STEM TODAY May 2017, No.20
STEM TODAY May 2017 , No.20
CONTENTS IM1: We do not know to what extent spaceflight alters various aspects of human immunity during spaceflight missions up to 6 months. A wealth of knowledge defined immune dysregulation postflight, including diminished cellular function, dysregulated cytokine production profiles and physiological stress. However, it was generally unknown if these observations reflected the inflight condition. Several narrowfocus, low ‘n’ inflight studies did indicate that immune dysregulation could be an inflight phenomenon, however proper investigation of the various aspects of immunity and stress (innate/adaptive, humoral/cellular, dysfunction among specific cell types, etc.) was lacking. The reactivation of latent herpesviruses, thought to be a direct consequence of diminished immune function, was well established during short duration spaceflight, but it was unknown if this phenomenon would persist or resolve during longduration spaceflight.
Editorial Editor: Mr. Abhishek Kumar Sinha Editor / Technical Advisor: Mr. Martin Cabaniss
STEM Today, May 2017, No.20
Cover Page This Week in NASA History: Hubble Space Telescope Deployed – April 25, 1990 This week in 1990, the Hubble Space Telescope was deployed from the cargo bay of space shuttle Discovery as part of STS-31. NASA’s Marshall Space Flight Center was responsible for the design, development, and construction of the Hubble Space Telescope and has played a significant role in the testing of Hubble’s successor, the James Webb Space Telescope. Scheduled to launch in October 2018, the Webb telescope will observe the most distant objects in the universe, provide images of the first galaxies formed and see unexplored planets around distant stars. Image Credit: NASA
Back Cover This Week in NASA History: Fourth Hubble Servicing Mission Launches – March 1, 2002 This week in 2002, space shuttle Columbia and STS-109 launched from NASA’s Kennedy Space Center to begin the fourth Hubble Space Telescope servicing mission. Here Hubble is berthed in Columbia’s cargo bay, silhouetted against the airglow of Earth’s horizon. During this mission, astronauts replaced Hubble’s solar panels and installed the Advanced Camera for Surveys, which took the place of Hubble’s Faint Object Camera, the telescope’s last original instrument. NASA’s Marshall Space Flight Center has been involved in development of many of the agency’s optical instruments. Notably, Marshall played a significant role in NASA’s Great Observatories, managing the development of Hubble and the Chandra X-ray Observatory, and the Burst and Transient Source Experiment for the Compton Gamma Ray Observatory. Marshall also manages Chandra’s flight, current operations and guest science observer program and has played a significant role in the testing of Hubble’s successor, the James Webb Space Telescope. Scheduled to launch in October 2018, the Webb telescope will observe the most distant objects in the universe, provide images of the first galaxies formed and see unexplored planets around distant stars. Image Credit: NASA
STEM Today , May 2017
Editorial Dear Reader
STEM Today, May 2017, No.20
All young people should be prepared to think deeply and to think well so that they have the chance to become the innovators, educators, researchers, and leaders who can solve the most pressing challenges facing our world, both today and tomorrow. But, right now, not enough of our youth have access to quality STEM learning opportunities and too few students see these disciplines as springboards for their careers. According to Marillyn Hewson, "Our children - the elementary, middle and high school students of today - make up a generation that will change our universe forever. This is the generation that will walk on Mars, explore deep space and unlock mysteries that we can’t yet imagine". "They won’t get there alone. It is our job to prepare, inspire and equip them to build the future - and that’s exactly what Generation Beyond is designed to do." STEM Today will inspire and educate people about Spaceflight and effects of Spaceflight on Astronauts. Editor Mr. Abhishek Kumar Sinha
Editorial Dear Reader The Science, Technology, Engineering and Math (STEM) program is designed to inspire the next generation of innovators, explorers, inventors and pioneers to pursue STEM careers. According to former President Barack Obama, " Science is more than a school subject, or the periodic table, or the properties of waves. It is an approach to the world, a critical way to understand and explore and engage with the world, and then have the capacity to change that world..." STEM Today addresses the inadequate number of teachers skilled to educate in Human Spaceflight. It will prepare , inspire and educate teachers about Spaceflight. STEM Today will focus on NASA’S Human Research Roadmap. It will research on long duration spaceflight and put together latest research in Human Spaceflight in its monthly newsletter. Editor / Technical Advisor Mr. Martin Cabaniss
STEM Today, May 2017, No.20
Human Health Countermeasures (HHC) IM1: We do not know to what extent space ight alters various aspects of human immunity during space ight missions up to 6 months At the 2005 inception of the HRP there was little known about the in- ight status of the human immune system. A wealth of knowledge de ned immune dysregulation post- ight, including diminished cellular function, dysregulated cytokine production pro les and physiological stress. However, it was generally unknown if these observations re ected the in- ight condition. Several narrow-focus, low 'n' in- ight studies did indicate that immune dysregulation could be an in- ight phenomenon, however proper investigation of the various aspects of immunity and stress (innate/adaptive, humoral/cellular, dysfunction among speci c cell types, etc.) was lacking. The reactivation of latent herpesviruses, thought to be a direct consequence of diminished immune function, was well established during short duration space ight, but it was unknown if this phenomenon would persist or resolve during long-duration space ight. It is generally believed that such dysregulation would not be a signi cant clinical risk for orbital ight (despite incidence of immune-related health events on orbit), but that persistent dysregulation could pose a crew health risk during exploration class deep-space missions. During the intervening period since HRP inception, Integrated Immune has thoroughly characterized certain aspects of adaptive immunity and viral reactivation during short- and long-duration space ight. The new ndings con rm that both immune dysregulation and latent herpesvirus reactivation persist during 6-month ISS missions. Other aspects of immunoreguation remain relatively uninvestigated during space ight.
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Latent virus reactivation in Astronauts at ISS
In this study , viral reactivation and shedding of EBV, VZV, CMV, HSV1, and human herpes virus 6 (HHV6) were measured in 23 astronauts (18 male and 5 female) before, during, and immediately following long duration spaceflight.
Results
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Viral reactivation Twenty-two of 23 astronauts shed one or more target viruses (Table 1). Fifteen astronauts shed VZV, 22 shed EBV, and 14 shed CMV at one or more time points before, during, or after spaceflight (Table 1). One astronaut did not shed any virus during any defined collection time. By contrast, none of the 20 control subjects shed VZV or CMV and only 2 of them shed EBV (Table 1). No astronauts or control subjects shed HSV1, HSV2, or HHV6 at any time throughout the study. Percent shedding among crewmembers with 95% binomial confidence intervals are shown for EBV, VZV, and CMV in Fig. 1. For these three viruses, there was considerable variation of the shedding percentages over the collection time points (Fig. 1) suggesting a possible overall mission effect on the reactivation of these viruses.
For VZV, no shedding occurred at both 180 days and 45 days before flight but shedding was found in early, mid, and late time points during flight as well as at landing and 30 days after landing (Table 1). The Friedman test comparing copy number distributions was significant for VZV (P < 0.0001). After adjustment for multiple comparisons, authors found significantly more VZV reactivation in the late-flight time period than either pre-
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flight (P = 0.011) or 30d post-flight (P = 0.035). There was no CMV shedding at 180 days before flight but there was CMV shedding 45 days before, during, and after flight. The Friedman test comparing copy numbers for CMV was also significant (P < 0.0001).
In particular, also after adjustment for multiple comparisons, authors found that this was due to increased CMV shedding during flight which was significantly greater than 180 days before flight (P = 0.013) and also greater than 30 days after flight (P = 0.041). Even when the time points with no shedding were excluded from the analysis, there were still significant differences between the remaining time points; P = 0.0027 (VZV) and P = 0.0008 (CMV) (Fig. 1).
For the same viruses as above, Figure 2 shows median number of copies for EBV, VZV, and CMV along with 95% confidence limits obtained from mixed-model regression analysis. The overall mission effect on copy numbers was evident for EBV (P < 0.001), VZV (P = 0.057), and CMV (P = 0.001). For EBV, post-hoc comparisons with Sidak - adjusted P-values reflected higher median viral copies during the last two flight periods (Mid, Late) relative to pre-flight (P < .001, both comparisons) and relative to 30 days post-flight (P < 0.001, both comparisons). Median copies for early recovery (at landing) were not significantly higher than preflight (unadjusted P = 0.08) (Fig. 2). For CMV, the median copy number was significantly higher during flight than before flight (P < 0.001), but not for landing day (unadjusted P = 0.56) or 30 days after landing (unadjusted P = 0.55). No post-hoc comparisons were made for median copies of VZV because the overall mission effect was not significant. EBV DNA levels in peripheral blood mononuclear cells (PBMCs) EBV DNA levels varied considerably: from below or at the detectable limit of 2 copies (55 cases) to a maximum of 71,500 copies, with 25 instances of at least 1000 copies being detected. Because of this high degree of variation, authors did not find a significant difference between median copy levels over the time points as a whole (P = 0.21, median regression analysis), although it did appear that median copies were somewhat elevated at the later time points (Fig. 3).
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Plasma cortisol Median plasma cortisol levels ranged from 15.2 Âľg/dL (late flight) to 24.8 Âľg/dL (late recovery). The overall test for differences in medians between the seven time points was not significant (bootstrapped median regression: P = 0.086). Estimates of medians and 95% confidence limits for each time point are shown in Fig. 4.
Salivary cortisol and DHEA With natural log cortisol concentration as outcome, there was some evidence that daily mean trajectories for each of the three during-flight periods differed from the trajectories before flight (unadjusted P = 0.069, 0.022, 0.086 by mixed-model regression on log cortisol concentration values for early-flight vs. before-flight, mid-flight vs. before-flight, and late-flight vs. before-flight, respectively).
However, the flight periods did not differ significantly between themselves (P = 0.132). After combining data across the three flight periods and comparing the mean daily trajectory during flight with pre-flight, authors found evidence for an effect of flight (P = 0.010 ). With one outlier subject removed , there was even stronger evidence of a flight effect (P = 0.0015). Figure 5a shows the predicted mean daily trajectory of log cortisol concentration before flight and during flight with 95% confidence limits. The difference was greatest around the middle of the waking period (about 10 h). A plot of this difference vs. hours since awakening with 95% confidence limits is shown in Fig. 5b. There was no evidence that the mean cortisol trajectory during the first period after flight was different from pre-flight (P = 0.42). There was some evidence of a difference between the second recovery period and before flight (unadjusted P = 0.048) . Estimated diurnal declines of DHEA did
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not change as a function of study phase. Figure 6a shows these trends (on a log scale) for the before and during flight periods and Fig. 6b shows the estimated difference in these trends between the "during flight" and "before flight" periods.
Anti-viral Antibodies After adjusting for differences between subjects, titer levels of anti-EBV viral capsid antigen (VCA) antibodies appeared increased relative to the first pre-flight period. However, because of high variability we found no significant difference between the periods (P = 0.53, repeated measures ordered logit analysis). Similarly authors could not detect any effect of flight on anti-CMV VCA antibodies (P = 0.79).
Findings Recent studies confirmed that astronauts on the ISS continue to experience dysregulation of immune and endocrine systems. Increased levels of plasma and urinary stress hormones (cortisol, and catecholamines) commonly accompany spaceflight. Shedding of EBV, CMV, and VZV did not abate during the longer missions onboard the ISS. Rather, virus shedding actually increased in frequency and amplitude (viral copy numbers) of all three viruses tested. As shown in Table 2, VZV shedding increased from 41% in short duration space shuttle to 65% in long duration ISS, EBV increased from 82 to 96%, and CMV increased from 47 to 61% of the crewmembers. Also, shedding for all three viruses persisted throughout the long duration (Early, Mid, and Late) missions. In addition, VZV and CMV shed up to 30 days after longer-duration spaceflight on the ISS unlike for the short-duration spaceflight on the space shuttle where VZV was shed only up to 5 days and CMV shed up to 3 days after flight. In a recent study, it was reported that immune changes observed in short duration spaceflight actually increased in astronauts after 6 months of ISS flight.
100% of all astronauts (n = 86) studied to date were seropositive for EBV. In this study, all but one of the 23 astronauts studied shed EBV at some time point. These findings could impact the design of exploration-class missions during which reactivation of latent viruses could result in an increased risk of wide-ranging adverse medical events.
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Decreases in thymopoiesis of astronauts returning from space flight
The thymus is responsible for production of the diverse repertoire of naive T cells that are critical for effective adaptive immunity . Naive T cells are continuously produced by the thymus and exported to the periphery where they expand in response to homeostatic signals or antigen exposure. Maintenance of this naive T cell receptor (TCR) repertoire is essential for control of endogenous pathogens, including latent herpesviruses, and other opportunistic viral and fungal pathogens. Space travel exposes astronauts to factors including microgravity, solar and cosmic radiation, and other significant stressors. Immune system dysregulation has been documented in astronauts during and after space flight. Space flight has been shown to result in suppression of antigen-specific T cell function, altered T cell memory subset distribution, altered cytokine production profiles, decreased delayed-type hypersensitivity, and herpesvirus reactivation . Physical and psychological stress (i.e., launch and landing stressors, microgravity, confinement, separation from family, and sleep deprivation) mediate these changes, presumably via activation of the hypothalamic-pituitary-adrenal axis. Accordingly, elevated levels of cortisol have been observed during and after spaceflight. Notably, glucocorticoids affect the immune system by altering leukocyte trafficking and migration as well as directly inhibiting cellular functions. Authors measured RTE within peripheral blood mononuclear cells (PBMCs) of astronauts returning from flight aboard the ISS and performed parallel assessments of stress-associated glucocorticoids.
STEM Today, May 2017, No.20
Results
Thymopoiesis, measured by assessment of TRECs, declines following return from space flight Thymopoiesis was measured at 3 time points beginning 180 days before scheduled launch by analysis of TRECs known to be present in RTE . Baseline thymopoiesis varied significantly (P < 0.001) within the group, consistent with prior studies in healthy adults, but remained relatively stable within individuals prior to launch (Figure 1). Astronauts remained in space for variable periods, with a median duration of 184 days, and no sampling was possible during this interval. Upon return, samples were taken within 2 to 4 hours of landing and thymopoiesis was assessed within purified mononuclear cells, given the potential occurrence of stress-associated neutrophilia.
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In all 16 astronauts studied, thymopoiesis was lower at the time of return than during any measured interval prior to flight (graphical representation of original data in Figure 1 and Table 1 as Log10 -transformed data). Mean levels declined from a baseline of 3,155 to 1,734 TRECs/million PBMCs (P < 0.001), corresponding to a 45% decrease in the output of RTE from starting levels.
STEM Today, May 2017, No.20
Following the consistent and significantly diminished thymopoiesis observed at landing, thymic output began a return to preflight levels within days to weeks of return to Earth and normalized to the preflight range, remaining there through the 180-day postflight observation period. Thymopoiesis rebounded from nadir at the time of return to 2,825 TRECs/million PBMCs (P < 0.001) postflight, and the difference between the baseline and postflight TREC levels was not significantly different (P = 0.12) (Table 2). Results were consistent across the entire group of ISS astronauts, and the relative decline in thymopoiesis appeared to be independent of the baseline values. These results demonstrate that thymic function is altered in ISS astronauts and suggest that T cell homeostasis may be impaired in long-duration space flight.
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Suppression of thymopoiesis is inversely correlated with a transient rise in cortisol levels In 15 of 16 astronauts, cortisol levels were strongly and inversely correlated to preflight to postflight changes in thymopoiesis (Table 2). In parallel to the significant declines in thymopoiesis seen at the time of return to Earth (P < 0.001), urine cortisol levels were significantly increased at return (36.2 to 73.5 Âľg/d; P = 0.004), with no differences between levels at baseline versus those after space flight (P = 0.43). Furthermore, stress responses in long-duration ISS astronauts were greater than those observed in shuttle astronauts spending relatively short amounts of time in space.
Findings
STEM Today, May 2017, No.20
Authors found consistent and significant decreases in thymopoiesis in all subjects upon return from space. The results demonstrate that the stresses of prolonged space flight result in a significant (P < 0.001) decrease in T cell production, with a median decrease of 45% from baseline. In this study, plasma and urinary cortisol were highly elevated in ISS crew members (50%-200% greater than Shuttle crew members) immediately after space flight. The results demonstrate that stress-induced increases in endogenous corticosteroids are strongly associated with reversible suppression of thymopoiesis and confirm that the thymus exhibits plasticity of function in healthy individuals subjected to stress. The data raise concerns that peripheral effects and central effects leading to suppression of thymopoiesis may together significantly increase the risk of infection during long-term space flight, as would be necessary for interplanetary travel. Additionally, loss of thymic function may be associated with increased risk of autoimmunity due to increased homeostatic proliferation or loss of regulatory T cells following lymphocyte depletion.
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T cell regulation in real and simulated microgravity experiments in vitro
Reduced T cell activation Several studies with isolated T lymphocytes were carried out in real (Table 1) and simulated (Table 2) microgravity. For the first time, in vitro activation of T lymphocytes isolated from human peripheral blood under microgravity conditions was conducted in space during the Spacelab 1 mission aboard the space shuttle Columbia (Space transportation system 9, STS-9) in 1983. The reactivity of peripheral blood lymphocytes to stimulation with the mitogen Concanavalin A (ConA) was almost completely lost. These results were confirmed in two subsequent experiments performed in the BIORACK facility of Spacelab D-1 aboard the space shuttle Challenger (STS-61-A) in 1985 which included 1g in-flight reference centrifuge controls. in vitro activation of human lymphocytes was significantly reduced by almost 100% in microgravity compared with control cells on an 1g onboard reference centrifuge . This reduced activation was confirmed in the ground-based facilities RWV, clinostat and RPM. The consideration that binding of the mitogen ConA is altered in microgravity, was examined in experiments performed on four sounding rockets providing 7 min resp. 13 min microgravity. Using fluorescent-labelled ConA, these experiments showed that binding of the mitogen to the membrane was principally not affected; only a slight delay of patching and capping was observed. Influence of cell-to-cell and cell-substrate interactions on T cell function In real and simulated microgravity, the lack of sedimentation may lead to reduced cell-to-cell and cell-substrate interactions, which in turn could contribute to the reduced proliferative response to mitogenic stimuli observed in altered gravity. However, phytohemagglutinin (PHA) stimulation of peripheral blood mononuclear cells (PBMCs) cultured in Teflon bags, which reduce cell-substratum interactions, did not significantly affect proliferation compared to cultures in standard cell culture flasks. Other experiments aboard STS-40 where PBMCs were immobilized on microcarrier beads prior to ConA stimulation showed that lymphocyte activation was almost doubled . Proliferation of T lymphocytes was totally inhibited in an RWV experiment where cells were stimulated with CD2/CD28 and CD3/CD28. These stimuli activate cells without requiring co-stimulatory signals by cell-to-cell interaction. Furthermore, experiments with human PBMCs and/or Jurkat T cells performed aboard the biosatellite BION-9, the space shuttle STS-65 and aboard three sounding rockets (MASER-3, -4, and MAXUS-1B) could show that cellular interactions occur in microgravity, since aggregates of lymphocytes were observed. Therefore, it is more likely that alterations in signal transduction rather than absence of cell-to-cell interactions are responsible for depression of T cell function.
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Microgravity changes the patterns of cytokine release IL-2 and IL-2 receptor (IL-2 R) interaction plays a critical role as a required co-stimulatory signal for full T cell activation. Thus, the reduced ability of T cells to proliferate and differentiate into functional effector cells upon activation in microgravity could also be caused by alterations in IL-2 secretion or IL-2 R surface expression, resulting in an impairment of the positive regulatory feedback loop. Experiments performed with human PBMCs during several space flights (STS-40, STS-60,STS- 81 and STS-84) revealed that IL-2 secretion as well as the level of IL-2 R expression are strongly reduced in microgravity . Moreover, experiments with PBMCs or primary human T cells in simulated microgravity provided by clinostat, RWV, or RPM could confirm these results and could further show that the genetic expression of IL-2 and its receptor was inhibited upon activation. However, co-stimulation with sub-mitogenic concentrations of phorbol-12-myristate-13-acetate (PMA) could restore proliferative response and surface expression of IL-2 R. Analyses of IFNγ secretion upon mitogenic stimulation of cells flown in space and cells from astronauts flown on several space shuttle missions revealed that in-flight stimulation of PBMCs with ConA led to increased IFNγ release in comparison to ground controls , whereas PMA/Ionomycin stimulation of samples which were taken directly after landing from astronauts exhibited significant reduction of IFNγ secretion by CD4+ T lymphocytes and unchanged secretion by CD8+ T lymphocytes. Furthermore, comparison of whole blood culture samples collected from astronauts of short- (space shuttle) and long-duration (ISS) flights showed that after the shortduration flight the percentage of T cells producing IFNγ was decreased, whereas after the long-duration flight T cell percentage producing IFNγ was unchanged. Exposure of lymphocytes to simulated microgravity in an RWV led to initial suppression of IFNγ secretion, which was restored to normal levels after three days. Bead-attached cells also showed markedly different cytokine profiles in comparison to cells in suspension culture: Especially IL-2, IFNγ and tumor necrosis factoralpha (TNFα) were significantly increased. Microgravity-induced changes in cytoskeletal structures and cell motility The cytoskeleton is responsible for giving a cell its shape and for generating the forces required for cell motility. It is an internal network of at least three types of cytosolic fibers: actin filaments, microtubules and intermediate filaments. Significant changes in the cytoskeletal structure, which plays an important role not only in cell motility but also in receptor signaling integrity, were observed. Structural changes of vimentin filaments, and the microtubule network were reported in several independent experiments in real microgravity. Since lymphocyte migration is fundamental to keep the organism under immunological surveillance and a crucial event in the systemic immune defense, investigating the impact of microgravity on this cellular function is important to understand the immune response in microgravity. Besides cell-to-cell interactions also cell motility is important for cell communication and signal transmission. Observation of cell motility under microgravity conditions revealed that T cells were motile but the motility did not decrease with increasing stimulation time, which indicates that cell cycle progression was inhibited. Simulating microgravity by RWV exhibited that locomotion of PBMCs was inhibited after 24 h but addition of PMA to RWV culture restored cell motility. Dysregulated distribution of PKC isoforms Since the cytoskeleton is involved in signal transduction, microgravity-induced disorganization of cytoskeletal structures could lead to disturbed localization of signaling molecules. Different protein kinase C (PKC) isoforms are associated with several cytoskeletal elements, in particular intermediate filaments and stress fibers. Upon T cell activation, the PKC isoforms normally are redistributed to distinct cellular compartments. In two experiments exposing Jurkat T cells and primary human T cells to microgravity during space flight, intracellular translocations of PKC isoforms were investigated. The results showed that the relative distribution of PKC isoforms in particular cell fractions was different from in-flight samples compared to 1g ground controls. These results were confirmed in an experiment with primary T cells in an RWV. Furthermore, mRNA as well as protein expression of specific calcium-independent PKC isoforms was inhibited in an RWV PBMC culture.
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Increased apoptosis Apoptosis may also contribute to the decreased proliferative response of lymphocytes in real microgravity. Actually, biochemical and microscopic studies revealed that the rate of apoptosis in Jurkat T lymphocytes was increased in microgravity conditions , which was reflected in time-dependent release of apoptosis-related factors like Fas/APO1 in the culture medium during exposure of approximately 2 days real microgravity aboard different space shuttle flights. Furthermore, microgravity led to increased DNA fragmentation, poly(ADP-ribose) polymerase (PARP) protein expression and p53 and calpain mRNA. These changes were paralleled by an early increase of 5-lipoxygenase (5-LOX) activity. In an experiment that authors performed during the 8th DLR (German Aerospace Center) parabolic flight campaign, authors could observe an increase of p53 phosphorylation after 20 s real microgravity. Risin and Pellis reported that radiation and activation-induced programmed cell death in T lymphocytes was inhibited under simulated microgravity conditions.
STEM Today, May 2017, No.20
Differential gene expression in microgravity The effect of microgravity on global gene expression was evaluated in cells cultured in real microgravity or in ground based simulations. Microarray analysis revealed that under microgravity conditions (ISS "Astrolab" and RPM) the expression of immediately early genes which are regulated primarily by transcription factors NF-ÎşB, CREB, ELK, AP-1 and STAT were down-regulated relative to 1g controls. The overall observed changes in gene expression induced by gravitational changes comprised a number of genes associated with cell stress response, cell proliferation and differentiation , cell cycle regulation , protein folding, DNA repair , transport and degradation, apoptosis, as well as differences in several cytoskeletal genes. These results demonstrated the broad spectrum of gene expression modulations in reduced gravity. Further experiments with primary human T cells revealed that microgravity induced epigenetic changes in DNA methylation and chromatin histone modifications. Influence of microgravity on membrane proximal T cell receptor signaling Authors investigated the impact of microgravity on key molecules of the early T cell activation signaling events. Experiments with primary human CD4+ T lymphocytes were conducted under real microgravity conditions on board of the sounding rocket MASER-12. In addition to the microgravity effects on CD4+ T lymphocytes, the influence of the hypergravity phase during the rocket launch (baseline (BL) samples) and of cultivating the cells in the experimental hardware (H/W) were investigated. Authors tested the influence of gravitational changes on key molecules involved in T cell receptor signaling of resting as well as of ConA/CD28 activated CD4+ T lymphocytes and quantified components of the T cell receptor, membrane proximal signaling events, MAPK signaling, IL-2 R, histone modifications and the cytoskeleton in non-activated as well as in ConA/CD28 activated T lymphocytes. The results are summarized in Table 3.
The hypergravity phase during the launch of MASER-12 resulted in a down-regulation of the both surface receptors IL-2R and CD3 and reduced overall intracellular tyrosine phosphorylation, p44/42-MAPK phosphorylation and histone H3 acetylation, whereas phosphorylation of the linker of activated T cells (LAT) protein
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was increased. Non-activated T cells showed a reduction of CD3 and IL-2R expression at the cell surface due to microgravity in comparison to the 1g H/W ground control. p44/42-MAPK-phosphorylation was also reduced after 6 min microgravity compared to the 1g H/W ground controls, but also in direct comparison between the in-flight microgravity samples and the 1g reference centrifuge control. In contrast, 5 min clinorotation and 20 s real microgravity led to an increase of phosphorylated p44/42 MAPK in nonactivated Jurkat T cells as well as in PMA or CD3/CD28-activated Jurkat T cells. In activated T cells, the reduced CD3 and IL-2 receptor expression after the rocket launch of MASER-12 recovered significantly under in-flight 1g conditions, but not in microgravity. Beta-tubulin increased significantly during the microgravity phase, but not when cells were re-exposed to 1g at the on-board reference centrifuge. Microgravity might not severely disturb key proteins of membrane proximal signaling in the first 6 min. Thus, it can be assumed that dysregulation of functional T cell activation occurs downstream of the T cell receptor signaling, such as at the level of gene expression regulation.
Findings
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A large number of studies on T lymphocytes were carried out in microgravity which clearly revealed, that individual cells are sensitive to changes of the gravitational force. The results of these experiments performed in space and in ground-based simulations contributed to the knowledge of how alterations in gravity influence basic cellular mechanisms. The influence of microgravity on T lymphocyte function was reflected in a variety of phenomenological cellular responses that can be grouped into different categories (Table 4).
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Microgravity was the causative factor for impaired T cell activation during spaceflight by inhibiting transactivation of key immediate early genes
The LEUKIN spaceflight experiment reported here determined the global gene expression pattern of human T cells after 1.5 h of stimulation by Con A and anti-CD28 to identify the immediate early genes whose transcription may be inhibited in microgravity. Important to the experimental design was the KUBIK facility aboard the ISS that provided simultaneously static microgravity positions and onboard 1g centrifuge positions. This approach avoided potential variability introduced by vibrational launch forces, temperature differences, and cosmic radiation that may confound the interpretation of biological differences between spaceflight and ground-control samples. Analysis of the microarray data characterized the earliest transcriptional events inhibited in microgravity and identified the upstream mediators affected. Detailed examination of the 47 genes most significantly down-regulated in microgravity after 1.5 h of activation demonstrated inhibition of key IER genes, as opposed to the secondary response genes determined at 4 h of activation (e.g., IL-2). Promoter analysis revealed that
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the Rel/NF- κB pathway was the principal transcription pathway inhibited in very early T cell activation in microgravity. Finally, pathway analysis of the down-regulated genes predicted that downstream TNF effector functions would be severely disrupted in microgravity.
Results
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T cells activated in 1g and µg have distinct gene expression patterns T cells were purified from four human volunteers and launched into orbit on board the Soyuz 13S rocket from Baikonur, Kazakhstan. To control for experimental variables, samples stimulated with Con A and anti-CD28 were located inside of the KUBIK facility on the static positions (µg) or on the centrifuge positions. The centrifuge was set up to apply a 1g force, as the earth’s gravity control, on the samples. In this way, all samples experienced the same launch forces, temperature variations, culture conditions, and cosmic radiation, and the only difference was the presence or absence of 1g gravitational force during the experiment. After 1.5 h of activation, cells were fixed in RNAlater while in orbit. Upon return to earth, the preserved RNA samples were analyzed with the Affymetrix Human U133 microarray gene chip. Data were normalized by the GC-RMA algorithm, and genes with statistically significant differential expression among the conditions of µg-nonactivated, µg-activated, and 1g-activated were identified by ANOVA analysis.
A total of 617 differentially expressed genes was identified (Fig. 1). Nonactivated T cells from the three donors demonstrated distinct gene expression patterns reflecting the environmental and genetic backgrounds of the individual donor. µg-Nonactivated samples from each donor constituted a separate individual branch in sample clustering analysis (horizontal clustering tree, yellow columns). Upon activation with Con A and anti-CD28
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in 1g, the gene expression patterns of the three donors became significantly more uniform and clustered together (blue columns). This indicated that, as expected, common pathways and genes were being induced in T cells of the three donors upon activation in 1g. Importantly, samples from the same three donors activated in µg clustered together separately from 1g-activated samples (red columns). The clustered µg-activated samples demonstrated statistically significant differences from 1g-activated samples in overall gene expression pattern. Common pathways and genes of T cell activation were inhibited or suppressed in µg compared with 1g in all three donors. Taken together, these results are powerful evidence that there are fundamental alterations in early T cell activation events in reduced gravity conditions.
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More detailed analysis showed that the differentially expressed genes may be segregated into six distinct groups by gene expression clustering (Fig. 1, vertical axis clustering tree). Group A consisted of 72 genes that demonstrated increased expression in µg and 1g-activated T cells compared with nonactivated T cells, and expression in 1g T cells was significantly higher than µg. Group B had 315 genes significantly induced in µg- and 1g-activated T cells compared with nonactivated, with no difference in level of expression between activated µg and 1g. Group C showed 77 genes in which expression was several-fold greater in 1g- compared with µg-activated T cells. Group D included six genes in which expression was increased in µg-activated T cells but not 1g-activated T cells. Group E consisted of 127 genes with significantly decreased expression in µg- and 1g-activated T cells compared with nonactivated and no difference between activated µg and 1g. Finally, Group F included 20 genes in which expression was slightly decreased in µg-activated samples but even more significantly decreased in 1gactivated samples compared with nonactivated µg samples. Overall, 72% (315 genes in Group B and 127 genes in Group E) of the differentially regulated genes in T cell activation was regulated similarly between T cells activated in µg and 1g. Many more genes were up-regulated after T cell activation in µg and 1g (75%; Groups A-C) than were down-regulated (24%; Groups E and F) compared with nonactivated T cells. Expression of 12% of the genes (Group A; 72 genes) was induced upon µg activation but significantly lower than T cells activated in 1g. This group may represent genes in pathways that were relatively inhibited in µg in a gradation of response. Another 12% of the genes (Group C; 77 genes) demonstrated expression that was minimally induced or not induced at all in µg-activated T cells compared with the highly up-regulated expression levels in 1g-activated T cells. These genes likely participated in pathways that were inhibited even more profoundly in the µg environment. T cell proliferation and differentiation genes are inhibited in T cells activated in µg To determine the genes most significantly modulated by the presence or absence of 1g during T cell activation, post hoc Tukey analysis was performed on the 617 genes that were identified by ANOVA analysis. Differentially regulated genes were further filtered for two-fold or greater expression difference between µg and 1g-activated samples. This resulted in 47 genes for which expression was two-fold or higher in 1g-activated compared with µg activated samples. There were no genes for which expression was two-fold or higher in µg compared with 1g. As presented in Table 1, the 47 genes are annotated through a combination of Gene Ontogeny Analysis, Online Mendelian Inheritance in Man, Uniprot, Entrez, and PubMed searches. The genes are broadly classified by biologic process, which includes apoptosis, antiapoptosis, cell cycle regulation, cytokine/chemokine, cell surface molecule, signal transduction, transcription factor/regulator, and unknown. Expression is presented as the ratio of 1g/µg. Therefore, the higher the fold difference, the more profoundly the expression of the gene is inhibited in µg during T cell activation. A substantial proportion of the genes most significantly inhibited in µg plays important roles in signal transduction and transcription regulation. Ten of the 47 genes (21%) encode members of signal transduction pathways, including the cRel pathway, small G-protein signaling, as well as phosphatases and inhibitors. Twenty-one of the 47 genes (45%) encode transcription factors or regulators, many of which are inducers of mitogenesis (e.g., EGRs, MYC, and REL). Other transcription factors significantly inhibited in µg are involved in Th1 (e.g., REL) or Th2 differentiation (e.g., FOSL2 and IRF4). These data from spaceflight indicate that gravity modulates the expression of genes that promote lymphocyte proliferation and inflammation, as well as those that may have a down-regulatory role. CD83 and CD69, both early surface markers of T cell activation, are expressed more than two-fold lower in µg compared with 1g (Table 1), providing further evidence that T cell activation is significantly inhibited in µg. Whereas antiapoptotic genes IER3, BCL2A1, and SERPINB9 are inhibited in µg, GRAMD4, an effector of apoptosis, is also inhibited. BTG2, which inhibits G1/S transition, is inhibited fivefold in µg; on the other hand, SERTAD1 promotes G1/S transition and is inhibited two-fold. Overall, taking into account the fold-expression differences, lymphocyte mitogenic and inflammatory responses appear to be inhibited most profoundly in µg. TNF, a critical proinflammatory cytokine, is inhibited 15-fold in µg, and CCL3, a chemokine important for neutrophil recruitment, is inhibited sixfold. EGR1, -2, and -3 are all
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inhibited more than tenfold in µg; NAB2, a repressor of EGR1 and EGR2, is inhibited two-fold. Several other transcription factors important for mitogenesis are significantly inhibited in µg, including MYC (4.62-fold), FOSL2 (4.38-fold), and REL (2.75-fold). JUNB, a transcription factor with antiproliferative properties, is inhibited 3.16-fold.
Authors selectively performed qRT-PCR on several genes to verify their differential expression in µg versus 1g. T cells that were µg nonactivated, µg-activated, or 1g-activated during spaceflight from all four human volunteers were tested for quantitative expression of MYC, IRF4, TNF, NFKBIA (IκBα), NR4A3, and TAGAP (Fig. 3). Each data point represents four biologically independent samples stimulated by Con A and anti-CD28
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during spaceflight in µg or 1g control. Fold expression was normalized to the housekeeping gene CPHI and then to µg-nonactivated values. Comparing µg-activated with 1g-activated samples, expression of these genes were all decreased significantly in µg-activated samples, as determined by qRT-PCR. These results, along with the microarray analysis, suggest that lymphocyte mitogenesis (as represented by MYC expression), cytokine production (IRF4), proinflammatory response (TNF), and the REL signaling pathway (NFKBIA) are decreased
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significantly in T cells activated in the absence of gravity. In addition, authors identified two novel, gravityresponsive genes that play a role in T cell activation. NR4A3 is a steroid hormone receptor family transcription factor involved in mediating the inflammatory response. TAGAP is a T cell activationspecific GTPase-activating protein genetically linked to inflammatory diseases, such as rheumatoid arthritis, Crohn’s disease, and celiac disease. NR4A3 and TAGAP represent novel pathways that are inhibited significantly in µg during T cell activation, as their roles were not identified by previous experiments in reduced gravity.
Transcription of immediate early genes are inhibited in T cells upon CD3/CD28 cross-linking in simulated µg To further investigate the effects of µg on early T cell activation signal transduction, authors determined whether
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expression of immediate early genes was inhibited in purified CD4+ T cells stimulated by CD3/CD28 crosslinking in simulated µg. CD4+ T cells were purified from the peripheral blood of four human volunteers. Simulated µg was created by RWVs that produced freefall conditions for cultured lymphocytes through rotation along a horizontal axis. Purified CD4+ T cells were activated in stationary culture vessels as 1g controls. After 1.5 h of incubation with CD3/CD28 activation beads, the CD4+ T cells were lysed for RNA isolation. Expression levels of immediate early genes, previously shown to be inhibited in µg in the spaceflight microarray experiment, were determined by qRT-PCR in nonactivated, activated-simulated µg, and activated 1g cultures. Expression of cREL, TNF, EGR1, EGR2, and JUNB was reduced significantly in simulated µg compared with 1g controls (Fig. 4). Expression level of cREL in activated simulated µg cultures was the same as nonactivated T cells and sixfold less than activated 1g cultures. Expression levels of TNF, EGR1, EGR2, and JUNB in activated-simulated µg T cells increased from nonactived T cells but were all significantly lower than 1g-activated T cells. Compared with 1g-activated T cells, expression of TNF was 3.1-fold less, EGR1 was 4.5-fold less, EGR2 was 2.9-fold less, and JUNB was 2.1-fold less in simulated µg-activated T cells. These immediate early genes are known to be transcribed within 30 min to 1 h of T cell activation and do not require de novo protein synthesis. The markedly reduced expression of these genes underscores that early signal transduction events are inhibited in CD4+ T cells activated in simulated µg.
Rel/NF-κB signaling is a key pathway inhibited in µg To identify the signaling pathways that were inhibited by µg during early T cell activation, authors performed promoter region analysis on the 47 genes that showed significant down-regulated expression after 1.5 h of activation in the spaceflight experiment (Table 1). The 47 genes were analyzed for statistically significant overrepresentation of TFBSs using the oPOSSOM algorithm. Remarkably, three of the top five over-represented TFBSs were in the Rel/NF-κB family (Table 2), indicating it to be a major pathway inhibited by µg in T cell activation. Of note, 20 of the 47 analyzed genes (43%) had cRel-binding sites in their promoter regions, making cRel the most highly represented promoter site in genes with down-regulated expression in µg. The overrepresentation of cRel sites is highly statistically significant with a Z-score of 25.2 and a Fisher score of 4.4 X 10−9 Likewise, the over-representation of RelA (p65) and NF-κB-1 (p50)-binding sites was also highly statistically significant with Z-scores of 27.3. and 23.4 and Fisher scores on the order of 10−8 and 10−6 , respectively. As Rel/NF-κB transcription factors positively regulate their own transcription , these data suggest that transcription of downstream effectors of the Rel/NF-κB pathway would be reduced greatly in T cells activated in µg. Indeed, expression of cREL itself is significantly inhibited in T cells activated in µg , as demonstrated by the spaceflight microarray data (Table 1) and CD3/CD28 bead activation experiment in simulated µg (Fig. 4). In addition to members of the Rel/NF-κB family, CREB1 and SRF-binding sites were highly represented in the promoter of genes inhibited in T cells activated in µg. CREB1 is a transcription factor that can be activated by cAMP/PKA, calcium/ calmodulin, and/or Ras/MAPK pathways. Authors previous work showed that phosphorylation of CREB1 was inhibited in µg at 4 h after T cell activation and was dependent on PKA activity. SRF is the major promoter response element of immediate early genes and an important target for the Ras/MAPK and Rho/actin pathways. Together, these data suggest that in early T cell activation, the Rel/NF-κB pathway may be the predominant pathway dysregulated in µg. Other signal transduction pathways, including cAMP/PKA, Ras/MAPK, and calcium/calmodulin, may also be affected. In contrast to the promoter analysis of genes downregulated in µg, no TFBSs for any particular family of transcription factors were over-represented in the 315 genes that were activated equally in µg and 1g (Group C in Fig. 1; data not shown). Pathway analysis predicts that TNF downstream signaling would be inhibited significantly in µg In addition to identifying the early gravity-responsive signals downstream of CD3 and CD28 engagement, authors performed a pathway analysis on the 47 genes down-regulated most significantly in µg to predict the T cell effector functions that are likely to be dysregulated most severely later in the response. Pathway analysis was performed with the GeneSpring GX 11.0.2 software, in which molecular interactions were curated from
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databases, such as IntAct and Natural Language Processing algorithms of published literature. Authors generated a pathway diagram of direct interactions between the molecules in the list of 47 genes from Table 1 and set the relation score to greater than or equal to nine on a scale of one to 10, to include only interactions that have the highest degree of support (Fig. 5).
Types of relations included binding, expression, member, metabolism, promoter binding, protein modification, regulation, and transport. Twentytwo genes were captured as part of this pathway in which TNF was the central element. These genes are also highlighted in Table 1 with bold type and outlines. The remaining 25 genes were not linked to TNF signals and were excluded by the analysis. The pathway diagram localized the molecules to the cell membrane, cytoplasm, or nucleus. The molecules of the input gene list involved in direct interactions with each other are denoted by ovals. MAPK (purple symbol) was not part of the input list, but its involvement in the pathway was strongly suggested by the other genes involved. Yellow circles represent signaling intermediaries not identified within the input gene list. The black lines connecting the molecules indicate various types of interactions with the direction of the arrowhead indicating a positive association or regulation. Black lines without arrowheads indicate a neutral association or participant. Blue lines indicate a negative association or regulation. The generated pathway diagram predicts that TNF is a key gravity-dependent effector function that would
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be inhibited in µg. TNF transcription is normally induced rapidly in T cells activated by CD3/CD28 cross-linking , and it is markedly reduced in T cells activated in µg (Fig. 4). In addition, the pathway analysis demonstrates that TNF downstream signals and interactions involve many cytokines, surface molecules, signaling molecules, and transcription factors that are also inhibited in µg. As TNF is an important proinflammatory effector cytokine, this analysis suggests that downstream inflammatory responses may be lowered significantly in µg.
Findings Data presented here prove that it is the µg environment of spaceflight that leads to impaired T cell activation and that transcription of immediate early genes is profoundly down-regulated. The transcription of immediate early genes is inhibited in T cells activated in µg and that disrupted activation of Rel/NF-κB, CREB1, and SRF transcription factors is involved.
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Spaceflight alters expression of microRNA during T-cell activation
MicroRNAs (miRNAs) were first discovered in 1993 in Caenorhabditis elegans when miRNA lin-4 was seen to downregulate expression of the gene lin-14; however, there were no homologs to lin-4 in other species. The later discovery of miRNAs in other species, including humans, showed that miRNAs are common in eukaryotes. Previous studies have shown that key miRNAs are up-regulated after activation of human T cells. Here authors present their discovery of dysregulation of miRNA gene expression by gravity during early T-cell activation in spaceflight. Authors recently proved that the lack of immune response inmicrogravity occurs at the cellular level and identified promoter regions, transcription factors, and signal transduction pathways regulating human Tcell gene activation under normal and altered gravity conditions.
Results
miR-21 is dysregulated in true ISS microgravity during T-cell activation Using bioinformatics, authors analyzed the gene expression T-cell activation of 3 individual human donors under normal gravity on board, and microgravity conditions that were incubated, activated, and fixed on the ISS. In initial bioinformatics analysis, authors found that there was 1 miRNA that had reduced gene expression in true microgravity; in normal gravity, miR-21 was increased 2-fold, while in microgravity miR-21 was not significantly changed compared to onboard normal gravity controls (n = 4). This gene array analysis was confirmed by mirVana qRT-PCR. Expression was corrected to internal standard 5SrRNA. A small amount of mRNA from 0.5g onboard samples (n = 4) was available for analysis. With a 0.5g fractional gravity, the expression of miR-21 was increased over the microgravity sample. Analysis of mRNA gene arrays for significantly dysregulated microgravity genes Global microarray analysis revealed significant suppression of 85 genesundermicrogravity conditions compared to gene up-regulation in onboard normal gravity-activated samples. Authors analyzed the altered gene expression of activated T cells in microgravity using software showing either proven miR-21 interaction (TarBase), predicted seed sequences (TargetScan), or both. Table 1 provides an abbreviated list of 13 key target genes that significantly changed gene expression within 1.5 h after activation in spaceflight. Of the 85 gravity-sensitive genes, 17 were defined as miR-21 targets. Of these, 13 were immune-related miR-21 target genes, including EGR3, CD69, FASLG, SPRY1, BTG2, SPRY2, and TAGAP. All the immune-related targets were biologically confirmed. Authors further confirmed via gene array analysis using qRT-PCR that FASLG, SPRY2, BTG2, and TAGAP gene expression was gravity sensitive. Heat map of gene expression Seventeen significant gene targets of miR-21 from flown individual donors (n = 3) were differentially regulated after 1.5 h of activation under normal gravity and microgravity conditions on board the ISS (P ≤ 0.05) (Fig. 1). Samples were incubated and activated in the same incubator and fixed on board the ISS. In some cases, the gene array contained multiple gene probe sets targeted to different regions of the gene. Here authors saw multiple probe sets for FASLG, TAGAP, and EGR1 showing the same trends of significant inhibition of gene expression
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under different gravities. The activation profile in the normal gravity-activated samples is fairly uniform across donors, indicating that common genes and pathways were stimulated in all 3 donors after activation with concanavalin A/anti-CD28. Gene expression in the microgravity flown samples was lower and less uniform than the activated normal gravity samples. In some cases, the microgravity profile was essentially the same as the nonactivated samples, indicating suppression of gene expression in all 3 donors. The 3 conditions had distinct profiles across the 17 predicted miR-21 gene targets, demonstrating clear differences in early activation gene expression between the onboard normal gravity-activated, microgravity-activated, and nontreated controls.
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qRT-PCR of immediate early genes in nonactivated, normal gravity onboard activated, and onboard microgravity-activated T cells with 3’-UTR miR-21 seed sequences Authors used qRT-PCR to analyze miR-21 targets BTG2, TAGAP, SPRY2, and FASLG in these ISS samples that were activated and fixed in orbit. These genes were selected because they contained the seed sequence in the 3’-UTR region. In addition, 4 genes (BTG2, TAGAP, SPRY2, and FASLG) had previously been confirmed to have interaction with miR-21. Authors also analyzed nuclear factor of κ light polypeptide gene enhancer in B-cell inhibitor α (NFKBIA) and CD40 ligand (CD40LG), 2 genes that are key in early T-cell activation. All 6 genes exhibit gravity-sensitive gene expression. It is notable that many of the miR-21 targets are co-up-regulated with miR-21 during the early hours of T-cell activation.
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Predictions of transcription factor locations in promoter regions of gravity-sensitive genes Authors used bioinformatics software, the Match program of TRANSFAC Pro, oPOSSUM, target genes to predict transcription factor locations in the promoter regions of gravity-sensitive mRNA. Key immune genes share common transcription factors with miR-21, showing that at the initiation of activation, both the positive immune genes share increased gene expression with the miR-21 precursor pre miR-21 (Fig. 2).
Authors show for the first time an altered profile of miRNA expression under true microgravity compared to its onboard normal gravity controls; all samples were isolated from donors on earth, incubated, activated, and fixed in ISS on-orbit operations. The miR-21 differential expression seen in the global gene analysis was confirmed by mirVana qRT-PCR (Fig. 3). Of interest, samples from onboard 0.5g samples demonstrated that fractional gravity is capable of restoring gene expression of miR-21. Global gene analysis of the ISS samples revealed altered gene expression in spaceflight vs. normal gravity onboard controls 1.5 h after activation, showing changes in 85 genes associated with T-cell activation. Of the 85 differentially expressed genes, 17 were computationally predicted or experimentally verified as miR-21 target genes. Table 1 provides an abbreviated list of 13 T-cell-relevant targets that are predicted miR-21 targets from
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the miR-21 seed sequence (UAGCUUAU) in the gene by TargetScan. Of the 17 predicted genes, EGR1, FASLG, SPRY2, BTG2, REL, and MYC have been biologically confirmed by others as true targets as described in the TarBase database. Using qRT-PCR, authors confirmed that microgravity-induced down-regulation of 4 of the miR-21 target genes, BTG2, TAGAP, SPRY2, and FASLG, as well as immune regulators NFKBIA and CD40LG genes (Fig. 4). Authors chose BTG2, TAGAP, and SPRY2 for qRT-PCR analysis because they had been previously shown to biologically interact with miR-21. BTG2 is a known factor in T-cell activation and supports proliferation in immune cells . Other genes important in immune function, such as NFKBIA and CD40LG, were selected because of their importance in early T-cell activation. CD40LG is a marker of T-cell activation, and here authors found that as early as 1.5 h of activation, it is induced in T cells that were normal gravity activated and was inhibited in microgravity activated cells. NF-κB-1A binds to REL, RELA, or RELB to formthe NF-κB complex. NF-κB-1A is associated with cells that express chemokines or cytokines. TAGAP is a member of the rho GTPase activator protein superfamily, and TAGAP loci have been associated with immune diseases such as rheumatoid arthritis and multiple sclerosis. SPRY2 protein down-regulation by miR-21 inhibits proliferation, and when SPRY2 is overexpressed, it can increase proliferation of HEK293T cells. Binding of FASLG(TNFSF6) to FAS results in cell apoptosis and cell death. FAS ligand is known to be expressed in activated splenocytes and may be part of an innate mechanism to limit the duration of cell activation.
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Transcriptomic changes occurring in the whole blood of astronauts in response to spaceflight
To evaluate the potential impact of the spaceflight environment on molecular pathways mediating cellular stress responses, authors performed a first-of-its-kind pilot study to assess spaceflight-related changes in the expression of stress-response genes in the whole blood of astronauts in response to spaceflight. Transcriptional profiling of 234 well-characterized stressresponse genes was performed using total RNA isolated from whole blood obtained from six consenting astronauts 10 days before launch aboard the space shuttle and 2-3 h after return to Earth.
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Four men between the ages of 38 and 47 (mean = 43 years) and two women between the ages of 38 and 44 (mean = 41 years) participated in the study. Each of these astronauts flew aboard one of four space shuttle missions ranging between 10 and 13 days duration that took place during a 2-year period. Previously published studies using blood and saliva samples from these same crew members before, during and just after spaceflight indicated that all six astronauts displayed increases in Epstein Barr virus reactivation both during and immediately post flight, which is consistent with these individuals being in a stressed state. Microarray analysis revealed some variation in the gene expression patterns displayed across individual crew members. This is not surprising given the smaller study size due to the exceptionally rare opportunity for this type of sample collection, as well as other studies that have shown that there can be a wide range of individual and temporal variability in gene-expression patterns in human blood. High variability was also observed in gene-expression studies using astronaut hair follicles. Authors found six transcripts that displayed significant (P<0.05) changes in expression in the crew in response to spaceflight when compared with pre-flight levels (Table 1). Two additional differentially regulated transcripts were identified following outlier removal. No gender-specific differences or relationship to number of missions flown were observed. It is important to note that as for many human spaceflight studies, the small number of human subjects available for analysis is a limiting factor to the statistical power. False discovery rate algorithms determined no significant genes were expressed, likely due to these low study group numbers.
The NASA Twins Study with astronauts Scott Kelly and Mark Kelly performed during the One-Year Mission may face similar challenges in their gene-expression data interpretation, as there is an n of 1 in each condition (spaceflight and ground). However, one advantage in the Twins Study over the present one is the longitudinal sample collection that could be performed during the year that Kelly spent in space. It will be intriguing to compare the results from gene-expression analyses between the two studies. Genes altered in expression encode proteins of known importance for DNA repair (XRCC1 and HHR23A), oxidative stress (GPX1), and chaperones which have key roles in protein folding and/or proteasomal degradation (HSP27 and HSP90AB1).
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The finding that genes encoding DNA-repair proteins are down regulated supports previous studies which showed post-flight increases in chromosomal aberrations in lymphocytes of astronauts and indications of increased DNA damage in astronauts after long-duration spaceflight. Exposure of human lymphocytes to simulated microgravity over the course of 7 days led to increased DNA damage. This damage was accompanied by progressive decreases in the expression of a number of representative DNA-repair genes, leading the authors to postulate that impaired DNA-repair capacity could lead to increased damage and mutations. In a separate study, it was reported that 1 week of hindlimb unloading in male BALB/c mice led to the downregulation of multiple DNA-repair genes including ERCC1, ERCC3, ERCC5, ERCC6, and XPC in the testes of BALB/c mice. Of particular interest was the downregulation of GPX1, which encodes for glutathione peroxidase (GPX1), an enzyme that protects cells from oxidative damage, modulates the immune response (including delayed type hypersensitivity/DTH), and has been associated with increased viral titers in herpes viral infections. Given that spaceflight depresses the DTH response, alters cellular oxidative functions, and increases herpes viral reactivation in astronauts on orbit, additional detailed studies related to glutathione and oxidative stress may fundamentally advance our understanding of mechanisms underlying risks to crew health during a mission.
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Several previous studies have indicated the potential for alterations in oxidative stress and antioxidant defense status in response to spaceflight and spaceflight-analog models in humans and animals. Although the reported magnitude and direction of the expression/activity levels of specific redox enzymes (including GPX1) were variable across these studies (likely due to differences in experimental parameters such as the use of human versus animal subjects, mission duration, etc) the overall findings support the potential for increased oxidative stress and/or decreased antioxidant defense capacity in response to spaceflight. It is also interesting to note that GPX1 is a selenoprotein and decreased serum selenium levels have been reported in astronauts post landing as compared with pre-launch. Therefore, additional insight into GPX1 and other oxidative defense mechanisms may serve as a guiding principle for establishing nutritional requirements to ensure health safety and performance of the crew during human exploration of space. When authors compared these transcriptomic findings from blood samples to a previously published study using hair samples, authors did not find similarities in the genes that were differentially regulated pre- and post flight, which could be due to a variety of experimental differences, including sample source (whole blood versus hair follicles) and mission duration (10-13 days in the present study compared with â&#x2C6;ź 6 months in the study by Terada et al). This study represents the first report of transcriptomic changes occurring in the blood of astronauts in response to spaceflight. Several transcripts encoding stress response genes were suppressed in the crew after exposure to the microgravity environment, including those important for DNA repair, oxidative stress, detoxification, and protein folding/degradation. These processes are vital for maintaining human health by mediating cellular pathways that serve to protect against both environmental and physiological stressors and have been implicated in a broad spectrum of diseases. Since changes in gene expression in peripheral blood may be attributed to a number of factors, including changes in the distribution of blood cellular subsets (which has been detected post flight in the crew), future studies designed to establish direct links between specific physiological/cellular adaptations underlying these transcriptomic changes in response to the microgravity environment will be important. It is important to note that no direct correlation can be made between gene-expression changes observed in this study and the disease status or potential for the development of disease in these individuals. This study provides an initial foundation into the molecular genetic response profiles of astronauts during spaceflight from which additional research into alterations in crew health and performance can be investigated. This kind of research could lead to human health countermeasures to mitigate risk during the transition from short-to-longduration flight and could potentially translate to health benefits for the general public.
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Martinez EM, Yoshida MC, Candelario TL, Hughes-Fulford M. Spaceflight and simulated microgravity cause a significant reduction of key gene expression in early T-cell activation. Am J Physiol Regul Integr Comp Physiol. 2015 Mar 15;308(6):R480-8.
Hughes-Fulford M, Chang TT, Martinez EM, Li CF. Spaceflight alters expression of microRNA during T-cell activation. FASEB J. 2015 Dec;29(12):4893-900.
Chang TT, Walther I, Li CF, Boonyaratanakornkit J, Galleri G, Meloni MA, Pippia P, Cogoli A, Hughes-Fulford M. The Rel/NF-κB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity. J Leukoc Biol. 2012 Dec;92(6):1133-45.
Hughes-Fulford M, Chang TT, Martinez EM, Li CF. Spaceflight alters expression of microRNA during T-cell activation. FASEB J. 2015 Dec;29(12):4893-900.
Jennifer Barrila, C Mark Ott, Carly LeBlanc, Satish K Mehta, Aurélie Crabbé, Phillip Stafford, Duane L Pierson & Cheryl A Nickerson , Spaceflight modulates gene expression in the whole blood of astronauts, npj Microgravity 2, Article number: 16039 (2016).
Terada, M. et al. Effects of a closed space environment on gene expression in hair follicles of astronauts in the International Space Station. PloS One 11, e0150801 (2016).
Swantje Hauschild, Svantje Tauber, Beatrice Lauber, Cora S. Thiel, Liliana E. Layer, Oliver Ullrich, T cell ˝ The current knowledge from in vitro experiments conducted in space, parabolic regulation in microgravity U flights and ground-based facilities, Acta Astronautica, Volume 104, Issue 1, November 2014, Pages 365-377, ISSN 0094-5765.
Cora S. Thiel, Beatrice A. Lauber, Jennifer Polzer, Oliver Ullrich, Time course of cellular and molecular regulation in the immune system in altered gravity: Progressive damage or adaptation ?, REACH - Reviews in Human Space Exploration, Volume 5, March 2017, Pages 22-32, ISSN 2352-3093.