Single-cell analysis reveals M. tuberculosis ESX-1-mediated accumulation of anti-inflammatory macrophages in infected mouse lungs

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 ...,

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.
F..., , # 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_scR...

Mycobacterium tuberculosis (MTB) ESX-1，一种VII型分泌系统，是MTB在肺单核吞噬细胞（MNPs）内生存的关键毒力决定因素，但它对MNP募集和分化的影响尚不清楚。这里，我们使用多种单细胞RNA测序技术，研究了ESX-1在小鼠和小鼠骨髓来源巨噬细胞（BMDM）中MNP异质性和应答中的作用。我们发现，ESX-1是MTB募集具有高MTB载量的多样化MNP亚群所必需的。此外，MTB以ESX-1依赖的方式在MNPs和BMDM中诱导抗炎转录特征。空间转录组学揭示了MTB病变内抗炎信号的上调，其中单核细胞来源的巨噬细胞集中在MTB感染细胞附近。总之，我们的研究结果表明，MTB ESX-1促进抗炎MNPs的募集和分化，MTB可感染并操纵这些MNPs以维持生存。重要的是，我们提供...

空间转录组学
通过右心室灌注10 mL PBS/2 mM EDTA，随后通过气管注入1 mL optimal cutting temperature（OCT）与PBS的1:1混合物进行肺膨胀。将气溶胶感染H37Rv-zsGreen（约75 CFU/小鼠）28天的C57BL/6小鼠处死。将肺包埋于OCT中，快速冷冻，并在-80°C下储存以备后续冷冻切片。为确认质量，通过从每个块中提取RNA（QIAGEN RNeasy Mini Kit，货号74104）并在Agilent Tapestation系统上分析数量，获得RNA完整性数（RIN）126。OCT包埋的组织块随后在-20°C下冷冻切片至10 μm厚度，并安装在MERSCOPE载玻片（Vizgen，PN 20400001）上。安装的组织立即用4% PFA在37°C下固定30分钟，用3x PBS洗涤，并储存在4°C的70%乙醇中不超过1个月，然后按照制造商的方案（MERSCOPE）进行后续操作，如先前所述83，但有以下例外。

# 单细胞分析揭示MTB ESX-1介导的感染小鼠肺中抗炎巨噬细胞的积累

[https://doi.org/10.5061/dryad.0p2ngf28s](https://doi.org/10.5061/dryad.0p2ngf28s)

该数据集包含使用Vizgen MERFISH收集的空间转录组学数据，并使用其标准数据处理流程进行预处理。本数据集包含本研究中用于空间分析的所有原始数据。

## 数据和文件结构描述

数据文件夹包含嵌套子文件夹，包括每个实验相关图像数据各部分的Merscope输出tar存档，内部按实验、组织切片和Z切片组织。

本文中所示的处理该数据所需的代码在本存档中以`TBSpatial_Analysis_code.ipynb`（Python笔记本）和html文件（`TBSpatial_Analysis_code.html`）呈现，也可在Github上找到：[https://github.com/bspeco/ESX1_macrophageheterogeneity_scR...