RNA-Seq Transcriptome Profiling Identifies CRISPLD2 as a Glucocorticoid Responsive Gene that Modulates Cytokine Function in Airway Smooth Muscle Cells

Asthma is a chronic inflammatory respiratory disease that affects over 300 million people worldwide. Glucocorticoids are a mainstay therapy for asthma because they exert anti-inflammatory effects in multiple lung tissues, including the airway smooth muscle (ASM). However, the mechanism by which glucocorticoids suppress inflammation in ASM remains poorly understood. Using RNA-Seq, a high-throughput sequencing method, we characterized transcriptomic changes in four primary human ASM cell lines that were treated with dexamethasone—a potent synthetic glucocorticoid (1 µM for 18 hours). Based on a Benjamini-Hochberg corrected p-value <0.05, we identified 316 differentially expressed genes, including both well known (DUSP1, KLF15, PER1, TSC22D3) and less investigated (C7, CCDC69, CRISPLD2) glucocorticoid-responsive genes. CRISPLD2, which encodes a secreted protein previously implicated in lung development and endotoxin regulation, was found to have SNPs that were moderately associated with inhaled corticosteroid resistance and bronchodilator response among asthma patients in two previously conducted genome-wide association studies. Quantitative RT-PCR and Western blotting showed that dexamethasone treatment significantly increased CRISPLD2 mRNA and protein expression in ASM cells. CRISPLD2 expression was also induced by the inflammatory cytokine IL1β, and small interfering RNA-mediated knockdown of CRISPLD2 further increased IL1β-induced expression of IL6 and IL8. Our findings offer a comprehensive view of the effect of a glucocorticoid on the ASM transcriptome and identify CRISPLD2 as an asthma pharmacogenetics candidate gene that regulates anti-inflammatory effects of glucocorticoids in the ASM

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

Intercepting IRE1 kinase-FMRP signaling prevents atherosclerosis progression.

Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis

Intercepting IRE1 kinase-FMRP signaling prevents atherosclerosis progression

Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis.ISSN:1757-4676ISSN:1757-468

Overview and Analysis of Vehicle Automation and Communication Systems from a Motorway Traffic Management Perspective

The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement n. 321132, project TRAMAN21.Summarization: During the last decade, there has been an enormous interdisciplinary effort by the automobile industry and numerous research institutions worldwide towards the development, testing and employment of a variety of Vehicle Automation and Communication Systems (VACS) with the main aims to improve road safety and driver convenience. Some VACS, however, have a direct impact on road efficiency as well and could therefore be exploited to relieve road networks from the significant congestion problems and their negative consequences for travel times, safety, fuel consumption, the environment and the quality of life in general. In other words, some of the available VACS could also be used as novel or innovative sensors, actuators and tools towards a new era of traffic management. This paper provides an overview of proposed and available VACS and discusses their perspectives from the motorway traffic management point of view. Classifications of the different systems in this respect are also provided, while SWOT (Strengths–Weaknesses–Opportunities–Threats) analyses are used to identify specific exploitation ways. Current trends and future perspectives of VACS within a motorway traffic management context are finally summarised.Presented on: Transportation Research Part A: Policy and Practic

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

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

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