Differential Effects of Limonene on Inflammation via Activation of A2A and A2B Adenosine Receptors in Asthma

Abstract

Limonene a mono-terpene flavonoid that is anti-inflammatory in asthma. We have shown that limonene induces bronchodilation and lowers inflammation via A2A receptor activation (FASEB J April 2017 31:820.1) in a mouse model of asthma. However, our data showed that limonene's effects on lowering neutrophils was occurring through a different pathway, as limonene reduced neutrophils in the absence of the A2A receptor. We hypothesized that limonene may also activate A2B receptors based on its similar binding interactions with both A2AAR and A2BAR using a structural homology model. We investigated the effects of limonene on lung inflammation and airway reactivity in A2AAR knock-out (A2AKO) and wild-type (WT) mice. Mice were divided into control (CON) and allergen sensitized-challenged (SEN) groups, and were sensitized i.p. on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13 (n=6 mice per group). Limonene was administered as an inhalation prior to allergen challenges in one group of allergic mice for both WT and KO (SEN+LIM). The role of A2B receptors was studied by treating SEN A2AKO mice with BAY 606583 (selective A2B agonist; 150mg/kg i.p.). Differential BAL cell analysis showed that limonene reduced levels of eosinophils in WT SEN (70.66±2.6% in SEN vs. 28.66±1.45% in SEN+LIM, p<0.05) while no difference was observed in A2AKO (76.33±1.35% in SEN vs 79±0.69% in SEN+LIM). However, neutrophils were significantly lowered in A2AKO (from 19±1.02 in SEN A2AKO to 10±0.4 in SEN+LIM) in BAL. SEN A2AKO treated with BAY 606583 showed similar reduction in the number of neutrophils (10±2.1%) compared to A2AKO SEN treated with limonene (10±0.4). No significant difference was seen in other inflammatory cells sensitized A2AKO mice treated with BAY606583. Tracheal ring reactivity was also studied using isolated organ bath system. Contraction to methacholine (MCh; 0.1μM) in isolated trachea was significantly higher in SEN tissues (225.99±67.34% vs 5.57±19.74% in CON). Treatment with limonene significantly reduced the tracheal reactivity (80.68±20% in SEN+LIM vs SEN; p<0.05). In A2AKO tissues, limonene did not have any effect to reduce MCh reactivity. SCH58651(A2A receptor antagonist) blunted the effects of limonene on tracheal reactivity. CGS-21680 (A2A receptor agonist) had very little effect on tracheal relaxation in SEN WT tissue while SEN+LIM trachea had relaxation to CGS 21680. These data suggest that limonene reduces tracheal reactivity to MCh and eosinophilia via an A2AAR-dependent pathway, while reduction in airway neutrophilia occurs vai an A2BAR-dependent pathway. Support or Funding Information Institutional Grant, Long Island University D-limonene binds to A2A adenosine receptor (A2AAR) and decreases the eosinophilia and airway hyper reactivity induced by methacholine. In the absence of A2AAR (A2AKO), d-limonene can bind to A2BAR and decreases airway neutrophil count in bronchoalveolar lavage This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.