Alarmin-loaded extracellular lipid droplets induce airway neutrophil infiltration during type 2 inflammation

Group 2 innate lymphoid cells (ILC2s) play a crucial role in allergic diseases by coordinating a complex network of various effector cell lineages involved in type 2 inflammation. However, their function in regulating airway neutrophil infiltration, a deleterious symptom of severe asthma, remains unknown. Here, we observed ILC2-dependent neutrophil accumulation in the bronchoalveolar lavage fluid (BALF) of allergic mice models. Chromatography followed by proteomics analysis identified the alarmin high mobility group box-1 (HMGB1) in the supernatant of lung ILC2s initiated neutrophil chemotaxis. Genetic perturbation of Hmgb1 in ILC2s reduced BALF neutrophil numbers and alleviated airway inflammation. HMGB1 was loaded onto the membrane of lipid droplets (LDs) released from activated lung ILC2s. Genetic inhibition of LD accumulation in ILC2s significantly decreased extracellular HMGB1 abundance and BALF neutrophil infiltration. These findings unveil a previously uncharacterized extracellular LD-mediated immune signaling delivery pathway by which ILC2s regulate airway neutrophil infiltration during allergic inflammation. Bone marrow neutrophils and lung ILC2s were isolated from wildtype (WT) mice in homeostasis for in vitro stimulation and Bulk RNA-seq. ILC-enriched population (CD45+CD3-CD19- cells) was purified from the lungs of Il5cre/+Hilpda+/+ and Il5cre/+Hilpdaflox/flox mice at day 3 after IIL-33 treatment for scRNA-seq.

2型固有淋巴细胞（Group 2 innate lymphoid cells，ILC2s）通过调控参与2型炎症的各类效应细胞谱系的复杂网络，在过敏性疾病中发挥关键作用。然而，其在调控气道中性粒细胞浸润——重症哮喘的有害病理特征——中的功能仍未明确。本研究在过敏性小鼠模型的支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）中观察到ILC2依赖性的中性粒细胞聚集现象。经色谱联用蛋白质组学分析，我们在肺ILC2的上清液中鉴定出警报素高迁移率族蛋白B1（high mobility group box-1，HMGB1）可启动中性粒细胞趋化。对ILC2中的Hmgb1进行基因扰动可降低BALF中的中性粒细胞数量，并缓解气道炎症。HMGB1被装载至活化肺ILC2释放的脂滴（lipid droplets，LDs）膜上。抑制ILC2内脂滴积累的基因操作可显著降低细胞外HMGB1的丰度与BALF中的中性粒细胞浸润。本研究揭示了一条此前未被阐明的细胞外脂滴介导的免疫信号传递通路，ILC2可通过该通路在过敏性炎症过程中调控气道中性粒细胞浸润。本研究从稳态下的野生型（wildtype，WT）小鼠体内分离骨髓中性粒细胞与肺ILC2，用于体外刺激实验与批量RNA测序（Bulk RNA-seq）。从经IL-33处理3天后的Il5cre/+Hilpda+/+与Il5cre/+Hilpdaflox/flox小鼠肺脏中纯化得到富集ILC的细胞群（CD45+CD3-CD19-细胞），用于单细胞RNA测序（scRNA-seq）。