The IL-33 and IL-4Ra blocking antibodies, itepekimab and dupilumab, modulate distinct and common inflammatory mediators in asthma - human mast cell and eosinophil data

Biologics that target IL-4Ra or IL-33 pathways have demonstrated clinical efficacy in asthma and chronic obstructive pulmonary disease (COPD), highlighting the importance of IL-4, IL-13 and IL-33 in respiratory diseases. Despite this, there are limited studies that link preclinical models to human disease and evaluate disease biology in the setting of clinical trials. To address these gaps, we evaluated transcriptional, cellular and pathophysiological processes driven by IL-4/ IL-13 and IL-33 using human innate cells in vitro, a mouse model of airway inflammation and a Bronchial Allergen Challenge (BAC) in house dust mite (HDM)-sensitized individuals with mild asthma. Using the HDM mouse model of airway inflammation, we show that blockade of either IL-4Ra or IL-33 at the peak of type 2 (T2) inflammation leads to inhibition of airway inflammation and remodeling. While the combination of dupilumab and itepekimab was not additive, blockade of both IL-33 and IL-4Ra in a severe, late-phase mixed T1/T2 inflammation reduced all measured parameters. Consistent with some of these observations in mice, blocking either IL-33 or IL-4Ra in the context of T2 airway inflammation suppressed sputum gene expression in individuals with mild persistent asthma post-BAC. Overall, these results highlight that IL-33 and IL-4/IL-13 are key drivers of airway inflammation and remodeling and provide insight into the differences in targeting IL-4Ra or IL-33 pathways in asthma independently or in combination. Overall design: Human mast cells were differentiated in vitro from bone marrow-CD133+ cells as previously described. Briefly, bone marrow-CD133+ cells were cultured in StemSpan SFEM media (StemCell) in the presence of 50ng/mL IL-6 (PeproTech), 100ng/mL SCF (PeproTech) and 1ng/mL IL-3 (R&D Systems) for 3 weeks, followed by treatment with IL-6 and SCF for 4 more weeks. Purity of in vitro-generated mast cells was confirmed by CD117 and FceRI expression by flow cytometry and mast cell morphology was confirmed by cytospin preparations and May-Grünwald/Giemsa staining (Sigma). Human eosinophils were isolated from buffy coats using MACSxpress Eosinophil Isolation Kit (Miltenyi) and red blood cells were removed by MACSxpress Erythrocyte Depletion Kit (Miltenyi). Cell purity was confirmed by CD45, CD15 and Siglec-8 surface staining and analysis by flow cytometry. Purified eosinophils and mast cells were cultured in RPMI-1640 (Gibco) with 10% Fetal Bovine Serum (Life Technologies) and treated with IL-4, IL-13, or IL-33 (R&D Systems) at 100nM in 2-5 donors overnight (eosinophils) or 48hrs (mast cells) at 37°C. Cells were harvested and suspended in TRIzol (Life Technologies) for RNA extraction. Untreated mast cells and eosinophil samples were also collected as control.