研究背景

哮喘是最常见的呼吸系统疾病之一，造成了沉重的医疗负担。哮喘属于慢性气道炎症疾病，免疫记忆是调节气道炎症的关键机制。中性粒细胞型哮喘跟重度哮喘和激素治疗抵抗相关，是造成哮喘医疗负担的主要临床表型，但是其机制仍有待进一步探究。

方法结果

为探究哮喘中促进气道炎症的关键细胞类型, 本研究通过单细胞转录组测序检测卵清蛋白诱导的慢性过敏性哮喘模型中肺组织细胞分群的改变，发现中性粒细胞在哮喘中增加显著，并存在4个不同的亚群，包括FcγRIIb⁺中性粒细胞和Cd177⁺中性粒细胞等。功能分析和细胞互作分析提示：Cd177⁺中性粒细胞为脱颗粒相关中性粒细胞，可以分泌中性粒细胞胞外陷阱促进气道炎症，并通过分泌肿瘤抑制素M（Oncostatin M, Osm）促进气道重塑。通过细胞轨迹和转录因子富集分析发现C/EBPβ和PU.1等转录因子可能调节中性粒细胞表型转换并介导中性粒细胞异质性。

其中FcγRIIb⁺中性粒细胞亚群在哮喘中特异性增加并与IL-4受体信号相关。功能分析提示：FcγRIIb⁺中性粒细胞可能为记忆性中性粒细胞，具有固有免疫记忆的分子特征，主要表现为转录因子激活介导的表观遗传修饰和Toll样受体通路的激活，以及炎症因子分泌通路的上调。为了明确FcγRIIb⁺中性粒细胞是否为记忆性中性粒细胞，分别构建慢性哮喘和急性哮喘模型，然后通过qPCR和Western-blot分析验证固有免疫记忆的关键特征分子，发现这些分子在流式分选的FcγRIIb⁺中性粒细胞中均为高表达，包括免疫记忆相关转录因子(C/EBPβ、Runx1)的激活，组蛋白修饰通路(Tet2，β-钙黏蛋白)、Toll样受体通路(Tlr2, Cd300lf)和cAMP通路(PDE4B)激活，以及炎症因子IL-1β和IL-6分泌通路的上调活化。最终推断FcγRIIb⁺中性粒细胞为促进气道炎症的关键细胞亚群。另外，FcγRIIb⁺中性粒细胞与尘螨加脂多糖诱导的中性粒细胞型哮喘也密切相关，提示记忆性中性粒细胞在中性粒细胞型哮喘中普遍存在。

为了研究哮喘与动脉粥样硬化共同的炎症机制，通过共同靶点网络分析等方法，发现YB1介导的mRNA稳定性可能在其中发挥了重要作用。通过慢病毒转染构建YB1磷酸化位点失义突变的小鼠细胞并进行转录组测序，发现YB1磷酸化可以影响CCL2等炎症因子表达以及糖皮质激素受体(GR)响应通路。通过Western-blot和qPCR验证发现：GR相关的复合物(GR、PNRC2、DCP1A、UPF1和HRSP12)可以与CCL2的mRNA结合调节其mRNA稳定性。

结论

本研究阐释了哮喘肺组织中中性粒细胞的异质性，其中FcγRIIb⁺中性粒细胞群为记忆性中性粒细胞，是促进哮喘的关键细胞亚群，另外Cd177⁺中性粒细胞通过分泌中性粒细胞胞外陷阱和Osm促进哮喘。本研究揭示了YB1是参与哮喘与动脉粥样硬化共同的炎症机制的分子。

Background

Asthma is one of the most common respiratory diseases, which imposes a heavy burden on healthcare systems. Asthma is a chronic airway inflammatory disease and immune memory is a key mechanism that regulate airway inflammation. Neutrophilic asthma is associated with severe asthma and glucocorticoid resistance and is the main clinical phenotype contributing to the medical burden of asthma, but its mechanisms remain to be further investigated.

Methods and Results

To investigate the key cell types that promote airway inflammation in asthma, this study used single-cell transcriptome sequencing to detect changes in cell subpopulations in the lung tissue of a chronic allergic asthma model induced by ovalbumin. It was found that neutrophils were significantly increased in asthma, and there were four different subgroups, including FcγRIIb⁺ neutrophils and Cd177⁺ neutrophils. Functional and cell interaction analyses suggested that Cd177⁺ neutrophils were degranulation-associated neutrophils that could secrete neutrophil extracellular traps to promote airway inflammation and promote airway remodeling by secreting Oncostatin M (Osm). Cell trajectory and transcription factor enrichment analysis found that transcription factors such as C/EBPβ and PU.1 may regulate neutrophil phenotypic switching-mediated neutrophil heterogeneity.

FcγRIIb⁺ neutrophil subpopulation was exclusively increased in asthma and associated with the IL-4 receptor signaling. Functional analysis suggested that FcγRIIb⁺ neutrophils may be memory neutrophils with molecular characteristics of innate immune memory, mainly manifested by transcription factor activation-mediated epigenetic modifications and Toll-like receptor pathway activation, as well as upregulation of the inflammation factor secretion pathway. To clarify whether FcγRIIb⁺ neutrophils are memory neutrophils, chronic and acute asthma models were constructed, and the key feature molecules of innate immune memory were validated by qPCR and Western-blot analysis. It was found that these molecules were highly expressed in FcγRIIb⁺ neutrophils sorted by flow cytometry, including activation of immune memory-related transcription factors (C/EBPβ, Runx1), histone modification pathways (Tet2, β-catenin), Toll-like receptor pathway (Tlr2, Cd300lf), cAMP pathway (PDE4B), as well as upregulation of the secretion pathways of inflammation factors IL-1β and IL-6. Finally, it was inferred that FcγRIIb⁺ neutrophils were a key cell subgroup that promoted airway inflammation. In addition, FcγRIIb⁺ neutrophils were closely related to neutrophilic asthma induced by house dust mite with lipopolysaccharide, suggesting that memory neutrophils may be commonly present in neutrophilic asthma.

To investigate the shared inflammatory mechanisms between asthma and atherosclerosis, we found that YB1-mediated mRNA stability may play an important role through co-target network analysis. We constructed mouse cells with YB1 phosphorylation site missense mutations through lentivirus transfection and performed transcriptome sequencing. The results showed that YB1 phosphorylation could affect the expression of inflammatory factors such as CCL2 and the glucocorticoid receptor (GR) response pathway. Western-blot and qPCR validation showed that the GR-related complex (GR, PNRC2, DCP1A, UPF1, and HRSP12) can bind to CCL2 mRNA to regulate its mRNA stability.

Conclusion

This study elucidates the heterogeneity of neutrophils in asthmatic lung tissue, in which the FcγRIIb⁺ neutrophil subgroup is a memory neutrophil and a key cell subset that promotes asthma, while Cd177⁺ neutrophils promote asthma through the secretion of neutrophil extracellular traps and Osm. This study revealed that YB1 as a shared target involved in the inflammatory mechanisms of asthma and atherosclerosis.