Rapid adaptation to microgravity in mammalian macrophage cells.

Despite the observed severe effects of microgravity on mammalian cells, many astronauts have completed long term stays in space without suffering from severe health problems. This raises questions about the cellular capacity for adaptation to a new gravitational environment. The International Space Station (ISS) experiment TRIPLE LUX A, performed in the BIOLAB laboratory of the ISS COLUMBUS module, allowed for the first time the direct measurement of a cellular function in real time and on orbit. We measured the oxidative burst reaction in mammalian macrophages (NR8383 rat alveolar macrophages) exposed to a centrifuge regime of internal 0 g and 1 g controls and step-wise increase or decrease of the gravitational force in four independent experiments. Surprisingly, we found that these macrophages adapted to microgravity in an ultra-fast manner within seconds, after an immediate inhibitory effect on the oxidative burst reaction. For the first time, we provided direct evidence of cellular sensitivity to gravity, through real-time on orbit measurements and by using an experimental system, in which all factors except gravity were constant. The surprisingly ultra-fast adaptation to microgravity indicates that mammalian macrophages are equipped with a highly efficient adaptation potential to a low gravity environment. This opens new avenues for the exploration of adaptation of mammalian cells to gravitational changes.

The influence of simulated microgravity on the proteome of Daphnia magna.

BACKGROUND: The waterflea Daphnia is an interesting candidate for bioregenerative life support systems (BLSS). These animals are particularly promising because of their central role in the limnic food web and its mode of reproduction. However, the response of Daphnia to altered gravity conditions has to be investigated, especially on the molecular level, to evaluate the suitability of Daphnia for BLSS in space. METHODS: In this study, we applied a proteomic approach to identify key proteins and pathways involved in the response of Daphnia to simulated microgravity generated by a two-dimensional (2D) clinostat. We analyzed five biological replicates using 2D-difference gel electrophoresis proteomic analysis. RESULTS: We identified 109 protein spots differing in intensity (P<0.05). Substantial fractions of these proteins are involved in actin microfilament organization, indicating the disruption of cytoskeletal structures during clinorotation. Furthermore, proteins involved in protein folding were identified, suggesting altered gravity induced breakdown of protein structures in general. In addition, simulated microgravity increased the abundance of energy metabolism-related proteins, indicating an enhanced energy demand of Daphnia. CONCLUSIONS: The affected biological processes were also described in other studies using different organisms and systems either aiming to simulate microgravity conditions or providing real microgravity conditions. Moreover, most of the Daphnia protein sequences are well-conserved throughout taxa, indicating that the response to altered gravity conditions in Daphnia follows a general concept. Data are available via ProteomeXchange with identifier PXD002096.

The oxidative burst reaction in mammalian cells depends on gravity.

Gravity has been a constant force throughout the Earth's evolutionary history. Thus, one of the fundamental biological questions is if and how complex cellular and molecular functions of life on Earth require gravity. In this study, we investigated the influence of gravity on the oxidative burst reaction in macrophages, one of the key elements in innate immune response and cellular signaling. An important step is the production of superoxide by the NADPH oxidase, which is rapidly converted to H2O2 by spontaneous and enzymatic dismutation. The phagozytosis-mediated oxidative burst under altered gravity conditions was studied in NR8383 rat alveolar macrophages by means of a luminol assay. Ground-based experiments in "functional weightlessness" were performed using a 2 D clinostat combined with a photomultiplier (PMT clinostat). The same technical set-up was used during the 13th DLR and 51st ESA parabolic flight campaign. Furthermore, hypergravity conditions were provided by using the Multi-Sample Incubation Centrifuge (MuSIC) and the Short Arm Human Centrifuge (SAHC). The results demonstrate that release of reactive oxygen species (ROS) during the oxidative burst reaction depends greatly on gravity conditions. ROS release is 1.) reduced in microgravity, 2.) enhanced in hypergravity and 3.) responds rapidly and reversible to altered gravity within seconds. We substantiated the effect of altered gravity on oxidative burst reaction in two independent experimental systems, parabolic flights and 2D clinostat / centrifuge experiments. Furthermore, the results obtained in simulated microgravity (2D clinorotation experiments) were proven by experiments in real microgravity as in both cases a pronounced reduction in ROS was observed. Our experiments indicate that gravity-sensitive steps are located both in the initial activation pathways and in the final oxidative burst reaction itself, which could be explained by the role of cytoskeletal dynamics in the assembly and function of the NADPH oxidase complex.

Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity.

In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.