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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) 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.
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pntd.0004069.g009: 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.

Mentions: Macrophage activation is dichotomous; classically activated macrophages (CAMΦ) are elicited by LPS or IFN-γ and have a generally pro-inflammatory phenotype whereas alternatively activated macrophages (AAMΦ), driven by IL-4 and IL-13, appear immunosuppressive or anti-inflammatory. Helminth infection is typically associated with the AAMΦ pathway although both CAMΦ and AAMΦ are involved in the immune response to, and immunopathology caused by, Brugia infection. Experiments demonstrate different Brugia preparations can generate both CAMΦ and AAMΦ activation phenotypes; dead and moribund worms and worm lysates produce CAMΦ [60] but live worms and complete excretory/secretory (ES) preparations drive AAMΦ [61–63]. To test the hypothesis that ELVs activate host macrophages, J774A.1 were treated with purified ELV preparations and their cytokine/chemokine responses monitored. J774A.1 were treated for 48 hrs with approximately 4 × 108 L3 stage vesicles, purified from in vitro culture medium by ultracentrifugation. The macrophage response was assayed using the Milliplex MAP Mouse Cytokine/Chemokine kit (EDM Millipore) interfaced with a Bio-Plex System (Bio-Rad) utilizing Luminex xMAP technology, a platform capable of simultaneously identifying and quantifying 32 cytokines/chemokines. Vesicle treatment effectively activated J774A.1 macrophages with significant increases in G-CSF, MCP-1, IL-6 and MIP-2 levels compared to control macrophages treated with naïve RPMI 1640 culture media, (p ≤ 0.001)(Fig 9A). Smaller increases in LIX, RANTES and TNF-α were also noted. Healthy, viable L3 stage parasites produced an almost identical response (Fig 9A), the only difference being a modest but significant enhancement of G-CSF stimulation by the viable parasites (p < 0.001), suggesting that the dominant parasite immunogen(s) are found in the vesicle pellet. Finally, parasite culture media from which the ELVs had been removed by centrifugation did not produce this response, nor did live schistosomes (S. mansoni cercaria) or their secreted vesicles (S2 Fig) suggesting the Brugia-associated activation is specific to this parasite and not a general response to helminths or their secreted vesicles.


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) 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581865&req=5

pntd.0004069.g009: 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.
Mentions: Macrophage activation is dichotomous; classically activated macrophages (CAMΦ) are elicited by LPS or IFN-γ and have a generally pro-inflammatory phenotype whereas alternatively activated macrophages (AAMΦ), driven by IL-4 and IL-13, appear immunosuppressive or anti-inflammatory. Helminth infection is typically associated with the AAMΦ pathway although both CAMΦ and AAMΦ are involved in the immune response to, and immunopathology caused by, Brugia infection. Experiments demonstrate different Brugia preparations can generate both CAMΦ and AAMΦ activation phenotypes; dead and moribund worms and worm lysates produce CAMΦ [60] but live worms and complete excretory/secretory (ES) preparations drive AAMΦ [61–63]. To test the hypothesis that ELVs activate host macrophages, J774A.1 were treated with purified ELV preparations and their cytokine/chemokine responses monitored. J774A.1 were treated for 48 hrs with approximately 4 × 108 L3 stage vesicles, purified from in vitro culture medium by ultracentrifugation. The macrophage response was assayed using the Milliplex MAP Mouse Cytokine/Chemokine kit (EDM Millipore) interfaced with a Bio-Plex System (Bio-Rad) utilizing Luminex xMAP technology, a platform capable of simultaneously identifying and quantifying 32 cytokines/chemokines. Vesicle treatment effectively activated J774A.1 macrophages with significant increases in G-CSF, MCP-1, IL-6 and MIP-2 levels compared to control macrophages treated with naïve RPMI 1640 culture media, (p ≤ 0.001)(Fig 9A). Smaller increases in LIX, RANTES and TNF-α were also noted. Healthy, viable L3 stage parasites produced an almost identical response (Fig 9A), the only difference being a modest but significant enhancement of G-CSF stimulation by the viable parasites (p < 0.001), suggesting that the dominant parasite immunogen(s) are found in the vesicle pellet. Finally, parasite culture media from which the ELVs had been removed by centrifugation did not produce this response, nor did live schistosomes (S. mansoni cercaria) or their secreted vesicles (S2 Fig) suggesting the Brugia-associated activation is specific to this parasite and not a general response to helminths or their secreted vesicles.

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