GSE123180
Deletion of myeloid IRS2 enhances adipose tissue sympathetic nerve function and limits obesity
Aim:  We generated mice lacking Irs2 which mediates signaling pathways from insulin and IL-4 specifically in lyzM Cre expressing cells. We  isolated BMDM from control and Irs2LyzM-/-  mice and compared their differential gene expression under veihicle, IL-4 and LPS treatment by performing mRNA sequencing studies. Methods: BMDM from control and Irs2LyzM-/-  mice were treated for 24 hours with vehicle,  IL-4 (10 ng/ml) or LPS (100 ng/ml). RNA integrity was confirmed and concentration measured using a Bioanalyzer. cDNA libraries were created from up to 5 µg total RNA. There were three (Irs2LyzM-/-) and four (control) biological replicates for each group and each biological replicate was divided into eight technical replicates and sequenced on individual lanes on the Illumina HiSeq 2000 (100 bp paired-end read length) at the Sanger Institute. Raw reads from the sequenced RNAseq libraries were mapped with Tophat splice junction aligner (Kim, Pertea et al. 2013), version 2.0.8 against Ensembl mouse genome reference sequence assembly (mm9) and transcript annotations. All parameters were set to default except inner distance between mate pairs (r = 130). Gene-based read counts were then obtained using the HTSeq count module (version 0.5.4p3). Differential expression analysis was performed on the counts data using the DESeq2 Bioconductor package (version 1.16.1). The GSEA  preranked tool from GSEA version 2.2.4 was used forGeneset enrichment analysis. Gene ontology enrichment analysis was also performed using the topGo Bioconductor package  separately for up and down-regulated genes in the comparisons analysed ( A. Alexa, J. Rahnenfuhrer, topGO: Enrichment Analysis for Gene Ontology. R package version 2.24.0 (2016)). Results: The mRNA seq. results reveal  an anti- inflammatory transcriptional profile  in LPS-treated Irs2LyzM-/-  BMDM  compared to control BMDM. Deletion of Irs2 in BMDM under all conditions (vehicle, IL-4 and LPS) manifest alteratons in genes involved in scavenging catecholamines and supporting increased sympathetic innervation. Overall design: BMDM were isolated from 10 week- old control and Irs2LyzM-/- male mice. RNA was isolated and cDNA libraries were prepared using the standard illumina library preparation protocol.