IL-36a and Pulmonary Inflammation and microwaved multiple times and washed in water to destain the gel. procedure was simultaneously monitored using electrocardio- grams. Western Immunoblotting Increasing amounts of IL-36a (5, 10 and 20 ng) were separated on a SDS-polyacrylamide gel under reducing and denaturing conditions. Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36a antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham). Tissue Collection and Analysis Mice were euthanized with an overdose of pentobarbital, and the treacheas were catheterized. Lungs were lavaged three times with 1 mL of ice cold-PBS. Bronchoalveolar lavage (BAL) fluid was centrifuged at 1,500 rpm for 15 minutes and the supernatants were stored at 280uC. Cells in the pellet were used to calculate total cell numbers (Cellometer, Nexelcom), as well as differential cell counts on cytospun slides stained with Hema-3 White Cell Differential Staining Kit (Fisher). More than 100 cells were counted per sample by light microscopy to calculate differential cell counts. Upper right lobe of the lung was collected for RNA analysis, and the left lung was inflation fixed with 10% neutral buffered formalin for histological analysis. RNA Isolation and Quantitative Real-time PCR Total RNA was isolated from lung tissue using Trizol (Invitrogen) and cell cultures using RNeasy (Qiagen) and treated with Amplification grade DNaseI (Invitrogen) according to manufacturer’s protocols. Equal concentrations of DNaseI treated RNA was reverse transcribed into cDNA using Taqman Reverse Transcription reagents (Applied Biosystems) and used as input for quantitative real-time PCR using Power SYBR Green kit (Applied Biosystems). Primers used in real-time PCR analyses were designed using the mouse qPrimerdepot (http:// mouseprimerdepot.nci.nih.gov/). Cycle threshold (CT) values obtained from the assays were analyzed and reported as copies of target genes per copy of GAPDH, a housekeeping gene. Measurement of Protein Levels in the BAL Fluid Cytometric bead array Flex-kits (BD Biosciences) for IL-1a, IL- 1b, TNFa and CXCL1 were used according to manufacturer’s protocols to evaluate the protein levels in the BAL fluid recovered from the mice. Measurement of Lung Function in Mice Six to eight week old mice were intratracheally administered 10 mg IL-36a or 50 ml PBS, and lung function measurements were performed 24 h later using the Flexivent system (Scireq, Montreal, Quebec, Canada), as described before [38]. Briefly, mice were anesthetized with an intraperitoneal injection of xylazine (12 mg/ kg) and pentobarbital (70 mg/kg). The trachea of anesthetized mice was cannulated and the mice were ventilated with 6 ml/kg tidal volume at 150 breaths per minute. To suppress spontaneous breathing during measurement of lung function, mice were intraperitoneally injected with pancuronium bromide (2 mg/kg). Incremental doses of neublized methacholine were used to determine total lung resistance and compliance according to the Snapshot-150 perturbation provided by the Flexivent equipment. Thirteen data points were collected for each methacholine dose, and only data with a coefficient of determination greater than 0.95 were included in analyses. The survival of mice during the Isolation and Culture of CD11c+ Cells Splenic CD11c+ cells were isolated from wildtype C57BL/6 using magnetic separation according to manufacturer’s (Miltenyi Biotec) protocol. Briefly, single cell suspensions prepared from spleens of wildtype C57BL/6 mice were incubated with red blood cell lysis solution (Invitrogen) to lyse RBCs. The resulting cell suspensions were incubated with mouse anti-CD11c microbeads (Miltenyi Biotec), and CD11c+ cells were isolated by two rounds of positive selection to enhance purity. The purity of isolated CD11c+ cells were routinely .90%. The isolated CD11c+ cells were resuspended in RPMI-1640 media containing 10% heat inacti- vated fetal bovine serum (FBS), L-glutamine and 1% penicillin- streptomycin. According to the experiment, aliquots of resus- pended cells were mixed with various concentrations of IL-36a (0.1 mg/mL, 1.0 mg/mL or 10.0 mg/mL) or an equivalent volume of PBS. For RNA isolation, cells mixed with IL-36a or PBS were plated at a concentration of 2.56105 cells per well in a 48-well plate in a volume of 250 ml. The cells were incubated for 2 hours to adhere. The media was gently removed from the plates by pipetting, and the cells were washed once with sterile, cold PBS. RNA was isolated from the adherent cells using RNeasy RNA isolation kit (Qiagen) according to manufacturer’s protocols. In some experiments, the media containing IL-36a was removed from the wells 2 hours after incubation, replaced with RPMI-1640 growth media and allowed to incubate for an additional 22 hours before evaluating cell surface expression of co-stimulatory molecules by flow cytometry. Isolation and Culture of CD4+ T Cells Spleens and pooled lymph nodes from wildtype C57BL/6 mice were used to isolate CD4+ cells using magnetic separation according to manufacturer’s (Miltenyi Biotec) protocol to a routine purity of .95%. Isolated CD4+ cells were stained with CFSE (Celltrace CFSE staining kit, Invitrogen) and resuspended in culture media. For CD4+ and CD11c+ co-culture studies, CD11c+ cells were isolated using magnetic separation protocols, mixed with the indicated concentrations of IL-36a, plated at a concentration of 16105 cells per well in a 96-well plate and allowed to adhere for 2 hours. Following incubation, media from the wells was removed by pipetting, cells were washed once with cold PBS, and 46105 CFSE-stained CD4+ T cells were incubated with the adherent CD11c+ cells for 96 hours. Following incubation for 96 h, cells were removed and analyzed by flow cytometry. To evaluate antigen-specific responses, isolated splenic CD11c+ cells were plated at a concentration of 16105 cells per well with indicated concentrations of IL-36a and 100 ng/mL OVA323-339 for 2 hours. Following incubation, media from the wells was removed by pipetting, cells were washed once with cold PBS, and 46105 CFSE-stained CD4+ T cells from OTII TCR transgenic mice were incubated with the adherent CD11c+ cells for 96 hours. Following incubation for 96 h, cells were removed and analyzed by flow cytometry. Flow Cytometry Cells isolated from BAL were incubated with Fc block, followed by incubation with fluorochrome conjugated antibodies against mouse CD11c, CD11b (BD Biosciences) or Ly6-G (R&D Systems) according to standard protocols. For CFSE-based cell proliferation studies, cells were collected and incubated with Fc block, followed by incubation with allophycocyanin-conjugated anti-CD4 (BD Biosciences) antibody. T cell proliferation responses were analyzed PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45784