Neutrophilic asthma modelling and translational markers

Abstract

Animal models of allergic airway inflammation are helpful tools to study the pathogenesis of asthma and potential therapeutic interventions. Despite the long use of experimental asthma models, the nature of allergen-driven experimental asthma and allergen delivery route with the use of adjuvants may be distant from human allergic sensitization and the physiopathology of actual asthma. The aim of this work has been to establish a murine asthma model through close-to-real pathogenic procedures that allow representing different airway inflammatory profiles as seen in clinical practice, with their associated cardinal disease traits, providing an extended set of tools for the comparative investigation of disease mechanisms and, eventually, identifying novel diagnostic or therapeutic targets. We chose for this purpose the C57BL/6J mouse, a non-Th2-biased inbred strain, house dust mite (HDM) as a relevant aeroallergen in human asthma, and intranasal (i.n.) instillations for an inhalational delivery route. We started with dose-finding experiments combined with allergen exposure length testing. We found evidence of a T-cell driven adaptive immune response in a High-dose, 4-week (4W) setup as reflected by increased CD3+ CD4+ T cells and the production of HDM-specific IgE, along with a mixed eosinophilic and neutrophilic inflammatory infiltrate, and airway hyperresponsiveness. Longer exposures (6 weeks) led to a declined T-cell response and abrogation of airway hyperresponsiveness and the eosinophilic infiltrates, suggesting immunoregulation on the Th2 arm. From the data of these experiments, we chose the High-dose 4W protocol for further development, although this model still failed to generate airway remodeling. Aiming at representing airway remodeling, we tested delivering HDM in combination with Staphylococcus aureus enterotoxin-B (SEB) or [Alfa]-galactosylceramide ([Alfa]-Galcer). The addition of SEB elicited airway remodeling but inhibited airway hyperresponsiveness and CD4+ T-cell infiltration, and [Alfa]-Galcer failed to reproduce all cardinal asthma traits. Thus, both strategies failed to generate competent asthma models encompassing airway remodeling. By Western blot analysis, we found that the DerP1 and DerP2 antigens undergo degradation in standardized, reconstituted HDM extract. Aeroallergen antigens may, therefore, undergo spontaneous degradation in environmental conditions leading to conformational changes that modify the immune response. By controlling the HDM preservation status, the use of partially degraded allergens (hereinafter termed H4 model) induced dominant neutrophilic inflammation, whereas preserved antigens (H20 model) elicited dominant eosinophilic inflammation. Both models showed evidence of a CD4+ T cell-driven adaptive immune response with Th2 functionality leading to HDM- specific IgE production and successfully developed airway inflammation and remodeling. The latter differed in its patterns, i.e., mucoid with increased airway contractile tissue mass in the H20 eosinophilic model versus fibrotic remodeling in the neutrophilic model. Upon kinetics analyses of immune response outcomes, these diverging models showed complex patterns that varied through time and were concordant with the eosinophilic versus neutrophilic profiles. This H20-eosinophilic versus H4-neutrophilic model diversion encompasses a broad representation of inflammometric profiles seen in real- world clinical practice. Employing these models, kinetic phenotyping of the infiltrating eosinophils and neutrophils by cell surface markers related to maturation, migration, and proinflammatory versus immunomodulatory activity (Ly6, CD11b, SiglecF) allowed us to identify subpopulations that varied between the models and fluctuated across time. These changing subpopulations likely involve functional implications not yet identified. Finally, we performed proteomics in murine BAL and induced sputum from asthmatics. Overall, the proteomics data support that the H4 and H20 models are driven by close-to- human's immune responses. We identify immune response-related gene expression patterns with some differences from proteins previously linked to atopic asthma and some proteins differing between eosinophilic and neutrophilic asthma. Further study of gene expression differing between the inflammatory profiles may point towards targets of potential diagnostic or therapeutic value.