DOI: 10.5061/dryad.zpc866thz
title: Spatial transcriptomics of an innate granuloma in a mouse infection model with Chromobacterium violaceum

description: From Amason et al., 2024. "Granulomas are defined by the presence of
 organized layers of immune cells that include macrophages. Granulomas are
 often characterized as a way for the immune system to contain an infection
 and prevent its dissemination. We recently established a mouse infection
 model where Chromobacterium violaceum induces the innate immune system to
 form granulomas in the liver. This response successfully eradicates the
 bacteria and returns the liver to homeostasis. Here, we sought to
 characterize the chemokines involved in directing immune cells to form the
 distinct layers of a granuloma. We use spatial transcriptomics to
 investigate the spatial and temporal expression of all CC and CXC
 chemokines and their receptors within this granuloma response. The
 expression profiles change dynamically over space and time as the
 granuloma matures and then resolves. To investigate the importance of
 monocyte-derived macrophages in this immune response, we studied the role
 of CCR2 during C. violaceum infection. Ccr2–/– mice had negligible numbers
 of macrophages, but large numbers of neutrophils, in the C.
 violaceum-infected lesions. In addition, lesions had abnormal architecture
 resulting in loss of bacterial containment. Without CCR2, bacteria
 disseminated and the mice succumbed to the infection. This indicates that
 macrophages are critical to form a successful innate granuloma in response
 to C. violaceum."
description: From Harvest et al., 2023. "Infected mouse liver tissues
 were harvested at various time points, embedded in OCT, and frozen. Frozen
 livers were cut to optimal section thickness and placed on a Tissue
 Optimization Slide to determine permeabilization conditions. Tissues were
 then placed within a 6.5mm<sup>2</sup> field on an expression
 slide that contained 5000 barcoded probes. The tissues were then fixed and
 stained with Hoechst and Eosin then permeabilized to release mRNA which
 binds to spatially barcoded capture probes, allowing for the capture of
 gene expression information. Captured mRNA from the slide surface was
 denatured, cleaved, and transferred into a PCR tube. From there, the cDNA
 was amplified, and standard NGS libraries were prepared. Adapters
 were ligated to each fragment followed by a sample index PCR. The
 libraries were sequenced to an average of 50,000 reads/probe on a
 paired-end, dual-indexed flow cell in the format of 28x10x10×90. Data was
 then uploaded to analysis packages for visualization. Visium spatial data
 was analyzed using 10xGenomincs Space Ranger software and visualization
 through Loupe Browser. Secondary statistical analysis was performed using
 a Seurat package in R(Studio)."
description: # Spatial transcriptomics of an innate granuloma in a mouse infection
 model with *Chromobacterium violaceum*
 [https://doi.org/10.5061/dryad.zpc866thz](https://doi.org/10.5061/dryad.zpc866thz) ## Description of the data and file structure The uploaded data file contains Visium spatial transcriptomics data extracted from *C. violaceum*-infected mouse liver tissues at various days post-infection. See Methods for further information on this file.  ## Code/software From Amason et al., 2024 "Spatial data were generated in Harvest et al., 2023 using the 10X Genomics Visium Platform. We were most interested in the immune cells present within the distinct zones of each lesion, and the adjacent healthy hepatocytes. Therefore, we used Loupe Browser v7.0 to visualize the H&E-stained tissues and manually select spots of interest. We deselected spots that were distant from infected lesions, while selecting the lesions and surrounding healthy hepatocytes. To account for cell-to-cell variation, especially across tissues, pre-processing included normalization using sctransform. To further analyze the spatial transcriptomics dataset of the selected spots, we used the Seurat package in RStudio to analyze gene expression over time and space. UMAP plots, SpatialDimPlots, SpatialFeaturePlots, ggplots, and Violin plots were all used to visualize normalized gene expression data." Software: RStudio 2024.04.0+735 "Chocolate Cosmos" Release (a00d0e775dbc93e0d79a1bf474e3e8e8de677383, 2024-04-24) for windows Mozilla/5.0 (Windows NT 10.0; Win64; x64) AppleWebKit/537.36 (KHTML, like Gecko) RStudio/2024.04.0+735 Chrome/120.0.6099.291 Electron/28.2.6 Safari/537.36, Quarto 1.4.553 The **Streamlined Code file** (Zenodo) contains all code relevant for generating plots found in Amason et al., 2024. Initial characterization of predominant cell types and/or location of each cluster was performed in Harvest et al., 2023. Relevant cluster abbreviations: Macrophage zone (M), hepatocyte (HEP), representative HEP (rep HEP), necrotic core center (NC-C), NC-periphery (NC-P), coagulative necrosis (CN), CN-macrophage (CN-M), endothelial cell (EC), outside granuloma (OG). ## Access information Data was derived from the following sources: * Harvest, C. K., Abele, T. J., Yu, C., Beatty, C. J., Amason, M. E., Billman, Z. P., DePrizio, M. A., Souza, F. W., Lacey, C. A., Maltez, V. I., Larson, H. N., McGlaughon, B. D., Saban, D. R., Montgomery, S. A., & Miao, E. A. (2023). An innate granuloma eradicates an environmental pathogen using Gsdmd and Nos2. *Nature Communications*, *14*(1), 6686. [https://doi.org/10.1038/s41467-023-42218-1](https://doi.org/10.1038/s41467-023-42218-1) * Amason M.E., Beatty C.J., Harvest C.K., Saban D.R., Miao E.A. Chemokine expression profile of an innate granuloma. bioRxiv [Preprint]. 2024 Jun 6:2024.01.30.577927. doi: 10.1101/2024.01.30.577927. PMID: 38352492; PMCID: PMC10862903. (publication pending through eLife)