Образцы крови были взяты у каждого астронавта в три разных момента времени: перед полетом, во время полета и по возвращении на Землю. Образцы были обработаны и хранились в соответствии со стандартными процедурами для предотвращения деградации РНК. Образцы были надежно хранятся при температуре -80°C до дальнейшего анализа.
Десять кровяных образцов были взяты у каждого космонавта в разные фазы своей миссии: до полета (PF), во время полета (IF) и после полета (R). Всего 139 кровяных образцов были взяты у 14 членов экипажа во время 12 миссий МКС.
Процесс анализа замороженных образцов крови для секвенирования РНК включает несколько этапов для обеспечения качества и целостности данных. Размораживание образцов, выделение общей РНК и подготовка библиотек для секвенирования — все это критически важные компоненты этого процесса. Давайте подробнее рассмотрим ключевые этапы:
Результаты анализа дифференциальной экспрессии были дополнительно визуализированы с помощью вулкановых графиков, тепловых карт и иерархической кластеризации для выявления паттернов экспрессии генов в разные временные точки. Вулкановые графики были созданы для отображения логарифма двойного изменения по отношению к -логарифму корректированного p-значения для каждого гена. Гены со статистически значимыми различиями в экспрессии были выделены красным цветом. Тепловые карты были созданы для отображения профилей экспрессии дифференциально экспрессированных генов во все временные точки, где строки представляют гены, а столбцы — образцы. Иерархическая кластеризация использовалась для группировки генов с похожими паттернами экспрессии.
The normalized read counts of gene candidates identified as differentially expressed between any of the 10 time points (temporal analysis) were averaged among all astronauts at each time point and scaled across genes as z-scores. These genes were then further analyzed to extract gene clusters displaying similar patterns of expression throughout the entire study. Briefly, the Euclidean distance between each gene candidate was calculated using their z-score scaled normalized read counts from each sample across time. These values were then hierarchically clustered using the hclust() function in R environment creating a tree dendrogram to visualize the gene clusters, which were resolved by a static tree cut (Supplementary Figure 3). Gene z-scores of temporal gene clusters were then plotted across time using lines and split violin plots overlayed by box plots to visualize the relative expression of genes over the course of the study (Figure 2).
As part of the temporal analysis, independent filtering of genes using DESeq2 excluded genes with mean normalized read counts <45 resulting in a profile of 15,410 genes representing the expressed transcriptome across time. The normalized read counts for this profile of expressed genes were scaled separately as z-scores. Gene z-scores for the 15,410 genes were then plotted across time as violin plots (Supplementary Figure 4).
RNA bio-typing
Gene candidate profiles identified from both temporal and time-point differential expression analyses were bio-typed according to the functionality of RNA transcribed from these genes. Bio-type annotation was done using the biomaRt package in R environment (18, 19), which utilized the Vega archive gene classifications (https://vega.archive.ensembl.org/info/about/gene_and_transcript_types.html). The relative proportions of RNA bio-types within each differentially expressed gene profile were then displayed as stacked bar plots (Figures 2, 3C, F).
Log fold change heatmap
The LFC values relative to PF were calculated for a subset of gene candidates identified from the differential expression analysis of selective time points. This subset of genes was identified from the Venn diagram overlap between the differentially expressed gene profiles for PF vs. IF2 and IF4 vs. R1 (Figure 4). LFC values relative to PF were then displayed as a heatmap across all IF and post-flight time points along with their gene identities (Figure 4).
Figure 4 Log2 fold change of 100 genes displaying downregulation when reaching space and upregulation when landing on Earth after 6 months in space. Heatmap illustration of the log2 fold changes (Log2FC) of expression relative to pre-flight across all in-flight and post-flight time points for the 100 genes both downregulated when reaching space and upregulated when landing on Earth. The Venn diagram (A) indicates the profile of genes being displayed in the heatmap (B). Expression levels of individual genes relative to pre-flight values are expressed across in-flight and post-flight time points as log2 fold changes displayed as a heatmap (B). The color bar represents values of log2 fold changes ranging from −2 (red) to +2 (green). Gene identities are shown by their ensembl ID and corresponding HGNC symbol (if applicable) in brackets. Asterisk (*) indicates non-coding genes.
Enrichment analysis
For insight into the broader biological functions of the differentially expressed gene profiles, functional enrichment of leukocyte transcriptomes using two separate overrepresentation analyses (ORAs) (20) of gene ontology (GO) terms was performed: one is to assess the temporal effect across all time points using the differentially expressed gene clusters and the other is to assess the spaceflight phase transitions using the differentially expressed genes from the selective time-point comparisons. A custom R script was used to detect significantly overrepresented GO terms between the list of differentially expressed gene profiles and the 15,410 expressed genes used as the reference list. ORA utilized clusterProfiler 4.0’s groupGO() function (21) to map genes to their associated level 4 GO terms grouped under “Biological Processes”. A Fisher exact test was applied to test for significantly overrepresented GO terms between genes and the reference list. After adjustment for multiple comparisons using the Benjamini–Hochberg correction, GO terms with FDR p-values <0.05 were considered statistically significant.
Results
Bio-typing of gene RNA revealed distinct bio-type proportions of the two temporal gene clusters. C1 consisted mostly of protein-coding genes (68.8%), 19.4% long non-coding RNAs (lncRNA), and 11.7% genes coding for other various RNA biotypes (Figure 2). C2 genes consisted mostly of protein-coding genes (93.1%), zero lncRNA, and 6.9% other RNAs (Figure 2).
The two temporal gene clusters differ in biological function
Gene expression between 2 and 6 months IF (IF3 and IF4) converged toward average levels. This pattern was observed for the 276 temporally differentially expressed genes (Figure 2) and was also evident with the profile of 15,410 genes obtained from independent filtering in DESeq2 (Supplementary Figure 4). The spread of average transcript levels for all expressed genes at IF3 and IF4 displayed the smallest IQR compared to all other time points (Supplementary Figure 4; Supplementary Table 1).
Leukocyte transcriptome at spaceflight phase transitions
To assess the effects of space transitions on astronauts’ transcriptomes, four time-point comparisons were selected on the basis of the most important changes in transcript levels displayed in the temporal profiles of clusters (Figure 2). The time-point comparisons included space phase transitions (PF vs. IF2 and IF4 vs. R1), transcriptional convergence after long-duration in space (IF3 vs. IF4), and 1 year after return from space (B vs. IF5). The differential expression results for PF vs. IF2 and IF4 vs. R1 are shown in (Figures 3A, B, respectively). All four differential expression results are summarized in Table 2.
Table 2 Summary of selective time-point differential expression results.
Differential expression was strongest during transitions to space and return to Earth
Comparing transcriptomes at PF and IF2 time points, we identified 112 downregulated genes and eight upregulated genes (Figure 3). The return to Earth was associated with 16 downregulated genes and 135 upregulated genes differentially expressed between IF4 and R1 transcriptomes. Substantial gene expression changes at space transitions were dominated by genes downregulated during early spaceflight (IF2) and upregulated during the return to Earth (R1). These results confirm the decrease-then-increase pattern of gene C1 identified in the temporal gene cluster analysis (Figure 2). In addition, RNA bio-typing of the differentially expressed genes at space transitions (PF vs. IF2 and IF4 vs. R1) replicated results from the temporal gene clusters with protein coding as most represented RNA (Figures 3C).
Space transition responses differ in biological function
Enrichment analysis of the 112 downregulated genes between PF and IF2 and 135 upregulated genes between IF4 and R1 identified biological functions differing between the transitions to and from space. GO terms are summarized in a cluster network on the basis of semantic similarity shown in Figures 3B, E. The transition to space enriched terms is related to cellular growth such as “cell population proliferation” (most enriched), “cell differentiation”, and “cellular component organization” (Figure 3). In contrast, the return to Earth resulted in two clusters of enriched terms both describing different biological processes than the transition to space (Figure 3). One cluster consisted of terms related to cellular transport such as “intracellular transport” and “protein transport and localization” (Figure 3). The other cluster included terms describing the regulation of immune system processes such as “leukocyte activation” and “lymphoid organ development” (Figure 3).
One hundred genes were both downregulated when reaching space and upregulated when landing on Earth
Among the 112 downregulated genes when reaching space and 135 upregulated genes when returning to Earth, 100 were the same genes (Figure 4). Figure 4 lists the 100 genes along with a heatmap displaying the LFC values relative to PF for each gene. The three most represented gene families were Zinc-Finger Protein (ZNF) genes (n = 6), Cluster of Differentiation (CD) genes (n = 3), and Long Intergenic Non-Protein Coding (LINC) RNA (n = 3).
Leukocyte transcriptome in-flight and 1-year post-flight
Our results found zero genes differentially expressed between 65–95 days IF and 30–1 day prior to return to Earth (IF3 vs. IF4) (Table 2). The convergence toward no changes later in flight is also evident from standardizing scaled expression to z-scores. The distribution of mean scaled expression (z-scores) for all genes at each time point are shown in Figure 2; Supplementary Figure 4. Late IF time points corresponding to IF3 and IF4 had the lowest IQRs compared to all 10 time points (0.29 and 0.32, respectively) (Supplementary Table 1).
Transcriptome 1-year post-flight is similar to pre-flight
Analysis between PF and 1-year post-flight time points (PF vs. R5) revealed zero differentially expressed genes (Table 2). From the 15,410 expressed genes, transcriptional variability between PF and 1 year after returning from space appeared similar on the basis of the spread of violin plots and IQR (Supplementary Table 1; Supplementary Figure 4). However, Figure 2 shows C1 and C2 having reversed expression 1-year post-flight when compared to PF. C1 genes had above average expression PF but had below average expression 1-year post-flight, and vice versa for C2.
Discussion
The first analysis of the leukocyte transcriptomes provided an overview of the relative transcriptional changes occurring at 10 time points across the three phases of a space mission: PF, IF, and R. Astronauts’ leukocyte transcriptomes showed opposite directions of gene expression changes upon reaching the space environment compared to the return on Earth. Cluster analysis grouped the differentially expressed genes into two clusters characterized by major changes in opposing directions: (C1) decrease-then-increase and (C2) increase-then-decrease.
The lower number of genes in C2 (n = 29) limited the conclusions for enrichment analysis and identification of represented biological processes. Of interest, the biological term “regulation of body fluid” represented in the short list of genes in C2 displayed a pattern of upregulation when reaching space. The gene SLC4A1 associated with the term “regulation of body fluid” encodes for an anion exchanger protein localized in the plasma membrane of erythrocytes and mediates carbon dioxide transport to the lungs (36). Increased expression of SLC4A1 gene when reaching space may respond to the increase of carbon dioxide levels in conditions of low red blood cell mass, with the latter being previously documented in astronauts (10). The gene AQP3 with changes in opposite directions at both phase transitions functions as a water and urea exit mechanism of antidiuresis in collecting duct cells —a mechanism regulating body fluids (37). Therefore, reaching space promoted leukocyte gene expression related to basic housekeeping cell functionality as well as specific space adaptations like headward body fluid shifts leading to loss in plasma volume and hemoconcentration (38). Restoring blood cells concentrations to homeostatic levels requires a decrease in the number of circulating leukocytes and red blood cells whose population is decreased by ~10% in the first 10 days in space (39). Therefore, in addition to immune functions, the leukocyte transcriptome identified cellular functions and physiological systems affected by spaceflight.
The opposite directions of expression changes in the gene clusters at space transitions replicated the results obtained from participants subjected to a microgravity analogue (40). The 6° head- down tilt bedrest model replicates the microgravity component of spaceflight with many of the physiological changes happening in space including fluid shift, muscle atrophy, bone loss, and hemolysis (41–44). Transcriptome composition changed in opposite directions at transitions between ambulation and bedrest and between bedrest and re-ambulation in 20 healthy participants submitted to 60 days of bedrest (40). While the space missions were longer with an average of 6 months compared to the 60 days period in bed, the transcriptome changes at phase transition coincided. Comparable changes in the leukocyte transcriptome may, therefore, indicate a characteristic response to the negative mechanotransduction, inactivity, and fluid shift brought about by prolonged exposure to both bedrest and space. Leukocyte transcriptomes are therefore highly sensitive to changes in the gravity vector and appear to mount an adaptive response toward restoring homeostasis.
The next characteristic of leukocyte transcriptome temporal changes observed was the transcriptional convergence toward average levels displaying no differential expression after 2 months of space exposure. This is a novel finding revealed through the temporal analysis. The biological meaning is unclear but indicative of global mechanisms yet to be identified that limit variations of mRNA levels in leukocytes in space. Interestingly, the gene expression convergence of astronauts replicated the results of participants to the 60-day bedrest study (40), supporting that gene expression convergence is related to inactivity and redirected gravity isolated from other space specific stressors. In addition, this might be compatible with a generalized loss of specialized cell functions upon removal of normally oriented gravity and activity. The lack of mechanotransduction and inactivity would then focus cellular activity on core housekeeping functions.
The comparison of transcriptomes between PF and 1-year post-flight showed that the two gene clusters were reversed in expression. This may suggest that some molecular space adaptations acquired while living in space for 6 months were maintained for at least 1 year after return to life on Earth. This may bear physiological significance given the ~20% increases in hemolysis in the same astronauts 1 year after returning from space (10, 39).
Shift in biological functions at spaceflight transitions to and from microgravity
Transiting to and from microgravity was associated with the differential expression of 120 and 151 genes from the reference list of 15,410 genes expressed in leukocytes. The majority (93.3%) of the differentially expressed genes when reaching space were down regulated and 95.7% were up regulated when returning. Differential expression measured at mission transitions is consistent with the temporal profile of C1 characterized by down- and up- expression. Downregulated genes identified between PF and early IF were associated with the biological term “regulation of cellular population proliferation”. This transcriptomic response is consistent with the head ward fluid shift and subsequent hemoconcentration of blood cells occurring when entering space (38). A decrease in circulating red and white blood cells restores blood cell concentrations to maintain homeostasis, consistent with the downregulation of genes involved in cellular proliferation (10). A suppression of blood cell proliferation represents an adaptation to the reduced blood volume in space.
At transition from space to Earth, transcriptomes were characterized by an up regulation of expression, opposite to changes measured when reaching space. Enrichment analysis of the upregulated genes between late IF and return to Earth resulted in biological processes describing the regulation of immune system, leukocyte activation, and lymphoid organ development. Returning to Earth’s surface gravity after ~6 months in microgravity reversed the down regulation of genes involved in immune processes. Many immune alterations persist during long-duration spaceflight (8). Reactivation of immune- related genes in response to the re-entry to Earth is needed to reverse immune dysregulation occurring during spaceflight. The composition of the leukocytes’ transcriptome was influenced by the transition to the different gravity environments.
Contributions and limitations
Our access to unique astronauts’ blood samples and RNA analysis of the leukocytes’ transcriptome using high- throughput sequencing technique represents the strength of this study. The finding of genes responding to both the transition to and from space with decreased and then increased profile of changes that related to immune processes represents a novel finding. Our study also identified additional expression changes at phase transitions in genes unrelated to specific immune functions, such as cell population regulation. This provides evidence of changes at the molecular level by which the body adapts to the headward fluid shift observed in microgravity environments. This study bears a number of limitations. Blood draws were taken at 10 different time points throughout astronaut missions; changes of interest to establish the onset of transcriptional convergence in space timed in-between blood draws may have been missed. Technical limitations onboard the ISS hampered sample acquisition, processing, and analysis. For instance, blood samples were collected within a window of days rather than on a specific day that introduced variability. Leukocyte and RNA isolation were not possible on the ISS and blood samples were frozen at −80°C for their journey back to Earth, leading to cell lysis and RNA degradation. This resulted in samples with inadequate RNA quality for sequencing, which were rejected, leading to an unbalanced final sample size. In addition, a potential contribution of altered leukocyte subpopulations to gene differential expression can not be excluded. RNA sequencing removed ribosomal RNA and was biased toward protein-coding genes; changes in other RNA biotypes would have been missed. The limited sample size and heterogeneous cohort of 14 astronauts with unequal sex distribution limited statistical power and prevented sex-specific comparisons.
Conclusion
The analysis of transcriptome composition identified changes during the transitions to and from space characterized mainly by a decrease and an increase of transcript levels respectively. When reaching space, the transcriptomic changes are indicative of decreased immune functions and increased basic cellular activities linked to adaptive changes. The transcriptomic changes egressing back to Earth were in opposite direction —increased expression, mainly for genes related to the immune system. These results shed light on immune modulation in space, the timing of differential expression at transition to and from space, and highlight the major adaptive changes in leukocyte activity engaged to adapt to extreme environments.
Ethics statement
The studies involving human participants were reviewed and approved by NASA Human Research Multilateral Review Board Johnson Space Center Institutional Review Board European Space Agency Medical Board Japanese Aerospace Exploration Agency Ottawa Health Science Network Research Ethics Board. The patients/participants provided their written informed consent to participate in this study.
Author contributions
GT and OL participated in the concept and design. All authors participated in the acquisition, analysis, or interpretation of data. DS, GT, OL participated in the drafting of the article. All authors participated in the critical revision of the article for important intellectual content. DS and OL participated in the statistical analysis. GT and OL participated in the funding acquisition. All authors contributed to the article and approved the submitted version.
Funding
This study was funded by the Canadian Space Agency (Contract No. 9F008-140254).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2023.1171103/full#supplementary-material
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