Materials and Methods

Mice
Female C57BL/6 mice were obtained from Shanghai Laboratory Animal Center, TLR2 deficient (TLR2−/−) mice on C57BL/6 background were presented by Dr. ZG. Tian (Institute of Immunology, School of Life Sciences, University of Science and Technology of China). All experimental protocols were approved by the Animal Care and Use Committee of Anhui Medical University.

OVA-Induced Allergic Airway Inflammation and Interventions
The murine model of allergic airway inflammation was established according to previous study (27). Briefly, the mice (6–8 weeks old) were sensitized on day 0 with an intra-peritoneal injection of OVA (Sigma, St. Louis, MO, USA) emulsified with 1 mg potassium aluminum sulfate (Sangon Biotech, Shanghai, China) in 500 μl of saline. Airway inflammation was induced by inhalation of 1% aerosolized OVA 30 min per day, for consecutive 7 days. Control mice received saline-sensitization and inhalation of nebulized saline solution. The mice from WT-OVA-Melatonin group were administered with melatonin (10 mg/kg, Sigma), and the mice from TLR2−/−-OVA-Luzindole group were administered with luzindole (30 mg/kg, Sigma) 1 h before allergen-challenge through intra-peritoneal injection, respectively, the dose of melatonin and luzindole was used according to previous studies (19, 28). Experiments were performed with six mice per group. Mice were harvested 24 h following the last challenge.

Bronchoalveolar Lavage Fluid (BALF) Collection and Cellular Analysis
The left lung bronchoalveolar lavage was performed with 500 μl ice-cold phosphate-buffered saline (PBS) for three times, and the resultant BALF was centrifuged to separate the cellular components. The cells were re-suspended in 200 μl PBS. Total cell counts were performed using a hemocytometer, differential cell counts were evaluated morphologically by staining cytospin slides of BALF samples with Wright Stain solution (Sigma).

Lung Histopathological Examination
Immediately after collection of the BALF samples, lung tissues were fixed in 4% paraformaldehyde, and then embedded in paraffin before sagittally cutting into 5 μm sections. Subsequently, the sections were stained with hematoxylin/eosin (H&E) or periodic acid-Schiff (PAS) to analyze the inflammatory cells infiltration and global cell hyperplasia, respectively. Finally, a semi-quantitative method as described previously was used to evaluate the peribronchial inflammation and global cell hyperplasia (29, 30).

Immunohistochemistry
Lung tissue sections (5 μM) were incubated with TLR2 antibody at 4°C overnight. Second day, the sections were incubated with HRP-conjugated secondary antibody (Beijing Golden Bridge Biotechnology, Beijing, China) for 1 h at 37°C. After rinsed for three times, sections were stained by diaminobenzidine (DAB) and counterstained by hematoxylin, and dark-brown staining was considered positive. The image were obtained under an Carl Zeiss Axio Scope.A1 (Carl Zeiss, German).

Cytokines, 5-HT and Melatonin Determined by Enzyme-Linked Immunosorbent Assay (ELISA)
The level of OVA-specific IgE in serum was determined by ELISA using specific kits from Cusabio (Wuhan, China). The levels of IL-4, IL-13, IL-1β, IL-18, and 5-HT in BALF, the content of melatonin in lung homogenate were measured by ELISA kits from Cloud-Clone Crop (Wuhan, China) according to the protocols from the manufacturer.

Western Blotting Analysis
Lung tissues were homogenized in RIPA in the presence of a protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and Phosphatase Inhibitor Cocktail (Roche). The protein concentrations were determined by a BCA kit (Pierce, Rockford, IL, USA). Equal concentrations of protein (25 μg) were separated by SDS-PAGE on 12% acrylamide gels. The primary antibodies used were as follow: NLRP3 (Adipogen, San Diego, CA, USA), caspase 1 (p20) (Adipogen), IL-1β, pro-IL-1β (R&D Systems, Minneapolis, MN, USA), TLR2 (Millipore, Billerica, MA, USA), AANAT (Sigma), ASMT (Abcam, Boston, MA, USA) and GAPDH (KANGCHEN Biotech, Shanghai, China) for 1 h at 37°C, followed by at 4°C overnight. Blots were washed thrice with TBST and probed with appropriate secondary antibodies for 1 h at room temperature. After washing three times, the immune-reaction was analyzed using the ECL detection system (Pierce). The band density was determined by ImageJ software (NIH, Bethesda, MD, USA).

Statistics
Results are showed as mean ± SEM. A one-way analysis of variance followed by Bonferroni multiple comparison test was used to determine the differences among the groups, except for analysis of TLR2 expression, which was performed using independent-sample t-test (SPSS 19.0). P < 0.05 was accepted significant.