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We would like to thank Dr. Cheryl Nickerson (Arizona State University) for technical discussions on simulated microgravity ground analogs, and Dr. Brian Crucian (NASA) for discussions on translational aspects of the immune system in space. Wewould like to thank Ryan Kwok and Ritesh Tiwari for technical assistance with flow cytometry. Figure 7A was created with BioRender.com."},{"name":"DOI","value":"10.26030/9pve-pg84"},{"name":"Data Source Accession","value":"GSE218937"},{"name":"Data Source Link","value":"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE218937"},{"name":"Experiment Platform","value":""},{"name":"Flight Program","value":""},{"name":"Funding","value":"This work was supported in part through funds derived from the Buck Institute for Research on Aging (D.A.W., D.F.), and the Huiying Memorial Foundation (D.A.W.). T.R.V. and J.B. are funded by a T32 NIH fellowship grant (NIA T32 AG000266). C.E.M. thanks the Scientific Computing Unit (SCU) at WCM, the WorldQuant Foundation, NASA (NNX14AH50G, NNX17AB26G, 80NSSC22K0254, NNH18ZTT001N-FG2, 80NSSC22K0254, NNX16AO69A), the National Institutes of Health (R01MH117406), and LLS (MCL7001-18, LLS 9238-16)."},{"name":"Identifiers","value":""},{"name":"Managing NASA Center","value":""},{"name":"Mission End","value":""},{"name":"Mission Link","value":""},{"name":"Mission Name","value":""},{"name":"Mission Start","value":""},{"name":"Project Identifier","value":"Simulated_Microgravity"},{"name":"Project Link","value":""},{"name":"Project Title","value":""},{"name":"Project Type","value":"Ground Study"},{"name":"Space Program","value":""},{"name":"Study Grant Number","value":""}],"description":"Microgravity is associated with immunological dysfunction, though the mechanisms are poorly understood. Here, using single-cell analysis of human peripheral blood mononuclear cells (PBMCs)\u00a0exposed to short term (25\u2009hours) 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. 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