Visualizing a lung neutrophil-platelet immunothrombosis cascade during sepsis in mice
Data files
Mar 18, 2026 version files 21.58 MB
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Neutrophil_Behavioromics_adv0377_Brown_et_al.xlsx
21.58 MB
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README.md
2.56 KB
Abstract
Sepsis is an immune paradox where host defence is necessary for survival but also contributes to organ damage and death. Using lung intravital microscopy we defined an immunothrombosis cascade of neutrophil and platelets in the microcirculation in response to E. coli sepsis. Neutrophil cathelicidin localized neutrophils to E. coli and initiated founder immunothrombi via formyl-peptide receptors. Immunothrombi captured vascular bacteria and cathelicidin enabled antimicrobial activities in platelets. Blocking cathelicidin prevented the immunothrombosis cascade and attenuated early sepsis death but resulted in delayed death with uncontrolled infection. LTB4 amplified the immunothrombi and inhibiting it diminished detrimental immunothrombi while preserving host defense, thus representing a discrete inflection point of sepsis disease progression. Therefore, targeting the immunothrombi cascade can mitigate immunopathology without suppressing host defence during sepsis.
Dataset DOI: 10.5061/dryad.0k6djhbfz
Description of the data and file structure
Neutrophil behavior was characterized using Imaris software (Oxford Instruments, v10.1). In brief, surfaces were applied to corresponding channels with absolute intensity thresholding. Voxels <40 were filtered out for neutrophils, and <10 for platelets, and endothelium. Spots were used for tracking bacteria. Neutrophil cellular characteristics, including area, sphericity (polarization), platelet surface overlap, distance to E. coli, etc., were measured.
2D video imaging was used for cellular behavioral analysis in the pulmonary microvasculature. Imaris surface and track parameters of neutrophils were exported with specific raw values into Microsoft Excel. Extracted raw values of morphometric and kinematic parameters of interest were exported into .csv files and loaded into R (v4.2.0). To facilitate ‘track-back’ analysis of cluster behaviors, unique neutrophil surface identification signatures (NeuID) along with unique animal IDs and genotypes were imprinted into the metadata and were maintained throughout the data analysis workflow.
Files and variables
File: Neutrophil_Behavioromics_adv0377_Brown_et_al.xlsx
Description: Descriptions of Imaris measurements can be found in the Imaris Reference Manual: https://www.bitplane.com/download/manuals/ReferenceManual9_2_0.pdf
Variables
- Area (µm2)
- Displacement delta length (µm)
- Ellipticity oblate (AU)
- Platelet overlap (µm2)
- Distance to E. coli (µm)
- Distance to platelets (µm)
- Speed (µm/s)
- Polarization (sphericity) (AU)
Code/software
Selected parameters were integrated into a single .csv file which was run through the Seurat (v.5.1.0) pipeline for data analysis. For analysis of the immunothrombi dataset, we transformed the original matrix into a Seurat object and normalized and scaled the parameters. We performed a principal component analysis and the cells were clustered based on k-nearest neighbor graphs using the Louvain algorithm. Finally, a non-linear reduction technique (t-SNE and UMAP) was performed to visualize the data in a low-dimensional space. Graphs were created using ggplot2.
Access information
Other publicly accessible locations of the data:
- NA
Data was derived from the following sources:
- NA