DOI: 10.5061/dryad.0p2ngf28s
title: Single-cell analysis reveals M. tuberculosis ESX-1-mediated accumulation of anti-inflammatory macrophages in infected mouse lungs

description: 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.
description: 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.
description: # Single-cell analysis reveals M. tuberculosis ESX-1-mediated accumulation
 of anti-inflammatory macrophages in infected mouse lungs
 [https://doi.org/10.5061/dryad.0p2ngf28s](https://doi.org/10.5061/dryad.0p2ngf28s) This dataset contains spatial transcriptomics collected using the Vizgen MERFISH and preprocessed using their standard data processing pipeline. This dataset contains all the raw data used for spatial analysis in this study. ## Description of the data and file structure The data folder contains nested subfolders including a tar archive of the Merscope output for each section of the image data pertaining to each experiment, internally organized by experiment, tissue section, and Z-slice. The code necessary to process this data as shown in the paper is presented in this archive as `TBSpatial_Analysis_code.ipynb` (python notebook) and an html file (`TBSpatial_Analysis_code.html`), and can also be found on Github at [https://github.com/bspeco/ESX1_macrophageheterogeneity_scRNA/blob/main/TBSpatial_Analysis_code.ipynb](https://github.com/bspeco/ESX1_macrophageheterogeneity_scRNA/blob/main/TBSpatial_Analysis_code.ipynb). After downloading the entire data folder, unzip the .tgz files with the command tar -xzvf filename.tgz. You should end up with a folder containing the following subfolders: ### Stats all the statistics calculated in this script, including genewise permutation statistics comparing monocyte/macrophage TB infected cells to their neighbors (`AllMonoMacInfectedVsNeighbors.csv`); comparing cells within 100 micrometers of a TB lesion to those farther away in bulk (`statsAllTB_regionAll.csv`) and divided by replicate (`statsAllTB_individualReplicates-excludingTB.csv`), which were compiled from the statistics shown in each included `region-specific csv` file; and comparing cells within 100 micrometers of a comparable lesion-like location within a control uninfected sample to those further away (`statsAllControl_regionAll.csv`).  ### Annotated\_h5ad\_individual Annotated H5AD (anndata) files for each spatial section. These files can be opened in python using the Anndata or Scanpy software packages. Each file is named according the experiment (listed above) and region within that experiment.  ### Annotated\_h5ad the mononuclear phagocyte subset of single cell (Smartseq2) sequencing data published in this paper for comparison (`scRNAseq_MNP_SS2only.h5ad`) as well as an annotated copy of all the vizgen data used in the form of an anndata (h5ad) file (`Total_annotatedvizgenTB20231120.h5ad`). A raw merged copy of all the files in Annotated_h5ad_individual is also included (`Total_concatenated_vizgen_h5ad.h5ad`). All h5ad files can be opened using anndata or scanpy in python.  ### cell\_annotations csv files with cell annotations defining whether a cell is positive for TB or not, as defined by the script, to load into the Merfish Vizualizer along with the .vzg files included in DOI:10.5061/dryad.08kprr5bx. Each file is named to correspond with a particular mouse lung section as detailed below.  ### avg\_signal\_df.csv csv file with all the TB antibody staining values per cell, provided so that you can skip running the most time-intensive code chunk generating this file. This file was used to determine the TB status of each cell.  ### postprocessed\_counts counts generated by Merscope post-processing. Each folder corresponds to one of the mouse lung samples listed below, and the h5ad file inside is the result of the automated Vizgen processing pipeline containing a summary of counts per gene for each identified cell.  Vizgen .tif image files and accompanying metadata are found in the following named folders: * **202305231432_TB-CTRL1-MB_VMSC02401:** Control uninfected mouse lung sample processed on 05/23/2023 by Michael Borja.  * **202305251147_TB-CTRL2-MB_VMSC02401:** Control uninfected mouse lung sample processed on 05/25/2023 by Michael Borja.  * **202305261458_TB-TEST3-MB_VMSC02401:** TB infected mouse lung sample processed on 05/26/2023 by Michael Borja.  * **202309251210_20230925-RitwicqA-TB_VMSC02401:** TB infected mouse lung sample processed on 09/25/2023 by Ritwicq Arjyal. * **202310091253_AmandaS-TB-02_VMSC02401:** TB infected mouse lung sample processed on 10/09/2023 by Amanda Seng. * **202310111428_RitwicqA-TB-4b_VMSC02401:** TB infected mouse lung sample processed on 10/11/2023 by Ritwicq Arjyal. Each folder contains the following:`dapi.png, polyt.png`: Image files showing DAPI signal and Poly-T signal for the prepared sample, including multiple regions. `experiment.json`: JSON file including details of image capture for all channels. **region_0, region_1, region_2**: Folders containing TIF images and information for each captured section. Only the regions used in this analysis were included. Regions were excluded if they were damaged or showed no evidence of MTB lesions. Each region folder contains the cell boundary coordinates in a csv and parquet format (`cellboundaries.parquet` and `cell_metadata.csv`), counts per gene per cell (`cell_by_gene.csv`), coordinates of all detected transcripts (`detected_transcripts.csv`), and a snapshot of the cell boundaries, transcripts detected, and automated qc graphs (`summary.png`). Each nested **images** folder contains a json (`manifest.json`) with image information, a csv file detailing how to convert pixel measurements into distance (`micron_to_mosaic_pixel_transform.csv`), and one compressed tif file per z-section with the TB antibody staining image ("Cellbound3"; `mosaic_Cellbound3_z[0,1,2,3,4,5,6]_lzw.tif`) ## Sharing/Access information The transcriptomic data associated with this study can be accessed on GEO, Series GSE263892, at [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE263892.](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE263892.) The vizgen .vzg files are available on Dryad at DOI: 10.5061/dryad.08kprr5bx.  ## 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](https://github.com/bspeco/ESX1_macrophageheterogeneity_scRNA/releases/tag/v1.0.0)