IL-36a and Pulmonary Inflammation Figure 1. Intratracheal instillation of IL-36a induced neutrophil influx in the lungs of wild-type C57BL/6 mice. A) Coomassie blue stained gel demonstrating the purity of IL-36a preparation. Ten mg IL-36a was loaded on the lane. B) Western immunoblotting of IL-36a protein preparation detects a band around 18 KDa, the predicted molecular weight of mouse IL-36a. C) Cytospin preparations demonstrating neutrophil influx in the BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 mg IL-36a. D) Total cell counts from BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 mg IL-36a. E) Differential cell count percentages and F) Differential cell count numbers in the BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 mg IL-36a. *Indicates significant differences (P,0.05) compared to PBS treated mice. Data represent mean6SEM from 4–5 mice/group. G) Flow cytometry on cells recovered from BAL fluid from mice 24 h following a single i.t. instillation of PBS or 10 mg IL-36a. A majority of cells from the IL-36a instilled lungs were CD11c2CD11b+Ly6G+ neutrophils. Depicted flow cytometry plots are representative of 4–5 mice/group. H) Hematoxylin & Eosin stained lung sections isolated from mice 24 h following a single i.t instillation of PBS or 10 mg IL-36a. Depicted sections are representative of 4–5 mice/group. doi:10.1371/journal.pone.0045784.g001 by gating on live CD4+ cells based on forward- and side-scatter properties. Flow cytometry data were acquired by an Accuri C6 flow cytometer (Accuri) and analyzed using CFlow software (Accuri) or Flowjo (Treestar). Isolation of Alveolar Macrophages from Na¨ıve Mice Na¨ıve C57BL/6 mice were euthanized with an overdose of pentobarbital. Lungs of mice were lavaged three times with 1 mL of ice cold-PBS. Pooled BAL fluids from three mice were centrifuged at 1,500 rpm for 15 minutes. An aliquot of cells were used for cytospin analysis. RNA was isolated from the remaining cells using RNeasy RNA isolation kit (Qiagen). Isolated RNA was treated with DNaseI, reverse transcribed and the resulting cDNA was used as the input for PCR. NF-kB Activation Assays in Mouse Macrophage Cell Lines A mouse macrophage cell line (RAW-ELAM) stably transfected with the NF-kB responsive E-Selectin promoter driving the expression of enhanced green fluorescent protein [48] was used to investigate if IL-36a induced NF-kB activity in mouse macrophages. Cells were cultured in RPMI-1640 with 10% fetal bovine serum and 1% penicillin and streptomycin. One day before the experiments, cells were plated at a density of 56105 cells per well. Media was removed the day of the experiment, and IL-36a mixed with media was added to the cells and incubated for 6 h. After incubation, media was removed and cells were washed twice with ice-cold PBS. Cells were collected, and eGFP fluorescence reflective of NF-kB activity was measured using an Accuri C6 flow cytometer. Statistical Analysis Data from BAL cell enumeration experiments and real-time PCR quantification on lung tissue were analyzed by two-tailed student’s t-test. Real-time PCR data from cell culture experiments were analyzed by two tailed student’s t-test. Lung resistance and compliance measurements were analyzed by two-way repeated measures ANOVA. Statistical significance was accepted at P,0.05. Results IL-36a Induces Neutrophil Influx in the Lungs of Mice We and others have previously demonstrated that IL-36c is predominantly expressed in airway epithelial cells and that i.t. instillation of IL-36c induces neutrophil influx in the lungs of mice [38,39]. IL-36a and IL-36c mRNA expression levels are increased in airway epithelial cells in response to various stimuli [39]. To investigate if IL-36a induces proinflammatory responses in the lung in vivo, we generated pure recombinant murine IL-36a protein (Fig. 1A & 1B). A single i.t. instillation of 10 mg IL-36a induced neutrophil influx (Fig. 1C) in the lungs of wild-type C57BL/6 mice 24 h following instillation. As expected, the majority of cells in the lungs of PBS instilled mice were macrophages (Fig. 1C). The total number of cells (Fig. 1D), percentage of neutrophils (Fig. 1E) and the total number of neutrophils (Fig. 1F) were significantly increased in the lungs of mice that received i.t. IL-36a compared to mice that received PBS. Flow cytometric evaluation of BAL cells also revealed that majority of cells recovered from 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, reflective of macrophages (Fig. 1G). Examination of Hematoxylin & Eosin stained lung sections revealed significant cellular infiltration in the lungs of IL-36a instilled mice (Fig. 1H) compared to the lungs of mice that received PBS. While we used polymyxin-treated, highly pure IL-36a, to ensure that the results we obtained are not due to trace amounts of contaminating LPS from the bacteria-derived IL-36a, we performed the same set of experiments in C3H/HeJ mice, which are unresponsive to LPS due to a spontaneous loss-of-function mutation in Toll like receptor 4 [49]. It has been previously demonstrated that LPS does not induce neutrophil influx or the production of neutrophil-specific chemokines CXCL1 and CXCL2 in the lungs of C3H/HeJ mice [50]. In the current study, a single i.t. instillation of 10 mg IL-36a induced neutrophil influx into the lungs (Fig. S1A), increased the total number of cells (Fig. S1B), percentage of neutrophils (Fig. S1C) and total number of neutrophils (Fig. S1D) in the lungs as assessed from BAL fluid recovered from C3H/HeJ mice 24 h following instillation. Macrophages were the main cell types present in the BAL fluid of PBS instilled C3H/HeJ mice. These results strongly suggest that the neutrophil influx observed in the lungs of mice is due to the agonist activity of IL-36a and not due to contaminants in the protein preparation. Taken together, these data demonstrate that neutrophil influx is the predominant response to overproduction of IL-36a in the lungs. IL-36a Induces the Expression of Early Response Cytokines and Neutrophil Specific Chemokines To determine IL-36a driven molecular mechanisms that induce neutrophil influx into the lungs, we collected lung tissue from wildtype C5BL/6 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 was significantly increased in the lungs of mice which received i.t. instillation of IL-36a, compared to saline controls (Fig. 2A). Similarly, the mRNA expression of IL-1a (Fig. 2B) and IL-b (Fig. 2C) were also significantly increased in the lungs of IL- 36a instilled mice, while the expression of IL-36c was unaltered (Fig. 2D). We did not detect the mRNA expression of other agonist IL-36 cytokines in the lungs under the conditions used (data not shown). While the mRNA expression of the classical IL-1 receptor (IL-1RI) was unchanged (Fig. 2E), the mRNA expression of the IL- 36R, the receptor for IL-36a and IL-36c, was significantly increased in the lungs of IL-36a instilled mice (Fig. 2F). In PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45784