Transcriptional Response in Human Jurkat T Lymphocytes to a near Physiological Hypergravity Environment and to One Common in Routine Cell Culture Protocols.

Cellular effects of hypergravity have been described in many studies. We investigated the transcriptional dynamics in Jurkat T cells between 20 s and 60 min of 9 g hypergravity and characterized a highly dynamic biphasic time course of gene expression response with a transition point between rapid adaptation and long-term response at approximately 7 min. Upregulated genes were shifted towards the center of the nuclei, whereby downregulated genes were shifted towards the periphery. Upregulated gene expression was mostly located on chromosomes 16-22. Protein-coding transcripts formed the majority with more than 90% of all differentially expressed genes and followed a continuous trend of downregulation, whereas retained introns demonstrated a biphasic time-course. The gene expression pattern of hypergravity response was not comparable with other stress factors such as oxidative stress, heat shock or inflammation. Furthermore, we tested a routine centrifugation protocol that is widely used to harvest cells for subsequent RNA analysis and detected a huge impact on the transcriptome compared to non-centrifuged samples, which did not return to baseline within 15 min. Thus, we recommend carefully studying the response of any cell types used for any experiments regarding the hypergravity time and levels applied during cell culture procedures and analysis.

Rapid Downregulation of H3K4me3 Binding to Immunoregulatory Genes in Altered Gravity in Primary Human M1 Macrophages.

The sensitivity of human immune system cells to gravity changes has been investigated in numerous studies. Human macrophages mediate innate and thus rapid immune defense on the one hand and activate T- and B-cell-based adaptive immune response on the other hand. In this process they finally act as immunoeffector cells, and are essential for tissue regeneration and remodeling. Recently, we demonstrated in the human Jurkat T cell line that genes are differentially regulated in cluster structures under altered gravity. In order to study an in vivo near system of immunologically relevant human cells under physically real microgravity, we performed parabolic flight experiments with primary human M1 macrophages under highly standardized conditions and performed chromatin immunoprecipitation DNA sequencing (ChIP-Seq) for whole-genome epigenetic detection of the DNA-binding loci of the main transcription complex RNA polymerase II and the transcription-associated epigenetic chromatin modification H3K4me3. We identified an overall downregulation of H3K4me3 binding loci in altered gravity, which were unequally distributed inter- and intrachromosomally throughout the genome. Three-quarters of all affected loci were located on the p arm of the chromosomes chr5, chr6, chr9, and chr19. The genomic distribution of the downregulated H3K4me3 loci corresponds to a substantial extent to immunoregulatory genes. In microgravity, analysis of RNA polymerase II binding showed increased binding to multiple loci at coding sequences but decreased binding to central noncoding regions. Detection of altered DNA binding of RNA polymerase II provided direct evidence that gravity changes can lead to altered transcription. Based on this study, we hypothesize that the rapid transcriptional response to changing gravitational forces is specifically encoded in the epigenetic organization of chromatin.

Gravitational Force-Induced 3D Chromosomal Conformational Changes Are Associated with Rapid Transcriptional Response in Human T Cells.

The mechanisms underlying gravity perception in mammalian cells are unknown. We have recently discovered that the transcriptome of cells in the immune system, which is the most affected system during a spaceflight, responds rapidly and broadly to altered gravity. To pinpoint potential underlying mechanisms, we compared gene expression and three-dimensional (3D) chromosomal conformational changes in human Jurkat T cells during the short-term gravitational changes in parabolic flight and suborbital ballistic rocket flight experiments. We found that differential gene expression in gravity-responsive chromosomal regions, but not differentially regulated single genes, are highly conserved between different real altered gravity comparisons. These coupled gene expression effects in chromosomal regions could be explained by underlying chromatin structures. Based on a high-throughput chromatin conformation capture (Hi-C) analysis in altered gravity, we found that small chromosomes (chr16-22, with the exception of chr18) showed increased intra- and interchromosomal interactions in altered gravity, whereby large chromosomes showed decreased interactions. Finally, we detected a nonrandom overlap between Hi-C-identified chromosomal interacting regions and gravity-responsive chromosomal regions (GRCRs). We therefore demonstrate the first evidence that gravitational force-induced 3D chromosomal conformational changes are associated with rapid transcriptional response in human T cells. We propose a general model of cellular sensitivity to gravitational forces, where gravitational forces acting on the cellular membrane are rapidly and mechanically transduced through the cytoskeleton into the nucleus, moving chromosome territories to new conformation states and their genes into more expressive or repressive environments, finally resulting in region-specific differential gene expression.

Rapid Cellular Perception of Gravitational Forces in Human Jurkat T Cells and Transduction into Gene Expression Regulation.

Cellular processes are influenced in many ways by changes in gravitational force. In previous studies, we were able to demonstrate, in various cellular systems and research platforms that reactions and adaptation processes occur very rapidly after the onset of altered gravity. In this study we systematically compared differentially expressed gene transcript clusters (TCs) in human Jurkat T cells in microgravity provided by a suborbital ballistic rocket with vector-averaged gravity (vag) provided by a 2D clinostat. Additionally, we included 9× g centrifuge experiments and rigorous controls for excluding other factors of influence than gravity. We found that 11 TCs were significantly altered in 5 min of flight-induced and vector-averaged gravity. Among the annotated clusters were G3BP1, KPNB1, NUDT3, SFT2D2, and POMK. Our results revealed that less than 1% of all examined TCs show the same response in vag and flight-induced microgravity, while 38% of differentially regulated TCs identified during the hypergravity phase of the suborbital ballistic rocket flight could be verified with a 9× g ground centrifuge. In the 2D clinostat system, doing one full rotation per second, vector effects of the gravitational force are only nullified if the sensing mechanism requires 1 s or longer. Due to the fact that vag with an integration period of 1 s was not able to reproduce the results obtained in flight-induced microgravity, we conclude that the initial trigger of gene expression response to microgravity requires less than 1 s reaction time. Additionally, we discovered extensive gene expression differences caused by simple handling of the cell suspension in control experiments, which underlines the need for rigorous standardization regarding mechanical forces during cell culture experiments in general.

Rapid Morphological and Cytoskeletal Response to Microgravity in Human Primary Macrophages.

The FLUMIAS (Fluorescence-Microscopic Analyses System for Life-Cell-Imaging in Space) confocal laser spinning disk fluorescence microscope represents a new imaging capability for live cell imaging experiments on suborbital ballistic rocket missions. During the second pioneer mission of this microscope system on the TEXUS-54 suborbital rocket flight, we developed and performed a live imaging experiment with primary human macrophages. We simultaneously imaged four different cellular structures (nucleus, cytoplasm, lysosomes, actin cytoskeleton) by using four different live cell dyes (Nuclear Violet, Calcein, LysoBrite, SiR-actin) and laser wavelengths (405, 488, 561, and 642 nm), and investigated the cellular morphology in microgravity (10-4 to 10-5 g) over a period of about six minutes compared to 1 g controls. For live imaging of the cytoskeleton during spaceflight, we combined confocal laser microscopy with the SiR-actin probe, a fluorogenic silicon-rhodamine (SiR) conjugated jasplakinolide probe that binds to F-actin and displays minimal toxicity. We determined changes in 3D cell volume and surface, nuclear volume and in the actin cytoskeleton, which responded rapidly to the microgravity environment with a significant reduction of SiR-actin fluorescence after 4-19 s microgravity, and adapted subsequently until 126-151 s microgravity. We conclude that microgravity induces geometric cellular changes and rapid response and adaptation of the potential gravity-transducing cytoskeleton in primary human macrophages.

Real-Time 3D High-Resolution Microscopy of Human Cells on the International Space Station.

Here we report the successful first operation of FLUMIAS-DEA, a miniaturized high-resolution 3D fluorescence microscope on the International Space Station (ISS) by imaging two scientific samples in a temperature-constant system, one sample with fixed cells and one sample with living human cells. The FLUMIAS-DEA microscope combines features of a high-resolution 3D fluorescence microscope based on structured illumination microscope (SIM) technology with hardware designs to meet the requirements of a space instrument. We successfully demonstrated that the FLUMIAS technology was able to acquire, transmit, and store high-resolution 3D fluorescence images from fixed and living cells, allowing quantitative and dynamic analysis of subcellular structures, e.g., the cytoskeleton. The capability of real-time analysis methods on ISS will dramatically extend our knowledge about the dynamics of cellular reactions and adaptations to the space environment, which is not only an option, but a requirement of evidence-based medical risk assessment, monitoring and countermeasure development for exploration class missions.

Expression of Hypoxia-Inducible Factor 1α (HIF-1α) and Genes of Related Pathways in Altered Gravity.

Immune system deterioration in space represents a major risk, which has to be mitigated for exploration-class missions into the solar system. Altered gravitational forces have been shown to regulate adaptation processes in cells of the immune system, which are important for appropriate risk management, monitoring and development of countermeasures. T lymphocytes and cells of the monocyte-macrophage system are highly migratory cell types that frequently encounter a wide range of oxygen tensions in human tissues and in hypoxic areas, even under homeostatic conditions. Hypoxia-inducible factor 1 and 2 (HIF's) might have an important role in activation of T cells and cells of the monocyte-macrophages system. Thus, we investigated the regulation of HIF-dependent and, therefore, hypoxia-signaling systems in both cell types in altered gravity and performed transcript and protein analysis from parabolic flight and suborbital ballistic rocket experiments. We found that HIF-1α and HIF-1-dependent transcripts were differently regulated in altered gravity, whereas HIF-1α-dependent gene expression adapted after 5 min microgravity. Inter-platform comparisons identified PDK1 as highly responsive to gravitational changes in human U937 myelomonocytic cells and in Jurkat T cells. We suggest HIF-1 as a potential pharmacological target for counteracting immune system deterioration during space flight.

Transcriptional Homeostasis of Oxidative Stress-Related Pathways in Altered Gravity.

Whereby several types of cultured cells are sensitive to gravity, the immune system belongs to the most affected systems during spaceflight. Since reactive oxygen species/reactive nitrogen species (ROS/RNS) are serving as signals of cellular homeostasis, particularly in the cells of the immune system, we investigated the immediate effect of altered gravity on the transcription of 86 genes involved in reactive oxygen species metabolism, antioxidative systems, and cellular response to oxidative stress, using parabolic flight and suborbital ballistic rocket experiments and microarray analysis. In human myelomonocytic U937 cells, we detected a rapid response of 19.8% of all of the investigated oxidative stress-related transcripts to 1.8 g of hypergravity and 1.1% to microgravity as early as after 20 s. Nearly all (97.2%) of the initially altered transcripts adapted after 75 s of hypergravity (max. 13.5 g), and 100% adapted after 5 min of microgravity. After the almost complete adaptation of initially altered transcripts, a significant second pool of differentially expressed transcripts appeared. In contrast, we detected nearly no response of oxidative stress-related transcripts in human Jurkat T cells to altered gravity. In conclusion, we assume a very well-regulated homeostasis and transcriptional stability of oxidative stress-related pathways in altered gravity in cells of the human immune system.

Signal transduction in primary human T lymphocytes in altered gravity - results of the MASER-12 suborbital space flight mission.

We investigated the influence of altered gravity on key proteins of T cell activation during the MASER-12 ballistic suborbital rocket mission of the European Space Agency (ESA) and the Swedish Space Cooperation (SSC) at ESRANGE Space Center (Kiruna, Sweden). We quantified components of the T cell receptor, the membrane proximal signaling, MAPK-signaling, IL-2R, histone modifications and the cytoskeleton in non-activated and in ConA/CD28-activated primary human T lymphocytes. The hypergravity phase during the launch resulted in a downregulation of the IL-2 and CD3 receptor and reduction of tyrosine phosphorylation, p44/42-MAPK phosphorylation and histone H3 acetylation, whereas LAT phosphorylation was increased. Compared to the baseline situation at the point of entry into the microgravity phase, CD3 and IL-2 receptor expression at the surface of non-activated T cells were reduced after 6 min microgravity. Importantly, p44/42-MAPK-phosphorylation was also reduced after 6 min microgravity compared to the 1g ground controls, but also in direct comparison between the in-flight µg and the 1g group. In activated T cells, the reduced CD3 and IL-2 receptor expression at the baseline situation recovered significantly during in-flight 1g conditions, but not during microgravity conditions. Beta-tubulin increased significantly after onset of microgravity until the end of the microgravity phase, but not in the in-flight 1g condition. This study suggests that key proteins of T cell signal modules are not severely disturbed in microgravity. Instead, it can be supposed that the strong T cell inhibiting signal occurs downstream from membrane proximal signaling, such as at the transcriptional level as described recently. However, the MASER-12 experiment could identify signal molecules, which are sensitive to altered gravity, and indicates that gravity is obviously not only a requirement for transcriptional processes as described before, but also for specific phosphorylation / dephosphorylation of signal molecules and surface receptor dynamics.