Upregulation and role of Ifng in RWE-induced allergic inflammation.

<p>(<b>A</b>) Sensitized Balb/c mice were challenged with either PBS (◊) or RWE (▪). Quantitative PCR was done on lung RNA four hours after challenge. (<b>B</b>) Sensitized WT or Ifng KO mice were challenged with either PBS or RWE. BAL was performed at different time points and eosinophils counted on Wright-Giemsa-stained cytospin slides. Data are represented as mean±SEM; * = P<0.05.</p

RWE-induced upregulation of GTPases, Socs1 and Gadd45g.

<p>(<b>A</b>) Sensitized Balb/c mice were challenged with either PBS (◊) or RWE (▪). Lungs were harvested from mice sacrificed at different time points. Total RNA was extracted from the lungs and subjected to real-time quantitative PCR analyses using gene-specific primers. Transcript abundance is presented here as a function of time after normalizing to 10⁶ β-actin transcripts. Data are represented as mean±SEM; * = P<0.05. (<b>B</b>) Schematic representation of the time frame for which the Th2 genes (upper group) and Th1 genes (lower group) remain upregulated after RWE challenge.</p

Heat map of genes significantly altered in the lungs by RWE challenge.

<p>Sensitized Balb/c mice were challenged with either PBS (n = 3) or RWE (n = 4) and gene micro-array was performed 4 hours post-challenge. Student's t-test was performed on the raw data from the micro-array analysis using the S+ Array Analyzer software. This analysis, at a P<0.01, identified 352 genes that were differentially expressed between the PBS and RWE challenge groups. These 352 genes were analyzed using Spotfire to generate this heat map. The numbers below the lanes represent data from individual mice.</p

Th2-associated genes upregulated by RWE challenge.

<p>Fold change analysis was done using the S+ Array Analyzer software.</p

A Th1-differentiating environment augments upregulation of Iigp, Gbp1, Gadd45g and Socs1.

<p>(<b>A</b>) Sensitized Balb/c mice were challenged with either PBS or RWE. To generate Th1- differentiating conditions, mice were pre-treated intranasally with either Il12 or CpG DNA 16 or 48 hrs before RWE challenge, respectively. Mice were sacrificed 4 hrs after challenge and quantitative PCR analysis was performed and analyzed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008172#pone-0008172-g001" target="_blank">Fig. 1A</a>. (<b>B</b>) Naïve Balb/c mice were treated intranasally with PBS, CpG or GpC DNA. Mice were sacrificed 48 hours later and quantitative PCR was performed on the lungs. Data are represented as mean±SEM; * = P<0.05.</p

Processes, functions and pathways altered by RWE challenge.

<p>Genes altered by RWE challenge were analyzed using the Ingenuity Pathway Analysis (IPA) knowledge database. Cellular Processes (A) and Immune Response (B) were the top two processes affected. Bar graphs show functions/pathways within each process significantly altered by RWE challenge (P<0.05). The Y-axis denotes the significance of altering that particular function; a higher bar means greater significance. The yellow line is the cut-off for the significance level and coincides with a P value of 0.05. The number above each bar denotes the number of genes in that particular function that were significantlyaltered by RWE challenge (P<0.01).</p

Biological processes altered by RWE challenge.

<p>Genes altered by RWE challenge were analyzed using the Ingenuity Pathway Analysis knowledge database and grouped into biological processes as shown.</p

AR inhibition prevents phosphorylation of STAT-6 in mouse lung epithelium.

<p>The mice were sensitized and challenged with PBS or RWE, without or with AR inhibitor and 20 h later lungs were perfused and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 5 µm. The sections were immunostained with p-STAT-6 specific antibodies using immunoflouroscence secondary antibodies (<b>A</b>) or DAB –based HRP conjugated antibodies counterstained with hematoxylin and eosin (<b>B</b>). Photomicrographs were acquired by fluorescence or light microscopes. A representative field for each group is shown (magnification: 200×). In (A) inset shows magnified view of the selected regions from representative photomicrographs (n = 4). RWE, ragweed pollen extract; ARI, aldose reductase inhibitor.</p

Inhibition of AR prevents IL-13-induced ROS production in SAEC.

<p>(<b>A</b>) Approximately 5×10⁴ cells were seeded on 2-chambered slides and starved in serum-free basal medium without or with fidarestat for overnight. The cells were washed with 1× HBSS and incubated with 10 µM H₂DCF-DA at 37°C for 30 min, washed again and treated with IL-13 (25 ng/ml) for 1 h. The cells were washed with cold 1× HBSS twice and mounted using floursave mounting medium with DAPI. Photomicrographs were acquired using a fluorescence microscope (Nikon). A representative image is shown (n = 4); Magnification 400×. (<b>B</b>) Approximately 10,000 SAEC were plated per well in a 96-well plate and serum-starved for 24 h without or with fidarestat. The cells were washed with 1× HBSS and incubated with 10 µM H₂DCF-DA at 37°C for 30 min. Cells were washed again to remove excess H₂DCF-DA and treated with Il-13 (25 ng/ml) in basal media for 1 h. At the end of the treatment, cells were washed twice with HBSS and fluorescence was determined at 485 nm excitation and 538 nm emission wavelengths. Relative ROS production is expressed as mean fluorescence intensity (MFI) (arbitrary units). The bars represent mean ± SD (n = 4–6); (^*<i>p</i><0.01 vs. Control; ^**<i>p</i><0.05 vs. IL-13).</p

Inhibition or deficiency of AR prevents RWE-induced goblet cell metaplasia in mice lungs.

<p>RWE-sensitized normal and AR-null mice were challenged with RWE and 72 h later the lungs were harvested from mice treated without or with AR inhibitor (<b>A</b>) or AR-null mice (<b>B</b>), perfused and fixed with 4% paraformaldehyde and embedded in paraffin. The sections were stained with PAS stain and observed under light microscope and photomicrographs were acquired. A representative photomicrograph from each group is shown (n = 4). Magnification 200× (A); 400× (B).</p