Experimental Design.

<p>Schematic representation of the timing of sensitization by intraperitoneal injection of OVA-Alum; intranasal administration of saline or antigen and PM_2.5 (OVA & PM_2.5); and the antibody injections administered intraperitoneally.</p

Markers of T (helper)h2 inflammation in the lungs.

<p>Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. <b>(A)</b> Legend. <b>(B-F)</b> Bar graphs show mean ± SEM and individual data points for <b>(B)</b> numbers of bronchoalveolar lavage (BAL) eosinophils; <b>(C)</b> mean fluorescent intensity (MFI) of major histocompatibility complex class II (MHCII) of BAL CD11c+ cells; <b>(D)</b> RELMβ; <b>(E)</b> IL-33; and <b>(F)</b> miR-135a expression in the lungs. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p<0.05) calculated with the Mann Whitney U test or the t-test with Welch’s correction (unpaired, two-tailed tests). Numbers below the bars indicate the numbers of mice per group.</p

Markers of Th17 inflammation in the lungs.

<p>Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. <b>(A)</b> Legend. <b>(B-E)</b> Bar graphs show mean ± SEM and individual data points for <b>(B)</b> numbers of BAL neutrophils; <b>(C)</b> S100a8; <b>(D)</b> S100a9; and <b>(E)</b> IL-6 gene expression. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p<0.05) calculated with the Mann Whitney U test or the t-test with Welch’s correction (unpaired, two-tailed tests). Numbers below the bars indicate the numbers of mice per group.</p

Severe thickening of pulmonary arteries and expression of pro-remodeling genes in the lungs.

<p>Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. <b>(A)</b> Legend. <b>(B-D)</b> Bar graphs show mean ± SEM and individual data for <b>(B)</b> severe arterial thickening by histological analysis and for expression of pro-remodeling genes (<b>C</b> RELMα, <b>D</b> MMP12) in the lungs. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p<0.05) calculated with the Mann Whitney U test or the t-test with Welch’s correction (unpaired, two-tailed tests). Numbers below the bars indicate the numbers of mice per group. <b>(E,F)</b> Photomicrographs show sections of OVA-PM2.5 exposed lungs of representative sensitized wild type <b>(E)</b> and B cell KO mice <b>(F</b>, given no injections with antibody). Sections were stained with hematoxylin & eosin. Scale bars indicate 100 μm; arrows point to severely thickened blood vessels. Note the irregular cell patterns in the remodeled vessels.</p

IL-13 & IL-17A response to exposure with OVA-PM_2.5 in wild type and B cell KO mice.

<p>Lymph node and lung tissues from groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. <b>(A)</b> Legend. <b>(B)</b> Representative dot plots were generated by flow cytometry of CD4+ T cells (CD3-CD4-dual-positive) showing staining for IL-13 vs. IL-17A. <b>(C-E)</b> The flow cytometry data were numerically analyzed to calculate the numbers for each cell type (mean ± SEM) per lung draining lymph node. <b>(F-H)</b> Gene expression in the lungs of IL-13, IL-17A, IL-17F is indicated (mean ± SEM) as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p<0.05) calculated with the Mann Whitney U test or the t-test with Welch’s correction (unpaired, two-tailed tests). Numbers below the bars indicate the numbers of mice per group.</p

Right ventricular systolic pressures in wild type and B cell KO mice.

<p>Wild type and B cell KO mice were challenged with saline, or antigen and PM_2.5 (OVA-PM_2.5) intranasally. Data were pooled from 3 experiments; circles represent the data from individual mice, n = 7–8 per group. OVA-PM_2.5 challenged B cell KO control mice were injected with control antibody (open circles) or given no injections (filled circles). Another group of B cell KO mice was injected with antigen specific IgG1 (anti-OVA monoclonal). Right ventricular systolic pressure (RVSP, mmHg) data are shown as box plot of the medians <b>(A)</b> or as bar graph of the numbers of mice with RVSP less or greater than 26 mmHg <b>(B)</b>. The left-most beginning of each horizontal line indicates the group with which pairwise comparisons were made using the Mann-Whitney U test <b>(A)</b> or Fisher’s exact test <b>(B)</b>. Significant P values (P<0.05) are indicated; ns: not significant.</p

OVA-specific IgG1 serum levels in sensitized wild type mice and in B cell KO mice following reconstitution with monoclonal anti-OVA IgG1 antibody.

<p>OVA-specific IgG1 levels were measured in sera of sensitized wild type mice that were either challenged intranasally with saline or OVA-PM, and in sera of sensitized B cell KO mice that were either controls or injected with anti-OVA IgG1 antibody. (A) Correlation of IgG1 serum titers with right ventricular systolic pressures in sensitized wild type mice. Values for P and rs (tie corrected, Spearman’s rank correlation test) are indicated. (B) Bar graphs show (mean ± SEM) and individual data points of OVA-specific IgG1 levels. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p<0.05) calculated with the t-test with Welch’s correction (unpaired, two-tailed). Numbers below the bars indicate the numbers of mice per group.</p

Right ventricular (RV) weight and RV-gene expression.

<p>Groups of wild type and B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. <b>(A)</b> Legend. <b>(B-G)</b> Bar graphs show mean ± SEM and individual data for right ventricular weight calculated relative to <b>(B)</b> the weight of the left ventricle and septum (RV/LV+S), or <b>(C)</b> body weight (RV/BW); and gene expression in the right ventricle of [BNP <b>(D)</b>, IL-33 <b>(E)</b>, RELMα <b>(F)</b>, RELMγ <b>(G)</b>]. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p<0.05) calculated with the Mann Whitney U test (unpaired, two-tailed). Numbers below the bars indicate the numbers of mice per group.</p

List of markers, their regulation, function related to tissue remodeling, and relation to T helper responses.

<p>List of markers, their regulation, function related to tissue remodeling, and relation to T helper responses.</p

Sequences of primers and probes.

<p>Abbreviations: F- forward, R-reverse</p><p>Sequences of primers and probes.</p