Abstract

Challenges associated with diving, such as increases in pressure, have been reported to alter cellular processes including several functions performed by immune cells such as macrophages and lymphocytes. Previous studies from our laboratory suggest phagocytosis is altered in beluga granulocytes and monocytes during and following in vitro pressure exposures. The purpose of this study was to measure the response of beluga lymphocytes, specifically proliferation and activation, following exposure to 2000 psi (1360 m) or 1000 psi (680 m), and evaluate the potential for stressors to alter that response. It is hypothesized that lymphocytes will respond differently between dive-simulated conditions and stressors. Beluga blood samples were obtained from animals at the Mystic Aquarium during monthly health checks (baseline) (n = 4), following a 30-minute out-of-water examination (OWE) (n = 3), and during a period of mild chronic inflammation (n = 2), as well as from free-ranging animals in Bristol Bay, AK (n = 9). In addition, human blood samples (n = 4) (purchased from Biological Specialty Corporation) were compared with belugas. Catecholamines and cortisol were measured in beluga samples in order to characterize the neuroendocrine response. Cell suspensions were exposed to simulated dives using a stainless steel pressure chamber and hydraulic pump containing mineral oil. Simulated dive profiles lasted for 30 minutes, 5 minutes or repeated 5-minute durations with 2 minutes or 15 seconds of compression and decompression. Lymphocyte activation was measured by monitoring IL2 receptor (IL2R) expression utilizing a human IL2 Fluorokine kit (R and D Systems), and concanavalin A-induced proliferation was measured using a BrdU incorporation assay (Roche Diagnostics). Mixed generalized linear models were used to compare the response of cells between controls (non-pressure exposed) and pressure-exposed cells, between conditions in belugas and humans, as well as between different dive profiles (α = 0.05). Cortisol was significantly higher in Bristol Bay belugas than aquarium animals (ANOVA F_3,14 = 27.524; p < 0.001). For aquarium animals the OWE had significantly higher cortisol levels than either baseline or inflammation conditions (ANOVA F_2,2 = 56.834; p = 0.017). Significant pressure induced changes in IL2R expression and lymphocyte proliferation were detected for all exposures to 2000 psi, as well as for all 30-minute exposures. Significantly different responses were found between conditions in belugas, between exposure profiles and between belugas and humans. Overall, both belugas and humans displayed increased IL2R expression following pressure exposures. Larger changes in IL2R expression were observed for humans than belugas except for 1000 psi dives with rapid compression. In contrast, decreased T lymphocyte proliferation was observed for baseline conditions in belugas, while pressure-induced increases in proliferation were suggested for human and wild belugas for exposures to 2000 psi with 2 minutes of compression and decompression. Results suggest that beluga lymphocyte function is altered during diving and changes may be part of normal dive adaptation as the response differs from that observed in humans, a non-dive adapted mammal. In addition, characteristics of a dive, including depth and duration, as well as stressors, can alter the response of beluga lymphocytes, impacting the ability of animals to fight infection or resist dive-related pathologies.

Acknowledgements

The authors would like to thank the research and veterinary teams at Mystic Aquarium for sample collection, and help with sample processing in the lab. We also thank the beluga husbandry team for help with sample collection. We acknowledge the Animal Health Diagnostic Center Endocrinology Laboratory at Cornell University for measuring cortisol concentrations in beluga plasma samples. This work was funded in part by the Office of Naval Research (ONR, #N00014-11-1-0437 and #N00014-13-1-0768), Mystic Aquarium and the University of Connecticut Marine Science Department Pre-Doctoral Fellowship. Additional funding was provided by NOAA's Oceans and Human Health, IRICH (Interdisciplinary Research and training Initiative on Coastal ecosystems and human Health) initiative.

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