PIP-seq identifies novel heterogeneous lung innate lymphocyte population activation after combustion product exposure. PIP-seq identifies novel heterogeneous lung innate lymphocyte population activation after combustion product exposure

Innate lymphoid cells (ILCs) are a heterogeneous population that play diverse roles in airway inflammation after exposure to allergens and infections. However, how ILCs respond after exposure to environmental toxins is not well understood. Here we show a novel method for studying the heterogeneity of rare lung ILC populations by magnetic enrichment for lung ILCs followed by particle-templated instant partition sequencing (PIP-seq). Using this method, we were able to identify novel group 1 and group 2 ILC subsets that exist after exposure to both fungal allergen and burn pit-related constituents (BPC) that include dioxin, aromatic hydrocarbon, and particulate matter. Toxin exposure in combination with fungal allergen induced activation of specific ILC1/NK and ILC2 populations as well as promoted neutrophilic lung inflammation. Oxidative stress pathways and downregulation of specific ribosomal protein genes (Rpl41 and Rps19) implicated in anti-inflammatory responses were present after BPC exposure. Increased IFNγ expression and other pro-neutrophilic mediator transcripts were increased in BPC-stimulated lung innate lymphoid cells. Further, the addition of BPC induced Hspa8 (encodes HSC70) and aryl hydrocarbon transcription factor activity across multiple lung ILC subsets. Overall, using an airway disease model that develops after occupational and environmental exposures, we demonstrate an effective method to better understand heterogenous ILC subset activation. Overall design: The mice received intranasal Alternaria alternata challange without or with the supplement of the environmental toxin cocktail (TCDD, BaP, and PM4) for continous 3 days. The next day, mouse lung ILCs were negatively enriched by Mouse Pan-ILC Enrichment Kit. The transcriptome was analyzed by scRNA-seq.

固有淋巴细胞（innate lymphoid cells, ILCs）是一类异质性群体，在暴露于过敏原与感染后可在气道炎症中发挥多种功能。然而，固有淋巴细胞在暴露于环境毒素后的应答机制尚未被充分阐明。本研究开发了一种研究稀有肺固有淋巴细胞群体异质性的新方法：先通过磁富集分离肺固有淋巴细胞，随后采用粒子模板即时分区测序（particle-templated instant partition sequencing, PIP-seq）进行分析。利用该方法，我们在暴露于真菌过敏原与垃圾焚烧场相关成分（burn pit-related constituents, BPC，包括二噁英、芳香烃与颗粒物）的样本中，鉴定出了新型1型固有淋巴细胞（ILC1）与2型固有淋巴细胞（ILC2）亚群。毒素暴露联合真菌过敏原刺激，可激活特定的1型固有淋巴细胞/自然杀伤细胞（ILC1/NK）与2型固有淋巴细胞（ILC2）亚群，并促进肺组织中性粒细胞性炎症的发生。在暴露于BPC后，样本中可检测到氧化应激通路激活，以及与抗炎应答相关的特定核糖体蛋白基因（Rpl41与Rps19）的表达下调。在BPC刺激的肺固有淋巴细胞中，干扰素γ（interferon gamma, IFNγ）的表达以及其他促中性粒细胞介质的转录本水平均有所升高。此外，添加BPC可在多种肺固有淋巴细胞亚群中诱导热休克蛋白70家族成员8（Hspa8，编码HSC70）的表达以及芳香烃转录因子的活性。综上，本研究利用职业与环境暴露诱导建立的气道疾病模型，证明了一种可有效解析异质性固有淋巴细胞亚群激活机制的方法。实验设计：小鼠连续3天经鼻内接种链格孢菌（Alternaria alternata），同时分别添加或不添加环境毒素混合物（TCDD、BaP与PM4）。次日，采用小鼠全固有淋巴细胞富集试剂盒（Mouse Pan-ILC Enrichment Kit）对小鼠肺固有淋巴细胞进行阴性富集。随后通过单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）对转录组进行分析。