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Differentially expressed genes in the blood, spleen, and liver of Peromyscus leucopus and Mus musculus with or without treatment with lipopolysaccharide

Citation

Barbour, Alan; Milovic, Ana; Balderrama-Gutierrez, Gabriela; Mortazavi, Ali (2021), Differentially expressed genes in the blood, spleen, and liver of Peromyscus leucopus and Mus musculus with or without treatment with lipopolysaccharide, Dryad, Dataset, https://doi.org/10.7280/D1VX0C

Abstract

Animals that are competent reservoirs of zoonotic pathogens commonly suffer little morbidity from the infections. To investigate mechanisms of this tolerance of infection, we used single-dose lipopolysaccharide (LPS) as an experimental model of inflammation and compared the responses by two rodents: Peromyscus leucopus, white-footed deermouse and reservoir for agents of Lyme disease and other zoonoses, and the house mouse Mus musculus. Four hours after injection with LPS or saline, blood, spleen and liver samples were collected and subjected to RNA-seq, metabolomics, and specific RT-qPCR. Differential expression analysis was at gene, pathway, and network levels. LPS-treated deermice showed similar signs of sickness as exposed mice and had similar increases in corticosterone levels and expression of interleukin (IL)-6, tumor necrosis factor, IL-1beta, and C-reactive protein. By network analysis the M. musculus response to LPS was characterized as cytokine-associated, while the P. leucopus response was dominated by neutrophil activity terms. In addition, dichotomies in expression of arginase 1 and nitric oxide synthase 2 and of IL-10 and IL-12 were consistent with type M1 macrophage responses in mice and type M2 in deermice.

This dataset comprises the list of differentially expressed genes from the RNA-seq analysis of 20 P. leucopus (10 females and 10 males) and 20 M. musculus (10 females and 10 males) that were inoculated intraperitoneally with 10 mg/kg of E. coli lipopolysaccharide (6 females and 6 males of each species) or with saline alone (4 females or 4 males of each species) at time 0 and then the animals were euthanized at 4 hours. The tissues were then processed for RNA extraction, cDNA library preparations, and sequencing and then analysis as described under Methods. 

Methods

Animals.  Adult outbred P. leucopus of the LL stock were obtained from the Peromyscus Genetic Stock Center (PGSC) of the University of South Carolina (101). The LL stock colony was founded with 38 animals captured near Linville, NC between 1982 and 1985 and has been closed since 1985. Animals were maintained in the AAALAC-accredited U.C. Irvine vivarium with 2-5 animals per cage according to sex and on 12 hours light-12 hours dark lighting schedule, temperature of 21-23° C, humidity of 30-70%, water ad libitum, and a diet of 8604 Teklad Rodent (Harlan Laboratories). 

Single dose LPS comparison. Animals were anesthetized with isoflurane and injected i.p. with a single dose of E. coli O111:B4 LPS at a concentration of 10 mg/kg body weight in a 50 µl volume as described above. The control group was anesthetized and then injected with the 0.9% saline alone. The experiment started at 0800 h with 10 min intervals between animals and with alteration of LPS and control injections. At 4.0 hr after their injection the animals were euthanized. After opening the chest, exsanguination was performed by cardiac puncture and blood was transferred to a heparin sulfate coated tube (Becton-Dickinson Microtainer). Anticoagulated blood was centrifuged to pellet blood cells for 3 min at 4600 x g at 4° C. Plasma and blood pellet was kept separately at -80° C until further analysis. Liver and spleen were extracted, flash-frozen in liquid nitrogen, and stored at -80 °C until RNA extraction. 

RNA extractions. RNA from blood was extracted using NucleoSpin RNA Blood Mini kit (Macherey-Nagel). Lysis buffer and proteinase K were added to the frozen pellet and shaken while thawing. RNA from liver and spleen was extracted from 30 mg of frozen tissues, mechanically homogenized and further lysed in RLT buffer with 2-mercaptoethanol on TissueLyser (Qiagen) with 3 mm beads then extracted according to the protocol using RNeasy Mini Kit (Qiagen).

RNA-seq. Library preparation with the Illumina TruSeq mRNA stranded kit was carried out. The libraries were normalized and then multiplexed to achieve 12 samples per flow cell on an Illumina HiSeq 4000 instrument and 100 or 150 cycles of paired-end read chemistry at the U.C. Irvine Genomic High Throughput Facility. The quality of sequencing reads was analyzed using FastQC (Babraham Bioinformatics). The reads were trimmed of low-quality reads (Phred score of <15) and adapter sequences, and corrected for poor-quality bases using Trimmomatic. After these steps there were overall for all the studies between 45 to 100 million reads for each of the samples. For the comparative study of 40 animals the mean and median numbers, respectively, of reads (x 10^6) for the three tissues were 62.9 and 62.9 for blood, 65.2 and 63.8 for spleen, and 60.5 and 59.4 for liver. Blood and liver RNA-seq were PE100, and spleen RNA-seq was PE150. Paired-end reads in fastq files were quantified using kallisto v. 0.46.1, using the GENCODE annotation v. 21 (https://www.gencodegenes.org) for mouse and GCF_004664715.1_Pero_o.1 _rna from NCBI for P. leucopus.

Differential expression. We used edgeR v. 3.28.1 for differential gene expression (DEG) analysis. Genes were called differentially expressed if their absolute fold-change between conditions was > 4.0 and the false-discovery rate (FDR) was <0.05. To compare fold-changes across species, we merged the output tables from the DEG analyses and retained 14,685 orthologous genes that were synonymously annotated between both species, out of total 24,295 annotated genes for P. leucopus and 35,805 for M. musculus. To screen for DEGs that varied in magnitude by species, we required an absolute fold change of > 5.0 (log2 = 2.5) in one species and < 5.0 in the other. For DEGs designated as "shared" between the species, the absolute fold change was > 5.0 in both.

The BioProject for this experiment is PRJNA643535. The BioSamples numbers for the samples for this experiment are SAMN15445905-SAMN15445964. SRA archive accession numbers for the fastq files of PE100 and PE150 reads are SRR12782328-SRR12782387.

Usage Notes

The data is a single spreadsheet entitled "Table of differentially expressed genes (DEG) of Peromyscus and Mus musculus in response to lipopolysaccharide".  The data are in 6 groups, each occupying four columns from the left to right: DEGs of the blood of P. leucopus, DEGs of the spleen of P. leucopus, DEGs of liver of P. leucopus, DEGs of blood of M. musculus, DEGs of spleen of M. musculus, and DEGs of liver of M. musculus. The four columns in each group are ascending rank by false-discovery rate (FDR) p value, gene or locus name, log2 value of the fold-change of LPS-treated over control animals, and FDR.

Funding

National Institutes of Health, Award: AI-136523