TNFα-induced cytokine levels in ASM.

<p>Cytokine levels in supernatant obtained from ASM derived from fatal asthma and non-asthma donors were determined by single ELISA. The data represent means ± standard error from ASM cells of 3 fatal asthma and 3 non-asthma donors for CCL2 and CXCL12; 5 fatal asthma and 6 non-asthma donors for CCL13; and 5 fatal asthma and 5 non-asthma donors for IL8 (numbers based on availability of cells used after RNA-Seq experiments). Each observation was performed in triplicate. Statistical significance was determined by Student’s one-tailed <i>t</i>-test with significance determined at p<0.05.</p

Top differentially expressed genes in fatal asthma-derived ASM at baseline vs. when treated with vitamin D.

<p>FPKM = fragments per kilobase of transcript per million mapped reads. All genes in list have q-value = 1.92E-03, magnitude Log₂(Fold Change) >1.30, and at least two individual samples with FPKM >5.</p

Top differentially expressed genes in fatal asthma- vs. non-asthma-derived ASM at baseline.

<p>FPKM = fragments per kilobase of transcript per million mapped reads. All genes in list have q-value = 1.92E-03, magnitude Log₂(Fold Change) >1.30, and at least two individual samples with FPKM >5.</p

Vitamin D inhibited TNFα-induced cytokine expression.

<p>Although the amount of inhibition differed by cytokine, vitamin D inhibited three cytokine levels to a comparable degree in fatal asthma-derived vs. non-asthma-derived ASM, even for IL8, whose TNFα-induced baseline secretion was significantly higher in fatal asthma- vs. non-asthma-derived ASM. Data represent means ± standard error of the mean for ASM cells derived from 5 fatal asthma donors and 10 non-asthma donors. Each condition was measured in triplicate. Statistical significance was determined using Student’s two-tailed <i>t</i>-test with a p<0.05 threshold. Bottom panels compare baseline TNFα-induced cytokine levels to those obtained with a maximal inhibitory vitamin D concentration (I_max).</p

Differential expression results for all comparison groups corresponding to four cytokine genes that were selected for further study.

<p>Genes were selected based on being (1) members of the <i>GO</i>:<i>0008009~chemokine activity</i> ontological category, which was significantly over-represented among genes differentially expressed in response to vitamin D treatment in both fatal asthma- and non-asthma-derived ASM, that were (2) differentially expressed in fatal asthma-derived vs. non-asthma-derived ASM at baseline. FPKM = fragments per kilobase of transcript per million mapped reads.</p

Top differentially expressed genes in non-asthma-derived ASM at baseline vs. when treated with vitamin D.

<p>FPKM = fragments per kilobase of transcript per million mapped reads. All genes in list have q-value = 1.92E-03, magnitude Log₂(Fold Change) >1.30, and at least two individual samples with FPKM >5.</p

RNA-Seq results expressed as FPKM for four cytokine genes (i.e., <i>CCL2</i>, <i>CCL13</i>, <i>CXCL12</i>, <i>IL8</i>) by condition status.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134057#pone.0134057.t005" target="_blank">Table 5</a> lists q-values and fold changes among comparison groups for these genes.</p

Characteristics of ASM donors.

<p>All donors were white non-smokers. There were no significant differences between sex, age, or BMI of fatal asthma- vs. non-asthma-derived donors. Medical examiner ruled cause of death for fatal asthma donors was “Asthma Attack/Anoxia,” or a significant asthma event is listed as preceding death.</p

Vitamin D and TNFα Responsiveness of <i>CCL2</i>, <i>CCL13</i>, <i>CXCL12</i>, and <i>IL8</i> by qRT-PCR using the <i>GABARAP</i> housekeeping gene as reference.

<p>Vitamin D and TNFα Responsiveness of <i>CCL2</i>, <i>CCL13</i>, <i>CXCL12</i>, and <i>IL8</i> by qRT-PCR using the <i>GABARAP</i> housekeeping gene as reference.</p

<i>CRISPLD2</i> is a GC- and IL1β-responsive gene.

<p>ASM cells were treated with 100<b>A</b>) increased <i>CRISPLD2</i> mRNA expression as measured by qRT-PCR, <b>B</b>) increased <i>CRISPLD2</i> protein expression as measured by immuno-blotting. ASM cells were treated with 5 ng/mL IL1β for 24 h, resulting in <b>C</b>) increased <i>CRISPLD2</i> mRNA expression as measured by qRT-PCR, and <b>D</b>) increased <i>CRISPLD2</i> protein expression as measured by immuno-blotting. <i>CRISPLD2</i> mRNA levels were measured in triplicate. CRISPLD2 protein levels are shown as normalized blot densitometry values, and the error bars are SE values across three independent experiments. * <i>P</i><0.05 (<i>t</i> test).</p