IL-36a and Pulmonary Inflammation 36a. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. *Indicates significant differences (P,0.05) compared to 0 mg/mL group. Data represent mean6SD of quadruplicate samples from one of two representative experiments. I) Diff-quik stained cells from cytospun BAL cells from the lungs of na¨ıve mice demonstrates that the majority of lung resident immune cells are alveolar macrophages. J) PCR on cDNA from na¨ıve mouse alveolar macrophages demonstrating the constitutive mRNA expression of an endogenous control (b-actin), IL-1R1, IL-36R and IL-1RAcP. Image of a DNA electrophoresis gel has been color-inverted for clarity. bp – base pairs. doi:10.1371/journal.pone.0045784.g006 above were obtained using splenic CD11c+ cells, we also demonstrate that alveolar macrophages isolated from the lungs of na¨ıve wild-type mice (Fig. 6I) express IL-1R1, IL-36R and IL- 1RAcP (Fig. 6J). In addition to previous reports demonstrating IL- 36R expression in BMDCs and T cells, here we show that alveolar macrophages also have the molecular machinery to respond to IL- 36 cytokines. Further studies are required to determine if other cells that infiltrate the lung during an immune response also express IL-36R. IL-36a Induces the Expression of co-stimulatory Molecules in Splenic CD11c+ Cells and Enhances CD4+ T Cell Proliferation Activation and excessive proliferation of T cells is a critical component in the pathogenesis of a multitude of inflammatory disorders, including lung disorders such as asthma. A recent report demonstrated that CD4+ T cells and CD11c+ cells express IL-36R, the receptor for IL-36a and the other IL-36 cytokines [51]. To determine if IL-36a regulated T cell proliferation by modulating costimulatory molecule expression in APCs, we incubated splenic CD11c+ cells with increasing concentrations of IL-36a and measured the mRNA expression of costimulatory molecules at earlier timepoints and cell surface expression of the costimulatory molecules at the later timepoints. While incubation of CD11c+ cells with IL-36a did not induce the mRNA expression of CD80 (Fig. 7A) or CD86 (Fig. 7B) in CD11c+ cells 2 h following incubation, the expression of the co-stimulatory molecule CD40 was significantly increased in a dose-dependent manner (Fig. 7C). Incubation of splenic CD11c+ cells with IL-36a for 2 h was sufficient to induce a modest, but dose-dependent increase in the percentage of CD80+CD86+ cells 24 hours later (Fig. 7D). Furthermore, incubation of splenic CD11c+ cells with IL-36a for 2 h also significantly increased cell surface CD80 expression and a modest, but significant increase in cell surface CD86 expression 24 hours later (Fig. 7E). In addition, the proliferation of CD4+ T cells was proportionally increased when co-cultured with CD11c+ cells pre-incubated with increasing concentrations of IL-36a (Fig. 8A). Antigen-specific CD4+ T cell proliferation was also proportionally increased when CD4+ T cells from OVA-specific OTII TCR transgenic mice were co-cultured with CD11c+ cells pre-incubated with increasing concentrations of IL-36a and a fixed concentration of OVA323–339 peptide, the cognate antigen. Since the IL-36a containing media was removed from the CD11c+ cells before addition of CD4+ cells, these data suggest that IL-36a induced expression of costimulatory molecules or mediators may indirectly contribute to T cell proliferation responses. IL-36a Induces the Activation of NF-kB in Mouse Macrophage Cell Lines Previous studies have reported that agonist IL-36 cytokines such as IL-36a and IL-36c induce NF-kB activation in Jurkat cells transfected with IL-36R [31]. We have previously demonstrated that IL-36c induces the activation of NF-kB in macrophage cell lines in a dose-dependent manner [38]. Here, we demonstrate that similar to IL-36c, IL-36a also induces the activation of NF-kB in a mouse macrophage cell line (Fig. 9). These data suggest that IL- 36a induced NF-kB activity may be a critical mechanism in driving the production of proinflammatory mediators by macro- phages and proliferation of T cells by enhancing the stimulatory properties of dendritic cells. Discussion The current study demonstrates that IL-36a acts as a pro- inflammatory cytokine in the lungs independent of both IL-1a and IL-1b. Intratracheal instillation of IL-36a induced neutro- phil influx and increased the expression of pro-inflammatory cytokines and chemokines in the lungs of wild-type C57BL/6 mice as well as mice deficient in both IL-1a and IL-1b. IL-36a acted directly on CD11c+ antigen presenting cells to induce the production of early response cytokines and neutrophil-specific chemokines. In addition, IL-36a increased the expression of T cell co-stimulatory molecules on CD11c+ cells and enhanced their ability to stimulate CD4+ T cell proliferation. Stimulation of a macrophage cells line with IL-36a induced NF-kB activation. Collectively, these data suggest that disease-induced overproduction of IL-36a in the lungs may play an important role in inflammatory lung disease. Intratracheal instillation of IL-36a induced neutrophil influx in the lungs of wildtype and IL-1ab2/2 mice. Transgenic mice that conditional overexpress IL-1b in the airway epithelium have neutrophil influx, increased mucus production and the develop- ment of emphysematous and fibrotic changes in the lung [52]. Prior reports also demonstrated that intratracheal administration of IL-1 induced neutrophil influx into the lungs of mice [53], and that IL-1 is necessary for lipopolysaccharide (LPS) mediated neutrophil influx in the murine lung [54,55]. We previously reported that intratracheal administration of IL-36c also induced neutrophil influx into the lungs and the expression of neutrophil- specific chemokines [38]. Here we show that IL-36a also resulted in neutrophil chemotaxis in the lungs, along with an increase in the mRNA expression of the classical IL-1 agonists IL-1a and IL- 1b in wild-type mice. This supports our previous findings [38] and those of others [39] demonstrating that agonist IL-36 cytokines induce the production of classical IL-1 cytokines. Importantly, in the current study intratracheal instillation of IL-36a induced neutrophil influx into the lungs of IL-1ab2/2 mice, suggesting that IL-36a driven neutrophil recruitment to the lungs is independent of IL-1a and IL-1b. The mRNA expression of early response cytokines such as TNFa, IL-1a and IL-1b were increased in the lungs of wildtype mice following intratracheal instillation of IL-36a. These results are consistent with our previous reports demonstrating that i.t. instillation of IL-36c into the lungs of mice induced the production of IL-1a in a dose-dependent manner [38]. While the mRNA expression of IL-1R1 was unchanged, the expression of IL-36R was significantly increased in the lungs of IL-36a treated mice, demonstrating that IL-36a can induce the expression of its own receptor in lung tissue either by directly acting on structural or immune cells in the lung, or indirectly by inducing the production of other mediators in the lung that increase IL-36R expression. Since the expression of IL-36R was also induced in the lungs of IL- 36a instilled IL-1ab2/2 mice, it is possible to speculate that neither IL-1a nor IL-1b contribute to IL-36a driven IL-36R expression. IL-1 induced the mRNA expression of multiple PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e45784