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 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

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

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

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 CELLBOXPRIME (= 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

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

<p>Abstract</p> <p>Background</p> <p>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 Δ9-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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

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

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

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