The TLR4/NF-κB signaling pathway-mediated type 2 skewing of T helper cell in cough variant asthma was counteracted by ethanol extract of Anacyclus pyrethrum root

Abstract

Type 2 T helper (Th2) cells-mediated immune response plays a pivotal role in the pathogenesis of cough variant asthma (CVA), and this study aims to determine the effect and mechanism of ethanol extract of Anacyclus pyrethrum root (EEAP) on regulating Th2 response in CVA. Peripheral blood mononuclear cells (PBMCs) collected from patients with CVA, and naive CD4+T cells induced by Th2-polarizing medium were administrated with EEAP. Interestingly, through conducting flow cytometry and enzyme linked immunosorbent assay method, we found that EEAP significantly alleviated Th2 skewing and increased Th1 response in these two kinds of cells. Results of western blot assay and quantitative reverse transcription PCR displayed that EEAP suppressed the expression of TLR4, total NF-κB p65, nuclear NF-κB p65 and the downstream genes. Subsequently, we proved that TLR4 antagonist E5564 played a similar improvement role to EEAP in Th1/Th2 imbalance, while combination of TLR4 agonist LPS and EEAP abolished the inhibitory effect of EEAP on Th2 polarization in Th2-induced CD4+T cells. Finally, CVA models induced by ovalbumin and capsaicin were established in cavies, and data showed that EEAP also improved Th1/Th2 imbalance in CVA in vivo, manifested in the increase of IL4+CD4+T cell ratio, Th2 cytokines (IL-4, IL-5, IL-6 and IL-13) and the decrease of Th1 cytokines (IL-2 and IFN-γ). Co-treatment of LPS and EEAP counteracted the inhibition of EEAP on Th2 response in CVA model cavies. Moreover, we found that EEAP mitigated airway inflammation and hyper-responsiveness in vivo, which was abolished by the combined application of LPS. In a word, EEAP restores Th1/Th2 balance in CVA through restraining the TLR4/NF-кB signaling pathway. This study may contribute to the clinical application of EEAP in CVA-related disease.