Gravitational stress during parabolic flights induce changes in the human leukocyte subsets

• ¹ Centrum für Translationale Medizin, Marien Hospital Herne, Universitätsklinikum der Ruhr-Universität Bochum, Germany
• ² Berlin-Brandenburgisches Zentrum für Regenerative Therapien, Charité Universitätsmedizin, Germany
• ³ Laboratoire d’hématologie, Hôpital Haut-Lévêque, France
• ⁴ Beckman Coulter (Germany), Germany
• ⁵ Beckman Coulter (France), France

The human immune system is made up of several different cell types with distinct functions. The intricate interactions of these immune cells are required for an efficient function of the immune system leading to an appropriate response to foreign pathogens. Dysfunctions of the immune system might lead to a hyper-activity resulting in auto-immune or allergic diseases or to a hypo-activity allowing aberrant cells to form tumors (Iranzo and Villoslada, 2014). Defense mechanisms against pathogens have evolved in all species of all taxonomic domains: from the restriction enzymes and loci of clustered, regularly interspaced, short palindromic repeat (CRISPR) protects bacteria and archaea from phage invasion to the germline encoded pattern recognition receptors of all invertebrates and vertebrates (Iwasaki and Medzhitov, 2010; Marraffini and Sontheimer, 2010). Vertebrates possess in addition a non-germline encoded adaptive subsystem, characterized by stochastic processes in generation of pathogen recognizing receptors (Boehm and Swann, 2014). The gravitational field of earth has accompanied and shaped the evolution of all species and especially terrestrial organisms (Dubinin and Vaulina, 1976). It is, in fact, not until recently that mankind has produced means of altering the gravitational force subjected to a body. Centrifuges can increase the forces on a body, while the free falling international space station orbiting Earth creates a zero g environment. In addition, changes to the gravitational field might be actively sought after through various thrills such as amusement rides, skydiving, or bungee jumping (Gomez and Rao, 2016; Liu et al., 2016). Here we evaluate the effects of repeated changes in the gravitational field on circulating blood cells. The data were collected from 20 volunteers (average age: 25.65 years, range: 19-43) during the 27th and the 28th DLR parabolic flight campaigns. The Airbus A310 performed one test parabola followed by 30 regular parabolas. The regular parabolas were performed in 6 sets of 5 parabolas, where each set was separated by steady flight for 5-8 minutes. Each parabola consisted of a single phase with µg, flanked by two phases of 1.8g. Each phase lasted about 20 seconds. A steady flight phase of 1g for about 2 minutes separated each parabola. Blood samples were drawn just before the test parable (P0) and immediately after the last parabola of the last set (P30). To control for circadian effects, blood was taken on the ground the day before (C0 and C30). Whole blood was processed within an hour after arrival of the plane, and the immune status was evaluated by multi-parametric flow cytometry using the DuraClone IM Phenotyping Basic Panel (Beckman Coulter) per manufacturer’s instructions. Briefly, all incubation steps were performed at room temperature for 15 minutes in the dark, and all centrifugation steps were performed at 200g, for 5 minutes at room temperature. Erythrocytes were lysed using 2 ml VersaLyse (Beckman Coulter) per reaction tube, and the samples were collected on a Navios Flow Cytometer (Beckman Coulter). We observed an increase in the frequency of Granulocytes (Figure 1A) during the parabolic flights, which was driven by an increase in Neutrophils (Figure 1B), as a decrease in the Eosinophils was observed (Figure 1C). These observations were also reflected in the cell count (Figure 2A-C), indicating an influx of Neutrophils during the cause of the parabolic flight. The population of NK cells was found to be decreased after the parabolic flights (Figure 1D). This decrease could be explained by a decrease in the mature NK cells, as the frequency of the immature NK cells remained constant (Figure E1-F). Similar observations were made for the cell count of the NK cell populations (Figure 2D-F). Taken together, the data presented here show that repeated alterations of the gravitational field can cause changes to blood circulating immune cells. We observed in particular changes related to the innate immune cells, which acts as a first line of defense against invading pathogens. This could indicate an impaired functional response to pathogens, which is in line with the observations from space flights (Cervantes and Hong, 2016). However, the true functional consequences remain to be confirmed. We have shown here, that gravitational stress as experienced on a parabolic flight, can lead to alterations of the composition of blood circulating immune cells. This could have consequences for the efficacy of the immune system and potentially leading to an inappropriate response self- and non-self-antigen. Figure 1: Gravitational stress disrupts the composition of circulating blood cells. The frequency of the indicated cell populations among all white blood cells were identified by flow cytometry. The boxes represent the 25th, 50th, and 75th percentile and the whiskers represent the range excluding outliers. Each point represents a single donor. Asterisks indicate p-values (** p < 0.01; * p < 0.05) for a Wilcoxon test. Figure 2: Gravitational stress disrupts the cell numbers of circulating blood cells. Total numbers of the indicated cell populations among all white blood cells were identified by flow cytometry. The boxes represent the 25th, 50th, and 75th percentile and the whiskers represent the range excluding outliers. Each point represents a single donor. Asterisks indicate p-values (* p < 0.05) of a Wilcoxon test.