, Ames, Iowa, United States of America; Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus