Fig 2: HES Suppression of IL-33(A) IL-33 levels (ELISA) in supernatants of naive murine lung cells (1 × 105 per well), cultured for 1 hr with Alternaria (Alt) allergen (200 μg/ml) and HES (10 μg/ml).(B) Propidium iodide (PI) and annexin V (AnnV) staining of cells from (A) was used to assess apoptosis (PI–AnnV+) versus necrosis (PI+AnnV+).(C) IL-33 levels (ELISA) in supernatants of naive murine lung cells, freeze-thawed in the presence of HES.All data shows SEM of 2–3 replicates, and are representative of 2–3 repeat experiments. Error bars show SEM.

Fig 3: HpARI Blocks IL-33-ST2 Interactions and Inhibits IL-33 Release(A) IL-33 western blot (non-reducing) of ST2-Fc fusion protein immunoprecipitation of recombinant murine IL-33 in the presence or absence of HpARI.(B) Lung ILC2 IL-5 production 24 hr after intranasal administration of recombinant murine IL-33 (200 ng/mouse) with 5 μg HpARI.(C) Lung ILC2 IL-5 production from mice described in (B).(D) Murine IL-33 levels (ELISA) in BAL 15 min after Alternaria allergen and HpARI were intranasally administered.(E) Murine IL-33 western blot (∼20 kDa band and densitometry analysis) of BAL from mice described in (D).(F) Human IL-33 levels (ELISA) in supernatants of human lung explants cultured for 1 hr with HpARI.(G) Human IL-33 western blot (∼20 kDa band and densitometry analysis) of supernatants from human lung explants cultures described in (F).(H) Human IL-33 levels (ELISA) in BAL fluid of human IL-33-transgenic mice, 30 min after Alternaria allergen and HpARI intranasal administration.(I) Human IL-33 western blot (∼20 kDa band and densitometry analysis) of BAL from human IL-33-transgenic mice described in (H).Mouse data (A–E, H–I) representative of 2–4 repeat experiments, each with 3–4 mice per group. Human data (C and D) shows 5 independent subjects. Error bars show SEM.

Fig 4: HpARI Binds Active Murine and Human IL-33(A) Murine IL-33 western blot (non-reducing) of HpARI immunoprecipitation of mouse lung homogenates, using anti-c-Myc antibody, or MOPC isotype control (iso).(B) Human IL-33 western blot (non-reducing) of HpARI immunoprecipitation of human lung homogenates, as in (A).(C) Characterization of the interaction of mouse IL-33 (mIL-33) with HpARI by surface plasmon resonance (SPR - BIAcore T200). Reference corrected single kinetic titration SPR binding curves (black), and a globally fitted 1:1 kinetic binding model (grey).(D) Characterization by SPR of the interaction of human IL-33 (hIL-33) with HpARI, as in (C).(E) IL-33 levels (ELISA) in supernatants of freeze-thawed murine lung cells, incubated at 37°C for 0, 1, 2, or 4 hr, before addition of 1 μg/ml HpARI, and a further incubation for 1 hr at 37°C.(F) Untreated or oxidized recombinant murine IL-33 immunoprecipitated with HpARI as in (A).(G) Untreated or oxidized recombinant human IL-33 immunoprecipitated with HpARI as in (B).(H) Immunoprecipitation experiments repeated with recombinant murine IL-1α, and probed with anti-murine IL-1α.Arrows indicate specific IL-33 or IL-1α bands, and IL-33 reduced (“red”) or oxidized (“ox”) bands. All data are representative of at least two independent repeats. Error bars show SEM.

Fig 5: Identification and Bioinformatic Characterization of HpARI Sequence and Structure(A) IL-33 suppression by HES size fractions.(B) IL-33 suppression by HES charge fractions.Data in (A) and (B) are percentage suppression of the IL-33 signal compared to Alternaria-only control. Dotted rectangles indicate peaks used for selection of candidates.(C) IL-33 levels (ELISA) in supernatants of naïve murine lung cells, freeze-thawed in the presence of supernatants of HEK293T cells transfected with four candidate genes. Mean and SEM are shown of three replicate wells, representative of three repeat experiments.(D) Alignment of HpARI CCP-like modules with complement receptor type 2 CCP2 (CR2-CCP2) and complement factor H CCP10 (FH-CCP10). The putative disulfide bonding pattern (CI-CIII; CII-CIV), conserved tryptophan (W) and structurally-important proline (P), glycine (G), and hydrophobic amino acid residues (h), characteristic of a CCP-module are indicated. Atypical insertions in CCP2/3 (green box), the hypervariable loop (cyan box), and beta-strands (pink arrows) are indicated, based on known CCP secondary structure of CR2-CCP2, as well as three potential N-linked glycosylation sites (light green box).(E) HpARI domain schematic, with putative disulfide bonding pattern and location of insertions indicated.(F) Structural models of the three HpARI CCP-like modules.Error bars show SEM.