Influence of altered gravity on the oxidative burst in macrophages

The recognition of pathogen patterns followed by the production of reactive oxygen species (ROS) during oxidative burst is one of the major functions in macrophages. This process is the first line of defence which is crucial for prevention of pathogen associated diseases. The immune system of astronauts is impaired during spaceflight, resulting in an increased susceptibility to infections. Several studies have shown that the oxidative burst of macrophages is highly impaired after spaceflight, but the underlying mechanism remained to be elucidated. Here, we investigated the characteristics of the reactive oxygen species production during oxidative burst after pathogen pattern recognition in hypergravity (Short-Arm Human Centrifuge) and microgravity (parabolic flight and ground-based Clinostat). Furthermore, the spleen tyrosine kinase Syk phosphorylation, which is required for ROS production, and the translocation of the transcription factor NF-κB to the nucleus were monitored to elucidate the influence of altered gravity on macrophage signalling. Hypergravity reveals an increase, whereas real and simulated microgravity leads to a significantly diminished ROS production upon zymosan recognition. The corresponding phagocytosis rate during altered gravity was only slightly reduced in microgravity, but hypergravity increased the phagocytosis drastically. Since the changes in ROS production occur within seconds and uncoupled of phagocytosis, the phosphorylation of Syk was examined, showing a significantly reduced phosphorylation in simulated microgravity. To address later signalling steps, the translocation of NF-κB to the nucleus was measured and remains normal. The results show that the ROS production in macrophages is a highly gravisensitive process which is caused by the diminished Syk phosphorylation. However, besides the impaired ROS production, the NF-κB signalling remains constant under simulated microgravity conditions, showing that early and fast responding steps are affected, whereas long-term signalling continues unaffected. Hypergravity seems to have an activating and stimulating effect on macrophages due to increased force environment, indicating that immune cells require certain force condition to be fully functional. Taken together, the study clearly demonstrates that macrophages display impaired signalling upon pattern recognition, when exposed to altered gravity, which can be a reason why astronauts display a higher susceptibility to infections

Ground-Based Facilities for Studies in Gravitational Biology

Our group provides experimental platforms to simulate the conditions of microgravity on ground. Clinostats, a Random Positioning Machine and a Rotating Wall Vessel have been adapted to allow investigations employing a variety of different model organisms like larval fish, plants, algae, and unicellular organisms as well as adherent or suspended cells in culture. Different types of clinostats have been developed which meet a broad variety of scientific requirements in providing various kinds of experimental applications such as parallel operation of up to ten sample containments in a defined environment, direct microscopical observations of sample, their fixation during clinorotation, bioluminescence kinetic measurements within cell cultures and submersion of aquatic systems during rotation. Correspondingly, various centrifuge devices complete our experimental scenario, enabling hypergravity studies from cells to humans. Ground-based data should ideally be validated in the course of experiments carried out under real microgravity, which is regularly performed by us. Currently, our studies focus on the effects of simulated microgravity on macrophage signaling and stem cell development and differentiation. Simulation of microgravity as well as increased gravitational stimulation provide new insights how Biosystems cope with altered gravity conditions, showing changes in signaling pathways. Our results support the necessity of a ground-based facility program, which – at low costs in comparison to space flight – give scientists the opportunity to intensively prepare their space experiments and get sufficient and statistically reliable and relevant data

Syk phosphorylation – a gravisensitive step in macrophage signalling

Background: The recognition of pathogen patterns followed by the production of reactive oxygen species (ROS) during the oxidative burst is one of the major functions of macrophages. This process is the first line of defence and is crucial for the prevention of pathogen-associated diseases. There are indications that the immune system of astronauts is impaired during spaceflight, which could result in an increased susceptibility to infections. Several studies have indicated that the oxidative burst of macrophages is highly impaired after spaceflight, but the underlying mechanism remained to be elucidated. Here, we investigated the characteristics of reactive oxygen species production during the oxidative burst after pathogen pattern recognition in simulated microgravity by using a fast-rotating Clinostat to mimic the condition of microgravity. Furthermore, spleen tyrosine kinase (Syk) phosphorylation, which is required for ROS production, and the translocation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) to the nucleus were monitored to elucidate the influence of altered gravity on macrophage signalling. Results: Simulated microgravity leads to significantly diminished ROS production in macrophages upon zymosan, curdlan and lipopolysaccharide stimulation. To address the signalling mechanisms involved, Syk phosphorylation was examined, revealing significantly reduced phosphorylation in simulated microgravity compared to normal gravity (1 g) conditions. In contrast, a later signalling step, the translocation of NF-κB to the nucleus, demonstrated no gravity-dependent alterations. Conclusions: The results obtained in simulated microgravity show that ROS production in macrophages is a highly gravisensitive process, caused by a diminished Syk phosphorylation. In contrast, NF-κB signalling remains consistent in simulated microgravity. This difference reveals that early signalling steps, such as Syk phosphorylation, are affected by microgravity, whereas the lack of effects in later steps might indicate adaptation processes. Taken together, this study clearly demonstrates that macrophages display impaired signalling upon pattern recognition when exposed to simulated microgravity conditions, which if verified in real microgravity this may be one reason why astronauts display higher susceptibility to infections