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Cited by 222Parasites and Host InteractionsHelminth infection and controlExtracellular vesicles in disease

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Extracellular Vesicles from a Helminth Parasite Suppress Macrophage Activation and Constitute an Effective Vaccine for Protective Immunity

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Coakley, Gillian, McCaskill, Jana L., Borger, Jessica G., Simbari, Fabio, Robertson, Elaine, Millar, Marissa, Harcus, Yvonne, McSorley, Henry J., Maizels, Rick M., Buck, Amy H.·2017·Cell Reports
DOI 10.1016/j.celrep.2017.05.001PMC PMC5457486
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Abstract

Recent studies have demonstrated that many parasites release extracellular vesicles (EVs), yet little is known about the specific interactions of EVs with immune cells or their functions during infection. We show that EVs secreted by the gastrointestinal nematode Heligmosomoides polygyrus are internalized by macrophages and modulate their activation. EV internalization causes downregulation of type 1 and type 2 immune-response-associated molecules (IL-6 and TNF, and Ym1 and RELMα) and inhibits expression of the IL-33 receptor subunit ST2. Co-incubation with EV antibodies abrogated suppression of alternative activation and was associated with increased co-localization of the EVs with lysosomes. Furthermore, mice vaccinated with EV-alum generated protective immunity against larval challenge, highlighting an important role in vivo. In contrast, ST2-deficient mice are highly susceptible to infection, and they are unable to clear parasites following EV vaccination. Hence, macrophage activation and the IL-33 pathway are targeted by H. polygyrus EVs, while neutralization of EV function facilitates parasite expulsion.

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Graphical abstract CC BY · PubMed Central
Figure 1 H. polygyrus EVs Are Efficiently Taken up by Bone Marrow-Derived Macrophages in a Cytochalasin D-Sensitive Manner (A) Percentage uptake of 2.5 μg PKH67-labeled EVs incubated with 2.5 × 105 BMDMs, RAW264.7, or MODE-K cells from 1 to 24 hr, as assessed by flow cytometry using the gating strategy in Figure S1A. Data are presented as mean values ± SD (n = 3). (B) Confocal microscopy image of PKH67-labeled EVs incubated with BMDMs from 1 to 24 hr. DAPI is used to stain nuclei and F4/80-Alexa Fluor-647 to label macrophages. (C) Percentage uptake of 2.5 μg PKH67-labeled EVs (Hp EVs) after 1 or 24 hr in the absence or presence of 4 hr pre-treatment of BMDMs with 2 μg/mL cytochalasin D, as assessed by flow cytometry. Data are presented as mean values ± SD (n = 5). (D) Confocal microscopy image of the cells treated as in (C) using the fluorescent markers described for (B). For (B) and (D), scale bars represent 15 μm, with representative images shown.
Figure 1 H. polygyrus EVs Are Efficiently Taken up by Bone Marrow-Derived Macrophages in a Cytochalasin D-Sensitive Manner (A) Percentage uptake of 2.5 μg PKH67-labeled EVs incubated with 2.5 × 105 BMDMs, RAW264.7, or MODE-K cells from 1 to 24 hr, as assessed by flow cytometry using the gating strategy in Figure S1A. Data are presented as mean values ± SD (n = 3). (B) Confocal microscopy image of PKH67-labeled EVs incubated with BMDMs from 1 to 24 hr. DAPI is used to stain nuclei and F4/80-Alexa Fluor-647 to label macrophages. (C) Percentage uptake of 2.5 μg PKH67-labeled EVs (Hp EVs) after 1 or 24 hr in the absence or presence of 4 hr pre-treatment of BMDMs with 2 μg/mL cytochalasin D, as assessed by flow cytometry. Data are presented as mean values ± SD (n = 5). (D) Confocal microscopy image of the cells treated as in (C) using the fluorescent markers described for (B). For (B) and (D), scale bars represent 15 μm, with representative images shown.CC BY · PubMed Central
Figure 2 EV Uptake Is Modulated by Macrophage Polarization and the Presence of Specific Antibodies (A) Percentage uptake of 2.5 μg PHK67-EVs into 2.5 × 105 BMDMs that were pre-treated for 24 hr with media alone, 500 ng/mL LPS, or 20 ng/mL IL-4/IL-13, as assessed by flow cytometry using the gating strategy in Figure S1A. (B) Quantification of uptake as in (A) but comparing incubation of PHK67-EVs ± polyclonal EV antisera (1:2,000). Data were analyzed 1 hr after incubation and are presented as mean values ± SD (n = 5–7; one-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; NS, not significant). (C) Confocal microscopy of PHK67-labeled EVs incubated with BMDMs as described in (B). (D) Imagestream analysis measuring uptake of 2.5 μg CellVue-labeled H. polygyrus or RAW264/7-derived EVs ± polyclonal parasite-EV antisera or naive rat sera (both 1:2,000). BMDMs incubated with EVs/sera were also stained with LysoTracker Green (n = 5; 10,000 live-cell images taken per replicate). (E) Quantitation of cells showing high co-localization (represented as a measure of bright detail similarity) of LysoTracker with CellVue-labeled EVs in double-positive cells, as determined by IDEAS software. Unless indicated, scale bars represent 15 μm, with representative images shown.
Figure 2 EV Uptake Is Modulated by Macrophage Polarization and the Presence of Specific Antibodies (A) Percentage uptake of 2.5 μg PHK67-EVs into 2.5 × 105 BMDMs that were pre-treated for 24 hr with media alone, 500 ng/mL LPS, or 20 ng/mL IL-4/IL-13, as assessed by flow cytometry using the gating strategy in Figure S1A. (B) Quantification of uptake as in (A) but comparing incubation of PHK67-EVs ± polyclonal EV antisera (1:2,000). Data were analyzed 1 hr after incubation and are presented as mean values ± SD (n = 5–7; one-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; NS, not significant). (C) Confocal microscopy of PHK67-labeled EVs incubated with BMDMs as described in (B). (D) Imagestream analysis measuring uptake of 2.5 μg CellVue-labeled H. polygyrus or RAW264/7-derived EVs ± polyclonal parasite-EV antisera or naive rat sera (both 1:2,000). BMDMs incubated with EVs/sera were also stained with LysoTracker Green (n = 5; 10,000 live-cell images taken per replicate). (E) Quantitation of cells showing high co-localization (represented as a measure of bright detail similarity) of LysoTracker with CellVue-labeled EVs in double-positive cells, as determined by IDEAS software. Unless indicated, scale bars represent 15 μm, with representative images shown.CC BY · PubMed Central
Figure 3 H. polygyrus EVs Suppress the Onset of Alternative Activation in Macrophages (A and B) Relative expression of (A) markers of alternative activation Retnla, Ym1, and Arg1 based on qRT-PCR analysis of extracted RNA or levels of (B) RELMα, Ym1, and CCL17 proteins measured by ELISA from BMDMs treated with 20 ng/mL IL-4/IL-13 ± 5 μg/mL EVs, EV-depleted HES (sup), or total HES (HES) or media alone for 24 hr. As a comparison, cells were incubated with 5 μg/mL EVs derived from MODE-K cells (MK). (C and D) The levels of (C) RELMα, Ym1, and IL-10 by ELISA and expression of (D) CD206 by flow cytometry in BMDMs that were pre-treated with IL-4/IL-13 for 24 hr prior to the addition of 5 μg/mL EVs. (E–H) Percentage uptake of (E) 2.5 μg PHK67-labeled EVs was determined by flow cytometry, and levels of (F) RELMα, (G) Ym1, and (H) CCL17 in the supernatant were assayed from BMDMs co-treated with IL-4/IL-13 and EVs ± 2 μg/mL cytochalasin D or EV antisera (1:2,000) for 24 hr. All data are pooled from two to three independent experiments and presented as mean values ± SD (n = 5–9; one-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; NS, not significant).
Figure 3 H. polygyrus EVs Suppress the Onset of Alternative Activation in Macrophages (A and B) Relative expression of (A) markers of alternative activation Retnla, Ym1, and Arg1 based on qRT-PCR analysis of extracted RNA or levels of (B) RELMα, Ym1, and CCL17 proteins measured by ELISA from BMDMs treated with 20 ng/mL IL-4/IL-13 ± 5 μg/mL EVs, EV-depleted HES (sup), or total HES (HES) or media alone for 24 hr. As a comparison, cells were incubated with 5 μg/mL EVs derived from MODE-K cells (MK). (C and D) The levels of (C) RELMα, Ym1, and IL-10 by ELISA and expression of (D) CD206 by flow cytometry in BMDMs that were pre-treated with IL-4/IL-13 for 24 hr prior to the addition of 5 μg/mL EVs. (E–H) Percentage uptake of (E) 2.5 μg PHK67-labeled EVs was determined by flow cytometry, and levels of (F) RELMα, (G) Ym1, and (H) CCL17 in the supernatant were assayed from BMDMs co-treated with IL-4/IL-13 and EVs ± 2 μg/mL cytochalasin D or EV antisera (1:2,000) for 24 hr. All data are pooled from two to three independent experiments and presented as mean values ± SD (n = 5–9; one-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; NS, not significant).CC BY · PubMed Central
Figure 4 EVs Suppress ST2/IL-33R Expression in Macrophages during Type 2 Responses (A and B) The percentage of (A) ST2+F4/80+CD11b+ cells as determined by flow cytometry and transcriptional levels of (B) Il1rl1 as determined by qRT-PCR analysis in BMDMs cultured with IL-4/IL-13 ± 5 μg/mL EVs, EV-depleted HES (sup), or total HES (HES) for 24 hr. MODE-K EVs (Mk exo) are used as a control. Gating strategy is shown in Figure S3A. (C) The proportion of ST2-expressing BMDMs co-treated with 5 μg/mL EVs ± cytochalasin D or EV antisera, as determined by flow cytometry. (D–F) Representative expression of (D) intracellular RELMα, (E) IL-10, or (F) surface CD206 in wild-type BALB/c (WT) and T1/ST2−/− (KO) BMDMs treated with 20 ng/mL IL-4/IL-13 ± 5 μg/mL EVs, as assessed by flow cytometry. Data are pooled from two to three independent experiments and presented as mean values ± SD (n = 6–12; one-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; NS, not significant).
Figure 4 EVs Suppress ST2/IL-33R Expression in Macrophages during Type 2 Responses (A and B) The percentage of (A) ST2+F4/80+CD11b+ cells as determined by flow cytometry and transcriptional levels of (B) Il1rl1 as determined by qRT-PCR analysis in BMDMs cultured with IL-4/IL-13 ± 5 μg/mL EVs, EV-depleted HES (sup), or total HES (HES) for 24 hr. MODE-K EVs (Mk exo) are used as a control. Gating strategy is shown in Figure S3A. (C) The proportion of ST2-expressing BMDMs co-treated with 5 μg/mL EVs ± cytochalasin D or EV antisera, as determined by flow cytometry. (D–F) Representative expression of (D) intracellular RELMα, (E) IL-10, or (F) surface CD206 in wild-type BALB/c (WT) and T1/ST2−/− (KO) BMDMs treated with 20 ng/mL IL-4/IL-13 ± 5 μg/mL EVs, as assessed by flow cytometry. Data are pooled from two to three independent experiments and presented as mean values ± SD (n = 6–12; one-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; NS, not significant).CC BY · PubMed Central
Figure 5 EVs Stimulate Protective Immunity against H. polygyrus Larval Challenge in C57BL/6 Mice, and They Induce Specific Antibody Responses In Vivo (A) Age-matched female C57BL/6 mice were vaccinated with EVs, HES supernatant, total HES, or PBS in alum adjuvant, prior to challenge with 200 H. polygyrus L3 larvae. (B) Egg counts per gram fecal matter were enumerated from H. polygyrus-challenged mice on days 14, 21, and 28. (C) Adult worm counts from the small intestine on day 28 in H. polygyrus-challenged mice. Data are pooled from three experiments and presented as mean values ± SD (n = 10–18 mice per group; one-way ANOVA). (D) Titers of IgM, IgG1, IgA, and IgE detectable from sera of immunized mice measured by ELISA coated with EVs. Data are representative of two independent experiments and presented as mean values ± SD (n = 5 mice per group; one-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; NS, not significant).
Figure 5 EVs Stimulate Protective Immunity against H. polygyrus Larval Challenge in C57BL/6 Mice, and They Induce Specific Antibody Responses In Vivo (A) Age-matched female C57BL/6 mice were vaccinated with EVs, HES supernatant, total HES, or PBS in alum adjuvant, prior to challenge with 200 H. polygyrus L3 larvae. (B) Egg counts per gram fecal matter were enumerated from H. polygyrus-challenged mice on days 14, 21, and 28. (C) Adult worm counts from the small intestine on day 28 in H. polygyrus-challenged mice. Data are pooled from three experiments and presented as mean values ± SD (n = 10–18 mice per group; one-way ANOVA). (D) Titers of IgM, IgG1, IgA, and IgE detectable from sera of immunized mice measured by ELISA coated with EVs. Data are representative of two independent experiments and presented as mean values ± SD (n = 5 mice per group; one-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; NS, not significant).CC BY · PubMed Central
Figure 6 ST2-Deficient Mice Are Fully Susceptible to H. polygyrus Infection (A) Female T1/ST2−/− and wild-type BALB/c mice were infected with 200 H. polygyrus L3 larvae, and egg counts per gram fecal matter were enumerated on days 14, 21, and 28 post-infection. (B and C) Intestinal adult (B) worm burden and (C) granulomas were enumerated on day 28. (D–F) MLNs recovered from H. polygyrus-infected BALB/c and T1/ST2−/− mice were isolated at day 28 post-infection, and absolute numbers of (D) macrophages (F4/80+CD11c−MHCII+), (E) ILC2s (Lineage−CD4−ICOS+), and (F) T helper cells (CD4+Lineage+) were determined by flow cytometry. (G and H) Absolute numbers of (G) macrophages isolated from the peritoneal lavage (gated as in D) were quantified by flow cytometry, and levels of myeloid-derived cytokines (H) Ym1, RELMα, and CCL17 were assessed by ELISA. (A and B) Data are pooled from two independent experiments (n = 10); (C–H) data are representative from one of two experiments and presented as mean values ± SD (n = 5 mice per group; Student’s t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; NS, not significant).
Figure 6 ST2-Deficient Mice Are Fully Susceptible to H. polygyrus Infection (A) Female T1/ST2−/− and wild-type BALB/c mice were infected with 200 H. polygyrus L3 larvae, and egg counts per gram fecal matter were enumerated on days 14, 21, and 28 post-infection. (B and C) Intestinal adult (B) worm burden and (C) granulomas were enumerated on day 28. (D–F) MLNs recovered from H. polygyrus-infected BALB/c and T1/ST2−/− mice were isolated at day 28 post-infection, and absolute numbers of (D) macrophages (F4/80+CD11c−MHCII+), (E) ILC2s (Lineage−CD4−ICOS+), and (F) T helper cells (CD4+Lineage+) were determined by flow cytometry. (G and H) Absolute numbers of (G) macrophages isolated from the peritoneal lavage (gated as in D) were quantified by flow cytometry, and levels of myeloid-derived cytokines (H) Ym1, RELMα, and CCL17 were assessed by ELISA. (A and B) Data are pooled from two independent experiments (n = 10); (C–H) data are representative from one of two experiments and presented as mean values ± SD (n = 5 mice per group; Student’s t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; NS, not significant).CC BY · PubMed Central
Figure 7 Vaccination against EVs Is Poorly Protective in H. polygyrus-Susceptible ST2-Deficient Mice (A) Female T1/ST2−/− and BALB/c mice were vaccinated with EVs or PBS in alum adjuvant prior to challenge with 200 H. polygyrus L3 larvae (as Figure 5A), and eggs counts per gram fecal matter were enumerated on days 14, 21, and 28 post-infection. (B) Intestinal adult worm burdens were measured on day 28. (C–E) EV-specific titers of (C) IgM, (D) IgG1, and (E) IgA detectable from sera of EV- or PBS-immunized mice measured by ELISA. (A–E) Data are pooled from two independent experiments (n = 6–7), presented as mean values ± SD (Student’s t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001; NS, not significant).
Figure 7 Vaccination against EVs Is Poorly Protective in H. polygyrus-Susceptible ST2-Deficient Mice (A) Female T1/ST2−/− and BALB/c mice were vaccinated with EVs or PBS in alum adjuvant prior to challenge with 200 H. polygyrus L3 larvae (as Figure 5A), and eggs counts per gram fecal matter were enumerated on days 14, 21, and 28 post-infection. (B) Intestinal adult worm burdens were measured on day 28. (C–E) EV-specific titers of (C) IgM, (D) IgG1, and (E) IgA detectable from sera of EV- or PBS-immunized mice measured by ELISA. (A–E) Data are pooled from two independent experiments (n = 6–7), presented as mean values ± SD (Student’s t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001; NS, not significant).CC BY · PubMed Central

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Metadata

Authors
Coakley, G., McCaskill, J. L., Borger, J. G., Simbari, F., Robertson, E., Millar, M., Harcus, Y., McSorley, H. J., Maizels, R. M., & Buck, A. H.
Affiliations
  • Centre for Immunity, Infection and Evolution (GB)
  • Centre for Inflammation Research (GB)
  • Wellcome Centre for Molecular Parasitology (GB)
Corresponding author
  • Maizels, Rick M.
  • Buck, Amy H.
ORCID
Venue
Cell Reports
Publisher
Elsevier BV
Published
2017-05
Volume
19
Issue
8
Pages
1545-1557
ISSN
2211-1247 (ISSN-L)
DOI
10.1016/j.celrep.2017.05.001
PMC
PMC5457486
OpenAlex
W2618954565
Cited by
219
References
53
Citations by year
2026: 14 · 2025: 20 · 2024: 32 · 2023: 23 · 2022: 21 · 2021: 22 · 2020: 38 · 2019: 23 · 2018: 21 · 2017: 4
FWCI
20.37
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Reports (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Discovery Research Portal (University of Dundee) (publishedVersion, cc-by)
  • ENLIGHTEN (Jurnal Bimbingan dan Konseling Islam) (acceptedVersion)
  • Monash University Research Portal (Monash University) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, cc-by)
Funders
  • Biotechnology and Biological Sciences Research Council (BB/J01446X/1)
  • Wellcome Trust (AHB-097394, RMM-106122, 095831, 104111)
Topics
  • Life Sciences › Immunology and Microbiology › Parasitology › Parasites and Host Interactions
  • Life Sciences › Immunology and Microbiology › Parasitology › Helminth infection and control
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
Keywords
extracellular vesicles, Heligmosomoides polygyrus, macrophage activation, alternative macrophage activation, vaccines, nematode infection, interleukin-6, TNF-α, Ym1, RELMα, protective immunity
MeSH terms
Extracellular Vesicles, Interleukin-1 Receptor-Like 1 Protein, Animals, Antibodies, Helminth, Antibody Formation, Bone Marrow Cells, Immunity, Macrophage Activation, Macrophages, Nematospiroides dubius, Parasites, Vaccination, Vaccines, Cytochalasin D, Receptors, Interleukin, Mice

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