Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China.
Eight-Year Medical Doctor Program, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China.
Neurobiol Dis. 2024 Oct 15;201:106695. doi: 10.1016/j.nbd.2024.106695. Epub 2024 Oct 5.
Spontaneous intracerebral hemorrhage (ICH) is associated with alarmingly high rates of disability and mortality, and current therapeutic options are suboptimal. A critical component of ICH pathology is the initiation of a robust inflammatory response, often termed "cytokine storm," which amplifies the secondary brain injury following the initial hemorrhagic insult. The precise sources and consequences of this cytokine-driven inflammation are not fully elucidated, necessitating further investigation.
To address this knowledge gap, our study conducted a comprehensive cytokine profiling using Luminex® assays, assessing 23 key cytokines. We then employed single-cell RNA sequencing and spatial transcriptomics at three critical time points post-ICH: the hyperacute, acute, and subacute phases. Integrating these multimodal analyses allowed us to identify the cellular origins of cytokines and elucidate their mechanisms of action.
Luminex® cytokine assays revealed a significant upregulation of IL-6 and IL-1β levels at the 24-h post-ICH time point. Through the integration of scRNA-seq and spatial transcriptomics in the hemorrhagic hemisphere of rats, we observed a pronounced activation of cytokine-related signaling pathways within the choroid plexus. Initially, immune cell presence was sparse, but it surged 24 h post-ICH, particularly in the choroid plexus, indicating a substantial shift in the immune microenvironment. We traced the source of IL-1β and IL-6 to endothelial cells, establishing a link to pyroptosis. Endothelial pyroptosis post-ICH induced the production of IL-1β and IL-6, which activated microglial polarization characterized by elevated expression of Msr1, Lcn2, and Spp1 via the NF-κB pathway in the choroid plexus. Furthermore, we identified neuronal populations undergoing apoptosis, mediated by the Lcn2-SLC22A17 pathway in response to IL-1β and IL-6 signaling. Notably, the inhibition of pyroptosis using VX-765 significantly mitigated neurological impairments.
Our study provides evidence that endothelial pyroptosis, characterized by the release of IL-1β and IL-6, triggers microglial polarization through NF-κB pathway activation, ultimately leading to microglia-mediated neuronal apoptosis in the choroid plexus post-ICH. These findings suggest that targeted therapeutic strategies aimed at mitigating endothelial cell pyroptosis and neutralizing inflammatory cytokines may offer neuroprotection for both microglia and neurons, presenting a promising avenue for ICH treatment.
自发性脑出血（ICH）与令人震惊的高残疾率和死亡率相关，而目前的治疗选择并不理想。ICH 病理的一个关键组成部分是启动强大的炎症反应，通常称为“细胞因子风暴”，它会在初始出血损伤后放大继发性脑损伤。这种细胞因子驱动的炎症的确切来源和后果尚未完全阐明，需要进一步研究。
为了弥补这一知识空白，我们使用 Luminex® 分析进行了全面的细胞因子谱分析，评估了 23 种关键细胞因子。然后，我们在 ICH 后三个关键时间点（超急性期、急性期和亚急性期）使用单细胞 RNA 测序和空间转录组学。整合这些多模态分析使我们能够识别细胞因子的细胞起源，并阐明其作用机制。
Luminex®细胞因子分析显示，在 ICH 后 24 小时，IL-6 和 IL-1β 水平显著上调。通过整合大鼠脑出血半球的 scRNA-seq 和空间转录组学，我们观察到在脉络丛中细胞因子相关信号通路的显著激活。最初，免疫细胞的存在很少，但在 ICH 后 24 小时内急剧增加，特别是在脉络丛中，表明免疫微环境发生了重大变化。我们将 IL-1β 和 IL-6 的来源追溯到内皮细胞，建立了与细胞焦亡的联系。ICH 后内皮细胞焦亡诱导 IL-1β 和 IL-6 的产生，通过 NF-κB 通路在脉络丛中激活小胶质细胞极化，特征是 Msr1、Lcn2 和 Spp1 的表达上调。此外，我们发现神经元群体在脉络丛中通过 IL-1β 和 IL-6 信号转导介导的凋亡，由 Lcn2-SLC22A17 通路介导。值得注意的是，使用 VX-765 抑制细胞焦亡可显著减轻神经损伤。
我们的研究提供了证据表明，内皮细胞焦亡，其特征是释放 IL-1β 和 IL-6，通过 NF-κB 通路激活触发小胶质细胞极化，最终导致 ICH 后脉络丛中小胶质细胞介导的神经元凋亡。这些发现表明，靶向治疗策略旨在减轻内皮细胞焦亡和中和炎症细胞因子，可能为小胶质细胞和神经元提供神经保护，为 ICH 治疗提供了有前途的途径。
