Hjalmar R. Bouma

Hibernation: the immune system at rest?

Mammalian hibernation consists of torpor phases when metabolism is severely depressed, and Tb can reach as low as approximately –2°C, interrupted by euthermic arousal phases. Hibernation affects the function of the innate and the adaptive immune systems. Torpor drastically reduces numbers of all types of circulating leukocytes. In addition, other changes have been noted, such as lower complement levels, diminished response to LPS, phagocytotic capacity, cytokine production, lymphocyte proliferation, and antibody production. Hibernation may therefore increase infection risk, as illustrated by the currently emerging WNS in hibernating bats. Unraveling the pathways that result in reduced immune function during hibernation will enhance our understanding of immunologic responses during extreme physiological changes in mammals.

Low body temperature governs the decline of circulating lymphocytes during hibernation through sphingosine-1-phosphate

Hibernation is an energy-conserving behavior consisting of periods of inhibited metabolism (‘torpor’) with lowered body temperature. Torpor bouts are interspersed by arousal periods, in which metabolism increases and body temperature returns to euthermia. In deep torpor, the body temperature typically decreases to 2–10 °C, and major physiological and immunological changes occur. One of these alterations constitutes an almost complete depletion of circulating lymphocytes that is reversed rapidly upon arousal. Here we show that torpor induces the storage of lymphocytes in secondary lymphoid organs in response to a temperature-dependent drop in plasma levels of sphingosine-1-phosphate (S1P). Regulation of lymphocyte numbers was mediated through the type 1 S1P receptor (S1P1), because administration of a specific antagonist (W146) during torpor (in a Syrian hamster at ∼8 °C) precluded restoration of lymphocyte numbers upon subsequent arousal. Furthermore, S1P release from erythrocytes via ATP-binding cassette (ABC)-transporters was significantly inhibited at low body temperature (4 °C) but was restored upon rewarming. Reversible lymphopenia also was observed during daily torpor (in a Djungarian hamster at ± 25 °C), during forced hypothermia in anesthetized (summer-active) hamsters (at ± 9 °C), and in a nonhibernator (rat at ∼19 °C). Our results demonstrate that lymphopenia during hibernation in small mammals is driven by body temperature, via altered plasma S1P levels. S1P is recognized as an important bioactive lipid involved in regulating several other physiological processes as well and may be an important factor regulating additional physiological processes in hibernation as well as in mediating the effects of therapeutic hypothermia in patients.

Induction of torpor: Mimicking natural metabolic suppression for biomedical applications†

Mammalian hibernation consists of periods of depressed metabolism and reduced body temperature called “torpor” that are interspersed by normothermic arousal periods. Numerous cellular processes are halted during torpor, including transcription, translation, and ion homeostasis. Hibernators are able to survive long periods of low blood flow and body temperature followed by rewarming and reperfusion without overt signs of organ injury, which makes these animals excellent models for application of natural protective mechanisms to human medicine. This review examines efforts to induce torpor‐like states in non‐hibernating species using pharmacological compounds. Elucidating the underlying mechanisms of natural and pharmacologically induced torpor will speed the development of new clinical approaches to treat a variety of trauma and stress states in humans. J. Cell. Physiol. 227: 1285–1290, 2012.

AMP-Activated Protein Kinase as a Target for Preconditioning in Transplantation Medicine

Graft quality before transplantation is a major factor influencing chronic rejection. Organ preservation and ischemia/reperfusion play an important role in the induction of organ injury. Although both suppression of metabolism by hypothermic preservation and preconditioning before ischemia limit injury, understanding the biochemical signaling pathways will allow us to optimize graft preservation further. Adenosine monophosphate-activated protein kinase (AMPK) is an important enzyme sensing cellular energy balance and regulating downstream signaling pathways, signaling toward an energy-conserving state. In this review, we summarize available literature regarding the protective signaling pathways activated by (hypothermic) ischemia and preconditioning and how they can be activated pharmacologically. Optimizing the graft quality before transplantation improves long-term graft survival. The major factor influencing organ quality is organ preservation, cold storage, currently, being a common practice. Loss of cellular homeostasis, inflammation, and endothelial dysfunction are the major factors inducing injury after cold storage. Adenosine triphosphate depletion and anaerobic metabolism during the cold ischemic period lead to mitochondrial dysfunction, disturbed osmoregulation, and cell death inducing inflammation. Ischemic preconditioning consists of brief periods of ischemia preceding preservation and protects organs against injury because of subsequent ischemia/reperfusion, in which endothelial nitric oxide synthase, nuclear factor-kB, and adenosine play a major role. After conversion of adenosine to AMP, AMPK can be activated, a central kinase involved in sensing cellular [AMP]:[adenosine triphosphate] levels and signaling toward an energy-conserving state. Pharmacologic activation of AMPK demonstrated its ability to activate endothelial nitric oxide synthase and inhibit nuclear factor-kB, thereby limiting endothelial dysfunction and inflammation. Further, studies in knock-out mice lacking ENTDP1 and NT5E (enzymes catalyzing formation and degradation of AMP, respectively) demonstrated a clear protective role for AMP in ischemia/reperfusion. AMPK activation before or during organ preservation might be a promising pharmacologic approach to limit organ injury and maintain graft quality before transplantation.

Signal Transduction Pathways Involved in Brain Death-Induced Renal Injury

Kidneys derived from brain death organ donors show an inferior survival when compared to kidneys derived from living donors. Brain death is known to induce organ injury by evoking an inflammatory response in the donor. Neuronal injury triggers an inflammatory response in the brain, leading to endothelial dysfunction and the release of cytokines in the circulation. Serum levels of interleukin‐6, ‐8, ‐10, and monocyte chemoattractant protein‐1 (MCP‐1) are increased after brain death. Binding with cytokine‐receptors in kidneys stimulates activation of nuclear factor‐kappa B (NF‐κB), selectins, adhesion molecules and production of chemokines leading to cellular influx. Mitogen‐activated protein kinases (MAP‐kinases) mediate inflammatory responses and together with NF‐κB they seem to play an important role in brain death induced renal injury. Altering the activation state of MAP‐kinases could be a promising drug target for early intervention to reduce cerebral injury related donor kidney damage and improve outcome after transplantation.

Serotonin and Dopamine Protect from Hypothermia/Rewarming Damage through the CBS/ H2S Pathway

Biogenic amines have been demonstrated to protect cells from apoptotic cell death. Herein we show for the first time that serotonin and dopamine increase H2S production by the endogenous enzyme cystathionine-β-synthase (CBS) and protect cells against hypothermia/rewarming induced reactive oxygen species (ROS) formation and apoptosis. Treatment with both compounds doubled CBS expression through mammalian target of rapamycin (mTOR) and increased H2S production in cultured rat smooth muscle cells. In addition, serotonin and dopamine treatment significantly reduced ROS formation. The beneficial effect of both compounds was minimized by inhibition of their re-uptake and by pharmacological inhibition of CBS or its down-regulation by siRNA. Exogenous administration of H2S and activation of CBS by Prydoxal 5′-phosphate also protected cells from hypothermic damage. Finally, serotonin and dopamine pretreatment of rat lung, kidney, liver and heart prior to 24 h of hypothermia at 3°C followed by 30 min of rewarming at 37°C upregulated the expression of CBS, strongly reduced caspase activity and maintained the physiological pH compared to untreated tissues. Thus, dopamine and serotonin protect cells against hypothermia/rewarming induced damage by increasing H2S production mediated through CBS. Our data identify a novel molecular link between biogenic amines and the H2S pathway, which may profoundly affect our understanding of the biological effects of monoamine neurotransmitters.

Reversible remodeling of lung tissue during hibernation in the Syrian hamster

SUMMARY During hibernation, small rodents such as hamsters cycle through phases of strongly suppressed metabolism with low body temperature (torpor) and full restoration of metabolism and body temperature (arousal). Remarkably, the repetitive stress of cooling–rewarming and hypoxia does not cause irreversible organ damage. To identify adaptive mechanisms protecting the lungs, we assessed histological changes as well as the expression and localization of proteins involved in tissue remodeling in lungs from Syrian hamsters at different phases of hibernation using immunohistochemical staining and western blot analysis. In torpor (early and late) phase, a reversible increased expression of smooth muscle actin, collagen, angiotensin converting enzyme and transforming growth factor-β was found, whereas expression of the epidermal growth factor receptor and caveolin-1 was low. Importantly, all these alterations were restored during arousal. This study demonstrates substantial alterations in protein expression mainly in epithelial cells of lungs from hibernating Syrian hamsters. These structural changes of the bronchial airway structure are termed airway remodeling and often occur in obstructive lung diseases such as asthma, chronic obstructive pulmonary disease (COPD) and lung fibrosis. Unraveling the molecular mechanism leading to reversal of airway remodeling by the end of torpor may identify possible therapeutic targets to reduce progression of this process in patients suffering from asthma, chronic obstructive pulmonary disease and lung fibrosis.

Blood cell dynamics during hibernation in the European Ground Squirrel

Hibernation is a unique natural model to study large and specific modulation in numbers of leukocytes and thrombocytes, with potential relevance for medical application. Hibernating animals cycle through cold (torpor) and warm (arousal) phases. Previous research demonstrated clearance of leukocytes and thrombocytes from the circulation during torpor, but did not provide information regarding the timing during torpor or the subtype of leukocytes affected. To study the influence of torpor-bout duration on clearance of circulating cells, we measured blood cell dynamics in the European Ground Squirrel. Numbers of leukocytes and thrombocytes decreased within 24h of torpor by 90% and remained unchanged during the remainder of the torpor-bout. Differential counts demonstrated that granulocytes, lymphocytes and monocytes are all affected by torpor. Although a decreased production might explain the reduced number of thrombocytes, granulocytes and monocytes, this cannot explain the observed lymphopenia since lymphocytes have a much lower turnover rate than thrombocytes, granulocytes and monocytes. In conclusion, although underlying biochemical signaling pathways need to be unraveled, our data show that the leukocyte count drops dramatically after entrance into torpor and that euthermic cell counts are restored within 1.5h after onset of arousal, even before body temperature is fully normalized.

Platelet dynamics during natural and pharmacologically induced torpor and forced hypothermia.

Hibernation is an energy-conserving behavior in winter characterized by two phases: torpor and arousal. During torpor, markedly reduced metabolic activity results in inactivity and decreased body temperature. Arousal periods intersperse the torpor bouts and feature increased metabolism and euthermic body temperature. Alterations in physiological parameters, such as suppression of hemostasis, are thought to allow hibernators to survive periods of torpor and arousal without organ injury. While the state of torpor is potentially procoagulant, due to low blood flow, increased viscosity, immobility, hypoxia, and low body temperature, organ injury due to thromboembolism is absent. To investigate platelet dynamics during hibernation, we measured platelet count and function during and after natural torpor, pharmacologically induced torpor and forced hypothermia. Splenectomies were performed to unravel potential storage sites of platelets during torpor. Here we show that decreasing body temperature drives thrombocytopenia during torpor in hamster with maintained functionality of circulating platelets. Interestingly, hamster platelets during torpor do not express P-selectin, but expression is induced by treatment with ADP. Platelet count rapidly restores during arousal and rewarming. Platelet dynamics in hibernation are not affected by splenectomy before or during torpor. Reversible thrombocytopenia was also induced by forced hypothermia in both hibernating (hamster) and non-hibernating (rat and mouse) species without changing platelet function. Pharmacological torpor induced by injection of 5′-AMP in mice did not induce thrombocytopenia, possibly because 5′-AMP inhibits platelet function. The rapidness of changes in the numbers of circulating platelets, as well as marginal changes in immature platelet fractions upon arousal, strongly suggest that storage-and-release underlies the reversible thrombocytopenia during natural torpor. Possibly, margination of platelets, dependent on intrinsic platelet functionality, governs clearance of circulating platelets during torpor.

The role of endogenous H2S formation in reversible remodeling of lung tissue during hibernation in the Syrian hamster

SUMMARY During hibernation, small mammals alternate between periods of metabolic suppression and low body temperature (‘torpor’) and periods of full metabolic recovery with euthermic temperatures (‘arousal’). Previously, we demonstrated marked structural remodeling of the lung during torpor, which is rapidly reversed during arousal. We also found that cooling of hamster cells increased endogenous production of H2S through the enzyme cystathionine-β-synthase (CBS). H2S suppresses the immune response and increases deposition of collagen. Therefore, we examined inflammatory markers and matrix metalloproteinase (MMP) activity in relation to CBS expression and H2S levels in lungs of euthermic and hibernating Syrian hamsters. Lung remodeling during torpor was confirmed by a strong increase in both collagenous and non-collagenous hydroxyproline content. The number of leukocytes in lung was unchanged in any phase of hibernation, while adhesion molecules VCAM-1 and ICAM-1, and the inflammatory marker NF-κB (P65) were modestly upregulated in torpor. Gelatinase activity was decreased in lungs from torpid animals, indicating inhibition of the Zn2+-dependent MMP-2 and MMP-9. Moreover, expression of CBS and tissue levels of H2S were increased in torpor. All changes normalized during arousal. Inhibition of gelatinase activity in torpor is likely caused by quenching of Zn2+ by the sulphide ion of H2S. In accord, inhibition of CBS normalized gelatinase activity in torpid animals. Conversely, NaHS decreased the gelatinase activity of euthermic animals, which was attenuated by excess Zn2+. Similar results were obtained on the activity of the Zn2+-dependent angiotensin converting enzyme. Our data indicate that increased production of H2S through CBS in hamster lungs during torpor contributes to remodeling by inhibition of gelatinase activity and possibly by suppression of the inflammatory response. Although administration of H2S is known to induce metabolic suppression in non-hibernating mammals (‘suspended animation’), this is the first report implying endogenous H2S production in natural hibernation.