Radar graphs of the different mouse strains.

<p>The radar graphs give a visual overview of all results combined for the complete control group (0/0) (blue field) versus the complete TDI-treated group (1/1) (red field). Experimental groups are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012581#pone-0012581-g001" target="_blank">figure 1</a>. The lower limit of each axis is always 0. The upper limit of each axis is the maximum average for a specific parameter measured in one strain and is presented as 100%. AHR  =  area under the curve (AUC) of the airway hyper-reactivity (0–36 AUC of airway resistance) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012581#pone-0012581-g001" target="_blank">figure 1H</a>); BAL cells  =  total BAL cell count (0–18.2×10⁴ cells); Macro  =  total number of BAL macrophages (0–11.5×10⁴ cells) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012581#pone-0012581-g002" target="_blank">figure 2A and B</a>); Neutro  =  total number of BAL neutrophils (0–5.7×10⁴ cells) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012581#pone-0012581-g002" target="_blank">figure 2A and B</a>); Eosino  =  total number of BAL eosinophils (0–1.3×10⁴ cells) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012581#pone-0012581-g002" target="_blank">figure 2A and B</a>); IgE  =  total serum IgE (0–8000 ng/ml) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012581#pone-0012581-g004" target="_blank">figure 4</a>); IFN-γ, IL-10, IL-13 and IL-4  =  cytokines measured in supernatant of cultured auricular lymphocytes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012581#pone-0012581-t001" target="_blank">table 1</a>), IFN-γ (0–3400 pg/ml), IL-10 (0–116 pg/ml), IL-13 (0–530 pg/ml) and IL-4 (0–10.2 pg/ml); CD19⁺  =  CD19⁺ B-lymphocytes per auricular lymph node (0–2.1×10⁶ cells) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012581#pone-0012581-g003" target="_blank">figure 3D</a>); CD8⁺  =  CD3⁺CD8⁺ Tc-lymphocytes per auricular lymph node (0–0.6×10⁶ cells) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012581#pone-0012581-g003" target="_blank">figure 3C</a>); CD4⁺CD25⁺  =  CD3⁺CD4⁺CD25⁺ activated/Treg-lymphocytes per auricular lymph node (0–0.145×10⁶ cells) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012581#pone-0012581-g003" target="_blank">figure 3B</a>); CD4⁺  =  CD3⁺CD4⁺ Th-lymphocytes per auricular lymph node (0–1.8×10⁶ cells) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012581#pone-0012581-g003" target="_blank">figure 3A</a>). For specific significant differences between the 0/0 and the 1/1 group, check the specific graphs.</p

Airway hyperresponsiveness (AHR) in different mouse strains.

<p>The airway resistance (R), after increasing concentrations of methacholine (0–10 mg/ml), was measured 24 hours after the challenge. Figures A) to G) reflect the airway hyperresponsiveness to increasing concentrations of methacholine per mouse strain. Figure H) represents the AUC of R per experimental condition and per mouse strain. Experimental groups are 0/0, 0/1 and 1/1. The first number identifies the agent used for the dermal applications (sensitizations) and the second number identifies the agent used for the oropharyngeal aspiration (challenge). A treatment with toluene-2,4-diisocyanate (TDI) is shown as 1 and a treatment with the vehicle (a mixture of acetone and olive oil), is shown as 0. Data are presented as mean ± S.D. (A–G) and mean with individual values (H), n = 5–11 per group, * p<0.05, ** p<0.01 and *** p<0.001 compared with the 0/0 group, # p<0.05 and ### p<0.001 compared with the 0/1 group.</p

Total serum IgE in different mouse strains.

<p>Total serum IgE was measured 24 hours after the challenge. Experimental groups are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012581#pone-0012581-g001" target="_blank">figure 1</a>. Data are presented as means ± SD, n = 6–14, ** p<0.01 and *** p<0.001 compared to the 0/0 group, ## p<0.01 and ### p<0.001 compared to the 0/1 group.</p

Cytokines in supernatants of lymphocytes obtained from auricular lymph nodes.

<p>Auricular lymph node cells were cultured (42 h) with concanavaline A (2.5 µg/ml). Concentrations (pg/ml) of IL-2 (data not shown), IL-4, IL-10, IL-13, IL-17 (data not shown) and IFN-γ were measured, by Cytometric Bead Array, in the supernatant. Experimental groups are identical to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012581#pone-0012581-g001" target="_blank">figure 1</a>. Data are presented as mean ± S.D., n = 4−11 values per group. * p<0.05, ** p<0.01 and *** p<0.001 compared to the 0/0 group and ^# p<0.05, ^## p<0.01 and ^### p<0.001 compared to the 0/1 group.</p

Lymphocyte subpopulations in the auricular lymph nodes of different mouse strains.

<p>Auricular lymph nodes were collected and FACS analyses were performed. A) CD3⁺CD4⁺ (Th-lymphocytes), B) CD3⁺CD4⁺CD25⁺ (activated/Treg-lymphocytes), C) CD3⁺CD8⁺ (Tc-lymphocytes) and D) CD19⁺ (B-lymphocytes) lymphocytes were characterized. Experimental groups are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012581#pone-0012581-g001" target="_blank">figure 1</a>. Data are presented as means ± S.D., n = 4−9, * p<0.05, ** p<0.01 and *** p<0.001 compared to the 0/0 group, # p<0.05, ## p<0.01 and ### p<0.001 compared to the 0/1 group.</p

Proteomic Alterations in B Lymphocytes of Sensitized Mice in a Model of Chemical-Induced Asthma

<div><p>Introduction and Aim</p><p>The role of B-lymphocytes in chemical-induced asthma is largely unknown. Recent work demonstrated that transferring B lymphocytes from toluene diisocyanate (TDI)-sensitized mice into naïve mice, B cell KO mice and SCID mice, triggered an asthma-like response in these mice after a subsequent TDI-challenge. We applied two-dimensional difference gel electrophoresis (2D-DIGE) to describe the “sensitized signature” of B lymphocytes comparing TDI-sensitized mice with control mice.</p><p>Results</p><p>Sixteen proteins were identified that were significantly up- or down-regulated in B lymphocytes of sensitized mice. Particularly differences in the expression of cyclophilin A, cofilin 1 and zinc finger containing CCHC domain protein 11 could be correlated to the function of B lymphocytes as initiators of T lymphocyte independent asthma-like responses.</p><p>Conclusion</p><p>This study revealed important alterations in the proteome of sensitized B cells in a mouse model of chemical-induced asthma, which will have an important impact on the B cell function.</p></div

Classification of the identified differentially expressed proteins.

<p>The differentially expressed proteins in CD19⁺ B lymphocytes from sensitized versus non-sensitized mice were classified according to biological function (retrieved from Gene Ontology).</p

Representation of the identified differentially expressed proteins on a raw 2D-DIGE gel image.

<p>Representation of the identified differentially expressed proteins on a raw 2D-DIGE gel image.</p

Differentially expressed proteins in CD19⁺ B cells from TDI-sensitized versus non-sensitized mice (p<0.01).

<p>Proteins are ranked according ontology, followed by most upregulated to most downregulated. Individual mass spectra can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138791#pone.0138791.s001" target="_blank">S1</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138791#pone.0138791.s015" target="_blank">S15</a> Figs.</p><p>* pI and Mw are depicted as theoretical values.</p><p>Differentially expressed proteins in CD19⁺ B cells from TDI-sensitized versus non-sensitized mice (p<0.01).</p

Production of an asthma-like response in naïve wild type BALB/c mice after having received B-lymphocytes.

<p>Experimental groups are D_TDIR_Veh, D_TDIR_TDI, D_TMAR_Veh, D_TMAR_TMA and D_TDIR_TMA. D represents donor (D) animals that received dermal applications of TDI (D_TDI) or TMA (D_TMA) on days 1 and 8. Their B-lymphocytes were transferred into naïve recipient (R) mice which received a challenge with vehicle (R_Veh), TDI (R_TDI) or TMA (R_TMA) three days after the transfer. Airway resistance (R), after increasing concentrations of methacholine (0-10 mg/ml), was measured using a forced oscillation technique, 22 hours after the challenge. Figure 2 A reflects the airway hyperreactivity (AHR) to increasing concentrations of methacholine after transferring B-lymphocytes of auricular lymph nodes into wild type BALB/c mice. Macrophages and neutrophils were identified in the BAL fluid (B) and in lung tissue (D) 24 hours after the challenge. Total serum IgE was measured (C). Figure 2 E, F, G and H represent AHR of the experiment assessing the transfer of the B-lymphocytes of TMA sensitized mice, the specificity of the B-lymphocytes to TDI, the transfer of serum and the transfer of B-lymphocytes of the spleen, respectively. Data are presented mean ± SEM, n = 4-10 per group, * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the DTDIRVeh group (A, B, C, G and H) and with DTMARTMA (E). Symbols: (↑) inflammation and (▲) epithelial damage.</p