Space travel presents unlimited opportunities for exploration and discovery, but requires better understanding of the biological consequences of long-term exposure to spaceflight. Immune function in particular is relevant for space travel. Human immune responses are weakened in space, with increased vulnerability to opportunistic infections and immune-related conditions. In addition, microorganisms can become more virulent in space, causing further challenges to health. To understand these issues better and to contribute to design of effective countermeasures, we used the Drosophila model of innate immunity to study immune responses in both hypergravity and spaceflight. Focusing on infections mediated through the conserved Toll and Imd signaling pathways, we found that hypergravity improves resistance to Toll-mediated fungal infections except in a known gravitaxis mutant of the yuri gagarin gene. These results led to the first spaceflight project on Drosophila immunity, in which flies that developed to adulthood in microgravity were assessed for immune responses by transcription profiling on return to Earth. Spaceflight alone altered transcription, producing activation of the heat shock stress system. Space flies subsequently infected by fungus failed to activate the Toll pathway. In contrast, bacterial infection produced normal activation of the Imd pathway. We speculate on possible linkage between functional Toll signaling and the heat shock chaperone system. Our major findings are that hypergravity and spaceflight have opposing effects, and that spaceflight produces stress-related transcriptional responses and results in a specific inability to mount a Toll-mediated infection response.

Competing interests: Jeff Alley is employed by a commercial company, Laverlam International, there are no products, patents, etc. that are connected to the authors' study. It is just that one of the authors is employed by the company, and this does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Funding: This work was funded by grants from the National Aeronautics and Space Administration, NNA04CC76A and NNA05CV40A to DAK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2014 Taylor et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The mechanisms of interactions within and between the Toll and Imd pathways and other systems are not fully understood, and unraveling the interrelationships will require many approaches. Here, we present genetic and transcriptional profiling experiments to address the response to infection in conditions related to space travel: Does hypergravity affect the response to fungal infection? Does development during spaceflight alter the response to bacterial and fungal infections?

Two signaling pathways are the main mediators of the response to bacterial and fungal infections in Drosophila [7] , [11] , [12] . The Toll pathway primarily responds to fungal and Gram-positive (Lys-type peptidoglycan (PGN)) infections, and the Imd pathway responds to Gram-negative (DAP-type PGN) infections [7] . Toll-like receptors (Tlrs) have been identified in mammals and are the direct mediators of responses to activators such as bacterial lipopolysacccharide and viral DNA [13] . Imd shares homology with the death domain of the mammalian Receptor Interacting Protein of the Tumor Necrosis Factor Receptor pathway [7] . Downstream, through the conserved NF-kB/Rel protein transcription factors relish (Imd signaling cascade), and DIF and dorsal (Toll signaling cascade), the AMPs and ∼400 other genes are involved in response to infection [7] , [14] , [15] . Recognition of the complexity of the Toll and Imd pathways continues to grow, for example with identification of new regulators, interactions with the nervous system, and modification with aging [16] , [17] , [18] , [19] . In contrast to mammals, in Drosophila only the original Toll was associated with infection response, through indirect sensing mediated by binding to Spätzle (Spz). More recently however, other Toll family members have been identified as mediating infection. Toll-8 regulates infection response in the airway epithelium [20] , and Toll-7 is involved in viral recognition and response [21] .

In our work, we have brought advances in understanding the host defense of Drosophila to bear on deciphering the immune alterations associated with altered gravity and spaceflight. Drosophila is a well-established model for human innate immune function, sharing elements in cellular and humoral immunity, clotting and wound healing, and signaling pathways [7] . Drosophila responds to microbial infection with 1) a systemic response, characterized by fat body production of antimicrobial proteins (AMPs), 2) tissue specific responses, such as production of AMPs in the gut and trachea, 3) phagocytosis by hemocytes, and 4) clotting and wound healing [7] , [8] , [9] , [10] .

Human space exploration, with its promise of unprecedented discoveries, excites the imagination. However, turning the exploration of space into a practical reality presents daunting challenges including conquering the compromised biological functions produced by spaceflight. In order to achieve space exploration, a better understanding of human biology, both on earth and in space, is required. Among the many aspects of biology affected by spaceflight, we have focused on the immune response. Immune dysfunction is a major health-related problem on earth and a major obstacle to long-term space missions [1] . As early as the Apollo and Skylab missions, immune dysfunction was recognized in astronauts, and later studies documented specific host cellular and humoral immune alterations induced by spaceflight [1] . Increased microbial growth and virulence in space have also been documented [2] . Spaceflight is associated with many stresses, with altered gravitational force (g) representing the most studied factor. Microgravity (µg) is constant in space, and hypergravity (hyper g) is experienced during launch and landing. Immune dysfunction in both µg and hyper g is well documented, but determination of the underlying cellular mechanisms and thus routes to appropriate countermeasures, remains unresolved [2] , [3] , [4] , [5] , [6] . Without normal immune function, many threats to long-term survival in space exist: fatal infections, failed immunosurveillance of cancer cells, aberrant inflammatory responses and reactivation of latent viruses are all potential hazards.

Results and Discussion

Hypergravity Increases Survival after Infection with Pathogenic Fungus The first goal was to test our hypothesis that the immune response of Drosophila would be affected by changes in gravity at the organismal level. The simplest immune function assay is post-infection survival, and a straightforward route for altering gravity is to achieve hyper g through use of centrifuges similar to the human centrifuges used for training pilots. We infected with B. bassiana, an entomopathogenic fungus that enters through the cuticle and is well studied with respect to survival kinetics and Toll pathway activation [7]. Infected and control flies were then exposed to hypergravity on a centrifuge maintained at the Chronic Acceleration Research Unit (CARU), UC Davis. The survival of wild type and immune response mutants (except Toll pathway mutants which do not survive infection long enough for prolonged hyper g experiments) was assessed. Strikingly, all strains showed increased post-infection survival at hyper g (Figure 1A bottom panel, 1B wild type, imd and Thor strains). Given that µg is associated with impaired immune function, one interpretation of this result is that hyper g exerts the opposite effect and boosts the host response. Opposite effects of opposing gravity vectors are not uncommon, for example for platelet functions [22]. However, microorganisms can become more virulent at µg [2], and an alternative explanation is that at hyper g the fungus itself is less virulent. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Effects of hyper g on post-infection survival and energy stores. A. Survival after infection with B. bassiana is increased by exposure to hyper g (4 g) in wild type (wt) and the rescued yuri strain, yuric263; UAS-yuri (UAS), but not in the gravitaxis mutant yuri, yuric263 (yuri). +infected, −uninfected. Error bars = SEM for 3 experiments. B. Additional strains tested also survive infection longer at hyper g: imd, using imd1, and for Thor, which encodes the Drosophila translational regulator 4E-BP, using Thor2, the null allele, and its control, the revertant strain Thor1rev1. P values for log rank. C. Post-infection energy stores of trigycerides, protein and carbohydrates are not significantly different at hyper g. Error bars = SEM for 3 experiments. https://doi.org/10.1371/journal.pone.0086485.g001 In an attempt to distinguish between host and fungal responses, we tested a gravitaxis mutation of the gene yuri gagarin (yuri) [23]. yuri encodes 3 isoforms of a coiled-coil protein that is ubiquitously expressed, and two mutations have separate tissue specific functions related to mechanotransduction [24], [25], [26]. The yuric263 allele, caused by a GAL4 enhancer trap insertion, has defective gravity responses. A UAS-yuri construct, driven by the c263 transposon, rescues this phenotype through expression limited to mechanosensory neurons [23], indicating defective gravity sensing in the yuric263 mutant. We hypothesized that if host response to hyper g were primary, then aberrant gravity sensing in yuric263 might modify the hyper g post-infection response, but if the fungal response were primary, then post-infection survival of yuric263would be comparable to that of wild type and the immune function mutants (Figure 1AB). On testing, yuric263 failed to show this increased post-infection survival, whereas the yuri rescue strain had the typical increased survival response (Figure 1AB). Thus, these data demonstrate a significant host component to the hyper g effect. How might hyper g increase post-infection survival? The yuri finding could indicate a neural route linking mechanical load sensation to immune response. Mechanical load also affects cell biological processes [27], and one possibility is that endocytosis, which is essential for Toll signaling [28], is enhanced at hyper g. Interestingly, Yuri protein appears to have membrane-associated functions [26]. The immune response is energetically expensive, and flies with greater energetic reserves may have greater post-infection survival. However, survival did not correlate with stores of triglycerides, carbohydrates or protein (Figure 1C).

The Fungus, Immunity, Tumorigenesis (FIT) Microgravity Experiment These results showing that the immune response of Drosophila responds to g force formed the basis for the space shuttle experiment Fungus, Immunity and Tumorigenesis (FIT). FIT is the first flight experiment to investigate µg effects on Drosophila immunity. The FIT experiment was flown on the shuttle Discovery (STS-121), and involved an experimental design adjusted for shuttle conditions. Ideally the design would have paralleled the hyper g work, with infection of Drosophila genotypes proceeding in space. But due to flight constraints, space infections were not possible and only a single genotype could be flown. However, the flight duration (12 days) allowed production and return to Earth of a small population of flies that had undergone their entire development in space (space flies). Upon return this population was divided into three groups and used for transcription profiling without infection and after infection with B. bassiana or E. coli. The fungal spores and E. coli used were grown on Earth. Earth-reared flies, grown at Kennedy Space Center, were used as controls (Earth flies). Recordings relayed from the shuttle ensured similar growth conditions for the space and Earth flies other than the change in g force. The experiments thus encompass humoral immunity in response to Toll and Imd mediated fungal and bacterial infections through transcriptional profiling after development in space. The uninfected space flies showed an altered transcriptional profile, and those changes will be presented last, in the context of the immune response data.