Abstract
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.
Highlights
A variety of gravity-sensing mechanisms evolved in multicellular and complex organisms to benefit from this constant force for orientation in three-dimensional space
In a combination of experiments using 2D clinorotation and real microgravity provided by several parabolic flight campaigns, we found that the oxidative burst reaction and phagocytosis in NR8383 macrophages depends on the gravitational force
Reactive oxygen species (ROS) release strongly depends on gravity conditions During the 13th DLR and the 51st ESA parabolic flight campaign, oxidative burst was studied in real microgravity conditions using a Photomultiplier tube (PMT) clinostat and a luminol assay
Summary
A variety of gravity-sensing mechanisms evolved in multicellular and complex organisms to benefit from this constant force for orientation in three-dimensional space. The innate immune system is characterized by a fast but unspecific immune reaction and activates the adaptive immune response. This is done mainly through interaction of antigen-presenting cells (APCs) and dendritic cells, and macrophages [7] with T lymphocytes. During the progress of phagocytosis after pattern recognition, an arsenal of killing agents is released, which includes the assembly of NADPH oxidase complexes on the phagolysosomal membranes. This catalyzes the production of oxygen-derived, highly toxic compounds, e.g. superoxide (O2-), hypochloride (HOCl), hydroxyl radicals or hydrogen peroxide (H2O2), a process which is known as the oxidative burst [8]. Especially H2O2, may be involved in signaling of the macrophage itself or other nearby cells after release to the extracellular medium [9,10]
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