A helminth-derived suppressor of ST2 blocks allergic responses

  1. Francesco Vacca
  2. Caroline Chauché
  3. Abhishek Jamwal
  4. Elizabeth C Hinchy
  5. Graham Heieis
  6. Holly Webster
  7. Adefunke Ogunkanbi
  8. Zala Sekne
  9. William F Gregory
  10. Martin Wear
  11. Georgia Perona-Wright
  12. Matthew K Higgins
  13. Josquin A Nys
  14. E Suzanne Cohen
  15. Henry J McSorley  Is a corresponding author
  1. Centre for Inflammation Research, University of Edinburgh, Queen’s Medical Research Institute, United Kingdom
  2. Department of Biochemistry, University of Oxford, United Kingdom
  3. Bioscience Asthma, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, United Kingdom
  4. Institute of Infection, Immunity and Inflammation, University of Glasgow, United Kingdom
  5. Division of Cell Signalling and Immunology, School of Life Sciences, Wellcome Trust Building, University of Dundee, United Kingdom
  6. Division of Microbiology & Parasitology, Department of Pathology, University of Cambridge, Tennis Court Road, United Kingdom
  7. The Edinburgh Protein Production Facility (EPPF), Wellcome Trust Centre for Cell Biology (WTCCB), University of Edinburgh, United Kingdom
8 figures, 1 table and 1 additional file

Figures

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+lineageCD45+) 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+lineageCD45+ 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+lineageCD45+ lung ILC2s. (E) Representative flow cytometry plots of IL-5 vs IL-13 on gated ICOS+lineageCD45+ cells. (F) Geometric mean fluorescence intensity of ST2 on ICOS+lineageCD45+ lung ILC2s. (G) Representative flow cytometry plots of ST2 on gated ICOS+lineageCD45+ 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+lineageCD45+ ILC2s from this experiment shown in Figure 3—figure supplement 1E and G.

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+lineageCD45+ 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+lineageCD45+ 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+LineageCD45+) 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 ICOSCD25Lineage non-ILC2s in CD45+ live lymphocytes. (B) Avidin-PE+ proportion of ILC2s. (C) Avidin-PE+ proportion of ICOSCD25-Lineage non-ILC2s. (D) Representative Avidin-PE versus ST2 bivariate plots on ICOS+CD25+LinCD45+ ILC2s (top row) or ICOSCD25LinCD45+ 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+lineageCD45+ ILC2s (A, B), lung ICOS+lineageCD45+ ILC2s (C, D), or peritoneal lavage SSChilineageCD45+ 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+lineageCD45+ ILC2s. (B) Geometric mean of ST2 on MLN KLRG1+CD25+lineageCD45+ ILC2s. (C) Representative histograms of ST2 staining on MLN KLRG1+CD25+lineageCD45+ ILC2s. (D) Gating strategy for peritoneal lavage (PL) SSChilineageCD45+ and KLRG1+CD25+lineageCD45+ 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+lineageCD45+ 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+lineageCD45+ 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
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background, Mus musculusST2-deficient; Il1rl1-/-(Mangan et al., 2007)BALB/c background
Cell line (Homo sapiens)Expi293FThermoFisher ScientificCat No: A14527
Recombinant DNA reagentpSecTAG2A vectorThermoFisher ScientificCat No: V90020
OtherAlternaria alternata allergen extractGreerCat No: XPM1D3A25
Peptide, recombinant proteinMouse IL-2BiolegendCat No: 575402
Peptide, recombinant proteinMouse IL-7BiolegendCat No: 577802
Peptide, recombinant proteinMouse IL-33BiolegendCat No: 580502
Peptide, recombinant proteinMouse IL-25BiolegendCat No: 587302
Peptide, recombinant proteinHuman IL-33BiolegendCat No: 581802
Peptide, recombinant proteinMouse ST2-Fc chimeraBiolegendCat No: 764902
Peptide, recombinant proteinHuman ST2-Fc chimeraBiolegendCat No: 557904
AntibodyAnti-CD3-FITC (Armenian hamster monoclonal)BiolegendCat No: 100306
RRID:AB_312671
Clone: 145–2 C11
(1:200)
AntibodyAnti-CD5-FITC (Rat monoclonal)BiolegendCat No: 100606
RRID:AB_312735
Clone: 53–7.3
(1:200)
AntibodyAnti-CD11b-FITC (Rat monoclonal)BiolegendCat No: 101224
RRID:AB_755986
Clone: M1/70
(1:200)
AntibodyAnti-CD19-FITC (Rat monoclonal)BiolegendCat No: 115506
RRID:AB_313641
Clone: 6D5
(1:200)
AntibodyAnti-CD49b-FITC (Rat monoclonal)ThermoFisher ScientificCat No: 11-5971-85
RRID:AB_465327
Clone: DX5
(1:200)
AntibodyAnti-GR1-FITC (Rat monoclonal)BiolegendCat No: 108406
RRID:AB_313371
Clone: RB6-8C5
(1:200)
AntibodyAnti-CD45-AlexaFluor700 (Rat monoclonal)BiolegendCat No: 103128
RRID:AB_493715
Clone: 30-F11
(1:200)
AntibodyAnti-CD45-Pacific Blue (Rat monoclonal)BiolegendCat No: 103126
RRID:AB_493535 Clone: 30-F11
(1:200)
AntibodyAnti-ICOS-PCP-Cy5.5 (Armenian hamster monoclonal)BiolegendCat No: 313518
RRID:AB_10641280
Clone: C398.4A
(1:100)
AntibodyAnti-CD4-PE-Dazzle (Rat monoclonal)BiolegendCat No: 100566
RRID:AB_2563685
Clone: RM4-5
AntibodyAnti-ST2-APC (Rat monoclonal)ThermoFisher ScientificCat No: 17-9335-82
RRID:AB_2573301 Clone: RMST2-2
(1:100)
Used throughout manuscript.
AntibodyAnti-ST2-APC (Rat monoclonal)BiolegendCat No: 145306
RRID:AB_2561917 Clone: DIH9
(1:100)
Used in Figure 7—figure supplement 1 only
AntibodyAnti-CD11c-AlexaFluor647 (Armenian hamster monoclonal)BiolegendCat No: 117312
RRID:AB_389328 Clone: N418
(1:200)
AntibodyAnti-SiglecF-PE (Rat monoclonal)MiltenyiCat No: 130-102-274
RRID:AB_2653451
Clone: ES22-10D8
(1:50)
AntibodyAnti-KLRG1-PCP-Cy5.5 (Syrian hamster monoclonalBiolegendCat No: 138417
RRID:AB_2563015 Clone: 2F1
(1:100)
AntibodyAnti-IL-5-PE (Rat monoclonal)BiolegendCat No: 504304
RRID:AB_315328
Clone: TRFK5
(1:200)
AntibodyAnti-IL-13-PE-Cy7 (Rat monoclonal)ThermoFisher ScientificCat No: 25-7133-82
RRID:AB_2573530
Clone eBio13A
(1:200)
Peptide, recombinant proteinStreptavidin-PEBiolegendCat No: 405203
KitLive/dead fixable blueThermoFisher ScientificCat No: L34962

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  1. Francesco Vacca
  2. Caroline Chauché
  3. Abhishek Jamwal
  4. Elizabeth C Hinchy
  5. Graham Heieis
  6. Holly Webster
  7. Adefunke Ogunkanbi
  8. Zala Sekne
  9. William F Gregory
  10. Martin Wear
  11. Georgia Perona-Wright
  12. Matthew K Higgins
  13. Josquin A Nys
  14. E Suzanne Cohen
  15. Henry J McSorley
(2020)
A helminth-derived suppressor of ST2 blocks allergic responses
eLife 9:e54017.
https://doi.org/10.7554/eLife.54017