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Long-duration spaceflight is known to cause immune dysregulation in astronauts. Biomarkers of immune system function are needed to determine both the need for and effectiveness of potential immune countermeasures for astronauts. Whereas plasma cytokine concentrations are a well-established biomarker of immune status, salivary cytokine concentrations are emerging as a sensitive indicator of stress and inflammation. For this study, to aid in characterizing immune dysregulation during spaceflight, plasma and saliva cytokines were monitored in astronauts before, during and after long-duration spaceflight onboard the International Space Station. Blood was collected from 13 astronauts at 3 timepoints before, 5 timepoints during and 3 timepoints after spaceflight. Saliva was collected from 6 astronauts at 2 timepoints before spaceflight, 2 timepoints during and 3 timepoints following spaceflight. Samples were analyzed using multiplex array technology. Significant increases in the plasma concentration of IL-3, IL-15, IL-12p40, IFN-α2, and IL-7 were observed during spaceflight compared to before flight baseline. Significant decreases in saliva GM-CSF, IL-12p70, IL-10 and IL-13 were also observed during spaceflight as compared to compared to before flight baseline concentrations. Additionally, plasma TGFβ1 and TGFβ2 concentrations tended to be consistently higher during spaceflight, although these did not reach statistical significance. Overall, the findings confirm an in-vivo hormonal dysregulation of immunity, appearing pro-inflammatory and Th1 in nature, persists during long-duration orbital spaceflight. These biomarkers may therefore have utility for monitoring the effectiveness of biomedical countermeasures for astronauts, with potential application in terrestrial research and medicine., Competing Interests: SSK was employed by KBR. SM was employed by JES Tech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Krieger, Zwart, Mehta, Wu, Simpson, Smith and Crucian.)

The National Aeronautics and Space Administration (NASA) Twins Study created an integrative molecular profile of an astronaut during NASA's first 1-year mission on the International Space Station (ISS) and included comparisons to an identical Earth-bound twin. The unique biochemical profiles observed when landing on Earth after such a long mission (e.g., spikes in interleukin-1 [IL-1]/6/10, c-reactive protein [CRP], C-C motif chemokine ligand 2 [CCL2], IL-1 receptor antagonist [IL-1ra], and tumor necrosis factor alpha [TNF-α]) opened new questions about the human body's response to gravity and how to plan for future astronauts, particularly around initiation or resolution of inflammation. Here, single-cell, multi-omic (100-plex epitope profile and gene expression) profiling of peripheral blood mononuclear cells (PBMCs) showed changes to blood cell composition and gene expression post-flight, specifically for monocytes and dendritic cell precursors. These were consistent with flight-induced cytokine and immune system stress, followed by skeletal muscle regeneration in response to gravity. Finally, we examined these profiles relative to 6-month missions in 28 other astronauts and detail potential pharmacological interventions for returning to gravity in future missions., Competing Interests: Declaration of Interests Although not relevant to this study, for full disclosure, C.E.M. is a cofounder and board member for Biotia and Onegevity Health as well as an advisor or grantee for Abbvie, ArcBio, Daiichi Sankyo, DNA Genotek, Tempus Labs, and Whole Biome., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Success of long duration space missions will depend upon robust immunity. Decreased immunity has been observed in astronauts during short duration missions, as evident by the reactivation of latent herpes viruses. Seventeen astronauts were studied for reactivation and shedding of latent herpes viruses before, during, and after 9-14 days of 8 spaceflights. Blood, urine, and saliva samples were collected 10 days before the flight (L-10), during the flight (saliva only), 2-3h after landing (R+0), 3 days after landing (R+3), and 120 days after landing (R+120). Values at R+120 were used as baseline levels. No shedding of viruses occurred before flight, but 9 of the 17 (designated "virus shedders") shed at least one or more viruses during and after flight. The remaining 8 astronauts did not shed any of the 3 target viruses (non-virus shedders). Virus-shedders showed elevations in 10 plasma cytokines (IL-1α, IL-6, IL-8, IFNγ, IL-4, IL-10, IL-12, IL-13, eotaxin, and IP-10) at R+0 over baseline values. Only IL-4 and IP-10 were elevated in plasma of non-virus shedders. In virus shedders, plasma IL-4 (a Th2 cytokine) was elevated 21-fold at R+0, whereas IFNγ (a Th1 cytokine) was elevated only 2-fold indicating a Th2 shift. The inflammatory cytokine IL-6 was elevated 33-fold at R+0. In non-shedding astronauts at R+0, only IL-4 and IP-10 levels were elevated over baseline values. Elevated cytokines began returning to normal by R+3, and by R+120 all except IL-4 had returned to baseline values. These data show an association between elevated plasma cytokines and increased viral reactivation in astronauts., (Copyright © 2012. Published by Elsevier Ltd.)

In this study, flow cytometry was used to positively identify the specific lymphocyte subsets exhibiting space flight-induced alterations in cytokine production. Whole blood samples were collected from 27 astronauts at three points (one preflight, two postflight) surrounding four space shuttle missions. Assays performed included serum/urine stress hormones, white blood cell (WBC) phenotyping, and intracellular cytokine production following mitogenic stimulation. Absolute levels of peripheral granulocytes were significantly elevated following space flight, but the levels of circulating lymphocytes and monocytes were unchanged. Lymphocyte subset analysis demonstrated a decreased percentage of T cells, whereas percentages of B cells and natural killer (NK) cells remained unchanged after flight. Nearly all the astronauts exhibited an increased CD4/CD8 T cell ratio. Assessment of naive (CD45RA+) vs. memory (CD45RO+) CD4+ T cell subsets was ambiguous, and subjects tended to group within specific missions. Although no significant trend was seen in absolute monocyte levels, a significant decrease in the percentage of the CD14+ CD16+ monocytes was seen following space flight in all subjects tested. T cell (CD3+) production of interleukin-2 (IL-2) was significantly decreased after space flight, as was IL-2 production by both CD4+ and CD8+ T cell subsets. Production of interferon-gamma (IFN-gamma) was not altered by space flight for the CD8+ cell subset, but there was a significant decrease in IFN-gamma production for the CD4+ T cell subset. Serum and urine stress hormone analysis indicated significant physiologic stresses in astronauts following space flight. Altered peripheral leukocyte subsets, altered serum and urine stress hormone levels, and altered T cell cytokine secretion profiles were all observed postflight. In addition, there appeared to be differential susceptibility to space flight regarding cytokine secretion by T cell subsets. These alterations may be the result of either microgravity exposure or the physiologic stresses of landing and readaptation to unit gravity. Future studies, including in-flight analysis or sampling, will be necessary to determine the cause of these alterations.

Significant changes have recently been described regarding circulating peripheral immune cells immediately following spaceflight. Existing methods for immunophenotype staining of peripheral blood in terrestrial labs do not meet the constraints for flight on the Space Shuttle. We have recently described the development and use of the Whole Blood Staining Device (WBSD), a simple device for staining flow cytometry specimens during spaceflight. When preparing samples with the WBSD, all liquids are safely contained as the cells are moved through staining, lysis and fixation steps. Here we briefly review the use of the WBSD, and then describe another versatile adaptation, a modification to perform intracellular staining of cytokines for detection by flow cytometry. Alterations in cytokine production have been reported both in ground-based simulated microgravity culture and in astronaut samples returning from spaceflight. Data regarding microgravity effects on cytokine production for specific subpopulations of cells is lacking. Flow cytometric cytokine analysis offers the unique ability to perform simultaneous surface marker analysis and positively identity cytokine producing subsets of cells. The utilization of the WBSD provides the ability to perform rapid and routine mitogenic activation during spaceflight coupled with the ability to perform simultaneous surface marker analysis. The only external requirements for this procedure are an in-flight 37-degree incubator and the capacity for 4-degree storage.

Interferon-gamma-inducing factor (IGIF) is a novel cytokine that stimulates T-cell proliferation, augments natural killer (NK) cell lytic activity, and induces interferon-gamma (IFN-gamma) production in established type 1 T-helper (Th1) cells in the presence of anti-CD3 antibody. The in vitro induction of IFN-gamma by recombinant murine IGIF in these cells was more potent than that induced by murine interleukin-12 (IL-12) and occurred apparently independent of murine IL-12. Here we report that subcutaneous injection into mice of tumor cells transfected with murine IGIF complementary DNA (cDNA) resulted in > or = 10-fold increase of mitogen-stimulated IFN-gamma production in cultured splenocytes. In addition, IGIF-transfected Renca and K1735 tumor cells can be rejected in vivo. The IGIF antitumor effect was abrogated in mice that were sublethally irradiated or depleted of both CD4+ and CD8+ T cells but not in mice depleted of either subpopulation alone. The antitumor effect mediated by IGIF appears to be dependent on IFN-gamma production, because in vivo neutralization of IFN-gamma was accompanied by growth of IGIF-transfected tumors in 100% of the animals. Taken together, our results show that murine IGIF can elicit T-cell-dependent antitumor immunity associated with IFN-gamma induction.

Production of T helper 1 and T helper 2 cytokines was investigated in peripheral blood mononuclear cells (PBMCs) from multiple sclerosis (MS) patients by a newly described technique, detection of intracellular cytokines by flow cytometry in conjunction with immunophenotype analysis. T-cell gamma interferon (IFN-gamma) production and interleukin 10 (IL-10) production were examined after PBMC activation with T-cell mitogens at 5 and 24 h, and monocyte spontaneous production of IL-10 and production after PBMC activation with lipopolysaccharide (LPS) for 24 h were also examined. The data indicate that MS patients have decreased percentages of T cells capable of secreting IFN-gama compared with healthy controls, and this change is detectable at 5 and 24 h. the patients displaying decreased T-cell production of IFN-gamma were essentially confined to a group being treated with the newly approved drug Betaseron (berlex Labs, Cedar Knolls, N.J.), a recombinant form of IFN-beta (rIFN-beta 1b). By gating of the entire lymphocyte population, analysis of IFN-gama production in T cells (CD3+ versus that in non-T cells (CD3+) was possible. The percentage of IFN-gamma-producing lymphocytes that was made up of T cells was essentially unchanged between the Betaseron-treated patients, non-Betaseron-treated patients, and controls, indicating that the suppression of IFN-gamma production displayed by betaseron-treated MS patients was a nonspecific suppression of all IFN-gamma-producing lymphocytes as opposed to a suppression of T-cell production only. The data seem to indicate that treatment of MS with Betaseron corresponds to an inhibition of the lymphocyte's ability to produce IFN-gamma. No changes were detected in T-cell production of IL-10 at either time point. We also observed that MS patients in general appear to have small percentages of peripheral blood monocytes spontaneously producing slight but detectable levels of IL-10. No difference was seen regarding monocyte production of IL-10 after PBMC activation with LPS between MS patients and controls. Both populations responded with high percentages of monocytes producing IL-10. The data seem to indicate that treatment of MS with Betaseron, known to decrease the exacerbation rate of relapsing-remitting MS, corresponds to a suppression of peripheral blood lymphocyte production of IFN-gamma. Monocyte production of IL-10 may also play a role in regulating the disease process.

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS). The disease is characterized by inflammatory lesions in the white matter of the CNS, consisting of a specific immune response to the myelin sheath. We investigated peripheral blood mononuclear cell (PBMC) cytokine production by enzyme-linked immunosorbent assays of 21 MS patients and 19 age-matched normal controls in response to the T-cell mitogen phytohemagglutinin (PHA). Peripheral blood mononuclear cells were cultured in medium alone or in medium with 5 micrograms of PHA per ml for 48 h, and culture supernatants were collected for analysis. Cytokines selected for study were interleukin-10 (IL-10), gamma interferon (IFN-gamma), IL-2, and IL-4. All cytokine activities described were expressed as concentrations per 500,000 cells. We found that 48% (10 of 21) of the MS patients produced small but detectable levels of IL-10 in medium alone, compared with 26% (5 of 18) of the controls. We found that the MS patients produced significantly higher quantities of IL-10 protein than the controls in response to PHA (mean supernatant concentrations of IL-10 for patients and controls, 421 and 204 pg/ml, respectively [P < 0.05]). No significant differences were detected in the production of IL-2, IFN-gamma, and IL-4 between patients and controls in response to PHA, although patients appeared to display a trend toward decreased production of IFN-gamma.