Vizgen MERFISH files for 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..., , # 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](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, ](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...

结核分枝杆菌（Mycobacterium tuberculosis, MTB）的ESX-1是一种VII型分泌系统，是介导MTB在肺部单核吞噬细胞（mononuclear phagocytes, MNPs）内存活的关键毒力决定因子，但其对MNP招募与分化的影响仍尚不明确。
本研究借助多种单细胞RNA测序技术，探究了ESX-1在小鼠及小鼠骨髓源性巨噬细胞（bone marrow-derived macrophages, BMDM）的MNP异质性与应答过程中的作用。研究发现，ESX-1是MTB招募携带高MTB载量的多种MNP亚群的必需因子。此外，MTB可通过ESX-1依赖的方式，在MNPs与BMDM中诱导抗炎转录特征。空间转录组学分析显示，在MTB感染病灶内，单核细胞源性巨噬细胞聚集于MTB感染细胞附近，且该处的抗炎信号表达上调。综上，本研究结果表明，MTB ESX-1可促进抗炎表型MNPs的招募与分化，而MTB可通过感染并调控这类细胞以实现自身存活。值得注意的是，本研究提供……

## 空间转录组学实验方法
通过右心室向小鼠肺部灌注10 mL PBS/2 mM乙二胺四乙酸（EDTA），随后经气管向肺内注入1 mL按1:1比例混合的最优切割温度包埋剂（optimal cutting temperature, OCT）与PBS。将C57BL/6小鼠以气溶胶方式感染约75 CFU的H37Rv-zsGreen菌株，感染28天后处死小鼠。摘取肺部组织并以OCT包埋，快速冷冻后于-80℃保存，用于后续冰冻切片制作。为验证样本质量，从每个包埋块中提取RNA（使用QIAGEN RNeasy Mini试剂盒，货号74104），并通过Agilent Tapestation系统检测RNA完整性，获得RNA完整性数（RNA integrity number, RIN）126。随后将OCT包埋的组织块于-20℃下制作厚度为10 μm的冰冻切片，并贴附于MERSCOPE玻片（Vizgen，货号PN 20400001）上。贴附完成的组织切片立即于37℃下用4%多聚甲醛（PFA）固定30分钟，经3×PBS洗涤后，置于70%乙醇中于4℃保存不超过1个月，后续按照厂商的MERSCOPE实验流程（参考文献83）进行操作，但存在以下几点调整。
F...,
# 用于单细胞分析的Vizgen MERFISH文件：揭示结核分枝杆菌ESX-1介导感染小鼠肺部抗炎巨噬细胞聚集
[https://doi.org/10.5061/dryad.08kprr5bx](https://doi.org/10.5061/dryad.08kprr5bx)

## 数据与文件结构说明
本数据集包含来自一项空间转录组学实验的Vizgen MERFISH .vzg格式图像文件，该实验探究了MTB感染后小鼠肺部巨噬细胞的转录应答情况。这些文件由Vizgen MERSCOPE平台生成，并通过其标准数据处理流程完成预处理。本数据集关联至Dryad数据库中的数据集[https://doi.org/10.5061/dryad.0p2ngf28s](https://doi.org/10.5061/dryad.0p2ngf28s)，该数据集包含所有已处理的计数数据及额外的实验细节。

### 文件与变量
#### 文件列表：202305231432_TB-CTRL1-MB_VMSC02401_region_0.vzg、202305231432_TB-CTRL1-MB_VMSC02401_region_1.vzg、202305231432_TB-CTRL1-MB_VMSC02401_region_2.vzg……