EXPERIMENT RECORD N° 8314
IMMUNO - Neuroendocrine and immune responses in humans during and after long term stay at ISS
  1. 2005 • ISS Increment 12
  2. 2006 • ISS Increment 13
  3. 2006 • ISS "Astrolab" - long-duration mission
  4. 2007 • ISS Increment 15
  5. 2007 • ISS Increment 16
  6. 2011 • ISS Increments 29-30
  7. 2012 • ISS Increments 31-32
  8. 2012 • ISS Increments 33-34
  9. 2013 • ISS Increments 35-36
Life Sciences:
  • Human Physiology
  • Immunology and Haematology
  • Medicine/Health
Jennifer Ngo-Anh
jennifer.ngo-anh@esa.int
A. Chouker (1), F. Christ (1), M. Thiel (1), I. Kaufmann (1), B. Morukov (2), I. Nichiporuk (2)
(1)  
Ludwig-Maximilians Universität
Klinikum Grosshadern München
Department of Anesthesiology
Marchioninistrasse 15
81377 München
GERMANY
Tel:  
+49(0)89.7095.64.22
Fax:  
+49(0)89.7095.88.86
e-mail:  
alexander.chouker@med.uni-muenchen.de
(2)  
Institute for Biomedical Problems (IBMP)
State Research Center of The Russian Federation
Khoroshevskoye Shosse 76 A
Moscow 123007
RUSSIA
Tel:  
+7.4991.95.23.63
Fax:  
+7.4991.95.15.00
e-mail:  
morukov@imbp.ru
BACKGROUND
Space flight can affect every organ system and may lead to several physiological changes (immune function, muscle atrophy, demineralisation of bones, changes in the circulation). Alterations in immune responses during and after space flight have been reported for humans which could be a consequence of microgravity and radiation. In addition environmental stress factors (confinement, artificial circadian rhythm) occur and altogether affect neuroendocrine stress and immune responses. In studies precluding the initiation of Russian, European and American space agencies to establish the long-term presence of people in Space (ISS-International Space Station, mission to Mars), ground-based simulation studies have allowed to investigate possible unfavourable changes of health under standardised conditions. Hence, ground based studies are carried out to mimic the conditions which may occur during space flight to a certain extent: the effects of low gravitation can be simulated during long-term hypokinesia in head down tilt at 6° (HDT 6°) or water immersion. Confinement can be induced operationally in submarines, polar stations, deep sea laboratories as well as during expeditions in the Antarctica. Ground-based investigations in normal pressurised space modules were also used to investigate neuroendocrine and immunological alterations which do occur during and/or after space flight . Even though these studies allow investigation of physiological adaptation to space related environmental conditions and are very useful in the development of „tools“ and methods for a subsequent use in space, „real-space-flight“ conditions remain the „golden standard“.

In previous studies performed in cooperation with the national German space agency (DLR) and the Russian partners form the IMBP indicate the impact of space flight stressors to the human immune systems. These changes appeared to be associated to the extent of the physical and psychic stress experienced by the participating individuals (publication from our group as follows:

A. Choukèr, M. Thiel, V. Baranov, D. Meshkov, A. Kotov, K. Peter, K. Messmer, F. Christ, (2001), "Simulated microgravity, psychic stress, and immune cells in men: observations during 120-day 6 degrees HDT", Journal of Applied Physiology, 90 (5), pp. 1736-1743.

F. Christ, J. Gamble, V. Baranov, A. Kotov, A. Chouker, M. Thiel, et al., (2001), "Changes in microvascular fluid filtration capacity during 120 days of 6 degrees head-down tilt", Journal of Applied Physiology, 91 (6), pp. 2517-2522.

A. Choukèr, L. Smith, F. Christ, I. Larina, I. Nichiporuk, V. Baranov, et al., (2002), "Effects of confinement (110 and 240 days) on neuroendocrine stress response and changes of immune cells in men", Journal of Applied Physiology (Bethesda, Md.: 1985), 92 (4), pp. 1619-27, doi:10.1152/japplphysiol.00732.2001.

A. Choukèr, I. Kaufmann, S. Kreth, D. Hauer, M. Feuerecker, D. Thieme, M. Vogeser, M. Thiel, G. Schelling, (2010) "Motion Sickness, Stress and the Endocannabinoid System", PLoS One, 5 (5), pp. e10752., doi:  10.1371/journal.pone.0010752, ed. Alejandro Lucia.

C. Strewe, M. Feuerecker, I. Nichiporuk, I. Kaufmann, D. Hauer, B. Morukov, G. Schelling, A. Chouker, (2012), "Effects of parabolic flight and spaceflight on the endocannabinoid system in humans", Nature Reviews Neuroscience, 23 (5-6), pp. 673-80.

M. Feuerecker, W. Mayer, I. Kaufmann, M. Gruber, F. Muckenthaler, Yi B., A.P. Salam, J. Briegel, G. Schelling, M. Thiel, A. Chouker, (2013), "A corticoid-sensitive cytokine release assay for monitoring stress-mediated immune modulation", Clinical & Experimental Immunology, 172, pp. 290-299.

B. Yi, M. Rykova, M. Feuerecker, B. Jäger, C. Ladinig, M. Basner, M. Hörl, S. Matzel, I. Kaufmann, C. Strewe, I. Nichiporuk, G. Vassilieva, K. Rinas, S. Baatout, G. Schelling, M. Thiel, D.F. Dinges, B. Morukov, A. Choukèr, (2014), "520-d Isolation and confinement simulating a flight to Mars reveals heightened immune responses and alterations of leukocyte phenotype", Brain, Behaviour, and Immunity, 40, pp. 203-210, doi: 10.1016/j.bbi.2014.03.018.

OBJECTIVES
On the basis of the experience and results obtained from previous experiments we now want to determine neuroendocrine and immunological changes during and after long-term stay at ISS.
Neuroendocinology: e. g psychic stress tests, stress hormones.
Immunology: e.g. numerical and functional analysis of unspecific (polymorphonuclear leukocytes, PMNL: expression of ß2-integrins, oxygen radicals) and specific immunity (lymphocytes-subpopulation and specific cytokine production, in vitro delayed type hypersensitivity reaction towards recall antigens, mRNA expressions).
Our routine methodology will be extended by parameters developed in our laboratory to determine cellular energy metabolism (purines) and cell signalling. These applications are focused to achieve:
i) additional information on the psycho-neuroendocrine and immunologic adoption of human physiology to space and
ii) a better insight how these processes are dependent on the cellular level of signal processing.

AIMS
The experiments aim to provide the basis for the development of pharmacological tools to countermeasure unwanted immunological side effects during long-term-stay in space.

BACKGROUND and JUSTIFICATION for the need of space environment:
Space flight can affect every organ system and may lead to several physiological changes (immune function, muscle atrophy, demineralisation of bones, changes in the circulation). Alterations in immune responses during and after space flight have been reported for humans, which could be a consequence of microgravity and radiation. In addition environmental stress factors (confinement, artificial circadian rhythm) occur and altogether affect neuroendocrine stress and immune responses.
In studies precluding the initiation of Russian, European and American space agencies to establish the long-term presence of people in Space (ISS-International Space Station, mission to Mars), ground-based simulation studies have allowed to investigate possible unfavourable changes of health under standardised conditions. Hence, ground based studies are carried out to mimic the conditions which may occur during space flight to a certain extent: the effects of low gravitation can be simulated during long-term hypokinesia in Head Down Tilt at 6° (HDT 6°) or water immersion.
Confinement can be induced operationally in submarines, polar stations, deep sea laboratories as well as during expeditions in the Antarctica. Ground-based investigations in normal pressurised space modules were also used to investigate neuroendocrine and immunological alterations which do occur during and/or after space flight. Even though these studies allow investigation of physiological adaptation to space related environmental conditions and are very useful in the development of „tools“ and methods for a subsequent use in space, „real-space-flight“ conditions remain the „golden standard.

RELATED RESEARCH
CoSi-500 Study - Confinement for 500 days - Evaluation of Stress and Immunity
Mars105 - 2009
Mars500 - 2010

Immunological changes during bed-rest: the role of countermeasures
BRAG - Bed Rest with Artificial Gravity - 2011

TEST SUBJECTS
number of subjects desired: 8
number of subjects required: 6

IMMUNO pre-flight measurements:

1 pre-flight mesurement will be performed at L-30 to L-7days. The following test and samplings will be performed:

  • Psychic Test: stress test (CST-paper test, at time of saliva collection). Test will be done twice, in the morning and in the evening.
  • Urine collection: For determination of dopamine, or epinephrine and epinephrine in urine collected within 24 hours.
  • Saliva collection: Cortisol (saliva-samples): Saliva is collected in the morning (8 a.m.) and in the evening (8 p.m.) by chewing on a cotton swab for 30-45 seconds.  Fluid is removed from swab by centrifugation and kept frozen at -20°C.
  • Blood sample: EDTA blood, heparinsed blood, special vacutainer Syringe (Becton Dickenson, CPT tube for immediate separation of leukocytes), Special prefilled syringe including Adenosin stopping solution.

IMMUNO in-flight measurements:

2 in-flight measurements will be performed:

  • The first one at L+3months +30 days
  • the second one between L+5months and R-2weeks.

The following test and samplings will be performed :

  • Psychic Test: Morning after longest sleeping session, perform stress test (CST-paper test, at time of saliva collection). Test will be done twice, in the morning and in the evening.
  • Urine collection: Start to collect urine with the second urination after wake-up.
  • Saliva collection: Saliva is collected in the morning (during stress test) and in the evening (during stress test) by chewing on a cotton swab for 30-45 and kept at room temperature (ie at 18°C to 25°C)
  • Blood sample

IMMUNO post-flight measurements:

2 post flight measures will be performed at R+1, R+7, R+20 days. The BDC program will be the same than the one performed during pre-flight measures.

The reference time for the R+1 session will be ISS time and for R+7 days session, the reference time will be either Moscow or Houston time, whatever is applicable depending on the country of return.

planned DATA ANALYSES
1.) Psycho-neuroendocrine regulation:
a)
Current stress test (CST-paper or computerized test):
The current-stress-test does not induce stress but evaluates the stress and (un-) comfort perceived on space flight as determined by a score.
b) Prolactin and Corticotropin Releasing Hormone (CRH) (EDTA-plasma): Determined by Enzyme Linked Immuno Assay
c) Catecholamines (urine): Determination of dopamine, norepinephrine and epinephrine in urine collected within 24 hours (subsequent 12hrs + 12hrs). Measurement of concentration by High Performance Liquid Chromatography (HPLC) on earth.
d) Cortisol – Amylase (saliva-samples): Enzyme Linked Immuno Assay
e) Anandamine, (endogenous cannabinoid) (EDTA blood) is determined by Fluorescence detection from whole blood.

2. Immune Monitoring:
a)
Relative Cell-Counts:
One drop of EDTA anti-coagulated whole blood (see 1 b) is placed on a blood smear glass device and a blood smear is conducted. The sample is dried thereafter at room air and stored in box at ambient room temperature until it is stained and analysed back on earth.
b) Cytokines and C-reactive protein (EDTA-Plasma). Multiplex-Analyses (up to 25 Pro and Anti-Inflammatory Cytokines), ELISA

3.) Energy metabolism and tissue perfusion (EDTA): 2ml
a)
Plasma concentrations of purines (xanthine and hypoxanthine):
Because adenosine and inosine measurement require the presence of a stopping solution which is has not been possible on orbit, the metabolites of adenosine, xanthine and hypoxanthine, are quantified from EDTA plasma without the need of “stopping solution using reverse phase HPLC.
b) Lactate: Colorimetric assay.

RESULTS
Long-duration spaceflight triggered a sustained stress dependent release of endocannabinoids combined with an aberrant immune activation mimicking features of people at risk for inflammation related diseases. These effects persisted in part 30 days after return to Earth. The currently available repertoire of in-flight testing as well as the post-flight observation periods need to be expanded to tackle the underlying mechanism for and consequences of these immune changes in order to develop corresponding mitigation strategies based on a personalized approach for future interplanetary space explorations.

Reference Document no 18 (see References section above or in the attachment section below) gives an comprehensive overview on the results of the research:
J.I. Buchheim, S. Matzel, M. Rykova, G. Vassilieva, S. Ponomarev, I. Nichiporuk, M. Hörl, D. Moser, K. Biere, M. Feuerecker, G. Schelling, D. Thieme, I. Kaufmann, M. Thiel, A. Choukèr, (2019), "Stress Related Shift Toward Inflammaging in Cosmonauts After Long-Duration Space Flight", Frontiers in Physiology, 10, DOI: 10.3389/fphys.2019.00085 ISSN=1664-042X, pp. 85.

PRELIMINARY RESULTS
The following preliminary results can be stated, considering that not all analyses are completed, especially from in-flight ISS samples:

A) The biological markers of stress showed a significantly increased psycho-neuroendocrine stress response.
B) The host defence against bacterial and fungal challenges was increased.

C) The Host defence against virus infection indicated a reduced antiviral, adaptive immune responses.

These findings are indicating a differentially “alerted” innate immune responses.

HYPOTHESIS

  1. Space flight induces psychic stress, affect neuro-endocrine regulation and initiate changes in the immune system.
  2. Exposure to microgravity causes modulation of unspecific (cytotoxic and bactericidal phagocyte properties) and specific immune responses (T-lymphocyte shift, immune-suppression).
  3. Exposure to microgravity affects the micro-circulation and hence lead to an altered tissue perfusion and energy metabolism as evidenced by increased concentrations of purines (e.g. adenosine). Adenosine affects functions of specific and unspecific immunity.

11 Russian cosmonauts and one ESA astronaut completed the IMMUNO experiment. A ground control of 5 healthy subjects living under normal conditions on earth has been conducted in parallel. 


BENEFITS FOR TERRESTRIAL RESEARCH / SPIN-OFF APPLICATIONS

At this stage (as of April 2014) it is too early to indicate benefits for terrestrial research as the analyses is not yet completed and could hence not compare to clinical setting and the results from other extreme but Earth-bound conditions (“space analogues”). However, the methodological needs and evolution of new methods to get advantage of a maximal data extraction of minimal amounts of blood which has led already to improvement in clinical research.

[1]  
L. Macho, R. Kvetnansky, M. Fickova, J. Kolena, J. Knopp, R.A. Tigranian, I.A. Popova, A.I. Grogoriev, (2001), "Endocrine responses to space flights", Journal of Gravitational Physiology, 8, 1, pp. 117-120.
[2]  
R.P. Stowe, S.K. Mehta, A.A. Ferrando, D.L. Feeback, D.L. Pierson, (2001), "Immune responses and latent herpesvirus reactivation in spaceflight", Aviation, Space, and Environmental Medicine, 72, pp. 884-891.
[3]  
R.P. Stowe, C.F. Sams, D.L. Pierson, (2003), "Effects of mission duration on neuroimmune responses in astronauts", Aviation, Space, and Environmental Medicine, 74, pp. 1281-1284.
[4]  
M.P. Rykova, E.N. Antropova, I.M. Larina, B.V. Morukov, (2008), "Humoral and cellular immunity in cosmonauts after the ISS missions", Acta Astronautica, 63, DOI: 10.1016/j.actaastro.2008.03.016., pp. 697-705.
[5]  
B.V. Morukov, M.P. Rykova, E. Antropova, T. Berendeeva, S. Ponomaryov, I. Larina, (2011), "T-cell immunity and cytokine production in cosmonauts after long-duration space flights", Acta Astronautica, 68, 7-8, http://dx.doi.org/10.1016/j.actaastro.2010.08.036, pp. 739-746.
[6]  
B.V. Morukov, M.P. Rykova, E.N. Antropova, T.A. Berendeeva, I.B. Morukov, S.A. Ponomarev, (2013), "Immunological aspects of a space flight to Mars", Human Physiology, 39, 2, pp. 126-135.
[7]  
I. Kaufmann, A. Hoelzl, F. Schliephake, T. Hummel, A. Choukèr, I. Lysenko, K. Peter, M. Thiel, (2007), "Effects of adenosine on functions of polymorphonuclear leukocytes from patients with septic shock.", Shock, 27, 1, pp. 25-31.
[8]  
A. Choukèr, A. Martignoni, R.J. Schauer, H.G. Rau, A. Volk, O. Heizmann, M. Dugas, K. Messmer, K. Peter, K.M. Thiel, (2005), "Ischemic preconditioning attenuates portal venous plasma concentrations of purines following warm liver ischemia in man", European Surgical Research, 37, 3, pp. 144-152.
[9]  
M. Thiel, A. Choukèr, A. Ohta, E. Jackson, C. Caldwell, P. Smith, D. Lukashev, I. Bittmann, M.V. Sitkovsky, (2005), "Oxygenation inhibits the physiological tissue-protecting mechanism and thereby exacerbates acute inflammatory lung injury", PLOS Biology, 3, 6.
[10]  
A. Choukèr, L. Smith, F. Christ, I. Larina, I. Nichiporuk, V. Baranov, E. Bobrovnik, L. Pastushkova, K. Messmer, K. Peter, M. Thiel, (2002), "Effects of confinement (110 and 240 days) on neuroendocrine stress response and changes of immune cells in men", Journal of Applied Physiology, 92, 4, pp. 1619-1627.
[11]  
F. Christ, J. Gamble, V. Baranov, A. Kotov, A. Choukèr, M. Thiel, I.B. Gartside, C.M. Moser, J. Abicht, K. Messmer, (2001), "Changes in microvascular fluid filtration capacity during 120 days of 6 degrees headdown tilt", Journal of Applied Physiology, 91, 6, pp. 2517-2522.
[12]  
A. Choukèr, M. Thiel, V. Baranov, D. Meshkov, A. Kotov, K. Peter, K. Messmer, F. Christ, (2001), "Simulated microgravity, psychic stress, and immune cells in men: observations during 120-day 6 degrees HDT", Journal of Applied Physiology, 90, 5, pp. 1736-1743.
[13]  
M. Thiel, J.D. Chambers, A. Choukèr, S. Fischer, C. Zourelidis, H.J. Bardenheuer, K.E. Arfors, K. Peter, (1996), "Effect of adenosine on the expression of beta(2) integrins and L-selectin of human polymorphonuclear leukocytes in vitro", Journal of Leukocyte Biology, 59, 5, pp. 671-682.
[14]  
M. Thiel, A. Choukèr, (1995), "Acting via A2 receptors, adenosine inhibits the production of tumor necrosis factor-alpha of endotoxin-stimulated human polymorphonuclear leukocytes", Journal of Laboratory and Clinical Medicine, 126, 3, pp. 275-282.
[15]  
C. Strewe, M. Feuerecker, I. Nichiporuk, I. Kaufmann, D. Hauer, B. Morukov, G. Schelling, A. Choukèr, (2012), "Effects of parabolic flight and spaceflight on the endocannabinoid system in humans", Nature Reviews Neuroscience, 23, 5-6, pp. 673-680.
[16]  
M. Feuerecker, W. Mayer, I. Kaufmann, M. Gruber, F. Muckenthaler, B. Yi, A.P. Salam, J. Briegel, G. Schelling, M. Thiel, A. Choukèr, (2013), "A corticoid-sensitive cytokine release assay for monitoring stress-mediated immune modulation", Clinical & Experimental Immunology, 172, pp. 290-299.
[17]  
A. Choukèr, F. Demetz, A. Martignoni, L. Smith, F. Setzer, A. Bauer, J. Holzl, K. Peter, F. Christ, M. Thiel, (2005), "Strenuous physical exercise inhibits granulocyte activation induced by high altitude", Journal of Applied Physiology, 98, 2, pp. 640-647.
[18]  
J.I. Buchheim, S. Matzel, M. Rykova, G. Vassilieva, S. Ponomarev, I. Nichiporuk, M. Hörl, D. Moser, K. Biere, M. Feuerecker, G. Schelling, D. Thieme, I. Kaufmann, M. Thiel, A. Choukèr, (2019), "Stress Related Shift Toward Inflammaging in Cosmonauts After Long-Duration Space Flight", Frontiers in Physiology, 10, DOI: 10.3389/fphys.2019.00085 ISSN=1664-042X, pp. 85.
click on items to display

Figure 1: Experiment Concept - Operational Overview

Figure 2: Experiment Concept - Operational Scenario Overview

Figure 3: Blood sample showing white blood cells: Lymphocytes (L) and Granulocytes (G).
http://eea.spaceflight.es
a.int/attachments/spacest
ations/ID5ca21d0253c5a.pd
f

ISS030-E-257690 (26 April 2012) European Space Agency astronaut Andre Kuipers, Expedition 30 flight engineer, prepares for IMMUNO venous blood sample draws in the Columbus laboratory of the International Space Station. Following the blood draws, the samples were temporarily stowed in the Minus Eighty Laboratory Freezer for ISS 1 (MELFI-1) and later packed together with saliva samples on the Soyuz TMA-22 for return to Earth for analysis. Credit: NASA/ESA

Reference Document [18] J.I. Buchheim, S. Matzel, M. Rykova, G. Vassilieva, S. Ponomarev, I. Nichiporuk, M. Hörl, D. Moser, K. Biere, M. Feuerecker, G. Schelling, D. Thieme, I. Kaufmann, M. Thiel, A. Choukèr, (2019), "Stress Related Shift Toward Inflammaging in Cosmonauts After Long-Duration Space Flight", Frontiers in Physiology, 10, DOI: 10.3389/fphys.2019.00085 ISSN=1664-042X, pp. 85.
 
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