Asthma-associated SNPs and H3K4me1 (enhancer) enriched regions in the human IL-33R locus of different cell/tissue types.

<p>From top to bottom, using the UCSC genome browser, are displayed: the gene track (genes), all the SNPs not associated with asthma, the SNPs associated with asthma (red are GWAS-identified SNPs, blue are SNPs in linkage disequilibrium), H3K4me1 ChIP-seq track (green) for different cell/tissue types (named on the left) underlined by the corresponding peak-calling track (black boxes). For the blood CD4+ T cells, peak calling tracks from seven samples/cell-types are displayed. The red box shows an H3K4me1 peak that is present only in CD4+ T cells.</p

Asthma SNPs enrichment in H3K27Ac of different tissues.

<p>Asthma SNPs enrichment in H3K27Ac of different tissues.</p

Distribution of the asthma-associated SNPs in TFBSs.

<p>Two sets of asthma-associated SNPs are shown. Asthma-associated SNPs that belong to any of H3K4me1 peaks called in this study are shown on the left. On the right, the distribution of asthma-associated SNPs that are located in any of H3K4me1 peaks. The distribution of SNPs into overlapping and non-overlapping TFBSs was done using TFBSs by ChIP-seq dataset from the ENCODE (Release 2) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054359#pone.0054359-The2" target="_blank">[55]</a>. The TFBSs ChIP-seq data were obtained from UCSC Genome Browser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054359#pone.0054359-Karolchik1" target="_blank">[34]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054359#pone.0054359-Karolchik2" target="_blank">[35]</a>.</p

Distribution of the SNPs in coding, 5′-UTR, 3′-UTR, introns and intergenic regions.

<p>Three sets of SNPs are shown. SNPs identified as being significantly associated with asthma according to the GWAS Integrator database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054359#pone.0054359-Yu1" target="_blank">[33]</a> are shown on the left. The middle shows the same set of SNPs extended by those that are in tight genetic linkage (r² = 0.8) according to HaploReg <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054359#pone.0054359-Ward1" target="_blank">[30]</a>. On the right, the distribution of common SNPs that are not associated with asthma is shown. The distribution of SNPs into coding, 5′-UTR, 3′-UTR, introns, and intergenic regions was done using RefSeq datasets from the UCSC Genome Browser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054359#pone.0054359-Karolchik1" target="_blank">[34]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054359#pone.0054359-Karolchik2" target="_blank">[35]</a>.</p

Distribution of enhancers in asthma-associated SNPs for different cell types.

<p>Plotted is the enrichment of asthma-associated SNPs compared to background SNPs in genomic regions in which there are CD4+ T enhancers, and anywhere from –0 to 7 additional cell types that also have a peak in that region.</p

The location of enhancers is cell-type specific.

<p>The plot depicts pairwise comparisons of the location of enhancers in different datasets using Matthew Correlation Coefficients (MCC). Black indicates a high correlation between enhancers in two cell types. The 37 studied datasets form distinct clusters that correspond to different cell- or tissue types.</p

Asthma SNPs enrichment in enhancers of different tissues.

<p>Asthma SNPs enrichment in enhancers of different tissues.</p

Asthma-associated SNPs and H3K4me1-enriched regions (enhancers) in the human Th2 cytokine locus of different cells and tissue types.

<p>From top to bottom, using the UCSC genome browser, are displayed: the conserved DNAse hypersensitivity regions identified in mouse T cells (HS regions), the gene track (genes), all the SNPs not associated with asthma, the SNPs associated with asthma, the species conservation track, the H3K4me1 ChIP-seq track (green) for the different cell and tissue types (named on the left) underlined by corresponding peak calling track (black boxes). For the blood CD4+ T cells, peak calling tracks from seven samples/cell-types are displayed. The red boxes show H3K4me1 peaks that are present only in CD4+ T cells (LCRO and HSV).</p

Effects of EGF on primary bronchial epithelial cell (PBEC) proliferation and mediating neutrophil chemotaxis.

<p>(A) PBECs were exposed to EGF and proliferation was assessed using a colorimetric assay based on methylene blue uptake and release (<i>see Results section; pilot experiments</i>). The stimulation index derived by dividing the methylene blue uptake of cells exposed to EGF (10 ng/ml, 24 h) by the uptake of unexposed cells were <1 in all subject groups, suggesting that, EGF did not increase the numbers of epithelial cells (<i>n</i> = 3–5 donors from each group). (B) Dose-dependency of chemotactic responses to PBEC conditioned media. PBECs were stimulated with EGF (10 ng/ml) to generate EGF-CM or left untreated (Basal-CM). Neutrophil chemotaxis induced by the CM was compared to SFM as described in <i>Methods</i>. The chemotactic activity was concentration-dependent with a maximal effect observed when using neat CM (<i>n</i> = 3 donors).</p

Neutrophil chemotactic and anti-apoptotic activity generated by EGF-induced bronchial epithelial cells.

<p>Conditioned media (CM) from 9 healthy control subjects, 8 mild and 11 Mod/Sev asthmatics were assessed for neutrophil chemotactic and anti-apoptotic activity as described in <i>Methods.</i> (A) Neutrophil chemotaxis was significantly induced by stimulation of PBECs with EGF in HC (p = 0.021) and Mod/Sev asthma (p = 0.010) but not in MA (0.093). The chemotactic activity in EGF-CM was related to the clinical severity of asthma (linear contrast test p = 0.010) (<i>n</i> = 8 donors). (B) Neutrophil apoptosis was assessed after 20 h of culture by FACS analysis of Annexin-V^FITC/PI staining and both basal and EGF-CM from the asthma-derived PBECs, but not from HC, contained significant neutrophil anti-apoptotic activity when compared to SFM (MA, p = 0.047 and Mod/Sev, p = 0.014; HC, p = 0.603, respectively). The anti-apoptotic activity in basal-CM and EGF-CM increased in relation to disease severity (p = 0.046 and 0.009, respectively, as assessed by trend analysis), (<i>n</i> = 7 donors). Repeated one-way ANOVA with post-hoc Bonferroni; ^†p<0.05, ^††p<0.01 and ^†††p<0.001 compared to SFM-treated neutrophils. *p<0.05 <i>vs.</i> basal-CM.</p