A study using single-cell analysis of human peripheral blood mononuclear cells (PBMCs) exposed to simulated microgravity for 25 hours identified conserved immune dysfunction features in microgravity and spaceflight. The research characterized altered genes and pathways in PBMCs at basal and stimulated states with a Toll-like Receptor-7/8 agonist. The findings were validated using RNA sequencing, super-resolution microscopy, and data from the Inspiration-4 (I4) mission, JAXA (Cell-Free Epigenome) mission, Twins study, and mouse spleens on the International Space Station. Microgravity altered pathways critical for optimal immunity, including the cytoskeleton, interferon signaling, pyroptosis, temperature-shock, innate inflammation, nuclear receptors, and sirtuin signaling. Microgravity directed monocyte inflammatory parameters and impaired T cell and NK cell functionality. Machine learning identified compounds linking microgravity to immune cell transcription, with quercetin reversing most abnormal pathways. These results define immune cell alterations in microgravity and provide opportunities for countermeasures to maintain normal immunity in space. Astronauts in low Earth orbit (LEO) experience immune dysfunction due to microgravity, with studies showing impaired T-cell responses and heightened innate immunity. The study found that simulated microgravity alters immune cell transcriptional landscapes, with significant changes in gene expression and pathways related to innate immunity, inflammation, and mitochondrial dysfunction. The research also identified overlapping immune dysfunction features between simulated microgravity and spaceflight, including reduced T-cell effector subset development, reduced oxidative phosphorylation, and increased innate immunity pathways. The findings suggest that microgravity induces immune dysfunction through mechanical forces, affecting immune cell function and signaling. The study used single-cell analysis to identify core genes and pathways altered by microgravity, and validated these findings using bulk RNA-seq and mouse spleen data. The results highlight the importance of understanding immune dysfunction in microgravity for developing countermeasures to maintain immune health in space.A study using single-cell analysis of human peripheral blood mononuclear cells (PBMCs) exposed to simulated microgravity for 25 hours identified conserved immune dysfunction features in microgravity and spaceflight. The research characterized altered genes and pathways in PBMCs at basal and stimulated states with a Toll-like Receptor-7/8 agonist. The findings were validated using RNA sequencing, super-resolution microscopy, and data from the Inspiration-4 (I4) mission, JAXA (Cell-Free Epigenome) mission, Twins study, and mouse spleens on the International Space Station. Microgravity altered pathways critical for optimal immunity, including the cytoskeleton, interferon signaling, pyroptosis, temperature-shock, innate inflammation, nuclear receptors, and sirtuin signaling. Microgravity directed monocyte inflammatory parameters and impaired T cell and NK cell functionality. Machine learning identified compounds linking microgravity to immune cell transcription, with quercetin reversing most abnormal pathways. These results define immune cell alterations in microgravity and provide opportunities for countermeasures to maintain normal immunity in space. Astronauts in low Earth orbit (LEO) experience immune dysfunction due to microgravity, with studies showing impaired T-cell responses and heightened innate immunity. The study found that simulated microgravity alters immune cell transcriptional landscapes, with significant changes in gene expression and pathways related to innate immunity, inflammation, and mitochondrial dysfunction. The research also identified overlapping immune dysfunction features between simulated microgravity and spaceflight, including reduced T-cell effector subset development, reduced oxidative phosphorylation, and increased innate immunity pathways. The findings suggest that microgravity induces immune dysfunction through mechanical forces, affecting immune cell function and signaling. The study used single-cell analysis to identify core genes and pathways altered by microgravity, and validated these findings using bulk RNA-seq and mouse spleen data. The results highlight the importance of understanding immune dysfunction in microgravity for developing countermeasures to maintain immune health in space.