Vitamin D Modulates Expression of the Airway Smooth Muscle Transcriptome in Fatal Asthma

Globally, asthma is a chronic inflammatory respiratory disease affecting over 300 million people. Some asthma patients remain poorly controlled by conventional therapies and experience more life-threatening exacerbations. Vitamin D, as an adjunct therapy, may improve disease control in severe asthma patients since vitamin D enhances glucocorticoid responsiveness and mitigates airway smooth muscle (ASM) hyperplasia. We sought to characterize differences in transcriptome responsiveness to vitamin D between fatal asthma- and non-asthma-derived ASM by using RNA-Seq to measure ASM transcript expression in five donors with fatal asthma and ten non-asthma-derived donors at baseline and with vitamin D treatment. Based on a Benjamini-Hochberg corrected p-value <0.05, 838 genes were differentially expressed in fatal asthma vs. non-asthma-derived ASM at baseline, and vitamin D treatment compared to baseline conditions induced differential expression of 711 and 867 genes in fatal asthma- and non-asthma-derived ASM, respectively. Functional gene categories that were highly represented in all groups included extracellular matrix, and responses to steroid hormone stimuli and wounding. Genes differentially expressed by vitamin D also included cytokine and chemokine activity categories. Follow-up qPCR and individual analyte ELISA experiments were conducted for four cytokines (i.e. CCL2, CCL13, CXCL12, IL8) to measure TNFα-induced changes by asthma status and vitamin D treatment. Vitamin D inhibited TNFα-induced IL8 protein secretion levels to a comparable degree in fatal asthma- and non-asthma-derived ASM even though IL8 had significantly higher baseline levels in fatal asthma-derived ASM. Our findings identify vitamin D-specific gene targets and provide transcriptomic data to explore differences in the ASM of fatal asthma- and non-asthma-derived donors

Nuclear morphology predicts cell survival to cisplatin chemotherapy

The emergence of chemotherapy resistance drives cancer lethality in cancer patients, with treatment initially reducing overall tumor burden followed by resistant recurrent disease. While molecular mechanisms underlying resistance phenotypes have been explored, less is known about the cell biological characteristics of cancer cells that survive to eventually seed the recurrence. To identify the unique phenotypic characteristics associated with survival upon chemotherapy exposure, we characterized nuclear morphology and function as prostate cancer cells recovered following cisplatin treatment. Cells that survived in the days and weeks after treatment and resisted therapy-induced cell death showed increasing cell size and nuclear size, enabled by continuous endocycling resulting in repeated whole genome doubling. We further found that cells that survive after therapy release were predominantly mononucleated and likely employ more efficient DNA damage repair. Finally, we show that surviving cancer cells exhibit a distinct nucleolar phenotype and increased rRNA levels. These data support a paradigm where soon after therapy release, the treated population mostly contains cells with a high level of widespread and catastrophic DNA damage that leads to apoptosis, while the minority of cells that have successful DDR are more likely to access a pro-survival state. These findings are consistent with accession of the polyaneuploid cancer cell (PACC) state, a recently described mechanism of therapy resistance and tumor recurrence. Our findings demonstrate the fate of cancer cells following cisplatin treatment and define key cell phenotypic characteristics of the PACC state. This work is essential for understanding and, ultimately, targeting cancer resistance and recurrence

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

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- 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

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 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

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