Spaceflight is known to affect immune cell populations. In particular splenic B-cell numbers decrease during spaceflight and in ground-based physiological models. Although antibody isotype changes have been assessed during and after spaceflight an extensive characterization of the impact of spaceflight on antibody composition has not been conducted in mice. Next Generation Sequencing and bioinformatic tools are now available to assess antibody repertoires. We can now identify immunoglobulin gene- segment usage junctional regions and modifications that contribute to specificity and diversity. Due to limitations on the International Space Station alternate sample collection and storage methods must be employed. Our group compared Illumina MiSeq xc2 xae sequencing data from multiple sample preparation methods in normal C57Bl/6J mice to validate that sample preparation and storage would not bias the outcome of antibody repertoire characterization. In this report we also compared sequencing techniques and a bioinformatic workflow on the data output when we assessed the IgH and Ig xce xba variable gene usage. Our bioinformatic workflow has been optimized for Illumina HiSeq xc2 xae and MiSeq xc2 xae datasets and is designed specifically to reduce bias capture the most information from Ig sequences and produce a data set that provides other data mining options.

Immune dysregulation is among the main adverse outcomes of spaceflight. Despite its crucial role in host protection effects on the human antibody repertoire are unknown. Consequently we examined the IgM repertoire of five cosmonauts 25 days before launch after 64 and 129 days spent on the International Space Station (ISS) and after 1 7 and 30 days of Earth reambulation using high-throughput sequencing. This is the first study of this kind. Our data revealed that the IgM repertoire of cosmonauts was different from the one of control subjects already prior to launch and that 2 out the 5 analyzed cosmonauts presented significant changes of their IgM repertoire during the mission. These modifications persisted up to 30 days after landing likely affected the specificities of IgM binding sites correlated with changes of the V(D)J recombination process in charge of creating functional antibody genes and coincided with a higher stress response. These data confirm that about half of the astronauts who spent six months on the ISS are subjected to immunological changes contribute to explain increased susceptibility to infection reveal individual responses and consequently that personalized approaches should be implemented during future deep-space exploration missions that will endure for an unprecedented amount of time.

Bone loss and immune dysregulation are among the main adverse outcomes of spaceflight challenging astronaut xe2 x80 x99s health and safety. However consequences on B cell development and responses are still under-investigated. Up to now most studies addressing these questions were performed using an amphibian species. Consequently we used advanced proteomics analysis of femur bone and marrow of mice flown for one month on board the BION-M1 biosatellite to determine whether extreme conditions encountered during a real spaceflight affect B cell development in mice and to examine reversibility of the effects upon return to Earth. Our data revealed that adverse effects on B lymphopoiesis were more marked one week after landing and that this phenomenon was associated with a 41% reduction of B cells in the spleen. Thus the effects of spaceflight persisted during at least one week after landing. These reductions may contribute to explain increased susceptibility to infection even if we confirmed that animals were able to mount a humoral immune response.

In the Rodent Research Reference Mission (RRRM-2), forty female C57BL/6NTac mice were flown on the International Space Station. To assess differences in outcomes due to age, twenty 12 week-old and twenty 29 week-old mice were flown, respectively. To directly assess spaceflight effects, half of the young and old mice (10 old, 10 young) were sacrificed on-orbit after 55-58 days (ISS Terminal, ISS-T), while the other half (10 old, 10 young) were returned live to Earth after 32 days and allowed to recover for 24 days (Live Animal Return, LAR) before sacrifice. ISS-T and LAR mice were the same age at sacrifice. Both the ISS-T and LAR animals had independent ground controls (10 mice per group housed in flight hardware in matched environmental conditions), basal controls (10 mice per group sacrificed 2 days before launch), and vivarium controls (10 mice per group housed within standard vivarium habitats). Thus RRRM-2 included a total of 160 mice This study includes single cell transcriptional profiling data from peripheral blood mononuclear cells (PBMCs) from 4 young LAR flight animals, 4 old LAR flight animals, 4 young LAR ground control animals, and 4 old LAR ground control animals.

Space travel presents unlimited opportunities for exploration and discovery, but requires a more complete understanding of the immunological consequences of long-term exposure to the conditions of spaceflight. To understand these consequences better and to contribute to design of effective countermeasures, we used the Drosophila model to compare innate immune responses to bacteria and fungi in flies that were either raised on earth or in outer space aboard the NASA Space Shuttle Discovery (STS-121). Microarrays were used to characterize changes in gene expression that occur in response to infection by bacteria and fungus in drosophila that were either hatched and raised in outer space (microgravity) or on earth (normal gravity). Whole Oregon R strain drosophila melanogaster fruit flies either raised on earth or in space that were (1) uninfected, (2) infected with bacteria (Escherichia coli), or (3) infected with fungus (Beauveria bassiana) were used for RNA extraction and hybridization on Affymetrix microarrays.

In the Rodent Research Reference Mission (RRRM-2), forty female C57BL/6NTac mice were flown on the International Space Station. To assess differences in outcomes due to age, twenty 12 week-old and twenty 29 week-old mice were flown, respectively. To directly assess spaceflight effects, half of the young and old mice (10 old, 10 young) were sacrificed on-orbit after 55-58 days (ISS Terminal, ISS-T), while the other half (10 old, 10 young) were returned live to Earth after 32 days and allowed to recover for 24 days (Live Animal Return, LAR) before sacrifice. ISS-T and LAR mice were the same age at sacrifice. Both the ISS-T and LAR animals had independent ground controls (10 mice per group housed in flight hardware in matched environmental conditions), basal controls (10 mice per group sacrificed 2 days before launch), and vivarium controls (10 mice per group housed within standard vivarium habitats). Thus RRRM-2 included a total of 160 mice. This study includes single cell transcriptional profiling data from the spleens from 4 young LAR flight animals, 4 old LAR flight animals, 4 young LAR ground control animals, and 4 old LAR ground control animals.

The objective of the Rodent Research-9 (RR-9) mission was to use mice to understand the molecular basis of phenomena that affect astronauts during long-duration spaceflight, particularly visual impairment, and joint tissue degradation. To this end, a flight group (FLT) of 10-week-old male C57BL/6J mice were launched from Kennedy Space Center (KSC) on 8/14/2017 and housed in Rodent Habitats on the ISS for 33 days before being returned alive to Earth. After splashdown in the Pacific Ocean, the animals were transported to Loma Linda University (LLU) for testing, euthanasia, and dissection on 9/18/2018. Ground Control (GC) studies were planned to commence at KSC approximately one-week after the conclusion of the flight experiments. However, all the GC mouse studies at KSC had to be cancelled due to Hurricane Irma and potential adverse effects on the animal housing facility. The GC studies were therefore rescheduled and begun in May 2018. The GC was euthanized and dissected 6/18/2018 - 6/20/2018. Because this resulted in a different cohort of mice being used for the GC controls as compared to the flight (FLT) groups, two cohort controls were included in the study. The first, Cohort Control 1 (CC_C1), was from the same cohort as the FLT animals and was sacrificed and dissected 4 days after the FLT group (9/22/2017). The second, Cohort Control 2 (CC_C2), was from the same cohort as the GC, and was sacrificed and dissected 2-8 days after the GC, (6/24/2018 - 6/26/2018). The CC_C1 and CC_C2 groups were housed in standard cages and fed standard chow in contrast to all other groups which received Rodent Foodbars. Upon dissection, spleen tissues were preserved in liquid nitrogen and stored at 80 C before RNA was extracted. Only the flight (FLT, n of 10) and Ground Control (GC, n of 10) samples were processed and analyzed in this study. Libraries were generated using a 3’ Tag-seq approach and sequenced at a targeted depth of 40 M clusters (SE 93 bp).

Future space missions will include return to the Moon and long duration deep space roundtrip missions to Mars. Leaving the protection that Low Earth Orbit provides will unavoidably expose astronauts to higher cumulative doses of space radiation, in addition to other stressors, e.g. microgravity. Immune regulation is known to be impacted and it remains to be seen whether prolonged effects will be encountered in deep space that can have an adverse impact on health. In this study we investigated the effects in overall metabolism of three different low dose radiation exposures (γ-rays, 16O, and 56Fe) in spleen from male C57BL/6 mice at 1, 2, and 4 months after exposure. Forty metabolites were identified with significant enrichment in purine metabolism, tricarboxylic acid cycle, fatty acids, acylcarnitines, and amino acids. Early perturbations were more prominent in the γ irradiated samples, while longer term responses shifted towards the high energy particle effects. Regression analysis showed a positive correlation of fatty acids with time and negative association with γ-rays, while degradation of purines were positively associated with time. Taken together, there is a strong suggestion of mitochondrial implication and the possibility of long term effects in DNA repair and nucleotide pools following radiation exposure.

We differentiated mouse bone marrow cells in the presence of recombinant macrophage colony stimulating (rM-CSF) factor for 14 days during the flight of space shuttle Space Transportation System (STS)-126. We tested the hypothesis that the receptor expression for M-CSF, c-Fms was reduced. We used flow cytometry to assess molecules on cells that were preserved during flight to define the differentiation state of the developing bone marrow macrophages; including CD11b, CD31, CD44, Ly6C, Ly6G, F4/80, Mac2, c-Fos as well as c-Fms. In addition, RNA was preserved during the flight and was used to perform a gene microarray. We found that there were significant differences in the number of macrophages that developed in space compared to controls maintained on Earth. We found that there were significant changes in the distribution of cells that expressed CD11b, CD31, F4/80, Mac2, Ly6C and c-Fos. However, there were no changes in c-Fms expression and no consistent pattern of advanced or retarded differentiation during space flight. We also found a pattern of transcript levels that would be consistent with a relatively normal differentiation outcome but increased proliferation by the bone marrow macrophages that were assayed after 14 days of space flight. There also was a surprising pattern of space flight influence on genes of the coagulation pathway. These data confirm that a space flight can have an impact on the in vitro development of macrophages from mouse bone marrow cells.

While it has been shown that astronauts suffer immune disorders after spaceflight, the underlying causes are still poorly understood and there are many variables to consider when investigating the immune system in a complex environment. Additionally, there is growing evidence that suggests that not only is the immune system being altered, but the pathogens that infect the host are significantly influenced by spaceflight and ground-based spaceflight conditions. In this study, we demonstrate that Serratia marcescens (strain Db11) was significantly more lethal to Drosophila melanogaster after growth on the International Space Station than ground-based controls, but that the host immune system is not significantly altered amongst known immune genes. High-throughput sequencing of wild-type (w1118) adult hosts infected with either space or ground-reared S. marcescens revealed few changes in gene expression, with 11 genes significantly differentially expressed (q-values less than 0.05) and only one gene related to the immune system. This data supports the main findings of the paper, which state that both spaceflight and low-shear modeled microgravity conditions increase the virulence of this pathogen, independent of the host immune system. This data, which shows that there are no significant immune-related changes to the host when infected with space-grown sample compared to ground-grown sample, provides further evidence that there are likely phenotypic changes to the pathogen itself that is causing increased virulence in spaceflight and in low-shear modeled microgravity. RNA was extracted in triplicate from 2 pooled adult (2-3 day old female) Drosophila melanogaster (w1118) per treatment, with 4 total treatment groups (no injection control, sham injection with PBS, ground bacteria-injected, and space bacteria-injected) with poly(A)+ RNA libraries. Samples were multiplexed and sequenced 100bp paired-end ready were sequenced on one lane of the Illumina HiSeq-4000.