DOI: 10.57745/z71cgc
title: Serum metabolomics of young and aged mice infected with influenza virus at day 0 and at day 7 post-infection

description: Data correspond to a metabolomic analysis of serum samples collected from young and aged mice infected with influenza virus at day 0 and at day 7 pos-infection, and are included in a publication entitled “Impact of Aging on Gut-Lung-Adipose Tissue Interactions and Lipid Metabolism during Influenza Infection in Mice”. Influenza remains a major threat to human health, especially for the elderly. Aging leads to substantial changes to lung function, gut microbiota, and white adipose tissue (WAT)—a key endocrine organ regulating energy balance and lipid metabolism. In the current study, we performed a multi-omics analysis to investigate how influenza impacts the gut-lung-adipose tissue axis differently with age. Compared to young-adult mice, aged mice experienced more severe short- and long-term outcomes following infection, along with distinct WAT alterations, including impaired browning, heightened inflammation, and reduced innate immune cell recruitment. Age-related differences were also evident in infection-driven shifts in gut microbiota. Akkermansia levels increased in young mice but not in aged mice, while Faecalibaculum and Muribaculum expanded only in aged mice and were correlated with lung pathology. Serum metabolomics also revealed age-dependent metabolic responses to infection. Compared to their non-infected counterparts, young mice had lower levels of p-Cresol-sulfate and Indoxyl-sulfate alongside higher triglycerides, whereas aged mice showed disrupted glycerophospholipid metabolism. By pinpointing specific gut bacteria as potential probiotics and identifying lipid pathways associated with disease progression, these findings could lead to the development of targeted, age-specific strategies to mitigate influenza severity in the elderly. Animals and ethics statement C57BL/6JRj male mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France). Young-adult mice (2-month-old) and aged mice (18-month-old) had ad libitum access to rodent chow (D12450B, with 10% kcal% fat, Research Diets, New Brunswick, NJ, USA) and water throughout experiments. All animal studies were performed in accordance with the ARRIVE guidelines recommendations and the care, use, and treatment of mice were in agreement with current national and institutional regulations and ethical guidelines (Animal Experimentation and High Technology Platform of the Institut Pasteur de Lille, Agreement # E59-350009). All mouse procedures were approved by the institutional regional ethics committee “Comité d’Ethique en Experimentation Animale” (CEEA)75. Animal studies were authorized by the French Ministry of Education, Research and Innovation (protocol APAFIS#: 2020013113414570_v2). Sample collection After a 7-day acclimatation in the BSL2 facility, mice were anesthetized with ketamine (1.3 mg) (Imalgene1000, Boehringer Ingelheim Animal Health France SCS, Lyon, France) and xylazine (0.26 mg) (Rompun 2%, Elanco GmbH, Cuxhaven, Germany) in PBS (100 µL) via intraperitoneal injection, then intranasally inoculated with either a sublethal dose (50 PFUs, diluted in 50 µL PBS) of human-derived, mouse-adapted influenza A/Scotland/20/1974 (H3N2) (infected groups) or PBS (mock-treated groups). Mice were monitored daily for 28 days post-infection (dpi) for signs of morbidity and changes in body weight. At specified sacrifice days (0, 2, 4, 7, 14, and 28 dpi), mice were euthanized with an intraperitoneal injection of euthasol (40 mg/kg), and blood, lungs, adipose tissues (subcutaneous (inguinal) adipose tissue (SCAT) and visceral (epididymal) adipose tissue (VAT)), and caecal contents were collected. Lung and adipose tissue samples were either snap-frozen for gene expression, embedded in paraffin for histology, or immediately processed for flow cytometry. Sera were stored at -80°C for cytokine and metabolomic analyses. Caecal contents were snap-frozen and stored at -80°C for microbiota analyses using 16S rRNA sequencing method. Metabolite extraction Briefly, 10 µL of each serum samples (n=5 young mock, n=5 young 7 dpi, n=5 aged mock, and n=5 aged 7 dpi) were loaded onto a filter containing internal standards for normalization. Filters were dried under a stream of nitrogen and a 5% solution of phenyl-isothiocyanate was added for derivatization. Dried analytes were subsequently extracted with 5 mmol/L ammonium acetate in methanol and analyzed by UPLC-MS/MS. The targeted analysis identified and quantified 1019 metabolites (https://biocrates.com/mxp-quant-500-xl/) detected by MS/MS after UPLC separation and flow injection analysis (FIA) (SCIEX 5500 Triple Quad System, SCIEX, Framingham, MA, USA). Data were recorded using the Analyst software (SCIEX). For each metabolite, peaks were quantified using area-under-the-curves. The analytical method has been fully validated by the kit’s manufacturer according to FDA and EMA guidelines. Metabolomic analysis To compare metabolite signatures in young and aged mice at baseline (mock-treated) and at 7 dpi, ultra-high pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used. Sera were processed using the MxP Quant 500 XL kit (BIOCRATES Life Sciences AG, Innsbruck, Austria). Metabolites with ≥ 40% missing values or values below the limit of detection (LOD) in all groups were excluded during initial data cleaning. Consequently, only metabolites with ≥ 60% of concentration values above the LOD in at least one of the three experimental groups were included for statistical analysis. Any remaining missing values were imputed using 1/5 of the minimum positive value of each variable. Given the metabolic interactions between adipose tissue and gut microbiota metabolism, the metabolomic analysis was performed using all metabolites retained after data processing, including lipids. At the end, 552 molecules were analyzed.