Experimental Studies On The Effect And Mechanism Of CD39in A Mouse Model Of Allergic Asthma

Objectives Recent studies have demonstrated that extracellular ATP (eATP) is involved in allergic asthma by binding to purinergic receptors. eATP can be hydrolyzed by ecto-nucleotidase triphosphate diphosphohydrolase (ENTPDase)-1/CD39, which is mainly expressed on CD4+Foxp3+regulatory cell (Treg) in mice. In this study, we investigate whether CD39is involved in the pathogenesis of allergic asthma and whether CD4+Foxp3+Treg attenuate allergic airway inflammation and decrease airway hyper-responsiveness via the expression of CD39.Methods A mouse model of allergic asthma was developed with chicken ovalbumin (OVA)-Imject Alum using female C57BL/6mice (6-8weeks old). The control mice were only challenged with OVA without OVA-Alum sensitization. Foxp3-GFP-knock in wild type (WT) and Cd39knock-out (KO) male C57BL/6mice (6-14weeks old) were sorted for CD4+GFP+regulatory cells (Tregs) using magnetic separation combination with flow cytometry. OVA-sensitized mice were treated with CD39inhibitor, or CD39inhibitor combination with apyrase, or PBS before each allergen challenge. OVA-sensitized mice were adoptively transferred with CD4+Foxp3+Tregs from WT and CD39KO mice prior to the first allergen challenge. Airway hyper-responsiveness and airway inflammation were evaluated at48h after the final challenge. The location of CD39in the lungs was detected by immunohistochemical analysis. CD39mRNA and protein expression in the lungs was determined by quantative polymerse chain reaction (qPCR) and western blot analysis, respectively. The mRNA levels of transcript factors (GATA3and RORyt) and purinergic receptor (P2Y2and P2Y6) were determined by qPCR. The frequencies of CD4+Foxp3+Tregs were measured by flow cytometry. Lung tissue inflammation and goblet cell hyperplasia were assessed by hematoxyin and eosin (H&E) and perioic-acid Schiff (PAS) staining, respectively. Cellular profiles in the BALF were counted by cytospin with Wright-Giemesa staining. OVA-specific IgE in the sera and cytokine levels (IL-4, IL-5, IL-13, IL-17A, and IFN-?) in the BALF were measured by enzyme-linked immunosorbent assay (ELISA). Results1) In a mouse model of allergic asthma, CD39mRNA and protein expression was down-regulated and the frequencies of CD4+Foxp3+Tregs were reduced.2) CD39inhibitor ARL67156aggravated these cardinal features of asthma, including lung eosinophilia, mucus overproduction, Th2-and Thl7-related cytokine production, and airway hyper-responsiveness, accompanied with an increase in the mRNA expresson of transcript factors (GATA3and RORyt) and purinergic receptor (P2Y2and P2Y6). Pre-treatment with apyrase improved the outcomes caused by ARL67156.3) In OVA-induced asthma models, CD39KO mice showed increased airway inflammation, which mainly showed lung eosinophilia, mucus overproduction, and an increase level in IL-4and IL-17.4) Adoptive transfer experiments demonstrated that CD39+Treg and CD39Treg cells inhibited allergic airway inflammation and airway hyper-responsiveness in a mouse model of asthma. By contrast to CD39"CD4+Foxp3+Tregs, CD39+CD4+Foxp3+Tregs displayed more potent suppressive capacities to the cytokine production.Conclusions1) CD39expression was reduced in the lungs of allergic asthmatic mice.2) CD39inhibitor increased allergic airway inflammation, which was related to P2R overexpression occurring in a mouse model of asthma.3) CD4+Foxp3+Tregs attenuated airway inflammation and airway hyper-responsiveness in asthmatic mice via the expression of CD39.