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Pathway enrichments from untargeted metabolomics of plasma of Peromyscus leucopus and Mus musculus with or without LPS treatment

Citation

Barbour, Alan; Islam, Nurul; Milovic, Ana; Belisle, John (2021), Pathway enrichments from untargeted metabolomics of plasma of Peromyscus leucopus and Mus musculus with or without LPS treatment, Dryad, Dataset, https://doi.org/10.7280/D1R70J

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. Untargeted metabolomics identified several pathways that deermice and mice have in common in their responses to LPS, but also pathways in which they differed. The accompanying dataset is of 72 pathways which were identified in the plasma of both P. leucopus and M. musculus and those that were enriched in one species or another in response to LPS and those that were not differentially enriched. The Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome.jp/kegg/) terms for the compounds of a particular pathway and that were identified in P. leucopus and/or in M. musculus are listed.

Methods

Adult outbred P. leucopus of the LL stock were obtained from the Peromyscus Genetic Stock Center (PGSC) of the University of South Carolina. The LL stock colony was founded with 38 animals captured near Linville, NC between 1982 and 1985 and has been closed since 1985. Sib-matings are avoided, and complete pedigree records are kept. 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). 

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. 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. 

Untargeted detection and analysis of metabolites in plasma of P. leucopus and M. musculus were carried out. In brief, 40 µl volumes of plasma, which had been stored frozen at -80° C, were extracted with 120 µl methanol containing as internal standards phenylalanine d5 (175 ng/mL), 1-methyl tryptophan (37.5 ng/mL), and arachidonoyl amide (30 ng/mL). After precipitated proteins were removed by centrifugation, the supernatant was dried under vacuum and then suspended in 50% methanol. Aliquots of 10 µl were subjected to high pressure liquid chromatography (HPLC) and quadrupole (Q) time-of-flight (TOF) mass spectrometry (MS) with periodic inclusion of pooled samples for quality control. Metabolites were separated on an Agilent Technologies Poroshell C8 column (100 x 2.1 mm, 2.7 µm) with a gradient of acetonitrile and water both containing 0.1% formic acid with an Agilent 1260 HPLC pump. The eluent was introduced into an Agilent Technologies 6520 Q-TOF-MS instrument equipped with an electrospray ionization source. The parameters for the analysis were a capillary voltage of 4000 V, fragmenter voltage of 120 V, gas at 310° C, gas flow of 10 L/min, and nebulizer pressure of 45 psig. Data were acquired in the positive ion mode at a scan range of 75-1700 for the mass-to-charge ratio (m/z) and at a rate of 1.67 spectra per second. The raw data were deposited at the Metabolomics Workbench repository (https://www.metabolomicsworkbench.org) under Study ID ST001654. 

The raw data files were converted to the XML-based mzML format with Proteowizard. The files were then processed for peak picking, grouping, and retention time correction with the XCMS suite of software. The centWave algorithm was applied to detect chromatographic peaks with these parameter settings: ≤ 30 ppm for m/z deviation in consecutive peaks, signal to noise ratio of 10, a prefilter of 3 scans with peak intensity of ≥ 750, and 10 to 45 s for peak width. Molecular features (MF), defined by m/z and retention time, were grouped across samples using a bandwidth of 15 and overlapping m/z slice of 0.02. Retention time correction was performed with the retcor algorithm of XCMS. Finally, peak area was normalized by median fold normalization method. 

Pathway enrichment analysis was performed with Mummichog network analysis software v. 2, which predicts functional activity from spectral feature tables without a priori identification of metabolites. This was implemented on-line at Metaboanalyst (http://www.metaboanalyst.ca). The parameter settings were mass accuracy of 20 ppm, positive ion mode, and cut-off p value of 0.05 for the Fisher's exact test of observed and expected hits of a given pathway. Pathway enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome.jp/kegg/) terms was determined separately for the P. leucopus and M. musculus sets of LPS-treated and control animals. 

The raw data were deposited at the Metabolomics Workbench repository (https://www.metabolomicsworkbench.org) under Study ID ST001654. Molecular features data for the 40 individual plasma samples was deposited with the Dryad data repository (https://doi.org/10.7280/D12M4N). 

Usage Notes

The dataset is a single Excel spreadsheet entitled "Table. Pathway enrichments from untargeted metabolomics of plasma of Peromyscus leucopus and Mus musculus with or without LPS treatment." The order of the column variables (with abbreviations) in the table from left to right are as follows: pathway, pathway total number of compounds, number of Mus musculus (Mus) hits, Mus significant hits, Mus expected hits, Mus enriched number (the number of observed hits above expected), Mus Fisher exact test (FET) p value, Peromyscus leucopus hits, Peromyscus significant hits, Peromyscus expected hits, Peromyscus enriched number, Peromyscus FET p value, list of KEGG terms for Mus musculus hits, list of KEGG terms for Peromyscus hits.

Funding

National Institutes of Health, Award: AI-136523

U.S. Department of Defense, Award: W81XWH-17-1-0481