Single Cell Analysis Identifies Conserved Features of Immune Dysfunction in Simulated Microgravity and Spaceflight [RNA-Seq]

Microgravity is associated with immunological dysfunction, though the underlying mechanisms are poorly understood. Here, using single cell analysis of human peripheral blood mononuclear cells (PBMC)s exposed to short term (25 hours) simulated microgravity, we characterize altered genes and pathways across immune cells under basal and stimulated states with a Toll like Receptor-7/8 agonist. At basal state, simulated microgravity altered the transcriptional landscape across immune cells, with monocyte subsets showing most pathway changes. Remarkably, short term simulated microgravity was sufficient to increase endogenous retroviral and mycobacterial transcripts. Under stimulation in simulated microgravity, nearly all immune cells demonstrated differences in functional pathways. Results from single cell analysis were validated against additional PBMC samples, including by RNA sequencing and super-resolution microscopy, and against data from the Inspiration-4 (i4) mission, JAXA6 mission, Twins study, and spleens from mice housed on the international space station. Combined results show significant impacts of microgravity on pathways essential for optimal immunity, including the cytoskeleton, interferon signaling, pyroptosis, temperature-shock, nuclear receptors, IL-6 signaling, HIF1α, and sirtuin signaling. Using machine learning, we identified numerous compounds linking microgravity to immune cell transcription, and demonstrate that the flavonol, quercetin, can reverse most abnormal pathways. These results offer insight into maladaptation of the immune system in microgravity, and provide opportunities to develop countermeasures that maintain normal immunity in space. Comparative gene expression profiling analysis of RNA-seq data for PBMCs under basal and quercetin treatment in simulated microgravity and normal gravity.

微重力可引发免疫功能紊乱，但其潜在分子机制尚未被充分阐明。本研究通过对暴露于短期（25小时）模拟微重力环境的人外周血单个核细胞（peripheral blood mononuclear cell, PBMC）进行单细胞分析，系统表征了静息状态与经Toll样受体7/8（Toll-like Receptor 7/8）激动剂刺激状态下，各类免疫细胞的基因与通路表达变化。在静息状态下，模拟微重力即可改变各类免疫细胞的转录谱，其中单核细胞亚群的通路变化最为显著。令人意外的是，短期模拟微重力即可上调内源性逆转录病毒与分枝杆菌相关转录本的表达。而在模拟微重力环境下接受刺激时，几乎所有免疫细胞的功能通路均出现显著差异。本研究通过额外的PBMC样本（包括RNA测序与超分辨率显微镜技术）以及多项已发表数据（包括Inspiration-4（i4）任务、JAXA6任务、双胞胎研究以及国际空间站饲养小鼠的脾脏数据），对单细胞分析结果进行了验证。综合分析结果表明，微重力对维持最佳免疫功能所必需的多条通路具有显著影响，包括细胞骨架、干扰素信号通路、细胞焦亡、热休克通路、核受体、IL-6信号通路、缺氧诱导因子1α（HIF1α）以及沉默蛋白（sirtuin）信号通路。本研究通过机器学习方法，筛选出多种可将微重力与免疫细胞转录调控关联起来的化合物，并证实黄酮醇类化合物槲皮素（quercetin）能够逆转多数异常通路的表达变化。本研究结果为理解微重力环境下免疫系统的适应不良机制提供了新视角，并为开发可在太空环境中维持正常免疫功能的对抗措施提供了潜在方向。此外，本研究还对模拟微重力与正常重力环境下，静息状态及经槲皮素处理的PBMC的RNA测序数据进行了比较基因表达谱分析。