Effects of cytokines on nuclear factor-kappa B, cell viability, and synaptic connectivity in a human neuronal cell line.

Maternal infection during pregnancy is associated with increased risk of psychiatric and neurodevelopmental disorders (NDDs). Experimental animal models demonstrate that maternal immune activation (MIA) elevates inflammatory cytokine levels in the maternal and fetal compartments and causes behavioral changes in offspring. Individual cytokines have been shown to modulate neurite outgrowth and synaptic connectivity in cultured rodent neurons, but whether clinically relevant cytokine mixtures similarly modulate neurodevelopment in human neurons is not known. To address this, we quantified apoptosis, neurite outgrowth, and synapse number in the LUHMES human neuronal cell line exposed to varying concentrations of: (1) a mixture of 12 cytokines and chemokines (EMA) elevated in mid-gestational serum samples from mothers of children with autism and intellectual disability; (2) an inflammatory cytokine mixture (ICM) comprised of five cytokines elevated in experimental MIA models; or (3) individual cytokines in ICM. At concentrations that activated nuclear factor-kappa B (NF-κB) in LUHMES cells, EMA and ICM induced caspase-3/7 activity. ICM altered neurite outgrowth, but only at concentrations that also reduced cell viability, whereas ICM reduced synapse number independent of changes in cell viability. Individual cytokines in ICM phenocopied the effects of ICM on NF-κB activation and synaptic connectivity, but did not completely mimic the effects of ICM on apoptosis. These results demonstrate that clinically relevant cytokine mixtures modulate apoptosis and synaptic density in developing human neurons. Given the relevance of these neurodevelopmental processes in NDDs, our findings support the hypothesis that cytokines contribute to the adverse effects of MIA on children.

Dental Pulp Stem Cells Model Early Life and Imprinted DNA Methylation Patterns.

Early embryonic stages of pluripotency are modeled for epigenomic studies primarily with human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSCs). For analysis of DNA methylation however, ESCs and iPSCs do not accurately reflect the DNA methylation levels found in preimplantation embryos. Whole genome bisulfite sequencing (WGBS) approaches have revealed the presence of large partially methylated domains (PMDs) covering 30%-40% of the genome in oocytes, preimplantation embryos, and placenta. In contrast, ESCs and iPSCs show abnormally high levels of DNA methylation compared to inner cell mass (ICM) or placenta. Here we show that dental pulp stem cells (DPSCs), derived from baby teeth and cultured in serum-containing media, have PMDs and mimic the ICM and placental methylome more closely than iPSCs and ESCs. By principal component analysis, DPSC methylation patterns were more similar to two other neural stem cell types of human derivation (EPI-NCSC and LUHMES) and placenta than were iPSCs, ESCs or other human cell lines (SH-SY5Y, B lymphoblast, IMR90). To test the suitability of DPSCs in modeling epigenetic differences associated with disease, we compared methylation patterns of DPSCs derived from children with chromosome 15q11.2-q13.3 maternal duplication (Dup15q) to controls. Differential methylation region (DMR) analyses revealed the expected Dup15q hypermethylation at the imprinting control region, as well as hypomethylation over SNORD116, and novel DMRs over 147 genes, including several autism candidate genes. Together these data suggest that DPSCs are a useful model for epigenomic and functional studies of human neurodevelopmental disorders. Stem Cells 2017;35:981-988.

Risk factors in autism: Thinking outside the brain.

Autism spectrum disorders (ASD) are complex neurodevelopmental conditions that have been rising markedly in prevalence for the past 30 years, now thought to affect 1 in 68 in the United States. This has prompted the search for possible explanations, and has even resulted in some controversy regarding the "true" prevalence of autism. ASD are influenced by a variety of genetic, environmental, and possibly immunological factors that act during critical periods to alter key developmental processes. This can affect multiple systems and manifests as the social and behavioral deficits that define these disorders. The interaction of environmental exposures in the context of an individual's genetic susceptibilities manifests differently in each case, leading to heterogeneous phenotypes and varied comorbid symptoms within the disorder. This has also made it very difficult to elucidate underlying genes and exposure profiles, but progress is being made in this area. Some pharmaceutical drugs, toxicants, and metabolic and nutritional factors have been identified in epidemiological studies as increasing autism risk, especially during the prenatal period. Immunologic risk factors, including maternal infection during pregnancy, autoantibodies to fetal brain proteins, and familial autoimmune disease, have consistently been observed across multiple studies, as have immune abnormalities in individuals with ASD. Mechanistic research using animal models and patient-derived stem cells will help researchers to understand the complex etiology of these neurodevelopmental disorders, which will lead to more effective therapies and preventative strategies. Proposed therapies that need more investigation include special diets, probiotics, immune modulation, oxytocin, and personalized pharmacogenomic targets. The ongoing search for biomarkers and better treatments will result in earlier identification of ASD and provide much needed help and relief for afflicted families.

The influence of sex, genotype, and dose on serum and hippocampal cytokine levels in juvenile mice developmentally exposed to a human-relevant mixture of polychlorinated biphenyls.

Polychlorinated biphenyls (PCBs) are pervasive environmental contaminants implicated as risk factors for neurodevelopmental disorders (NDDs). Immune dysregulation is another NDD risk factor, and developmental PCB exposures are associated with early life immune dysregulation. Studies of the immunomodulatory effects of PCBs have focused on the higher-chlorinated congeners found in legacy commercial mixtures. Comparatively little is known about the immune effects of contemporary, lower-chlorinated PCBs. This is a critical data gap given recent reports that lower-chlorinated congeners comprise >70% of the total PCB burden in serum of pregnant women enrolled in the MARBLES study who are at increased risk for having a child with an NDD. To examine the influence of PCBs, sex, and genotype on cytokine levels, mice were exposed throughout gestation and lactation to a PCB mixture in the maternal diet, which was based on the 12 most abundant PCBs in sera from MARBLES subjects. Using multiplex array, cytokines were quantified in the serum and hippocampus of weanling mice expressing either a human gain-of-function mutation in ryanodine receptor 1 (T4826I mice), a human CGG premutation repeat expansion in the fragile X mental retardation gene 1 (CGG mice), or both mutations (DM mice). Congenic wildtype (WT) mice were used as controls. There were dose-dependent effects of PCB exposure on cytokine concentrations in the serum but not hippocampus. Differential effects of genotype were observed in the serum and hippocampus. Hippocampal cytokines were consistently elevated in T4826I mice and also in WT animals for some cytokines compared to CGG and DM mice, while serum cytokines were usually elevated in the mutant genotypes compared to the WT group. Males had elevated levels of 19 cytokines in the serum and 4 in the hippocampus compared to females, but there were also interactions between sex and genotype for 7 hippocampal cytokines. Only the chemokine CCL5 in the serum showed an interaction between PCB dose, genotype, and sex. Collectively, these findings indicate differential influences of PCB exposure and genotype on cytokine levels in serum and hippocampal tissue of weanling mice. These results suggest that developmental PCB exposure has chronic effects on baseline serum, but not hippocampal, cytokine levels in juvenile mice.