STEM Today

STEM TODAY June 2017, No.21

STEM TODAY June 2017 , No.21

CONTENTS IM1: We do not know to what extent spaceflight alters various aspects of human immunity during spaceflight missions up to 6 months.

Editorial Editor: Mr. Abhishek Kumar Sinha Editor / Technical Advisor: Mr. Martin Cabaniss

STEM Today, June 2017, No.21

Cover Page Space Station’s View of Florida at Night This nighttime photo of Florida was taken from the International Space Station by Expedition 51 Flight Engineer Thomas Pesquet of the European Space Agency, in March 2017. Bright lights of cities stand out, including the Miami-Fort Lauderdale metropolitan area, the Tampa Bay region along the Gulf Coast, and in the middle, Orlando.

Visible on Florida’s Atlantic coast is the Cape Canaveral area where, currently, launch preparations are underway at NASA’s Kennedy Space Center for the eleventh SpaceX cargo resupply mission to the International Space Station, targeted for liftoff at 5:55 p.m. EDT on Thursday, June 1, from Launch Complex 39A. Image Credit: ESA/NASA

Back Cover Lake Powell and Grand Staircase-Escalante This panorama, photographed by an astronaut aboard the International Space Station, shows nearly the full length of Lake Powell, the reservoir on the Colorado River in southern Utah and northern Arizona. Note that the ISS was north of the lake at the time, so in this view south is at the top left of the image. Astronaut photograph ISS048-E-73279 was acquired on September 6, 2016, with a Nikon D4 digital camera using an 800 millimeter lens, and is provided by the ISS Crew Earth Observations Facility and the Earth Science and Remote Sensing Unit, Johnson Space Center. The image was taken by a member of the Expedition 48 crew. The image has been cropped and enhanced to improve contrast, and lens artifacts have been removed.

Image Credit: NASA Caption: M. Justin Wilkinson, Texas State University, and Andi Hollier, Hx5, Jacobs Contract at NASA-JSC

STEM Today , June 2017

Editorial Dear Reader

STEM Today, June 2017, No.21

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

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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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Effect of Space Flight on Immunological Signal Transduction

Experimentation with crew members of space flights showed reduced anti-viral immunity and a landmark experiment conducted in Spacelab 1 in 1983 demonstrated a near total absence of activation of peripheral blood leukocytes upon exposure to concanavalin A (Con-A) when compared to identical ground controls. The mechanistic basis of this effect however, remains largely obscure. A recent study employing DNA array analysis of Con-A and anti-CD28-stimulated T cells, on board of the international space station, showed that microgravityexposed T cells were deficient in transcription of immediate early genes associated with the tumour necrosis factor alpha pathway. However, T cells stimulated in an on-board centrifuge, artificially generating a 1 g condition, did not show this effect. And thus the effects of spaceflight on the immune system are related to microgravity and are indeed not just a mere artefact resulting from stress. The molecular pathways mediating these effects remain to be elucidated, but their knowledge could provide important clues on how to device better therapies for conditions associated with exaggerated activation of the immune system on earth, e.g. autoimmune disease or leukaemia.

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To identify the interaction of the immune system with spaceflight and microgravity in particular, authors decided to investigate the effect of stimulation of primary isolated human peripheral blood monocytes with LPS in 0g versus 1g conditions during space flight. LPS is a relatively invariant surface structure of gram negative bacteria. Its recognition by TLR-4 forms an important part of the innate immune response and provokes highly reproducible responses and signal transduction in peripheral blood monocytes, which have been characterized in great detail.

Results Activation of p38 map kinase is not affected by space flight associated microgravity It is conceivable that activation of p38 MAPK is impaired during microgravity. As expected, the onboard 1g control displayed p38 MAP kinase activation (Figure 2). In contrast, activation of p38 MAP kinase was not impaired in microgravity-exposed monocytes, if anything p38 MAP kinase activation appears to be increased. Thereby

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suggesting that space flight-associated microgravity does not interfere with LPS-induced pro-inflammatory signal transduction per se and its effects, if present, are restricted to specific signaling pathways.

Jun-n-terminal kinase is not activated by lps during space flight associated microgravity During microgravity the capacity of LPS to stimulate Jun-N-terminal kinase activation is lost, whereas monocytes stimulated with LPS during the onboard control condition were completely Jun-N-terminal kinase activation proficient (Figure 3). Thus, microgravity evokes a dichotomy in immunological signaling, allowing p38 MAP kinase activation to proceed, but incompatible with LPS dependent Jun-N-terminal kinase activation. Although it does not involve immunologically relevant cells, the observation that the induction of c-Jun in A431 cells by epidermal growth factor is absent in microgravity, fits well with authors findings. Especially since it has been well established that Jun-N-terminal kinase is involved in the induction of c-Jun and p38 MAPK is not.

Effects of space flight-associated microgravity on erk activation The effect on Jun-N-terminal kinase, an inhibitory influence of microgravity on the capacity of LPS to provoke activation of the extracellular regulated kinase (ERK) was observed (Figure 4). This corresponds with recent findings in T cells that show a minor effect of microgravity on ERK phosphorylation in unstimulated T cells.

Thus, the effect of microgravity is not solely restricted to Jun-N-terminal kinase. Diminished activity of the immune system is among the foremost effects of space flight on the human body. Importantly, this diminished activity of the immune system is well-tolerated. Over forty years of experience with manned space flight have now conclusively demonstrated that man can easily survive and work in weightless conditions, despite chronic repression of the body’s immune functions.

The observed dichotomy between p38 MAP kinase and Jun-N-terminal kinase is consistent with these observations: p38 MAP kinase is important for fighting immediate bacterial threats and thus its inhibition causes undesirable side effects in these and other respects. On the other hand, Jun-N-terminal kinase is more associated with chronic inflammatory responses whose inhibition represents a clear and for now unresolved clinical need.

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Phenotypic and Functional Immune System Alterations in Astronauts participating in 6-month Orbital Spaceflight

It remains unknown, that the phenomenon observed during short-duration flights would persist during longduration spaceflight or resolve, having merely been owing to the stressors of launch and early adaptation to spaceflight. In a very limited number of subjects, it was reported that cell-mediated immunity was depressed during flights on board the Russian Mir Space Station.

In this study authors performed an integrated assessment of immune function, cytokine profiles, and viral immunity during flight on board the ISS. 23 ISS astronauts participated in this study. To determine sample size, existing subject incidence rates for detectable immune system changes were used for power calculations (based on previous spaceflight experiments). Sixteen of the subjects flew on the Russian ’Soyuz’ vehicle, and mission durations were nearly 6 months. The remaining seven subjects flew on the US Space Shuttle. For the Shuttle-ISS astronauts, five had mission durations of >100 days, still considered ’long-duration’ spaceflight. The remaining two astronauts had mission durations of < 60 days. Of the 23 subjects, 18 were male and 5 were female, and their mean age was 53 years. Blood samples were collected at three intervals during flight, always shortly before undock of a visiting vehicle, and were immediately returned (ambient) for analysis.

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Results Peripheral leukocyte distribution

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Among the bulk leukocyte subsets (WBC, differential, lymphocyte subsets), significant differences were observed during spaceflight. Relative to L180 baseline data, both the absolute WBC and granulocyte levels increased during flight, whereas levels of lymphocytes and monocytes were unaltered during flight (Figure 1a). Levels of T cells, CD4 and CD8 subsets, and memory (CD45RO+ ) T cells were also unaltered during spaceflight (Figure 1b, c). The level of NK cells was elevated at the late in-flight timepoint only, whereas B-cell levels were unaltered during flight (Figure 1b).

In-flight alterations were observed among specific subsets of CD8+ T cells. The absolute level of active cytotoxic (CD28+ /CD244+ ) CD8+ T cells was significantly increased only at the early in-flight time point, whereas the corresponding late senescent (CD28− /CD244+ ) T-cell population were significantly decreased during flight at the mid-mission timepoint (Figure 1d). For both subsets, nonsignificant in-flight data time points had mean data trending in a similar manner, and the mean levels of undifferentiated (true naive) CD8+ T cells (CD28+ /CD244− ) trended downward through all in-flight data time points (Figure 1d).

Central memory-effector T-cell subsets were also evaluated; however, because of large standard errors, significant in-flight differences were observed only for the central memory subset of cells (CD62L− /CD45RA+ ), which decreased early in-flight but trended similarly throughout flight (Figure 1e). Levels of early senescent (CD57+ ) T-cell subsets (both CD4+ and CD8+ ) trended negatively during flight; however, the decrease reached significance for only the CD4+ /CD57+ subset immediately after landing (Figure 1f).

Interestingly, among constitutively activated T-cell subsets disparity was observed between early-activated ( CD69+ ) and late-activated ( HLA-DR+ ) subsets. For both CD4 and CD8+ T-cell subsets, early-activated T cells trended to be elevated, but reached significance only at R+0 for the CD8+ subset (Figure 1g). In contrast, levels of late-activated T cells (both subsets) trended downward from baseline during flight, reaching significance for the CD8 population at the mid-mission timepoint and for the CD4 population at R+0 (Figure 1h). Levels of T cells specific for viral peptides from EBV and CMV were unaltered during flight (Figure 1i).

Intracellular cytokine profiles After 6 h of stimulation in the presence of PMA+ionomycin and monensin as described, the percentage of T-cell subsets capable of being stimulated to produce cytokine was assessed. The percentage of CD8+ T cells capable of being stimulated to produce IFNγ was unchanged during spaceflight (Figure 2). The percentage of CD4+ T cells capable of being stimulated to produce IL-2 was also unchanged during spaceflight, but it was significantly reduced immediately after landing (Figure 2).

Early T-cell function After 24 h of T-cell stimulation in the presence of SEA+SEB, the percentage of CD69+ and CD69+ /CD25+ cells decreased during flight for both CD4+ and CD8+ T-cell subsets (Figure 3a). All four parameters were significantly decreased at the early timepoint. Although there seemed to be a general trend towards recovery through the mid-mission and late in-flight time points, all values remained significantly depressed except for the CD8+ subset, which recovered function by the late timepoint (Figure 3a). When T cells were stimulated directly using antibodies to CD3 and CD28, no in-flight alteration was observed among either early-activated subset (CD69+ ), but the CD4+ subset reflecting full activation (CD69+ /CD25+ ) was significantly decreased at the early timepoint only (Figure 3b).

Virus-specific T-cell function Function of both EBV and CMV-specific T cells was determined by peptide stimulation followed by intracellular determination of IFNγ. For both subsets, quantity was determined by the MHC-tetramer method. The functional percentage of both EBV and CMV peptide-specific T cells was calculated as described above. The absolute levels

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of both EBV and CMV peptide-specific T cells were not significantly altered at any phase of flight, nor post flight (Figure 4).

Mitogen-stimulated cytokine profiles Secreted cytokine profiles were determined in culture supernatants following mitogen stimulation for 48 h.

After T-cell-specific stimulation (anti-CD3 and anti-CD28 antibodies), production of IFNγ, IL-4, IL-5, IL-10, and IL-17A were all significantly reduced during flight at all three in-flight time points (Figure 5a). Following culture in the presence of PMA and ionomycin, a stronger mitogenic stimulus which bypasses some early intracellular signaling events and may activate most leukocyte populations, production of IFNγ, IL-4, IL-5, IL-10, IL-17A, TNFα, and IL-6 was significantly decreased at all three in-flight time points (Figure 5b). Following stimulation with LPS, production of both IL-1b and IL-10 was depressed at least one time point during flight, whereas production of IL-8 was actually elevated during flight at the early and mid-mission time points (Figure 5c).

Space Shuttle missions are typically extremely busy and stressful, more so than a typical work day on board ISS, where crew schedules are managed relative to the longer mission duration. Because of these conditions, short-duration data may not reflect how immunity may function during long-duration spaceflight. With limited exceptions, the status of the humanimmune system during long-duration spaceflight has remained uncharacterized. On board, the Russia Mir space station, the ’Multitest’ assay of cell-mediated immunity was performed on three long-duration subjects, and the data indicated that cell-mediated immunity was depressed during spaceflight.

In this study , authors assessed a variety of adaptive immune parameters at three points (at ∼ 2 weeks, between 2 and 4 months, and at 6 months) during long-duration orbital spaceflight. Given a lack of analytical equipment supporting immunology or hematology parameters on board ISS, the study was designed to col-

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lect samples on board ISS shortly before a vehicle undocking, so that ambient viable blood samples could be returned for terrestrial analysis. Blood collected in nutrient and anticoagulant-containing ACD tubes was validated in the laboratory to maintain viability for the relevant cell populations and culture assays for up to 48-72 h, which was the nominal delay until processing and analysis (data not shown). For each subject, in-flight data were compared with baseline data collected 180 days before launch.

The data revealed that with respect to the peripheral leukocyte distribution, the WBC count increases during spaceflight. A previously observed surge in the WBC count after landing was confirmed, but this finding has been purported to be solely a postflight effect mediated by stress-induced demargination of neutrophils.

Terrestrial space analog studies have suggested that altered leukocyte distribution in astronauts may result from stress and not microgravity conditions.

For ISS crews the WBC count was significantly elevated during missions at both the early and late time points, relative to the L-180 baseline data. No in-flight alterations were observed among the bulk lymphocyte, T and T-memory subsets, with the exception of an increased NK-cell concentration at the late in-flight timepoint.

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’Fine’ T-cell subsets were assessed using a variety of markers. An in-flight increase was observed for the ’cytotoxic’ CD8+ subset (CD28+ /CD244+ ), whereas a decrease was observed for the central memory subset (CD62L+ /CD45RA− ). Several markers of senescence were assessed. A decrease in senescent CD8+ T cells was observed during flight by using both the CD244+ /CD28− phenotype and expression of CD57 as senescence markers. CD69 and HLA-DR were used to assess constitutively activated T cells.

Although it trended upwards relative to preflight baseline data, the number of CD69+ T cells did not increase during flight for either the CD4+ or CD8+ subset. However, the number of CD8+ /HLA-DR+ T cells decreased during flight, reaching significance at the mid-mission timepoint. No alteration in the level of EBV- or CMVspecific T cells was observed during flight, although being HLA restricted, this assay was performed on only 10 crewmembers.

This leukocyte distribution pattern suggests that some in vivo immunological processes are occurring and persisting during long-duration spaceflight, including an elevated WBC count, largely because of an increased number of neutrophils and a shift within the CD8+ T-cell compartment toward a more active phenotype. It is possible that these alterations represent innate immunity compensating for diminished adaptive immune capacity. It is also possible that maturation shifts within CD8+ T cells may parallel persistent latent herpes virus reactivation.

During flight, activation responses for both the CD4+ and CD8+ T-cell subsets following SEA+SEB stimulation were significantly reduced, a finding which, although it trended toward baseline, remained largely significant for the duration of the 6-month spaceflight (Figure 3a). Conversely, in assessment of total CD69+ , no reduction in T-cell function was observed during spaceflight following CD3 and CD28 stimulation (Figure 3b). When CD25+ was assessed, however, a significant decrease was observed at the early in-flight timepoint only.

Crewmembers on board ISS do possess significant functional immune alterations that persist for the duration of a 6-month orbital spaceflight. These alterations may partly explain, from a mechanistic perspective, the root cause of the persistent reactivation of latent herpesviruses reported to occur during spaceflight. Although an attempt was made to investigate changes specific to various adaptive immune biases (Th1, Th2, Th17, and so on), no ’bias shifts’ were observed.

Instead, the functional depressions seem to be across the various aspects of adaptive T-cell immunity. Although this study did not investigate innate immunity in a comprehensive fashion, the lack of cytokine suppression following LPS stimulation may indicate that innate parameters are unaffected. In fact, the increase in IL-8, coupled with other reports of a mild ’pro-inflammatory’ state persisting during ISS missions,38 may indicate compensatory increases in innate immune function. Authors also interpret that the increase in IL-8 production supports the acceptability of the sampling architecture, including delays in processing, used for this study.

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Clinical Events during Long-Duration Orbital Spaceflight

Clinical events potentially related to immune system dysregulation may include a variety of bacterial or viral infections (skin, upper respiratory, urinary tract, etc.), clinical viral reactivation, documented hypersensitivities, or increased incidence of allergies. During prolonged missions (Mars, asteroids, etc.), more prolongeddevelopment diseases such as autoimmunity or malignancies may become possible. During exploration missions, the risk for malignancies may be directly related to immune dysregulation (diminished NK cell function) synergizing with radiation exposure. Basic understanding of the incidence of clinical symptoms during orbital spaceflight is important for anticipation of potential clinical risks during exploration class deep space missions.

This is especially true, considering the persistent immune dysregulation during spaceflight.

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For this study, authors examined medical records from 46 long-duration US astronaut missions on board the ISS. Of the 46 US crew members completing ISS missions as of the date of this manuscript, all were included in this study. No symptoms or immunological events were reported by 8 crew members. During the analysis period, 5 astronauts actually repeated long-duration space missions on board the ISS, for which each longduration mission was counted as a separate "flight/ mission". Therefore, the records from 46 long-duration ISS crew members were examined, which actually consisted of 41 flown US astronauts. Among the 46 US astronauts, there were 34 male and 12 female astronauts, with an average age of 47.51 years at time of flight. The average mission duration was 162.95 days; the sum total for the 46 crew members (representing 38 missions) was 7058 flight days or 20.57 flight years. The primary data review, consisting of ISS crew electronic medical records (EMR), was performed using the evaluation categories as described below.

The NASA integrated medical model (IMM) is an operational NASA tool that helps to align science, technology, and operational activities. The IMM is intended for use in optimizing crew health and safety and to ensure mission success by quantifying the probability and consequences of medical risks The IMM has identified various

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medical conditions that have occurred, or have the potential to occur, in-flight. From this list, authors identified 13 IMM conditions that could be associated with dysregulated immunity during spaceflight.

For purposes of this review, otitis media and otitis externa were combined into a single analysis category of ear-related symptoms: pain, congestion, and itchiness.

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Additionally, the condition of upper respiratory infections was removed and the related symptoms, prolonged congestion, rhinitis, and sneezing were added instead. The IMM category identified as "skin rashes" was interpreted to also apply to hypersensitivities. Two other conditions not contained within the IMM, malignancy and autoimmunity, were also evaluated during this activity.

Finally, one additional category identified as "Infections, other" was created to account for those infectious disease conditions that occurred during spaceflight but were not associated with any specific IMM condition (Table 1).

By far, the majority of the adverse events observed on board the ISS were skin rashes (23 events), followed by upper respiratory symptoms, including congestion, rhinitis and/or sneezing (20 events). Tabulating the various types of infectious disease observed during spaceflight (including pharyngitis, skin infection, etc.) indicates at least 13 infectious disease events occurred during the reporting period.

A more detailed evaluation of the 70 reported medical events further classified 42 events as "notable". Notable events included such characteristics as prolonged duration, repeated or recurring and/or, being unresponsive to treatment. These 42 notable events were observed in 46% of the crew members, whereas the remaining 54% either experienced no notable events or only events that were not classified as "notable" (Figure 1). The most

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likely diagnosis breakdown of these 42 noteworthy events, based on the reported symptoms, is presented in Figure 2.

Almost one-half were found to be prolonged rashes, followed by infectious disease, atypical allergies, and cold sores. An evaluation of the 42 notable events in the context of mission kinetics (month) is presented in Table 2.

The highest concentration of notable adverse medical events occurred during the first month of flight (18 events); however, beyond the first month, there was a generally even spread of events between months 2 and 6 of flight. An evaluation of the distribution of the 42 notable medical events among the 46 crew members is presented in Figure 3.

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Eleven crew members experienced a single notable in-flight medical event, whereas 10 individual crew members experienced 2 or more events. One unique crew member experienced 9 in-flight notable medical events.

The appearance of the rashes generally consisted of bumps/nodules and/or small brown scaly patches, with or without petechiae, redness/hyperemia, and itching. The prevalence of persistent rashes in the US population is about 0.044 cases/year, and our observed incidence of rashes on board the ISS was elevated in comparison at 1.12 events per flight year.

The incidence for rashes on board the ISS is therefore 25-fold higher than terrestrial incidence. The body locations for rash occurrence on board the ISS seemed to vary; however, the appearance was generally consistent, manifesting as bumps, nodules, small scaly patches, and itchy redness. Presumed diagnoses for such rashes could include eczema, contact dermatitis, psoriasis, allergic cholinergic urticaria, and/or simple acne.

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During spaceflight, rashes were observed to occur in the following locations: scalp, face, neck, chest, back, trunk, abdomen, arms, and hands. A representative photograph from a persistent rash incident on board the ISS is presented in Figure 4.

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Plasma Cytokine Concentration

Plasma cytokine assessment has been demonstrated to have clinical utility as a biomarker for various specific immunologic diseases or other disruptions in physiological homeostasis. Elevated levels of IL-6, IL-7, IL-10, and IFN-γ have been detected in the plasma of HIV-infected patients and correlated well with prognosis. Plasma levels of cytokines have also been found to correlate with disease presence or prognosis in rheumatoid arthritis , myelofibrosis, Sjogren’s syndrome , COPD , and pelvic inflammatory disease.

It has been suggested that persistent immune dysregulation may increase specific clinical risks for astronaut crewmembers participating in exploration-class deep space missions . A broad human survey of human immunity during long-duration spaceflight has not yet been performed. To determine in vivo immune homeostasis during long-duration orbital spaceflight, authors investigated astronaut plasma cytokine levels as a biomarker of immune status during missions to the International Space Station (ISS).

Twenty-eight ISS astronaut crewmembers participated in this study. Mission durations were approximately 6 months, which is considered long-duration spaceflight. Of the 28 subjects, 21 were male and 7 were female, and their mean age was 49 ±4 years.

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Results Inflammatory/anti-inflammatory

Mean L-180 baseline levels of IL-1α, IL-1β, TNFα, IL-6, and IL-8 were all below the limit of sensitivity for each cytokine (Table 2). There were no statistically significant inflight alterations for IL-1α, IL-1β, or IL-6. The concentration of TNFα was increased during spaceflight (main effect P < 0.01); however, the more conservative post hoc analysis did not indicate significant increases for any specific in-flight time points. A main-effect increase in IL-8 was observed during spaceflight (P < 0.001), with several in-flight time points achieving individual statistical significance when compared to L-180 (Table 2). Although no in-flight increase was observed for plasma IL-6, there appeared to be a trend toward elevated concentration immediately postflight. Among all premission samples (3 time points per crewmember, 28 crewmembers), only 2 samples had detectable levels of

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IL-6 above the limit of sensitivity, whereas immediately following the spaceflight, 10 crewmembers possessed IL-6 concentrations above the limit (data not shown). There was a fairly stable preflight baseline concentration of IL-1ra between L-180 and L-45 (378 and 363 pg/mL, respectively). By L-10, IL-1ra concentration was trending upward and was significantly higher than baseline (L-180) for all in-flight time points (R + 0), resulting in a significant main-effect difference (P < 0.001; Table 2). Individual analysis of the IL-1ra increase achieved significance at FD120 and R+0 (P < 0.001).

Adaptive immunity cytokines Mean L-180 baseline levels of IFN-Îł, IL-2, IL-17, IL-4, IL-5, and IL-10 were all below the limit of sensitivity (Table 2). There were no statistically significant differences in any adaptive immunity cytokines at any measured in-flight or postflight time points.

Growth factors The mean baseline concentration for the cytokines identified as "growth factors" varied from < 1.0 pg/mL (GMCSF) to 141 pg/mL for thrombopoietin (Tpo) (Table 2). During spaceflight, there were no significant alterations in the plasma concentration of G-CSF, GM-CSF, or FGF basic. Main-effect in-flight increases were detected in the plasma concentration of both Tpo and vascular endothelial growth factor (VEGF)(P < 0.001), with Tpo

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significantly elevated at all 5 individual in-flight time points. In-flight alterations for all growth factors returned to baseline immediately after landing.

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Chemokines Generally, the baseline concentration for chemokines was much higher than for all other measured cytokines.

The mean baseline concentration for chemokines ranged from 9.4 pg/mL (CCL3) to 3427 pg/mL (CCL5) (Table 2). During spaceflight, a main-effect increase was observed for CCL2, CCL4, and CXCL5 (P < 0.001), with single point significance detected for CXCL5 at all 5 in-flight points, and on FD120 for CCL4. No single-time point significant in-flight increases were observed for CCL2, but a postflight significant increase was detected at R+0 (Table 2). No deviations from baseline were observed for CCL3 or CCL5.

For this study, 22 plasma cytokines were assessed at 5 time points during 6-month flight onboard the ISS. Time points were spaced to include the early adaptation phase (FD15, 30) and subsequent long-adaptation points through the entire 6-month mission (FD60, 120, 180). All in-flight data were compared with the baseline sample collected ∼180 days before launch. A second prelaunch sample (L-45) allows another correlative prelaunch sampling and a general measure of intrasubject variability, and a third preflight sample opportunity was available at 10 days before launch.

Individual subject data analysis indicated that where statistically significant alterations occurred, crewmember data were remarkably consistent. Setting the L-180 baseline concentration to zero to observe deviations from baseline, single-subject analysis of IL-1ra, IL-8, CXCL5, VEGF, and Tpo shows that most crewmembers generally manifested a positive alteration during flight, with few obvious negative outliers (Fig. 1A-E). Plotting the individual data also may resolve in-flight effects from postflight trends such as an apparent increase in plasma IL-6 present for most crewmembers after landing, which unfortunately remained below the threshold of sensitivity for the assay for most in-flight data points (Fig. 1F). When tracking individual crew data, occasional outlier crewmember data may be observed that show, usually at a few specific time points only, relatively high concentrations of certain cytokines.

Examples are shown in Fig. 1G-I, where clear single-subject outliers are visible for IL-1β, TNFα, and IL-4. Although these alterations were not frequent enough to influence statistical significance, on a case-by-case basis, these shifts may be clinically relevant. Crewmembers do occasionally experience adverse medical events during spaceflight, including infectious disease or hypersensitivity responses. For this study, authors cannot know if these particular outliers correlated with adverse medical events; however, such a correlation would appear possible even if the underlying mechanistic cause was subclinical. Unfortunately, individual crew health data were not captured as part of the current study.

During spaceflight, there was a significant increase in plasma IL-8 at multiple in-flight time points, whereas TNFα demonstrated a significant main-effect increase associated with spaceflight (Table 1). Although the levels of other inflammatory cytokines (IL-1a, IL-1b, IL-6) were not elevated during spaceflight, the increase in IL-8 and TNFα is indicative of mild inflammation and would be consistent with sensitized innate immunocyte function associated with persistent low-level inflammation during spaceflight. Persistent inflammation during flight could derive from flight-associated alterations in the gut microbiome or the consistent exposure to increased environmental radiation. It is interesting that plasma levels of IL-1ra, an inhibitor of the proinflammatory effects of IL-1, were also consistently elevated during spaceflight. IL-1ra demonstrated both a maineffect increase associated with flight and a specific increase at the FD120 time point (Table 2).

Further supporting an inflammatory state during flight are the observed elevations in CCL2, CCL4, and CXCL5 during spaceflight (Table 2). Levels of CXCL5 increased by nearly 10-fold during flight, remained elevated for

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an entire 6-month spaceflight, and return to baseline almost immediately upon landing (Table 2). CXCL5 (ENA78), a potent neutrophil chemoattractant, is produced by cells subsequent to stimulation with inflammatory cytokines, such as IL-1 or TNFα . CCL4 (MIP-1b) is a proinflammatory molecule produced by macrophages and an attractant for monocytes, NK cells, and other immunocytes . CCL2, also known as monocyte chemotactic protein-1, recruits monocytes, memory T cells, and dendritic cells to a site of localized inflammation. CCL4 (and other MIP-1 family members) upregulates the process of inflammation via the recruitment of inflammatory cells, inducing the release of proinflammatory mediators, and modulating Thelper cell differentiation.

In this study, no increases were observed for any Th1, Th17, or Th2 cytokines during spaceflight (Table 2).

It is noteworthy that the in-flight concentration of 9 plasma cytokines has been previously reported for shortduration Space Shuttle missions . Significant in-flight increases, compared to preflight baseline, were reported for IL-1β, TNFα, IFNα, IFNg, IL-17, IL-4, IL-10, and IL-12. For IL-6, concentration was not increased during flight, but a significant postflight increase was observed. Only 7 of these cytokines were also measured during the current ISS study. For ISS astronauts, an in-flight increase was observed only for TNFα (Table 2), and a trend toward increasing concentration of IL-6, although not significant, was observed postflight (Fig. 1).

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Effects of prolonged stress on Immune System

A long-term spaceflight simulation study (Mars520) provided an extraordinary chance for investigating the effects of prolonged stress. Six healthy males lived under the condition of social isolation and confinement for 520 days, similar to the real Mars voyage. The isolation study was conducted at the Institute for Biomedical Problems (IBMP) in Moscow and approved by several ethical boards of the Russian Federation and European Space Association authorities. Six healthy male volunteers were selected based on modified astronaut selection criteria. They provided written informed consent after due approval to spend 520 days in an enclosed habitat consisting of hermetically sealed interconnecting modules.

Results Persistent high-level cortisol To determine the level of chronic stress induced by long-term isolation, authors have analyzed the results from both subjective ratings and objective physiological stress marker cortisol. The POMS-SF showed no signs of elevation in total mood disturbance (P = 0.7510 for differences in measurement periods), and the results from visual analog scale ratings indicated low levels of feeling stressed, unhappy, mentally fatigued, or tired, and they showed no reliable changes during the isolation (P-values for differences in measurement periods 0.5228, 0.7394, 0.0909, and 0.5629, respectively), which is consistent with previous isolation studies showing that socially isolated young adults did not report more frequent everyday stress.

The participants reported lower workload (all P < 0.001) and correspondingly lower physical exhaustion (all P < 0.05) in the study period compared with baseline condition, reflecting a lower activity during this period.

Interestingly, subjective feelings of sickness were increased during the mission compared to baseline, although statistically significantly only for measurements taken 360 days (P = 0.0006) and 510 days (P = 0.0054) into the mission (Table S5). Medical records did not indicate they were physically sick, suggesting psychologically the prolonged isolation had an influence on the subjects. From a physiological perspective, authors collected morning saliva samples and measured cortisol concentrations. Surprisingly, in comparison with baseline levels, a cortisol increase was detected on the saliva samples from the 360th day of the isolation (P = 0.004). As shown in Fig. 1A, this increase was observed in all subjects, and this high level cortisol persisted on the following time points (410th d and 510th d) during the isolation.

Authors also measured cortisol in evening saliva samples. Similarly, evening cortisol concentrations were increased in the study period (Table S6). To check the reliability of cortisol baseline, morning saliva cortisol was measured continuously for 7 days shortly after the confinement started (from day 29 to day 35). No statistical difference was detected between baseline levels (collected before the isolation) and the average cortisol levels from these 7 days (data not shown).

In addition to the saliva collection time points which were identical to blood sampling, to limit the risk of cortisol random changes, authors have measured morning saliva cortisol in a blind way continuously for 7 days in saliva samples from the period of day 411 - day 420 and day 471 - day 480, respectively. Significant difference from baseline level was observed in most days from these two periods (Fig. 1B).

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Leukocyte distribution pattern is changed with increased lymphocyte numbers The immunologic profiles of the subjects during the study period are shown in Table 1. The total numbers of leukocytes were marginally increased, but these changes were not statistically significant.

In contrast, leukocyte distribution pattern displayed significant changes during the isolation.

On the 360th day, 410th day and 510th day of isolation, the lymphocyte percentages were all increased to ∼ 50% of the total leukocyte, showing a significant change from baseline.

Accordingly, the percentage of neutrophils was reduced, while neutrophil numbers remained unchanged. The changes of leukocyte distribution pattern were largely due to the increase of lymphocyte numbers during the isolation period. Among the peripheral blood lymphocyte subpopulations, both CD3+ T cells and CD19+ B cell displayed an increase during the isolation, while NK cells did not vary.

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Notably, although both CD8+ cytotoxic T cells and CD4+ helper T cells increased, the ratio of helper/cytotoxic T cells was unchanged in the isolation period.

Heightened antiviral immune responses Authors assayed all the participants during the isolation for antiviral responses against EBV, one of the most common virus affecting humans, as defined by the ability to upregulate IFN-γ, IL-2, TNF-α, authors used IFN-γ production as a basal readout of activation while it plays a critical role in antiviral response . During the study period, on all the 3 time points (360th day, 410th day and 510th day), the IFN-γ productions were up-regulated against EBV stimulation (P < 0. 05) (Fig. 2A) and in response to EBV, authors could also observe significant increase of TNF-α production during the study period.

Interestingly, IL-2 showed no increase from the baseline levels across the three sampling times (Fig. 2B). In non-infected control samples, no changes of IFN-γ, IL-2 and TNF-α were detected throughout the study interval (data not shown).

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No increased virus infection or latent virus reactivation were detected Circulating proinflammatory cytokines (IFN-Îł, TNF-Îą, IL-2 and IL-6) from plasma showed no significant changes during the study period (Table S7), suggesting no increased in vivo viral infection or latent virus reactivation. In the plasma samples authors also examined the EBV antibodies directed to the "Viral Capsid Antigen" (VCA) and the nuclear antigen (EBNA), while the detections of these two antibodies are diagnostically often performed to determine EBV infection. No significant difference has been detected for both VCA and EBNA antibody titers comparing the time points during the study period with baseline level (Fig. 3).

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Furthermore, authors tested for the level of EBV in saliva at the same time points as for the blood sampling.

Interestingly, although more than 2000 geq/ml saliva virus shedding were detected in a few subjects in the baseline samples, during the study period the levels of viral DNA found in saliva were largely negative (Table 2).

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