A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation
A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation
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DOI：
10.1074/jbc.c115.700492
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发表时间：
2016-01-01
影响因子：
4.8
通讯作者：
Hornung, Veit
中科院分区：
文献类型：
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作者：
Schmid-Burgk, Jonathan L.;Chauhan, Dhruv;Hornung, Veit
炎性小体是高分子量蛋白质复合物，其在遇到病原体时在胞质溶胶中组装。这导致半胱天冬酶-1依赖性促炎细胞因子成熟，以及一种特殊类型的细胞死亡，称为焦亡。Nlrp 3炎性体在病原体防御中起着关键作用，但同时，其活性也涉及许多常见的无菌炎症状况。为此，几项研究已经鉴定了Nlrp 3炎性体参与许多常见的人类疾病，如动脉粥样硬化、2型糖尿病、阿尔茨海默病或痛风。尽管已经表明已知的Nlrp 3刺激物会聚于Nlrp 3激活上游的钾离子流出，但Nlrp 3激活的确切分子机制仍然难以捉摸。在这里，我们描述了永生化小鼠巨噬细胞中的全基因组CRISPR/Cas9筛选，旨在无偏地鉴定参与Nlrp 3炎性小体激活的基因产物。我们采用了基于FACS的屏幕Nlrp 3依赖性细胞死亡，使用离子载体化合物尼日利亚菌素作为钾流出诱导刺激。使用全基因组指导RNA（gRNA）文库，我们发现靶向Nek 7将巨噬细胞从尼日利亚菌素诱导的致死性中拯救出来。随后的研究显示，Nek 7缺陷的鼠巨噬细胞显示出很大程度上钝化的Nlrp 3炎性小体应答，而Aim 2介导的炎性小体活化被证明是完全完整的。虽然Nek 7在Nlrp 3上游发挥作用的机制仍然难以捉摸，但这些研究提供了Nlrp 3上游特异性发挥作用的组分的第一个遗传处理。
Inflammasomes are high molecular weight protein complexes that assemble in the cytosol upon pathogen encounter. This results in caspase-1-dependent pro-inflammatory cytokine maturation, as well as a special type of cell death, known as pyroptosis. The Nlrp3 inflammasome plays a pivotal role in pathogen defense, but at the same time, its activity has also been implicated in many common sterile inflammatory conditions. To this effect, several studies have identified Nlrp3 inflammasome engagement in a number of common human diseases such as atherosclerosis, type 2 diabetes, Alzheimer disease, or gout. Although it has been shown that known Nlrp3 stimuli converge on potassium ion efflux upstream of Nlrp3 activation, the exact molecular mechanism of Nlrp3 activation remains elusive. Here, we describe a genome-wide CRISPR/Cas9 screen in immortalized mouse macrophages aiming at the unbiased identification of gene products involved in Nlrp3 inflammasome activation. We employed a FACS-based screen for Nlrp3-dependent cell death, using the ionophoric compound nigericin as a potassium efflux-inducing stimulus. Using a genome-wide guide RNA (gRNA) library, we found that targeting Nek7 rescued macrophages from nigericin-induced lethality. Subsequent studies revealed that murine macrophages deficient in Nek7 displayed a largely blunted Nlrp3 inflammasome response, whereas Aim2-mediated inflammasome activation proved to be fully intact. Although the mechanism of Nek7 functioning upstream of Nlrp3 yet remains elusive, these studies provide a first genetic handle of a component that specifically functions upstream of Nlrp3.