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Abstract：
BACKGROUND: The immunologic features of nontuberculous mycobacterial pulmonary disease (NTM-PD) are largely unclear. This study investigated the immunologic features of NTM-PD using digital spatial profiling techniques.
METHODS: Lung tissues obtained from six patients with NTM-PD between January 1, 2006, and December 31, 2020, at Seoul National University Hospital were subjected to RNA sequencing. Cores from the peribronchial areas were stained with CD3, CD68, and DNASyto13, and gene expression at the whole-transcriptome level was quantified using PCR amplification and Illumina sequencing. Lung tissues from six patients with bronchiectasis collected during the same period were used as controls. The RNA sequencing results were validated using immunohistochemistry (IHC) in another cohort (30 patients with NTM-PD and 15 patients with bronchiectasis).
RESULTS: NTM-PD exhibited distinct gene expression patterns in T cells and macrophages. Gene set enrichment analysis revealed that pathways related to antigen presentation and processing were upregulated in NTM-PD, particularly in macrophages. Macrophages were more prevalent and the expression of genes associated with the M1 phenotype (CD40 and CD80) was significantly elevated. Although macrophages were activated in the NTM-PD group T cell activity was unaltered. Notably, expression of the costimulatory molecule CD28 was decreased in NTM-PD. IHC analysis showed that T cells expressing Foxp3 or TIM-3, which facilitate the regulatory functions of T cells, were increased.
CONCLUSIONS: NTM-PD exhibits distinct immunologic signatures characterized by the activation of macrophages without T cell activation.
METHODS: Lung tissues obtained from six patients with NTM-PD between January 1, 2006, and December 31, 2020, at Seoul National University Hospital were subjected to RNA sequencing. Cores from the peribronchial areas were stained with CD3, CD68, and DNASyto13, and gene expression at the whole-transcriptome level was quantified using PCR amplification and Illumina sequencing. Lung tissues from six patients with bronchiectasis collected during the same period were used as controls. The RNA sequencing results were validated using immunohistochemistry (IHC) in another cohort (30 patients with NTM-PD and 15 patients with bronchiectasis).
RESULTS: NTM-PD exhibited distinct gene expression patterns in T cells and macrophages. Gene set enrichment analysis revealed that pathways related to antigen presentation and processing were upregulated in NTM-PD, particularly in macrophages. Macrophages were more prevalent and the expression of genes associated with the M1 phenotype (CD40 and CD80) was significantly elevated. Although macrophages were activated in the NTM-PD group T cell activity was unaltered. Notably, expression of the costimulatory molecule CD28 was decreased in NTM-PD. IHC analysis showed that T cells expressing Foxp3 or TIM-3, which facilitate the regulatory functions of T cells, were increased.
CONCLUSIONS: NTM-PD exhibits distinct immunologic signatures characterized by the activation of macrophages without T cell activation.
摘要：
背景：非结核性分枝杆菌肺病（NTM-PD）的免疫学特征尚不清楚。本研究使用数字空间分析技术调查了NTM-PD的免疫学特征。
方法：从2006年1月1日至2020年12月31日在首尔国立大学医院的6名NTM-PD患者获得的肺组织进行RNA测序。用CD3、CD68和DNASyto13染色支气管周围区域的核,并使用PCR扩增和Illumina测序定量全转录组水平的基因表达。在同一时期收集的6名支气管扩张患者的肺组织用作对照。在另一个队列（30名NTM-PD患者和15名支气管扩张患者）中使用免疫组织化学（IHC）验证RNA测序结果。
结果：NTM-PD在T细胞和巨噬细胞中表现出不同的基因表达模式。基因集富集分析显示，NTM-PD中与抗原呈递和加工相关的通路上调，特别是在巨噬细胞中。巨噬细胞更为普遍，与M1表型相关的基因（CD40和CD80）的表达显着升高。尽管巨噬细胞在NTM-PD组中被激活,但T细胞活性没有改变。值得注意的是,共刺激分子CD28在NTM-PD中的表达降低。IHC分析显示，表达Foxp3或TIM-3的T细胞促进T细胞的调节功能，增加了。
结论:NTM-PD表现出不同的免疫学特征,其特征在于巨噬细胞的活化而没有T细胞活化。
方法：从2006年1月1日至2020年12月31日在首尔国立大学医院的6名NTM-PD患者获得的肺组织进行RNA测序。用CD3、CD68和DNASyto13染色支气管周围区域的核,并使用PCR扩增和Illumina测序定量全转录组水平的基因表达。在同一时期收集的6名支气管扩张患者的肺组织用作对照。在另一个队列（30名NTM-PD患者和15名支气管扩张患者）中使用免疫组织化学（IHC）验证RNA测序结果。
结果：NTM-PD在T细胞和巨噬细胞中表现出不同的基因表达模式。基因集富集分析显示，NTM-PD中与抗原呈递和加工相关的通路上调，特别是在巨噬细胞中。巨噬细胞更为普遍，与M1表型相关的基因（CD40和CD80）的表达显着升高。尽管巨噬细胞在NTM-PD组中被激活,但T细胞活性没有改变。值得注意的是,共刺激分子CD28在NTM-PD中的表达降低。IHC分析显示，表达Foxp3或TIM-3的T细胞促进T细胞的调节功能，增加了。
结论:NTM-PD表现出不同的免疫学特征,其特征在于巨噬细胞的活化而没有T细胞活化。
