IL-36a and Pulmonary Inflammation Figure 5. Intratracheal instillation of IL-36a increased the mRNA expression of proinflammatory mediators in the lungs of IL-1ab2/2 mice. A–F) Transcript expression of early response cytokines (TNFa and IL-36c), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R and the neutrophil specific chemokines CXCL1 and CXCL2 in the lungs of IL-1ab2/2 mice 24 h following a single i.t instillation of PBS or 10 mg IL-36a. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. G–H) Protein expression of TNFa and CXCL1 in the BAL fluid recovered from IL-1ab2/2 mice 24 h following a single i.t instillation of PBS or 10 mg IL-36a. Protein expression was quantified by multiplexed cytometric bead arrays. *Indicates significant differences (P,0.05) compared to PBS treated mice. Data represent mean6SEM from 3–4 mice/group. doi:10.1371/journal.pone.0045784.g005 IL-36a Induces the mRNA Expression of Early Response Cytokines and Neutrophil-specific Chemokines in Splenic CD11c+ Cells IL-36a and IL-36c have been reported to be expressed and inducibly upregulated in airway epithelial cells in response to various stimuli [39]. In addition, recent reports demonstrated that bone marrow derived dendritic cells express IL-36R, the receptor for IL-36a and IL-36c [51]. Since more than 95% of immune cells in a na¨ıve mouse lung are CD11c+ alveolar macrophages, we hypothesized that under inflammatory conditions, overproduced IL-36a may activate CD11c+ cells, leading to the production of inflammatory mediators that orchestrate the cellular immune responses in the lung. To determine if IL-36a has direct effects on cytokine production and antigen-presenting abilities of CD11c+ cells, we first isolated splenic CD11c+ cells and incubated them with increasing concentrations of IL-36a for 2 h. The mRNA expression of TNFa (Fig. 6A), IL-36c (Fig. 6B), IL-1a (Fig. 6C) and IL-1b (Fig. 6D) were increased in a dose-dependent manner upon incubation with IL-36a. However, the expression of neither IL-1RI (Fig. 6E) nor IL-36R (Fig. 6F) was increased in CD11c+ cells upon incubation with IL-36a. It is important to note that the baseline expression of IL-36R in CD11c+ cells was relatively high (,0.3 copies/copy GAPDH, Fig. 6F), suggesting that CD11c+ cells may readily respond to IL-36a and IL-36c. In addition, IL-36a also induced a dose-dependent increase in the mRNA expression of neutrophil recruiting chemokines such as CXCL1 (Fig. 6G) and CXCL2 (Fig. 6H) in CD11c+ cells. These data also demonstrate that IL-36a directly acts on CD11c+ cells to induce the production of neutrophil recruiting chemokines, elucidating a molecular mechanism that potentially drives the influx of neutrophils to the lung following i.t. instillation of IL-36a. While the results described PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e45784