Vizgen MERFISH files for Single-cell analysis reveals M. tuberculosis ESX-1-mediated accumulation of anti-inflammatory macrophages in infected mouse lungs
Data files
Dec 30, 2024 version files 72.98 GB
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202305231432_TB-CTRL1-MB_VMSC02401_region_0.vzg
427.32 MB
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202305231432_TB-CTRL1-MB_VMSC02401_region_1.vzg
1.84 GB
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202305231432_TB-CTRL1-MB_VMSC02401_region_2.vzg
6.62 GB
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202305251147_TB-CTRL2-MB_VMSC02401_region_0.vzg
2.94 GB
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202305251147_TB-CTRL2-MB_VMSC02401_region_1.vzg
7.57 GB
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202305261458_TB-TEST3-MB_VMSC02401_region_1.vzg
4.18 GB
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202309251210_20230925-RitwicqA-TB_VMSC02401_region_1.vzg
9.88 GB
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202309251210_20230925-RitwicqA-TB_VMSC02401_region_2.vzg
4.49 GB
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202310091253_AmandaS-TB-02_VMSC02401_region_0.vzg
1.22 GB
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202310091253_AmandaS-TB-02_VMSC02401_region_1.vzg
13.29 GB
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202310091253_AmandaS-TB-02_VMSC02401_region_2.vzg
3.85 GB
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202310111428_RitwicqA-TB-4b_VMSC02401_region_1.vzg
7.62 GB
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202310111428_RitwicqA-TB-4b_VMSC02401_region_2.vzg
9.05 GB
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README.md
2.56 KB
Abstract
Mycobacterium tuberculosis (MTB) ESX-1, a type VII secretion system, is a key virulence determinant contributing to MTB’s survival within lung mononuclear phagocytes (MNPs), but its effect on MNP recruitment and differentiation remains unknown. Here, using multiple single-cell RNA sequencing techniques, we studied the role of ESX-1 in MNP heterogeneity and response in mice and murine bone marrow-derived macrophages (BMDM). We found that ESX-1 is required for MTB to recruit diverse MNP subsets with high MTB burden. Further, MTB induces an anti-inflammatory transcriptional signature in MNPs and BMDM in an ESX-1-dependent manner. Spatial transcriptomics revealed an upregulation of anti-inflammatory signals within MTB lesions, where monocyte-derived macrophages concentrate near MTB-infected cells. Together, our findings suggest that MTB ESX-1 facilitates the recruitment and differentiation of anti-inflammatory MNPs, which MTB can infect and manipulate for survival. Importantly, we provide a comprehensive transcriptomic dataset across various models and methods, which could contribute to the broader understanding of recruited cell heterogeneity during MTB lung infection.
This data is associated with processed data and code found in https://doi.org/10.5061/dryad.0p2ngf28s.
README: Vizgen MERFISH files for Single-cell analysis reveals M. tuberculosis ESX-1-mediated accumulation of anti-inflammatory macrophages in infected mouse lungs
https://doi.org/10.5061/dryad.08kprr5bx
Description of the data and file structure
This dataset contains Vizgen MERFISH .vzg images files from a spatial transcriptomics experiment investigating macrophage transcriptional responses in the mouse lung following MTB infection. These files were generated with the Vizgen MERSCOPE and preprocessed using their standard data processing pipeline. This data is linked to the Dryad dataset https://doi.org/10.5061/dryad.0p2ngf28s, which contains all of the processed count data and additional experimental details.
Files and variables
Files: 202305231432_TB-CTRL1-MB_VMSC02401_region_0.vzg, 202305231432_TB-CTRL1-MB_VMSC02401_region_1.vzg, 202305231432_TB-CTRL1-MB_VMSC02401_region_2.vzg
Description: Control mouse lung not infected with MTB, three sections on one slide.
Files: 202305251147_TB-CTRL2-MB_VMSC02401_region_0.vzg, 202305251147_TB-CTRL2-MB_VMSC02401_region_1.vzg
Description: Additional control mouse lung not infected with MTB, two sections on one slide.
Files: 202309251210_20230925-RitwicqA-TB_VMSC02401_region_1.vzg, 202309251210_20230925-RitwicqA-TB_VMSC02401_region_2.vzg
Description: MTB infected mouse lung from mouse 2, two sections on one slide.
File: 202305261458_TB-TEST3-MB_VMSC02401_region_1.vzg
Description: MTB infected mouse lung from mouse 3, slide 1, region 1.
File: 202310091253_AmandaS-TB-02_VMSC02401_region_0.vzg, 202310091253_AmandaS-TB-02_VMSC02401_region_1.vzg, 202310091253_AmandaS-TB-02_VMSC02401_region_2.vzg
Description: MTB infected mouse lung from mouse 4, 3 sections on one slide.
File: 202310111428_RitwicqA-TB-4b_VMSC02401_region_1.vzg, 202310111428_RitwicqA-TB-4b_VMSC02401_region_2.vzg
Description: MTB infected mouse lung from mouse 4, 2 sections on an additional slide.
Code/software
The code associated with the processing of this dataset for the linked manuscript is available within this dataset and on github at https://github.com/bspeco/ESX1_macrophageheterogeneity_scRNA/releases/tag/v1.0.0
Access information
This data is associated with processed data and code found in https://doi.org/10.5061/dryad.0p2ngf28s
Methods
Spatial Transcriptomics
Lungs were perfused with 10 mL of PBS/2 mM EDTA via right ventricle, followed by inflation through the trachea with 1mL of optimal cutting temperature (OCT) mixed with PBS at 1:1. C57BL/6 mice aerosol infected with H37Rv-zsGreen at ~75 CFU/mouse for 28 days were sacrificed. Lungs were embedded in OCT, flash frozen, and stored at -80°C for later cryosectioning. To confirm quality, RNA integrity number (RIN)126 was obtained by extracting RNA from each block (QIAGEN RNeasy Mini Kit, Cat. No. 74104) and analyzing quantity on an Agilent Tapestation system. OCT-embedded tissue blocks were later cryosection at -20°C to a thickness of 10 μm and mounted onto MERSCOPE Slides (Vizgen, PN 20400001). The mounted tissues were immediately fixed with 4% PFA at 37°C for 30 minutes, washed with 3x PBS and stored in 70% ethanol at 4°C for no more than 1 mo before proceeding following the manufacture’s protocol (MERSCOPE) as previously described83 with the following exceptions.
First, cell boundaries of mounted infected tissues including MTB bacterium were stained with the Vizgen Cell boundary Staining Kit (Cat. no. 10400009) in conjunction with a polyclonal MTB antibody (Abcam, ab905) raised in rabbit diluted to 1:100. After washing, the Secondary Staining Mix (Vizgen, PN 20300011) was diluted with a blocking solution in two step dilution (1st - 1:100, 2nd - 1:33) to ensure correct ratio of primary to secondary staining since a custom primary MTB antibody was used.
Imaging: The MERFISH gene panel consisted of 122 genes, designed by selecting top differentially expressed genes in each cell type from the myeloid scRNA sequence atlas, canonical markers, and genes of interest. The probes were hybridized, and samples washed prior to gel embedding without deviation from manufacter protocol. Samples were then treated with a clearing solution and imaged 1 day later per MERSCOPE User Guide 91600001. Only one gel-embedded and cleared sample was prepared per imaging run. The gel-embedded and cleared sample was washed prior to adding the DAPI / Poly T Staining Reagent, and again prior to assembling the onto the MERSCOPE Flow Chamber (Vizgen, PN 10300003). Once fully thawed, the MERSCOPE 140 Gene Imaging Cartridge was activated and a layer of mineral oil was carefully added on top of the imaging buffer. The assembled flow chamber with sample and imaging cartridge was placed in the MERSCOPE, image mosaic was acquired from the regions of interest, and imaging of the sample was performed with multiple rounds of hybridization of fluorescent probes which resulted in a raw images stack that contained about 1 TB of data.