The role of microgravity and stress-related humoral factors in dysregulated NK-cell function during spaceflight (First Award Fellowship)
Austin Bigley, Ph.D.
University of Houston
Before we can ethically send astronauts to distant locations in space, such as Mars or asteroids, it is critical that we understand how spaceflight affects the human immune system. For years, post-flight data has suggested that spaceflight has a negative impact on the immune system. Unfortunately, the majority of this data was obtained following short-duration missions after the astronauts had returned to Earth, meaning that no in-flight data was collected. The recent ‘Integrated Immune’ study filled many of these knowledge gaps by analyzing blood obtained from astronauts on the Space Shuttle or International Space Station (ISS) while they were still in space. Of particular interest were the findings that anti-viral immune responses were compromised during spaceflight. Consequently, latent viral reactivation can result in many negative consequences including Shingles, reduced vaccine efficacy, and increased susceptibility to infection.
While the ‘Integrated Immune’ study focused primarily on the adaptive immune system, our ‘Salivary Markers’ flight experiment has focused on the innate immune system. The current proposal focuses on natural killer (NK)-cells, which are able to kill virally-infected and malignant cells without prior exposure. Our in-flight data shows that NK-cell anti-tumor activity is greatly reduced during spaceflight, while cytomegalovirus (CMV)-driven responses are amplified. The current proposal will explore two plausible mechanisms for these observations; microgravity and stress. The effects of microgravity can be simulated with a rotating wall vessel that keeps the cells in a constant state of freefall, while stress can be simulated using serum (with natural stress hormones) from astronauts on ISS. It is hypothesized that treatment with simulated microgravity and spaceflight-derived serum will mimic the deleterious effects of spaceflight on NK-cells. Once we have determined the mechanisms underpinning the adverse effects of spaceflight on NK-cells, we can begin to develop countermeasures that will protect future space explorers from becoming immunocompromised during long-duration missions.
NASA Taskbook Entry
HRP Risk Title: Risk of Crew Adverse Health Event Due To Altered Immune Response
The specific relevant HRP Gaps for this proposal are as follows:
Gap IM1: Does spaceflight alter immune function?
Gap IM2: Is an improved standard needed?
Gap IM8: What is the correlation of immune risks with other risks, particularly psychological stress, physical deconditioning, nutrition and/or radiation?
Rationale: Post-flight data suggests that immune functions, including NK-cell cytotoxicity, are dysregulated following spaceflight. Unfortunately, these prior data are confounded by the acute stress of high-G entry and acclimation to terrestrial gravity. Our lab, however, has collected blood from astronauts on the ISS and measured the real-time effects of spaceflight on NK-cell activity and phenotype. Our data show that spaceflight is associated with major phenotypic and functional perturbations, including decreases in NK-cell Perforin/Granzyme B expression and anti-tumor cytotoxicity as well as cytomegalovirus (CMV)-associated expansion of the NKG2C+ NK-cell subset. Through this proposal, we aim to provide insight into the mechanisms underlying these phenomena by dissecting the roles of stress-induced humoral factors and microgravity itself.
Overarching Aim: The aim of this project is to determine the effects of spaceflight-derived serum and simulated microgravity on NK-cell phenotypic and functional responses to CMV-infected cells and tumor cell lines. The overarching hypothesis is that spaceflight-derived serum (via cortisol) and simulated microgravity will reduce anti-tumor NK-cell activity and perforin/Granzyme B expression, and enhance CMV-driven expansion of NKG2C+/NKG2A- NK-cells similarly to spaceflight. The specific aims to test this hypothesis are:
Specific Aim 1: Determine the effects of spaceflight-derived serum and simulated microgravity on NK-cell phenotype and NK-cell activity (NKCA) against CMV-infected fibroblasts and tumor cell lines with varying patterns of Human Leukocyte Antigen (HLA) expression.
Hypothesis 1a: Treatment of NK-cells with simulated microgravity will decrease anti-tumor NKCA and perforin/Granzyme B expression relative to a static control. Approach: NK-cell phenotypes (i.e. Perforin/Granzyme B expression) and function (NKCA against CMV-infected fibroblast, K562, U266, 721.221, and 221.AEH cell lines) will be determined using flow cytometry before and after 24h culture in a rotating microgravity-bioreactor system [0 rpm (static) or 30 rpm (microgravity)]. The change in NK-cell phenotype and function following 24h culture in microgravity will be compared to 24h static culture.
Hypothesis 1b: Treatment of NK-cells with spaceflight-derived serum will decrease NKCA and Perforin/Granzyme B expression relative to an autologous non-flight control through a cortisol-dependent mechanism. Approach: NK-cell phenotype and function will be determined using flow cytometry before and after 24h culture with either 50% spaceflight-derived serum (with or without Mifepristone to block Cortisol) or autologous non-flight/control serum. The change in NK-cell phenotype and function following 24h culture with spaceflight-derived serum will be compared to 24h culture with autologous non-flight/control serum.
Specific Aim 2: Determine the effect of simulated microgravity on “CMV-specific” NKG2C+/NKG2A- NK-cell expansion relative to a static control.
Hypothesis 2: It is hypothesized that the expansion rate of NKG2C+/NKG2A- NK-cells will be highest in CMV+ subjects with a low initial proportion of NKG2C+ NK-cells and this preferential expansion of the NKG2C+/NKG2A- subset will be enhanced when cells are cultured in simulated microgravity. Approach: We will expand NK-cells in a rotating microgravity-bioreactor system spun at 30 rpm (simulated microgravity) and 0 rpm (static) over 14-days using CMV-infected fibroblasts and Interleukin-15 (IL-15). NK-cell phenotypes (i.e. NKG2C+/NKG2A-) and NKCA against tumor cell lines and CMV-infected fibroblasts will be determined at weekly intervals by flow cytometry.
Summary, Timeline and Long-Term Goals:
We anticipate that it will take 1 year to complete this project. The studies in SA1 and SA2 will determine the effects of microgravity and spaceflight-derived humoral stress factors on NK-cell phenotype, cytotoxicity, and CMV responses. This will provide us with novel insight into the potential mechanisms underlying the striking loss of NK-cell activity that is observed during spaceflight. These aims will largely overlap, cross-informing each other regarding interpretation. Both aims will strongly contribute to our long-term goals of developing sensible countermeasures to protect the immune system of astronauts during long-duration missions, such as traveling to Mars or an asteroid.
Findings to date suggest that spaceflight inhibits NK-cell function, but the mechanism underlying this inhibition remains unknown. The relevance of this research for NASA is that decreased NK-cell function has been linked to increased incidence of cancer and latent viral reactivation. The alterations observed include decreased anti-tumor NK-cell cytotoxicity and inhibited CMV-specific responses. The implication of these findings is that innate immunity on long-duration space missions might be compromised in astronauts and presents a potential threat to mission effectiveness and astronaut health. By isolating the contributions of factors such as microgravity and stress to decreased NK-cell-mediated immunity, we can begin to develop countermeasures that will protect future space explorers from becoming immunocompromised during long-duration missions.
The impact of our findings for Earth-based medical practice are that periods of prolonged unloading and/or stress (e.g. bed rest, surgery, and military deployment) may result in impaired NK-cell-mediated immunity. In addition, this work will lead to greater insights into the mechanisms underlying NK-cell interactions with tumor cells and CMV effects on NK-cells. This information may have relevance toward a better understanding of the underlying causes of impaired immunity in patients experiencing paralysis, chronic immobility or extended bed rest. Ultimately, this project will explore potential countermeasures for decreased NK-cell function in space and may also offer potential treatments for maintaining immunity in Earth-bound, non-weight-bearing patients and highly stressed individuals.