Transcriptomic analysis unveils bona fide molecular signatures of microglia under conditions of homeostasis and viral encephalitis

Microglia provide a front-line defense against neuroinvasive viral infection, however, determination of their bona fide transcriptional profiles under conditions of health and disease is challenging. Here, we used a combination of experimental tools to delineate the overall transcriptional landscape of microglia during viral infection. By exploiting the ribosomal tagging approach, we developed the concept of enrichment of relevant marker genes by comparing immunoprecipitated RNA with total RNA. Enriched transcripts corresponding to genes expressed in target cells were instrumental in defining bona fide signatures of microglia. With this approach, we generated a comprehensive and accurate transcriptome of microglia at the in situ environment under conditions of health and virus infection. These unified microglial signatures may serve as a benchmark to retrospectively assess ex vivo artefacts from available atlases. Leveraging the microglial translatome, we found enrichment of genes implicated in T-cell activation and cytokine production during the course of VSV infection. These data linked microglia with T-cell re-stimulation and further underscored that microglia shape anti-viral T-cell responses in the brain. Collectively, this study faithfully defines the transcriptional landscape of microglia in steady state and during viral encephalitis and highlights cellular interactions between microglia and T cells that contribute to the control of virus dissemination. Mixed gender CX3CR1-CreERT2+/- Rpl22wt/HA mice aged 6 weeks were subcutaneously injected with 4 mg of tamoxifen for two consecutive days. 8 weeks after initial tamoxifen injection, mice weeks were intranasally instilled with 10 µL of VSV. Controls animals received same volume of PBS. Six days post infection the animals were sacrificed by cervical dislocation prior to brain harvesting. Thereafter, brain was homogenized and lysate used to pull down HA-tagged ribosomes and to isolate RNA from “input” and RiboTag-IP fractions for bulk RNA-sequencing.

小胶质细胞（Microglia）是抵御神经侵袭性病毒感染的一线防御细胞，然而在健康与疾病状态下解析其真实转录谱仍颇具挑战。本研究联合多种实验手段，刻画了病毒感染过程中小胶质细胞的整体转录景观。通过利用核糖体标记法，我们通过对比免疫沉淀RNA与总RNA，建立了相关标记基因富集的分析思路。靶细胞中表达的基因所对应的富集转录本，有助于明确小胶质细胞的真实特征谱。依托该方法，我们获得了健康及病毒感染状态下原位环境中小胶质细胞的全面且精准的转录组数据。这些统一的小胶质细胞特征谱可作为基准，用于回溯评估现有图谱中的离体人工假象。我们借助小胶质细胞翻译组数据，发现VSV（水疱性口炎病毒）感染过程中与T细胞活化及细胞因子产生相关的基因呈现富集。该数据将小胶质细胞与T细胞再激活建立关联，并进一步证实小胶质细胞可调控脑内抗病毒T细胞应答。综上，本研究明确了稳态及病毒性脑炎状态下小胶质细胞的转录景观，并揭示了小胶质细胞与T细胞间的细胞互作，该互作有助于控制病毒的扩散。
本研究选用6周龄的混合性别CX3CR1-CreERT2+/- Rpl22wt/HA小鼠，连续2天皮下注射4 mg他莫昔芬。初始他莫昔芬注射8周后，对小鼠经鼻腔滴注10 μL VSV；对照组小鼠则注射等体积磷酸盐缓冲液（PBS）。感染6天后，采用颈椎脱位法处死小鼠并取材脑组织。随后将脑组织匀浆，取裂解液用于免疫沉淀HA标记的核糖体，并分别从"输入"组分及RiboTag免疫沉淀组分中分离RNA，用于批量RNA测序。