Differential cell counts.

<p>Differential cell counts.</p

Reduced amount of inflammatory cell aggregates in gremlin-1 transgenic lung.

<p>A. Histological sections of gremlin-1 transgenic and wild type lung showing pleural thickening (original magnification 400x) and alveolar space enlargement (original magnification 100x) at 6 months of age. Results of histological scoring are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159010#pone.0159010.t001" target="_blank">Table 1</a>. B. Mice were treated with silica for 2 months and sacrificed at 6 months of age. Collagen 1 (<i>Col1A1</i>), collagen 3 (<i>Col3A1</i>) and tenascin-C (<i>Tnc</i>) mRNA expression levels analyzed from lung tissue RNA using quantitative RT-PCR (n = 4). The results are presented as box blots. The p values were calculated using the Kruskal-Wallis test or the Mann-Whitney U-test when comparing two groups. WT = wild type mice; TG = gremlin-1 transgenic mice. C. Representative histological pictures showing decreased amount of inflammatory cell aggregates in silica-treated gremlin-1 transgenic mice. Original magnification 200x, inset original magnification 400x. D. Immunofluorescence staining of wild type frozen lung tissue sections using CD4 and CD8 T-cell markers. DAPI staining was used to visualize the nuclei. Immunohistochemical staining of CD45R/B220 (brown color) is shown on the right.</p

Top genes differentially regulated in gremlin-1 transgenic lung.

<p>Top genes differentially regulated in gremlin-1 transgenic lung.</p

Generation of gremlin-1 transgenic mice.

<p>A. Schematic representation of the breeding strategy. B. SPC-lox-gremlin-1 mice were crossbread with Rosa26CreERT mice. Part of the mice were treated with tamoxifen for five days before they were sacrificed. Tissue DNA was isolated followed by genotyping for SPC-loxP-gremlin1 (floxed) and R26CreERT2. The recombination event was monitored using primers surrounding the NEO cassette (deleted). Results of SPC-lox-gremlin-1/ R26CreERT2 positive (+/+) and SPC-lox-gremlin-1/- positive (+/-) mice are shown. Expression of gremlin-1 protein was analyzed using Western blotting of lung and kidney tissue lysates. C. Gremlin-1 protein expression was analyzed by immunofluorescence staining of frozen lung tissue sections. Original magnification 200x. WT = wild type mice; TG = gremlin-1 transgenic mice.</p

Histological scoring.

<p>Histological scoring.</p

Gremlin-1 does not alter the overall innate immune response to silica.

<p>A. Immunohistochemical staining of lung tissue sections using CD11b antibody after two-week silica-exposure. Original magnification 200x. Staining scores are indicated below the photomicrographs (mean ± SEM, n = 8). B. Quantitative RT-PCR analyses of macrophage migration inhibitory factor (<i>Mif</i>) and tumor necrosis factor-α (<i>Tnf</i>) after two-week (n = 8) or two-month (n = 4) silica-exposure. The results are presented as box blots. The p values were calculated using the Mann-Whitney U-test. WT = wild type mice; TG = gremlin-1 transgenic mice.</p

Reduced inflammatory gene response to silica.

<p>A. Gene expression microarray was performed using lung tissue mRNA isolated from 6 months old mice (n = 4 in each group). The number of upregulated or downregulated genes are indicated. B. Bubble plots for all immune-related annotations. It compares the most significant Gene Ontology (GO) terms from the “Immune-related Biological Process” ontology found across the different experimental conditions. The same selection strategy was applied for all conditions, which was a significance threshold of 0.05 for the adjusted enrichment p-value, at least five genes from the input list in the enriched category and the whole genome as reference background. C. and D. Quantitative RT-PCR analyses of selected genes identified as differentially expressed in the microarray. The results are presented as box blots. The p values were calculated using the Mann-Whitney U-test (at 2 weeks n = 8; at 2 months n = 4). WT = wild type mice; TG = gremlin-1 transgenic mice.</p

CXCL10 chemokine levels correlate negatively with gremlin-1 levels in mouse and human lung.

<p>A. Inflammatory cytokines in BAL fluid of wild type and gremlin-1 transgenic mice exposed to silica for two weeks were analyzed using a mouse cytokine array. B. Quantification of positive array signals. Mean pixel density of the signal in transgenic BAL fluid is divided by the signal in wild type BAL fluid. The error bars represent standard deviation (n = 2). C. Quantitative RT-PCR analyses of <i>Cxcl10</i> after two-week or two-month silica-exposure. The results are presented as box blots. The p value was calculated using the Mann-Whitney U-test (n = 8). WT = wild type mice; TG = gremlin-1 transgenic mice. D. Quantitative RT-PCR analyses of human gremlin-1 (<i>GREM1</i>) and <i>CXCL10</i> in control (ctrl) and idiopathic pulmonary fibrosis patient (IPF) lung tissue. E. Cultured human fibroblasts isolated from control (ctrl) and IPF patient lung tissue (IPF) were analyzed for <i>GREM1</i> and <i>CXCL10</i> expression by quantitative RT-PCR. The error bars represent standard deviation (n = 3).</p

Gene-gene interactions between NPSR1 and NPS.

<p>Location of six <i>NPSR1</i> SNPs with significant interactions with <i>NPS</i> rs10830123 in the multiplicative model for epistasis in connection with their p-value for interaction and linkage disequilibrium. The most significant interaction signals in BAMSE (green) and MAGIC/ISAAC (orange) are marked in bold. Statistics on LD between the functional rs324981 and other interacting SNPs are provided as D’ and r².</p

Haplotype association of NPS polymorphisms with asthma at 8 years.

<p>Haplotype association of NPS polymorphisms with asthma at 8 years.</p