Immunologic features of nontuberculous mycobacterial pulmonary disease based on spatially resolved whole transcriptomes

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. 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 and fibrotic stromal 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 four 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). 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. From these, NTM-PD exhibits distinct immunologic signatures characterized by the activation of macrophages without T cell activation. RNA sequencing of lung tissue from patients underwent surgical resection because of nontuberculous mycobacterial lung disease or bronchiectasis using Illumina TruSeq Stranded mRNA Library prep and sequenced on Illumina HiSeq 2500.

非结核分枝杆菌肺病（nontuberculous mycobacterial pulmonary disease, NTM-PD）的免疫学特征目前尚未完全阐明。本研究采用数字空间分析技术（digital spatial profiling），对NTM-PD的免疫学特征展开了探究。2006年1月1日至2020年12月31日期间，首尔国立大学医院收集的6例NTM-PD患者的肺组织样本均接受了RNA测序。对支气管周围及纤维化间质区域的组织芯实施CD3、CD68及DNASyto13染色，并通过PCR扩增与Illumina测序对全转录组水平的基因表达量进行定量分析。同期收集的4例支气管扩张症患者的肺组织样本作为对照。本研究通过另一独立队列（30例NTM-PD患者与15例支气管扩张症患者）的免疫组化（immunohistochemistry, IHC）实验，验证了RNA测序结果。NTM-PD在T细胞与巨噬细胞中均呈现出独特的基因表达谱模式。基因集富集分析（gene set enrichment analysis）结果显示，抗原呈递与加工相关通路在NTM-PD中显著上调，其中巨噬细胞亚群的上调趋势尤为明显。巨噬细胞在NTM-PD组织中的浸润丰度更高，且与M1表型相关的基因（CD40与CD80）的表达水平显著升高。尽管NTM-PD组的巨噬细胞已被激活，但T细胞的活性并未出现明显改变。值得注意的是，共刺激分子（costimulatory molecule）CD28的表达在NTM-PD样本中有所下调。免疫组化分析显示，表达Foxp3或TIM-3的T细胞（二者可辅助T细胞发挥免疫调节功能）的数量显著增加。综上，NTM-PD具有独特的免疫学特征，其核心表现为巨噬细胞激活而T细胞未被活化。本研究针对因非结核分枝杆菌肺病或支气管扩张症接受手术切除的患者的肺组织，采用Illumina TruSeq Stranded mRNA Library Prep进行文库构建，并在Illumina HiSeq 2500测序平台上完成转录组测序。