Enhanced resistance to <em>Listeria</em> infection in mice surviving sepsis: The role of lipid metabolism and myeloid cell reprogramming

This dataset contains raw and processed lipidomics data from an experiment investigating the lipid mechanisms of CD11b+ myeloid cells in mice surviving sepsis, through a murine cecal ligation and puncture (CLP) model. Single-cell RNA sequencing data of splenic myeloid cells are accessible in the GEO database (GSE249839). The data encompass untargeted plasma lipidomics, confocal microscopy of lipid droplets, quantitative RT-PCR, and survival and bacterial burden outcomes following Listeria monocytogenes infection. Lipid species were quantified using LC-MS/MS, and lipid class composition was analyzed. This dataset includes normalized lipid abundance tables, instrument settings, and group metadata. Methods Animal Model and Experimental Design Male C57BL/6J mice (8 weeks old) were used to model sepsis via cecal ligation and puncture (CLP), conducted under isoflurane anesthesia. Postoperative care included buprenorphine analgesia, subcutaneous resuscitation fluids, and one dose of imipenem/cilastatin. Sham controls underwent laparotomy without cecal manipulation. Mice were monitored daily, and moderate sepsis severity resulted in 20–30% mortality within 3–5 days. CLP survivors were studied at 4 and 12 weeks post-surgery. Listeria monocytogenes Infection and Bacterial Load Analysis At 12 weeks post-CLP or sham surgery, mice were intravenously challenged with 1×10⁷ CFU of Listeria monocytogenes (Lm). Survival was monitored for 7 days. For bacterial load quantification, mice were injected with 2×10⁶ CFU and sacrificed at 72 hours post-infection. Liver and spleen tissues were harvested, homogenized, serially diluted, plated on Brain Heart Infusion (BHI) agar, and incubated at 37 °C overnight. Colony-forming units (CFU) were normalized per gram of tissue. Flow Cytometry and Cell Sorting Splenocytes were harvested 4 weeks post-CLP and stained for CD11b, Ly6C, and Ly6G markers. CD11b⁺Ly6ChighLy6G⁻ monocytes were sorted using BD FACSAria II. Downstream assays included qRT-PCR, scRNA-seq, and immunofluorescence. Single-Cell RNA Sequencing (scRNA-seq) Sorted CD11b⁺Ly6Chigh cells were barcoded with TotalSeq™ hashtags and processed with Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3.1 (10x Genomics). Sequencing was performed on an Illumina HiSeq platform. Data processing included Cell Ranger pipeline alignment to the GRCm38 genome, UMAP-based clustering, and differential gene expression (DEG) analysis via Seurat (v5.0.1). DEGs with log2 fold change > 2 and FDR < 0.05 were used for KEGG pathway enrichment. Immunohistochemistry and Lipid Droplet Analysis Spleens were cryosectioned (10 μm) and stained for CD11b (Alexa Fluor 488) and LipidTOX (Deep Red or Green). Confocal images were acquired on a Zeiss LSM900 with Airyscan. LD accumulation in CD11b⁺ myeloid cells was quantified using Imaris software’s 3D colocalization module. Plasma Lipidomics Plasma was collected 4 weeks post-CLP following 2-hour fasting. Lipids were extracted from 20 μL plasma using methylene chloride:methanol:isopropanol (25:10:65 v/v/v + 0.1% BHT) and spiked with SPLASH® Lipidomix®. UHPLC was performed using a Waters CSH C18 column with a TripleTOF 5600 MS (AB Sciex). Data were normalized to internal standards and analyzed using MetaboAnalyst 6.0. Significant lipid shifts were determined via PLS-DA and t-tests. Lipids were annotated via LIPID MAPS and NIST references. Quantitative RT-PCR RNA from sorted monocytes was extracted with Direct-zol (Zymo), reverse transcribed, and pre-amplified using TaqMan PreAmp Master Mix. qPCR was performed using TaqMan assays for Lpl, Abca1, and Abca4, normalized to Gapdh expression using the2 -ΔΔCt method. Statistical Analysis Data are expressed as mean ± SEM. Comparisons between two groups used two-tailed unpaired Student’s t-tests or Mann–Whitney U tests where appropriate. Log-rank tests were used for survival analysis. A p-value < 0.05 was considered statistically significant.