Biobehavioral organization shapes the immune epigenome in infant rhesus Macaques (Macaca mulatta).

How individuals respond to and cope with stress is linked with their health and well-being. It is presumed that early stress responsiveness helps shape the health of the developing organism, but the relationship between stress responsiveness and early immune function during development is not well-known. We hypothesized that stress responsiveness may shape epigenetic regulation of immune genes in infancy. We investigated whether aspects of behavioral responsiveness and hypothalamic-pituitary adrenal stress-response were associated with epigenome-wide immune cell DNA methylation patterns in 154 infant rhesus monkeys (3-4 months old). Infants' behavioral and physiological responses were collected during a standardized biobehavioral assessment, which included temporary relocation and separation from their mother and social group. Genome-wide DNA methylation was quantified using restricted representation bisulfite sequencing (RRBS) from blood DNA collected 2-hours post-separation. Epigenome-wide analyses were conducted using simple regression, multiple regression controlling for immune cell counts, and permutation regression, all corrected for false discovery rate. Across the variables analyzed, there were 20,368 unique sites (in 9,040 genes) at which methylation was significantly associated with at least one behavioral responsiveness or cortisol measure across the three analyses. There were significant associations in 442 genes in the Immune System Process ontology category, and 94 genes in the Inflammation mediated by chemokine and cytokine signaling gene pathway. Out of 35 candidate genes that were selected for further investigation, there were 13 genes with at least one site at which methylation was significantly associated with behavioral responsiveness or cortisol, including two intron sites in the glucocorticoid receptor gene, at which methylation was negatively correlated with emotional behavior the day following the social separation (Day 2 Emotionality; ß = -0.39, q < 0.001) and cortisol response following a relocation stressor (Sample 1; ß = -0.33, q < 0.001). We conclude that biobehavioral stress responsiveness may correlate with the developing epigenome, and that DNA methylation of immune cells may be a mechanism by which patterns of stress response affect health and immune functioning.

Epigenetic plasticity following early stress predicts long-term health outcomes in rhesus macaques.

Early life stress has been linked with poorer lifelong health outcomes across species, including modern and ancient humans. Epigenetic mechanisms, such as DNA methylation patterning of stress pathway genes in stress-responsive tissue, may play an important role in the long-term health effects of early stress across species. The relationships among early maternal care quality, DNA methylation patterns in a candidate stress pathway gene (serotonin transporter, 5-HTT) linked region in blood DNA, and adult health outcomes were examined in male and female rhesus macaques, excellent models of human health. Male (n = 12) and female (n = 32) infants were observed with their mothers for the first 12 weeks of life and 5-HTT linked DNA methylation was measured in blood at 12 weeks of age. Approximately 8 years later, health-related measures were collected for the 25 animals (6 male and 19 female) that were available for study. Health composite scores were generated using factor analysis of body condition, body weight, and diagnosis of diarrhea during the lifespan. Better quality maternal care predicted lower 5-HTT linked methylation at 12 weeks of age. Lower 5-HTT methylation, in turn, predicted better health composite scores in adulthood, including better body condition, greater body weight and absence of lifetime diarrhea. These data suggest that the epigenetic regulation of 5-HTT may be one biomarker of the link between early stress and lifetime health trajectories. Future studies will examine whether epigenetic signatures in modern and ancient human DNA lends insight into stress and health and natural selection in human evolutionary history.

Oxytocin receptor binding in the titi monkey hippocampal formation is associated with parental status and partner affiliation.

Social cognition is facilitated by oxytocin receptors (OXTR) in the hippocampus, a brain region that changes dynamically with pregnancy, parturition, and parenting experience. We investigated the impact of parenthood on hippocampal OXTR in male and female titi monkeys, a pair-bonding primate species that exhibits biparental care of offspring. We hypothesized that in postmortem brain tissue, OXTR binding in the hippocampal formation would differ between parents and non-parents, and that OXTR density would correlate with frequencies of observed parenting and affiliative behaviors between partners. Subjects were 10 adult titi monkeys. OXTR binding in the hippocampus (CA1, CA2/3, CA4, dentate gyrus, subiculum) and presubiculum layers (PSB1, PSB3) was determined using receptor autoradiography. The average frequency of partner affiliation (Proximity, Contact, and Tail Twining) and infant carrying were determined from longitudinal observations (5-6 per day). Analyses showed that parents exhibited higher OXTR binding than non-parents in PSB1 (t(8) = - 2.33, p = 0.048), and that OXTR binding in the total presubiculm correlated negatively with Proximity (r = - 0.88) and Contact (r = - 0.91), but not Tail Twining or infant carrying. These results suggest that OXTR binding in the presubiculum supports pair bonding and parenting behavior, potentially by mediating changes in hippocampal plasticity.

Serotonin transporter expression is predicted by early life stress and is associated with disinhibited behavior in infant rhesus macaques.

Serotonin transporter (5-HTT) expression patterns may contribute to the risk for adverse psychological outcomes following early life stress. The present study investigated whether two types of early life stress, maternal and social aggression, and a serotonin transporter gene promoter polymorphism (rh5-HTTLPR) predicted lower post-stressor peripheral blood mononuclear cell (PBMC) 5-HTT expression in infant rhesus macaques. We further probed the relationships among these factors and infant behavioral disinhibition within a stressful situation. Fifty-three infants residing with mothers in large, complex social groups were observed over the first 12 postnatal weeks, during which time the rate of aggression received by the infant from their mothers and social group members was recorded. At 90-120 days of age, infants underwent a 25-h maternal separation/biobehavioral assessment, which included standardized behavioral assessments and blood sampling. Infants' rh5-HTTLPR genotypes were determined, and infant 5-HTT expression was quantified from PBMCs collected 8 h after separation. Receipt of aggression from the mother, but not from social group members, was associated with lower post-stressor 5-HTT expression. Lower post-stressor 5-HTT expression, but not receipt of aggression, was associated with disinhibited behavior during assessment. Rh5-HTTLPR genotype was unrelated to any measure. We conclude that 5-HTT regulation is linked with specific, presumably stressful early experiences in infant rhesus macaques. Further, 5-HTT expression predicted behavioral disinhibition, presumably via parallel processes that operate in the brain.

Epigenetic regulation of serotonin transporter expression and behavior in infant rhesus macaques.

Epigenetic mechanisms may moderate genetic and environmental risk (GxE) for mood disorders. We used an experimental rhesus macaque model of early life stress to test whether epigenetic regulation of serotonin transporter (5-HTT) may contribute to GxE interactions that influence behavior and emotion. We hypothesized that peripheral blood mononuclear cell (PBMC) DNA methylation within an 800 bp cytosine-phosphate-guanosine (CpG) island that overlaps with the 5-HTT transcription initiation start site, a hypothesized model of the same genomic region in brain tissue, would mediate or moderate the effects of early life stress and a functional 5-HTT promoter polymorphism (rh5-HTTLPR) on two outcomes: PBMC 5-HTT expression and behavioral stress reactivity. Eighty-seven infant rhesus macaques (3-4 months of age) were either mother reared in large social groups (n = 70) or nursery reared (n = 17). During a maternal/social separation, infants' blood was sampled and behavioral stress reactivity recorded. PBMC DNA and RNA samples were used to determine rh5-HTTLPR genotype, 5-HTT mRNA expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and 5-HTT CpG methylation status using sodium bisulfite pyrosequencing. Consistent with human data, carriers of the low-expressing rh5-HTTLPR alleles exhibited higher mean 5-HTT CpG methylation, which was associated with lower PBMC 5-HTT expression. Higher 5-HTT CpG methylation, but not rh5-HTTLPR genotype, exacerbated the effects of early life stress on behavioral stress reactivity in infants. 5-HTT CpG methylation may be an important regulator of 5-HTT expression early in development and may contribute to the risk for mood disorders observed in 'high-risk'5-HTTLPR carriers.

Effects of early experience and genotype on serotonin transporter regulation in infant rhesus macaques.

The moderating effect of early experience on gene-behavior associations has been well characterized. The molecular events that allow for such moderation are not well understood, however. We assessed the impact of early experience and serotonin transporter linked promoter polymorphism (rh5-HTTLPR) genotype on peripheral serotonin transporter (5-HTT) regulation in response to a maternal/social separation and relocation stressor in infant rhesus macaques. We further tested the hypothesis that modulation of 5-HTT regulation by rearing and/or genotype is mediated by glucocorticoid (GC) availability. Fifty-three infant (3-4 months of age) rhesus macaques that were either nursery reared (NR) or mother reared (MR) were genotyped for rh5-HTTLPR. Infants were blood sampled within 2.5 h of maternal or social separation/relocation and again 5 h later. Infants were then administered dexamethasone, a synthetic GC and blood sampled 16.5 h later. 5-HTT RNA was quantified from peripheral blood mononuclear cells. Plasma cortisol was measured at all time points. The MR individuals upregulated 5-HTT significantly during maternal/social separation, while NR individuals did not. Concomitant increases in cortisol were not observed, but dexamethasone treatment stimulated 5-HTT expression regardless of genotype/rearing group, and 5-HTT expression in the post-stressor sample was correlated with plasma cortisol levels at all time points. Our data indicate that early experience exerted a strong influence on 5-HTT regulation during a stressor in infant rhesus macaques independent of rh5-HTTLPR genotype. We also showed that GCs may stimulate 5-HTT expression but that there likely exist faster-acting transcriptional regulators of 5-HTT that are in place as a function of experience.

Molecular cloning of rat alpha1,3-fucosyltransferase IX (Fuc-TIX) and comparison of the expression of Fuc-TIV and Fuc-TIX genes during rat postnatal cerebellum development.

Fucosylated glycoconjugates play an essential role in central nervous system development, but the regulation of expression of these molecules is not well understood. The final biosynthetic step for a major group of cerebellar fucosylated glycoconjugates (those bearing the developmentally regulated epitope 3-fucosyl-N-acetyllactosamine, CD15, and related fucosylated epitopes) is catalyzed by an alpha-1,3-fucosyltransferase (FucT). The major FucT activity in postnatal rat cerebellum has a specificity consistent with that encoded by either a Fuc-TIV- or Fuc-TIX-like gene, and thus the expression of these genes was investigated during postnatal rat cerebellar development. A rFuc-TIX cDNA was cloned and a comparison of its enzymatic activity with rFuc-TIV revealed similar results on oligosaccharides, but strikingly higher activity on lipid acceptors, suggesting a greater role for rFuc-TIX than rFuc-TIV in the synthesis of CD15 glycolipids. rFuc-TIX mRNA levels were much higher than those of rFuc-TIV in neonatal cerebellum. Whereas rFuc-TIX mRNA levels remained relatively constant, rFuc-TIV mRNA levels declined during postnatal cerebellar development. In situ hybridization of postnatal rat cerebella also revealed different patterns of expression for these two genes. The rFuc-TIV gene was expressed predominantly in Purkinje cells and the deep cerebellar nuclei throughout postnatal development, but was expressed in granule neurons only in the neonatal, and not the adult, rat. In contrast, the rFuc-TIX gene was expressed in cells in the granule cell layers in both neonatal and in the adult rat. The potential implications of the different enzymatic activities and cell localization of rFuc-TIV and rFuc-TIX expression for the regulation of fucosylated glycoconjugates during cerebellar development are discussed.

Restriction of high CD15 expression to a subset of rat cerebellar astroglial cells can be overcome by transduction with adenoviral vectors expressing the rat alpha 1,3-fucosyltransferase IV gene.

Glycoconjugates bearing the epitope 3-fucosyl-N-acetyllactosamine (CD15) are believed to be involved in cell-cell interactions and are temporally and spatially regulated in the brain. In the rat postnatal cerebellum, CD15 is predominantly expressed in the molecular layer by Bergmann glial cells, but little CD15 expression is seen in other astroglia, and the basis for this restricted expression is not known. Adenoviral vectors were shown to efficiently deliver transgenes to cerebellar glial cells and were used to determine whether manipulation of glycosyltransferase activities could enhance the expression of CD15 in these cells. In dissociated cerebellar cell cultures, few glial cells normally express CD15. However, transduction of these cells with an adenoviral vector (AdGFPCMVFucT) that expressed both green fluorescent protein (GFP) and FLAG-tagged rat alpha 1, 3-fucosyltransferase IV (rFuc-TIV) resulted in high CD15 expression on the surface of all transduced glial cells. Likewise, infection of cerebellar slice cultures caused the appearance of CD15-positive transduced cells of glial cell morphology in the internal granule cell layer. Thus, enhancement of Fuc-T activity caused robust CD15 expression in cerebellar glial cells that normally show little expression of CD15, suggesting a role for Fuc-T levels in regulating CD15 expression in this cell type. The manipulation of levels of glycosyltransferases using adenoviral vectors may prove a useful tool to investigate questions of glycoconjugate regulation in glial cells in the developing rodent cerebellum.