antigen-reactive Th17 cells are enriched in bronchoalveolar lavage fluid in severe equine asthma.

Abstract

INTRODUCTION: Equine asthma (EA) is a common disease of adult horses with chronic respiratory pathology and common neutrophilic airway inflammation. It presents with hyperreactivity to hay dust components such as molds, and underlying dysregulated T cell responses have been suggested. Thus far, T cells have been analysed in EA with conflicting results and the antigen reactivity of T cells has not been demonstrated. Serological and epidemiological data point to the relevance ofas an antigen source in EA. Here, we aimed to identify and characteriseantigen-reactive T cells in EA. METHODS: Cryopreserved bronchoalveolar lavage cells (BALC) and peripheral blood mononuclear cells (PBMC) from healthy horses (HE, n=9) and those with mild-moderate (MEA, n=3) or severe asthma (SEA, n=8) were stimulatedwith the recombinantantigens Asp f 1, or Asp f 7 combined with Asp f 8, to assess antigen reactivity, and with phorbol-12-myristat-13-acetate and ionomycin (P/i) to assess overall T cell reactivity. Stimulated cells were analysed by flow cytometry for CD4, CD8, IL-17, IL-4, and IFN-γ. Cytokine expression in all lymphocytes, and in CD4or CD8T cells, was quantified and compared between the groups. In BAL fluid (BALF), soluble cytokines and chemokines were quantified by bead-based assays. RESULTS: Antigen restimulation of BALC with Asp f 1 or Asp f 7/8 provoked higher frequencies of IL-17lymphocytes, CD4IL-17Th17 cells, and CD4IL-4Th2 cells in SEA than in HE, whereas MEA and HE were similar. Antigen stimulation of PBMC did not result in group differences. P/i stimulation of BALC resulted in increased IL-17lymphocyte and CD4IL-17Th17 cell frequencies in MEA compared with HE but the limited number of horses with MEA must be considered. P/i-stimulated PBMC from MEA or SEA contained more IL-17lymphocytes compared with HE. Cytokines were hardly detected in BALF and similar between the groups but CCL2 and CCL5 concentrations were increased in BALF from SEA or MEA, respectively, compared with HE. CONCLUSION: Horses with SEA have increasedantigen-reactive Th17 cells in their airways, emphasising local T cell responses to this mold, which were quantified in EA for the first time here.