Phase specific suppression of neutrophil function in hibernating Syrian hamster.

Hibernation consists of alternating periods of reduced metabolism (torpor) with brief periods of metabolism similar to summer euthermia (arousal). The function of the innate immune system is reduced during hibernation, of which the underlying mechanisms are incompletely understood. Here, we studied neutrophil functionality during hibernation in Syrian hamsters. The inflammatory response to LPS-induced endotoxemia is inhibited in hibernation, partly mediated by reduced IL-6 production in early arousal. Furthermore, neutrophil pathogen binding, phagocytosis and oxidative burst is profoundly reduced in early arousal. Functionality of both summer and early arousal neutrophils was repressed in plasma from early arousal and mixed plasma from early arousal and summer euthermic, but restored by summer euthermic plasma, signifying that a plasma factor in early arousal inhibits TLR-recognition. Identification of the inhibiting factor may offer a target to modulate neutrophil function with relevance to (auto-)inflammatory diseases.

Determinants of defence strategies of a hibernating European bat species towards the fungal pathogen Pseudogymnoascus destructans.

Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome in North America, has decimated bat populations within a decade. The fungus impacts bats during hibernation when physiological functions, including immune responses, are down-regulated. Studies have shown that Pd is native to Europe, where it is not associated with mass mortalities. Moreover, genomic and proteomic studies indicated that European bats may have evolved an effective immune defence, which is lacking in North American bats. However, it is still unclear which defence strategy enables European bats to cope with the pathogen. Here, we analyzed selected physiological and immunological parameters in torpid, Pd infected European greater mouse-eared bats (Myotis myotis) showing three different levels of infection (asymptomatic, mild and severe symptoms). From a subset of the studied bats we tracked skin temperatures during one month of hibernation. Contrasting North American bats, arousal patterns remained unaffected by Pd infections in M. myotis. In general, heavier M. myotis aroused more often from hibernation and showed less severe disease symptoms than lean individuals; most likely because heavy bats were capable of reducing the Pd load more effectively than lean individuals. In the blood of severely infected bats, we found higher gene expression levels of an inflammatory cytokine (IL-1ß), but lower levels of an acute phase protein (haptoglobin), reactive oxygen metabolites (ROMs) and plasma non-enzymatic antioxidant capacity (OXY) compared to conspecifics with lower levels of infection. We conclude that M. myotis, and possibly also other European bat species, tolerate Pd infections during torpor by using selected acute phase response parameters at baseline levels, yet without arousing from torpor and without synthesizing additional immune molecules.

Expression of Paracrine Effectors in Human Adipose-Derived Mesenchymal Stem Cells Treated With Plasma From Brown Bears (Ursus arctos).

Adipose-derived mesenchymal stem cells (ADSCs) are promising candidates for novel cell therapeutic applications. Hibernating brown bears sustain tissue integrity and function via unknown mechanisms, which might be plasma borne. We hypothesized that plasma from hibernating bears may increase the expression of favorable factors from human ADSCs. In an experimental study, ADSCs from patients with ischemic heart disease were treated with interventional media containing plasma from hibernating and active bears, respectively, and with control medium. Extracted RNA from the ADSCs was sequenced using next generation sequencing. Statistical analyses of differentially expressed genes were performed using fold change analysis, pathway analysis, and gene ontology. As a result, we found that genes associated with inflammation, such as IGF1, PGF, IL11, and TGFA, were downregulated by > 10-fold in ADSCs treated with winter plasma compared with control. Genes important for cardiovascular development, ADM, ANGPTL4, and APOL3, were upregulated in ADSCs when treated with winter plasma compared with summer plasma. ADSCs treated with bear plasma, regardless if it was from hibernating or active bears, showed downregulation of IGF1, PGF, IL11, INHBA, IER3, and HMOX1 compared with control, suggesting reduced cell growth and differentiation. This can be summarized in the conclusion that plasma from hibernating bears suppresses inflammatory genes and activates genes associated with cardiovascular development in human ADSCs. Identifying the involved regulator(s) holds therapeutic potential.

Immune responses in hibernating little brown myotis (Myotis lucifugus) with white-nose syndrome.

White-nose syndrome (WNS) is a fungal disease responsible for decimating many bat populations in North America. Pseudogymnoascus destructans (Pd), the psychrophilic fungus responsible for WNS, prospers in the winter habitat of many hibernating bat species. The immune response that Pd elicits in bats is not yet fully understood; antibodies are produced in response to infection by Pd, but they may not be protective and indeed may be harmful. To understand how bats respond to infection during hibernation, we studied the effect of Pd inoculation on the survival and gene expression of captive hibernating Myotis lucifugus with varying pre-hibernation antifungal antibody titres. We investigated gene expression through the transcription of selected cytokine genes (Il6, Il17a, Il1b, Il4 and Ifng) associated with inflammatory, Th1, Th2 and Th17 immune responses in wing tissue and lymph nodes. We found no difference in survival between bats with low and high anti-Pd titres, although anti-Pd antibody production during hibernation differed significantly between infected and uninfected bats. Transcription of Il6 and Il17a was higher in the lymph nodes of infected bats compared with uninfected bats. Increased transcription of these cytokines in the lymph node suggests that a pro-inflammatory immune response to WNS is not restricted to infected tissues and occurs during hibernation. The resulting Th17 response may be protective in euthermic bats, but because it may disrupt torpor, it could be detrimental during hibernation.

Immunoreactivities of NF-κB, IL-1ß and IL-1R in the skin of Chinese brown frog (Rana dybowskii).

The cytokine interleukin-1 beta (IL-1ß) is an evolutionarily conserved molecule that was originally identified in the immune system. Nuclear factor κB (NF-κB) plays a critical role in the activation of immune cells by upregulating the expression of many cytokines. In this study, we investigated the localization and expression level of IL-1ß, its functional membrane receptor type I (IL-1R1) and NF-κB in the skin of Rana dybowskii during the breeding period and pre-hibernation. Histologically, the skin of Rana dybowskii consists of epidermis and dermis, and four kinds of cells were identified in the epidermis during the breeding period and pre-hibernation, while the dermis was composed of homogenous gel, mucous glands and granular glands. IL-1ß, IL-1R1 and NF-κB were immunolocalized in the epithelial and glandular cells in both periods. Western blotting showed that there was no significant difference in the expression of IL-1ß between the breeding period and pre-hibernation, whereas IL-1R1 and NF-κB were significantly higher in the pre-hibernation compared to the breeding period. These results suggested that IL-1ß and NF-κB may collectively play important roles in the skin immune system of Rana dybowskii during the breeding period and pre-hibernation.

The White-Nose Syndrome Transcriptome: Activation of Anti-fungal Host Responses in Wing Tissue of Hibernating Little Brown Myotis.

White-nose syndrome (WNS) in North American bats is caused by an invasive cutaneous infection by the psychrophilic fungus Pseudogymnoascus destructans (Pd). We compared transcriptome-wide changes in gene expression using RNA-Seq on wing skin tissue from hibernating little brown myotis (Myotis lucifugus) with WNS to bats without Pd exposure. We found that WNS caused significant changes in gene expression in hibernating bats including pathways involved in inflammation, wound healing, and metabolism. Local acute inflammatory responses were initiated by fungal invasion. Gene expression was increased for inflammatory cytokines, including interleukins (IL) IL-1ß, IL-6, IL-17C, IL-20, IL-23A, IL-24, and G-CSF and chemokines, such as Ccl2 and Ccl20. This pattern of gene expression changes demonstrates that WNS is accompanied by an innate anti-fungal host response similar to that caused by cutaneous Candida albicans infections. However, despite the apparent production of appropriate chemokines, immune cells such as neutrophils and T cells do not appear to be recruited. We observed upregulation of acute inflammatory genes, including prostaglandin G/H synthase 2 (cyclooxygenase-2), that generate eicosanoids and other nociception mediators. We also observed differences in Pd gene expression that suggest host-pathogen interactions that might determine WNS progression. We identified several classes of potential virulence factors that are expressed in Pd during WNS, including secreted proteases that may mediate tissue invasion. These results demonstrate that hibernation does not prevent a local inflammatory response to Pd infection but that recruitment of leukocytes to the site of infection does not occur. The putative virulence factors may provide novel targets for treatment or prevention of WNS. These observations support a dual role for inflammation during WNS; inflammatory responses provide protection but excessive inflammation may contribute to mortality, either by affecting torpor behavior or causing damage upon emergence in the spring.

Hibernation induces immune changes in the lung of 13-lined ground squirrels (Ictidomys tridecemlineatus).

During hibernation, significant changes occur in the systemic and intestinal immune populations. We found that the lungs of hibernating 13-lined ground squirrels (Ictidomys tridecemlineatus) also undergo shifts in immune phenotype. Within the population of mononuclear cells, the percentage of T cells increases and the percentage of CD11b/c(+) cells decreases in hibernators. E-selectin, which promotes endothelial attachment, increases during arousal from torpor. Levels of the anti-inflammatory cytokine interleukin (IL)-10 in the lung are lower during hibernation while levels of the pro-inflammatory cytokine, tumor necrosis factor (TNF)-α remain constant. Expression of suppressor of cytokine signaling (SOCS) proteins is also decreased in torpid hibernators. Our data point to a unique immune phenotype in the lung of hibernating ground squirrels in which certain immunosuppressive proteins are downregulated while some potentially inflammatory proteins are maintained or amplified. This indicates that the lung houses an immune population that can potentially respond to antigenic challenge during hibernation.

Body temperature during hibernation is highly correlated with a decrease in circulating innate immune cells in the brown bear (Ursus arctos): a common feature among hibernators?

BACKGROUND: Hibernation involves periods of severely depressed metabolism (torpor) and decreases in body temperature (Tb). Small arctic mammals (<5kg), in which Tb generally drop drastically, display leukopenia during hibernation. This raised the question of whether the decreased leukocyte counts in mammalian hibernators is due to torpor per se or is secondary to low Tb. The present study examined immune cell counts in brown bears (Ursus arctos), where torpor is only associated with shallow decreases in Tb. The results were compared across hibernator species for which immune and Tb data were available. METHODS AND RESULTS: The white blood cell counts were determined by flow cytometry in 13 bears captured in the field both during summer and winter over 2 years time. Tb dropped from 39.6±0.8 to 33.5±1.1°C during hibernation. Blood neutrophils and monocytes were lower during hibernation than during the active period (47%, p= 0.001; 43%, p=0.039, respectively), whereas no change in lymphocyte counts was detected (p=0.599). Further, combining our data and those from 10 studies on 9 hibernating species suggested that the decline in Tb explained the decrease in innate immune cells (R(2)=0.83, p<0.0001). CONCLUSIONS: Bears have fewer innate immune cells in circulation during hibernation, which may represent a suppressed innate immune system. Across species comparison suggests that, both in small and large hibernators, Tb is the main driver of immune function regulation during winter dormancy. The lack of a difference in lymphocyte counts in this context requires further investigations.

Hibernating little brown myotis (Myotis lucifugus) show variable immunological responses to white-nose syndrome.

White-nose syndrome (WNS) is an emerging infectious disease devastating hibernating North American bat populations that is caused by the psychrophilic fungus Geomyces destructans. Previous histopathological analysis demonstrated little evidence of inflammatory responses in infected bats, however few studies have compared other aspects of immune function between WNS-affected and unaffected bats. We collected bats from confirmed WNS-affected and unaffected sites during the winter of 2008-2009 and compared estimates of their circulating levels of total leukocytes, total immunoglobulins, cytokines and total antioxidants. Bats from affected and unaffected sites did not differ in their total circulating immunoglobulin levels, but significantly higher leukocyte counts were observed in bats from affected sites and particularly in affected bats with elevated body temperatures (above 20°C). Bats from WNS-affected sites exhibited significantly lower antioxidant activity and levels of interleukin-4 (IL-4), a cytokine that induces T cell differentiation. Within affected sites only, bats exhibiting visible fungal infections had significantly lower antioxidant activity and levels of IL-4 compared to bats without visible fungal infections. Overall, bats hibernating in WNS-affected sites showed immunological changes that may be evident of attempted defense against G. destructans. Observed changes, specifically elevated circulating leukocytes, may also be related to the documented changes in thermoregulatory behaviors of affected bats (i.e. increased frequencies in arousal from torpor). Alterations in immune function may reflect expensive energetic costs associated with these processes and intrinsic qualities of the immunocapability of hibernating bats to clear fungal infections. Additionally, lowered antioxidant activity indicates a possible imbalance in the pro- versus antioxidant system, may reflect oxidative tissue damage, and should be investigated as a contributor to WNS-associated morbidity and mortality.

Hibernation is associated with depression of T-cell independent humoral immune responses in the 13-lined ground squirrel.

Mammalian hibernation consists of periods of low metabolism and body temperature (torpor), interspersed by euthermic arousal periods. The function of both the innate and adaptive immune system is suppressed during hibernation. In this study, we analyzed the humoral adaptive immune response to a T-cell independent (TI-2) and a T-cell dependent (TD) antigen. Thirteen-lined ground squirrels were immunized in summer or during hibernation with either a TI-2 or TD antigen on day 0 and day 14. Blood was drawn on day 0, 7, 14, 21 and 28. Both types of antigens induced a significant rise in antibody titer in summer animals. Much to our surprise, however, only immunization with the TD antigen, and not with the TI-2 antigen induced a humoral response in hibernators. Flow cytometric analysis of CD4 (helper T-lymphocytes), CD8 (cytotoxic T-lymphocytes) and CD45RA (B-lymphocytes) in blood, spleen and lymph nodes ruled out massive apoptosis as explanation of the absent TI humoral response during hibernation. Rather, reduced TI-2 stimulation of B-lymphocytes, possibly due to lowered serum complement during torpor, may explain the reduced antibody production in response to a TI-2 antigen. These results demonstrate that hibernation diminishes the capacity to induce a TI-2 humoral immune response, while the capacity to induce a humoral response to a TD antigen is maintained.

Immunoreactivity of c-kit receptor protein during the prehibernation period in the oviduct of the Chinese brown frog, Rana chensinensis.

The objective of this study was to investigate immunoreactivity of the c-kit receptor in the oviduct of Rana chensinensis during the prehibernation period. Histological examination of oviducts was performed during the prehibernation period. The sections of oviduct were immunostained by the avidin-biotin-peroxidase complex method using rabbit polyclonal antisera raised against the rat c-kit receptor and PCNA. Total proteins were extracted from oviducal tissues and used for Western blotting analysis. Immunohistochemistry revealed the presence of the c-kit receptor and PCNA in the oviduct tissues during the prehibernation period. Also, positive signals for the c-kit receptor and PCNA by Western blotting were observed in oviduct tissues during the prehibernation period. These results suggested that the c-kit receptor might play a regulatory role in oviducal hypertrophy in the brown frog, Rana chensinensis.

Specific alterations in complement protein activity of little brown myotis (Myotis lucifugus) hibernating in white-nose syndrome affected sites.

White-nose syndrome (WNS) is the most devastating condition ever reported for hibernating bats, causing widespread mortality in the northeastern United States. The syndrome is characterized by cutaneous lesions caused by a recently identified psychrophilic and keratinophylic fungus (Geomyces destructans), depleted fat reserves, atypical behavior, and damage to wings; however, the proximate cause of mortality is still uncertain. To assess relative levels of immunocompetence in bats hibernating in WNS-affected sites compared with levels in unaffected bats, we describe blood plasma complement protein activity in hibernating little brown myotis (Myotis lucifugus) based on microbicidal competence assays using Escherichia coli, Staphylococcus aureus and Candida albicans. Blood plasma from bats collected during mid-hibernation at WNS-affected sites had higher bactericidal ability against E. coli and S. aureus, but lower fungicidal ability against C. albicans when compared with blood plasma from bats collected at unaffected sites. Within affected sites during mid-hibernation, we observed no difference in microbicidal ability between bats displaying obvious fungal infections compared to those without. Bactericidal ability against E. coli decreased significantly as hibernation progressed in bats collected from an affected site. Bactericidal ability against E. coli and fungicidal ability against C. albicans were positively correlated with body mass index (BMI) during late hibernation. We also compared complement activity against the three microbes within individuals and found that the ability of blood plasma from hibernating M. lucifugus to lyse microbial cells differed as follows: E. coli>S. aureus>C. albicans. Overall, bats affected by WNS experience both relatively elevated and reduced innate immune responses depending on the microbe tested, although the cause of observed immunological changes remains unknown. Additionally, considerable trade-offs may exist between energy conservation and immunological responses. Relationships between immune activity and torpor, including associated energy expenditure, are likely critical components in the development of WNS.

Hibernation: the immune system at rest?

Mammalian hibernation consists of torpor phases when metabolism is severely depressed, and T(b) 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.

Seasonal changes in the intestinal immune system of hibernating ground squirrels.

Hibernation is associated with a prolonged fast (5-8 mo) which has the potential to affect intestinal immunity. We examined several aspects of the intestinal immune system in summer (non-hibernating) and hibernating ground squirrels. Peyer's patches were largely unaffected by hibernation, but numbers of intraepithelial lymphocytes (IEL) and lamina propria leukocytes (LPL) were greater in hibernators compared with summer. Hibernator IEL were less mature as demonstrated by low numbers of cells expressing activation-associated markers and co-receptors. Compared with summer, the percentage of B cells was higher and percentage of T cells was lower in the hibernator LPL. Hibernation was associated with greater mucosal levels of IFN-gamma, TNF-alpha, IL-10 and IL-4, but IL-6 and TGF-beta were unchanged. Mucosal IgA levels were greater in entrance and torpid hibernators compared with summer. The results suggest that modifications of the intestinal immune system during hibernation may help preserve gut integrity throughout the winter fast.

Ground squirrel splenic macrophages bind lipopolysaccharide over a wide range of temperatures at all phases of their annual hibernation cycle.

This study evaluates binding of bacterial lipopolysaccharide (LPS) by splenic macrophages from golden-mantled ground squirrels (Spermophilus lateralis, GMGS), a hibernating mammal, at a variety of in vitro incubation temperatures to determine whether this aspect of immune function is effective at low body temperatures. LPS-binding by ground squirrel macrophages was compared to that of rat splenic macrophages. Macrophages were collected from squirrels at discreet stages in their annual cycle and incubated with fluorescein-labeled LPS (LPS-FITC). The percentage of GMGS that bound LPS-FITC did not change as a function of hibernation season or as a function of incubation temperature. The total amount of LPS-FITC bound per cell was similarly unaffected by season or temperature, however, macrophages from torpid squirrels bound more LPS-FITC than cells from normothermic squirrels. Macrophages of golden-mantled ground squirrels bind LPS at a wide range of temperatures throughout their annual cycle; an ability shared between hibernators and non-hibernators alike.

Classical pathway serum complement activity throughout various stages of the annual cycle of a mammalian hibernator, the golden-mantled ground squirrel, Spermophilus lateralis.

Little is known about the changes in the immune system that coincide with the annual cycle of hibernating mammals. This study investigates classical pathway complement activity in the serum of the golden-mantled ground squirrel, a mammalian hibernator. Complement activity varied significantly among discreet stages of the annual cycle and is lowest during torpor and greatest during stages of arousal. C3 mRNA levels follow a pattern similar to that of complement-mediated hemolysis throughout the year but do not vary significantly among hibernation states. The classical pathway of the serum complement system is able to function in vitro at 5 degrees C, although at a slower rate than at 34 degrees C. The classical pathway of the serum complement system is active throughout all phases of the annual cycle of the golden-mantled ground squirrel.

Periodic arousal from hibernation is necessary for initiation of immune responses in ground squirrels.

Golden-mantled ground squirrels (Spermophilus lateralis) undergo seasonal hibernation during which core body temperature (T(b)) values are maintained 1-2 degrees C above ambient temperature. Hibernation is not continuous. Squirrels arouse at approximately 7-day intervals, during which T(b) increases to 37 degrees C for approximately 16 h; thereafter, they return to hibernation and sustain low T(b)s until the next arousal. Over the course of the hibernation season, arousals consume 60-80% of a squirrel's winter energy budget, but their functional significance is unknown and disputed. Host-defense mechanisms appear to be downregulated during the hibernation season and preclude normal immune responses. These experiments assessed immune function during hibernation and subsequent periodic arousals. The acute-phase response to bacterial lipopolysaccharide (LPS) was arrested during hibernation and fully restored on arousal to normothermia. LPS injection (ip) resulted in a 1-1.5 degrees C fever in normothermic animals that was sustained for > 8 h. LPS was without effect in hibernating squirrels, neither inducing fever nor provoking arousal, but a fever did develop several days later, when squirrels next aroused from hibernation; the duration of this arousal was increased sixfold above baseline values. Intracerebroventricular infusions of prostaglandin E(2) provoked arousal from hibernation and induced fever, suggesting that neural signaling pathways that mediate febrile responses are functional during hibernation. Periodic arousals may activate a dormant immune system, which can then combat pathogens that may have been introduced immediately before or during hibernation.

Production of tumor necrosis factor in cells of hibernating ground squirrels Citellus undulatus during annual cycle.

TNF production has been studied in peritoneal macrophages and splenic T cells of Arctic Yakutian ground squirrel (Citellus Undulatus Pallas) in hibernating and awake animals in winter and in prehibernating autumn as well as in active euthermic spring-summer animals. A high level of TNF production in macrophages of ground squirrel is observed over the active period and during arousals in winter. There are no significant season variations in TNF production in splenic T lymphocytes of ground squirrels. This suggests the major role of activated macrophages in the arousals of hibernating animals. T lymphocyte proliferation in ground squirrels in the active period is higher than in winter, and the most significant seasonal variations are found in T cell mitogenic response, which increases in spring-summer period. Evidence is presented that functional activity of macrophages of squirrel in autumn has much in common with that in winter rather than in spring-summer period.