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

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

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 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