Microbial modulation via cross-fostering prevents the effects of pervasive environmental stressors on microglia and social behavior, but not the dopamine system.

Environmental toxicant exposure, including air pollution, is increasing worldwide. However, toxicant exposures are not equitably distributed. Rather, low-income and minority communities bear the greatest burden, along with higher levels of psychosocial stress. Both air pollution and maternal stress during pregnancy have been linked to neurodevelopmental disorders such as autism, but biological mechanisms and targets for therapeutic intervention remain poorly understood. We demonstrate that combined prenatal exposure to air pollution (diesel exhaust particles, DEP) and maternal stress (MS) in mice induces social behavior deficits only in male offspring, in line with the male bias in autism. These behavioral deficits are accompanied by changes in microglial morphology and gene expression as well as decreased dopamine receptor expression and dopaminergic fiber input in the nucleus accumbens (NAc). Importantly, the gut-brain axis has been implicated in ASD, and both microglia and the dopamine system are sensitive to the composition of the gut microbiome. In line with this, we find that the composition of the gut microbiome and the structure of the intestinal epithelium are significantly shifted in DEP/MS-exposed males. Excitingly, both the DEP/MS-induced social deficits and microglial alterations in males are prevented by shifting the gut microbiome at birth via a cross-fostering procedure. However, while social deficits in DEP/MS males can be reversed by chemogenetic activation of dopamine neurons in the ventral tegmental area, modulation of the gut microbiome does not impact dopamine endpoints. These findings demonstrate male-specific changes in the gut-brain axis following DEP/MS and suggest that the gut microbiome is an important modulator of both social behavior and microglia.

Proteobacteria abundance during nursing predicts physical growth and brain volume at one year of age in young rhesus monkeys.

Over the last decade, multiple studies have highlighted the essential role of gut microbiota in normal infant development. However, the sensitive periods during which gut bacteria are established and become associated with physical growth and maturation of the brain are still poorly defined. This study tracked the assembly of the intestinal microbiota during the initial nursing period, and changes in community structure after transitioning to solid food in infant rhesus monkeys (Macaca mulatta). Anthropometric measures and rectal swabs were obtained at 2-month intervals across the first year of life and bacterial taxa identified by 16S rRNA gene sequencing. At 12 months of age, total brain and cortical regions volumes were quantified through structural magnetic resonance imaging. The bacterial community structure was dynamic and characterized by discrete maturational phases, reflecting an early influence of breast milk and the later transition to solid foods. Commensal microbial taxa varied with diet similar to findings in other animals and human infants; however, monkeys differ in the relative abundances of Lactobacilli and Bifidobacteria, two taxa predominant in breastfed human infants. Higher abundances of taxa in the phylum Proteobacteria during nursing were predictive of slower growth trajectories and smaller brain volumes at one year of age. Our findings define discrete phases of microbial succession in infant monkeys and suggest there may be a critical period during nursing when endogenous differences in certain taxa can shift the community structure and influence the pace of physical growth and the maturational trajectory of the brain.

Lyticase Facilitates Mycobiome Resolution Without Disrupting Microbiome Fidelity in Primates.

BACKGROUND: Microbiome research has expanded to consider contributions of microbial kingdoms beyond bacteria, including fungi (i.e., the mycobiome). However, optimal specimen handling protocols are varied, including uncertainty of how enzymes utilized to facilitate fungal DNA recovery may interfere with bacterial microbiome sequencing from the same samples. METHODS: With Institutional Animal Care and Use Committee approval, fecal samples were obtained from 20 rhesus macaques (10 males, 10 females; Macaca mulatta). DNA was extracted using commercially available kits, with or without lyticase enzyme treatment. 16S ribosomal RNA (bacterial) and Internal Transcribed Spacer (ITS; fungal) sequencing was performed on the Illumina MiSeq platform. Bioinformatics analysis was performed using Qiime and Calypso. RESULTS: Inclusion of lyticase in the sample preparation pipeline significantly increased usable fungal ITS reads, community alpha diversity, and enhanced detection of numerous fungal genera that were otherwise poorly or not detected in primate fecal samples. Bacterial 16S ribosomal RNA amplicons obtained from library preparation were statistically unchanged by the presence of lyticase. CONCLUSIONS: We demonstrate inclusion of the enzyme lyticase for fungal cell wall digestion markedly enhances mycobiota detection while maintaining fidelity of microbiome identification and community features in non-human primates. In restricted sample volumes, as are common in limited human samples, use of single sample DNA isolation will facilitate increased rigor and controlled approaches in future work.

Maternal and Breast Milk Influences on the Infant Gut Microbiome, Enteric Health and Growth Outcomes of Rhesus Monkeys.

OBJECTIVES: Gut bacteria play an essential role during infancy and are strongly influenced by the mode of birth and feeding. A primate model was used to investigate the benefits of exposure to the mother or conversely the negative impact of early nursery rearing on microbial colonization. METHOD: Rectal swabs were obtained from rhesus macaques born vaginally and mother-reared (MR, N = 35) or delivered primarily via cesarean-section and human-reared (HR, N = 19). Microbiome composition was determined by rRNA gene amplicon sequencing at 2, 4, and 8 weeks of age and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs used to assess influences on functional metabolic pathways in the gut. Growth trajectories and incidence of diarrheic symptoms were evaluated. RESULTS: The microbial community structure was different between MR and HR infants with respect to phylogeny and abundance at all 3 ages. When examining dominant phyla, HR infants had a higher Firmicutes-to-Bacteroidetes ratio. At the genus level, breast milk-dependent commensal taxa and adult-typical genera were more abundant in MR infants. This difference resulted in a corresponding shift in the predicted metabolic effects, specifically for microbial genes associated with metabolism and immune function. HR infants had faster growth trajectories (P < 0.001), but more diarrheic symptoms by 6 months postnatal (P = 0.008). CONCLUSIONS: MR infants acquired adult-typical microbiota more quickly, and had higher levels of several beneficial commensal taxa. Cesarean-delivered and formula-fed infants had different developmental trajectories of bacterial colonization. Establishment of the gut microbiome was associated with an infant's growth trajectory, and implicated in the subsequent vulnerability to Campylobacter infections associated with diarrhea in infant monkeys.

Maternal Perceived Stress during Pregnancy Increases Risk for Low Neonatal Iron at Delivery and Depletion of Storage Iron at One Year.

OBJECTIVE: To investigate the impact of maternal stress during pregnancy on newborn iron and stage 1 iron deficiency at 1 year of age. STUDY DESIGN: In total, 245 mothers and their newborn infants (52% male; 72% white) were recruited at the Meriter Hospital Birthing Center on the basis of known risk factors for iron deficiency. Umbilical cord blood hemoglobin and zinc protoporphyrin/heme (ZnPP/H) were determined to evaluate erythrocyte iron and plasma ferritin was determined to reflect storage iron. Mothers retrospectively reported stress experienced previously during pregnancy on a 25-item questionnaire. Blood was also was collected from 79 infants who were breastfed at 1 year of age. RESULTS: Maternal recall of distress and health concerns during pregnancy correlated with cord blood ZnPP/H indices (r = 0.21, P < .01), even in the absence of major traumatic events. When concurrent with other known risks for iron deficiency, including maternal adiposity, socioeconomic status, and race, maternal stress had a summative effect, lowering cord blood iron. At 1 year, 24% of infants who were breastfed had moderate iron deficiency (plasma ferritin <12 µg/L). Higher cord blood ZnPP/H was predictive of this moderate iron deficiency (95% CI 0.26-1.47, P = .007). When coincident with maternal reports of gestational stress, the likelihood of low plasma ferritin at 1 year increased 36-fold in breastfed infants as compared with low-stress pregnancies (95% CI 1.33-6.83, P = .007). CONCLUSIONS: Maternal recall of stress during pregnancy was associated with lower iron stores at birth. High cord blood ZnPP/H, reflecting low erythrocyte iron, was correlated with the likelihood of stage 1 iron deficiency at 1 year, when rapid growth can deplete storage iron in breastfed infants.

Daughter dearest: Sex-biased calcium in mother's milk among rhesus macaques.

Mother's milk provides building blocks necessary for infant development and growth postnatally. Minerals in milk are particularly important for infant skeletal development and may reflect maternal characteristics that are associated with the capacity to synthesize milk and sex-specific developmental priorities of the infant. Using a large sample of mother-infant dyads assigned to the outdoor breeding colony at the California National Primate Research Center (N=104), we investigated the relationship of milk calcium (Ca) and phosphorus (P) concentrations and the ratio of Ca/P to maternal and infant characteristics and to other milk variables. Ca and P are largely associated with casein micelles, and as expected, both Ca and P were positively correlated with protein concentrations in milk. Neither Ca nor P concentrations were associated with maternal parity. Mothers rearing daughters tended to produce higher mean Ca concentration in milk, and consequently a higher Ca/P ratio, than did mothers rearing sons, even though protein concentration was not elevated. These results suggest that the Ca/P ratio in rhesus milk may have been under separate selective pressure from protein content to facilitate the accelerated rate of skeletal calcification that has been observed in female Macaca mulatta infants.

Significant Microbial Changes Are Evident in the Reproductive Tract of Pregnant Rhesus Monkeys at Mid-Gestation but Their Gut Microbiome Does Not Shift until Late Gestation.

Vaginal and rectal specimens were obtained from cycling, pregnant, and nursing rhesus monkeys to assess pregnancy-related changes in the commensal bacteria in their reproductive and intestinal tracts. Using 16S rRNA gene amplicon sequencing, significant differences were found only in the vagina at mid-gestation, not in the hindgut. To verify the apparent stability in gut bacterial composition at mid-gestation, the experiment was repeated with additional monkeys, and similar results were found with both 16S rRNA gene amplicon and metagenomic sequencing. A follow-up study investigated if bacterial changes in the hindgut might occur later in pregnancy. Gravid females were assessed closer to term and compared to nonpregnant females. By late pregnancy, significant differences in bacterial composition, including an increased abundance of 4 species of Lactobacillus and Bifidobacterium adolescentis, were detected, but without a shift in the overall community structure. Progesterone levels were assessed as a possible hormone mediator of bacterial change. The relative abundance of only some taxa (e.g., Bifidobacteriaceae) were specifically associated with progesterone. In summary, pregnancy changes the microbial profiles in monkeys, but the bacterial diversity in their lower reproductive tract is different from women, and the composition of their intestinal symbionts remains stable until late gestation when several Firmicutes become more prominent.

Microbial modulation prevents the effects of pervasive environmental stressors on microglia and social behavior, but not the dopamine system.

Environmental toxicant exposure, including air pollution, is increasing worldwide. However, toxicant exposures are not equitably distributed. Rather, low-income and minority communities bear the greatest burden, along with higher levels of psychosocial stress. Both air pollution and maternal stress during pregnancy have been linked to neurodevelopmental disorders such as autism, but biological mechanisms and targets for therapeutic intervention remain poorly understood. We demonstrate that combined prenatal exposure to air pollution (diesel exhaust particles, DEP) and maternal stress (MS) in mice induces social behavior deficits only in male offspring, in line with the male bias in autism. These behavioral deficits are accompanied by changes in microglial morphology and gene expression as well as decreased dopamine receptor expression and dopaminergic fiber input in the nucleus accumbens (NAc). Importantly, the gut-brain axis has been implicated in ASD, and both microglia and the dopamine system are sensitive to the composition of the gut microbiome. In line with this, we find that the composition of the gut microbiome and the structure of the intestinal epithelium are significantly shifted in DEP/MS-exposed males. Excitingly, both the DEP/MS-induced social deficits and microglial alterations in males are prevented by shifting the gut microbiome at birth via a cross-fostering procedure. However, while social deficits in DEP/MS males can be reversed by chemogenetic activation of dopamine neurons in the ventral tegmental area, modulation of the gut microbiome does not impact dopamine endpoints. These findings demonstrate male-specific changes in the gut-brain axis following DEP/MS and suggest that the gut microbiome is an important modulator of both social behavior and microglia.

Precipitous Dehydroepiandrosterone Declines Reflect Decreased Physical Vitality and Function.

Dehydroepiandrosterone (DHEA) and its sulfated form, DHEA-S, peak in young adulthood and then decrease dramatically with age. However, there is extensive variation in this age-related hormone decline, suggesting an early decrement may be associated with lower vitality and be prognostic of poor health in old age. To determine whether DHEA-S and DHEA are correlated with physical indices of vitality, hormone levels were analyzed with respect to clinical health histories, physical functioning including grip strength, gait speed and repetitive standing, and self-reported chronic pain. The participants (N = 1,214) were 35-86 years of age from a nationally representative survey, Midlife Development in the United States. DHEA-S and DHEA below age-expected levels were associated with more chronic illness conditions and self-reported persistent pain and pain sensitivity upon manual palpation. Additionally, lower DHEA-S and DHEA correlated with poorer performance on tests of physical functioning by middle age suggesting a more precipitous decline is already indicative of reduced vigor and physical strength. When considered with respect to age- and gender-typical norms, larger decrements in DHEA-S and DHEA may be causally related to the loss of physical vitality. Conversely, when hormone secretion is sustained in older adults, it conveys reduced risk for the physical weakness and ailments that precede frailty.

Gestational Timing of Prenatal Disturbance and Fetal Sex Determine the Developmental Outcomes.

BACKGROUND: Maternal stress during pregnancy can have deleterious consequences, increasing risk for prematurity and low birth weight, as well as postnatal effects on emotional regulation and neuromotor development. It is less clear, however, whether moderate and brief gestational disturbances have similar effects. OBJECTIVE: To determine the impact of a delimited period of moderate maternal stress on infant growth, emotional reactivity, and neurobehavioral maturity in a nonhuman primate model. METHODS: Eighty-three infant rhesus monkeys were generated from disturbed pregnancies, either early or late gestation, and compared with 51 undisturbed infants. Maternal stress was induced with an acoustical startle protocol for 25% of gestation. Infant weights, anthropometrics, and neurobehavioral data were obtained. Analyses focused on differential effects of prenatal stress on male and female infants. RESULTS: The disturbance manipulation elevated cortisol levels acutely in the gravid females and they gained less weight by term. Nevertheless, female infants from the early stress condition were significantly larger at birth. This differential growth trajectory was then sustained through 6 months of age. Infants from stress conditions were more emotionally reactive and evinced immature neuromotor reflexes, especially when gestated by late stress mothers. CONCLUSIONS: Even moderate maternal disturbance impacted infant temperament and neuromotor development in this nonhuman primate model. Effects on fetal and infant growth differed from typical reports of growth inhibition, both in other animal species and human studies. The findings convey the importance of considering the duration and severity of prenatal insults, and the potential for fetal plasticity and recovery, permitting compensatory growth responses.