Event Abstract

Back to Event

Altered Anandamide Metabolism in Microgravity: the “RESLEM” experiment

Natalia Battista1, Monia Di Tommaso1, Aleandro Norfini2, Marco Passerai2, Valerio Chiurchiù3, Mauro Maccarrone3* and Monica Bari4*
  • 1 Facoltà di Bioscienze e tecnologie agro-alimentari e ambientali, Università degli studi di Teramo, Italy
  • 2 Kayser (Italy), Italy
  • 3 Facoltà di Medicina e Chirurgia, Università Campus Bio-Medico di Roma, Italy
  • 4 Dipartimento di Medicina Sperimentale e Chirurgia, Università degli Studi di Roma Tor Vergata, Italy

To date it is widely accepted that human immune response is impaired during Space flight conditions, thus prognostic, diagnostic and therapeutic markers of microgravity-induced immunodepression are highly desirable (Hughes-Fulford, 20011). Our previous studies, performed in the course of the 28th parabolic flight campaign of the European Space Agency (ESA) (Maccarrone et al., 2001), under simulated microgravity (Maccarrone et al., 2003), as well as in the frame of our previous “ROALD” (Role of Apoptosis in Lymphocyte Depression) project (Maccarrone et al., 2008), have highlighted the key-role of 5-lipoxygenase (5-LOX) as a gravity responder that triggers apoptosis of human peripheral blood mononuclear cells (PBMCs), and hence immunodepression, under authentic microgravity conditions (Battista et al., 2012). In this context, we should recall that anandamide (AEA), the first endocannabinoid discovered more than 25 years ago, is considered a peripheral marker for immune disorders, and that during neuroinflammatory processes the expression of the main AEA-synthesizing enzyme (N-acylphosphatidyl-ethanolamine specific phospholipase D, NAPE-PLD) and AEA-degrading enzyme (Fatty Acid Amide Hydrolase, FAAH) are altered in immune cells (Maccarrone, 2017). The results obtained during the ROALD experiment have demonstrated that microgravity induces in PBMCs an early increase in 5-LOX activity and, subsequently, in leukotrienes B4 (LTB4) production, speaking in favour of an early engagement of 5-LOX along the signaling pathway that leads to apoptosis (Battista et al., 2012). It is worth reminding that AEA, unlike other endocannabinoids, inhibits FAAH activity by promoting the release of arachidonate, to be converted by LOXs into hydroperoxides, which act as competitive inhibitors of FAAH. The observation that all LOX inhibitors (MK886, caffeic acid), as well as the hydro(pero)xides of AEA generated by LOX (Maccarrone et al., 1998), yield the same effects on FAAH seems to stress the hypothesis that LOX pathway might regulate FAAH activity (Maccarrone et al., 2000). Additionally, previous findings showed a significant increase of circulating AEA levels in humans exposed to brief or prolonged periods of weightlessness (Chouker et al., 2010; Strewe et al., 2012). On this basis, we speculated that under real microgravity conditions AEA immunosuppressive effects might be associated with inhibition of lymphocyte proliferation throughout the involvement of 5-LOX (Maccarrone and Finazzi Agrò, 2003) and that this regulatory mechanism might be involved in controlling AEA levels, due to an unbalanced expression of the main enzymes that regulate its metabolism. Therefore, we proposed the “RESLEM” (Role of the Endocannabinoid System in human Lymphocytes Exposed to Microgravity) project, that has been selected by ESA following the 2009 International Life Science Research Announcement (ILSRA-2009) (Battista et al., 2011). Our proposal was aimed at studying the gene and protein expression of the enzymes that regulate the endogenous tone of AEA, in order to provide new insights in the immune response of lymphocytes in Space, and to identify its role as modulator of cell survival and death. The experiment was part of the International Space Station (ISS) Increment 29-30 (PromISSe) mission organized by the European Space Agency (ESA) in 2011. RESLEM was launched on December 21, 2011 from the Baikonur Cosmodrome (Kazakhstan) onboard Soyuz rocket mission 29S, operated by the Russian Space Agency. After 126 days onboard the ISS, the RESLEM samples were recovered from the returning Soyuz 28S landed in Kazakhstan on April 27, 2012, kept at 25 °C in a yellow box and transported to the University of Teramo, where they were received on April 29, 2012 by science team for postflight analysis. Human PBMCs were isolated from whole blood, taken from two human healthy donors, and were purified by gradient concentration, using the density separation medium Histopaque-1077. Cells were resuspended in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing L-glutamine (2 mM), gentamicin sulfate (50 µg/ml), HEPES (40 mM), and 10% fetal bovine serum, all purchased from Sigma-Aldrich (St. Louis, MO, USA). Cells were loaded into a specific hardware developed by Kayser Italia composed of 8 experimental units (EUs), each fitting into a KIC-SL container. After arrival on the ISS, RESLEM experiment containers were transferred into KUBIK 3 facility, pre-conditioned at 37°C, in the Columbus module. One set of samples was in a static position (0g) and another was put in the centrifuge (1g). During inflight operations, human PBMCs were activated automatically for different times (0, 3, 24, and 48 hrs), following a well-established procedure to investigate the effect of microgravity on immune function by using Concanavalin A (15 µg/ml solution in each culture chamber; Invitrogen) (Walther et al., 1998). Then, cell suspensions were fixed with RNAlater (900 µl/culture chamber; Ambion, Austin, TX, USA), and were immediately moved by the astronauts into the minus 80°C laboratory freezer for ISS (MELFI) facility, where they were stored until download with Soyuz 28S. The experimental design and timeline are reported in Supplementary Figure S1. Briefly, RNA was extracted from human PBMCs using the RNeasy extraction kit (Qiagen, Crawley, UK) and quantitative real-time RT-PCR (qRT-PCR) reactions were performed using the RT-PCR SuperScript III Platinum Two-Step qRT-PCR Kit (Invitrogen, Carlsbad, CA, USA), as already described (Battista et al., 2012). NAPE-PLD and FAAH protein expression were evaluated by using both an ELISA assay and Western blot analysis (Battista et al., 2012). Our results demonstrate that microgravity enhances the expression, both at transcriptional and translational level, of the main enzymes responsible for AEA endogenous tone (Figure 1). Interestingly, our data highlight that human PBMCs are active in synthesizing AEA, showing a NAPE-PLD:FAAH ratio >1 within 48 hrs of exposure to microgravity (Figure 2). These findings provide a possible explanation for the increased levels of circulating endocannabinoids and are in line with the data obtained during parabolic flights as well as in long-duration space missions to the ISS. In the perspective of Space exploration and colonization, the RESLEM project has disclosed an unprecedented potential engagement of endocannabinoid signaling in lymphocyte apoptosis and immunodepression, two events that have been already documented in Space. Additionally, endocannabinoid-based drugs can serve as novel therapeutics to be exploited as countermeasures against weightlessness sickness. Figure legends Figure 1. (A) NAPE-PLD and (B) FAAH mRNA and protein expression in microgravity (0g) and inflight control (1g) PBMCs samples onboard the ISS. Figure 2. Overall scheme of hypothetical effect of microgravity conditions on AEA metabolism in lymphocytes. Inset graph shows NAPE-PLD:FAAH ratio. Figure S1. Timeline and experimental procedures of RESLEM project during pre-flight, on orbit and post-flight operations.

Figure 1
Image
Figure 2
Image
Figure 3
Image

Acknowledgements

The authors gratefully acknowledge KI team for their kind and expert support, and Ing. Dr. Raimondo Fortezza (Mars Center, Naples, Italy). The RESLEM project was made possible by Italian Space Agency, European Space Agency and the Russian Space Agency, Energia.

References

Battista, N., Rapino, C., Di Tommaso, M., Bari, M., Gasperi, V., Finazzi Agrò, A., et al. (2011) Role of the Endocannabinoid System in human Lymphocytes Exposed to Microgravity (ROALD-2/RESLEM). ESA Grant – Proposal: ILSRA-2009-0928. Missions/Campaigns: 2011 - ISS Increment 29-30 (PromISSe). Battista, N., Di Sabatino, A., Di Tommaso, M., Biancheri, P., Rapino, C., Vidali, F., et al. (2012). Abnormal anandamide metabolism in celiac disease. J. Nutr. Biochem. 23, 1245-1248. Battista, N., Meloni, M.A., Bari, M., Mastrangelo, N., Galleri, G., Rapino, C., et al. (2012). 5-Lipoxygenase-dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment. FASEB J. 26, 1791-1798. Choukèr, A., Kaufmann, I., Kreth, S., Hauer, D., Feuerecker, M., Thieme, D., et al. (2010). Motion sickness, stress and the endocannabinoid system. PLoS One. 5, e10752. Hughes-Fulford, M. (2011). To infinity ... and beyond! Human spaceflight and life science. FASEB J. 25, 2858-2864. Maccarrone, M., Fiorucci, L., Erba, F., Bari, M., Finazzi-Agrò, A., Ascoli, F. (2000). Human mast cells take up and hydrolyze anandamide under the control of 5-lipoxygenase and do not express cannabinoid receptors. FEBS Lett. 46, 8176-8180. Maccarrone, M., Tacconi, M., Battista, N., Valgattarri, F., Falciani, P., Finazzi-Agrò A. (2001). Lipoxygenase activity during parabolic flights. J. Gravit. Physiol. 8, 123-124. Maccarrone, M., Battista, N., Meloni, M., Bari, M., Galleri, G., Pippia, P., et al. (2003). Creating conditions similar to those that occur during exposure of cells to microgravity induces apoptosis in human lymphocytes by 5-lipoxygenase-mediated mitochondrial uncoupling and cytochrome c release. J. Leukoc. Biol. 73, 472-481. Maccarrone, M., Finazzi-Agrò, A. (2003). The endocannabinoid system, anandamide and the regulation of mammalian cell apoptosis. Cell Death Differ. 10, 946-955. Maccarrone, M., Gasperi, V., Bari, M., Finazzi-Agrò, A., Cogoli-Greuter, M., et al. (2008). Role of programmed cell death (apoptosis) in the depression of human T lymphocytes activation in microgravity. NASA-ESA-ASI Grant – Proposal: 98-HEDS-02-027 (LSRA). Missions/Campaigns: 2008 - ISS Increment 18. Maccarrone, M. (2017). Metabolism of the Endocannabinoid Anandamide: Open Questions after 25 Years. Front. Mol. Neurosci. 10, 166. Strewe, C., Feuerecker, M., Nichiporuk, I., Kaufmann, I., Hauer, D., Morukov, B., et al. (2012). Effects of parabolic flight and spaceflight on the endocannabinoid system in humans. Rev. Neurosci. 23, 673-680. Walther, I., Pippia, P., Meloni, M. A, Turrini, F., Mannu, F., Cogoli, A. (1998). Simulated microgravity inhibits the genetic expressionofinterleukin-2anditsreceptorinmitogen-activated T lymphocytes. FEBS Lett. 436, 115–118.

Keywords: anandamide, Immunosuppression, microgravity, Lymphocytes, Lipoxygenase

Conference: 39th ISGP Meeting & ESA Life Sciences Meeting, Noordwijk, Netherlands, 18 Jun - 22 Jun, 2018.

Presentation Type: Extended abstract

Topic: Immune System and Radiations

Citation: Battista N, Di Tommaso M, Norfini A, Passerai M, Chiurchiù V, Maccarrone M and Bari M (2019). Altered Anandamide Metabolism in Microgravity: the “RESLEM” experiment. Front. Physiol. Conference Abstract: 39th ISGP Meeting & ESA Life Sciences Meeting. doi: 10.3389/conf.fphys.2018.26.00013

Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.

The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.

Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.

For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.

Received: 02 Dec 2018; Published Online: 16 Jan 2019.

* Correspondence:
Prof. Mauro Maccarrone, Facoltà di Medicina e Chirurgia, Università Campus Bio-Medico di Roma, Rome, Lazio, 00128, Italy, mauro.maccarrone@univaq.it
Dr. Monica Bari, Dipartimento di Medicina Sperimentale e Chirurgia, Università degli Studi di Roma Tor Vergata, Rome, Italy, Bari@med.uniroma2.it