which consists of agonists (IL-1a, IL-1b) and an antagonist (IL- 1Ra) which binds to a functional heterodimeric receptor complex containing IL-1 receptor I (IL-1R1) and IL-1RAcP [35]. Both the classical and novel IL-1 cytokine cluster agonists induce the activation of nuclear factor kappa B (NF-kB) and mitogen associated protein kinase (MAPK) to initiate pro-inflammatory pathways [31,35]. While the role of classical IL-1 cytokines have been extensively characterized in the pathogenesis of inflamma- tory disorders [35,36], the disease-relevant functions of the novel IL-1 like cytokines (IL-36 cytokines) remain poorly characterized. We have previously demonstrated that IL-36c is constitutively expressed and inducibly upregulated in the lungs of mice following allergic inflammation [37,38]. Airway epithelial cells express IL- 36a and IL-36c [38,39], and the mRNA expression of IL-36a and IL-36c in airway epithelial cells is increased in response to several inflammatory stimuli [39,40]. IL-36a and IL-36c directly act on IL-36R expressing lung fibroblasts to activate NF-kB and MAPK pathways [39]. We have demonstrated that intratracheal in- stillation of recombinant IL-36c increased airway hyperrespon- siveness, induced neutrophil influx and neutrophil-specific che- mokine production the lungs in vivo [38]. We also demonstrated that IL-36c activated NF-kB in whole lung tissue and alveolar macrophage cell lines [38]. Reports from other groups have demonstrated that the mRNA expression of IL-36a and IL-36c were also increased in human bronchial epithelial cells exposed to Pseudomonas aeruginosa [40] and in recurrent respiratory papillomas [41]. Recent reports have also demonstrated the critical role of IL- 36a, IL-36Ra and IL-36R in the development of psoriasis [42– 45], and that a single amino acid mutation in IL-36Ra can lead to unchecked cytokine production and pustular psoriasis [46]. Accumulating evidence thus points to the existence of a novel, underexplored signaling pathway in inflammatory diseases, driven by the IL-36 cytokine cluster. Despite the recent progress in elucidating the role of IL-36 cytokines in inflammatory diseases, the in vivo role of IL-36a has not been extensively investigated in inflammatory lung disorders. Since multiple cytokines contribute to shaping the overall immune response in inflammatory disorders, it is necessary to characterize the proportional contribution and functional redundancy of individual cytokines to comprehensively understand disease pathogenesis and devise therapeutic strategies. Since it has been demonstrated that the mRNA expression of IL-36a in the airway epithelial cells are increased in response to multiple pro- inflammatory stimuli [39], we hypothesized that the presence of IL-36a in the lungs may induce pulmonary inflammation in vivo. Here we demonstrate that intratracheal instillation of recombinant mouse IL-36a induces neutrophil influx and the production of neutrophil-specific chemokines in wild-type mice as well as mice that lack IL-1a and IL-1b. Furthermore, we also demonstrate that IL-36a acts directly on CD11c+ cells to induce the expression of neutrophil-specific chemokines, T cell costimulatory molecules and the enhancement of CD4+ T cell proliferation. Finally, we also demonstrate that IL-36a activates NF-kB in mouse macrophages. These results suggest that IL-36a acts as a pro-inflammatory cytokine in the lung, and that IL-36a could increase inflammatory responses in disease conditions which involve release of IL-36a into the lungs. Materials and Methods Mice and Procedures Six to eight week old mice (C57BL/6, C3H/HeJ and OTII TCR transgenic strains) were purchased from Jackson Laboratory (Bar Harbor, ME). Mice deficient in IL-1a and IL-1b (IL-1a/b2/ IL-36a and Pulmonary Inflammation 2) were obtained from Dr. Yoichiro Iwakura [47] and backcrossed to C57BL/6 background. All mice were housed or bred in specific pathogen free facility at Massachusetts General Hospital. For intratracheal instillations, mice were anesthetized with a mixture of xylazine (8 mg/kg) and ketamine (45 mg/kg). Recombinant protein preparation or phosphate buffered saline (PBS) controls in a 50 ml volume were intratracheally (i.t.) instilled using a non- invasive method. Mice were euthanized with an overdose of pentobarbital, and tissues were collected at indicated time points following the procedure. For cell isolation protocols that do not require isolation of lung tissue, mice were euthanized by carbon- dioxide asphyxiation. Ethic Statement Mice were housed in a temperature controlled specific-pathogen free facility at Massachusetts General Hospital, and provided unrestricted access to food and water. All protocols involving the use of mice were approved by Subcommittee on Research Animal Care at Massachusetts General Hospital. Generation of Recombinant IL-36a A cDNA clone containing full length mouse Il36a (Il1f6, Clone ID: MMM1013-99829107) was obtained (Open Biosystems). A PCR product encoding a fusion protein comprised of an N- terminal FLAG tag followed by the full length coding sequence of Il36a was generated from this clone using the following restriction- site engineered primers: Forward 59-CGGAATTCCgattacaaggat- gacgatgacaagAATAAGGAGAAAGAACTAAGAG-39 and re- verse 59-AAGGAAAAAAGCGGCCGCTTAATGTACCA- CAATCATCTC-39. The PCR product and pETDuet-1 vector (Clontech) were digested with EcoRI and NotI. The digested PCR product was cloned in-frame with the Histidine purification tags into the pETDuet-1 vector. The ligation reactions were trans- formed into E. coli and positive clones were identified. Expression of His_FLAG_IL-F6 fusion protein was induced in positive clones by addition of 1 mM Isopropyl b-D-1-thiogalactopyranoside (IPTG) to log-phase bacterial cultures for 4 h. Following in- duction, bacterial pellets were frozen at 220uC until protein isolation. IL-36a was purified from bacterial lysate by Ni2+ column chromatography (B-PER 6xHis Fusion Protein Kit, Pierce), and dialyzed against PBS. Purified, dialyzed IL-36a was treated with enterokinase (EK-Max, Invitrogen) to cleave His_FLAG tags from IL-36a protein. Enterokinase was removed from the tag-cleaved IL-36a preparation by treating with EK-Away (Invitrogen), according to the manufacturer’s protocols. The purified, tag- cleaved, enterokinase-removed recombinant IL-36a preparation was dialyzed and the dialyzed preparation was passed through a Ni2+ chromatography column. Cleaved tags bound to the column and the tag-free IL-36a preparations recovered from the flow-through were dialyzed against PBS, treated with polymyxin-B agarose beads (Detoxi-Gel, Pierce) to remove LPS contamination. LPS concentration in the purified IL-36a preparation was ,0.01 EU/mg protein, as measured by Limulus Amebocyte Lysate assay (Lonza). Tag-removed, polymyxin-treated recombinant mouse IL- 36a was used for in vitro and in vivo studies. Coomassie Staining Ten mg of IL-36a was separated on a SDS-polyacrylamide gel under reducing and denaturing conditions. The gel was soaked in Fairbank’s Coomassie solution A (25% isopropanol, 10% acetic acid and 0.05% Coomassie R), microwaved until before boiling and shaken for 10 minutes at room temperature to stain the gel. The stain was drained, and the gel was soaked in distilled water PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45784