Figure 3. Intratracheal instillation of IL-36a does not induce airway hypresponsiveness. A) Total lung resistance and B) lung compliance were not significantly different in the lungs of IL-36a challenged mice compared to PBS controls. Airway responses in mice were measured using invasive plethysmography 24 h following i.t instillation of IL-36a or PBS. Data presented are percentage changes from baseline (0mg/mL) measurements. Data represent mean6SEM from 5–7 mice/group. doi:10.1371/journal.pone.0045784.g003 addition, the mRNA expression of the neutrophil recruiting chemokines CXCL1 (Fig. 2G) and CXCL2 (Fig. 2H) were also significantly increased in the lungs of IL-36a instilled mice compared to PBS controls. The protein levels of IL-1a (Fig. 2J) and CXCL1 (Fig. 2L) were significantly increased in the BAL fluid recovered from IL-36a instilled mice, however the protein levels of TNFa and IL-1b were not significantly increased. These results suggest that by inducing the expression of its receptor (IL-36R), IL-36a may potentially contribute to the initiation of a feedback signaling loop to enhance proinflammatory responses. Further- more, these results also suggest that IL-36a acts on structural or immune cells in the lung to induce the production of CXCL1 and CXCL2, leading to neutrophil influx in the lungs. A Single i.t. Instillation of IL-36a does not Induce Airway Hyperresponsiveness in Mice To determine if IL-36a induced proinflammatory effects in the lungs results in differences in physiological responses, wildtype C57BL/6 mice were intratracheally instilled with a single dose of IL-36a. Pulmonary function parameters evaluated 24 h later using invasive plethysmography revealed that the total lung resistance (Fig. 3A) and lung compliance (Fig. 3B) were not significantly different between IL-36a and PBS instilled mice. IL-36a and Pulmonary Inflammation IL-36a Induces Neutrophil Influx in the Lungs of IL-1a and IL-1b Deficient Mice Agonist members of the IL-1 cytokine network often induce overlapping and redundant pro-inflammatory responses. For example, IL-1a and IL-1b can induce neutrophil influx into the lung and induce the production of a variety of proinflammatory mediators that are induced by IL-36a and IL-36c. We have demonstrated that IL-36a and IL-36c induce the production of IL-1a and IL-1b ([38] and the current study). Therefore, to determine if IL-1a and IL-1b may also contribute in part to the IL-36a induced pro-inflammatory responses in the lung, we performed a single i.t. instillation of IL-36a into the lungs of mice genetically deficient in both IL-1a and IL-1b (IL-1ab2/2 mice). A single i.t. instillation of 10 mg IL-36a induced neutrophil influx (Fig. 4A) in the lungs of IL-1ab2/2 mice 24 h following instillation. The majority of cells in the lungs of PBS instilled mice were macrophages. The total number of cells (Fig. 4A), percentage of neutrophils (Fig. 4B) and the total number of neutrophils (Fig. 4C) were significantly increased in the lungs of IL-1ab2/2 mice that received i.t. IL-36a compared to IL-1ab2/2 mice that received PBS. Flow cytometric evaluation of BAL cells also revealed that majority of cells recovered from the BAL fluid of IL-36a instilled mice were CD11c2CD11b+Ly6G+ neutrophils, while a majority of cells recovered from the BAL fluid of PBS instilled mice were CD11c+CD11b2Ly6G2 cells (Fig. 4D). Examination of hematoxylin & eosin stained lung sections also revealed significant cellular infiltration in the lungs of IL-36a instilled mice (Fig. 4E) compared to the lungs of mice that received PBS. These data demonstrate that IL-36a can induce neutrophil influx in the lungs in an IL-1a/b-independent manner. IL-36a Induces the Expression of Early Response Cytokines and Neutrophil Specific Chemokines in the Lungs of IL-1a and IL-1b Deficient Mice To determine if IL-36a induced cytokine and chemokine expression is altered in the absence of IL-1a and IL-1b, we collected lung tissue from IL-1ab2/2 mice 24 h following a single i.t. instillation of IL-36a and profiled the mRNA expression of selected cytokines, chemokines as well as agonists and receptors in the IL-1 cytokine network. The mRNA expression of the early response cytokine TNFa (Fig. 5A) as well as IL-36c Fig. (5B) was significantly increased in the lungs of IL-1ab2/2 mice i.t. instilled with IL-36a, compared to saline controls. As expected, mRNA expression of neither IL-1a nor IL-1b was detected in the lungs of IL-1ab2/2 mice (data not shown). While the mRNA expression of the classical IL-1 receptor (IL-1RI) was unchanged (Fig. 5C), the mRNA expression of IL-36R was significantly increased in the lungs of IL-36a instilled IL-1ab2/2 mice (Fig. 5D). In addition, the mRNA expression of the neutrophil recruiting chemokines CXCL1 (Fig. 5E) and CXCL2 (Fig. 5F) were also increased in the lungs of IL-36a instilled IL-1ab2/2 mice compared to PBS controls. While TNFa protein levels were increased in the BAL fluid recovered from the lungs of IL-36a instilled IL-1ab2/2 mice, the increase was not statistically significant (Fig. 5G). However, the protein levels of CXCL1 were significantly increased in the BAL fluid recovered from the lungs of IL- 36a instilled IL-1ab2/2 mice (Fig. 5H). These results suggest that IL-36a can induce the expression of its receptor (IL-36R) and the neutrophil chemokines CXCL1 and CXCL2 in the absence of both IL-1a and IL-1b. PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e45784