(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin [7,33]. Cytokine receptor activation by IL-4, IL-5 and IL-13 induces PI3K/Akt signaling cascade [47,48], and IL-4 and IL-13 are potent inducers of eotaxin and RANTES expression in human bronchial epithelial cells [49]. PI3Kc plays an imperative role in mediating eotaxin-induced eosinophil chemotaxis in vitro [18]. Inhibition of class IA PI3K in eosinophil has been shown to block IL-5-induced b2-integrin adhesion of human eosinophils to ICAM-1 [48]. In addition, selective PI3Kd inhibition in mice suppressed aeroallergen-induced VCAM-1 and ICAM-1 expres- sion in lungs [43]. We have demonstrated that artesunate strongly suppressed ICAM-1, VCAM-1 and E-selectin mRNA expression and eotaxin production in OVA-challenged lungs, and IL-8, RANTES and MCP-1 mRNA expression in EGF-stimulated normal human bronchial epithelial cells. Taken together, the observed reduction in airway eosinophilia by artesunate may be a result of combined inhibitory effects on IL-4, IL-5, IL-13, eotaxin and RANTES production, and on adhesion molecule expression, secondary to inhibition of PI3K/Akt. We have also demonstrated a dramatic reduction in airway mucus production in artesunate-treated mice as compared with DMSO control. Cumulative evidence indicates that IL-4, IL-5, IL- 6, IL-13, IL-17 and IL-33 induce goblet cell hyperplasia and mucin production in mice [25,28,29,37]. Mice deficient in p110d or p110c PI3K had impaired mucus production in response to aeroallergen challenge [21,22]. Muc5ac gene expression is also dependent on the transcriptional activity of NF-kB [25,50]. We also observed a substantial drop in Muc5ac mRNA expression by artesunate in OVA-challenged lungs. As such, the marked decrease in mucus production in the lungs of artesunate-treated mice may be attributable to a significant reduction of Th2 cytokine levels, together with inhibition of NF-kB transactivation in airway epithelium. A family of chitinase proteins including AMCase, Ym1, Ym2 and YKL-40 has recently been found to be markedly elevated in allergic airway inflammation in human and in mouse asthma models [30,31,32]. AMCase level is increased in a mouse asthma model and in asthmatic subjects. When given intratracheally, IL- 13 elevates Ym1 and Ym2 levels in BAL fluid from mice in vivo [51]. Besides, YKL-40 serum level correlates positively with asthma severity, airway remodeling and deterioration of pulmo- nary function in asthmatic subjects [32]. Overall, chitinases may play a role in airway inflammation and remodeling. Our data show that artesunate markedly down-regulated AMCase, Ym2 and YKL-40 mRNA expression in the lungs of OVA-challenged mice. These may be a consequence of the major drop in IL-4 and IL-13 levels in the airways with artesunate treatment and may contribute to the diminished pulmonary eosinophilia. Patients with bronchial asthma produce higher level of exhaled NO as compared with healthy controls, and the NO level may reflect the severity of asthma [52]. It appears that increased exhaled NO is associated with increased iNOS expression in the lung epithelium of asthma patients [53]. IL-13 has been shown to induce iNOS expression in normal human bronchial epithelial cells leading to elevate NO production [26]. PI3K plays an important role in the dimerization of iNOS for the NO production [54]. In addition, iNOS gene expression is regulated by the NF- kBtranscriptional activity [50]. Our results show that artesunate markedly suppressed the OVA-induced iNOS expression in the lungs, which may be due to inhibition of PI3K/Akt pathway and of the downstream NF-kB activity, and the reduced level of IL-13 in the allergic airways. Activation of PI3K/Akt and its downstream molecules tuberin, p70S6K and 4E-BP1 lead to airway smooth muscle hypertrophy Artesunate Attenuates Asthma and hyperplasia [55,56]. More recently, p110d PI3K has been shown to mediate IL-13-induced mouse tracheal smooth muscle hyperreactivity to methacholine [57]. We report here that artesunate significantly inhibited OVA-induced AHR to increas- ing concentrations of methacholine. Thus, the observed reduction of AHR by artesunate may be associated with the reduction in Th2 cytokine production, tissue eosinophilia, and airway smooth muscle contractile machinery via inhibition of PI3K/Akt signaling pathway. We report here for the first time that the anti-malarial agent artesunate effectively reduced OVA-induced inflammatory cell recruitment into BAL fluid, IL-4, IL-5, IL-13 and eotaxin production, pulmonary eosinophilia, mucus hypersecretion and AHR in a mouse asthma model potentially via inhibition of the PI3K/Akt signaling pathway. Artesunate is a clinically effective drug for both uncomplicated and severe malaria [9,10,11]. Our present findings support a novel therapeutic use of artesunate in the treatment of asthma. Materials and Methods Animalsand ethics statement All animal experiments were performed according to the Institutional guidelines for Animal Care and Use Committee of the National University of Singapore. Female BALB/c mice, 6 to 8 weeks old (Interfauna, East Yorkshire, UK), were sensitized and challenged with OVA as described [58]. Briefly, mice were sensitized by i.p. injections of 20 mg OVA and 4 mg Al(OH)3 suspended in 0.1 ml saline on days 0 and 14. On days 22, 23 and 24, mice were challenged with 1% OVA aerosol for 30 minutes. Artesunate (3, 10, and 30 mg/kg; Sigma, St. Louis, MO) or vehicle (DMSO) in 0.1 ml saline was given by i.p. injections 1 hour before each OVA aerosol challenge. Saline aerosol was used as a negative control. For the development of house dust mite mouse asthma model, female BALB/c mice were anaesthetized using isoflurane (Halocarbon Products Corporation, River Edge, NJ, USA) and then administered through intratracheal route with either 40 ml of Dermatophagoidespteronyssinus extracts (100 mg, Greer Laborato- ries, Lenoir, NC, USA) or saline as a negative control on days 0, 7 and 14 as described [38,39]. Artesunate (30 mg/kg) or vehicle in 0.1 ml saline was given by i.p on days 6, 7 and 8, and days 13, 14 and 15. Mice were sacrificedon day 17 and BAL fluid was collected for total and differential cell countsas described below. BAL fluid and serum analysis Mice were anesthetized 24 hours after the last aerosol challenge and BAL was performed as described [58]. Briefly, tracheotomy was performed, and a cannula was inserted into the trachea. Ice- cold PBS (0.5 ml63) was instilled into the lungs, and BAL fluid was collected. BAL fluid total and differential cell counts, and cytokine and chemokine levels were determined as described [58]. IL-4, IL-5 and IL-12 ELISA were obtained from BD PharMingen (San Diego, CA, USA). Eotaxin, IL-13 and IFN-c ELISA were purchased from R&D Systems (Minneapolis, MN, USA). Histologic analysis Lungs were fixed in 10% neutral formalin, paraffinized, cut into 5-mm sections, and stained with hematoxylin and eosin (H&E) for examining cell infiltration and with periodic acid-Schiff stain (PAS) for measuring mucus production. Quantitative analyses of cell infiltration and mucus production were performed blind as previously described [56]. PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e20932