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Lung function, airway and peripheral basophils and eosinophils are associated with molecular pharmacogenomic endotypes of steroid response in severe asthma.

ABSTRACT: Lung function, airway and peripheral basophils and eosinophils are associated with molecular pharmacogenomic endotypes of steroid response in severe asthma.

PROVIDER: PRJNA847238 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR19588751_1.fastq.gz	Fastqsanger.gz
	SRR19588751_2.fastq.gz	Fastqsanger.gz
	SRR19588752_1.fastq.gz	Fastqsanger.gz
	SRR19588752_2.fastq.gz	Fastqsanger.gz
	SRR19588753_1.fastq.gz	Fastqsanger.gz

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Eosinophils and basophils

Project description:Ten male 4get mice were infected with N. brasiliensis and single-cell suspensions of total lung tissue were prepared 9 days after infection. Cells were stained for CD3, CD19, c-kit and CCR3 and sorted for CD3-CD19-c-kit-GFP+CCR3+SSChi (eosinophils) and CD3-CD19-c-kit- GFP+CCR3-SSClo (basophils) using a MoFlo high-speed cell sorter (Cytomation, Fort Collins, CO). Total RNA was isolated from 2 x 106 eosinophils and 1.75 x 105 basophils using the Total RNA Isolation Kit (Fluka, Buchs, Switzerland) and amplified by two rounds of in vitro transcription using the Amino Allyl MessageAmpTM aRNA kit (Ambion, Austin, TX). Universal Mouse Reference RNA (#740100; Stratagene, La Jolla, CA) was amplified in the same way. Aminoallyl-UTP was incorporated during the second round of amplification and 5 µg of amplified RNA was coupled to Cy3 and Cy5 fluorescent dyes (CyScribeTM dye labeling kit, Amersham Biosciences, Peapack, NJ). Probes were hybridized to spotted glass oligonucleotide (70-mer) arrays which cover just over 16,400 unique genes (Mouse Genome Set Version 2.0, Qiagen, Germany). The arrays were prehybridized in 1% BSA (Invitrogen), 5 x SSC, 0.1% SDS for 2 hr at 42°C, washed in water and hybridized with the Cy3/Cy5-labeled probes in 2.8 x SSC, 0.2% SDS, 0.6 mg/ml Cot-1 DNA (Invitrogen), and 0.8 mg/ml yeast tRNA, at 63°C for 45 hr. Slides were washed sequentially in 0.03%SDS/1 x SSC, 0.2 x SSC and 0.05 x SSC, dried and scanned on an Axon 4000B scanner using Genepix 3.0 software (Axon Instruments, Inc.). Data were normalized using the R package Bioconductor software and ?lowess? normalization on the pixel medians without background subtraction (Ihaka and Gentleman, 1996). Keywords = Eosinophils Keywords = Basophils Keywords = Nippostrongylus Keywords = Lung Keywords = Type 2 immunity Keywords: other

2004-02-21 | GSE984 | GEO

Lung function, airway and peripheral basophils and eosinophils are associated with molecular pharmacogenomic endotypes of steroid response in severe asthma.

Project description:IntroductionAsthma is a complex disease with heterogeneous expression/severity. There is growing interest in defining asthma endotypes consistently associated with different responses to therapy, focusing on type 2 inflammation (Th2) as a key pathological mechanism. Current asthma endotypes are defined primarily by clinical/laboratory criteria. Each endotype is likely characterised by distinct molecular mechanisms that identify optimal therapies.MethodsWe applied unsupervised (without a priori clinical criteria) principal component analysis on sputum airway cells RNA-sequencing transcriptomic data from 19 asthmatics from the Severe Asthma Research Program at baseline and 6-8 weeks follow-up after a 40 mg dose of intramuscular corticosteroids. We investigated principal components PC1, PC3 for association with 55 clinical variables.ResultsPC3 was associated with baseline Th2 clinical features including blood (rank-sum p=0.0082) and airway (rank-sum p=0.0024) eosinophilia, FEV1 change (Kendall tau-b R=-0.333 (-0.592 to -0.012)) and follow-up FEV1 albuterol response (Kendall tau-b R=0.392 (0.079 to 0.634)). PC1 with blood basophlia (rank-sum p=0.0191). The top 5% genes contributing to PC1, PC3 were enriched for distinct immune system/inflammation ontologies suggesting distinct subject-specific clusters of transcriptomic response to corticosteroids. PC3 association with FEV1 change was reproduced in silico in a comparable independent 14-subject (baseline, 8 weeks after daily inhaled corticosteroids (ICS)) airway epithelial cells microRNAome dataset.ConclusionsTranscriptomic PCs from this unsupervised methodology define molecular pharmacogenomic endotypes that may yield novel biology underlying different subject-specific responses to corticosteroid therapy in asthma, and optimal personalised asthma care. Top contributing genes to these PCs may suggest new therapeutic targets.

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Lung function, airway and peripheral basophils and eosinophils are associated with molecular pharmacogenomic endotypes of steroid response in severe asthma.

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