高可Coco

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2022/11/14阅读:21主题:默认主题

001-文献分析分享:普通转录组+单细胞分析-巨噬细胞分群 (2)

001-文献分析分享:普通转录组+单细胞分析-巨噬细胞分群 (2)

行动和思考都很重要, 但是行动和思考之间本身就是在争抢时间。


Cell Metabolism 2022 IF: 31
Cell Metabolism 2022 IF: 31

继续上篇的Figure1

Figure1

  • Figure 1E 单细胞轨迹分析:RNA velocity analysis.

随着这张图进入结果的第2个section Regulation of NASH-associated liver macrophages

单细胞RNA-seq数据区分未成熟的mRNA(unspliced)和成熟的RNA(spliced)

教程可参考简书单细胞轨迹分析-1

细胞轨迹:从经典的单核-->NAM s和KC (其实这部分发在section1里也可,放在这里的原因探索NAM的上游,从细胞到分子机制,使的第二部分更完整)

we observed notable cell state transitions from classical monocytes to NAMs and KC subclusters

  • Figure 1F 小鼠移植后肝脏巨噬细胞的FCM

探索NAM的起源骨髓,用了host CD45.2和 donor CD45.1,发现表达NAM的marker共表达CD45.1,说明是BM产生的NAM (低配版是不是也可用CSFE细胞标记?)

+ 背景介绍
- Donor 1: CD45.1
- Host 2: CD45.2
小鼠的CD45蛋白有两个等位基因,
可用CD45.1和CD45.2作为
受供受之间的细胞过继转移,
从而研究细胞的增殖分化分布特征


Figure 1 G and H

  • (G) Bubble plot illustrating relative mRNA expression for genes encoding secreted factors (top) and membrane proteins (bottom) with enriched expression in NAMs.

However, the nature of the extracellular signals that trigger the induction of this macrophage subpopulation remains largely unknown. To address this, we performed differential gene expression analysis for secreted factors and membrane proteins among five macrophage subclusters. This analysis identified a set of putative ligands exhibiting enriched expression in NAMs, including Pf4, Apoe, Spp1, and Ccl4

  • (H) Violin plot of gene expression among different macrophage subclusters.

Violin plots indicate that mRNA expression of Trem2, Tgfbr1, C3ar1, and C5ar1 exhibited higher expression in NAMs compared with other sub clusters

气泡图和小提琴图可视化:为探索NAM上游分子机制提供证据

  • (l) qPCR analysis of Tgfb expression in chow and NASH liver.

qPCR analysis revealed that hepaticmRNAexpression of all three TGF-b ligands was elevated upon diet-induced NASH in mice

  • (J) qPCR analysis of gene expression in cultured BMDMs treated with vehicle or 2.5 ng/mL TGF-b for 24 h.

To examine whether TGF-b promotes NAM gene expression in a cell-autonomous manner, we performed treatments on bone marrow-derived macrophages (BMDMs) and observed that TGF-b robustly stimulated mRNA expression of Trem2, Gpnmb, Apoe, and Tgfbr1 in cultured macrophages (Figure 1J).

体外实验验证分子机制,验证成功

通过TGF-b信号通路促进NAM的产生和TAM在肿瘤发生的促进作用,相当于找NAM的上游(暂时这么理解)

进入Section3部分 NASH pathogenesis triggers CD8+ T cell exhaustion in the liver

  • Figure 2A 上:UMAP-NPCs;下:T细胞被分为CD8+,CD4+,NKT UMAP illustrating T cells among liver NPCs (top) and three T cell subclusters
  • Figure 2B CD8+T细胞亚群显示出NASH的一系列基因下调
  • Figure 2C NASH鼠的CD8+T细胞,涉及到TCR信号和IFN-r信号 关于细胞质蛋白转化和淋巴分化的相关基因被下调
  • Figure 2E NASH饮食诱导后mRNA水平增高 用PD-1免疫荧光抗体和流式评估T细胞耗竭
  • Figure 2F PD1阳性逐渐增多
  • Figure 2G FCM显示和Chow组相比,高PD1的T细胞在CD8+T细胞亚群从9.6%到54%在NASH组
  • Figure 2H 在CD4+T细胞组,PD-1表达也增高
  • Figure 2I CD8+T细胞的CFSE增殖
  • Figure 2J 人的样本验证

CD4+T细胞亚群分为四群:naive CD4+T,Tregs,Th17,Th1细胞

DC分群:Cd209+,plasacytoid DC,Xcr1 high,Ccr7high,Xcr1lowCCR7high,DCs.Cd209+DC在NASH的肝脏扩增。

NASH肝脏 Trem2+巨噬细胞和耗竭的CD+8T细胞耗竭

进入Section 4部分 NRG4 shapes the liver microenvironment and serves as a checkpoint for NASH-associated HCC

之前的研究结果表明NRG4调节肝脏代谢等发挥重要作用。但是NRG4对免疫微环境的影响并不明确

因此测了NRG4-KO和WT小鼠bulk RNA-seq,发现了1929个272个差异基因

Figure3

富集分析

NRG4缺乏提高了NASH相关诱导基因涉及的T细胞耗竭

结果提示NRG4在肝脏免疫微环境中发挥了重要作用,并且提示了NRG4在NASH-相关HCC的发展中起到了重要作用

DEN/NASH protocol

WT和KO鼠比较体重 WT和KO鼠比较疾病进展

进入Section 5部分 NRG4 restrains tumor-prone liver immune microenvironment during NASH

从WT和NRG4 KO分离的NPC又进行了一次scRNA-seq,和先前的结果一样,UMAP分析显示了16个clusters,展示了8个主要的活细胞群(内皮细胞、巨噬细胞、T、B、DC、hepatocyte,cholangiocyte,HSC)

Figure4 A

巨噬细胞cluster包含了12,824个细胞,是NPCs最大的亚群。

亚群分析显示4个主要的巨噬细胞群 KC、MDM、NAM和dividing cells

比较了WT和NRG4 KO小鼠巨噬细胞亚群的比例,NAM还是在NRG4 KO小鼠中多见

进入下一个Section

Accordingly, immunofluorescence indicated that F4/80, GPNMB, and MHC-II were markedly increased in NRG4 KO livers

背景介绍
F4/80, GPNMB, and MHC-II
巨噬细胞的marker

一些T细胞亚群展示了CD4+, CD8+ NKT,少见T cells

CD8+ T cells isolated from NRG4 KO mouse livers exhibited reduced proliferative response upon activation by CD3/CD28 Dynabeads

Flow cytometry analysis of PD1 expression in intrahepatic CD8+ T cells from NASH-diet-fed WT (n = 8) and NRG4 KO (n = 9) mice.
;CFSE proliferation assay of CD8+ T cells from WT (n = 8) and NRG4 KO (n = 10) mice fed NASH diet for 5 months.
Flow cytometry analysis of PD1 expression in intrahepatic CD8+ T cells from NASH-diet-fed WT (n = 8) and NRG4 KO (n = 9) mice. ;CFSE proliferation assay of CD8+ T cells from WT (n = 8) and NRG4 KO (n = 10) mice fed NASH diet for 5 months.
Anti-PDL1 treatment study. WT and NRG4 KO mice were subjected to the DEN/NASH tumor induction protocol followed by treatments twice a week: WT
IgG2b (n = 10), WT anti-PDL1 (n = 15), Nrg4 KO IgG2b (n = 11), and Nrg4 KO anti-PDL1 (n = 10).
Anti-PDL1 treatment study. WT and NRG4 KO mice were subjected to the DEN/NASH tumor induction protocol followed by treatments twice a week: WT IgG2b (n = 10), WT anti-PDL1 (n = 15), Nrg4 KO IgG2b (n = 11), and Nrg4 KO anti-PDL1 (n = 10).
Anti-PDL1 treatment study. WT and NRG4 KO mice were subjected to the DEN/NASH tumor induction protocol followed by treatments twice a week: WT
IgG2b (n = 10), WT anti-PDL1 (n = 15), Nrg4 KO IgG2b (n = 11), and Nrg4 KO anti-PDL1 (n = 10).
Anti-PDL1 treatment study. WT and NRG4 KO mice were subjected to the DEN/NASH tumor induction protocol followed by treatments twice a week: WT IgG2b (n = 10), WT anti-PDL1 (n = 15), Nrg4 KO IgG2b (n = 11), and Nrg4 KO anti-PDL1 (n = 10).

这些结果提示在NRG4缺乏的情况下,T细胞功能不足可能这有助于肝脏成瘤。

PD-1治疗效果,提示受损的免疫检查点有助于NRG4 KO小鼠中NASH-HCC的发生

巨噬细胞-NRG4-CD8T细胞-PD-1 感觉pd-1这部分硬拉啊

绕了好多圈圈~~~这部分需要画个思维导图

本部分总结

  • 通过巨噬细胞分群找到NAM
  • NAM在疾病进展过程中数量逐渐增多
  • NAM与T细胞的细胞通讯(细胞互作)找下游
  • PD-1治疗干预回复实验——临床意义提升
  • 明星分子:NRG4在其中的作用(这个才是最大亮点。一般人猜不到分子)

PS: 分享一个科研大佬说的话(临床狗 vs 科学家思维的区别)

当我们要做什么事情的时候,并不是因为没人做过我们才做。而是这件事真的很有意义,需要有人去完成

我们追求的创新,也许是为了更好的解决问题。从另一个角度,窥见未知世界。

谢谢所有让我成长的机会。

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高可Coco
V1

喜欢抹茶冰淇淋的医学生