Mast cell-dependent adjuvant activity is a key component of the respiratory immune response to inhaled Alternaria

Abstract

Exposure and sensitization to Alternaria alternata (Alternaria) is strongly associated with asthma development, persistence, and exacerbations. Alternaria elicits murine and human mast cell (MC) activation, independent of IgE receptor signaling, but the relevant fungal ligand, mechanism of activation, and potential for adjuvanticity have not been characterized. Alternaria fractions were obtained using sequential salting-out fractionation, ion-exchange, and size-exclusion chromatography, and tested for activity on murine bone marrow-derived MCs (BMMCs). Putative MC ligands from active fractions were identified using Mass-spectroscopy and cloned for recombinant expression. WT and Mcpt5/DTA mice, with diptheria toxin-mediated MC deletion, were treated with intranasal Alternaria and dendritic cell (DC) activation and lung inflammation was assessed at serial timepoints. In response to Alternaria inhalation, IgE-independent MC activation not only potentiates pulmonary inflammation but also regulates the migration of antigen-bearing DCs to regional lymph nodes to mediate the earliest events in aeroallergen sensitization. Partial purification of Alternaria extracts demonstrates that MC degranulation and CysLT generation is elicited by a weakly-anionic heat-labile protein (30-50kDa in size) and is protease-independent. Mass-spectroscopy and cloning have identified several novel fungal candidate proteins that have not been previously characterized. Although several Alternaria allergens have been identified, the adjuvant activities of most fungal proteins remain poorly characterized. We find that a novel Alternaria Ag acts as a danger signal to initiate innate MC-dependent sensitization and subsequent pulmonary inflammation. These findings highlight the role of innate MC sensing in the respiratory tract and the importance of MC-DC crosstalk in priming for allergen-elicited inflammation.