Institut Curie, PSL Research University, INSERM U932, France.
Department of Medicine, Cambridge Institute for Medical Research, Cambridge Biomedical Campus, UK.
Innate Immun. 2020 Aug;26(6):459-472. doi: 10.1177/1753425920915507. Epub 2020 Apr 4.
A subset of TLRs is specialised in the detection of incoming pathogens by sampling endosomes for nucleic acid contents. Among them, TLR3 senses the abnormal presence of double-stranded RNA in the endosomes and initiates a potent innate immune response via activation of NF-κB and IRF3. Nevertheless, mechanisms governing TLR3 regulation remain poorly defined. To identify new molecular players involved in the TLR3 pathway, we performed a genome-wide screen using CRISPR/Cas9 technology. We generated TLR3 reporter cells carrying a NF-κB-responsive promoter that controls GFP expression. Cells were next transduced with a single-guide RNA (sgRNA) library, subjected to sequential rounds of stimulation with poly(I:C) and sorting of the GFP-negative cells. Enrichments in sgRNA estimated by deep sequencing identified genes required for TLR3-induced activation of NF-κB. Among the hits, five genes known to be critically involved in the TLR3 pathway, including TLR3 itself and the chaperone UNC93B1, were identified by the screen, thus validating our strategy. We further studied the top 40 hits and focused on the transcription factor aryl hydrocarbon receptor (AhR). Depletion of AhR had a dual effect on the TLR3 response, abrogating IL-8 production and enhancing IP-10 release. Moreover, in primary human macrophages exposed to poly(I:C), AhR activation enhanced IL-8 and diminished IP-10 release. Overall, these results reveal AhR plays a role in the TLR3 cellular innate immune response.
TLR 家族的一个亚类专门通过内体对核酸内容进行采样来检测入侵病原体。其中，TLR3 在内体中检测到双链 RNA 的异常存在，并通过激活 NF-κB 和 IRF3 引发强烈的先天免疫反应。然而，TLR3 调节的机制仍未得到明确界定。为了鉴定参与 TLR3 途径的新分子，我们使用 CRISPR/Cas9 技术进行了全基因组筛选。我们生成了携带 NF-κB 反应性启动子的 TLR3 报告细胞，该启动子控制 GFP 的表达。然后，细胞被转导了一个单指导 RNA(sgRNA)文库，用 poly(I:C)进行连续刺激并对 GFP 阴性细胞进行分选。通过深度测序估计 sgRNA 的富集，鉴定出了 TLR3 诱导 NF-κB 激活所必需的基因。在命中物中，筛选鉴定出了五个已知在 TLR3 途径中起关键作用的基因，包括 TLR3 本身和伴侣蛋白 UNC93B1，从而验证了我们的策略。我们进一步研究了前 40 个命中物，并将重点放在转录因子芳香烃受体(AhR)上。AhR 的耗竭对 TLR3 反应具有双重影响，既消除了 IL-8 的产生，又增强了 IP-10 的释放。此外，在暴露于 poly(I:C)的原代人巨噬细胞中，AhR 的激活增强了 IL-8 的释放，同时减少了 IP-10 的释放。总之，这些结果表明 AhR 在 TLR3 细胞先天免疫反应中发挥作用。
