Fungal exposure exacerbates allergen-driven asthma in a two-hit mouse model.

BACKGROUND

Severe and exacerbation-prone asthma is often triggered by environmental fungi, yet the immunopathologic impact of live spore exposure remains unclear. Traditional murine models using fungal extracts do not reflect the complexity of inhaled spores during real-world exposures. We sought to establish a clinically relevant model of fungal-driven asthma exacerbation and to define its inflammatory, structural, and molecular features.

METHODS

Ovalbumin (OVA)-sensitized mice were challenged intratracheally with live Aspergillus fumigatus or Alternaria alternata spores; spore-only and OVA-only groups served as controls. Airway inflammation and remodeling were assessed by histology and morphometric analysis, including epithelial and peribronchial smooth muscle thickness. Pulmonary mechanics were evaluated using invasive lung function testing. Immune responses were characterized by flow cytometry and cytokine quantification in bronchoalveolar lavage fluid and serum. Tissue inhibitor of metalloproteinase-1 (TIMP1) levels were measured to evaluate matrix-remodeling–associated responses.

RESULTS

Live spore exposure alone caused modest inflammation and airway hyperresponsiveness. In contrast, spore challenge in OVA-primed mice elicited a markedly amplified phenotype with dense peribronchial/perivascular infiltrates, extensive goblet-cell metaplasia, epithelial disruption, increased epithelial and smooth muscle thickness, and excessive mucus accumulation. Lung function demonstrated substantial increases in airway resistance and lung elastance, consistent with an exacerbation-like state. Co-challenged mice showed increased frequencies of IL-4⁺ CD4⁺ T cells, an early increase in an ILC2-like Lin⁻CD44⁺CD25⁺ population, and elevated IL-4, IL-13, and IL-17 in bronchoalveolar lavage fluid and serum, indicating a mixed type 2 and type 17 cytokine milieu. TIMP1 was robustly increased and was associated with the most marked inflammatory, remodeling, and physiologic abnormalities. This two-hit model recapitulates key hallmarks of fungus-associated exacerbation-like allergic airway disease and provides a translational platform for testing interventions and evaluating remodeling-linked biomarkers.