production of IL-1b, IL-6 and IL-8 from TNF-a-stimulated human rheumatoid arthritis fibroblast-like synoviocytes [14]. In addition, artesunate inhibits lipopolysaccharide-induced production of TNF- a, IL-6 and nitric oxide (NO), and expression of toll-like receptor 4 (TLR4) and TLR9 from macrophages [15,16]. The exact molecular mechanism that mediates these anti-inflammatory effects by artesunate has not been unequivocally determined. There are some evidence pointing to the inhibition of nuclear factor NF-kB transcriptional activity by artesunate and other artemisinin derivatives [15,16,17]. More recently, artesunate has been found to possess strong inhibitory activity against the phosphoinositide 3- kinase (PI3K)/Akt signaling pathway [12,13,14]. As PI3K/Akt signaling pathway plays a critical role in the activation and immune responses of eosinophils, T and B lymphocytes, and mast cells [18,19,20], and genetic ablation of PI3Kd or PI3Kc isoform in mice has been shown to dampen Th2 immune responses, eosinophilic lung infiltration, and airway hyperresponsiveness and remodeling [21,22,23], we investigated the effects of artesunate on various aspects of ovalbumin (OVA)- induced Th2-mediated allergic airway inflammation in an in vivo mouse asthma model and explored the anti-inflammatory mechanism of action of artesunate. We also verified the anti- inflammatory effect of artesunate in a house dust mite mouse asthma model. Our results clearly indicate that artesunate attenuates allergic airway inflammation and it is likely mediated through inhibition of the PI3K/Akt signaling pathway. Results Artesunate suppresses OVA-induced inflammatory cell recruitment and mucus production Bronchoalveolar lavage (BAL) fluid was collected 24 hours after the last OVA or saline aerosol challenge, and total and differential cell counts were performed. OVA inhalation markedly increased total cell and eosinophil counts, and slightly yet significantly (p,0.05) increased macrophage, lymphocyte and neutrophil counts, as compared with saline aerosol control. Artesunate (3, 10 and 30 mg/kg) drastically decreased the total cell and eosinophil counts in BAL fluid in a dose-dependent manner as compared with the DMSO vehicle control (Figure 1A). We have conducted flow cytometric analysis of peripheral blood leukocytes obtained from saline-challenged, OVA-challenged, vehicle con- trol, and artesunate-treated mice. Similar percentages of CD3+, CD4+, CD8+ T cells, B cells (B220), NK cells (NK 1.1), neutrophils and monocytes were observed in all mice (data not shown). Hence, artesunate-induced reduction of eosinophil and lymphocyte pulmonary recruitment is unlikely due to any potential nonspecific cytotoxic effects of the drug. Lung tissue was also collected 24 hours after the last OVA or saline aerosol challenge. OVA aerosol challenge induced marked infiltration of inflammatory cells into the peribronchiolar and perivascular connective tissues as compared with saline aerosol challenge. Artesunate (30 mg/kg) markedly diminished the eosinophil-rich leukocyte infiltration as compared with DMSO control (Figure 1B and 1D). On the other hand, OVA- challenged mice, but not saline-challenged mice, developed marked goblet cell hyperplasia and mucus hypersecretion in the bronchi. OVA-induced mucus hypersecretion was significantly halted by artesunate (30 mg/kg) (Figure 1C and 1E). Artesunate reduces OVA-induced BAL fluid Th2 cytokine levels OVA inhalation in sensitized mice caused a notable increase in IL-4, IL-5, IL-13 and eotaxin levels in BAL fluid as compared with Artesunate Attenuates Asthma saline aerosol control (Figure 2). In contrast, BAL fluid level of IFN-c and IL-12, two Th1 cytokines, dropped slightly in OVA- challenged mice. Artesunate drastically reduced IL-13 and eoxtain, and to a lesser extent, IL-4 and IL-5 levels in BAL fluid in a dose-dependent manner as compared with DMSO control (Figure 2). Noticeably, artesunate at 10 and 30 mg/kg could up- regulated IFN-c and IL-12 levels in BAL fluid levels similar to those in saline control mice. This finding implies that artesunate is able to modify the Th2-predominant immune activity in our OVA-induced mouse asthma model. Alternatively, the increase in IL-12 BAL fluid level in artesunate-treated mice may be due to enhanced IL-12 production by dendritic cells upon inhibition of PI3K [24]. The exact mechanism that mediates up-regulation of IFN-c and IL-12 BAL fluid levels remains to be determined. Artesunate suppresses OVA-specific lymphocyte responses in vitro To assess whether artesunate treatment could suppress lympho- cyte cytokine production, we examined OVA-specific immune responses in thoracic lymph node cultures. The in vitro OVA-specific production of IL-4, IL-5 and IL-13 was markedly higher in lymphocytes isolated from OVA-challenged mice than those from saline-challenged mice (Figure 3). Artesunate (30 mg/kg) pretreat- ment significantly (p,0.05) lowered the levels of IL-4, IL-5 and IL- 13. In contrast, in vitro OVA-specific IFN-c production was found to be elevated in mice treated with artesunate (30 mg/kg). The observed immune modulation by artesunate in vitro was OVA- specific because Con A-induced production of IL-4, IL-5, IL-13 and IFN-c in parallel cultures was not affected (data not shown). Artesunate reduces OVA-induced airway hyperresponsiveness in mice To investigate the effect of artesunate on airway hyperrespon- siveness (AHR) in response to increasing concentrations of methacholine, we measured both RI and Cdyn in mechanically ventilated mice. Rl is defined as the pressure driving respiration divided by flow. Cdyn refers to the distensibility of the lung and is defined as the change in volume of the lung produced by a change in pressure across the lung. OVA-challenged mice developed AHR which is typically reflected by high RI and low Cdyn (Figure 4). Artesunate (30 mg/kg) dramatically reduced RI and restored Cdyn in OVA-challenged mice in response to metha- choline aerosol, suggesting that immune-mediated airway pathol- ogy in vivo was modified. Artesunate inhibits OVA-induced inflammatory gene expression and PI3K/Akt activation in allergic airway inflammation OVA aerosol challenge markedly up-regulated lung mRNA level of Muc5ac, which is essential for mucus hypersecretion [25]; inducible nitric oxide synthase (iNOS), the enzyme responsible for nitric oxide (NO) production in allergic airway inflammation [26]; thymic stromal lymphopoietin (TSLP), a cytokine key to the initiation of Th2 immune response [27]; IL-17 and IL-33, two effector cytokines that have recently been shown essential for airway inflammation and remodeling [28,29]; of chitinase family members including acidic mammalian chitinase (AMCase), Ym2 and YKL- 40, which have recently been shown to play critical roles in airway inflammation and remodeling [30,31,32]; and of adhesion mole- cules such as ICAM-1, VCAM-1 and E-selectin, which are pivotal for pulmonary recruitment of inflammatory cells like eosinophils and lymphoctyes [7,33]. Pretreatment with artesunate (30 mg/kg) demonstrated strong suppression of Muc5ac, iNOS, TSLP, IL-17, PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e20932