ITGAM-巨噬细胞调控作为治疗中性粒细胞型哮喘的潜在策略：来自生物信息学分析和体内实验的见解
2023/11/23
摘要
鉴定与中性粒细胞型哮喘（NA）表型相关的分子生物标志物可能有助于发现新的病理生理学机制和诊断标志的开发。我们利用来自哮喘患者诱导痰液的三个mRNA转录组数据集，筛选了NA中与巨噬细胞相关的分子机制和靶点。此外，预测的靶点还在独立数据集（N = 3）和动物模型（N = 5）上进行了验证。在由卵清蛋白/弗氏佐剂完全（OVA/CFA）诱导的NA小鼠的支气管肺泡灌洗（BAL）液中观察到总细胞数、中性粒细胞和巨噬细胞的显著增加。我们还发现在外部数据集中，NA亚型中中性粒细胞和巨噬细胞的浸润水平升高。NA小鼠的血清和BAL液中IgE、IL-1β、TNF-α和IL-6的分泌增加。在NA小鼠中，中性粒细胞中存在的酶MPO的表达也很高。然后，通过加入与巨噬细胞相关的基因，加权基因共表达网络分析（WGCNA）识别了与NA最强相关的684个靶点，我们在NA中获得了609个巨噬细胞相关的特异性差异表达基因（DEGs）。通过RT-qPCR和受试者操作特征（ROC）分析确定了具有高度值的前10个基因的mRNA水平和诊断性能。在所有关键靶点和巨噬细胞之间找到了统计显著的相关性，其中在NA中ITGAM和巨噬细胞之间的相关性最强。双重免疫荧光染色进一步确认了ITGAM和F4/80在NA中的共定位。ITGAM被确定为一个关键靶点，可以区分NA与健康/非NA个体，这可能为进一步揭示NA的机制和治疗提供了新途径。
ITGAM-macrophage modulation as a potential strategy for treating neutrophilic Asthma: insights from bioinformatics analysis and in vivo experiments
Qian Yan, Zixing Liu Yujing Chen, Xinxin Zhang, Wenjiang Zheng, Xiaohong Liu, Huiting Huang, Qiong Liu, Yong Jiang, Shaofeng Zhan, Xiufang Huang
Abstract
Identification of molecular biomarkers associated with neutrophilic asthma (NA) phenotype may inform the discovery of novel pathobiological mechanisms and the development of diagnostic markers. Three mRNA transcriptome datasets extracted from induced sputum of asthma patients with various inflammatory types were used to screen for macrophage-related molecular mechanisms and targets in NA. Furthermore, the predicted targets were also validated on an independent dataset (N = 3) and animal model (N = 5). A significant increase in total cells, neutrophils and macrophages was observed in bronchoalveolar lavage (BAL) fluid of NA mice induced by ovalbumin/freund's adjuvant, complete (OVA/CFA). And we also found elevated levels of neutrophil and macrophage infiltration in NA subtype in external datasets. NA mice had increased secretion of IgE, IL-1β, TNF-α and IL-6 in serum and BAL fluid. MPO, an enzyme present in neutrophils, was also highly expressed in NA mice. Then, weighted gene co-expression network analysis (WGCNA) identified 684 targets with the strongest correlation with NA, and we obtained 609 macrophage-related specific differentially expressed genes (DEGs) in NA by integrating macrophage-related genes. The top 10 genes with high degree values were obtained and their mRNA levels and diagnostic performance were then determined by RT-qPCR and receiver operator characteristic (ROC) analysis. Statistically significant correlations were found between macrophages and all key targets, with the strongest correlation between ITGAM and macrophages in NA. Double-Immunofluorescence staining further confirmed the co-localization of ITGAM and F4/80 in NA. ITGAM was identified as a critical target to distinguish NA from healthy/non-NA individuals, which may provide a novel avenue to further uncover the mechanisms and therapy of NA.
鉴定与中性粒细胞型哮喘（NA）表型相关的分子生物标志物可能有助于发现新的病理生理学机制和诊断标志的开发。我们利用来自哮喘患者诱导痰液的三个mRNA转录组数据集，筛选了NA中与巨噬细胞相关的分子机制和靶点。此外，预测的靶点还在独立数据集（N = 3）和动物模型（N = 5）上进行了验证。在由卵清蛋白/弗氏佐剂完全（OVA/CFA）诱导的NA小鼠的支气管肺泡灌洗（BAL）液中观察到总细胞数、中性粒细胞和巨噬细胞的显著增加。我们还发现在外部数据集中，NA亚型中中性粒细胞和巨噬细胞的浸润水平升高。NA小鼠的血清和BAL液中IgE、IL-1β、TNF-α和IL-6的分泌增加。在NA小鼠中，中性粒细胞中存在的酶MPO的表达也很高。然后，通过加入与巨噬细胞相关的基因，加权基因共表达网络分析（WGCNA）识别了与NA最强相关的684个靶点，我们在NA中获得了609个巨噬细胞相关的特异性差异表达基因（DEGs）。通过RT-qPCR和受试者操作特征（ROC）分析确定了具有高度值的前10个基因的mRNA水平和诊断性能。在所有关键靶点和巨噬细胞之间找到了统计显著的相关性，其中在NA中ITGAM和巨噬细胞之间的相关性最强。双重免疫荧光染色进一步确认了ITGAM和F4/80在NA中的共定位。ITGAM被确定为一个关键靶点，可以区分NA与健康/非NA个体，这可能为进一步揭示NA的机制和治疗提供了新途径。
(中日友好医院呼吸与危重症医学科 顾宪民 摘译 林江涛 审校)
(Apoptosis. 2023 Nov 11. doi: 10.1007/s10495-023-01914-5.)
(Apoptosis. 2023 Nov 11. doi: 10.1007/s10495-023-01914-5.)
ITGAM-macrophage modulation as a potential strategy for treating neutrophilic Asthma: insights from bioinformatics analysis and in vivo experiments
Qian Yan, Zixing Liu Yujing Chen, Xinxin Zhang, Wenjiang Zheng, Xiaohong Liu, Huiting Huang, Qiong Liu, Yong Jiang, Shaofeng Zhan, Xiufang Huang
Abstract
Identification of molecular biomarkers associated with neutrophilic asthma (NA) phenotype may inform the discovery of novel pathobiological mechanisms and the development of diagnostic markers. Three mRNA transcriptome datasets extracted from induced sputum of asthma patients with various inflammatory types were used to screen for macrophage-related molecular mechanisms and targets in NA. Furthermore, the predicted targets were also validated on an independent dataset (N = 3) and animal model (N = 5). A significant increase in total cells, neutrophils and macrophages was observed in bronchoalveolar lavage (BAL) fluid of NA mice induced by ovalbumin/freund's adjuvant, complete (OVA/CFA). And we also found elevated levels of neutrophil and macrophage infiltration in NA subtype in external datasets. NA mice had increased secretion of IgE, IL-1β, TNF-α and IL-6 in serum and BAL fluid. MPO, an enzyme present in neutrophils, was also highly expressed in NA mice. Then, weighted gene co-expression network analysis (WGCNA) identified 684 targets with the strongest correlation with NA, and we obtained 609 macrophage-related specific differentially expressed genes (DEGs) in NA by integrating macrophage-related genes. The top 10 genes with high degree values were obtained and their mRNA levels and diagnostic performance were then determined by RT-qPCR and receiver operator characteristic (ROC) analysis. Statistically significant correlations were found between macrophages and all key targets, with the strongest correlation between ITGAM and macrophages in NA. Double-Immunofluorescence staining further confirmed the co-localization of ITGAM and F4/80 in NA. ITGAM was identified as a critical target to distinguish NA from healthy/non-NA individuals, which may provide a novel avenue to further uncover the mechanisms and therapy of NA.
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