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.

Astronauts in low earth orbit (LEO), such as on the international space station (ISS), experience immune dysfunction associated with the microgravity environment. Multiple studies have

described immune dysregulation in short or long-term simulated1,2,3,4 or actual microgravity5,6,7,8,9. For the most part, such studies have described impaired T-cell responses, coupled with

some form of heightened innate immunity7,10, though some innate immune cells, like natural killer (NK) cells, also show impaired function11. Consistent with altered adaptive immunity,

potentially due to impaired cytotoxic and Th1 T cell function, and reduced NK cell function, astronauts develop increased reactivation of latent viruses, including herpes viruses (EBV, CMV,

VZV)3,7,12,13,14,15. In one study, viral shedding after 9–14 days of spaceflight was linked to changes in serum cytokines, including a preferential large increase in IL-4 compared to

interferon (IFN)γ, indicating a possible shift away from Th1 immunity towards Th2 immunity16. Consistently, some astronauts report heightened hypersensitivity reactions, such as increased

allergic and Th2-like responses in space7.

Multiple studies using higher throughput approaches have started to add insight into pathways impacted by spaceflight. In the Twins study17, a one-year ISS mission altered innate, adaptive,

and NK cell-mediated immunity across bulk RNA sequencing analysis. In T cells, increases in DNA methylation were seen in the promoters of notch3 for CD4+ T cells, linked to T cell

differentiation, and in scl1a5/asct2, linked to activation, for CD8+ T cells. A total of 50 of 62 assayed cytokines were also altered by spaceflight or landing17. During a recent multi-omic

analysis, including bulk RNA and DNA methylation sequencing, of astronauts and mice in space, mouse organs such as the liver and kidney demonstrated reduced IFN signatures, coupled to

altered methylation patterns of these gene sets, while muscles had increased IFNγ, IL-1, and TNF10. Serum inflammatory markers from 59 astronauts in this study (and in a similar companion

study) showed increased VEGF-1, IGF-1, and IL-1 during spaceflight, which resolved upon returning to Earth4,10. This same study also identified mitochondrial dysfunction as a major response

of different non-hematolymphoid tissues to spaceflight10. More recently, another study using a NASA-developed Rotating Wall Vessel, which was employed in our current work, utilized a

41-parameter mass cytometry approach to show that short-term (18–22 hours) simulated microgravity can dampen NK cell, CD4+, and CD8+ T cell responses to Concanavalin A/anti-CD28 stimulation,

but potentiates STAT5 signaling to boost Tregs18.

Despite these important advances, the core fundamental mechanisms, genes, and pathways that are directly altered by microgravity to adversely impact immunity, including at single-cell

resolution, are largely unknown. Interestingly, mechanical forces are emerging as critical orchestrators of immune cell function, whereby mechanotransduction tunes immune cell responsiveness

to danger signals19. Some of these effects occur through environmental modulation of mechanosensing pathways that alter ion currents in cells, metabolism, or directly act on the

cytoskeleton19. Thus, a spaceflight environment, which alters forces such as gravity, associated hydrostatic pressure, and shear force20,21 onto immune cells likely directly contributes to

immune system dysfunction.

Here, using a common ground-based analog, the NASA developed low shear modeled microgravity Rotating Wall Vessel (RWV)2,18,22, we examine in depth how short-term (25 hours) exposure to

simulated microgravity impacts the human peripheral blood mononuclear immune system in detail at single-cell resolution. Combining this data with validation experiments from mice and

crewmembers in LEO, as well as machine learning algorithms, we identify numerous core genes and pathways in immune cells that are altered by simulated microgravity or spaceflight, and

identify numerous potential compounds that directly map onto immune cell transcriptional signatures in simulated microgravity.

To begin understanding how simulated microgravity impacts immune cell function, we loaded PBMC samples from two young healthy CMV+ donors, one male, and one female, into either RWV simulated

microgravity (uG) or normal gravity (1G) static controls for 16 hours of conditioning. The 16-hour-conditioning time point was chosen based on prior work that used approximately the same

time and tracked transcriptional or proteomic changes on immune cells to simulated microgravity1,18. PBMCs were either left unstimulated or stimulated for an additional 9 hours with R848, a

standard TLR7/8 agonist. We chose TLR7/8 as a putative target since it mimics viral infection, and is expressed on most human immune cells, including T cells23. Using this methodology, we

next developed a single-cell atlas of 55,648 human PBMCs exposed to these conditions.

In the unstimulated state, after 25 hours of simulated microgravity, we identified 28 clusters of immune cells visualized by UMAP (Uniform Manifold Approximation and Projection), including

cell types such as mucosal associated invariant T cells (MAIT cells), double negative T cells, γδ T cells, innate lymphoid cells, and plasmacytoid dendritic cells, which have rarely been

studied in simulated microgravity (Fig. 1A). Simulated microgravity altered proportions of immune cell clusters to a mild extent, with B intermediate cells, and MAIT cell proportions being

most negatively impacted, and CD14+ monocytes, and CD4+ T effector memory (TEM) cells being most increased based on percent change (Fig. 1B). Across all immune populations, simulated

microgravity altered expression of over 4500 genes with adj P cutoff of 0.1. This list was further condensed to visualize on a Volcano plot with |log2FC|> 0.25 (Fig. 1C), showing only the

very top positively and negatively altered genes. Volcano plots of DEGs for individual immune cell clusters are shown in Supplementary Fig. 1. Across all immune cells, some of the most

induced genes in simulated microgravity included acute response genes such as s100a8, s100a9, s100a12, thbs1, heat-shock genes such hsp90ab1, chemokines like ccl2, ccl4, iron storage genes

(fth1, ftl), and matrix metalloproteinases (mmp9). The most reduced genes in simulated microgravity included interferon response (stat1) and associated guanylate binding proteins (gbp1), and

cold shock genes (rbm3, cirbp). Expression of the top DEGs (with mitochondrial encoded genes excluded for visual simplicity) across 22 populations of immune cells are shown in Fig. 1D.

CD14+ classical monocytes, CD16+ nonclassical monocytes, and natural killer (NK) cells exhibited the most pronounced changes across major gene sets, consistent with short term simulated

microgravity’s direct effect at reprogramming transcriptional changes most prominently in innate immunity. Consistently, using single-cell trajectory analysis, we identify numerous

trajectories mainly in the innate immune cell clusters, especially the monocyte cluster, in response to simulated microgravity. Trajectory analysis is used to construct a path that describes

how cells move through different states, and the numerous states seen in the monocyte cluster in simulated microgravity may reflect an increased capacity to generate distinct

transcriptional states to simulated microgravity (Fig. 1E).

A UMAP plot of unstimulated PBMCs single-cell transcriptomes (10X Genomics), pooled together from a male (36 years old) and a female (25 years old) donor, that underwent either 1G or

simulated microgravity (uG) for 25 hours total. Cells were resolved into 28 distinct clusters. B Quantification of relative abundance of each cluster of single PBMCs by percentage, or

log2Fold Change (FC) between simulated uG and 1G conditions. Source data are provided with this paper. C Volcano plot of differentially expressed genes (DEGs) across all immune cell types

between uG and 1G; DEGs (including log2FC and adj. p) were calculated by the MAST method. The Benjamini–Hochberg (B-H) method was used for multiple comparison adjustments. Adjusted p value

(adj. p) cutoff is 0.05, and log2FC cutoff is 0.25. D Dot plot showing the top DEGs from C and their expression levels across 22 immune cell populations. DEGs (including log2FC and adj. p)

were calculated by the MAST method. P values were adjusted by the B-H method. Spot color reflects Log2FC of uG vs 1G, while spot size shows −log10 (adj. p). E UMAP of trajectory analysis of

1G and simulated uG unstimulated PBMCs. White circles represent the root nodes of the trajectory. Black circles indicate branch nodes, where cells can travel to a variety of outcomes. Light

gray circles designate different trajectory outcomes. F Canonical pathway enrichment analysis obtained from Ingenuity Pathway Analysis (IPA) is shown across 19 immune cell clusters. Spot

color reflects IPA z-score enrichment of simulated uG vs 1G, with red meaning predicted activation of the pathway in uG and blue meaning repression of the pathway in uG. Spot size shows the

level of significance via −log10 (adj. p). Adj. p was calculated by the Fisher’s Exact Test (right-tailed) followed by B-H adjustment.

Ingenuity pathway analysis (IPA) (Fig. 1F, Supplementary Data 2) generated using our core list of 375 genes from the overall populations, as well as the DEGs in major immune cell types

(Supplementary Data 3) revealed that monocytes, conventional dendritic cells type 2 (cDC2)s, double negative (dn)T cells and NK cells show the most notable pathway alterations. Major

pathways altered by simulated microgravity across immune cells included reductions in oxidative phosphorylation, interferon signaling like protein kinase R (PKR) in interferon response,

nuclear receptor signaling (LXR/RXR, PPAR, AHR), RHOA and pyroptosis signaling, as well as increases in BAG2 (heat-shock protein 70 interactor) signaling, fibrosis signaling, actin-based

motility, RAC, HIF1 signaling, acute phase response, oxidative stress and sirtuin signaling, amongst others.

Given that multiple pathways we detected were associated with inflammatory processes linked to aging (i.e., increased innate immunity coupled to reduced adaptive immunity), we next

determined whether acute exposure to simulated microgravity mimicked inflammatory aging processes in immune cells. We mapped the gene expression signatures of individual immune cells, and

overall immune signatures, against two recently developed inflammatory signatures of aging, the inflammatory age (iAge) clock24, and the SenMayo list of senescence associated secretory

inflammatory products25. Simulated microgravity induced a significant enrichment in inflammaging related genes, consistent with the notion that short term simulated microgravity can induce

aging-like inflammatory changes in unstimulated immune cells (Fig. 2A, B, Supplementary Fig. 2A). Next, because both spaceflight and aging are associated with reactivation of latent viruses,

we mined the meta-transcriptome of our single-cell analysis with meta-transcriptome detector (MTD) pipeline26. Surprisingly, we saw that as little as 25 hours of simulated microgravity

could induce the transcription of latent retroviruses and mycobacteria within human immune cells (Fig. 2C, Supplementary Figs. 2B, C and 3), directly implicating microgravity itself as a

contributing trigger for latent pathogen activation. We confirmed the meta-transcriptome results with a different alignment tool, and we could still detect increases in Gammaretrovirus and

Mycobacterium canettii transcripts seen with MTD pipeline (Supplementary Fig. 2B, C).

A Differences in iAge index between all cell types (left) and across 22 individual immune cell types (right) at 1G or simulated uG. Each box spans from the 25th to 75th percentiles

(interquartile range, IQR), and features a median value denoted by a horizontal line. The whiskers extend to values within 1.5 times the IQR range from the 25th and 75th percentiles. 1G

group (n = 11,934 cells examined over 2 independent experiments) is shown in blue and uG group (n = 16,568 cells examined over 2 independent experiments) is shown in yellow. The median for

1G group is 9.1, with min = −5.8 and max = 22.4. The median for uG group is 12.5, with min = −1.1 and max = 26.2. Two-tailed Mann–Whitney test (p value