Ex vivo allergen specific type 2 immune responses in house dust mite-driven acute airway inflammation in mice depend on allergen extract characteristics

House dust mite (HDM) is frequently used in animal models of allergic airway diseases. HDM extracts differ in allergen concentration and endotoxin contamination, requiring model optimalization for each batch. This study aimed to refine and validate our murine model of HDM-driven acute allergic airway inflammation with a new batch of HDM extract and comparing different administration doses. We evaluated three HDM extracts, HDM1 (old batch), HDM2 and HDM3, containing varying Der p1 concentrations (33.7, 71.4 and 9.0 mg/g protein) and endotoxin levels (94.8, 53.9 and 24.7 EU/g protein). Male BALB/c mice were intranasally sensitized (day 0) with 1 µg (HDMlow) or 5 µg (HDMhigh), and challenged (day 7–11) with 10 µg (HDMlow) or 15 µg (HDMhigh) of the same HDM extract. Bronchoalveolar lavage fluid (BALF), lung tissue and serum were collected on day 14. High doses of all extracts significantly increased BALF total cell influx, including eosinophils and lymphocytes, and elevated lung Th2 and CD69 + Th2 cell frequencies. Eosinophils and lymphocytes induction varied across extracts (6–17 × 104 and 3–8 × 104 cells respectively). HDM3high tended (p < 0.1) to increase serum HDM-specific IgE. Upon ex-vivo HDM-restimulation of lung cells comparing HDM3 high with HDM2 high, only cells of HDM3high-treated mice showed increased IL-5 and IL-13 production. Overall, the high dose protocol most effectively induced HDM-driven acute allergic airway inflammation, based on BALF eosinophil and lymphocyte influx. Even though having low Der p1 and endotoxin levels, only HDM3high enhanced cytokine production upon ex-vivo HDM-restimulation, indicating the value of this functional assay to discriminate between HDM batches. House dust mite (HDM) extracts are widely used to induce allergic airway inflammation in mice, but differences in allergen and endotoxin content of these extracts might affect model outcomes. This study demonstrates that variations in HDM extract composition and dosing could lead to distinct inflammatory and immunological responses in mice. These findings highlight the importance of characterizing and optimizing each HDM extract batch to ensure reproducibility and comparability across experimental allergy studies and prevent the use of large groups of animals in suboptimal models.