Structural basis for IL-33 recognition and its antagonism by the helminth effector protein HpARI2

Abstract

IL-33 plays a significant role in inflammation, allergy, and host defence against parasitic helminths. The model gastrointestinal nematode Heligmosomoides polygyrus bakeri secretes the Alarmin Release Inhibitor HpARI2, an effector protein that suppresses protective immune responses and asthma in its host by inhibiting IL-33 signalling. Here we reveal the structure of HpARI2 bound to mouse IL-33. HpARI2 contains three CCP-like domains, and we show that it contacts IL-33 primarily through the second and third of these. A large loop which emerges from CCP3 directly contacts IL-33 and structural comparison shows that this overlaps with the binding site on IL-33 for its receptor, ST2, preventing formation of a signalling complex. Truncations of HpARI2 which lack the large loop from CCP3 are not able to block IL-33-mediated signalling in a cell-based assay and in an in vivo female mouse model of asthma. This shows that direct competition between HpARI2 and ST2 is responsible for suppression of IL-33-dependent responses.

Fig. 1. The CCP1 domain does not contribute to the effector function of HpARI2.

a Schematic representation of the three CCP-like domains of HpARI2 (CCP1-3). b Surface plasmin resonance analysis of the binding of full-length HpARI2 (upper panel), HpARI2_CCP1/2 (middle panel) and HpARI_CCP2/3 (lower panel) (red dashed lines) to mIL-33 were generated by flowing 2-fold serial dilutions of mIL-33 starting from 40 nM (over HpARI2), 200 nM (over HpARI2_CCP2/3), and 1000 nM (over HpARI2_CCP1/2) over surfaces immobilised with HpARI2 truncations. Indicated K_D values were deduced from Langmuir fit (red dashed lines) and the experiments were conducted twice (n = 2). c Overview of the cellular assay for assessing effector function of HpARI2s. Mouse bone marrow cells were isolated and cultured with IL-2, IL-7, and IL-33 in the presence of HpARI2 truncations at various concentrations, followed by measurement of mIL-5 levels in cultured supernatants. d Concentration-response curves for the inhibition of signalling by various HpARI2 forms as determined by IL-5 production. Error bars indicate mean +/- standard error of the mean of 4 technical replicates. Data are representative of 3 repeat experiments (n = 3). Source data are provided as a Source Data file.

Fig. 2. The structure of the HpARI2_CCP2/3:IL-33 complex.

a Two views of HpARI2_CCP2/3 (CCP2 in cyan and CCP3 in blue) bound to mIL-33 (green). b The structure of HpARI2_CCP2/3 (CCP2 in cyan and CCP3 in blue) with disulphide bonds (DS1-4) shown as yellow sticks. The domain schematic on the left gives an overview of the structure with precise domain boundaries and patterns of disulphide bonds within each domain derived from the crystal structure. c Two views of the HpARI2_CCP2/3:mIL-33 complex with boxes below showing zoomed views of these interfaces. Interfacial residues are shown as sticks and hydrogen or polar interactions are indicated with a dashed line. d Binding of mIL-33 to HpARI2 variants determined by surface plasmon resonance analysis at a HpARI2 concentration of 100 nM. Bars represent the average and standard deviation for three independent measurements (n = 3). e Surface plasmon resonance analysis of HpARI2-LD binding to mIL-33. Multicycle response curves for two-fold mIL-33 dilutions starting from 2 µM (n = 2). Source data are provided as a Source Data file.

Fig. 3. HpARI2 competes for binding to IL-33 with the receptor ST2.

a Assessment of the binding of the mST2 ectodomain (orange curve) to mIL-33 (green), HpARI2:mIL-33 (blue) and HpARI2_CCP2/3:mIL-33 (cyan) in a size exclusion column. The SEC curves are representative of two independent experiments (n = 2). Coomassie-strained gels show fractions highlighted on the size exclusion traces by dotted blue lines. b Overlay of the HpARI2_CCP2/3:mL33 crystal structure on that of the mIL-33:mST2 complex (5VI4 (IL33:ST2:IL-1RAcP)), revealing a steric clash between CCP3 and ST2. ST2 is orange, mIL-33 is green and the two CCP domains of HpARI2 are cyan (CCP2) and blue (CCP3). The dotted box to the right shows a close view of the complex. c Measurement by surface plasmon resonance of the binding of mIL-33 to immobilised ST2-Fc in the presence of HpARI2 and variants. Bars show the mean from three independent measurements (n = 3) with error bars showing standard deviation. Source data are provided as a Source Data file.

Fig. 4. Assessing the effect of HpARI2 mutants on IL-33 signalling and function.

a Mouse bone marrow cells were cultured for 5 days with IL-2 (10 ng/ml), IL-7 (10 ng/ml) and IL-33 (1 ng/ml) in the presence of a range of concentrations of HpARI2 or its N69, N70, N152 or LD mutants. Levels of IL-13 (left) and IL-5 (right) in culture supernatants were measured by ELISA. Error bars indicate mean +/- SEM of 6 technical replicates per condition. Data is representative of 3 repeat experiments. b The left panel is a schematic showing that Alternaria allergen (50 μg), together with HpARI2 WT, N70 or LD mutants (10 μg) were intranasally administered to BALB/c female mice. The right depicts calculated cell numbers of SiglecF^hiCD11c^–CD45⁺ live cells in right lobes of lung. Data are pooled from 2 repeat experiments for n = 7, except for the PBS control which has n = 3. Error bars indicate mean +/- SEM. One-way ANOVA with Sidak’s multiple comparisons test was used to compare each group to Alt control ****p < 0.0001, ns = p > 0.05. Source data are provided as a Source Data file.