DOI: 10.5061/dryad.0p2ngf2cj
title: Data from: Enhanced resistance to <em>Listeria</em> infection in mice surviving sepsis: The role of lipid metabolism and myeloid cell reprogramming

description: 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.
description: <strong>Animal Model and Experimental
 Design</strong><br> 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. <strong>Listeria
 monocytogenes Infection and Bacterial Load
 Analysis</strong><br> At 12 weeks post-CLP or sham surgery,
 mice were intravenously challenged with 1×10⁷ CFU of <em>Listeria
 monocytogenes</em> (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. <strong>Flow
 Cytometry and Cell Sorting</strong><br> Splenocytes were
 harvested 4 weeks post-CLP and stained for CD11b, Ly6C, and Ly6G markers.
 CD11b⁺Ly6C<sup>high</sup>Ly6G⁻ monocytes were sorted using BD
 FACSAria II. Downstream assays included qRT-PCR, scRNA-seq, and
 immunofluorescence. <strong>Single-Cell RNA
 Sequencing (scRNA-seq)</strong><br> Sorted
 CD11b⁺Ly6C<sup>high</sup> 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 &gt; 2
 and FDR &lt; 0.05 were used for KEGG pathway enrichment.
 <strong>Immunohistochemistry and Lipid Droplet
 Analysis</strong><br> 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.
 <strong>Plasma Lipidomics</strong><br> 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. <strong>Quantitative
 RT-PCR</strong><br> 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
 <em>Lpl</em>, <em>Abca1</em>, and
 <em>Abca4</em>, normalized to <em>Gapdh</em>
 expression using the2 <sup>-ΔΔCt</sup> method.
 <strong>Statistical Analysis</strong><br> 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
 &lt; 0.05 was considered statistically significant.
description: # README: Enhanced resistance to listeria infection in mice surviving
 sepsis: The role of lipid metabolism and myeloid cell reprogramming
 **Dataset
 DOI: [10.5061/dryad.0p2ngf2cj](https://datadryad.org/submission/10.5061/dryad.0p2ngf2cj)** **Description of the data and file structure** This dataset contains raw and processed data supporting the manuscript: "Enhanced resistance to *Listeria* infection in mice surviving sepsis: the role of lipid metabolism and myeloid cell reprogramming." EXPERIMENTAL GROUPS Group: Sham \- Surgical exposure without ligation or puncture. Group: CLP (Sepsis Survivors) \- Cecal ligation and puncture performed; samples taken 4–12 weeks post-surgery. **Files and variables** FILE: RE_Figure_1_Suvial_and_bacterial_load.csv **Description:** Survival (%) and CFU counts (log-transformed) in spleen and liver for sham and CLP mice 3 days post-infection. **Figure 1B**: Monocyte Analysis This section contains data on the percentage (%, left) and absolute counts (number of cells x 10^5) of monocytes. Each row represents a single mouse. *Variables* Sham: The monocyte percentage or absolute count for an individual mouse in the sham control group. CLP: The monocyte percentage or absolute count for an individual mouse in the CLP sepsis model group. **Figure 1C**: Survival Data This section contains survival data for mice used to generate a Kaplan-Meier survival curve. Note the stacked layout: The data for the Sham group is listed first, followed by the data for the CLP group. The time point at which survival status was assessed for an individual mouse (0-3 days).  Value 1: Indicates the animal was alive on the specified day.  Value 0: Indicates the animal was deceased or culled on the specified day. *Variables* Time (days): The day post-procedure (0, 1, 2, or 3) on which the survival status was recorded. Sham: The survival status for a mouse in the sham group on the corresponding day (1 = alive; 0 = deceased/culled). CLP: The survival status for a mouse in the CLP group on the corresponding day (1 = alive; 0 = deceased/culled). **Figure 1D**: Bacterial Load This section contains bacterial load data from the liver (left) and spleen (right) of mice following infection. Each row represents a single mouse.  Unit: CFU (Colony-Forming Units), a measure used to quantify the number of viable bacteria per liver or spleen. *Variables* Sham: The bacterial load (CFU) for an individual mouse in the sham group. CLP: The bacterial load (CFU) for an individual mouse in the CLP group.   FILE: RE_Figure_2_Imaging_and_Cytokine_Data.csv Description: This file contains the raw data for generating panels 2B and 2D in Figure 2. The data for each panel is organized as a distinct table within the same CSV file, separated by empty rows. **Figure 2B**: Colocalized Voxel Analysis Description: This table contains quantitative data from imaging analysis. Each value represents the total count of colocalized voxels in each field, which is a measure of the spatial overlap between two different signals in microscopy images (Imaris). Each row represents a single mouse/sample.         Unit: Count of voxels (dimensionless number) *Variables* Sham: The total count of colocalized voxels for a sample from the sham group. CLP: The total count of colocalized voxels for a sample from the CLP group.   **Figure 2D**: Interferon-gamma (IFNγ) Concentration This table contains concentration measurements of the cytokine Interferon-gamma (IFNγ) from serum. Each row represents a single mouse.         Unit: pg/mL *Variables* Sham: The IFNγ concentration (pg/mL) for an individual mouse in the sham group. CLP: The IFNγ concentration (pg/mL) for an individual mouse in the CLP group.    FILE: Figure_3_scRNAseq_DEGs.csv    - Differential gene expression results for CD11b+Ly6Chigh cells (CLP vs. sham), including log2 fold-change, p-values, and FDR-adjusted q-values. *Variables* gene_name: The official symbol for the differentially expressed gene. log2FoldChange: The log2 of the fold change in expression (CLP relative to sham). pvalue: The nominal p-value for the differential expression test. padj: The p-value adjusted for multiple comparisons using FDR.   FILE: Figure_3_qPCR_validation.csv    - RT-qPCR expression values for genes LPL, ABCA1, and ABCA4. Normalized to GAPDH using the 2^−ΔΔCt method. *Variables* LPL Sham: Relative expression of the LPL gene in a mouse from the sham group. LPL CLP: Relative expression of the LPL gene in a mouse from the CLP group. ABCA1 Sham: Relative expression of the ABCA1 gene in a mouse from the sham group. ABCA1 CLP: Relative expression of the ABCA1 gene in a mouse from the CLP group. ABCA4 Sham: Relative expression of the ABCA4 gene in a mouse from the sham group. ABCA4 CLP: Relative expression of the ABCA4 gene in a mouse from the CLP group.   FILE:  Figure_4_lipidomics_data.csv    - Normalized plasma concentration for lipid species (PS, PI, PG, PC), with mouse IDs and group labels. *Variables* PS Sham: Normalized concentration of the PS lipid class in a mouse from the sham group (nmol/mL). PS CLP: Normalized concentration of the PS lipid class in a mouse from the CLP group (nmol/mL). PG Sham: Normalized concentration of the PG lipid class in a mouse from the sham group (nmol/mL). PG CLP: Normalized concentration of the PG lipid class in a mouse from the CLP group (nmol/mL). PC Sham: Normalized concentration of the PC lipid class in a mouse from the sham group (nmol/mL). PC CLP: Normalized concentration of the PC lipid class in a mouse from the CLP group (nmol/mL). PI Sham: Normalized concentration of the PI lipid class in a mouse from the sham group (nmol/mL). PI CLP: Normalized concentration of the PI lipid class in a mouse from the CLP group (nmol/mL). PI36:2 Sham: Normalized concentration of the PI(36:2) lipid species in a mouse from the sham group (nmol/mL). PI36:2 CLP: Normalized concentration of the PI(36:2) lipid species in a mouse from the CLP group (nmol/mL). PI36:1 Sham: Normalized concentration of the PI(36:1) lipid species in a mouse from the sham group (nmol/mL). PI36:1 CLP: Normalized concentration of the PI(36:1) lipid species in a mouse from the CLP group (nmol/mL).   FILE: RE_Supplementary_Table_1._Lipid_species.csv **Description:** pooled concentration of lipid species of mouse plasma (nmol/mL). *Variables* Samples: The name of the lipid species being measured in that row. S1 to S6: The concentration for Sham group samples 1 through 6. C1 to C6: The concentration for CLP group samples 1 through 6.   FILE: RE_Supplementary_Table_2._Lipid_class.csv **Description:** Calculated concentrations from the lipidomics data (nmol/mL). *Variables* Samples: The name of the lipid class being measured in that row. S1 to S6: The total concentration for the lipid class in Sham group samples 1 through 6. C1 to C6: The total concentration for the lipid class in CLP group samples 1 through 6.   **Code/software** The dataset can be viewed and analyzed using the following free or open-source software: Data are provided in standard formats (CSV, XLSX, TXT). For re-analysis, tools such as GraphPad Prism, Seurat (R), MetaboAnalyst, and Microsoft Excel were used in the original analysis. GraphPad Prism (v9.5.1) – for statistical tests, survival curves, bar plots, and scatter plots. MetaboAnalyst (v6.0, web-based) – for lipidomics data analysis.    Used for normalization, Pareto scaling, volcano plots, PLS-DA, and heatmaps.    Input: Normalized lipid intensity values from LC-MS analysis (CSV format) Microsoft Excel – Used to organize and summarize raw data from lipidomics, qPCR, CFU counts, and flow cytometry; processed data include embedded metadata and sample group identifiers for clarity.