DOI: 10.57745/vnv5j0
title: 16S rRNA sequencing of the gut microbiome of young and aged mice infected with influenza virus at day 0 and at days 4, 7, 14 and 28 post-infection

description: Data correspond to a gut microbiome analysis through 16S rRNA sequencing of caecal samples collected from young and aged mice infected with influenza virus at day 0 and at days 4, 7, 14 and 28 post-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. 16S rRNA gene sequencing Biomnigene (https://www.biomnigene.fr/fr/, Besançon, France) conducted the DNA extraction from frozen caecal samples using the E.Z.N.A® Stool DNA kit (Omega Bio-tek, Norcross, GA, USA). PCR reactions to amplify the V3-V4 hypervariable region of the 16S rRNA gene were performed using the AccuStartTM II PCR SuperMix (VWR International, Radnor, PA, USA). PCR products were analyzed using a QIAxcel DNA High Resolution Cartridge (QIAGEN). For preparation of libraries, PCR product concentrations were determined using Qubit 4.0, and samples were pooled equimolarly before purification by electrophoresis on PippinHT using a 1.5% agarose cassette (Sage Sciences, Beverly, MA, USA). Sequencing of V3-V4 amplicons was performed on MiSeq Illumina in 2X251 bp by using the Illumina MiSeq Reagent Kit v2 (500 cycles) (Illumina, San Diego, CA, USA). Alpha diversity indices (number of observed OTUs and Shannon diversity index) were computed using QIIME v1.9.1. Gene and metabolic pathway abundances were inferred using PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) software (2020), based on the 16S rRNA gene sequencing results obtained in QIIME 2.