Einfluss von veränderter Schwerkraft auf den oxidativen Burst in Makrophagen

Die Hauptaufgabe von Makrophagen besteht in der Erkennung und Verdauung aufgenommener Pathogene durch die Produktion von reaktiven Sauerstoff Spezies (ROS) innerhalb des oxidativen Burst. Dieser Prozess stellt die erste Immunantwort dar und verhindert den Ausbruch von Erkrankungen durch Bakterien und Viren. Astronauten leiden unter einem beeinträchtigten Immunsystem, welches zu einer erhöhten Anfälligkeit für Erkrankungen im Weltraum führen kann. Einige Studien weisen darauf hin, dass der oxidative Burst von Makrophagen durch Weltraumbedingungen stark beeinträchtigt ist, jedoch der molekulare Mechanismus noch unbekannt ist. In der vorliegenden Studie wurden die Charakteristika des Pathogen-induzierten oxidativen Bursts unter veränderten Schwerkraftbedingungen, Hypergravitation (humane Kurzarmzentrifuge) und Mikrogravitation (Parabelfug und Klinostat) untersucht. Des Weiteren wurde die Phosphorylierung des für die ROS Produktion wichtigen Proteins Syk und die Aktivierung des Transkriptionsfaktors NF-kB untersucht, um den Einfluss veränderter Schwerkraft auf die Signalwege innerhalb von Makrophagen aufzuklären. Hypergravitation führt zu einer Zunahme, Mikrogravitation zur einer signifikanten Abnahme der radikalen Sauerstoff-Produktion, nach Stimulation durch Zymosan. Die damit verbundene Phagozytose-Rate war in Mikrogravitation leicht reduziert, jedoch in Hypergravitation signifikant erhöht. Die Veränderungen in der ROS Produktion verlaufen sehr schnell (innerhalb von Sekunden), wodurch eine Kopplung mit der Phagozytose ausgeschlossen werden kann. Aufgrund dessen wurde ein schneller Prozess, die Syk Phosphorylierung, untersucht, die eine signifikante Verringerung in simulierter Mikrogravitation zeigte. Ein späterer Schritt innerhalb der Signalkaskade, die Aktivierung des Transkriptionsfaktors NF-kB, zeigte keine Veränderung in simulierter Mikrogravitation. Die Ergebnisse zeigen, dass die ROS Produktion in Makrophagen ein gravisensitiver Prozess ist, bei dem die verringerte Syk Phosphorylierung eine Rolle spielt. Jedoch bleibt die Aktivierung des Transkriptionsfaktors NF-kB erhalten, was darauf hindeutet, dass Gravitation lediglich schnelle und frühe Prozesse beeinflusst, aber keinen Einfluss auf spätere Signalschritte ausübt. Hypergravitation hat einen stimulierenden Effekt auf die Zellen, offensichtlich ausgelöst durch die Erhöhung der Kräfte die auf die Zellen wirken und zeigt, dass Immunzellen nur unter gewissen Kräfteverhältnissen vollständige Funktionalität zeigen. Daraus lässt sich schließen, dass Makrophagen bei der Stimulation mit einem Pathogen-Analogon unter reduzierter Schwerkraft Veränderung in den Signalwegen zeigen, was ein Grund für die Beeinträchtigungen des Immunsystems von Astronauten sein kann

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

Short radius centrifuges - a new approach for life science experiments under hyper-g conditions for applications in space and beyond

A broad variety of countermeasures on the effects of weightlessness on human physiology have been developed and applied in the course of space exploration. Devices like treadmills, stretch ropes etc. have several disadvantages in common: they require a significant amount of crew time and they may not efficiently counteract the degradation of physiological structures and cellular functions. Some methods even include potentially painful or uncomfortable procedures for the astronauts. Thus, the application of Artificial Gravity (AG) generated by short radius centrifuges (they fit into space vessels) has been discussed and proposed by a number of scientists and space agencies as an alternative countermeasure during long-term space missions. Although there is a profound knowledge concerning, e.g., the cardiovascular system and immune responses acquired on long radius centrifuges, there is a remarkable lack of knowledge concerning the same issues on devices operating with short radius. In strict contrast to long radius centrifuges, there is a significant gravity gradient in the head-to-toe axis which comes along with the short radius and higher relative rotation velocity. Thus it is of utmost importance to continue investigating the effects of AG, especially by use of short radius centrifuges. The Short Arm Human Centrifuge (SAHC) at the German Aerospace Center (DLR) in Cologne, Germany, is the most advanced type of short radius centrifuges presently commercially available. Experience gained so far using the SAHC at DLR revealed that future projects on centrifuge devices with short radius should aim at a clear identification of the threshold level of the g-load, which is necessary to efficiently counteract the degradation of physical structures and an efficient support of cellular functions. A satisfying result would be combined countermeasure methods applied at a threshold concerning g-load and exposition time in the course of long-term sojourn in microgravity. Another future control or monitoring method to exactly dose AG training is heart rate variability, which offers an insight into neurovegetative and cardiovascular regulation. Centrifuges like the SAHC are also useful platforms to accommodate small biological experiments, e.g., experiments addressing the response of cultured cells to hypergravity. Here, we briefly review the issue of short radius centrifuges and also address our experience hitherto gained during a number of scientific projects carried out at the SAHC at DLR