Table 1. Primer sets for reverse transcriptase-polymerase chain reaction analysis. Targets m AMCase m Ym2 m YKL-40 m ICAM-1 m VCAM-1 m E-selectin m Muc5ac m iNOS m TSLP m IL-17A m IL-33 h IL-6 h IL-8 h RANTES h MCP-1 h/m b-Actin Sequences Forward 59-TGGGTTCTGGGCCTACTATG-39 59-CAGAACCGTCAGACATTCATTA-39 59-GTACAAGCTGGTCTGCTACT-39 59-CATCGGGGTGGTGAAGTCTGT-39 59-CAAGGGTGACCAGCTCATGAA-39 59-AACGCCAGAACAACAATTCC-39 59-GAGTGACATTGCAGGAAGCA-39 59-GTCAACTGCAAGAGAACGGAGAC-39 59-CGACAGCATGGTTCTTCTCA-39 59-CCGCAATGAAGACCCTGATAGA-39 59-GATGGGAAGAAGGTGATGGGTG-39 59-CAGGAGAAGATTCCAAAGAT-39 59-ATGACTTCCAAGCTGGCCGTGGCT-39 59-ATGAAGGTCTCCGCGGCACGCCT-39 59-GATCTCAGTGCAGAGGCTCG-39 59-TCATGAAGTGTGACGTTGACATCCGT-39 doi:10.1371/journal.pone.0020932.t001 Measurements of AHR Mice were anesthetized and tracheotomy was performed as described [58]. The trachea was intubated with a cannula that was connected to the pneumotach, ventilator and nebulizer. Lung resistance (Rl) and dynamic compliance (Cdyn) in response to increasing concentrations of nebulized mechacholine (0.5– 8.0 mg/ml) were recorded using FinePointeTM data acquisition and analysis software (Buxco, Wilmington, NC, USA) as described [58]. Results are expressed as a percentage of the respective basal values in response to phosphate-buffered saline (PBS). Cell cultures To determine the effects of artesunate on OVA-specific immune responses in lymphocytes, thoracic lymph node cells were prepared as described [58]. Cells were exposed to 200 mg/ml OVA for 72 hours. Concanavalin A (Con A, 10 mg/ml) was used as a positive control. Supernatants from parallel triplicate cultures were analyzed for cytokine levels by ELISA. Normal human bronchial epithelial cells (Lonza, Basel, Switzerland) were cultured in optimized bronchial epithelial bulletkit medium with supple- ments. Cells of passage numbers 3–6 were pretreated with 10 mM artesunate or vehicle (0.01% DMSO) 2 hours before stimulation with 100 ng/ml epidermal growth factor (EGF). Total and nuclear proteins, and mRNA were extracted from cells at specified time intervals. References 1. Galli SJ, Tsai M, Piliponsky AM (2008) The development of allergic inflammation. Nature 454: 445–454. 2. Medoff BD, Thomas SY, Luster AD (2008) T cell trafficking in allergic asthma: The ins and outs. Annu Rev Immunol 26: 205–232. 3. Li-Weber M, Krammer PH (2003) Regulation of IL4 gene expression by T cells and therapeutic perspectives. Nat Rev Immunol 3: 534–543. 4. Galli SJ, Grimbaldeston M, Tsai M (2008) Immunomodulatory mast cells: negative, as well as positive, regulators of immunity. Nat Rev Immunol 8: 478- U414. Artesunate Attenuates Asthma Reverse 59-GCTTGACAATGCTGCTGGTA-39 59-ATGGTCCTTCCAGTAGGTAATA-39 59-GTTGGAGGCAATCTCGGAAA-39 59-TGTGGGGGAAGTGTGGTC-39 59-TGTGCAGCCACCTGAGATCC-39 59-TGAATTGCCACCAGATGTGT-39 59-CAGAGGACAGGAAGGTGAGC-39 59-GAGCTCCTCCAGACGGGTAGGCTTG-39 59-CGACAGCATGGTTCTTCTCA-39 59-CAGCATCTTCTCGACCCTGAAA-39 59-TTGTGAAGGACGAAGAAGGC-39 59-ACTGGTTCTGTGCCTGCAGC-39 59-TCTCAGCCCTCTTCAAAAACTTCTC-39 59-CTAGCTCATCTCCAAAGAGTTG-39 59-TGCTTGTCCAGGTGGTCCAT-39 59-CCTAGAAGCATTTGCGGTGCACGATG-39 Immunoblotting, mRNA expression and NF-kB DNA- binding Lung and cell culture protein lysates (20 mg per lane) were separated by 10% SDS-PAGE and immunoblots were developed as described [58]. Immunoblots were probed with anti-Akt, anti- phospho-Akt (Ser473), anti-tuberin, anti-phospho-tuberin (Ser1462), anti-p70S6K, anti-phospho-p70S6K (Ser389), anti-4E-BP1, anti- phospho-4E-BP1 (Ser65), and anti-b-actin antibodies (Cell Signal- ing Technology, Beverly, MA). Primers for inflammatory genes are shown in Table 1 and mRNA expression was analyzed by quantitative real time PCR (ABI 750 Cycler, Applied Biosystems, Carlsbad, CA, USA). Nuclear proteins were analyzed for NF-kB DNA-binding activity using TransAM NF-kB transcription factor assay kit (Active Motif, Carlsbad, CA). Statistical analysis Data are presented as means 6 SEM. One-way ANOVA followed by Dunnett’s test was used to determine significant differences between treatment groups. Significant levels were set at p,0.05. Author Contributions Conceived and designed the experiments: WSFW BPL. Performed the experiments: CC WEH SPG LRK WL BPL FYG. Analyzed the data: CC BPL WSFW. Contributed reagents/materials/analysis tools: WSFW BPL. Wrote the paper: CC BPL WSFW. 5. Takatsu K, Nakajima H (2008) IL-5 and eosinophilia. Curr OpinImmunol 20: 288–294. 6. Wills-Karp M (2004) Interleukin-13 in asthma pathogenesis. Immunol Rev 202: 175–190. 7. Hogan SP, Rosenberg HF, Moqbel R, Phipps S, Foster PS, et al. (2008) Eosinophils: Biological properties and role in health and disease. Clin Exp Allergy 38: 709–750. 8. Cockcroft DW, Davis BE (2006) Mechanisms of airway hyperresponsiveness. J Allergy Clin Immunol 118: 551–559. PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e20932