Monitoring functional immune responses with a cytokine release assay: ISS flight hardware design and experimental protocol for whole blood cultures executed under microgravity conditions.

Introduction: Long-term space missions trigger a prolonged neuroendocrine stress response leading to immune system dysregulation evidenced by susceptibility to infections, viral reactivation, and skin irritations. However, due to existing technical constraints, real-time functional immune assessments are not currently available to crew inflight. The in vitro cytokine release assay (CRA) has been effectively employed to study the stimulated cytokine response of immune cells in whole blood albeit limited to pre- and post-flight sessions. A novel two-valve reaction tube (RT) has been developed to enable the execution of the CRA on the International Space Station (ISS). Methods: In a comprehensive test campaign, we assessed the suitability of three materials (silicone, C-Flex, and PVC) for the RT design in terms of biochemical compatibility, chemical stability, and final data quality analysis. Furthermore, we thoroughly examined additional quality criteria such as safety, handling, and the frozen storage of antigens within the RTs. The validation of the proposed crew procedure was conducted during a parabolic flight campaign. Results: The selected material and procedure proved to be both feasible and secure yielding consistent and dependable data outcomes. This new hardware allows for the stimulation of blood samples on board the ISS, with subsequent analysis still conducted on the ground. Discussion: The resultant data promises to offer a more accurate understanding of the stress-induced neuroendocrine modulation of immunity during space travel providing valuable insights for the scientific community. Furthermore, the versatile nature of the RT suggests its potential utility as a testing platform for various other assays or sample types.

Gravity-Related Immunological Changes in Human Whole Blood Cultured Under Simulated Microgravity Using an In Vitro Cytokine Release Assay.

Although immune dysfunction by space conditions has been reported postflight, as well as during ground-based experiments, the cause(s) and nature of the immunological changes are not completely understood. Microgravity has been suggested as one of the factors responsible for the observed immune dysregulation. The goal of this study was to assess immune changes in simulated microgravity (s-µG) using an in vitro cytokine release assay. The effect of s-µG provided by the desktop random positioning machine on cell-mediated immunity was examined by analyzing interleukin 2 (IL-2), interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 10 (IL-10), in response to immune cell stimulation in whole blood samples (n = 10). Stimuli used were bacterial recall antigens, pokeweed mitogen (PWM), lipopolysaccharide (LPS), or heat-killed Listeria monocytogenes (HKLM). S-µG caused an overall inhibition of the IL-2 and IFN-γ responses to recall antigen and mitogen stimulation. More specifically, s-µG most strongly influenced the levels of all four cytokines elicited by bacterial recall antigen stimulation. In contrast, HKLM-induced TNF-α secretion was elevated. The average concentrations of TNF-α in response to PWM and LPS and IL-10 release stimulated by PWM, LPS, and HKLM were not significantly altered by s-µG. However, a variable response between individual subjects could be observed. In conclusion, our results demonstrate that the in vitro cytokine release assay can detect gravity-related immune alterations. Furthermore, the use of multiple stimuli and the associated changes in cytokine secretion has the potential to reveal information on the underlying mechanisms affected by s-µG.