Calcaratarin D reduces airway inflammation and oxidative damage by modulating alveolar macrophages via Michael acceptor system

Abstract

Our laboratory has recently shown that Calcaratarin D (CalD), a labdane diterpenoid, attenuated house dust mite (HDM)-induced acute mouse asthma model. The objective of the present study was to investigate the mechanisms of action of CalD using a series of <i>in vivo, ex vivo and in vitro</i> strategies. CalD abrogated both the mRNA and protein levels of a wide array of cytokines, chemokines and mediators in lung homogenates, and oxidative damage markers in lung sections. In primary alveolar macrophages (AMs) isolated from asthmatic mouse lungs, both M1 (iNOS) and M2 (Arg1) markers were up-regulated, and CalD suppressed both markers and further reduced polyamine level and blocked methacholine-induced airway resistance, indicative of prevention of airway remodeling and hyperresponsiveness. In addition, CalD inhibited T cell chemoattractants CCL17 and CCL22 levels in primary AMs both <i>ex&nbsp;vivo</i> and <i>in vitro</i>. To delineate the mechanisms of action of CalD, we observed that CalD blocked HDM-induced FoxO1/IRF4 pathway and promoted endogenous Nrf2/HO-1 antioxidant pathway in allergic airways. Our structure-activity relationship study revealed that the α, β-unsaturated γ-butyrolactone Michael acceptor system in CalD capable of forming covalent bonds with cellular protein targets is critical for its anti-inflammatory effects. Saturation of the system or removal of C-14 hydroxyl from CalD abolished its inhibitory effects on IL-4/IL-13-stimulated gene targets and chemokine production in primary AMs. Taken together, CalD is a novel anti-inflammatory agent for allergic asthma with actions targeting at alveolar macrophages probably <i>via</i> Michael acceptor system.