Single-cell analysis identifies conserved features of immune dysfunction in simulated microgravity and spaceflight

Microgravity is associated with immunological dysfunction, though the mechanisms are poorly understood. Here, using single-cell analysis of human peripheral blood mononuclear cells (PBMCs) exposed to short term (25 hours) simulated microgravity, we characterize altered genes and pathways at basal and stimulated states with a Toll-like Receptor-7/8 agonist. We validate single-cell analysis by RNA sequencing and super-resolution microscopy, and against data from the Inspiration-4 (I4) mission, JAXA (Cell-Free Epigenome) mission, Twins study, and spleens from mice on the International Space Station. Overall, microgravity alters specific pathways for optimal immunity, including the cytoskeleton, interferon signaling, pyroptosis, temperature-shock, innate inflammation (e.g., Coronavirus pathogenesis pathway and IL-6 signaling), nuclear receptors, and sirtuin signaling. Microgravity directs monocyte inflammatory parameters, and impairs T cell and NK cell functionality. Using machine learning, we identify numerous compounds linking microgravity to immune cell transcription, and demonstrate that the flavonol, quercetin, can reverse most abnormal pathways. These results define immune cell alterations in microgravity, and provide opportunities for countermeasures to maintain normal immunity in space.

微重力可引发免疫功能异常，但其具体分子机制尚未阐明。本研究针对暴露于短期（25小时）模拟微重力环境的人外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）开展单细胞分析，在基础状态与Toll样受体7/8（Toll-like Receptor-7/8）激动剂刺激状态下，解析了基因表达与信号通路的改变特征。我们通过RNA测序与超分辨率显微镜技术对单细胞分析结果进行了验证，并将结果与Inspiration-4（I4）任务、日本宇宙航空研究开发机构（Japan Aerospace Exploration Agency, JAXA）无细胞表观基因组任务、双胞胎研究以及国际空间站实验小鼠脾脏的相关数据进行了比对。总体而言，微重力会干扰维持最佳免疫功能的特定信号通路，涵盖细胞骨架、干扰素信号通路、细胞焦亡、热休克反应、固有炎症通路（如冠状病毒致病通路与IL-6信号通路）、核受体以及去乙酰化酶（sirtuin）信号通路。微重力会改变单核细胞的炎症相关参数，并损伤T细胞与自然杀伤细胞（natural killer cell, NK cell）的功能。本研究借助机器学习方法，筛选出大量可将微重力与免疫细胞转录调控过程关联起来的化合物，并证实黄酮醇类物质槲皮素（quercetin）能够逆转多数异常的信号通路。本研究明确了微重力环境下免疫细胞的改变特征，为开发可维持太空环境中正常免疫功能的干预措施提供了理论依据与可行方向。