Investigating the Effect of Microgravity on Adaptive Immune Response to Viral Infection

Microgravity conditions associated with space flight have been shown to cause immune deficiencies in a number of ways, such as decreasing hematopoietic differentiation, cytokine production, and lymphocyte proliferation1. Space flight has also been shown to cause changes in DNA fragmentation and changes in lymphoid organ size in mice models2. Research on immunology in spaceflight became a prevalent topic when NASA published results of a significantly high re-emergence of latent viruses in astronauts returning from space missions, with additional studies showing decreases in cytokine activation and virus-specific T cells3, 4. The evidence for immune system impairment in microgravity conditions is particularly concerning given the plans for longer duration spaceflights for humans. Further research needs to be conducted to better understand the effect of microgravity on immune function. This proposed experiment will investigate the effect of microgravity on adaptive immune response to viral infection by inoculating mice with a virus and studying the immune responses of mice in simulated microgravity conditions. It is hypothesized that the mice in the simulated microgravity condition will show a lower antibody response to LCMV than the control group. Data will be obtained through the use of plaque assays, intracellular cytokine staining, tetramer staining, and ELISAs. Based on previous findings, is anticipated that the hypothesis will be supported; however, aberrant results would still provide insight to the role of microgravity on immune function and response. Introduction The first landing on the moon was in 1969, and NASA proposes to have humans land on Mars by 2030. Outer space is an exciting scientific frontier to explore, but the factors associated with spaceflights of any duration can lead to significant impairment of health. Unsurprisingly, leaving the earth’s orbit brings about a number of unfamiliar circumstances to the individuals who venture out of the atmosphere. The rocket’s takeoff results in a massive exposure to G forces, and the missions themselves tend to result in severe sleep deprivation, nutritional deficiencies, and anxiety or depression1. In addition, research shows that the immune system suffers from the microgravity conditions experienced. Microgravity refers to conditions where the force of gravity is so low that feelings of weightlessness occur. Gravity is generally measured by the speed at which an object would fall in free fall. On Earth, gravity is 9.807m/s2. This value significantly decreases once Earth’s atmosphere is exited—on Mars the force of gravity is 3.711m/s2, and on the moon, gravity is 1.622m/s2 (5). These significant decreases in gravity lead to observable impairment in immune function for a number of species. Drosophila melanogaster, the common fruit fly, showed a complete inability to activate Toll-mediated responses to fungal infections. The stress of microgravity on the body resulted in a faulty transcriptional response. Interestingly, hypergravity conditions, where the force of gravity was higher than experienced on earth, actually improved the signaling strength of Toll-responses, resulting in a faster clearing of the fungal infection6. In mice models, a number of immune changes have been observed. Lymphocyte proliferation is decreased due to a downregulation of T-cell activation markers CD25, CD69, and CD71. Decreased lymphocyte function and proliferation, especially for CD4 T-cells, occurred in a time-dependent manner with exposure to microgravity conditions7. Microgravity causes a significant unloading of mammalian tissues, which decreases tissue growth and regeneration. As a result, mice showed decreased hematopoiesis due to down-regulation of gene-expression markers for early mesenchymal and hematopoietic differentiation by at least two-fold. Cultures of bone marrow cells after microgravity conditions showed increase of mesenchymal differentiation to mineralized bone nodules, whereas hemotopoietic differentiation primarily resulted in osteoclasts. This indicates an increase of undifferentiated progenitor cells after microgravity8. Mice also show a decrease in lymphoid organ size relative to their body mass, with a 13-day spaceflight showing a significant decrease in spleen size. Thymus size was not significantly affected, but an increase in DNA fragmentation in the thymus was observed. T-cell and cancer gene expression markers were highly altered, with 30 out of 84 T-cell genes altered, and 15 out of 84 cancer related genes altered2. This evidence suggests that there could be an increased risk of infection and cancer development associated with spaceflight. IFNγ production is of particular importance, as it is the first response to viral infections. Rodents showed a decrease in IFNγ production in response to T-cell mitogen ConA9. The decrease in IFNγ present in mice causes Swiss/Webster mice that are normally immune to the D variant of encephalomyocarditis to become unusually susceptible in microgravity conditions10. A further investigation of rodent models also indicated an overproduction of cytokines IL-6 and IL-10, and a decrease in TNFα in microgravity conditions11. Humans also show an impaired immune response during microgravity conditions experienced during spaceflight. Astronauts show decreased functionality of their monocytes, with reduced ability to engulf E. coli, induce oxidative burst or degranulate. In addition, responses to gram-negative LPS endotoxins have decreased responsiveness12. These alterations in immunity are likely due to decreased expression of CD14 and increased expression of TLR4, due to LPS responsiveness depending on the association of the CD14-TLR4 myeloid differentiation protein 2 complex1. Cytotoxicity of non-MHC-restricted killer cells was found to be depressed in spaceflight as well, with a 40% decrease in lytic activity after landing13. Cytokine expression changes have also been observed in astronauts, suggesting further indication of decreased natural immune response. Astronauts returning from a 7day spaceflight showed low NK cell activity and decreased IFN secretion, which would reduce the ability to respond to viral infections14. The study of microgravity conditions on immune function became an especially prevalent topic when NASA observed a higher re-emergence of latent viruses in astronauts on short-duration space flights of 12 to 16 days. Astronauts showed increased re-emergence of Epstein-Barr virus, cytomegalovirus, herpes simplex I, and varicella virus3. NASA conducted further studies to investigate the alarming immune deficiencies in astronauts with simulated microgravity experiments. Research participants were found to have a decrease in activation of CD8+, CD69+, and virus-specific T-cells for both CMV and EBV4. Research on immune function in microgravity conditions is highly relevant due to NASA’s plans to send humans to Mars by mid 2030s—this mission would be a duration of a minimum of 520 days15. Additionally, bacteria actually survive better in microgravity conditions, leaving humans at an even greater risk for infections. Bacteria are found to proliferate faster, improve gene expression and pathogenesis, and require higher doses of antibiotics to kill. The increased radiation in space also subjects bacteria to higher levels of mutations, which could lead to increased virulence1. Spaceflight also includes inherent risks that could increase susceptibility to infections, such as the emotional stress due to confinement or fear of failure. Air, food, water, and waste are all recycled on board the space crafts, and the confinement of the passengers has been shown to increases in the transfer of microorganisms16, 17. The role of gravity on immune cells needs to be established before humans are sent on space missions lasting such an exorbitant amount of time, especially considering the evidence of decreased immune function in shortduration spaceflights. Further research could definitively establish a connection between mechanical unloading of tissues negatively impacting hematopoiesis, cytokine production, and adaptive and innate immune responses. Despite the compelling research suggesting significant immune impairment in microgravity conditions, gaps in scientific knowledge exist on the adaptive immune response to specific viral infections in microgravity conditions. This research will investigate the hypothesis that adaptive immune responses will be deficient in microgravity conditions when exposed to viral infection. It is suspected that mice subjected to microgravity conditions will show a lower antibody response of IFNγ and TNF at 30 days than control groups.