重症哮喘的代谢及细胞特征研究

Metabolic and Cellular Characterization of Severe Asthma

目的哮喘是常见的慢性气道炎性疾病,重症哮喘是哮喘诊治中关注的重点。哮喘发病中,免疫细胞参与并发生改变,几种脂类代谢物可作为其诊断标志物。本研究在代谢和细胞层面,探讨了重症哮喘的特征。方法使用多因素统计学分析和独立样本t检验筛选了重症哮喘(41例)和对照(18例)血液样本中的差异代谢物,通过KEGG富集分析鉴定关键通路,基于ROC曲线进行生物标志物的鉴定;基于细胞类型注释结果,识别了血液中免疫细胞的种类和比例(5例重症和3例对照);使用单样本基因富集分析(ssGSEA)研究了差异代谢通路在单细胞中的特征。结果相比于对照(健康),28种代谢物的丰度在重症哮喘患者血液中增加,13种代谢物丰度降低(P<0.05);差异代谢物富集于4条通路:鞘脂代谢、甘油磷脂代谢、烟酸和烟酰胺代谢、组氨酸代谢。其中,有13种差异代谢物可以作为重症哮喘诊断的标志物,如L-谷氨酸(AUC=0.809)、烟酰胺(AUC=0.886)、植物鞘氨醇(AUC=0.882)、鞘氨醇(AUC=0.893)等。在单细胞分析中,鉴定出了5种主要细胞类型(CD4^(+)T细胞、CD8^(+)T细胞、NK细胞、B细胞和单核细胞),NK细胞的数量在重症哮喘患者中增加;重症哮喘具有更加频繁的细胞通讯,其CD8^(+)T细胞与其他细胞通讯密集,而在健康样本中,密集通讯的细胞主要为单核细胞。ssGSEA表明,重症样本中,4条富集了差异代谢物的通路,在CD4^(+)T和CD8^(+)T细胞中具有更低的得分(P<0.01),推测这些通路相关的基因表达在这两种细胞中被抑制,被抑制的基因包括DGKA、NT5C3A等,这些基因与免疫过程相关,在调节T细胞信号转导、活化、分化及免疫应答中发挥关键作用。结论L-谷氨酸、烟酰胺、植物鞘氨醇、鞘氨醇可以作为重症哮喘诊断的生物标志物;重症哮喘相关通路的基因在CD4^(+)T和CD8^(+)T细胞中被抑制表达。

Objective Asthma is a common chronic inflammatory airway disease,and severe asthma poses a significant challenge in its diagnosis and management.Immune cells are involved in and altered by asthma pathogenesis,and several lipid metabolites can serve as diagnostic markers for the disease.In this study,we investigated the characterization of severe asthma at the metabolic and cellular level.Methods Differential metabolites in blood samples from severe asthma(41 cases)and controls(18 cases)were screened using multifactorial statistical analysis and independent samples t-tests;key pathways were identified by KEGG enrichment analysis,and biomarkers were characterized based on ROC curves;immune cell types and proportions in the blood were identified based on the results of cell-type annotations(5 severe and 3 control cases);and singlesample Gene Enrichment Analysis(ssGSEA)to investigate the characterization of differential metabolic pathways in single cells.Results Compared with controls,the abundance of 28 metabolites was increased and the abundance of 13 metabolites was decreased in the blood of patients with severe asthma(P<0.05);the differential metabolites were enriched in 4 pathways:sphingolipid metabolism,glycerophospholipid metabolism,nicotinate and nicotinamide metabolism,and histidine metabolism.Among them,13 differential metabolites could be used as biomarkers for the diagnosis of severe asthma,including L-glutamic acid(AUC=0.809),nicotinamide(AUC=0.886),phytosphingosine(AUC=0.882),and sphinganine(AUC=0.893).In single-cell transcriptome analysis,5 key cell types were identified:CD4^(+)T cells,CD8^(+)T cells,NK cells,B cells,and monocytes.The number of NK cells was increased in patients with severe asthma,and severe asthma exhibited more frequent cellular communication,particularly dense communication between CD8^(+)T cells and other cell types.In healthy samples,monocytes were the primary cells engaging in dense communication.Single-sample gene enrichment analysis(ssGSEA)showed that 4 pathways enriched for differential metabolites had lower scores(P<0.01)in CD4^(+)T and CD8^(+)T cells in severe patients,and it was hypothesized that the expression of genes associated with these pathways was suppressed in these two types of cells.The suppressed genes included DGKA and NT5C3A,which are associated with immune processes.We observed that these genes play key roles in the regulation of T cell signaling,activation,differentiation,and immune responses.Conclusion L-glutamic acid,nicotinamide,phytosphingosine,and sphinganine can be used as biomarkers for the diagnosis of severe asthma;genes of the severe asthma-associated pathway were suppressed in CD4^(+)T cells and CD8^(+)T cells.