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.

Metabolic Dynamics in Short- and Long-Term Microgravity in Human Primary Macrophages.

Microgravity acts on cellular systems on several levels. Cells of the immune system especially react rapidly to changes in gravity. In this study, we performed a correlative metabolomics analysis on short-term and long-term microgravity effects on primary human macrophages. We could detect an increased amino acid concentration after five minutes of altered gravity, that was inverted after 11 days of microgravity. The amino acids that reacted the most to changes in gravity were tightly clustered. The observed effects indicated protein degradation processes in microgravity. Further, glucogenic and ketogenic amino acids were further degraded to Glucose and Ketoleucine. The latter is robustly accumulated in short-term and long-term microgravity but not in hypergravity. We detected highly dynamic and also robust adaptative metabolic changes in altered gravity. Metabolomic studies could contribute significantly to the understanding of gravity-induced integrative effects in human cells.

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.

"No Innovation Without Cooperation" - How Switzerland Innovation Promotes Cooperation Between Industry, Research and Startups.

Switzerland Innovation, the Swiss innovation park with its five branches, is facilitating collaborations for companies, startups, and universities to find solutions to some of the world's most pressing challenges in the fields of health and the life sciences, in particular in the areas of chemistry, biochemistry, biomedicine, biotech, medtech and digital health. Together with its numerous and diverse partners, Switzerland Innovation creates an ecosystem for universities and research-based companies, accelerating the transformation of research results into marketable products and services.

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.

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.

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.

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 during parabolic flight and clinostat experiments.

BACKGROUND/AIMS: Several limiting factors for human health and performance in microgravity have been clearly identified arising from the immune system, and substantial research activities are required in order to provide the basic information for appropriate integrated risk management. The gravity-sensitive nature of cells of the immune system renders them an ideal biological model in search for general gravity-sensitive mechanisms and to understand how the architecture and function of human cells is related to the gravitational force and therefore adapted to life on Earth. METHODS: We investigated the influence of altered gravity in parabolic flight and 2D clinostat experiments on key proteins of activation and signaling in primary T lymphocytes. We quantified components of the signaling cascade 1.) in non-activated T lymphocytes to assess the "basal status" of the cascade and 2.) in the process of activation to assess the signal transduction. RESULTS: We found a rapid decrease of CD3 and IL-2R surface expression and reduced p-LAT after 20 seconds of altered gravity in non-activated primary T lymphocytes during parabolic flight. Furthermore, we observed decreased CD3 surface expression, reduced ZAP-70 abundance and increased histone H3-acetylation in activated T lymphocytes after 5 minutes of clinorotation and a transient downregulation of CD3 and stable downregulation of IL-2R during 60 minutes of clinorotation. CONCLUSION: CD3 and IL-2R are downregulated in primary T lymphocytes in altered gravity. We assume that a gravity condition around 1g is required for the expression of key surface receptors and appropriate regulation of signal molecules in T lymphocytes.

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.

Expression of hypoxia-inducible genes is suppressed in altered gravity due to impaired nuclear HIF1α accumulation.

Extravehicular activities, the backbone of manned space exploration programs, set astronauts into mild hypoxia. Unfortunately, microgravity aggravates threatening symptoms of hypoxia such as vision impairment and brain edema. Hypoxia-inducible factors (HIFs) sense cellular hypoxia and, subsequently, change the cells' expression profile instantaneously by rapidly translocating-most likely cytoskeleton-dependently-into the nucleus and subsequently forming transcription complexes with other proteins. We tested the hypothesis that this fundamental process could be altered by sudden changes in gravitational forces in parabolic flights using a newly developed pocket-size cell culture lab that deoxygenizes cells within 15 min. Sudden gravity changes (SGCs 1g-1.8g-0g-1.8g-1g) during hypoxic exposure suppressed expression of the HIF1α-dependent genes investigated as compared with hypoxia at constant 1g. Normoxic cells subjected to SGCs showed reduced nuclear but not cytoplasmatic HIF1α signal and appeared to have disturbed cytoskeleton architecture. Inhibition of the actin-dependent intracellular transport using a combination of myosin V and VI inhibitors during hypoxia mimicked the suppression of the HIF1α-dependent genes observed during hypoxic exposure during SGCs. Thus, SGCs seem to disrupt the cellular response to hypoxia by impairing the actin-dependent translocation of HIF1α into the nucleus.

Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity.

In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.

The cannabinoid receptors agonist WIN55212-2 inhibits macrophageal differentiation and alters expression and phosphorylation of cell cycle control proteins.

In this study we investigated if and how cannabinoid receptor stimulation regulates macrophageal differentiation, which is one of the key steps in the immune effector reaction. For that reason, we used a well established differentiation model system of human U937 myelocytic leukemia cells that differentiate along the monocyte/macrophage lineage upon stimulation with the phorbol ester PMA. Constant cannabinoid receptor (CB) stimulation was performed using WIN55212-2, a potent synthetic CB agonist. We found that WIN55212-2 inhibited CB1/2-receptor-dependent PMA-induced differentiation of human myelocytic U937 cells into the macrophageal phenotype, which was associated with impaired vimentin, ICAM-1 and CD11b expression. In the presence of WIN55212-2, cdc2 protein and mRNA expression was progressively enhanced and Tyr-15-phosporylation of cdc2 was reduced in differentiating U937 cells. Additionally, p21Waf1/Cip1 expression was up-regulated. PMA-induced apoptosis was not enhanced by WIN55212-2 and differentiation-associated c-jun expression was not altered. In conclusion, we suppose that WIN55212-2-induced signals interferes with cell-cycle-arrest-signaling in differentiating myelocytic cells and thus inhibits macrophageal differentiation. Thus, it is possible that the cannabinoid system is able to influence one of the key steps in the immune effector function, the monocytic-macrophageal differentiation by alteration of cell cycle control proteins cdc2 and p21, and is therefore representing a promising option for therapeutic intervention in exacerbated immune reactions.

Cannabinoid receptor CB1 mediates baseline and activity-induced survival of new neurons in adult hippocampal neurogenesis.

BACKGROUND: Adult neurogenesis is a particular example of brain plasticity that is partially modulated by the endocannabinoid system. Whereas the impact of synthetic cannabinoids on the neuronal progenitor cells has been described, there has been lack of information about the action of plant-derived extracts on neurogenesis. Therefore we here focused on the effects of Delta9-tetrahydrocannabinol (THC) and Cannabidiol (CBD) fed to female C57Bl/6 and Nestin-GFP-reporter mice on proliferation and maturation of neuronal progenitor cells and spatial learning performance. In addition we used cannabinoid receptor 1 (CB1) deficient mice and treatment with CB1 antagonist AM251 in Nestin-GFP-reporter mice to investigate the role of the CB1 receptor in adult neurogenesis in detail. RESULTS: THC and CBD differed in their effects on spatial learning and adult neurogenesis. CBD did not impair learning but increased adult neurogenesis, whereas THC reduced learning without affecting adult neurogenesis. We found the neurogenic effect of CBD to be dependent on the CB1 receptor, which is expressed over the whole dentate gyrus. Similarly, the neurogenic effect of environmental enrichment and voluntary wheel running depends on the presence of the CB1 receptor. We found that in the absence of CB1 receptors, cell proliferation was increased and neuronal differentiation reduced, which could be related to CB1 receptor mediated signaling in Doublecortin (DCX)-expressing intermediate progenitor cells. CONCLUSION: CB1 affected the stages of adult neurogenesis that involve intermediate highly proliferative progenitor cells and the survival and maturation of new neurons. The pro-neurogenic effects of CBD might explain some of the positive therapeutic features of CBD-based compounds.

Rapid coupling between gravitational forces and the transcriptome in human myelomonocytic U937 cells.

The gravitational force has been constant throughout Earth's evolutionary history. Since the cell nucleus is subjected to permanent forces induced by Earth's gravity, we addressed the question, if gene expression homeostasis is constantly shaped by the gravitational force on Earth. We therefore investigated the transcriptome in force-free conditions of microgravity, determined the time frame of initial gravitational force-transduction to the transcriptome and assessed the role of cation channels. We combined a parabolic flight experiment campaign with a suborbital ballistic rocket experiment employing the human myelomonocytic cell line U937 and analyzed the whole gene transcription by microarray, using rigorous controls for exclusion of effects not related to gravitational force and cross-validation through two fully independent research campaigns. Experiments with the wide range ion channel inhibitor SKF-96365 in combination with whole transcriptome analysis were conducted to study the functional role of ion channels in the transduction of gravitational forces at an integrative level. We detected profound alterations in the transcriptome already after 20 s of microgravity or hypergravity. In microgravity, 99.43% of all initially altered transcripts adapted after 5 min. In hypergravity, 98.93% of all initially altered transcripts adapted after 75 s. Only 2.4% of all microgravity-regulated transcripts were sensitive to the cation channel inhibitor SKF-96365. Inter-platform comparison of differentially regulated transcripts revealed 57 annotated gravity-sensitive transcripts. We assume that gravitational forces are rapidly and constantly transduced into the nucleus as omnipresent condition for nuclear and chromatin structure as well as homeostasis of gene expression.

Immune control by endocannabinoids - new mechanisms of neuroprotection?

The endocannabinoid system consists of cannabinoid receptors, their endogenous ligands and enzymes for synthesis and degradation of endocannabinoids and represents a local messenger system within and between the nervous and immune system. Apparently, the endocannabinoid system is involved in immune control and neuoprotection. In this review, we discuss possible mechanisms of immune control by endocannabinoids, which include the modulation of innate immunity, effects on cytokin networks, downregulation of adaptive immune response and apoptosis in immune cells.

Cytoskeletal stability and metabolic alterations in primary human macrophages in long-term microgravity.

The immune system is one of the most affected systems of the human body during space flight. The cells of the immune system are exceptionally sensitive to microgravity. Thus, serious concerns arise, whether space flight associated weakening of the immune system ultimately precludes the expansion of human presence beyond the Earth's orbit. For human space flight, it is an urgent need to understand the cellular and molecular mechanisms by which altered gravity influences and changes the functions of immune cells. The CELLBOX-PRIME (= CellBox-Primary Human Macrophages in Microgravity Environment) experiment investigated for the first time microgravity-associated long-term alterations in primary human macrophages, one of the most important effector cells of the immune system. The experiment was conducted in the U.S. National Laboratory on board of the International Space Station ISS using the NanoRacks laboratory and Biorack type I standard CELLBOX EUE type IV containers. Upload and download were performed with the SpaceX CRS-3 and the Dragon spaceship on April 18th, 2014 / May 18th, 2014. Surprisingly, primary human macrophages exhibited neither quantitative nor structural changes of the actin and vimentin cytoskeleton after 11 days in microgravity when compared to 1g controls. Neither CD18 or CD14 surface expression were altered in microgravity, however ICAM-1 expression was reduced. The analysis of 74 metabolites in the cell culture supernatant by GC-TOF-MS, revealed eight metabolites with significantly different quantities when compared to 1g controls. In particular, the significant increase of free fucose in the cell culture supernatant was associated with a significant decrease of cell surface-bound fucose. The reduced ICAM-1 expression and the loss of cell surface-bound fucose may contribute to functional impairments, e.g. the activation of T cells, migration and activation of the innate immune response. We assume that the surprisingly small and non-significant cytoskeletal alterations represent a stable "steady state" after adaptive processes are initiated in the new microgravity environment. Due to the utmost importance of the human macrophage system for the elimination of pathogens and the clearance of apoptotic cells, its apparent robustness to a low gravity environment is crucial for human health and performance during long-term space missions.

Stability of gene expression in human T cells in different gravity environments is clustered in chromosomal region 11p15.4.

In the last decades, a plethora of in vitro studies with living human cells contributed a vast amount of knowledge about cellular and molecular effects of microgravity. Previous studies focused mostly on the identification of gravity-responsive genes, whereas a multi-platform analysis at an integrative level, which specifically evaluates the extent and robustness of transcriptional response to an altered gravity environment was not performed so far. Therefore, we investigated the stability of gene expression response in non-activated human Jurkat T lymphocytic cells in different gravity environments through the combination of parabolic flights with a suborbital ballistic rocket and 2D clinostat and centrifuge experiments, using strict controls for excluding all possible other factors of influence. We revealed an overall high stability of gene expression in microgravity and identified olfactory gene expression in the chromosomal region 11p15.4 as particularly robust to altered gravity. We identified that classical reference genes ABCA5, GAPDH, HPRT1, PLA2G4A, and RPL13A were stably expressed in all tested gravity conditions and platforms, while ABCA5 and GAPDH were also known to be stably expressed in U937 cells in all gravity conditions. In summary, 10-20% of all transcripts remained totally unchanged in any gravitational environment tested (between 10-4 and 9 g), 20-40% remained unchanged in microgravity (between 10-4 and 10-2 g) and 97-99% were not significantly altered in microgravity if strict exclusion criteria were applied. Therefore, we suppose a high stability of gene expression in microgravity. Comparison with other stressors suggests that microgravity alters gene expression homeostasis not stronger than other environmental factors.

Dynamic gene expression response to altered gravity in human T cells.

We investigated the dynamics of immediate and initial gene expression response to different gravitational environments in human Jurkat T lymphocytic cells and compared expression profiles to identify potential gravity-regulated genes and adaptation processes. We used the Affymetrix GeneChip® Human Transcriptome Array 2.0 containing 44,699 protein coding genes and 22,829 non-protein coding genes and performed the experiments during a parabolic flight and a suborbital ballistic rocket mission to cross-validate gravity-regulated gene expression through independent research platforms and different sets of control experiments to exclude other factors than alteration of gravity. We found that gene expression in human T cells rapidly responded to altered gravity in the time frame of 20 s and 5 min. The initial response to microgravity involved mostly regulatory RNAs. We identified three gravity-regulated genes which could be cross-validated in both completely independent experiment missions: ATP6V1A/D, a vacuolar H + -ATPase (V-ATPase) responsible for acidification during bone resorption, IGHD3-3/IGHD3-10, diversity genes of the immunoglobulin heavy-chain locus participating in V(D)J recombination, and LINC00837, a long intergenic non-protein coding RNA. Due to the extensive and rapid alteration of gene expression associated with regulatory RNAs, we conclude that human cells are equipped with a robust and efficient adaptation potential when challenged with altered gravitational environments.

Rapid adaptation to microgravity in mammalian macrophage cells.

Despite the observed severe effects of microgravity on mammalian cells, many astronauts have completed long term stays in space without suffering from severe health problems. This raises questions about the cellular capacity for adaptation to a new gravitational environment. The International Space Station (ISS) experiment TRIPLE LUX A, performed in the BIOLAB laboratory of the ISS COLUMBUS module, allowed for the first time the direct measurement of a cellular function in real time and on orbit. We measured the oxidative burst reaction in mammalian macrophages (NR8383 rat alveolar macrophages) exposed to a centrifuge regime of internal 0 g and 1 g controls and step-wise increase or decrease of the gravitational force in four independent experiments. Surprisingly, we found that these macrophages adapted to microgravity in an ultra-fast manner within seconds, after an immediate inhibitory effect on the oxidative burst reaction. For the first time, we provided direct evidence of cellular sensitivity to gravity, through real-time on orbit measurements and by using an experimental system, in which all factors except gravity were constant. The surprisingly ultra-fast adaptation to microgravity indicates that mammalian macrophages are equipped with a highly efficient adaptation potential to a low gravity environment. This opens new avenues for the exploration of adaptation of mammalian cells to gravitational changes.