Figures and data
HpARI family members have differing effects against IL-33 release.
A. Experimental setup for B and C.
B. Quantification of mouse IL-33 levels by ELISA in the cell-free BAL fluids of mice treated as shown in A.
C. Quantification of IL-33 by western blot of the samples shown in B.
D. EMSA assay of HpARI1, HpARI2 and HpARI3.
E. Quantification of % free DNA (i.e. low molecular weight band) in the presence of each protein in EMSA assay shown in D.
F. HpARI3:2 and HpARI2:3 fusion protein design.
G. EMSA assay of HpARI2, HpARI3:HpARI2 fusion, or HpARI2:HpARI3 fusion.
Data in B-C pooled from two repeat experiments each with 3 mice per group for a total n=6. Error bar shows SEM. ns = not significant, * = p<0.05, **** = p<0.0001.
IL-33 western blot in BAL supernatant.
Western blot for IL-33 in BAL supernatant. Mature IL-33 band at ∼18 kDa used for densitometry measurements shown in Fig 1C. Representative of 2 repeat experiments, data from both experiments pooled in Fig 1C.
HpARI2:HpARI3 chimeras indicate CCP2/3 domains are central to IL-33 amplification versus suppression.
A. IL-5 production from bone marrow cells in response to IL-2, IL-7 and freeze-thawed CMT-64 supernatants, in the presence of range of concentrations of HpARI2, HpARI3, HpARI2:3 or HpARI3:2. Data pooled from 3 biological replicates.
B. Alternaria allergen with HpARI2, HpARI3 or fusions were administered to mice, cell-free BAL fluid prepared 15 min later, and IL-33 measured by western blot.
C. Experimental set up for D-H.
D. BAL eosinophil numbers (Siglecf+CD11−CD45+) from mice treated as shown in B.
E. Eosinophil (SiglecfhiCD11−CD45+) numbers in lung tissue from mice treated as shown in B.
F. CD25 expression level on lung ILC2 (ICOS+Lin−CD45+) from mice treated as shown in B.
G. FSC mean in lung ILC2 (ICOS+Lin−CD45+) from mice treated as shown in B.
H. BAL IL-5 levels (ELISA) from mice treated as shown in B.
All in vivo data pooled from two repeat experiments each with 4 mice per group for a total n=8. Error bar shows SEM. NS= not significant, *= p<0.05 ** = p<0.01, *** = p<0.001, **** = p<0.0001.
HpARI fusion proteins show CCP1 domain determines half-life in vivo.
A. Experimental setup for B-E. HpARI2 or HpARI3:2 fusion were administered intranasally 7, 3 or 1 day prior to Alternaria allergen. Mice were culled 24 h after Alternaria administration, and BAL and lung tissue taken for analysis.
B. BAL eosinophil numbers (Siglecf+CD11−CD45+) from mice treated as shown in A.
C. Eosinophil (SiglecfhiCD11−CD45+) numbers in lung tissue from mice treated as shown in A.
D. CD25 geometric mean fluorescence intensity on lung ILC2 (ICOS+CD90+Lin−CD45+) from mice treated as shown in A.
E. FSC mean in lung ILC2 from mice treated as shown in A.
F. BAL IL-5 levels (ELISA) from mice treated as shown in A.
Data from day −7 timepoint from a single experimental repeat, all other groups pooled from 2 experiments. Total biological repeats at day 7 = 4, all other timepoints n=8. Error bar shows SEM. NS= not significant, *= p<0.05 ** = p<0.01, *** = p<0.001, **** = p<0.0001. Analysed by 2 way ANOVA with Dunnet’s post test.
Gating strategy and representative flow plots for bronchoalveolar lavage (BAL) eosinophils
A. Gating strategy for BAL cells. Cells were gated on FSC versus zombie UV viability stain to exclude debris and dead cells, then gated for all cells by FSC versus SSC, then on CD45-positive cells, and finally for SiglecF+CD11c− eosinophils.
B. Representative flow cytometry plots for BAL SiglecF+CD11c− eosinophils, gated on live CD45+ cells, as shown in (A). Data used to calculate total BAL eosinophil cell numbers shown in Figure 3B.
Representative flow plots for lung tissue cells.
Representative flow cytometry plots for lung SiglecFhiCD11c− eosinophils, gated on live CD45-positive cells. Data shown here was used to calculate total lung eosinophil cell numbers shown in Figure 3C.
Gating strategy and representative flow plots for lung tissue type 2 innate lymphoid cells (ILC2)
A. Gating strategy for lung ILC2s. Cells were gated on FSC versus zombie UV viability stain to exclude debris and dead cells, then on CD45-positive cells, lymphocytes by FSC versus SSC, and finally for CD90.2+ICOS+Lineage− ILC2s.
B. Representative histograms for CD25 expression in gated ILC2s. Samples representative of data shown in Figure 3C. Geometric Mean Fluorescence Intensity (GeoMFI) shown in brackets. CD25 fluorescence minus one (FMO) control also shown.
C. Representative histograms for FSC signal in gated ILC2s. Samples representative of data shown in Figure 3D. Median Fluorescence Intensity (MFI) shown in brackets.
HpARI proteins have variable levels of heparin sulphate binding.
A. 50 µg of HpARI1, HpARI2 or HpARI3 were added to 50 µg Heparin Sulphate (HS) and ran on a Superdex 200 Increase 10/300 GL gel filtration column. A280 trace shown.
B. Coomassie gel of HpARI1, HpARI2 or HpARI3 pull down using HS-coated beads. Input, unbound (ie supernatant from beads) and pull-down elution shown. Representative of 3 repeat experiments.
C. Flow cytometry staining of HpARI1, HpARI2 or HpARI3 tetramers with streptavidin-PE, on naive mouse lung cells gated on live CD45+ or live CD45-cells. Representatives 2 repeat experiments.
D. Isothermal calorimetry of HpARI1 +/- HS
E. Isothermal calorimetry of HpARI2 +/- HS
F. Isothermal calorimetry of HpARI3 +/- HS
Molecular modelling of HpARI2 interaction of Heparin oligosaccharide.
A. The top panel shows electrostatic surface rendering of alpha fold models of the CCP1 domains of HpARI1-3. Blue and red surfaces indicate positive and negative surfaces respectively. The lower panel shows an amino acid sequence alignment of HpARI family proteins with residues contributing to electropositive patch highlighted in cyan.
B and C. Electrostatic surface representation of an AlphaFold model of heparin tetrasaccharide docked on full-length HpARI1 (B) and HpARI2 (C). Three different docking solutions are shown. The right-hand panels show the spread of these models with heparins shown as coloured sticks on a surface representation of the CCP1 domain.
HpARI2 pentaR mutant effectively blocks IL-33 responses in vitro, but has a short half-life in vivo.
A. IL-5 production from bone marrow cells in response to IL-2, IL-7 and freeze-thawed CMT-64 supernatants, in the presence of range of concentrations of HpARI2_WT or HpARI2_pentaR. Data pooled from 3 biological replicates.
B. Coomassie gel of HpARI1, HpARI2, HpARI3 or HpARI2-pentaR (A2pentaR) pull down using heparin-coated beads. Input, unbound (ie supernatant from beads) and pull-down elution shown. Representative of 3 repeats.
C. EMSA assay of HpARI2 or HpARI2_pentaR. Representative of 2 repeats.
D. Experimental setup for C-F. HpARI2_WT or HpARI2_pentaR (10 µg of each) were administered intranasally 3 days or 1 day prior to Alternaria (Alt) allergen. Mice were culled 24 h after Alternaria administration, and BAL and lung tissue taken for analysis.
E. BAL eosinophil numbers (Siglecf+CD11−CD45+) from mice treated as shown in B.
F. Eosinophil (SiglecfhiCD11−CD45+) numbers in lung tissue from mice treated as shown in B.
G. CD25 geometric mean fluorescence intensity on lung ILC2 (ICOS+Lin−CD45+) from mice treated as shown in B.
H. BAL IL-5 levels (ELISA) from mice treated as shown in B.
Data in E-H from a single experiment, for a total of 4 biological replicates per timepoint. Error bar shows SEM. NS= not significant, *= p<0.05 ** = p<0.01, *** = p<0.001, **** = p<0.0001. Analysed by 1 way ANOVA with Dunnet’s post test, comparing each condition to Alternaria-only control.
