Transcriptomic responses of human monocyte-derived dendritic cells to HIV and other innate stimuli. Transcriptomic responses of human monocyte-derived dendritic cells to HIV and other innate stimuli

Transcriptional programming of the innate immune response is pivotal for host protection. The transcriptional mechanisms that link pathogen sensing with innate activation remain poorly understood. During infection with HIV-1, human dendritic cells (DCs) can detect the virus through an innate sensing pathway leading to antiviral type I interferon and DC maturation. Here, we have developed an iterative experimental and computational approach to map the innate response circuitry during HIV-1 infection. By integrating genome-wide chromatin accessibility with expression kinetics, we have inferred a gene regulatory network that links 542 transcription factors (TFs) with 21,862 target genes. Through genetic perturbation and drug treatments we identify PRDM1 and RARA as essential regulators of the interferon response and DC maturation, respectively. This work provides a resource for interrogation of regulators of HIV replication and innate immunity, highlighting the complexity and cooperativity in the regulatory circuit controlling the DC response to HIV-1 infection. Overall design: DCs were derived from the peripheral blood from seven deidentified human donors and infected with HIV or stimulated with innate immune agonists. Conditions include infection with HIV-1-GFP in the presence or absence of Vpx, infection with wild type HIV-1, HIV-2, mutated HIV-2 capsid, or stimulation with LPS ( 100ng/ml), polyIC (10ug/ml) , cGAMP, recombinant IFN, R848, and polydCdG. Bulk RNA-Seq was performed on cells at 0, 2, 6, 24, and 48 hours after infection or stimulation as indicated. For some samples, cells were sorted by flow cytometry prior to sequencing based on GFP or CD86 expression as indicated.

固有免疫应答的转录编程对于宿主防御至关重要。将病原体感知与固有免疫激活相联系的转录调控机制，目前仍不甚明晰。在HIV-1感染过程中，人类树突状细胞（dendritic cells, DCs）可通过固有感知通路识别病毒，进而触发抗病毒I型干扰素应答与DC成熟过程。本研究开发了一套迭代式实验与计算结合的方法，用于解析HIV-1感染过程中的固有免疫应答调控网络。通过整合全基因组染色质可及性数据与表达动力学数据，本研究推断出一个基因调控网络，该网络将542个转录因子（transcription factors, TFs）与21862个靶基因关联起来。通过基因扰动与药物处理实验，我们分别确定PRDM1与RARA为干扰素应答与DC成熟的关键调控因子。本研究为探究HIV复制与固有免疫的调控因子提供了宝贵资源，同时揭示了调控DC对HIV-1感染应答的调控网络所具备的复杂性与协同性。

实验设计：从7名匿名人类供者的外周血中分离培养DCs，分别用HIV感染或固有免疫激动剂进行刺激。实验组别包括：在存在或不存在Vpx的条件下感染HIV-1-GFP；感染野生型HIV-1、HIV-2以及突变型HIV-2衣壳；或用脂多糖（LPS，100ng/ml）、聚肌胞苷酸（polyIC，10μg/ml）、环鸟苷酸-腺苷酸（cGAMP）、重组干扰素（recombinant IFN）、R848以及聚dC-dG（polydCdG）进行刺激。按照实验设置，分别在感染或刺激后的0、2、6、24以及48小时收集细胞，进行批量RNA测序（bulk RNA-Seq）。部分样本将按照实验要求，在测序前通过流式细胞术根据GFP或CD86的表达水平对细胞进行分选。