# 2.2.10 研究| 2021-单细胞转录组分析COVID-19重症患者肺泡巨噬细胞亚型

## 前言

题目：Single-cell transcriptome identifies *FCGR3B* upregulated subtype of alveolar macrophages in patients with critical COVID-19

日期：2021-09-24

期刊：iScience

链接：<https://doi.org/10.1016/j.isci.2021.103030>

## 一句话概括

数据分析文章：scRNA分析重症COVID-19患者多个样本，得到一种单核细胞衍生的肺泡巨噬细胞 (MoAMs) 亚型，并且FCGR3B基因在其中特异性表达，提供了一个新的biomarker。

## 使用的数据

* 对照1：10例健康气管样本（ [Deprez et al., 2020](https://www.cell.com/iscience/fulltext/S2589-0042\(21\)00998-6?rss=yes\&utm_source=dlvr.it\&utm_medium=twitter#bib14)）
* 对照2：4例健康肺样本（[Madissoon et al., 2019](https://www.cell.com/iscience/fulltext/S2589-0042\(21\)00998-6?rss=yes\&utm_source=dlvr.it\&utm_medium=twitter#bib30)）
* BALF单细胞数据（支气管肺泡灌洗液, broncho-alveolar lavage fluid）：总共14个样本，包括对照、轻度和重症患者样本 ([Liao et al., 2020](https://www.cell.com/iscience/fulltext/S2589-0042\(21\)00998-6?rss=yes\&utm_source=dlvr.it\&utm_medium=twitter#bib29))

## BALF单细胞群初步注释

利用Liao文章的marker基因，大致分成macrophages, myeloid dendritic cells, and T-cells，但发现很多cluster的细胞类型不好确定【A) Control. B) Moderate. C) Severe BALF clusters using Liao et al markers】

![image-20211018212214987](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-10-18-132215.png)

于是自己又细分亚群，分别得到19, 17, and 18个cluster， 21939, 7316 and 37197 cells

![image-20211018212407400](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-10-18-132407.png)

然后又找了一些marker自行细胞注释：<https://www.cell.com/cms/10.1016/j.isci.2021.103030/attachment/24e25182-6d40-41fa-a4f5-fc33e365f86f/mmc2>

![image-20211018212524960](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-10-18-132525.png)

## 重症样本注释

包括了：basal cells, vascular cells, dendritic cells, ionocytes, monocyte-derived alveolar macrophages, plasma cells, and alveolar epithelial cells

![image-20211018212755234](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-10-18-132755.png)

![image-20211018212928624](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-10-18-132928.png)

## 重症样本的cluster11与COVID-19并发症基因之间的关联

并发症包括了：encoding cytokines and cytokine receptors, or associated with rare infectious diseases, rare syndromes, chronic obstructive pulmonary disease, cardiovascular disease, hypertension, obesity, and diabetes

基因列表：<https://www.cell.com/cms/10.1016/j.isci.2021.103030/attachment/0b421468-3a53-48b6-8c28-3eee4d7e06f7/mmc4>

发现重症的17个cluster中，**cluster11表现非常突出，9个基因列表中表达了8个，并且表达量还主要是上调**

因此，这个cluster11就被标记为monocyte-derived alveolar macrophages (MoAMs)，它的marker 基因就是*CCl3L1* ，而且这个MoAMs亚型在 moderate or control样本中都没发现

![image-20211018213458204](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-10-18-133458.png)

之后也看了一下富集分析，主要集中在：

* host immune response signaling networks related to TNFα
* cytokine and interferon gamma responses
* response to type1 interferon and biotic stimulus
* innate immune and inflammatory responses

![image-20211018213759747](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-10-18-133800.png)

## MoAM亚型top20基因分析

既然感兴趣的cluster找到，那么接下来就看其中的top基因（这里选择前20）

做了一个气泡图【 A) Control. B) Moderate and C) Severe BALF】，不过感觉没啥必要，既然选择cluster11的前20，那么肯定这些基因（横坐标）就主导啊

![image-20211018214139670](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-10-18-134140.png)

然后就定位到了其中一个差异最大的基因：*FCGR3B*

![image-20211018214440837](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-10-18-134441.png)

然后开始研究这个基因了：

* regulate both adaptive and innate immune responses which are crucial for the **defense against infection** and prevention of chronic inflammation or autoimmune diseases
* FcRs mediate important immune responses such as **release of cytokines or phagocytosis** ([Ben Mkaddem et al., 2019](https://www.cell.com/iscience/fulltext/S2589-0042\(21\)00998-6?rss=yes\&utm_source=dlvr.it\&utm_medium=twitter#bib5))

并且图C中cluster11表达的FCGR3B，比其他FcR族基因更多

## FCGR3B的数据集验证

在bulk PBMC data进行验证 ([Arunachalam et al., 2020](https://www.cell.com/iscience/fulltext/S2589-0042\(21\)00998-6?rss=yes\&utm_source=dlvr.it\&utm_medium=twitter#bib2)) ，并且发现在non-classical monocytes中表达更多

![image-20211018214849262](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-10-18-134849.png)

在 bulk data set from nasopharyngeal swabs数据集验证（左图），另外之前看cluster11特异性表达CCL3L1，那么同样在重症的CCL3L1高表达细胞中，FCGR3B表达同样高

![image-20211018215022234](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-10-18-135022.png)

不过，不同于*FCGR3B*，*CCL3L1*和*TNFAIP6* (indicator of COVID-19 severity) 在其他数据集中并非一直是重症表达量高于对照组。所以最后的目光又集中于*FCGR3B* 这一个基因了

![image-20211018215544716](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-10-18-135545.png)

## FCGR3B的实验验证

qPCR positive COVID-19 patients (n = 31) and qPCR negative controls (n = 11)，发现：

* 左图（单纯covid）：**50% had greater than 1.5-fold change** of *FCGR3B* compared with 28.1% in controls
* 右图（covid+并发症）：**57.1% of patients with severe COVID-19 with comorbidity had greater** than 1.5-fold change of *FCGR3B* compared with 28.1% in controls

![image-20211018215754823](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-10-18-135754.png)

当然，最后还拓展了一下，做了个体外并发症（肥胖）关联实验，发现：*FCGR3B* as a potential modulator of COVID-19 severity in patients with obesity

![image-20211018220058328](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-10-18-140058.png)

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