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Assessing polar bear (Ursus maritimus) immune function in relation to environmental stressors, including habitat change, nutritional stress, pathogen prevalence, and pollution, has been identified as critical for improved understanding of the species' health. The objectives of this study were two-fold: 1) to assess the role of climate-associated factors (habitat use, body condition) in explaining the plasma concentrations of contaminants in southern Beaufort Sea (SB) polar bears, and 2) to investigate how climate-associated factors, contaminant concentrations, and pathogen sero-prevalence influence the plasma concentrations of immune-signaling proteins called cytokines. A commercially available multiplex canine cytokine panel was validated for the quantification of five pro- and anti-inflammatory cytokines in polar bear plasma: tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), IL-8, IL-10, and interferon gamma-induced protein 10 (IP-10). This panel was then used to measure cytokine concentrations in 49 SB polar bears sampled in the springs of 2013 and 2014. Mean ∑PCBs (plasma), ∑OCs (plasma), and THg (hair) were 13.01 ± 1.52 ng g -1 w.w. (range: 0.17-52.63), 19.46 ± 1.17 ng g -1 w.w. (range: 6.63-45.82), and 0.49 μg g -1 d.w. (range: 0.99-15.18), respectively. Top models explaining variation in concentrations of plasma PCBs, plasma OC pesticides, and hair THg in SB polar bears included body mass index and/or habitat use (onshore versus offshore), with higher contaminant concentrations in leaner and/or offshore bears. Plasma cytokine concentrations were influenced most strongly by plasma PCBs and age, with little to no influence found for plasma OCs or hair THg concentrations, habitat use, or pathogen sero-prevalence. The lack of association between cytokines and these latter variables is likely due to a temporal disconnect between measured endpoints. The change of polar bear habitat use, feeding ecology, and body condition with ongoing climate warming is affecting exposure to contaminants and pathogens, with potential adverse consequences on a well-balanced immune system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Killer whales ( Orcinus orca ) are among the most highly polychlorinated biphenyl (PCB)-contaminated mammals in the world, raising concern about the health consequences of current PCB exposures. Using an individual-based model framework and globally available data on PCB concentrations in killer whale tissues, we show that PCB-mediated effects on reproduction and immune function threaten the long-term viability of >50% of the world's killer whale populations. PCB-mediated effects over the coming 100 years predicted that killer whale populations near industrialized regions, and those feeding at high trophic levels regardless of location, are at high risk of population collapse. Despite a near-global ban of PCBs more than 30 years ago, the world's killer whales illustrate the troubling persistence of this chemical class., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Threatened loggerhead sea turtles (Caretta caretta) face numerous environmental challenges, including exposure to anthropogenic chemicals such as polychlorinated biphenyls (PCBs). Despite being banned by the USA in the 1970s, PCBs persist in the environment and produce immunotoxic effects in a wide range of marine vertebrate species. This is of particular concern, as the modulation of the immune system may enhance the susceptibility to a variety of pathogens. Blood samples were collected from 19 immature, captive-reared loggerhead sea turtles. Functional immune assays phagocytosis and natural killer (NK) cell activity were used to quantify the direct effects of PCB congeners 105, 138, and 169 on innate immune functions upon in vitro exposure of sea turtle cells to increasing concentrations (control (0), 0.5, 1, 2.5, 5, 10, 15, or 20 ppm) of each PCB. PCB 105 significantly elevated eosinophil phagocytosis at 10 and 15 ppm and PCB 138 at 15 ppm compared to unexposed (0 ppm). The effects of PCB 169 on phagocytosis were not evaluated. PCB 138 and 105 significantly decreased NK cell activity at 15 and 20 ppm, compared to unexposed (0 ppm) controls. PCB 169 did not markedly modulate NK activity. This constitutes the first study to investigate the in vitro effects of these three PCBs on sea turtle innate immune functions. These results add to our understanding of PCB-induced immunotoxicity in sea turtles and may provide a framework for establishing the relationships between chemical levels and turtle immunity.

To better elucidate the potential immune-related health effects of exposure to environmentally persistent organic pollutants (POP), such as polychlorinated biphenyls (PCBs) and perfluoroalkyl substances (PFASs), in ringed seals (Pusa hispida), a sentinel Arctic species, we assessed 1) associations between mitogen-induced lymphocyte proliferation and in vivo tissue contaminant burdens, and 2) the concentration-response effects of in vitro exposure to PFASs and PCB congeners on mitogen-induced lymphocyte proliferation. Upon in vitro contaminant exposure, the non-coplanar PCB congeners CB 138, 153, and 180, but not the coplanar CB 169, significantly reduced lymphocyte proliferation between 10 and 20µgg -1 ww. The respective in vitro EC 50 values for these congeners were 13.3, 20.7, 20.8, and 54.6µgg -1 ww. No modulation of lymphocyte proliferation was observed upon in vitro exposure to two individual PFASs, perfluorooctane sulphonic acid (PFOS) and perfluorooctanoic acid (PFOA), at concentrations up to 1000ngg-1. In addition, no significant correlations were found between lymphocyte proliferation and any blood or blubber contaminant measured. Taken together, these data suggest this population of ringed seals is not currently at high risk of altered lymphocyte proliferation from exposure to the POPs or PFASs in this study., (Copyright © 2016. Published by Elsevier Inc.)

Polychlorinated biphenyls (PCBs) have been demonstrated to modulate marine mammal immune functions; however, the underlying mechanisms involved are poorly understood. Cytosolic calcium (Ca2+) is an important second messenger involved in numerous leukocyte functions. The direct effects of in vitro exposure to PCBs on Ca2+ mobilization were evaluated in leukocytes isolated from bottlenose dolphins, beluga whales, and B6C3F1 mice. Concentration- and time-response experiments with three non-coplanar PCBs (138, 153, 180), one coplanar PCB (169), and TCDD were tested. Exposure to the three non-coplanar PCBs significantly increased cytosolic Ca2+ in dolphin neutrophils, while PCB 180 significantly increased cytosolic Ca2+ in beluga neutrophils. Two non-coplanar PCBs (138 and 153) significantly increased Ca2+ in beluga monocytes, yet the response was delayed compared to that in neutrophils. Neither PCBs nor TCDD increased cytosolic Ca2+ in mouse neutrophils or monocytes. In experiments with Ca2+-free medium, only PCB 153 increased cytosolic Ca2+ in dolphin neutrophils, though the increase was less than that observed with Ca2+-supplemented medium, suggesting that extracellular Ca2+ was the predominant source for the rise in cytosolic Ca2+. Furthermore, in cells incubated with Ca2+-free medium, a significant increase in cytosolic Ca2+ was induced by thapsigargin following PCB exposure, indicating that intracellular Ca2+ was available, yet not mobilized by the PCBs, and further suggesting that PCBs mobilize extracellular Ca2+. These results demonstrate for the first time the direct effects of non-coplanar PCBs on Ca2+ mobilization in marine mammals, which may be involved in the modulation of phagocytosis previously observed in these species.

The effects of organochlorines (OC) on the immune systems of marine mammals and humans are poorly understood. One important innate immune function of peripheral blood neutrophils and monocytes is the respiratory burst, which generates reactive oxygen species (ROS) used to kill engulfed microorganisms. The present study characterized the immunomodulatory potential for mixtures of OCs, compared to that of individual OCs, on the respiratory burst in several marine mammals, humans, and B6C3F1 mice. The effects of three non-coplanar polychlorinated biphenyls (PCBs) (138, 153, 180), one coplanar PCB (169), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and all possible mixtures were tested upon in vitro exposure for 1 h, and their effects on the generation of a respiratory burst were measured by flow cytometry. The final concentration for each congener, alone or in a mixture, was 5 ppm for PCBs and 0.05 ppb for TCDD. Both significant enhancement and suppression of the respiratory burst occurred in all species tested, but the pattern was different between species and cell types (neutrophils vs. monocytes). Both coplanar and non-coplanar OCs were involved in the modulation of the respiratory burst. Regression analysis was not able to elucidate which OCs were involved in modulating the responses, highlighting the difficulty of developing models to predict the immunotoxic effects attributed to OC mixtures. The traditional mouse model and toxic equivalency (TEQ) approach both failed to consistently predict the toxicity of OCs in all species tested, questioning their applicability in the risk assessment process for all species. Elucidating the relative sensitivities to the immunomodulatory effects of OC mixtures between different species may have important implications for risk assessment as well as conservation and management strategies.

Marine mammals bioaccumulate various environmental contaminants such as organochlorines (OCs), which biomagnify via the food web. While the immunomodulatory effects of individual OCs have been studied, the effects of mixtures are not well understood. The immunomodulatory effects of polychlorinated biphenyl (PCB) 138, 153, 169, and 180 as well as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and all possible mixtures were examined in marine mammals and mice. Lymphocyte proliferation was significantly modulated by OCs in all species tested, mostly by non-coplanar PCBs, as shown using regression analyses. Correlation analyses showed significant correlations (interpreted as additive effects) between OCs in mice, killer whales, and Steller sea lions. Nonadditive synergistic and antagonistic interactions between OCs were detected in most of the species tested. Toxic equivalency (TEQ) values used for OC toxicity assessment failed to predict the immunomodulatory effects measured in mice and marine mammals. The commonly used mouse model failed to predict immunomodulatory effects in other species. Clustering data suggested that phylogeny does not predict toxicity of OCs. Overall, our data suggest the presence of species-specific sensitivities to different mixtures, in which OCs interactions may be complex and that may exert their effects through dioxinlike or dioxin-independent pathways. Lastly, lymphocyte proliferation, an important part of adaptive immunity, was significantly modulated in mice and marine mammals, suggesting the possibility of increased susceptibility to diseases. These findings will be useful to better characterize the risk associated with OC exposure and possibly lead to new conservation and management strategies.

Organochlorine (OC) contaminants, notably polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are ubiquitous in all ecosystems and found in the tissues of humans and wildlife. Although the immunotoxicity of coplanar, dioxinlike PCBs is well documented, the adverse effects exerted by non-coplanar, non-dioxinlike PCBs have received little attention. Direct causal relationship between PCB and dioxin exposure and the observed detrimental effects on the immune system has yet to be fully established in humans. The immunomodulatory potential of toxic coplanar PCB 169 and TCDD and abundant non-coplanar PCBs 138, 153, and 180 on human leukocyte phagocytosis, an important innate immune function that initiates the clearance of pathogens, was tested upon in vitro exposure. Mixture and concentration-response experiments demonstrated a suppression of phagocytosis by non-coplanar PCBs suggesting a previously unrecognized aryl hydrocarbon receptor (AhR)-independent pathway. Regression analysis revealed that reduction of phagocytosis was mostly explained by the non-coplanar congeners. The effects on phagocytosis could not be accurately predicted by either the currently used toxic equivalence (TEQ) approach or the mouse model, thus undermining the use of the traditional models in the risk assessment for OC mixtures containing non-coplanar congeners. Our results are cause for concern as they suggest an AhR-independent pathway through which non-coplanar PCBs modulate phagocytosis, the immune system's first line of defense, possibly increasing the risk to developing infectious disease.

Recent pinniped die-offs have led to the speculation that persistent organic pollutants (POPs) are immunomodulatory, making individuals more susceptible to viral infections. Eighteen healthy harbor seal (Phoca vitulina) pups (aged 3-4 weeks) were live-captured from southern British Columbia, Canada, and maintained temporarily in captivity for an immunotoxicological assessment. The relationships between mitogen-induced peripheral blood lymphocyte proliferation and blubber concentrations of three major immunotoxic POP classes (the polychlorinated biphenyls [PCBs], polychlorinated dibenzo-p-dioxins [PCDDs], and the polychlorinated dibenzofurans [PCDFs]) were evaluated. A significant body weight-independent positive correlation was observed between both T-cell mitogen (phytohemagglutinin [PHA])- and B-cell mitogen (lipopolysaccharide [LPS])-induced lymphocyte proliferation and the blubber concentrations of total PCB. Best subset regression analysis revealed that total PCBs, and not total PCDD or total PCDF, explained 24 and 29% of the changes in both T-cell mitogen-and B-cell mitogen-induced lymphocyte proliferation, respectively. Further regression analysis performed on the PCB classes measured in this study showed that di-ortho PCBs accounted for 25 and 30% of the changes in both T-cell and B-cell lymphocyte proliferation, respectively. Results suggest that POPs, and PCBs in particular, are associated with changes in lymphocyte proliferation, something that could result in increased susceptibility to infections in harbor seal pups. Further research is needed to evaluate the relative roles of natural and contaminant-related influences on the immune system of marine mammals.

Increasing evidence has supported the general hypothesis that organochlorines (OC) can produce immunotoxic effects in marine mammals. One important innate defense mechanism is phagocytosis, the ability of cells to ingest extracellular macromolecules. The present study is aimed at characterizing the immunomodulatory potential of mixtures of OCs on phagocytosis compared to that of individual compounds in different species of marine mammals and mice, the traditional model to study mammalian immunotoxicity. The ability of peripheral blood neutrophils and monocytes to engulf fluorescent microspheres was evaluated using flow cytometry. The immunomodulatory effects of three non-coplanar polychlorinated biphenyl (PCB) congeners, 138, 153, 180, one coplanar PCB, 169, as well as 2,3,7,8-TCDD, and all possible mixtures (26) were tested upon in vitro exposure. All species were not equally sensitive to the adverse effects of OCs on either neutrophils or monocytes phagocytosis. With the exception of harbor seals, all mixtures that significantly modulated neutrophil or monocyte phagocytosis contained at least one non-coplanar PCB. Regression analysis revealed that the non-coplanar congeners, more than the coplanar congeners, explained the variability in phagocytosis. Dendrograms revealed that phylogeny could not predict immunotoxicity. The currently used toxic equivalency (TEQ) approach and the traditional mouse model both failed to predict experimentally induced immunomodulatory effects in marine mammals tested, leading us to question the reliability of both TEQs and mouse model in risk assessment of OC mixtures. Testing the relative sensitivity to immunomodulatory effects of contaminants and contaminant mixtures between different species of marine mammals may have important implications for risk assessment as well as conservation and management strategies.

Contaminant-induced immunosuppression by organochlorines (OC), particularly polychlorinated biphenyls (PCBs), has been suspected as a cofactor in the deaths of thousands of marine mammals. One important innate defense mechanism is phagocytosis, the ability of cells to ingest extracellular macromolecules. The present study was aimed at characterizing the immunomodulatory potential of representative OCs on phagocytosis in bottlenose dolphins and beluga whales. The ability of peripheral blood leukocytes to engulf fluorescent microspheres was evaluated using flow cytometry. The immunomodulatory effects of three non-coplanar PCB congeners, 138, 153, and 180, one coplanar PCB, 169, and 2,3,7,8-TCDD and all possible mixtures (26) were tested upon in vitro exposure. In both species, all mixtures containing at least two non-coplanar PCBs significantly reduced both neutrophil and monocyte phagocytosis, with effects more marked in dolphins than in belugas. Coplanar OCs, on their own or when added to non-coplanar congeners, did not further modulate phagocytosis, suggesting an Ah receptor-independent mechanism. Concentration-response experiments with individual congeners further demonstrated a non-coplanar PCB-induced suppression of phagocytosis, while coplanar congeners produced no consistent effects. Our results suggest simple additive interactions of chemicals in a mixture. However, calculation of toxic equivalency (TEQs) failed to predict the experimentally induced immunomodulatory effects of OCs on dolphin and beluga phagocytosis, confirming the Ah receptor-independent nature of the effects on phagocytosis. Overall, our results suggest that non-AhR mechanisms may explain one facet of immunotoxicity (phagocytosis), something that is not captured using the TEQ approach. This is the first report demonstrating the immunomodulatory effects of OCs on dolphin and beluga phagocytosis, and the first overall demonstration of immunomodulatory effects on phagocytosis mediated specifically by non-coplanar PCBs.

Considerable efforts have been made to better understand the immune system of bottlenose dolphins in view of the common environmental challenges they encounter, such as exposure to polychlorinated biphenyls, oil spills, or harmful algal bloom biotoxins. However, little is known about the identity and functionality of the Th1, Th2, and Treg T helper cell subsets in bottlenose dolphins. The present study aimed at validating assays and reagents to identify T helper cell subsets and their functions in a subset of dolphins from Sarasota Bay, Florida, USA, which have been long studied and often used as a reference population. A population of CD4+ FOXP3+ lymphocytes was identified representing an average <1% of blood lymphocyte population, which is in the range observed in for Treg cells in other species. The use of porcine reagents to measure TGFß, one of the key Treg cytokines, was further validated using the relatively high-throughput and highly standardized Luminex technology. The proportion of circulating Treg cells was not correlated with the serum concentrations of the Treg effector cytokines TGFß and IL-10, nor could it significantly contribute to predicting the variability of T lymphocyte proliferation, suggesting that not all dolphin circulating Treg cells are functional and active. However, stimulation of dolphin lymphocytes with TGFß and IL-2 increased the expression of the gene for TGFß and IL-10, and stimulation with IL-12 and IFNγ induced a robust increase in the expression of the gene for IFNγ, suggesting the potential for polarization and differentiation of dolphin T helper cells toward a Treg and Th1 response, respectively. The lack of an increase in the expression of the genes for the Th2 cytokines IL-4 and IL-13 upon stimulation with IL-4 may be due to the requirement for IL-2 for a Th2 polarization as described in mice. However, regression analysis and PCA suggested the potential ability of both the Th1 and Th2 response to be triggered upon acute inflammatory signals. These results may be useful in better understanding the mechanisms by which the dolphin immune system is affected upon exposure to environmental challenges and how it responds to pathogen challenges. [ABSTRACT FROM AUTHOR]

Organochlorines (OC) are lipophilic and stable, and therefore accumulate in tissues of top predators, such as marine mammals. While the immunomodulatory effects of individual OC have been studied in lab animals, their effects in other species (such as marine mammals) and the possible interactions between chemicals in mixtures are not well understood. This study investigated the immunomodulatory effects of four polychlorinated biphenyls (PCB, IUPAC numbers 138, 153, 169, and 180), as well as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), individually and in mixtures, in marine mammals and mice. Mitogen-induced B lymphocyte proliferation was mostly modulated by non-coplanar PCBs, for which general mechanisms underlying toxicity are poorly understood. Simple additive effects of OC in mixtures were found only in mice, while both synergistic and antagonistic interactions between OC were found in marine mammals. The toxic equivalency (TEQ) approach, which is currently used to assess the dioxin-like toxicity of OC mixtures, failed to predict immunotoxicity in mice and marine mammals, likely due to the complexity of interactions between OC and effects via dioxin-independent pathways. The commonly used mouse model failed to predict the immunotoxicity due to OC in the marine mammals tested. In addition, clustering data suggested that phylogeny might not help predict the toxicity of OC. Lymphoproliferative response was modulated in most species tested suggesting the possibility of increased susceptibility to infectious diseases in these animals. These findings may be helpful in more accurately characterizing the immunotoxic potential of OC in different target species and help in more relevant risk assessment. [ABSTRACT FROM AUTHOR]

Due to their marine ecology and life-history, marine mammals accumulate some of the highest levels of environmental contaminants of all wildlife. Given the increasing prevalence and severity of diseases in marine wildlife, it is imperative to understand how pollutants affect the immune system and consequently disease susceptibility. Advancements and adaptations of analytical techniques have facilitated marine mammal immunotoxicology research. Field studies, captive-feeding experiments and in vitro laboratory studies with marine mammals have associated exposure to environmental pollutants, most notable polychlorinated biphenyls (PCBs), organochlorine pesticides and heavy metals, to alterations of both the innate and adaptive arms of immune systems, which include aspects of cellular and humoral immunity. For marine mammals, reported immunotoxicology endpoints fell into several major categories: immune tissue histopathology, haematology/circulating immune cell populations, functional immune assays (lymphocyte proliferation, phagocytosis, respiratory burst, and natural killer cell activity), immunoglobulin production, and cytokine gene expression. Lymphocyte proliferation is by far the most commonly used immune assay, with studies using different organic pollutants and metals predominantly reporting immunosuppressive effects despite the many differences in study design and animal life history. Using combined field and laboratory data, we determined effect threshold levels for suppression of lymphocyte proliferation to be between < 0.001–10 ppm for PCBs, 0.002–1.3 ppm for Hg, 0.009–0.06 for MeHg, and 0.1–2.4 for cadmium in polar bears and several pinniped and cetacean species. Similarly, thresholds for suppression of phagocytosis were 0.6–1.4 and 0.08–1.9 ppm for PCBs and mercury, respectively. Although data are lacking for many important immune endpoints and mechanisms of specific immune alterations are not well understood, this review revealed a systemic suppression of immune function in marine mammals exposed to environmental contaminants. Exposure to immunotoxic contaminants may have significant population level consequences as a contributing factor to increasing anthropogenic stress in wildlife and infectious disease outbreaks. [ABSTRACT FROM AUTHOR]