Spatial mapping of SARS-CoV-2 and H1N1 Lung Injury Identifies Differential Transcriptional Signatures

Here, we analyzed specific ARDS regions (lower left lung lobe) of interest utilizing a new spatial transcriptomic platform (Nanostring GeoMx) on autopsy-derived lung tissue from patients with SARS-CoV-2 (n=3), H1N1 (n=3), and a unique dual- infected individual (n=1). Paraffin embedded tissues were processed and analyzed at NanoString techonology laboratories using a combination of fluorescently labeled antibodies, anti-CD68 (Santa Cruz, sc-20060 AF647, RRID: KP1), anti-EpCAM (Abcam, ab213500, RRID:EPR20532-222), anti-smooth muscle actin (Invitrogen, 53-9760-82, RRID:1A4) and the GeoMX COVID-19 Immune Response Atlas gene set with custom probe set specific for SARS-CoV-2 lung infection and tissue responses. Selection of regions of interests (ROI, 12 per patient) was performed based on the immunofluorescent viral staining, the cellular immunofluorescent profile and the pathological features of ARDS (i.e. presence of hyaline membranes and diffused alveolar damage) observed in the H&E stained sections. To ensure even and representative selection of ROIs, only patients lower left lung tissue was analyzed. Each patient had 2-3 total lung areas selected in regions of ARDS (confirmed by Dr. Benson, pathologist), 2-4 ROIs for epithelial cells (normal epithelium vs hyperplastic), 2-3 vascular beds selected, and 2-4 macrophage populations (infiltrate and clusters). For cell-specific profiling, at least 50 cells per ROI were utilized for analyses. The 1860 gene profile not only identified increased regional coagulopathy, but also robust transcriptional signatures of enhanced extracellular remodeling, alternative macrophage activation, and squamous metaplasia of type II pneumocytes in SARS- CoV-2. These gene signatures were expressed and enhanced in alveolar epithelium, vascular tissue, and lung macrophages. Both the H1N1 and dual infected transcriptome demonstrated an enhanced antiviral response compared to SARS-CoV-2.本研究针对新型冠状病毒（SARS-CoV-2，n=3）、甲型H1N1流感病毒（H1N1，n=3）及1例罕见双重感染患者（n=1）的尸检肺组织，利用Nanostring GeoMx这一空间转录组学平台（spatial transcriptomic platform），分析了特定的急性呼吸窘迫综合征（ARDS）感兴趣区域——左下肺叶。 石蜡包埋组织在NanoString技术实验室完成处理与分析，所用试剂包括荧光标记抗体：抗CD68（Santa Cruz，sc-20060 AF647，RRID: KP1）、抗EpCAM（Abcam，ab213500，RRID:EPR20532-222）、抗平滑肌肌动蛋白（Invitrogen，53-9760-82，RRID:1A4），以及GeoMX COVID-19免疫应答图谱基因集，搭配针对新型冠状病毒肺部感染与组织应答的定制探针组。 感兴趣区域（ROI，每名患者12个）的选取基于苏木精-伊红（H&E）染色切片中观察到的免疫荧光病毒染色结果、细胞免疫荧光谱及急性呼吸窘迫综合征（ARDS）病理特征（即透明膜形成与弥漫性肺泡损伤）。 为确保感兴趣区域选取的均匀性与代表性，本研究仅分析患者的左下肺组织。每名患者选取2-3个经病理学家本森博士确认的ARDS受累肺区域、2-4个针对上皮细胞的ROI（分为正常上皮与增生上皮两类）、2-3个血管床区域，以及2-4个巨噬细胞群区域（包括浸润性巨噬细胞与巨噬细胞簇）。 针对细胞特异性谱分析，每个ROI至少选取50个细胞用于后续分析。 这套包含1860个基因的表达谱不仅揭示了局部凝血功能异常的上调，还在新型冠状病毒感染样本中检测到显著的转录特征：细胞外基质重塑增强、巨噬细胞替代性活化，以及II型肺上皮细胞的鳞状上皮化生。上述基因特征在肺泡上皮、血管组织与肺巨噬细胞中均有表达且表达水平显著上调。 与新型冠状病毒感染组相比，甲型H1N1流感病毒感染组与双重感染组的转录组均呈现出更强的抗病毒应答。