Airway inflammation after epicutaneous sensitization of mice requires protease activity of low-dose allergen inhalation

Recent studies demonstrated that antigen-specific ST2+ but not ST2− memory TH2 cells were responsible for airway eosinophilia in mice8 and also that IL-33 stimulated CD4+ T cells from the nasal polyps of patients with eosinophilic chronic rhinosinusitis to upregulate the expression of IL-5 and/or IL-13.8,9 The delivery of exogenous proteases evokes an enzyme-mediated “alarm,” such as the release of IL-33, presumably via damage to the lung epithelium.5 In intranasal models with the inhalation of 10 to 30 μg of papain, the IL-33-ILC2 axis has been reported to contribute to TH2 differentiation, IgE production, the recruitment of TH2 cells, and airway eosinophilia.5-7 We analyzed effector-phase responses in a tissue (lung) different and distant from the sensitization site (ear skin). Recently we reported that in the epicutaneous sensitization-intranasal challenge model with the inhalation of a low dose (2.5-3 μg) of papain, airway eosinophilia and the maintenance of IgE levels induced by the subsequent inhalation of a minute amount of papain were dependent on IL-33, whereas IL-33 was dispensable for skin inflammation and antibody responses in the epicutaneous sensitization phase.4 In the present study, we demonstrated that (1) the onset of allergic airway inflammation after epicutaneous presensitization was critically dependent on allergen protease activity contained in the low-dose airway encounter (Fig 2, A, and Fig E3, A) and (2) 10-fold more ST2+ TH2 cells than ILC2s infiltrated bronchial alveolar lavage fluid with abundant amounts of TH2-attracting chemokines (Fig 2, C and D) and the combination of the antigen and IL-33 synergistically stimulated DLN cells to upregulate cytokine production (Fig 2, E), suggesting the contribution of the “antigen plus IL-33-TH2 axis.” [...]we herein demonstrated that the effector-phase responses of the onset of allergic airway inflammation after the establishment of epicutaneous sensitization in mice not only need intact T/B-cell epitope structures but also the crucial commitment of the protease activity of the low-dose inhalation and the possible contribution of IL-33–responsive, antigen-specific TH2 cells. In the covalent complex between E-64 and papain (E64-papain) and dialyzed papain, purity, no degradation, and no aggregation were confirmed by electrophoresis, protease activity was analyzed using synthetic fluorogenic substrates in the presence or absence of a reducing reagent,E7,E8 and protein concentrations were asse