A helminth-derived suppressor of ST2 blocks allergic responses
- Centre for Inflammation Research, University of Edinburgh, Queen’s Medical Research Institute, United Kingdom
- Department of Biochemistry, University of Oxford, United Kingdom
- Bioscience Asthma, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, United Kingdom
- Institute of Infection, Immunity and Inflammation, University of Glasgow, United Kingdom
- Division of Cell Signalling and Immunology, School of Life Sciences, Wellcome Trust Building, University of Dundee, United Kingdom
- Division of Microbiology & Parasitology, Department of Pathology, University of Cambridge, Tennis Court Road, United Kingdom
- The Edinburgh Protein Production Facility (EPPF), Wellcome Trust Centre for Cell Biology (WTCCB), University of Edinburgh, United Kingdom
Figures
Figure 1
HES contains a factor, distinct from HpARI, which suppresses detection of ST2.
(A–B) HpARI (5 μg) or HES (10 μg) were coadministered with 25 μg of Alternaria allergen by the intranasal route, and lung cell ST2 staining assessed 24 hr later. Geometric mean fluorescence intensity (MFI) of ST2 staining on ILC2 (ICOS+lineage–CD45+) is shown in (A), with representative histograms shown in (B). Representative of 2 replicate experiments, each with 3–5 mice per group. Error bars show SEM. (C) Naive murine lung cells were cultured for 24 hr in the presence of HES at the concentrations indicated, after which ST2 MFI on ILC2 was assessed. Data representative of >3 repeat experiments, n = 3 per group.
Figure 2 with 3 supplements
HpBARI suppresses ST2 detection, and suppresses IL-33 responses in vitro.
(A) Naive murine lung cells were cultured at 37°C overnight with HES or recombinant HpBARI, and ST2 expression measured by flow cytometry. (B–D) Naive murine bone marrow cells cultured for 5 days with IL-2, IL-7 and IL-33 (all at 10 ng/ml) followed by ELISA of cell-free supernatants for IL-5 (B), IL-6 (C) and IL-13 (D). Dotted line indicates levels with IL-2, IL-7 and IL-33 alone. All data are representative of >3 repeat experiments, with three technical replicates per measurement. Error bars show SEM.
Figure 2—figure supplement 1
Identification of HpBARI.
(A) Selection strategy for ST2-suppressive candidate. (B) Candidate ST2-suppressive proteins. (C) SDS-PAGE gel of expressed, purified HpBARI (Hp_I25642_IG17586_L548) protein. Molecular weights in kDa.
Figure 2—figure supplement 2
HpBARI DNA, protein and genomic sequence.
(A) CCP domain structure of HpBARI, aligned with HpARI, human complement factor H CCP10 (Hs CFH CCP10) and human complement receptor 1 CCP2 domain. (B) HpBARI DNA sequence aligned with nHp.2.0.scaf01207:7865–9131 from the published H. polygyrus genome (Wormbase ParaSite). (C) DNA and amino acid sequence showing exon and domain structure of HpBARI.
Figure 2—figure supplement 3
C-terminus tagged HpBARI shows no suppressive activity.
(A–C) Expressed amino acid sequence and diagrammatic representation of HpBARI expressed with N-terminal tags (HpBARI_Nterm) (A), HpBARI expressed with C-terminal tags (HpBARI_Cterm) (B) and HpBARI expressed without tags (HpARI_notag) (C). (D–F) Purified HpBARI_Nterm or HpBARI_Cterm, or unpurified Expi293 supernatants containing HpBARI_notag were added to cultures of murine bone marrow cells stimulated with IL-2, IL-7 and IL-33 (all at 10 ng/ml), and levels of IL-5 (D), IL-6 (E) and IL-13 (F) assessed in culture supernatants 5 days later.
Figure 3 with 2 supplements
HpBARI suppresses Alternaria-induced innate immune responses.
Alternaria allergen (10 μg) and HpBARI (10 μg) were intranasally administered and mice culled 24 hr later. Eosinophils were enumerated in the BAL (A) and lung (B), and intracellular cytokine staining carried out for IL-13 in ICOS+lineage–CD45+ lung ILC2s (C). IL-5 was measured in cell-free BAL supernatants (D). Data representative of 2 repeat experiments, each with 3–4 mice per group. Error bars show SEM.
Figure 3—figure supplement 1
C-terminus tagged HpBARI shows abrogated suppressive activity, while no suppression is seen with control protein administration.
Alternaria allergen (20 μg), N-terminal tagged HpBARI (HpBARI_N), C-terminal tagged HpBARI (HpBARI_C) or control protein (HpAChE) (10 μg of each recombinant protein) were intranasally administered and mice culled 24 hr later. (A–B) Eosinophils numbers in the BAL (A) or lung (B), measured by flow cytometry. (C–D) Intracellular cytokine staining carried out for IL-5 (C) and IL-13 (D) in ICOS+lineage–CD45+ lung ILC2s. (E) Representative flow cytometry plots of IL-5 vs IL-13 on gated ICOS+lineage–CD45+ cells. (F) Geometric mean fluorescence intensity of ST2 on ICOS+lineage–CD45+ lung ILC2s. (G) Representative flow cytometry plots of ST2 on gated ICOS+lineage–CD45+ cells. Data representative of at least two repeat experiments, each with 3–4 mice per group, apart from Alt+HpAChE group, which is from a single experiment. Error bars show SEM.
Figure 3—figure supplement 2
Gating strategy for lung ILC2s.
A single cell suspension of lung cells was prepared, stimulated for 4 hr in PMA, Ionomycin and Brefeldin A, and stained for flow cytometry. Gating strategy for a lung sample from an Alternaria-treated positive control sample shown. IL-5, IL-13 and ST2 staining in ICOS+lineage–CD45+ ILC2s from this experiment shown in Figure 3—figure supplement 1E and G.
Figure 4
HpBARI suppresses Alternaria-induced adaptive immune responses.
Alternaria allergen (10 μg) and OVA protein (20 μg) were coadministered in the presence or absence of HpBARI (10 μg) at day 0 (D0), and the type two immune response recalled two weeks later by three daily administrations of OVA protein, on days 14–16 (D14-16). Eosinophils were enumerated in the BAL (A) and lung (B), and intracellular cytokine staining carried out for IL-13 in ICOS+lineage–CD45+ lung ILC2s (C) and CD4+lineage+CD45+ Th cells (D). Data representative of 2 repeat experiments, each with 3–4 mice per group. Error bars show SEM.
Figure 5 with 1 supplement
HpBARI administration results in persistent suppression of ST2 in vivo.
(A–C) Alternaria allergen and HpBARI was intranasally administered and mice culled 24 hr later, as in Figure 3A–D. Representative ST2 versus lineage bivariate plots (gated on live CD45+ cells) are shown in (A). ST2 geometric mean fluorescence intensity on the surface of ICOS+lineage–CD45+ ILC2s was measured by flow cytometry (B). ST2 transcript was measured by qPCR in lung homogenates (C). PBS, Alt and Alt+HpBARI groups are representative of 2 repeat experiments, each with 3–4 mice per group; HpBARI alone group from a single experiment, n = 3. (D–E) Biotinylated HpBARI was intranasally administered 7 days, 4 days, 2 days or 1 day before Alternaria allergen, or co-administered (co-ad) with it. 24 hr after Alternaria allergen administration, BAL eosinophil (Siglecf+CD11c–) proportions of CD45+ cells (D), and mean fluorescence intensity of ST2 (E) on lung ILC2s (ICOS+Lineage–CD45+) were calculated. Representative results from two repeat experiments, each with three mice per group. Error bars show SEM.
Figure 5—figure supplement 1
HpBARI is detectable on ILC2 in vivo.
Biotinylated HpBARI was intranasally administered 7 days, 4 days, 2 days or 1 day before Alternaria allergen, or co-administered (co-ad) with it. 24 hr after Alternaria administration, single-cell suspensions of lung cells were stained for flow cytometry. (A) Gating strategy for ICOS+CD25+Lineage– ILC2s and ICOS–CD25–Lineage– non-ILC2s in CD45+ live lymphocytes. (B) Avidin-PE+ proportion of ILC2s. (C) Avidin-PE+ proportion of ICOS–CD25-Lineage– non-ILC2s. (D) Representative Avidin-PE versus ST2 bivariate plots on ICOS+CD25+Lin–CD45+ ILC2s (top row) or ICOS–CD25–Lin–CD45+ non-ILC2s. Representative results from two repeat experiments, each with three mice per group. Error bars show SEM.
Figure 6 with 1 supplement
ST2 suppression in the peritoneal lavage and lung during Heligmosomoides polygyrus infection.
Mice were infected with 200 H. polygyrus L3 larvae, and at day seven post-infection peritoneal lavage (PL) and lung cells were stained for flow cytometry of ST2 on peritoneal lavage KLRG1+CD25+lineage–CD45+ ILC2s (A, B), lung ICOS+lineage–CD45+ ILC2s (C, D), or peritoneal lavage SSChilineage–CD45+ cells (E, F). Geometric mean fluorescence intensity of ST2 is shown in A, C and E, representative ST2 histograms on gated cells is shown in B, D and F. Data from a single experiment with five mice per group. Error bars show SEM.
Figure 6—figure supplement 1
Gating strategy for mesenteric lymph node and peritoneal lavage cells from Heligmosomoides polygyrus-infected mice.
(A) Gating strategy for mesenteric lymph node (MLN) KLRG1+CD25+lineage–CD45+ ILC2s. (B) Geometric mean of ST2 on MLN KLRG1+CD25+lineage–CD45+ ILC2s. (C) Representative histograms of ST2 staining on MLN KLRG1+CD25+lineage–CD45+ ILC2s. (D) Gating strategy for peritoneal lavage (PL) SSChilineage–CD45+ and KLRG1+CD25+lineage–CD45+ ILC2s. Data from a single experiment with five mice per group. Error bars show SEM.
Figure 7 with 3 supplements
HpBARI binds to murine ST2, blocking IL-33 ligation.
(A) HpBARI or HpARI (1 μg/ml each) were coated onto wells of an ELISA plate, followed by addition of mouse ST2-Fc (ST2) (top panel) or mouse IL-33 TRAP (bottom panel) or IgG controls, followed by detection using anti-human IgG-HRP. Representative of 3 repeat experiments. Two-way ANOVA comparing HpBARI-mST2 to HpARI-mST2. Error bars show SEM. (B) Mouse ST2-Fc, mouse IL-33 TRAP or IgG were bound to protein G-coated beads, and used to immunoprecipitate HpBARI. Anti-myc western blots shown. Representative of 2 repeat experiments. (C–D) Bone marrow cells of wild type (C–D) or ST2-deficient mice (C) were cultured for 5 days with IL-2, IL-7 and IL-25 (all 10 ng/ml), then incubated with biotinylated HpARI or HpBARI (0.1 μg/ml) for 20 min at 4°C, followed by detection of biotin with avidin-PE. Representative plots gated on ICOS+lineage–CD45+ ILC2. In (D), where indicated samples were treated with HES (10 μg/ml) or unlabelled HpBARI (10 μg/ml) for 20 min prior to HpBARI staining (D). Representative of at least two repeat experiments. (E) Surface plasmon resonance of HpBARI binding to chip-coated mouse ST2 (left panel) or mouse IL-33 TRAP (right panel). (F) Anti-IL-33 western blot, after immunoprecipitation of mouse IL-33 with mouse ST2-Fc or mouse IL-33 TRAP-Fc on protein G dynalbeads, in the presence of HpBARI or heat-treated HpBARI (HT). Representative of 2 repeat experiments.
Figure 7—figure supplement 1
Gating strategy for bone marrow ILC2s.
(A) Bone marrow cells were cultured for 5 days in IL-2, IL-7 and IL-25, then treated with HES, HpARI-Bio and HpBARI-Bio as indicated in Figure 7D. Gating strategy and overlay of stained cells. (B) Inhibition of ST2 detection using anti-ST2-APC clone RM-ST2-2 (ThermoFisher) or clone DIH9 (Biolegend) on gated bone marrow ICOS+lineage–CD45+ ILC2. Representative of 3 repeats.
Figure 7—figure supplement 2
C-terminus tagged HpBARI has decreased affinity for the IL-33 receptor.
Surface plasmon resonance of HpBARI_Nterm or HpBARI_Cterm binding to immobilised IL-33-TRAP.
Figure 7—figure supplement 3
HpBARI blocks mouse IL-33-TRAP/IL-33 interaction.
Surface plasmon resonance of mouse IL-33 binding to chip-bound mouse IL-33-TRAP-Fc in the presence (grey line) or absence (black line) of HpBARI. Inset shows larger image of mIL-33 binding signal.
Figure 8 with 3 supplements
HpBARI_Hom2 binds to human ST2, blocking human IL-33 responses.
(A) HpBARI or HpBARI_Hom2 (1 μg/ml each) were coated onto wells of an ELISA plate, followed by addition of mouse ST2-Fc (mST2), human ST2-Fc (hST2) or IgG controls, followed by detection using anti-human IgG-HRP. Representative of 2 repeat experiments. Two-way ANOVA comparing hST2 to mST2 binding by HpBARI and HpBARI_Hom2. (B–C) Surface plasmon resonance of HpBARI binding to chip-coated mouse (B) or human ST2-Fc (C). (D) An oxidation-resistant mutant of human IL-33 (1 μg/ml) was coated onto wells of an ELISA plate. Human ST2-Fc was then added to the plate, either alone or after incubation with HpBARI or HpBARI_Hom2, followed by detection of IL-33 binding by ST2-Fc using anti-human IgG-HRP. Representative of 2 repeat experiments. (E) Human PBMCs were stimulated with 5 ng/ml human IL-12, 0.1 ng/ml oxidation resistant human IL-33 and HpBARI, HpBARI_Hom2 or a control protein (HpAChE) for 44 hr. IFN-γ in the supernatant was measured by ELISA with values expressed as % maximal response (IFN-γ release) where 100% is IFN-γ release in response to IL-12 and IL-33 co-stimulation, corrected for background levels in each plate/donor. The graph shows curves pooled from three donors where identical dose-response curves were performed. Two-way ANOVA compares HpBARI and HpBARI_Hom2 to control protein treatment. Error bars show SEM.
Figure 8—figure supplement 1
Alignment of HpBARI and HpBARI_Hom2 amino acid sequence, and wester blot of HpBARI, HpBARI_Hom2 and HES.
(A) Alignment of HpBARI and HpBARI_Hom2 amino acid sequences. (B) Western blot of HpBARI (50 ng), HpBARI_Hom2 (50 ng) and HES (10 μg) probed with rat anti-HpBARI polyclonal IgG, or naive rat IgG (5000 ng/ml), followed by anti-rat IgG-HRP.
Figure 8—figure supplement 2
HpBARI_Hom2 inhibits human ST2/IL-33 interaction.
(A) HpBARI_Hom2 binding to negative control of chip-bound hIgG (same concentration series as in Figure 7B–C). (B) Human IL-33 binding to chip-bound human ST2-Fc in the presence (red line) or absence (black line) of HpBARI_Hom2.
Figure 8—figure supplement 3
Stimulation of PBMC IFN-y production by IL-12 and IL-33.
Human PBMCs were stimulated with oxidation resistant IL-33, IL-12 or both, and IFNγ in the supernatant measured after 44 hr culture. Error bars show SEM.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background, Mus musculus | ST2-deficient; Il1rl1-/- | (Mangan et al., 2007) | BALB/c background | |
| Cell line (Homo sapiens) | Expi293F | ThermoFisher Scientific | Cat No: A14527 | |
| Recombinant DNA reagent | pSecTAG2A vector | ThermoFisher Scientific | Cat No: V90020 | |
| Other | Alternaria alternata allergen extract | Greer | Cat No: XPM1D3A25 | |
| Peptide, recombinant protein | Mouse IL-2 | Biolegend | Cat No: 575402 | |
| Peptide, recombinant protein | Mouse IL-7 | Biolegend | Cat No: 577802 | |
| Peptide, recombinant protein | Mouse IL-33 | Biolegend | Cat No: 580502 | |
| Peptide, recombinant protein | Mouse IL-25 | Biolegend | Cat No: 587302 | |
| Peptide, recombinant protein | Human IL-33 | Biolegend | Cat No: 581802 | |
| Peptide, recombinant protein | Mouse ST2-Fc chimera | Biolegend | Cat No: 764902 | |
| Peptide, recombinant protein | Human ST2-Fc chimera | Biolegend | Cat No: 557904 | |
| Antibody | Anti-CD3-FITC (Armenian hamster monoclonal) | Biolegend | Cat No: 100306 RRID:AB_312671 Clone: 145–2 C11 | (1:200) |
| Antibody | Anti-CD5-FITC (Rat monoclonal) | Biolegend | Cat No: 100606 RRID:AB_312735 Clone: 53–7.3 | (1:200) |
| Antibody | Anti-CD11b-FITC (Rat monoclonal) | Biolegend | Cat No: 101224 RRID:AB_755986 Clone: M1/70 | (1:200) |
| Antibody | Anti-CD19-FITC (Rat monoclonal) | Biolegend | Cat No: 115506 RRID:AB_313641 Clone: 6D5 | (1:200) |
| Antibody | Anti-CD49b-FITC (Rat monoclonal) | ThermoFisher Scientific | Cat No: 11-5971-85 RRID:AB_465327 Clone: DX5 | (1:200) |
| Antibody | Anti-GR1-FITC (Rat monoclonal) | Biolegend | Cat No: 108406 RRID:AB_313371 Clone: RB6-8C5 | (1:200) |
| Antibody | Anti-CD45-AlexaFluor700 (Rat monoclonal) | Biolegend | Cat No: 103128 RRID:AB_493715 Clone: 30-F11 | (1:200) |
| Antibody | Anti-CD45-Pacific Blue (Rat monoclonal) | Biolegend | Cat No: 103126 RRID:AB_493535 Clone: 30-F11 | (1:200) |
| Antibody | Anti-ICOS-PCP-Cy5.5 (Armenian hamster monoclonal) | Biolegend | Cat No: 313518 RRID:AB_10641280 Clone: C398.4A | (1:100) |
| Antibody | Anti-CD4-PE-Dazzle (Rat monoclonal) | Biolegend | Cat No: 100566 RRID:AB_2563685 Clone: RM4-5 | |
| Antibody | Anti-ST2-APC (Rat monoclonal) | ThermoFisher Scientific | Cat No: 17-9335-82 RRID:AB_2573301 Clone: RMST2-2 | (1:100) Used throughout manuscript. |
| Antibody | Anti-ST2-APC (Rat monoclonal) | Biolegend | Cat No: 145306 RRID:AB_2561917 Clone: DIH9 | (1:100) Used in Figure 7—figure supplement 1 only |
| Antibody | Anti-CD11c-AlexaFluor647 (Armenian hamster monoclonal) | Biolegend | Cat No: 117312 RRID:AB_389328 Clone: N418 | (1:200) |
| Antibody | Anti-SiglecF-PE (Rat monoclonal) | Miltenyi | Cat No: 130-102-274 RRID:AB_2653451 Clone: ES22-10D8 | (1:50) |
| Antibody | Anti-KLRG1-PCP-Cy5.5 (Syrian hamster monoclonal | Biolegend | Cat No: 138417 RRID:AB_2563015 Clone: 2F1 | (1:100) |
| Antibody | Anti-IL-5-PE (Rat monoclonal) | Biolegend | Cat No: 504304 RRID:AB_315328 Clone: TRFK5 | (1:200) |
| Antibody | Anti-IL-13-PE-Cy7 (Rat monoclonal) | ThermoFisher Scientific | Cat No: 25-7133-82 RRID:AB_2573530 Clone eBio13A | (1:200) |
| Peptide, recombinant protein | Streptavidin-PE | Biolegend | Cat No: 405203 | |
| Kit | Live/dead fixable blue | ThermoFisher Scientific | Cat No: L34962 |
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A helminth-derived suppressor of ST2 blocks allergic responses
eLife 9:e54017.
https://doi.org/10.7554/eLife.54017
