Microgravity alters the immune response to in vitro LPS assault engineered in spaceflight: A multi-omics study Microgravity can facilitate creation of a potent environment for opportunistic infection by augmenting virulence and suppressing the host defense. Presumably extraterrestrial infection may trigger potentially novel bionetworks different from the terrestrial equivalent which could only be probed by investigating the host-pathogen relationship with minimum terrestrial bias. Towards this objective we strategically engineered a cell culture module equipped with a feedback controlled semi-automated platform to expose human endothelial cells to lipopolysaccharide (LPS). The assay was carried out in the STS-135 space shuttle and a concurrent ground study constituted the baseline. Transcriptomic investigation revealed an immune blunting in microgravity; Lbp MyD88 and MD-2 failed to encode proteins responsible for early LPS uptake. Longer exposure results implied that there was a delayed response potentially ineffectual in preventing pathogens from opportunistically modulating the infection network. Lack of recruitment of growth factors and a debilitated apoptosome supported this potential explanation. Certain cytokines such as IL-6 and IL-8 surged in response to LPS insult in microgravity. Contrasting expressions of B2M TIMP-1 and VEGRs suggested impaired pro-survival adaptation and healing mechanisms. The susceptibility of oxidative stress and immune regulation to microgravity compelled further investigation of the respective microRNA modulators such as miR-200a and miR-146b. These miRNAs were expressed differently in response to LPS assaults in different gravitational limits. In conclusion despite a serious drawback attributed to the small sample size we delineated some of the important aspects of the extraterrestrial etiology; more comprehensive follow up studies are warranted. Present study though compromised by the small sample size was able to shade lights on several aspects of immunological responses to the endotoxic assault mediated by uG. Implementing the host-pathogen interactions in the spaceflight and subsequently lysing the cells onboard presented the critical distinguishing features of the present study from the past reports. We identified the CCM of Tissue Genesis Inc. HI as the suitable hardware system to carry out the experiment in the spaceflight. CCM is an automated feedback controlled module that can concurrently support 24 bioreactors following protocols exclusively programmed for individual bioreactor. For this experiment we use samples EA41 EA 47 EA45 and EA155 that were exposed to LPS for 4 hours. Samples EA123 EA165 EA127 EA126 were exposed to LPS for 8Hrs. Samples EA33 EA 125 EA79 and EA 39 were controls in this experiment.

This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen Candida albicans grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 454 genes compared to synchronous ground controls which represented 8.4% of the analyzed ORFs. Spaceflight-cultured C. albicans induced genes involved in cell aggregation (similar to flocculation) which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to more normal bipolar budding patterns for ground samples in accordance with the gene expression data. Furthermore genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight including induction of ABC transporters and members of the major facilitator family downregulation of ergosterol-encoding genes and upregulation of genes involved in oxidative stress resistance. Finally downregulation of genes involved in the actin cytoskeleton was observed. Interestingly the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested as this regulator is involved in random budding cell aggregation actin cytoskeleton and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype no increased virulence in a murine intraperitoneal (i.p.) infection model was observed. This study represents an important basis for the assessment of the risk that commensal flora could play during spaceflight missions. Furthermore since the low fluid-shear environment of microgravity is relevant to physical forces encountered by pathogens during the infection process insights gained from this study could identify novel infectious disease mechanisms with downstream benefits for the general public. Cells were grown for 24 hours on the space shuttle or as ground-based controls preserved in RNALater and stored at -80C. Four samples of each flight- and ground-based controls were harvested for microarray analysis. GAP is Group Activation Pack and each GAP contains 8 FPAs. The numbers represent the # assigned to the particular GAP and the number assigned to the specific FPA (1-8) within the indicated GAP. The same hardware is used for the flight samples and the ground samples.

This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen Candida albicans grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 454 genes compared to synchronous ground controls which represented 8.4% of the analyzed ORFs. Spaceflight-cultured C. albicans induced genes involved in cell aggregation (similar to flocculation) which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to more normal bipolar budding patterns for ground samples in accordance with the gene expression data. Furthermore genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight including induction of ABC transporters and members of the major facilitator family downregulation of ergosterol-encoding genes and upregulation of genes involved in oxidative stress resistance. Finally downregulation of genes involved in the actin cytoskeleton was observed. Interestingly the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested as this regulator is involved in random budding cell aggregation actin cytoskeleton and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype no increased virulence in a murine intraperitoneal (i.p.) infection model was observed. This study represents an important basis for the assessment of the risk that commensal flora could play during spaceflight missions. Furthermore since the low fluid-shear environment of microgravity is relevant to physical forces encountered by pathogens during the infection process insights gained from this study could identify novel infectious disease mechanisms with downstream benefits for the general public. Cells were grown for 24 hours on the space shuttle or as ground-based controls preserved in RNALater and stored at -80C. Four samples of each flight- and ground-based controls were harvested for microarray analysis. GAP is Group Activation Pack and each GAP contains 8 FPAs. The numbers represent the # assigned to the particular GAP and the number assigned to the specific FPA (1-8) within the indicated GAP. The same hardware is used for the flight samples and the ground samples.

Irradiated cell lines exposed to 1-10 Gy 2 Lymphoblastoid cell lines (GM15510 and GM15036) irradiated 1 2.5 5 7.5 10 Gy RNA is isolated and labeled using a T7 amplification Arcturus kit for hybridization on triplicate arrays.

Spaceflight has been shown to suppress the adaptive immune response, altering the distribution and function of lymphocyte populations. B lymphocytes express highly specific and highly diversified receptors, known as immunoglobulins (Ig), that directly bind and neutralize pathogens. Ig diversity is achieved through the enzymatic splicing of gene segments within the genomic DNA of each B cell in a host. The collection of Ig specificities within a host, or Ig repertoire, has been increasingly characterized in both basic research and clinical settings using high-throughput sequencing technology (HTS). We utilized HTS to test the hypothesis that spaceflight affects the B-cell repertoire. To test this hypothesis, we characterized the impact of spaceflight on the unimmunized Ig repertoire of C57BL/6 mice that were flown aboard the International Space Station (ISS) during the Rodent Research One validation flight in comparison to ground controls. Individual gene segment usage was similar between ground control and flight animals, however, gene segment combinations and the junctions in which gene segments combine was varied among animals within and between treatment groups. We also found that spontaneous somatic mutations in the IgH and Igκ gene loci were not increased. These data suggest that space flight did not affect the B cell repertoire of mice flown and housed on the ISS over a short period of time.

One of the most likely risks astronauts on long duration space missions face is exposure to ionizing radiation associated with highly energetic and charged heavy (HZE) particles. Since access to medical expertise on such a mission is limited at best early diagnosis and mitigation of such exposure is critical. In order to accurately determine the dosage within 1 hour post-exposure dose-dependent biomarkers are needed. Therefore we performed a dose-course transcriptional analysis for radiation exposure at 0 0.3 1.5 and 3.0 Gy with corresponding time point at 1 hour (hr) post-exposure using Affymetrix GeneChip Human Gene 1.0 ST v1 Array chips. The analysis of our data suggests a set of sensitive genetic biomarkers specific to each radiation level as well as generic radiation response biomarkers. Upregulated biomarkers can then be used within lab-on-a-chip (LOC) systems to detect exposure to ionizing radiation. A total of sixteen human samples representing radiation exposure at levels 0 Gy 0.3 Gy 1.5 Gy and 3.0 Gy at time point 1 hour (hr) post-exposure were constructed. Blood samples were extracted from four human volunteers and were irradiated. Leukocytes were extracted and gene expression was measured. Samples for all four volunteers were measured at 1 hr for all four dose levels resulting in four replicates at each dose level. Thus a total of 4 samples at each of the four radiation levels were sampled yielding the total of 16 samples.

One of the most likely risks astronauts on long duration space missions face is exposure to ionizing radiation associated with highly energetic and charged heavy (HZE) particles. Since access to medical expertise on such a mission is limited at best early diagnosis and mitigation of such exposure is critical. In order to accurately determine the dosage within 1 hour post-exposure dose-dependent biomarkers are needed. Therefore we performed a dose-course transcriptional analysis for radiation exposure at 0 0.3 1.5 and 3.0 Gy with corresponding time point at 1 hour (hr) post-exposure using Affymetrix GeneChip Human Gene 1.0 ST v1 Array chips. The analysis of our data suggests a set of sensitive genetic biomarkers specific to each radiation level as well as generic radiation response biomarkers. Upregulated biomarkers can then be used within lab-on-a-chip (LOC) systems to detect exposure to ionizing radiation. A total of sixteen human samples representing radiation exposure at levels 0 Gy 0.3 Gy 1.5 Gy and 3.0 Gy at time point 1 hour (hr) post-exposure were constructed. Blood samples were extracted from four human volunteers and were irradiated. Leukocytes were extracted and gene expression was measured. Samples for all four volunteers were measured at 1 hr for all four dose levels resulting in four replicates at each dose level. Thus a total of 4 samples at each of the four radiation levels were sampled yielding the total of 16 samples.

Astronauts are exposed to a unique combination of stressors during spaceflight which leads to alterations in their physiology and potentially increases their susceptibility to infectious pathogens. Here we report the first microarray evaluation of any astronaut tissue sample specifically whole blood before and after spaceflight using an array comprising 234 well-characterized stress response genes. Differentially regulated genes included those important for DNA repair oxidative stress and protein folding/degradation. Microarrays comprising 234 well characterized stress-related genes were used to profile transcriptomic changes in six astronauts before and after short-duration spaceflight. Blood samples were collected for analysis from each eastronaut 10 days prior and 2-3 hours after return from spaceflight. Data submitted for platform GPL140 contain genes that have been pre-filtered by the analytical software to remove values of low certainty resulting in missing values for some samples. Unfortunately these original data are no longer available due to physical damage at Tulane University during hurricane Katrina but the processed values were retained in redundant locations and these are submitted for upload to GEO.

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.

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.