建立全基因組干擾型CRISPR系統篩選參與白介素-4所誘導巨噬細胞M2型極化途徑之調控基因

To establish CRISPR interference-based whole-genome screening platform for identification of novel genes in IL4-mediated macrophage alternative polarization

陳威羽(Wei-Yu Chen)

指導教授 ： 蔡欣祐

國立臺灣大學/醫學院/分子醫學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202103353

摘要

巨噬細胞是一群對於先天性免疫以及組織恆定性相當重要的免疫細胞，透過接受環境中不同的刺激物，而有不同的活化型態。極化的巨噬細胞可分為典型活化型態(M1)與另類活化型態(M2)兩個極端型態，前者具有促進發炎反應的功能，而後者則與抗發炎反應息息相關。目前的文獻證實在哺乳動物的惡性腫瘤中，有一群與另類活化型態(M2)相似的巨噬細胞，他們受CD4+T細胞分泌的白介素-4（Interleukin-4，IL-4）所誘導並參與癌症轉移(metastasis)的過程。MCPIP1是一個具有核醣核酸酶活性的蛋白，曾被報導在IL-4所誘導之M2巨噬細胞極化過程中扮演著不可或缺的角色。儘管受IL-4所誘導的M2巨噬細胞對於癌症的了解極其重要，但是詳細的機制，像是參與在訊號傳導途徑中的基因卻尚未清楚。因此，我們的研究目的為確認MCPIP1是否確實參與在巨噬細胞另類極化過程，並且建立CRISPR干擾(CRISPR interference，CRISPRi)的全基因篩選平台，找出IL-4所誘導之M2巨噬細胞極化過程中的調控基因。期望透過流式細胞儀(flow cytometry)以及次世代定序(next generation sequencing，NGS)，我們能夠篩選出受CRISPRi所干擾，導致在IL-4誘導下無法成功進行M2極化的基因，即為潛在的M2極化的調控者。
在本篇研究中，首先我們製備野生型(wild-type)與MCPIP1突變型之永生化老鼠骨髓源性巨噬細胞 (immortalized bone marrow-derived macrophages，iBMDMs) 還有可誘導過量表現MCPIP1基因之iBMDMs，來確認MCPIP1對於M2巨噬細胞極化過程的重要性，不過結果顯示其與先前研究並不完全一致。為了利用全基因篩選找出參與在M2巨噬細胞極化過程的重要基因，我們測試iBMDMs的M2表現型並優化能夠區分M2及未誘導(M0)巨噬細胞的條件，發現當細胞在IL-4誘導48小時下，透過流式細胞儀共同偵測M2巨噬細胞表面標誌CD206與CD301b， 能夠最佳區分M2及M0巨噬細胞。同時，我們也測試了其他的巨噬細胞株，像是J774A.1與Raw264.7。目前我們成功製備穩定表現dCas9-KRAB的iBMDMs，也測試了在iBMDMs中sgRNA文庫的多樣性。簡而言之，我們製備了不同的細胞株; 測試了適合的巨噬細胞株; 優化了能區分M2/M0巨噬細胞的條件，來提供CRISPRi全基因篩選的重要材料。

關鍵字

另類活化型態巨噬細胞 ； 介白素-4 ； MCPIP1 ； CRISPR干擾 ； sgRNA文庫 ； 全基因篩選

並列摘要

Macrophages are immune cells playing important roles in innate immunity and tissue homeostasis. By responding to a wide spectrum of stimuli, macrophages are polarized to pro-inflammatory macrophages (classically activated /M1 macrophages) and anti-inflammatory macrophages (alternative activated/M2 macrophages). Studies of tumor-associated macrophages (TAMs) in mammary carcinomas have shown that M2-like TAMs promote metastasis mediated by CD4+ T cell secreted IL-4. MCPIP1 is a ribonuclease reported with an essential role in M2 (IL-4) polarization pathway. Despite the importance of IL-4 stimulated M2 macrophages, very little is known about the signaling cascade or genes that participated in the polarization processes. Thus, the aim of our study is to confirm the role of MCPIP1 in M2 (IL-4) polarization and establish a CRISPR interference (CRISPRi)-based whole-genome screening platform to identify novel genes that are required for IL-4 polarized M2 macrophages.
In this study, we first generated wild-type and Mcpip1mt/mt immortalized bone marrow-derived macrophages (iBMDMs) as well as the inducible MCPIP1 overexpression iBMDMs for confirmation of MCPIP1 roles in M2 macrophage polarization. However, the role of MCPIP1 in M2 macrophages in our hand was not completely consistent with the previous study. We then tested iBMDMs and optimized the condition to distinguish M2 (IL-4)/M0 (uninduced) macrophages for establishing a whole-genome screen. By detecting the surface expression of CD206 and CD301b together upon 48 h IL-4 induction, we were able to obtain the best M2 (IL-4)/M0 (uninduced) distinction. We also tested other macrophage cell lines J774a.1 and Raw264.7 for backup. Meanwhile, we generated the stably expressing dCas9-KRAB iBMDMs and also tested the diversity of the sgRNA library in iBMDMs. In sum, we generated multiple cell lines; testing the feasible macrophage cell model; optimizing the procedures for M2 (IL-4)/M0 (uninduced) macrophages distinction; to provide components in CRISPRi screening assay.

並列關鍵字

macrophage alternative polarization ； interleukin-4 ； MCPIP1 ； CRISPR interference ； sgRNA library ； genome-wide screening assay

參考文獻

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Orecchioni, M., et al., Macrophage Polarization: Different Gene Signatures in M1(LPS+) vs. Classically and M2(LPS-) vs. Alternatively Activated Macrophages. Front Immunol, 2019. 10: p. 1084.