Single-cell RNA sequencing reveals non-canonical brain macrophage activation states in murine neurotrauma

Background: Macrophage polarization programs, commonly referred to as “classical” and “alternative” activation, are widely considered as distinct states that are exclusive of one another, and are associated with different functions such as inflammation and wound healing, respectively. In a number of disease contexts, such as traumatic brain injury (TBI), macrophage polarization influences the extent of pathogenesis, and efforts are underway to eliminate pathogenic subsets. However, previous studies have not distinguished whether the simultaneous presence of both classical and alternative activation signatures represents the admixture of differentially polarized macrophages, or if they have adopted a unique state characterized by components of both classical and alternative activation.Results: We analyzed the polarization of individual macrophages responding to TBI using single-cell RNA sequencing. Analysis of signature polarization genes revealed diverse activation states, including M(IL4), M(IL10), and M(LPS, IFNγ). However, the expression of a given polarization marker was no more likely than at random to predict simultaneous expression or repression of markers of another polarization program within the same cell, suggesting a lack of exclusivity in macrophage polarization states in vivo in TBI. Also unexpectedly, individual TBI macrophages simultaneously expressed high levels of signature polarization genes across two or three different polarization states, and in several distinct and seemingly incompatible combinations.Conclusions: Single-cell gene expression profiling demonstrated that monocytic macrophages in TBI are not comprised of distinctly polarized subsets, but are uniquely and broadly activated. TBI macrophage activation in vivo is deeply complex, with individual cells concurrently adopting both inflammatory and reparative features. These data provide physiologically relevant evidence that the early macrophage response to TBI is comprised of novel activation states that are discordant with the current paradigm of macrophage polarization—a key consideration for therapeutic modulation.

研究背景：巨噬细胞极化程序通常被分为「经典激活」与「替代激活」两种模式，长期以来被认为是彼此互斥的独立状态，分别对应炎症反应与伤口愈合等不同生理功能。在创伤性脑损伤（traumatic brain injury, TBI）等多种疾病场景中，巨噬细胞极化状态会影响疾病的发病进程，目前已有多项研究致力于清除致病的巨噬细胞亚群。然而，既往研究尚未明确：同时存在经典与替代激活特征，究竟是不同极化状态的巨噬细胞发生混合，还是巨噬细胞本身获得了兼具两类激活特征的独特状态。研究结果：本研究通过单细胞RNA测序（single-cell RNA sequencing）分析了创伤性脑损伤中响应损伤的单个巨噬细胞的极化状态。对极化特征基因的分析显示，巨噬细胞存在多种激活状态，包括M(IL4)、M(IL10)以及M(LPS, IFNγ)。但进一步分析发现，单一极化标志物的表达，并不能随机预测同一细胞内另一极化程序标志物的同时表达或抑制，这提示在创伤性脑损伤的体内环境中，巨噬细胞极化状态并不存在互斥性。此外，本研究还获得了意外发现：单个创伤性脑损伤巨噬细胞可同时高表达两种或三种不同极化状态的特征性基因，并呈现出多种迥异且看似互斥的组合模式。研究结论：单细胞基因表达谱分析证实，创伤性脑损伤中的单核细胞源性巨噬细胞并非由独立极化的亚群构成，而是处于一种独特且广泛的激活状态。体内创伤性脑损伤的巨噬细胞激活过程极为复杂，单个细胞可同时兼具促炎与修复特性。本研究数据为创伤性脑损伤早期巨噬细胞应答存在不同于当前巨噬细胞极化范式的新型激活状态提供了生理学相关证据，这一发现对于巨噬细胞相关治疗调控具有重要参考价值。