Towards human exploration of space: The THESEUS review series on immunology research priorities

Abstract

Dysregulation of the immune system occurs during spaceflight and may represent a crew health risk during exploration missions because astronauts are challenged by many stressors. Therefore, it is crucial to understand the biology of immune modulation under spaceflight conditions in order to be able to maintain immune homeostasis under such challenges. In the framework of the THESEUS project whose aim was to develop an integrated life sciences research roadmap regarding human space exploration, experts working in the field of space immunology, and related disciplines, established a questionnaire sent to scientists around the world. From the review of collected answers, they deduced a list of key issues and provided several recommendations such as a maximal exploitation of currently available resources on Earth and in space, and to increase increments duration for some ISS crew members to 12 months or longer. These recommendations should contribute to improve our knowledge about spaceflight effects on the immune system and the development of countermeasures that, beyond astronauts, could have a societal impact.

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REVIEW ARTICLE OPEN

Towards human exploration of space: The THESEUS review series on immunology research priorities

Jean-Pol Frippiat1, Brian E Crucian2, Dominique J-F de Quervain3, Daniela Grimm4, Nicola Montano5, Siegfried Praun6, Benno Roozendaal7, Gustav Schelling8, Manfred Thiel9, Oliver Ullrich10 and Alexander Choukr8

Dysregulation of the immune system occurs during spaceight and may represent a crew health risk during exploration missions because astronauts are challenged by many stressors. Therefore, it is crucial to understand the biology of immune modulation under spaceight conditions in order to be able to maintain immune homeostasis under such challenges. In the framework of the THESEUS project whose aim was to develop an integrated life sciences research roadmap regarding human space exploration, experts working in the eld of space immunology, and related disciplines, established a questionnaire sent to scientists around the world. From the review of collected answers, they deduced a list of key issues and provided several recommendations such as a maximal exploitation of currently available resources on Earth and in space, and to increase increments duration for some ISS crew members to 12 months or longer. These recommendations should contribute to improve our knowledge about spaceight effects

on the immune system and the development of countermeasures that, beyond astronauts, could have a societal impact.

npj Microgravity (2016) 2, 16040; doi:http://dx.doi.org/10.1038/npjmgrav.2016.40

Web End =10.1038/npjmgrav.2016.40 ; published online 1 December 2016

INTRODUCTIONSpaceight is a unique stress model impacted consistently or intermittently by a myriad of stressors of psychosocial and physical origin, high G forces at the time of launch and landing, increased radiation, sleep deprivation and persistent circadian misalignment, microgravity, and nutritional factors while in space. This multitude of factors has profound immune modulatory effects on humans and animals and can lead to certain types of immune dysregulation with compromised defenses against infections, thereby representing a potential barrier to long-term space exploration (for more information consult recent reviews14). For example, 15 of the 29 Apollo astronauts contracted bacterial or viral infections either during the mission or within a week of returning.5 Moreover, recent results after mission to the International Space Station revealed that during spaceight some aspects of human immunity may be oversensitized, as evidenced by some astronauts who experienced prolonged allergic/skin hypersensitivity symptoms.6,7

Several studies have shown that spaceight conditions induce frequently hypoplasia of the spleen in rats and mice.8,9 Variations in peripheral blood leukocyte subsets were also reported at landing. These modications could be mediated by changes in the expression of adhesion molecules10,11 and/or the redistribution of body uids in microgravity.12 An increase in the number of peripheral blood neutrophils has been often observed. Results for other cell types are variable thereby revealing the sensitivity of immune cell populations to differences in spaceight conditions, post-ight procedures, environmental inuences, and experimental designs.

In the same way, it has been shown that the phagocytic and oxidative functions of neutrophils are affected by spaceight conditions.13,14 Astronauts monocytes exhibit phenotypic and cytokine-production deregulations and a reduced ability to engulfE. coli, elicit an oxidative burst and degranulate.1416 The response

of astronauts monocytes to Gram-negative endotoxins is modulated by spaceight-associated factors.17 Low natural killer cell cytotoxicity and a delay in responses to hypersensitivity skin tests were also observed.1820 Moreover, reactivation of latent herpes viruses (e.g., varicella zoster virus, cytomegalovirus, and Epstein-Barr virus) has frequently been reported and is correlated with a drop in interferon production and elevated levels of stress hormones, suggesting that spaceight-associated stressors may be responsible for these reactivations.2128 Latent virus reactivation can therefore be considered as a good biomarker of spaceight-induced weakening of cell-mediated immunity.

Similarly, the activation of T lymphocytes is severely depressed under low-gravity conditions as highlighted by Cogolis team, which performed many investigations on this topic following their discovery of this phenomenon in 1984.2933 This lower response

has several explanations. First, changes in gene expression as lower expressions of Interleukin-2 (IL-2) and IL-2 receptor were observed under real and simulated microgravity3140 and it was shown that

gravity can regulate T-cell activation by blocking translation via noncoding microRNA.41 Second, reduced cellcell interactions and cytoskeleton structure changes. Indeed, T lymphocytes motility was found to be affected, the motility of monocytes was severely reduced and the structure of their cytoskeleton was modied.30,34,40,4246 These motility and cytoskeleton changes

1Stress Immunity Pathogens Laboratory, EA7300, Lorraine University, Nancy, France; 2NASA Johnson Space Center, Houston, TX, USA; 3Departments of Medicine and Psychology, University of Basel, Basel, Switzerland; 4Department of Biomedicine, Pharmacology, Aarhus University, Aarhus, Denmark; 5Cardiovascular Neuroscience Laboratory, Department of Biomedical and Clinical Sciences, University of Milan, Milan, Italy; 6VF Services GmbH, Absam, Austria; 7Department of Cognitive Neuroscience, Radboud University Medical Centre and Donders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The Netherlands; 8Department of Anaesthesiology, Stress and Immunity Laboratory, University of Munich, Munich, Germany; 9Department of Anaesthesiology and Surgical Intensive Care Medicine, University Medical Center Mannheim, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany and 10Institute of Anatomy, Faculty of Medicine, University of Zurich, Zurich, Switzerland.

Correspondence: J-P Frippiat (mailto:[email protected]

Web End [email protected])

Received 20 February 2016; revised 11 October 2016; accepted 13 October 2016

Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA

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Stressors encountered during spaceflight (microgravity, radiation, chronic stress, fluid shear, hydrostatic pressure, etc.) weaken the immune system and have a positive impact on some pathogens

virulence, resistance & proliferation

immune performance

Figure 1. Numerous dysregulations of the immune system have been reported during and following spaceight. In parallel, spaceight has been shown to increase the virulence, antibiotic resistance, and proliferation of some pathogens suggesting that susceptibility to infections could be enhanced during space missions.

could reduce the interaction between T lymphocytes and monocytes, essential for delivering the costimulatory signal. Because the cytoskeleton is involved in signal transduction47 and could be the structure through which cells sense gravity,48 inhibition of T-cell response could thirdly result from alterations in signaling events. HughesFulfords group analyzed differential gene expression in Concanavalin A and anti-CD28-activated human T cells36 and

discovered that the impaired induction of early genes regulated primarily by transcription factors NF-B, CREB, ELK, AP-1, and STAT1 contributes to T-cell dysfunction under altered gravity. They also showed that the protein kinase A (PKA) signaling pathway is downregulated under microgravity. As NF-B, AP-1, and CREB are all regulated by PKA, these ndings indicate that PKA is a key player in gravity-mediated modulation of T-cell activation. In accordance with these results, a recent study conrmed that the Rel/NF-B signaling pathway and the transcription of key immediate early genes involved in T-cell activation are inhibited by microgravity.49

Fourth, the disturbed expression of cell cycle regulatory proteins50

and the induction of apoptosis51,52 could also contribute to

impaired T-cell activation in microgravity, whereas key proteins of T-cell signal modules seemed not to be severely disturbed in microgravity.53 Finally, post-ight cytokine data collected from crew members54 revealed a decrease in TH1 cytokine expression, which can contribute to decreased natural immunity and suggests a TH2 cytokine shift. This TH2 shift represents a signicant clinical risk for TH2-related autoimmune diseases, disease susceptibility related to diminished cell-mediated immunity, as well as allergies and hypersensitivities as recently demonstrated.6,7 Although a well-characterized post-ight phenomenon, immune dysregulation occurs, and persists during spaceight, conrming in-ight dysregulation distinct from the inuences of landing and read-aptation following deconditioning.55,56

Humoral immunity has rarely been investigated in astronauts. According to the few studies available, no signicant changes in plasma levels of immunoglobulin were observed after short-term spaceights,10,14,57 but inconsistent results were reported after long-duration missions. Konstantinova et al.58 reported increased levels of serum immunoglobulin, particularly total IgA and IgG, whereas Rykova et al.14 indicated that the total amounts of serum IgA, IgG, and IgM were unchanged after prolonged missions. Furthermore, nothing is known about B-cell activation under spaceight conditions. Only studies performed using ground-based analogs have been reported.32,59 Studies performed with

the urodele amphibian Pleurodeles waltl as animal model conrmed the increase of neutrophils in peripheral blood at landing.60 As for

astronauts, this increase can be attributed to stress because it was shown that neutrophilia occurs in the urodele amphibian Notophthalamus viridescens when it is treated with hydrocortisone or adrenocorticotropic hormone (ACTH), two stress hormones, or when it is subjected to environmental stress.61 Amphibians are interesting models for analyzing the effects of spaceight on the immune system because cardinal elements of the adaptive immune system are shared by all gnathostomes.1,6269 Later on, adult P. waltl

were immunized with protein onboard the Mir space station. The analysis of these animals revealed an increase of IgY heavy chain mRNAs (IgY is the physiological counterpart of human IgA66) in the spleen of own animals.70 This increase supports previous observations made in astronauts by Konstantinova et al.58

Furthermore, these animals allowed demonstrating that spaceight affects antibody production in response to an antigenic stimulation. Indeed, the use of the different VH gene subgroups69 and the

expression of individual VH gene segments71 were observed to be modied under spaceight conditions. In addition, these animals enabled, for the rst time, the demonstration that somatic hypermutations, which diversify antibody binding sites to improve their afnity, occur in space following immunization but at a lower frequency.72 This observation suggests that antibody afnity maturation could be less efcient in space, thereby decreasing the efciency of the immune response. Finally, it was noted that the transcription levels of IgM heavy chains and of an early B-cell transcription factor are modied when P. waltl embryos are subjected to gravity changes, suggesting a modication in lymphopoiesis.73 This hypothesis was then conrmed in mice subjected to ground-based models.74,75

In parallel, changes in microbial growth characteristics and pathogenicity have been observed for several microorganisms during spaceight.7680 These data, coupled to dysregulations of the immune system, suggest that opportunities for microbes to establish foci of infection could be enhanced during space missions (Figure 1). Furthermore, some data suggest that antibiotics could be less effective in space.81

Finally, it was shown that spaceight induces signicant changes in the thymic mRNA expression of genes that regulate stress and glucocorticoid receptor metabolism.82 To better approach the consequences of physical and emotional stressors in man exposed to spaceight, investigations have been undertaken by applying

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Cortisol Endocannabinoids

Adenosine

Adrenaline Noradrenaline

Endocannabinoids

Control of Immune Responses

IL-1

TNF-

Figure 2. Interlinks between the central nervous system and the immune system. ANS, autonomic nervous system; IL, interleukin; PNS, peripheral nervous system; SNS, sympathetic nervous system; TNF-, tumor necrosis factor .

complementary and elaborated blood, urine and saliva sampling, and analyses, together with questionnaires based emotional stress monitoring. These investigations in man ranged from very acute gravitational challenges (parabolic ight) to short (12 weeks) and long (47 months) duration ISS missions and showed that there is a shift from an alert state of primed innate immune cells after acute gravitational stress to a decrease of their activity after spaceight. Adrenocortical stress-responses suggest that sympathetic nervous system (SNS) responses predominate after short spaceights, although long ights are characterized by glucocorticoid-mediated changes. Furthermore, central activation of SNS, even via non-adrenomedullary pathways, such as sleep deprivation,83

has recently been shown to have a pivotal role in the regulation of inammation84 and innate immunity.85 The immunotropic endocannabinoid system is also activated under acute gravitational stress and in space.86 Further, Earth-bound studies have provided evidence of emotional stress-sensitive immune changes in human.54,8688 These data highlight the complex systemic com

promise of the immune system in response to exposure to multiple stressors during spaceight (Figure 2).

Altogether, these data indicate that spaceight-induced alterations of the immune system have the potential to be a serious clinical risk to astronauts and cosmonauts during exploration-class deep space missions beyond Earth orbit.3,89 This represents an area that should be considered more thoroughly, via both ground-analog studies and investigations onboard the ISS, to ensure long-term survival in space stations and sustain habitation during future missions to the moon, Asteroids, or on any planet i.e., Mars.

IDENTIFIED KEY ISSUESConsequently, experts working in the eld of space immunology and experts working in related areas (e.g., psychoneuroendocrine and autonomous nervous system regulation) established a questionnaire that was sent to scientists around the world to identify key issues that should be addressed for the preparation of future long-duration space missions. The analysis of collected answers highlighted the following points:

The identication and the quantication of stress factors encountered during a space mission and their impact on the immune system should be better studied.

The extension of ISS increments to 1 year or longer is of importance to address the following questions: (i) are immune

system development, response, and regulation as efcient in space (ISS/moon/Mars) as on Earth? (ii) What are the consequences of chronic immune changes on disease during and after long-duration missions? (iii) What are the consequences of long-duration (1 year) missions on the degree of immunosuppression/modulation? (iv) What effects Lunar or Mars dusts, habitat environment and other chemicals, have on immune system performance?

Are stress-dependent virus reactivation patterns linked to cancer development?

The interaction between the immune system and other stress-sensitive systems (neurophysiological and others) should be further studied.

The denition and testing of countermeasures.

In order to understand and track the footprint of space exploration on the immune system, and its interactions with other organs, it appears crucial that these key issues are addressed through an appropriate technical and structural framework. In this context, three interlinked approaches emerged: (i) pathophysio-logical cellular pathways and complex molecular mechanisms analysis using isolated cell systems. (ii) Implementation of animal experiments to investigate the compromise of the immune system after exposure to multiple stressors encountered during space-ight. (iii) Coordinated investigations in man to better approach the consequences of multifactorial effects of physical stressors and emotional stress induced by spaceight.

KNOWLEDGE GAPS AND RESEARCH NEEDS IDENTIFIED BY THE ONLINE SURVEY

In light of future manned spaceight for exploration, extension of ISS increments for some ISS astronauts/cosmonauts to one year or longer has been considered by all Expert Group (EG) members, and the majority of investigators participating to the online consultation, of very high importance. It is fortunate that the rst 1-year ISS expedition has been successfully accomplished and may pave the way for further repeat or an even further prolongation.

The development of inight hardware to perform easy-to-handle and noninvasive exams to monitor immune and hemostatic changes was considered to be of very high importance by EG as well as by 90% of the consulted specialists. Indeed, up to now, due to technical constrains and limited astronaut time

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dedicated to each experiment, most inight assessments resulted from the analysis of inight xed cells.

To achieve a higher relevance of gathered data, especially for critical investigations, a need for (i) repeating inight experiments at least twice and (ii) some standardization of experiment design, methods, assays, and so on is requested. Both points were considered of high or very high importance by 490% of consulted scientists.

Before going to space, test and control experiments should be performed using the full scale of Earth-bound models that can adequately simulate specic spaceight conditions. Fifty percent of the respondents considered this point as very highly relevant and 40% as highly relevant. The appropriate terrestrial analog may vary based on the space-physiological system of interest. For immune dysregulation Arctic, undersea, and Antarctica deployment have all be demonstrated to have similarities to ight. Prolonged head-down tilt bed rest can also be used. However, conicting results have been reported using this model; some studies reported limited changes in the immune system,90,91

whereas others reported changes in cytokine production, immune cell activation and innate immunity.9297

The establishment of a bank of tissue and plasma samples available to the scientic community was dened by the majority of EG members and external experts (490%) to be very important to extract maximal information as well as to reanalyze samples when new tools become available.

Taking advantage of health data les from ISS crew members, with retrospective and prospective access, is necessary to estimate clinically relevant changes in regard to immune alterations.

PROPOSED INVESTIGATIONS AND RECOMMENDATIONS Given these knowledge gaps and research needs, the expert group established a list of recommendations:

The immune system is a target and a sensor of environmental changes affecting the organism as a whole. Therefore, interdisciplinary approaches are required to understand how home-ostasis and immune functions are affected through other organs under acute or chronic stress conditions, both in space and on Earth.

Multidisciplinary approaches should include the full-scale use of available in vitro and in vivo methods using rodents or other animal models, as well as clinical and space related Earth-bound studies.

It is highly recommended to standardize some methodologies used for immune research as well as repeating space experiments.

The establishment of an all ISS-partners international biological sample archive (samples collected from space crew members and animals) is mandatory to allow the extraction of a maximum of information from these samples. Such a sample archive would be most benecial if multiple types of biological samples, such as saliva, urine, plasma, and blood cells could be appropriately preserved for various soluble, cellular and/or molecular analyses.

The access to anonym health data from crew members, retrospectively and prospectively, should be allowed to complete space experiments results.

The development of new technologies to diagnostic immune changes in minute amounts of blood should be encouraged. Preliminary efforts to develop such microgravity-compatible laboratory instruments have been initiated both in Europe and in North America and should be continued because such hardware would be a benet for space and Earth medicine.

TRANS- AND CROSS-DISCIPLINARY ASPECTSThe understanding of stress related immune challenges in space is of high relevance to understand the biology of cancer, immunology and inammation in astronauts as well as in the young and ageing population on Earth. The translational aspects of technological developments for space research constitute also a potentially highly attractive spin-off for, e.g., the non-invasive monitoring of immune and health conditions in minute amounts of blood in space and on Earth. Finally, the immune system is one of the largest and most widespread systems with hormonal and neuronal connections (Figure 2). The immune system is strongly linked with almost every organ homeostasis and can be affected, together with other systems, upon stress challenges. As an example, stress induces the secretion of hormones which can modulate immune responses and also promote bone turnover. Moreover, immune responses are susceptible to exercise and nutritional factors as well as to environmental factors like radiation, the microbial load and the oxygen tension in the habitat. Therefore, there are manifold non-selective interactions with other elds and topics such as bone, muscle, kidney, lung, neurophysiology, cognitive performance, nutrition, exercise, the cardiovascular system, habitat environment and design, radiation, and health (prevention, diagnosis, and therapy). Consequently, several types of countermeasures could be tested that affect directly or indirectly the immune system such as vaccination, nutrition, exercise but also pharmacological and psychosocial approaches dedicated to reducing stress because it is an important contributor to immune dysfunction. These countermeasures are also interesting for other conditions in which the function of the immune system is compromised on Earth such as people subjected to acute or chronic stress.

CONCLUSION AND EARTH BENEFITSIt is an international scope to gather more knowledge on how the immune system is webbed in the pathology and clinical appearance of disease, from it cellular functions to an orchestrated action of immune functions in the complex human system. In the elds of fundamental and clinical immunology, the analyses of the links with other organs are important to address societal questions such as aging and the consequences of a sedentary life style. From clinical investigations in the healthy and the sick, the lifesaving importance of adequate immunity at the switch between the host and the environment was never clearer before. On-orbit immune dysregulation has the potential to be of either a hyper or a hypo nature, or even both as various innate and adaptive cellular components may respond differently during ight. Beside controlling infections and eliminating germs, immunological responses are also capable of eliminating non-functional or dysfunctional cells, e.g., tumor cells. Failure to establish adequate immune answers and to distinguish between self and non-self can result in autoimmune diseases (e.g., rheumatoid arthritis), acute and life-threatening infections and overwhelming systemic immune responses (e.g., septicemia) or the development of cancer. Changes to this endogenous balance between the immune and other systems due to psychological or environmental factors can impact this nely balanced, endo-, para-, and autocrine-controlled immune function, resulting in immune dysfunction and disease. The understanding of these deregulations, together with the development of appropriate countermeasures, can help space travelers and people on Earth.

ACKNOWLEDGMENTS

This review is a product of the Theseus Project: Towards Human Exploration of Space: a European Strategy of the European Science Foundation and was supported by European Communitys 7th Framework Programme (FP7/20072013) under grant agreement #242482. We also thank the French (CNES), the German (DLR), the Italian

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(ASI), the Russian (IBMP, Rocosmos) and the American (NASA) space agencies for their support. We are particularly grateful to Brigitte Gaillard for outstanding work in coordinating the heavy editorial processes of the overall Theseus series dedicated to integrated physiology system.

24. Mehta, S. K. et al. Reactivation of latent viruses is associated with increased plasma cytokines in astronauts. Cytokine 61, 205209 (2013).

25. Mehta, S. K., Stowe, R. P., Feiveson, A. H., Tyring, S. K. & Pierson, D. L. Reactivation and shedding of cytomegalovirus in astronauts during spaceight. J. Infect. Dis. 182, 17611764 (2000).

26. Pierson, D. L., Stowe, R. P., Phillips, T. M., Lugg, D. J. & Mehta, S. K. Epstein-Barr virus shedding by astronauts during space ight. Brain Behav. Immun. 19, 235242 (2005).

27. Stowe, R. P., Mehta, S. K., Ferrando, A. A., Feeback, D. L. & Pierson, D. L. Immune responses and latent herpesvirus reactivation in spaceight. Aviat. Space Environ. Med. 72, 884891 (2001).

28. Stowe, R. P., Pierson, D. L. & Barrett, A. D. T. Elevated stress hormone levels relate to Epstein-Barr virus reactivation in astronauts. Psychosom Med 63, 891895 (2001).

29. Cogoli, A., Tschopp, A. & Fuchsbislin, P. Cell sensitivity to gravity. Science 225, 228230 (1984).

30. Cogoli, A. The effect of hypogravity and hypergravity on cells of the immune system. J. Leukoc. Biol. 54, 259268 (1993).

31. Cogoli, A. The effect of space ight on human cellular immunity. Environ. Med. 37, 107116 (1993).

32. Gaignier, F. et al. Three weeks of murine hindlimb unloading induces shifts from B to T and from Th to Tc splenic lymphocytes in absence of stress and differentially reduces cell-specic mitogenic responses. PLos ONE 9, e92664 (2014).

33. Gridley, D. S. et al. Spaceight effects on T lymphocyte distribution, function and gene expression. J. Appl. Physiol. 106, 194202 (2009).

34. Cogoli-Greuter, M. Effect of gravity changes on the cytoskeleton in human lymphocytes Gravit. Space Biol. Bull. 17, 2737 (2004).

35. Sciola, L., Cogoli-Greuter, M., Cogoli, A., Spano, A. & Pippia, P. Inuence of microgravity on mitogen binding and cytoskeleton in Jurkat cells. Adv. Space Res. 24, 801805 (1999).

36. Boonyaratanakornkit, J. B. et al. Key gravity-sensitive signaling pathways drive T-cell activation. FASEB J. 19, 20202022 (2005).

37. Cogoli, A. et al. Mitogenic signal-transduction in T-lymphocytes in microgravity.J. Leukoc. Biol. 53, 569575 (1993).38. Pippia, P. et al. Activation signals of T lymphocytes in microgravity. J. Biotechnol.

47, 215222 (1996).

39. Walther, I. et al. Simulated microgravity inhibits the genetic expression of interleukin-2 and its receptor in mitogen-activated T lymphocytes. FEBS Lett. 436, 115118 (1998).

40. Cogoli, A. Signal transduction in T lymphocytes in microgravity. Gravit. Space Biol. Bull. 10, 516 (1997).

41. Hughes-Fulford, M., Chang, T. T., Martinez, E. M. & Li, C. F. Spaceight alters expression of microRNA during T-cell activation. FASEB J. 29, 48934900 (2015).

42. Meloni, M. A. et al. Space ight affects motility and cytoskeletal structures in human monocyte cell line J-111. Cytoskeleton 68, 125137 (2011).

43. Meloni, M. A., Galleri, G., Pippia, P. & Cogoli-Greuter, M. Cytoskeleton changes and impaired motility of monocytes at modelled low gravity. Protoplasma 229, 243249 (2006).

44. Meloni, M. A. et al. Modeled microgravity affects motility and cytoskeletal structures. J. Gravit. Physiol. 11, P197P198 (2004).

45. Cogoli-Greuter, M. et al. Movements and interactions of leukocytes in micro-gravity. J. Biotechnol. 47, 279287 (1996).

46. Pellis, N. R. et al. Changes in gravity inhibit lymphocyte locomotion through type I collagen. In Vitro Cell Dev. Biol. Anim. 33, 398405 (1997).

47. Janmey, P. A. The cytoskeleton and cell signaling: component localization and mechanical coupling. Physiol. Rev. 78, 763781 (1998).

48. Ingber, D. How cells (might) sense microgravity. FASEB J. 13 (Suppl), S3S15 (1999).

49. Chang, T. T. et al. The Rel/NF-kappa B pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity. J. Leukoc. Biol. 92, 11331145 (2012).

50. Thiel, C. S. et al. Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity. Cell Commun. Signal. 10, 1 (2012).

51. Battista, N. et al. 5-Lipoxygenase-dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment. FASEB J. 26, 17911798 (2012).

52. Maccarrone, M. et al. Creating conditions similar to those that occur during exposure of cells to microgravity induces apoptosis in human lymphocytes by 5-lipoxygenase-mediated mitochondrial uncoupling and cytochrome c release.J. Leukoc. Biol. 73, 472481 (2003).53. Tauber, S. et al. Signal transduction in primary human T lymphocytes in altered gravityresults of the MASER-12 suborbital space ight mission. Cell Commun. Signal. 11, 32 (2013).

54. Crucian, B. E., Stowe, R. P., Pierson, D. L. & Sams, C. F. Immune system dysregulation following short- vs long-duration spaceight. Aviat. Space Environ. Med. 79, 835843 (2008).

CONTRIBUTIONS

Conceived and designed the report based on the expert group (EG) inputs: J.P.F. (EG rapporteur) and A.C. (EG chair); wrote the paper: J.P.F.; revised the paper: J.P.F., A.C. and B.E.C. Paper was reviewed by all authors before submission.

COMPETING INTERESTS

The authors declare no conict of interest.

REFERENCES1. Frippiat, J. P. Contribution of the urodele amphibian Pleurodeles waltl to the analysis of spaceight-associated immune system deregulation. Mol. Immunol. 56, 434441 (2013).

2. Grimm, D., Wise, P., Lebert, M., Richter, P. & Baatout, S. How and why does the proteome respond to microgravity? Expert Rev. Proteomics 8, 1327 (2011).

3. Gueguinou, N. et al. Could spaceight-associated immune system weakening preclude the expansion of human presence beyond Earth's orbit? J. Leukoc. Biol. 86, 10271038 (2009).

4. Pietsch, J. et al. The effects of weightlessness on the human organism and mammalian cells. Curr. Mol. Med. 11, 350364 (2011).

5. Kimzey, S. L. in Biomedical Results from Skylab 248282 (eds Johnson, R. S. & Dietlein, L. F.) (NASA, 1977).

6. Crucian, B. et al. A case of persistent skin rash and rhinitis with immune system dysregulation onboard the International Space Station. J. Allergy Clin. Immunol. Pract. 4, 759762 (2016).

7. Crucian, B. et al. Incidence of clinical symptoms during long-duration orbital spaceight. Int. J. Gen. Med., (in the press).

8. Baqai, F. P. et al. Effects of spaceight on innate immune function and antioxidant gene expression. J. Appl. Physiol. 106, 19351942 (2009).

9. Gridley, D. S. et al. Genetic models in applied physiologySelected contribution: Effects of spaceight on immunity in the C57BL/6 mouse. II. Activation, cytokines, erythrocytes, and platelets. J. Appl. Physiol. 94, 20952103 (2003).

10. Grove, D. S., Pishak, S. A. & Mastro, A. M. The effect of a 10-day space-ight on the function, phenotype, and adhesion molecule expression of splenocytes and lymph-node lymphocytes. Exp. Cell Res. 219, 102109 (1995).

11. Stowe, R. P. et al. Leukocyte subsets and neutrophil function after short-term spaceight. J. Leukoc. Biol. 65, 179186 (1999).

12. Vernikos, J. Human physiology in space. Bioessays 18, 10291037 (1996).13. Kaur, I., Simons, E. R., Castro, V. A., Ott, C. M. & Pierson, D. L. Changes in neutrophil functions in astronauts. Brain Behav. Immun. 18, 443450 (2004).

14. Rykova, M. P., Antropova, E. N., Larina, I. M. & Morukov, B. V. Humoral and cellular immunity in cosmonauts after the ISS missions. Acta Astronaut 63, 697705 (2008).

15. Crucian, B., Stowe, R., Quiriarte, H., Pierson, D. & Sams, C. Monocyte phenotype and cytokine production proles are dysregulated by short-duration spaceight. Aviat. Space Environ. Med. 82, 857862 (2011).

16. Kaur, I., Simons, E. R., Castro, V. A., Ott, C. M. & Pierson, D. L. Changes in monocyte functions of astronauts. Brain Behav. Immun. 19, 547554 (2005).

17. Kaur, I., Simons, E. R., Kapadia, A. S., Ott, C. M. & Pierson, D. L. Effect of spaceight on ability of monocytes to respond to endotoxins of gram-negative bacteria. Clin. Vaccine Immunol. 15, 15231528 (2008).

18. Lesnyak, A. T. et al. Immune changes in test animals during spaceight. J. Leukoc. Biol. 54, 214226 (1993).

19. Meshkov, D. & Rykova, M. The natural cytotoxicity in cosmonauts on board space stations. Acta Astronaut 36, 719726 (1995).

20. Taylor, G. R. & Janney, R. P. In vivo testing conrms a blunting of the human cell-mediated immune mechanism during space-ight. J. Leukoc. Biol. 51, 129132 (1992).

21. Cohrs, R. J., Mehta, S. K., Schmid, D. S., Gilden, D. H. & Pierson, D. L. Asymptomatic reactivation and shed of infectious varicella zoster virus in astronauts. J. Med. Virol. 80, 11161122 (2008).

22. Crucian, B. E., Cubbage, M. L. & Sams, C. F. Altered cytokine production by specic human peripheral blood cell subsets immediately following space ight. J. Interf. Cytok. Res. 20, 547556 (2000).

23. Meehan, R., Whitson, P. & Sams, C. The role of psychoneuroendocrine factors on spaceight-induced immunological alterations. J. Leukoc. Biol. 54, 236244 (1993).

Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2016) 16040

Space immunology research priorities J-P Frippiat et al

6

55. Crucian, B. et al. Alterations in adaptive immunity persist during long-duration spaceight. Npj Micrograv. 1, 15013 (2015).

56. Crucian, B. E. et al. Plasma cytokine concentrations indicate that in vivo hormonal regulation of immunity is altered during long-duration spaceight. J. Interferon Cytok. Res. 34, 778786 (2014).

57. Voss, E. W. Prolonged weightlessness and humoral immunity. Science 225, 214215 (1984).

58. Konstantinova, I. V., Rykova, M. P., Lesnyak, A. T. & Antropova, E. A. Immune changes during long-duration missions. J. Leukoc. Biol. 54, 189201 (1993).

59. Gueguinou, N. et al. Stress response and humoral immune system alterations related to chronic hypergravity in mice. Psychoneuroendocrinology 37, 137147 (2012).

60. Michurina, T. V., Domaratskaya, E. I., Nikonova, T. M. & Khrushchov, N. G. Blood and clonogenic hematopoietic-cells of newts after the space-ight. Adv. Space Res. 17, 295298 (1995).

61. Bennett, M. F. & Daigle, K. R. Temperature, stress and the distribution of leukocytes in red-spotted newts, notophthalmus-viridescens. J. Comp. Physiol. 153, 8183 (1983).

62. Bascove, M. & Frippiat, J. P. Molecular characterization of Pleurodeles waltl activation-induced cytidine deaminase. Mol. Immunol. 47, 16401649 (2010).63. Boudarra, N., Frippiat, C., Dournon, C. & Frippiat, J. P. An alternative internal splicing site denes new Ikaros isoforms in Pleurodeles waltl. Dev. Comp. Immunol. 26, 659673 (2002).

64. Fonte, C., Gruez, A., Ghislin, S. & Frippiat, J. P. The urodele amphibian Pleurodeles waltl has a diverse repertoire of immunoglobulin heavy chains with polyreactive and species-specic features. Dev. Comp. Immunol. 53, 371384 (2015).

65. Frippiat, C., Kremarik, P., Ropars, A., Dournon, C. & Frippiat, J. P. The recombination-activating gene 1 of Pleurodeles waltl (urodele amphibian) is transcribed in lymphoid tissues and in the central nervous system. Immunogenetics 52, 264275 (2001).

66. Gueguinou, N., Huin-Schohn, C., Ouzren-Zarhloul, N., Ghislin, S. & Frippiat, J. P. Molecular cloning and expression analysis of Pleurodeles waltl complement component C3 under normal physiological conditions and environmental stresses. Dev. Comp. Immunol. 46, 180185 (2014).

67. Schaerlinger, B., Bascove, M. & Frippiat, J. P. A new isotype of immunoglobulin heavy chain in the urodele amphibian Pleurodeles waltl predominantly expressed in larvae. Mol. Immunol. 45, 776786 (2008).

68. Schenten, V., Gueguinou, N., Baatout, S. & Frippiat, J. P. Modulation of Pleurodeles waltl DNA Polymerase mu expression by extreme conditions encountered during spaceight. PLos ONE 8, e69647 (2013).

69. Schaerlinger, B. & Frippiat, J. P. IgX antibodies in the urodele amphibian Ambystoma mexicanum. Dev. Comp. Immunol. 32, 908915 (2008).

70. Boxio, R., Dournon, C. & Frippiat, J. P. Effects of a long-term spaceight on immunoglobulin heavy chains of the urodele amphibian Pleurodeles waltl.J. Appl. Physiol. 98, 905910 (2005).71. Bascove, M., Huin-Schohn, C., Gueguinou, N., Tschirhart, E. & Frippiat, J. P. Spaceight-associated changes in immunoglobulin VH gene expression in the amphibian Pleurodeles waltl. FASEB J. 23, 16071615 (2009).

72. Bascove, M., Gueguinou, N., Schaerlinger, B., Gauquelin-Koch, G. & Frippiat, J. P. Decrease in antibody somatic hypermutation frequency under extreme, extended spaceight conditions. FASEB J. 25, 29472955 (2011).

73. Huin-Schohn, C. et al. Gravity changes during animal development affect IgM heavy-chain transcription and probably lymphopoiesis. FASEB J. 27, 333341 (2013).

74. Ghislin, S., Ouzren-Zarhloul, N., Kaminski, S. & Frippiat, J. P. Hypergravity exposure during gestation modies the TCRb repertoire of newborn mice. Sci. Rep. 5, e92664 (2015).

75. Lescale, C. et al. Hind limb unloading, a model of spaceight conditions, leads to decreased B lymphopoiesis similar to aging. FASEB J. 29, 455463 (2015).

76. Wilson, J. W. et al. Space ight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq. Proc. Natl Acad. Sci. USA 104, 1629916304 (2007).

77. Wilson, J. W. et al. Media ion composition controls regulatory and virulence response of Salmonella in spaceight. PLos ONE 3, e3923 (2008).

78. Crabb, A. et al. Spaceight enhances cell aggregation and random budding in Candida albicans. PLos ONE 8, e80677 (2013).

79. Leys, N. et al. The response of Cupriavidus metallidurans CH34 to spaceight in the international space station. Anton. Leeuw. Int. J. G. 96, 227245 (2009).

80. Crabbe, A. et al. Transcriptional and proteomic responses of Pseudomonas aeruginosa PAO1 to spaceight conditions involve Hfq regulation and reveal a role for oxygen. Appl. Environ. Microb. 77, 12211230 (2011).

81. Juergensmeyer, M. A., Juergensmeyer, E. A. & Guikema, J. A. Long-term exposure to spaceight conditions affects bacterial response to antibiotics. Micrograv. Sci. Tech. 12, 4147 (1999).

82. Lebsack, T. W. et al. Microarray analysis of spaceown murine thymus tissue reveals changes in gene expression regulating stress and glucocorticoid receptors. J. Cell Biochem. 110, 372381 (2010).

83. Tobaldini, E. et al. One night on-call: Sleep deprivation affects cardiac autonomic control and inammation in physicians. Eur. J. Intern. Med. 24, 664670 (2013).

84. Flierl, M. A. et al. Phagocyte-derived catecholamines enhance acute inammatory injury. Nature 449, 721725 (2007).

85. Sternberg, E. M. Neural regulation of innate immunity: a coordinated nonspecic host response to pathogens. Nat. Rev. Immunol. 6, 318328 (2006).

86. Chouker, A. et al. Motion Sickness, Stress and the Endocannabinoid System. PLos ONE 5, e10752 (2010).

87. Kaufmann, I. et al. Parabolic ight primes cytotoxic capabilities of polymorpho-nuclear leucocytes in humans. Eur. J. Clin. Invest. 39, 723728 (2009).

88. Stowe, R. P., Sams, C. F. & Pierson, D. L. Effects of mission duration on neuroimmune responses in astronauts. Aviat. Space Environ. Med. 74, 12811284 (2003).

89. Crucian, B. & Sams, C. Immune system dysregulation during spaceight: clinical risk for exploration-class missions. J. Leukoc. Biol. 86, 10171018 (2009).

90. Mehta, S. K., Crucian, B., Pierson, D. L., Sams, C. & Stowe, R. P. Monitoring immune system function and reactivation of latent viruses in the Articial Gravity Pilot Study. J. Gravit. Physiol. 14, P21P25 (2007).

91. Crucian, B. E. et al. Immune status, latent viral reactivation, and stress during long-duration head-down bed rest. Aviat Space Environ. Med. (2009); 80, A37A44 (2009).

92. Gmnder, F. K. et al. Effect of head-down tilt bedrest (10 days) on lymphocyte reactivity. Acta Physiol. Scand. Suppl. 604, 131141 (1992).

93. Schmitt, D. A. et al. Head-down tilt bed rest and immune responses. Pugers Arch. 441, R79R84 (2000).

94. Choukr, A. et al. Simulated microgravity, psychic stress, and immune cells in men: observations during 120-day 6 degrees HDT. J. Appl. Physiol. 90, 17361743 (2001).

95. Shearer, W. T. et al. Immune responses in adult female volunteers during the bed-rest model of spaceight: antibodies and cytokines. J. Allergy Clin. Immunol. 123, 900905 (2009).

96. Kelsen, J. et al. 21 Days head-down bed rest induces weakening of cell-mediated immunitySome spaceight ndings conrmed in a ground-based analog. Cytokine 59, 403409 (2012).

97. Xu, X. et al. Changes of cytokines during a spaceight analog--a 45-day head-down bed rest. PLos ONE 8, e77401 (2013).

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