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Pharmacologic IRE1/XBP1s Activation Promotes Systemic Adaptive Remodeling in Obesity

ABSTRACT: RNA was isolated from livers of DIO mice treated with IXA4 or vehicle (n=4 per condition) using the Zymo Research Quick-RNA Miniprep Kit according to the manufacturer’s instructions. RNA sequencing was performed by BGI Americas on the BGI proprietary platform (DNBseq), providing single-end 50 bp reads at 20 million reads per sample. Alignment of the sequencing data to the Mus musculus MGSCv37 NCBI build 37.2 was performed using DNAstar Lasergene SeqManPro. Assembled data were then imported into ArrayStar 12.2 with QSeq (DNAStar Inc) to quantify gene expression and normalized reads per kilobase million (RPKM). Differential expression analysis and statistical significance calculations between conditions were assessed using DESeq in R using a standard negative binomial fit for the aligned counts data and are described relative to the indicated control

ORGANISM(S): Mus musculus

PROVIDER: GSE162567 | GEO | 2022/01/08

REPOSITORIES: GEO

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Project description:Purpose: T. gondii is an obligate intracellular parasite that remodels host transcriptome during infection. How T. gondii provokes these changes is not fully understood. In the present study we found that GRA18 is secreted in the infected host cell and associates with the β-catenin destruction complex to induce β-catenin levels, notably in the host nuclei. Therefore, we hypothesized that GRA18 could modulate the expression of β-catenin-dependent genes. To determine whether GRA18 induces changes in the host cell gene expression, we performed a transcriptomic analysis of BMDMs infected by Wild-Type and gra18 mutant strains of T. gondii. Methods: RNA profiles were generated from BMDMs (BALB/c mice) that were left uninfected or infected with the Pru ku80, Pru ku80 Δgra18, and Pru ku80 Δgra18, GRA18+++ strains (MOI = 6) for 18 hours and subsequently subjected or not to LPS stimulation for 4 hours.Total RNAs were extracted and purified using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy Plus Mini Kit (Qiagen). RNA-sequencing was performed by GENEWIZ (South Plainfield, NJ, USA). The RNA quality was checked with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California, USA) and Illumina TruSEQ RNA library prep and sequencing reagents were used following the manufacturer's recommendations (Illumina, San Diego, CA, USA). The samples were paired-end multiplex sequenced (2 × 125 bp) on the Illumina Hiseq 2500 platform and generated at least 70 million reads for each sample. The RNA-Seq reads (FASTQ) were processed and analyzed using the Lasergene Genomics Suite version 14 (DNASTAR, Madison, WI, USA) using default parameters. The paired-end reads were uploaded onto the SeqMan NGen (version 14, DNASTAR. Madison, WI, USA) platform for reference-based assembly using either the Mus musculus genome package (GRCm38.p3) or the Toxoplasma Type II ME49 strain (ToxoDB-24, ME49 genome) as reference template. The ArrayStar module (version 14, DNASTAR. Madison, WI, USA) was used for normalization, differential gene expression and statistical analysis of uniquely mapped paired-end reads using the default parameters. The expression data quantification and normalization were calculated using the RPKM (Reads Per Kilobase of transcript per Million mapped reads) normalization method. Conclusion: This approach identifies a specific set of genes that are regulated in a GRA18-dependent fashion in BMDM upon T. gondii infection. Particularly, GRA18 promotes the expression of chemokines Ccl17, Ccl22, and Ccl24.

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Pharmacologic IRE1/XBP1s Activation Promotes Systemic Adaptive Remodeling in Obesity

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