Withaferin A reduces pulmonary eosinophilia and IL-25 production in a mouse model of allergic airways disease

Abstract

Several studies report that ashwagandha, a traditional Ayurvedic supplement, has anti-inflammatory properties. Type 2 (T2) asthma is characterized by eosinophilic airway inflammation. We hypothesized that allergen-induced eosinophilic airway inflammation in mice would be reduced following administration of Withaferin A (WFA), the primary active phytochemical in Ashwagandha. C57BL/6J mice were given 10 total intra-peritoneal injections of 2 mg/kg WFA or vehicle control, concurrent with 6 total intranasal administrations of 50 μg house dust mite extract (HDM) or saline control over 2 weeks. We observed that treatment with WFA reduced allergen-induced peribronchial inflammation and airway eosinophil counts compared to mice treated with controls. In addition, we observed that treatment with WFA reduced lung levels of interleukin-25 (IL-25) but increased lung gene expression levels of its co-receptor, Il17ra, in HDM-challenged mice compared to HDM-challenged mice that received the vehicle control. This study pinpoints a potential mechanism by which WFA modulates allergen-induced airway eosinophilia via the IL-25 signaling pathway. Future studies will investigate the effects of WFA administration on lung eosinophilia and IL-25 signaling in the context of chronic allergen-challenge.

Figure 1.

Airway eosinophilic inflammation is reduced with WFA in HDM-challenged mice. (A) Schematic diagram of the mouse model of acute intranasal house dust mite allergen (HDM)-induced allergic airways disease combined with intraperitoneal Withaferin A (WFA) or vehicle control (VC) injection. Control mice were challenged with intranasal phosphate buffered saline (PBS). Bronchoalveolar lavage fluid (BALF) and lung tissue harvest occurred 24 h following the last administrations of HDM/PBS and WFA/VC. Created with BioRender.com. (B) Representative images of H&E-stained lung sections from all 4 treatment groups. (C) Mean H&E scores ± SEM depicting depth of peribronchial staining for n=2 mice per treatment group. (D) BALF differential cell counts in HDM-challenged mice treated with VC or WFA (n = 2–4 mice per group). Data are presented as mean percentage of total BALF cells ± SEM. **p<0.005 for eosinophils in BALF of both treatment groups in HDM-challenged mice.

Figure 2.

In lung tissue of HDM-challenged mice, WFA reduces IL-25 and increases lung Il17ra mRNA expression. (A) IL-33 was measured in whole lung homogenates using ELISA. Data are presented as mean pg/mL IL-33 ± SEM. (n = 3 mice per group). *p<0.050 in HDM-challenged mice compared to saline-challenged mice for both VC and WFA treatment groups. (B) IL-25 was measured in whole lung homogenates using ELISA. Data are presented as mean pg/mL IL-25 ± SEM. *p<0.050 in VC-treated vs WFA-treated HDM-challenged mice (n = 3 mice per group). *p<0.050 in WFA-treated mice comparing saline-challenge to HDM-challenge. (C and D) Il17ra and Il17rb mRNA expression was measured in whole lung RNA using RT-PCR. Data are presented as fold change in Il17ra (C) or Il17rb (D) mRNA expression ± SEM relative to Gapdh mRNA expression and to the mean of the saline-challenged VC-treated mice. (n = 2–3 mice per group). *p<0.050 Il17ra mRNA expression in WFA-treated vs VC-treated HDM-challenged mice.