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 的一个子集专门用于通过对核酸内容物的核内体取样来检测传入的病原体。其中，TLR3 感知核内体中双链 RNA 的异常存在，并通过激活 NF-κB 和 IRF3 启动有效的先天免疫反应。然而，TLR3 监管的机制仍然不明确。为了识别参与 TLR3 通路的新分子参与者，我们使用 CRISPR/Cas9 技术进行了全基因组筛选。我们生成了 TLR3 ⁺携带 NF-κB 反应启动子的报告细胞，该启动子控制 GFP 表达。接下来用单向导 RNA (sgRNA) 文库转导细胞，用 poly(I:C) 进行连续多轮刺激并分选 GFP 阴性细胞。通过深度测序估计的 sgRNA 富集鉴定了 TLR3 诱导的 NF-κB 激活所需的基因。在命中中，屏幕识别出五个已知与 TLR3 通路密切相关的基因，包括 TLR3 本身和伴侣 UNC93B1，从而验证了我们的策略。我们进一步研究了前 40 个匹配项，并专注于转录因子芳烃受体 (AhR)。AhR 的消耗对 TLR3 反应具有双重影响，即取消 IL-8 的产生并增强 IP-10 的释放。此外，在暴露于 poly(I:C) 的原代人类巨噬细胞中，AhR 激活增强了 IL-8 并减少了 IP-10 的释放。总的来说，这些结果表明 AhR 在 TLR3 细胞先天免疫反应中发挥作用。