Dichotomal effect of space flight-associated microgravity on stress-activated protein kinases in innate immunity

Abstract

Space flight strongly moderates human immunity but is in general well tolerated. Elucidation of the mechanisms by which zero gravity interacts with human immunity may provide clues for developing rational avenues to deal with exaggerated immune responses, e.g. as in autoimmune disease. Using two sounding rockets and one manned Soyuz launch, the influence of space flight on immunological signal transduction provoked by lipopolysaccharide (LPS) stimulation was investigated in freshly isolated peripheral blood monocytes and was compared to samples obtained from on-board centrifuge-loaded 1 g controls. The effect of microgravity on immunological signal transduction is highly specific, since LPS dependent Jun-N-terminal kinase activation is impaired in the 0 g condition, while the corresponding LPS dependent activation of p38 MAP kinase remains unaffected. Thus our results identify Jun-N-terminal kinase as a relevant target in immunity for microgravity and support using Jun-N-terminal kinase specific inhibitors for combating autoimmune disease.

Figure 1. Summary of the experimental procedure and the timeline.

The experiment was repeated three times. The first experiment was performed as part of the 2004 Soyuz TMA-4 campaign. Under the name Kappa it was executed during a manned flight to the International Space Station. The experiment was repeated in 2005 during the Maser 10 mission (Amuse) and again in 2012 during the Maser 12 mission (Micimmun). Both missions used sounding rockets to carry the experiment into space. During a parabolic flight the rocket reaches an altitude of approximately 260 km allowing for 5 to 6 minutes of microgravity. The experiments are performed in automated climate chambers termed Flush Units. The Flush Units are placed within the BIM module. The module regulates ambient temperature aboard the rocket and provides electricity to the Flush Units during flight. As an onboard control the Flush Units can be placed in a centrifuge which spins at a speed equal to the force of gravity. The other units are placed on a static rack and are allowed to experience microgravity. Prior to the flight the monocytes are placed inside a rectangular chamber containing two glass slides to which the monocytes attach. At the start of microgravity a timed mechanism releases a spring plunger which replaces the medium in the culture chamber with medium containing LPS. At the end of microgravity a similar mechanism replaces the LPS containing medium with fixative. We would like to acknowledge NASA and Dutch Space for the use of their illustrations in this figure.

Figure 2. Microgravity does not influence activation of p38 MAP Kinase following LPS stimulation.

The figure shows phospho-p38 MAP kinase analysis of 100 ng/ml LPS stimulated monocytes in the presence (1 g) or relative absence of gravity (0 g). Fixed monocytes were stained fluorescently (green) and the nuclei were visualized by DAPI (blue) (Figure 2A). Staining was measured at different exposure times (Figure 2B) and mean fluorescence was finally determined at 2000 ms (Figure 2D). Although differences between the conditions are apparent, the absence of gravity does not affect activation of p38 MAP kinase. The nuclear localization of p38 MAPK is also not different from the control condition (Figure 2C). The graphs are based on 36 determinations.

Figure 3. LPS-dependent Jun-N-terminal kinase activation is sensitive to space flight-associated microgravity.

The figure shows phospho-Jnk analysis of LPS stimulated monocytes in the presence (1 G) or relative absence of gravity (0 G). Fixed monocytes were stained fluorescently (green) and the nuclei were visualized by DAPI (blue) (Figure 3A). Comparison between individual cells clearly shows a brighter staining when stimulated with LPS in the presence of gravity. Staining intensity was measured at different exposure times (Figure 3B) and mean fluorescence was finally determined at 2000 ms (Figure 3D). Microgravity significantly impairs the activation of Jnk in monocytes. Analysis of nuclear localization of activated Jnk shows no change (Figure 3C). The graphs are based on 32 determinations. *P < 0.05.

Figure 4. Effects of microgravity on ERK activation.

The figure shows phospho-Erk analysis of LPS stimulated monocytes in the presence (1 G) or relative absence of gravity (0 G). Fixed monocytes were stained fluorescently (green) and the nuclei were visualized by DAPI (blue) (Figure 4A). Comparison between individual cells clearly shows a brighter staining when stimulated with LPS in the presence of gravity. Staining intensity was measured at different exposure times (Figure 4B) and mean fluorescence was finally determined at 2000 ms (Figure 4D). Microgravity impairs the activation of Erk in monocytes. Analysis of nuclear localization of activated Erk shows no change (Figure 4C). The graphs are based on 32 determinations.