Figure 9. IL-36a directly induced NF-kB activation in mouse macrophage cell lines. A) Cells from a mouse macrophage NF-kB reporter cell line (RAW-ELAM cells) were stimulated with increasing concentrations of IL-36a. Green fluorescent protein (GFP) expression, indicative of NF-kB activation, was increased in a dose-dependent manner upon incubation with IL-36a. Flow cytometry plot presented is representative of triplicate samples in one out of two independent experiments. doi:10.1371/journal.pone.0045784.g009 IL-36a and Pulmonary Inflammation IL-36a acted directly on splenic CD11c+ cells to induce the production of neutrophil-specific chemokines. A recent report demonstrated that IL-36R was expressed in murine BMDCs and CD4+ T cells and that the stimulation of BMDCs with IL-36a, IL- 36b and IL-36c induced the production of multiple cytokines and chemokines [51]. In the current study we demonstrate that IL-36a acts directly on splenic CD11c+ cells to induce the production of the neutrophil-specific chemokines CXCL1, CXCL2 as well as other early response cytokines such as TNFa, IL-1a and IL-1b. Interestingly, the mRNA expression of neither IL-1R1 nor IL-36R was increased in IL-36a stimulated splenic CD11c+ cells under the conditions tested. Since we observed increased IL-36R expression in whole lung tissue following a single IL-36a challenge, we speculate that IL-36a either directly increases IL-36R in non- CD11c+ cells in the lung, or induces the production of another endogenous mediator from an IL-36a responsive, non-CD11c+ cell type in the lungs to increase IL-36R mRNA expression in vivo. Furthermore, we have also demonstrated that similar to splenic CD11c+ cells, alveolar macrophages from na¨ıve mice also express IL-36R mRNA at levels higher than IL-1R1, suggesting that alveolar macrophages are poised to respond to IL-36 cytokines released into the lung airspaces. IL-36a acted directly on CD11c+ cells to induce the expression of co-stimulatory molecules that regulate T cell activation and proliferation. While activation and proliferation of T cells are essential to confer protection against certain pathogens, aberrant activation and proliferation of T cells leads to detrimental responses in lung diseases such as asthma [60]. Apart from T cell receptor activation, engagement of co- stimulatory molecules expressed on T cells (such as CD28 and CD40L) with those expressed on antigen presenting cells (such as CD80, CD86 and CD40) are critical for T cell activation and proliferation [61]. Previous reports have demonstrated that IL-1b induces antigen-specific proliferation of CD4+ T cells during the primary and secondary (memory) immune responses [62], and that CD4+ T cells from IL-1ab2/2 mice are defective in proliferation and the production of T helper 2 (Th2) cytokines [13]. It has also been demonstrated that IL-1a induced CD40 expression in human dendritic cells in vitro [63]. Consistent with a recent report [51], we also found that IL-36a directly induced the mRNA expression of CD40 in CD11c+ cells. Although both CD80 and CD86 mRNA were constitu- tively expressed in CD11c+ cells in our experiments, mRNA expression was not significantly increased 2 h following stimulation with IL-36a. However, we found that the cell surface expression of CD80 and CD86 were increased in splenic CD11c+ cells 24 hours following 2 hours of stimulation with IL- 36a. We also found that CD4+ T cell proliferation responses were enhanced when co-cultured with IL-36a stimulated CD11c+ cells, suggesting that IL-36a mediated upregulation of costimulatory molecules may be important for T cell pro- liferation. Interaction of CD40 with CD40L is important for Th1 cell differentiation [64]. Vigne et al., recently demonstrated that IL-36b (IL-1F8) increased CD40 mRNA expression and enhanced Th1 responses in vivo [51]. Collectively, these data support an important role of novel IL-1 cytokine agonists in regulating T cell activation and proliferation by modulating antigen presentation and co-stimulatory abilities of APCs. Experiments in the current study were performed using splenic CD11c+ cells, which may have different cytokine secretion and antigen-presenting capabilities than CD11c+ cells that reside in or are recruited to the lungs during pulmonary inflammation [65]. We have shown that alveolar macrophages isolated from na¨ıve wild-type mice express IL-36R and IL-1RAcP, suggesting PLOS ONE | www.plosone.org 14 September 2012 | Volume 7 | Issue 9 | e45784