Species-specific self-DNA detection mechanisms by mammalian cyclic GMP-AMP synthases.

The mechanisms by which innate immune receptors mediate self-nonself discrimination are unclear. In this study, we found species-specific molecular determinants of self-DNA reactivity by cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase (cGAS). Human cGAS contained a catalytic domain that was intrinsically self-DNA reactive and stimulated interferon responses in diverse cell types. This reactivity was prevented by an upstream amino (N)-terminal domain. The cGAS proteins from several nonhuman primate species exhibited a similar pattern of self-DNA reactivity in cells, but chimpanzee cGAS was inactive even when its amino-terminal domain was deleted. In contrast, the N terminus of mouse cGAS promoted self-DNA reactivity. When expressed within tumors, only self-DNA-reactive cGAS proteins protected mice from tumor-induced lethality. In vitro studies of DNA- or chromatin-induced cGAS activation did not reveal species-specific activities that correlate with self-DNA reactivity observed in macrophages. Cell biological analysis revealed that self-DNA reactivity by human cGAS, but not mouse cGAS, correlated with localization to mitochondria. We found that epitope tag positions affected self-DNA reactivity in cells and that DNA present in cell lysates undermines the reliability of cGAS biochemical fractionations. These studies reveal species-specific diversity of cGAS functions, even within the primate lineage, and highlight experimental considerations for the study of this innate immune receptor.

Regional variations and relationships among cytokine profiles, white blood cell counts, and blood mercury concentrations in Steller sea lion (Eumetopias jubatus) pups.

The Steller sea lion (SSL) population west of 144°W longitude experienced a significant population decline. While there appears to be a stable or increasing population trend in rookeries in the Gulf of Alaska (GOA) and Southeast Alaska (SEA), some rookeries within the Aleutian Islands (AI) have failed to recover. Previous studies found regional differences in whole blood total mercury concentrations ([THg]) showing more than 20% of AI pups had [THg] above critical thresholds for increased risk of immunological effects and other adverse outcomes. Measurements of immune cell-signaling proteins can be used to evaluate the immune status of marine mammals in relation to [THg]. We compared serum cytokine and chemokine concentrations in pups among regions (AI, eastern GOA, SEA), and examined associations among cytokines, chemokines, white blood cell (WBC) counts, and [THg]. Considering liver is an important target organ for mercury and immune protein synthesis we additionally examined the relationship of [THg] with liver-related enzymes serum aspartate (AST) and alanine aminotransferase (ALT). We observed regional differences in cytokine and chemokine measurements and immune protein associations. There was a positive association between total WBC counts and [THg] in AI pups, whereas a negative association between lymphocytes and [THg] in SEA pups. These findings may indicate regional variation in proliferation and differentiation of hematopoietic cells, differences in immune system development, and/or a difference in antigenic stimuli. No associations between [THg] and cytokines, chemokines, AST or ALT were found. Observed regional differences in cytokine and chemokine milieu during gestational and early development in SSL pups could lead to an imbalance in cell differentiation that could impact immunological resiliency in juvenile and adult life stages. We report concentration ranges of a suite of cytokines and chemokines which may prove to be a useful metric for ecotoxicology and risk assessment studies in SSLs and other wildlife.

Assessing oxidative stress in Steller sea lions (Eumetopias jubatus): Associations with mercury and selenium concentrations.

Selenium (Se) bioavailability is required for synthesis and function of essential Se-dependent antioxidants, including the enzyme glutathione peroxidase (GPx). Strong interactions between monomethyl mercury and Se impair the critical antioxidant role of Se. Approximately 20% of Steller sea lion (Eumetopias jubatus, SSL) pups sampled in the western Aleutian Islands, Alaska, had total Hg concentrations ([THg]) measured in hair and whole blood above thresholds of concern for adverse physiologic effects in pinnipeds. Importantly, low molar ratios of TSe:THg, in some cases < 1 in several tissues (hair, liver, pelt, muscle, spleen, intestine, heart, lungs, brain) were documented for one SSL pup with [THg] above threshold of concern, which may lead to antioxidant deficiency. Our aim with this study was to evaluate the relationship between circulating [THg], [MeHg+], [TSe] and TSe:THg molar ratio status relative to oxidative stress and antioxidants measured during general anesthesia in free-ranging SSL. We captured, anesthetized and sampled newborn SSL pups at rookeries located in the Aleutian Islands or Gulf of Alaska. Biomarkers analyzed for oxidative stress included 4-hydroxynenonal and thiobarbituric acid reactive substances (4-HNE and TBARS, respectively, lipid peroxidation), protein carbonyl content (PCC, protein oxidation), and GPx activity as a key indicator for Se-dependent antioxidant defense levels. We found a negative association between TBARS and [TSe], and SSL with low [TSe] had higher concentrations of 4-HNE than those with intermediate [TSe]. These results suggest that SSL with lower [TSe] experience increased lipid peroxidation potentially associated with [THg] status.

REGIONAL AND AGE-RELATED VARIATIONS IN HAPTOGLOBIN CONCENTRATIONS IN STELLER SEA LIONS ( EUMETOPIAS JUBATUS) FROM ALASKA, USA.

Varying concentrations of the highly conserved acute phase response protein, haptoglobin, can indicate changes to the health and disease status of mammals, including the Steller sea lion (SSL; Eumetopias jubatus). To better understand factors relating to acute phase response in SSLs, circulating haptoglobin concentrations (Hp) were quantified in plasma collected from 1,272 individuals sampled near rookeries and haulouts off the coast of Alaska, US. We compared Hp in SSLs between sexes and among different age classes (young pups, young-of-the-year, yearlings, subadults, and adults) sampled within distinct regions in Alaska (Aleutian Islands, Gulf of Alaska, Southeast Alaska). Regional and age-related differences were observed, particularly in younger SSLs. No sex-related differences were detected. We identified weakly significant relationships between Hp and hematology measurements including white blood cell counts and hematocrit in young pups from the Aleutian Islands and Southeast Alaska. No relationship between Hp and body condition was found. Lastly, a nonlinear relationship of plasma Hp and whole blood total mercury concentrations (THg) was observed in SSLs from the endangered western distinct population segment in Alaska. These results demonstrated that regional variation in Hp, especially in younger SSLs, may reflect regional differences in health and circulating THg.

Enhanced quantification of serum immunoglobulin G from a non-model wildlife species, the Steller sea lion (Eumetopias jubatus), using a protein A ELISA.

Immunoglobulins (Ig) are proteins that preserve immune homeostasis and are quantified to infer changes to the acquired humoral immune response in mammals. Measuring Ig in non-model wildlife for immune surveillance often requires ingenuity, and rigorous standardization of methodologies to provide reliable results especially when lacking species-specific reagents. We modified and optimized existing ELISA methodology utilizing the binding properties of Staphylococcus-derived Protein A (PrtA) to immunoglobulin G (IgG). We enhanced the assay for quantifying IgG in Steller sea lion (SSL) serum using critical quality control measures including dilution linearity, spike and percent recoveries, and internal controls. Of the modifications made, heat treatment of SSL serum enhanced accuracy and precision of IgG measurements by improving linearity and percent recovery in parallel dilutions and serum spikes. Purified canine IgG standard was not affected by heat inactivation. These results support that confounding serum proteins interfere with binding of PrtA with IgG demonstrating the need for heat treatment of serum to optimize IgG quantification using the PrtA-ELISA. Further, essential validation measures ensure proper assay performance. Consequently, the improved PrtA-ELISA provides species-independent IgG detection with validation criteria to enhance accuracy and precision for addressing future immunological questions in non-model wildlife in clinical, ecological, and conservation contexts.

Evaluating Hair as a Predictor of Blood Mercury: The Influence of Ontogenetic Phase and Life History in Pinnipeds.

Mercury (Hg) biomonitoring of pinnipeds increasingly utilizes nonlethally collected tissues such as hair and blood. The relationship between total Hg concentrations ([THg]) in these tissues is not well understood for marine mammals, but it can be important for interpretation of tissue concentrations with respect to ecotoxicology and biomonitoring. We examined [THg] in blood and hair in multiple age classes of four pinniped species. For each species, we used paired blood and hair samples to quantify the ability of [THg] in hair to predict [THg] in blood at the time of sampling and examined the influence of varying ontogenetic phases and life history of the sampled animals. Overall, we found that the relationship between [THg] in hair and blood was affected by factors including age class, weaning status, growth, and the time difference between hair growth and sample collection. Hair [THg] was moderately to strongly predictive of current blood [THg] for adult female Steller sea lions (Eumetopias jubatus), adult female California sea lions (Zalophus californianus), and adult harbor seals (Phoca vitulina), whereas hair [THg] was poorly predictive or not predictive (different times of year) of blood [THg] for adult northern elephant seals (Mirounga angustirostris). Within species, except for very young pups, hair [THg] was a weaker predictor of blood [THg] for prereproductive animals than for adults likely due to growth, variability in foraging behavior, and transitions between ontogenetic phases. Our results indicate that the relationship between hair [THg] and blood [THg] in pinnipeds is variable and that ontogenetic phase and life history should be considered when interpreting [THg] in these tissues.