Fig 1: (A) Torudokimab completely inhibited 30ng/mL human IL-33 (95–270) from binding to ST2 in a blocking ELISA assay with an IC50 of 3.7nM. Results are expressed as IC50 (half maximal inhibitory concentration) and calculated using a 4-parameter sigmoidal fit of the data (GraphPad, Prism). (B) Torudokimab completely inhibited the cleaved mature form of human- and cynomolgus monkey-, but not mouse-, rat- or rabbit- IL-33 induced NFκB activity in a dose-dependent manner. The GEC-NFkB luciferase cell line was treated in biological triplicates with 2.5 ng/mL IL-33 (95–270) in the presence of the antibodies for 6 hours before luciferase reading was done. IC50 showed that torudokimab inhibited human IL-33 (95–270) at 0.330 nM and cynomolgus monkey IL-33 (95–270) at 1.918 nM. Whereas soluble ST2 (sST2), the positive control, inhibited human IL-33 at 0.426 nM and cynomolgus monkey IL-33 at 0.175 nM. Results were calculated using a 4-parameter sigmoidal fit of the data (GraphPad, Prism). Results are representative of three independent experiments.
Fig 2: IL-33 is inactivated by disulphide bonding.(a) Concentration of IL-33 (mean±s.e.m.) in bronchoalveolar lavage fluid (BALF) following intranasal Alternaria (ALT) challenge of BALB/c mice (n=3 per group), representative of 2 independent studies. (b) Western blot analysis of the same samples (pooled per group) under reducing and non-reducing conditions. Controls are as follows: cell lysate, lysate of HEK cells transfected with full length mouse IL-33; R&D, truncated mouse IL-33 (R&D systems); pep, truncated mouse IL-33 (Peprotech); PBS (30 min), BALF from vehicle (PBS) challenged mice at 30 min timepoint. (c) Non-reduced SDS-PAGE of IL-33112–270 either untreated (−) or post treatment with cell culture media (Iscoves Modified Dulbeccos Media) (+). Monomeric IL-33 was purified prior to analysis. (d) The human IL-33 linear sequence illustrated with the position of the cysteines and the proposed disulphide bridges formed after media treatment. Disulphide mapping was performed on purified monomeric protein. Numbering is based on the full length IL-33 sequence. (e) Hydrogen-exchange mass spectrometry (HX-MS) analysis of monomeric IL-33 and DSB IL-33. Comparison of fractional hydrogen exchange (for deuterium) in IL-33 (left panel) and DSB IL-33 (right panel). Data are mapped onto the published IL-33 structure22 in both cases for comparison purposes. Gaps in sequence coverage where no HX-MS data could be obtained are highlighted in slate blue. Side chains of cysteine residues are displayed as sticks. (f) Structural model displaying the difference in fractional hydrogen exchange between IL-33 and DSB IL-33 overlaid with the ST2 binding sites (red and magenta)23. Only increased hydrogen exchange was observed for DSB IL-33 versus IL-33, with regions exhibiting the greatest differences of hydrogen exchange depicted in dark blue. (g) ST2 binding of human IL-33 (upper panel) and purified DSB IL-33 (lower panel) measured by surface plasmon resonance. (h) Signalling in human umbilical vein endothelial cells (HUVEC) stimulated by human IL-33 and purified DSB IL-33. NFκB p65/RelA translocation was measured at 30 min post stimulation. Data points are mean±s.e.m. of duplicate determinations, representative of 3 independent experiments.
Fig 3: BMP (bone morphogenetic protein) 9 protects from IL (interleukin)-33–induced endothelial-to-mesenchymal transition (EndMT) and induces sST2 (soluble supression of tumorigenicity 2) expression in pulmonary arterial endothelial cells (PAECs) from patients with pulmonary arterial hypertension (PAH) in vitro. A, Representative immunofluorescent staining of PAH PAEC for CD31 (endothelial marker), SM22α (smooth muscle protein 22-alpha; mesenchymal marker), and 4′,6-diamidino-2-phenylindole (DAPI). Cells were treated with BMP9 (1 ng/mL), IL-33 (100 ng/mL), both, or left untreated (control [CTR]) for 3 days. Bar graphs show quantification of CD31 and SM22α intensity (technical replicates [t.n.]=3). B, sST2, ST2L, CTGF, and Pai-1 gene expressions in PAH PAECs s after 16-hour stimulation with TGF (transforming growth factor)-β (1 ng/mL), activin A (50 ng/mL), or untreated (CTR; t.n.=3). C, sST2, ST2L, ID1, and ID3 gene expressions in PAH PAECs after 3-hour stimulation with BMP4 (50 ng/mL), BMP6 (50 ng/mL), BMP9 (1 ng/mL), BMP10 (1 ng/mL), or untreated (CTR; t.n.=3). Statistical analysis: 1-way ANOVA with the Tukey post hoc test; P<0.05, *P<0.01, **P<0.001, and ***P<0.0001. Data are shown as mean±SD.
Fig 4: BMP (bone morphogenetic protein) 9 protects from IL (interleukin)-33–induced endothelial-to-mesenchymal transition (EndMT) and induces sST2 (soluble supression of tumorigenicity 2) expression in control pulmonary arterial endothelial cells (PAECs) in vitro. A, Representative immunofluorescent staining of PAEC for CD31 (endothelial marker), SM22α (smooth muscle protein 22-alpha; mesenchymal marker), and 4′,6-diamidino-2-phenylindole (DAPI; nuclei). Cells were treated with BMP9 (1 ng/mL), IL-33 (100 ng/mL), both, or left untreated (control [CTR]) for 3 days. Bar graphs show CD31 and SM22α intensity quantification (n=3). B, sST2, ST2L, CTGF, and PAI-1 gene expressions in PAECs after 16-hour stimulation with TGF (transforming growth factor)-β (1 ng/mL), activin A (50 ng/mL), or untreated (CTR; technical replicates [t.n.]=3). C, sST2, ST2L, ID1, and ID3 gene expressions in PAECs after 3-hour stimulation with BMP4 (50 ng/mL), BMP6 (50 ng/mL), BMP9 (1 ng/mL), BMP10 (1 ng/mL), or untreated (CTR; t.n.=3). Statistical analysis: 1-way ANOVA with the Tukey post hoc test; *P<0.05, **P<0.01, and ****P<0.0001. Data are shown as mean±SD.
Fig 5: BMP (bone morphogenetic protein) 9 prevents IL (interleukin)-33–induced endothelial-to-mesenchymal transition (EndMT) in pulmonary arterial endothelial cells (PAECs) in vitro, similar to rsST2 (recombinant soluble ST2), thereby inhibiting IL-33 target gene expression. A, Representative immunofluorescent staining of for CD31 (endothelial marker), SM22α (smooth muscle protein 22-alpha; mesenchymal marker), and 4′,6-diamidino-2-phenylindole (DAPI; nuclei). Cells were pretreated with BMP9 (1 ng/mL, 3 hours) or rsST2 (1 μg/mL, 30 minutes) before IL-33 (100-ng/mL) stimulation for 3 days. Bar graphs show CD31 and SM22α intensity quantification (n=3). B and C, Representative immunoblots of p-p38 (phosphorylated p38 mitogen-activated protein kinase) and p-IκBα (phosphorylated inhibitor of nuclear factor kappa-B alpha) in PAECs pretreated overnight with BMP9 (1 ng/mL), rST2 (1 μg/mL, 30 minutes), or left untreated before IL-33 (100-ng/mL) stimulation for 5, 15, or 60 minutes. Densitometric analysis was performed using ImageJ, with protein levels normalized to vinculin and expressed as fold change relative to the 0-minute control (n=3). D, MyD88, IL1RAP, and IL-8 gene expressions in PAEC pretreated with BMP9 (1 ng/mL, 3 hours) before IL-33 (100 ng/mL, 24 hours) stimulation or left untreated (control [CTR]; n=3). Statistical analysis: 1-way ANOVA (A and D) or 2-way ANOVA (B and C) with the Tukey post hoc test; P<0.05, *P<0.01, and **P<0.001. Data are shown as mean±SD (A and D) or mean±SEM (B and C). ns indicates not significant.
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