Paired single-cell and spatial transcriptional profiling reveals a central osteopontin macrophage response mediating tuberculous granuloma formation in humans and zebrafish [Spatial Transcriptomics]

Mycobacterial granulomas are categorical manifestations of tuberculosis pathogenesis. They result from an ensemble of immune responses to Mycobacterium tuberculosis infection, but the identities, arrangement, cellular interactions and regulation of the cells that comprise them has thus far been incompletely understood. We conducted spatial and single-cell RNA sequencing of clinical granuloma biopsy specimens from tuberculosis patients. We found that granulomas consist of concentric transcriptional laminae surrounding areas of central necrosis. Gene expression programs associated with regional architecture were conserved among samples. We identified distinct populations of granuloma-associated stromal cells, fibroblasts, lymphocytes, mast cells, dendritic cells, neutrophils and macrophages. Populations further differentiate into multiple granuloma-specific states that correlate with location. We used inferential analysis to predict dominant granuloma cell-cell interactions, the activity of major signaling pathways, and differential transcription factor activities. Using spatial deconvolution, we mapped a conserved pattern of cellular organization dominated by macrophages rich in osteopontin/SPP1 expression. Trajectory analysis of macrophage subtypes mapped their differentiation and supported the importance of SPP1 to granuloma macrophage polarization. Cumulatively, we have identified a dominant macrophage granuloma population as well as its central regulatory gene in human samples and confirmed its importance to granuloma biology in vivo. To examine the interdependence of lesional architecture and gene expression, we evaluated the whole spatial transcriptomes of 4 samples from four different patients at 55 µM resolution (~5-10 cells) using the 10x Genomics Visium v2 with CytAssist assay.

分枝杆菌肉芽肿（Mycobacterial granulomas）是结核病发病机制的特征性病理表现。其本质是机体针对结核分枝杆菌（Mycobacterium tuberculosis）感染产生的一系列免疫应答的结果，但迄今为止，构成肉芽肿的细胞的身份、排布方式、细胞间相互作用及调控机制仍未被完全阐明。我们对结核病患者的临床肉芽肿活检标本开展了空间转录组测序与单细胞RNA测序。研究发现，肉芽肿由围绕中央坏死区域的同心转录分层结构构成；与区域组织结构相关的基因表达程序在不同标本中具有保守性。我们鉴定出了多种特征各异的肉芽肿相关细胞群，包括基质细胞、成纤维细胞、淋巴细胞、肥大细胞、树突状细胞、中性粒细胞与巨噬细胞。这些细胞群可进一步分化为多种与空间位置相关的肉芽肿特异性细胞状态。我们通过推断分析预测了肉芽肿内主要的细胞间相互作用、核心信号通路活性以及差异转录因子的活性。借助空间反卷积分析，我们绘制了以高表达骨桥蛋白/SPP1（osteopontin/SPP1）的巨噬细胞为主导的保守细胞组织模式。对巨噬细胞亚型的轨迹分析揭示了其分化过程，并证实了SPP1在肉芽肿巨噬细胞极化中的关键作用。综上，我们在人类样本中鉴定出了占主导地位的肉芽肿巨噬细胞群及其核心调控基因，并证实了其在体内肉芽肿生物学过程中的重要性。为探究病灶组织结构与基因表达的相互依存关系，我们采用10x Genomics Visium v2结合CytAssist检测技术，对4名不同患者的4份标本开展了55微米分辨率（约5-10个细胞）的全空间转录组分析。