卞修武老师去年发的文章，板板让我学习一下～
1. Transcriptomic and proteomic analyses of COVID-19 pathology
Fig 1a：作者对4例正常对照和5例新冠患者的post-mortem肺组织进行了bulk RNA seq、LCC-MS/MS和snRNA-seq。
Fig 1b：组织学检测提示患者存在广泛的肺泡损伤，包括肺泡上皮剥脱和粘液栓形成；血管也存在广泛损伤，可以看到弥散的血管内凝血；此外，组织中还存在fulminant肺泡内巨噬细胞浸润，严重的肺纤维化和凋亡增加。
Fig 1c：作者首先分析了bulk RNA seq数据和data-independent acquisition (DIA) mass spectrometry-based proteomics数据。
Fig 1d：作者一共鉴定出3,972个差异基因和2,299个差异表达蛋白，而且这些基因和蛋白高度重叠。
Fig 1e：随后作者对差异基因和差异蛋白的交集做了功能富集。和肺组织广泛破坏和功能减低相一致，cytoskeleton organization 和 cell-junction assembly通路出现下调，凋亡通路、病毒基因表达、核糖体和内质网定位通路则出现上调。
Fig 1f, g：转录因子网络分析结果显示主要参与未折叠蛋白反应 (UPR; 比如ATF4 和 DDIT3) 的激活和诱导凋亡反应。提示新冠感染可以触发URP和细胞凋亡。
Fig 1h：炎症反应通路也存在广泛激活。其中上调最显著的基因/蛋白是CRP和SAA1 (serum amyloid A)，两者都是新冠的潜在诊断marker。
Fig 1i：对肺组织和外周血的整合分析发现CRP, SAA1 和 serpin serine protease inhibitors (SERPINA3, SERPING1 和 SERPINA1)在外周血中也升高，提示它们的潜在诊断意义。
2. A single-cell transcriptional atlas of the lungs of patients with COVID-19
为了得到更精度的信息，作者对单细胞的数据进行了分析。注释得到28种细胞类型，表达新冠受体ACE2和TMPRSS2的主要是AT2细胞。而且新冠患者的肺泡上皮表达ACE2 and/or TMPRSS2比HC显著要高，提示SARS-CoV-2 通过诱导正反馈效应增加了新冠患者的infectivity。
3. Molecular characterization of COVID-19 pathology
Fig 3a, b：为了探究细胞特异性转录变化，作者计算了28种细胞类型的差异基因，得到3,264个差异基因 (a)。上调基因和下调基因分别聚成了6个module (b)
上调的模块：
> Module 1: ubiquitin-dependent protein catabolic process (commonly upregulated pathways);
> Module 2: diseases associated with surfactant metabolism and lung fibrosis (mainly attributed to epithelial cells);
> Module3: angiogenesis (mainly attributed to endothelial cells);
> Module 4: extracellular-matrix organization (mainly attributed to stromal cells; Fig. 3b);
> Module 5: which was mainly enriched in myeloid cells (macrophage, monocytes and mast cells), as reflected by upregulated gene expression associated with myeloid leukocyte activation;
> Module 6: which was mainly enriched in lymphocytes, as reflected by interferon type I signalling pathways and SARS-CoV infection.
下调的模块：主要与tissue morphogenesis，structural integrity 和 homeostasis 有关。比如Module 7 (mainly attributed to epithelial cells) 和Module 12 (mainly attributed to immune cells)。
模块计算方法：
这个module计算方法还真是简单粗暴
Fig 3c, d：To dissect the transcriptional regulons underlying COVID-19 pathogenesis，随后作者使用SCENIC进行了转录因子预测。和预期一致，上调基因的regulatory nodes包括促炎转录因子如NFKB1, REL, STAT1, -3, -4 和 -5A 以及hypoxia regulator HIF1A, UPR regulator ATF6 和 apoptosis regulator BCL6。下调基因的转录因子中，最关键的是FOXO3 (a gene often implicated in tissue morphogenesis and regeneration)。
These analyses highlight the multi-faceted consequences of COVID-19 and provide a molecular portrait of the pathology of the lungs of patients with COVID-19.
4. Senescence as a pathological characteristic of the lungs of patients with COVID-19
许多先前的研究提示SARS-CoV-2感染触发了免疫系统的衰老。
Fig 4a, b, Extended Data Fig. 4f-h：作者发现新冠患者存在p16, p21, IL-6 和 p53 以及DNA oxidation marker 8-hydroxy-2′-deoxyguanosine (8-OHdG)的上调。此外，作者还检测到降低的lamina-associated polypeptide 2 (LAP2)表达，heterochromatin-associated HP1γ的表达也有降低趋势，而LINE1-ORF1p的表达有升高趋势。indicative of exacerbated lung senescence in patients with COVID- 19
基于前面的结果，作者推测：lung ageing might be aggravated by SARS-CoV-2 infection。
Fig 4c：因此作者通过将正常肺脏与正常young individuals (Control-Y)相比，得到了ageing-associated DEGs (ageing DEGs) 。将ageing DEGs和新冠DEGs取交集，得到了91个基因（包括上调和下调）。These DEGs were primarily associated with epithelial and endothelial cells, suggesting that these two cell types are more prone to manifest ageing-related changes in SARS-CoV- 2-infected lungs.
Fig 4d：上调的基因主要与炎症反应相关，下调的基因主要与tissue morphogenesis有关。
Fig 4e：此外作者发现，SASP基因表达were further elevated by COVID-19, indicating that SARS-CoV-2 infection amplifies the pro-inflammatory microenvironment of the aged lung.
Fig 4f：作者还鉴定出20个基因在新冠患者和aged individuals都上调的基因, and genes underlying a variety of ageing-related lung disorders, 提示COVID-19 增强了 pulmonary senescence programmes, which in turn contribute to lung ageing and related disorders.
Extended Data Fig. 5：SASP基因的表达
5. Molecular signatures of epithelial damage in the lungs of patients with COVID-19
Fig 5a：AT2和AT1表达新冠受体，是病毒的主要攻击对象。作者发现了这两种细胞的比例下降，和组织学上的上皮细胞崩解以及肺泡灌洗液中出现肺泡上皮组织并检测出AT1和AT2的标志物相一致。
Fig 5b-d：剩余的AT1和AT2细胞表达细胞marker下降，细胞死亡评分则增加。
Fig 5e：AT1和AT2细胞上调基因主要与免疫反应相关
Fig 5f：此外，AT2的感染引起regeneration-related genes的显著下调，这些基因参与肺脏损伤修复。
Fig 5g：在air–liquid interface，肺泡上皮细胞分泌surfactants以降低肺表面张力。作者在新冠患者肺组织的不同上皮细胞类型中仅检测到了低水平的SFTPC, SFTA3 和 SFTPA1。而黏液蛋白表达则出现增加。
Fig 5h：上面的变化可能是由NFKB1 和 STAT3 调控的。
Fig 5i：在肺脏组织中也验证了粘液蛋白的上调，HE染色也提示粘液栓的存在(Figs. 1b)
6. Dissection of the immune-cell disorders in the lungs of patients with COVID-19
这一部分作者对免疫细胞进行了分析
Fig 6a-d：此前的研究显示，Macrophages are known to play a pivotal role in COVID-19 lethality. 在这里作者也发现了肺组织中巨噬细胞，尤其是促炎性M1巨噬细胞的增多。
Fig 6e：其他免疫细胞也转化成一种更活化的状态，高表达myeloid-leukocyte activation, cytokine signalling 和 SARS-CoV infections 相关基因。其中，一组对免疫细胞活化和机体免疫应答启动的糖基化基因如MGAT1, MGAT4A, MGAT5, PARP8, PARP14, RPN2, ST6GALNAC3 和 ST3GAL1，在COVID-19免疫细胞中高水平上调。
Fig 6f-g：随后作者做了所有细胞的互作分析，结果提示增强的促炎反应和促纤维化反应。尤其是IL1B, IL6, 和 TGFB1, -2 , -3与它们的配体互作显著增强。
Together, these data suggest that an imbalanced host immune system worsens lung damage by releasing excessive cyto- kine factors that drive diffuse alveolar damage.
细胞互作是CellPhoneDB做的
7. Characterization of the endotheliopathy in the lungs of patients with COVID-19
Endothelial cell dysfunction and impaired vascular function contribute to the COVID-19-associated complications with a high mortality risk such as coagulopathy and thrombosis.
Fig 7a：为了进一步探究新冠相关的血管病变，作者将肺泡上皮分为6个亚群。包括1群动脉内皮，1群静脉内皮，1群淋巴管内皮和3群毛细血管内皮(a, g, i)。
Fig 7b：新冠患者的Cap.EC.a 和 Cap.EC.g 都出现下降。与患者常出现的氧气交换障碍和缺氧相一致。
Fig 7c：不同肺泡上皮的凋亡通路都出现了高度激活，提示新冠患者的肺脏内皮细胞的high vulnerability。
Fig 7d：新冠患者内皮细胞的损伤marker也出现了高表达。
Fig 7e：内皮细胞的IL-1相关信号通路，多种趋化因子和细胞粘附分子都出现了表达上调。
Fig 7f：SCENIC分析提示NFKB1, REL 和 CEBPD 是SARS-CoV-2 血管损伤的主要调节者。
Fig 7g：细胞互作分析的结果提示内皮细胞和髓系细胞之间VEGFA 与其受体 FLT1 (VEGFR1), NRP1 和 NRP2 (essential for angiogenesis and vascular permeability) 的互作增强。delineating how SARS-CoV-2 leads to dysregulated angiogenesis of endothelial cells, a signature feature of COVID-19。
Fig 7h-g：内皮感染与增强的凝血激活有关。作者发现损伤的内皮细胞表达更高的促凝血分子如VWF，因而可以介导血小板粘附和凝血反应。
These data point to a working model of how SARS-CoV-2 infection progressively causes endothelial injury and widespread endotheliopathy.
8. Accumulation of myofibroblasts in the lungs of patients with COVID-19
最后一部分作者对肌成纤维细胞进行了分析。
Fig 8a-b：肌成纤维细胞哦的比例在新冠患者增加
Fig 8c：肌成纤维细胞主要由 fibroblasts (还有少部分平滑肌、周皮和AT2细胞) 分化而来。fate2代表了内皮间质转化过程。
Fig 8d：作者鉴定出了与肌成纤维细胞形成有关的560个上调的拟时基因，其中有224个在新冠患者肺组织中也是上调的。
Fig 8e, f：在这些基因中，HIF-1α was identified as a top upregulated transcription factor in the lung fibroblasts of patients with COVID-19。而且，在肺成纤维细胞中，作者也观测到了FOXO3的下调。
Fig 8g：When we used short interfering RNA (siRNA) to knock down FOXO3 in primary human lung fibroblasts, we observed increased cell apoptosis.
Fig 8h, i：FOXO3表达抑制后的差异基因包括extracellular-structure organization, collagen-fibril organization和regulation of the immune response。
Fig 8j：Notably, a series of myofibroblast-marker genes (COL14A1 and COL3A1) were induced in FOXO3-deficient fibroblasts.
These findings support the role of FOXO3 silencing in mediating pro-fibrotic and pro-inflammatory effects in the lungs of patients with COVID-19.
总结图
