Microarray Analysis of Space-flown Murine Thymus Tissue
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ABSTRACT: Microarray Analysis of Space-flown Murine Thymus Tissue Reveals Changes in Gene Expression Regulating Stress and Glucocorticoid Receptors. We used microarrays to detail the gene expression of space-flown thymic tissue and identified distinct classes of up-regulated genes during this process. We report here microarray gene expression analysis in young adult C57BL/6NTac mice at 8 weeks of age after exposure to spaceflight aboard the space shuttle (STS-118) for a period of 13 days. Upon conclusion of the mission, thymus lobes were extracted from space flown mice (FLT) as well as age- and sex-matched ground control mice similarly housed in animal enclosure modules (AEM). mRNA was extracted and an automated array analysis for gene expression was performed. Examination of the microarray data revealed 970 individual probes that had a 1.5 fold or greater change. When these data were averaged (n=4), we identified 12 genes that were significantly up- or down-regulated by at least 1.5 fold after spaceflight (p≤0.05). Together, these data demonstrate that spaceflight induces significant changes in the thymic mRNA expression of genes that regulate stress, glucocorticoid receptor metabolism, and T cell signaling activity. These data explain, in part, the reported systemic compromise of the immune system after exposure to the microgravity of space.
Project description:Efforts to understand the impact of spaceflight on the human body stem from the growing interest in long-term space travel. Multiple organ systems are affected by micro-gravity and radiation, including the cardiovascular system. Previous transcriptomic studies have sought to reveal the changes in gene expression after spaceflight. However, little is known about the impact of long-term spaceflight on the mouse heart in vivo. This study focused on transcriptomic changes in the hearts of female C57BL/6J mice flown on the International Space Station (ISS) for 30 days. RNA was isolated from the hearts of three flight and three comparable ground control mice and RNA sequencing was performed. Our analysis showed that 1,147 transcripts were significantly regulated after spaceflight. MAPK, PI3K-Akt and GPCR signaling pathways were predicted to be activated. Transcripts related to cytoskeleton breakdown and organization were upregulated, but no significant change in the expression of extracellular matrix (ECM) components or oxidative stress pathway associated transcripts occurred. Our results indicate an absence of cellular senescence, and a significant upregulation of transcripts associated with the cell cycle. Transcripts related to cellular maintenance and survival were most affected by spaceflight, suggesting that the cardiovascular transcriptome initiates an adaptive response to long-term spaceflight
Project description:This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in microgravity (mg). Immunosuppression during spaceflight is a major barrier to safe long-term human space habitation and travel. The goals of these experiments were to prove that mg was the cause of impaired T cell activation during spaceflight as well as understand the mechanisms controlling early T cell activation. T cells from 4 human donors were stimulated with concanavalin A (ConA) and anti-CD28 onboard the International Space Station (ISS). An onboard centrifuge was used to generate a 1g simultaneous control to isolate the effects of mg from other variables of spaceflight. Microarray expression analysis after 1.5 hours of activation demonstrated that mg- and 1g-activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly differentially down-regulated in mg. Importantly, several key immediate early genes were inhibited in mg. T cells were isolated from human volunteers. T cells from each donor were kept separate and loaded into individual chambers in separate cassettes for the following treatments: mg non-activated, mg activated, and 1g activated. Therefore, samples represent biological triplicates. Experimental units were launched into space and placed into the KUBIK facility onboard the International Space Station. The 1g units were placed in the central centrifuge positions and centrifuged with an applied 1g force. The mg units were place in the static positions for continued mg exposure. After 30 minutes of pre-incubation, mg non-activated units were fixed by addition of RNALater (QIAGEN, Valencia, CA), removed from the incubator, and stored in 4M-BM-0C. The mg and 1g activated units were injected with final concentration 10mg/ml Con A and 4mg/ml anti-CD28. These cassettes were replaced into KUBIK on either the centrifuge or static positions and activated for 1.5 hours. Activation was stopped with the addition of RNALater and the units were then moved to 4M-BM-0C storage. All units were returned to Earth for analysis.
Project description:This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in microgravity (mg). Immunosuppression during spaceflight is a major barrier to safe long-term human space habitation and travel. The goals of these experiments were to prove that mg was the cause of impaired T cell activation during spaceflight as well as understand the mechanisms controlling early T cell activation. T cells from 4 human donors were stimulated with concanavalin A (ConA) and anti-CD28 onboard the International Space Station (ISS). An onboard centrifuge was used to generate a 1g simultaneous control to isolate the effects of mg from other variables of spaceflight. Microarray expression analysis after 1.5 hours of activation demonstrated that mg- and 1g-activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly differentially down-regulated in mg. Importantly, several key immediate early genes were inhibited in mg.
Project description:The mechanism of egress of mature regulatory T cells (Tregs) from the thymus to the periphery remains enigmatic, as does the nature of those factors expressed in the thymic environment. Here, we examined the fate of thymic Tregs in TNFα/RelA double-knockout (TA-KO) mice, because TA-KO mice retain a Treg population in the thymus but have only a small Treg population at the periphery. Transplantation of whole TA-KO thymus to under the kidney capsule of Rag1 null mice failed to induce the production of donor-derived splenic Tregs expressing neuropilin-1 (Nrp1), which was reported to be a marker of naturally occurring Tregs, indicating that TA-KO thymic Tregs either do not leave the thymus or are lost at the periphery. We next transplanted enriched TA-KO thymic Tregs to the peripheries of TA-KO mice and traced mouse survival. Transplantation of TA-KO thymic Tregs rescued the lethality in TA-KO mice, demonstrating that TA-KO thymic Tregs remain functional at the periphery. The TA-KO thymic Treg population had highly demethylated CpG motifs in the foxp3 locus, indicating that the cells were arrested at a late-mature stage. Also, the population included a large subpopulation of Tregs expressing IL-7Rα, which is a possible marker of late-mature Tregs. Finally, TA-KO fetal liver chimeric mice developed an Nrp1+ splenic Treg population from TA-KO cells, suggesting that Treg arrest is caused by a lack of RelA in the thymic environment. Together, these results suggest that egress of mature Tregs from the thymus depends on RelA in the thymic environment. For the isolation of thymic Tregs, CD4+CD8α-CD25hi thymocytes were isolated from five 1.5- to 2-week-old TNFα-KO or TA-KO mice by using a FACSAria cell sorter. For the isolation of thymic stromal cells, 10 thymi from 1.5- to 2-week-old TNFα-KO or TA-KO mice were minced with scissors and treated with RPMI 1640 supplemented with 2% FCS, 0.2 mg/ml collagenase (Roche, Basel, Switzerland), 0.2 mg/ml dispase I (Roche), and 100 U/ml DNase I (Life Technologies) for 30 min with stirring. Digested thymi were centrifuged in a Percoll (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) gradient (density, 1.115, 1.065, and PBS) at 1400g for 30 min. Cells in the upper layer were collected, and the CD45-EpCAM+ (thymic epithelial cells) and CD45+EpCAM- populations (enriched thymic stromal cells containing macrophages or dendritic cells) were sorted.
Project description:Adult and neonatal human cardiovacular progenitor cell clonal populations were flown aboard the ISS for 12 days prior to fixation in RNAProtect, total RNA purification, and microRNA expression analysis against clone-, patient-, and passage-matched ground control samples.
Project description:Bone loss and immune dysregulation are among the main adverse outcomes of spaceflight challenging astronaut’s health and safety. However, consequences on B cell development and responses are still under-investigated. Up to now, most studies addressing these questions were performed using an amphibian species. Consequently, we used advanced proteomics analysis of femur bone and marrow of mice flown for one month on board the BION-M1 biosatellite, to determine whether extreme conditions encountered during a real spaceflight affect B cell development in mice and to examine reversibility of the effects upon return to Earth. Our data revealed that adverse effects on B lymphopoiesis were more marked one week after landing and that this phenomenon was associated with a 41% reduction of B cells in the spleen. Thus, the effects of spaceflight persisted during at least one week after landing. These reductions may contribute to explain increased susceptibility to infection even if we confirmed that animals were able to mount a humoral immune response.
Project description:Adult and neonatal human cardiovacular progenitor cell clonal populations were flown aboard the ISS for 12 days prior to fixation in RNAprotect. Gene expression analysis was performed against clone- and passage-matched ground control samples.
Project description:Adult and neonatal human cardiovacular progenitor cell clonal populations were flown aboard the ISS for 12 days prior to fixation in RNAprotect. Gene expression analysis was performed against clone-, patient-, and passage-matched ground control samples.