Global metabolic profiles in a non-human primate model of maternal immune activation: implications for neurodevelopmental disorders.

Epidemiological evidence implicates severe maternal infections as risk factors for neurodevelopmental disorders, such as ASD and schizophrenia. Accordingly, animal models mimicking infection during pregnancy, including the maternal immune activation (MIA) model, result in offspring with neurobiological, behavioral, and metabolic phenotypes relevant to human neurodevelopmental disorders. Most of these studies have been performed in rodents. We sought to better understand the molecular signatures characterizing the MIA model in an organism more closely related to humans, rhesus monkeys (Macaca mulatta), by evaluating changes in global metabolic profiles in MIA-exposed offspring. Herein, we present the global metabolome in six peripheral tissues (plasma, cerebrospinal fluid, three regions of intestinal mucosa scrapings, and feces) from 13 MIA and 10 control offspring that were confirmed to display atypical neurodevelopment, elevated immune profiles, and neuropathology. Differences in lipid, amino acid, and nucleotide metabolism discriminated these MIA and control samples, with correlations of specific metabolites to behavior scores as well as to cytokine levels in plasma, intestinal, and brain tissues. We also observed modest changes in fecal and intestinal microbial profiles, and identify differential metabolomic profiles within males and females. These findings support a connection between maternal immune activation and the metabolism, microbiota, and behavioral traits of offspring, and may further the translational applications of the MIA model and the advancement of biomarkers for neurodevelopmental disorders such as ASD or schizophrenia.

Increased Monocyte Production of IL-6 after Toll-like Receptor Activation in Children with Autism Spectrum Disorder (ASD) Is Associated with Repetitive and Restricted Behaviors.

The prevalence of autism spectrum disorder (ASD) has starkly increased, instigating research into risk factors for ASD. This research has identified immune risk factors for ASD, along with evidence of immune dysfunction and excess inflammation frequently experienced by autistic individuals. Increased innate inflammatory cytokines, including interleukin (IL)-6, are seen repeatedly in ASD; however, the origin of excess IL-6 in ASD has not been identified. Here we explore specific responses of circulating monocytes from autistic children. We isolated CD14+ monocytes from whole blood and stimulated them for 24 h under three conditions: media alone, lipoteichoic acid to activate TLR2, and lipopolysaccharide to activate TLR4. We then measured secreted cytokine concentrations in cellular supernatant using a human multiplex bead immunoassay. We found that after TLR4 activation, CD14+ monocytes from autistic children produce increased IL-6 compared to monocytes from children with typical development. IL-6 concentration also correlated with worsening restrictive and repetitive behaviors. These findings suggest dysfunctional activation of myeloid cells, and may indicate that other cells of this lineage, including macrophages, and microglia in the brain, might have a similar dysfunction. Further research on myeloid cells in ASD is warranted.

Translational outcomes relevant to neurodevelopmental disorders following early life exposure of rats to chlorpyrifos.

BACKGROUND: Neurodevelopmental disorders (NDDs), including intellectual disability, attention deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD), are pervasive, lifelong disorders for which pharmacological interventions are not readily available. Substantial increases in the prevalence of NDDs over a relatively short period may not be attributed solely to genetic factors and/or improved diagnostic criteria. There is now a consensus that multiple genetic loci combined with environmental risk factors during critical periods of neurodevelopment influence NDD susceptibility and symptom severity. Organophosphorus (OP) pesticides have been identified as potential environmental risk factors. Epidemiological studies suggest that children exposed prenatally to the OP pesticide chlorpyrifos (CPF) have significant mental and motor delays and strong positive associations for the development of a clinical diagnosis of intellectual delay or disability, ADHD, or ASD. METHODS: We tested the hypothesis that developmental CPF exposure impairs behavior relevant to NDD phenotypes (i.e., deficits in social communication and repetitive, restricted behavior). Male and female rat pups were exposed to CPF at 0.1, 0.3, or 1.0 mg/kg (s.c.) from postnatal days 1-4. RESULTS: These CPF doses did not significantly inhibit acetylcholinesterase activity in the blood or brain but significantly impaired pup ultrasonic vocalizations (USV) in both sexes. Social communication in juveniles via positive affiliative 50-kHz USV playback was absent in females exposed to CPF at 0.3 mg/kg and 1.0 mg/kg. In contrast, this CPF exposure paradigm had no significant effect on gross locomotor abilities or contextual and cued fear memory. Ex vivo magnetic resonance imaging largely found no differences between the CPF-exposed rats and the corresponding vehicle controls using strict false discovery correction; however, there were interesting trends in females in the 0.3 mg/kg dose group. CONCLUSIONS: This work generated and characterized a rat model of developmental CPF exposure that exhibits adverse behavioral phenotypes resulting from perinatal exposures at levels that did not significantly inhibit acetylcholinesterase activity in the brain or blood. These data suggest that current regulations regarding safe levels of CPF need to be reconsidered.

Genetic variants drive altered epigenetic regulation of endotoxin response in BTBR macrophages.

The BTBR T+Itpr3tf/J (BTBR) mouse has been used as a complex genetic model of Autism Spectrum Disorders (ASD). While the specific mechanisms underlying BTBR behavioral phenotypes are poorly understood, prior studies have implicated profound differences in innate immune system control of pro-inflammatory cytokines. Innate immune activation and elevated pro-inflammatory cytokines are also detected in blood of children with ASD. In this study, we examined how underlying BTBR genetic variants correspond to strain-specific changes in chromatin accessibility, resulting in a pro-inflammatory response specifically in BTBR bone marrow derived macrophages (BMDM). In response to repeated lipopolysaccharide (LPS) treatments, C57BL/6J (C57) BMDM exhibited intact endotoxin tolerance. In contrast, BTBR BMDM exhibited hyper-responsive expression of genes that were normally tolerized in C57. This failure in formation of endotoxin tolerance in BTBR was mirrored at the level of chromatin accessibility. Using ATAC-seq, we specifically identified promoter and enhancer regions with strain-specific differential chromatin accessibility both at baseline and in response to LPS. Regions with strain-specific differences in chromatin accessibility were significantly enriched for BTBR genetic variants, such that an average of 22% of the differential chromatin regions had at least one variant. Together, these results demonstrate that BTBR genetic variants contribute to altered chromatin responsiveness to endotoxin challenge resulting in hyper-responsive innate immunity in BTBR. These findings provide evidence for an interaction between complex genetic variants and differential epigenetic regulation of innate immune responses.

T cell populations in children with autism spectrum disorder and co-morbid gastrointestinal symptoms.

Children with ASD are more likely to experience gastrointestinal (GI) symptoms than typically-developed children. Numerous studies have reported immune abnormalities and inflammatory profiles in the majority of individuals with ASD. Immune dysfunction is often hypothesized as a driving factor in many GI diseases and it has been suggested that it is more apparent in children with ASD that exhibit GI symptoms. In this study we sought to characterize peripheral T cell subsets in children with and without GI symptoms, compared to healthy typically-developing children. Peripheral blood mononuclear cells were isolated from participants, who were categorized into three groups: children with ASD who experience GI symptoms (n â€‹= â€‹14), children with ASD who do not experience GI symptoms (n â€‹= â€‹10) and typically-developing children who do not experience GI symptoms (n â€‹= â€‹15). In order to be included in the GI group, GI symptoms such as diarrhea, constipation, and/or pain while defecating, had to be present in the child regularly for the past 6 months; likewise, in order to be placed in the no GI groups, bowel movements could not include the above symptoms present throughout development. Cells were assessed for surface markers and intracellular cytokines to identify T cell populations. Children with ASD and GI symptoms displayed elevated TH17 populations (0.757% â€‹± â€‹0.313% compared to 0.297% â€‹± â€‹0.197), while children with ASD who did not experience GI symptoms showed increased frequency of TH2 populations (2.02% â€‹± â€‹1.08% compared to 1.01% â€‹± â€‹0.58%). Both ASD groups showed evidence of reduced gut homing regulatory T cell populations compared to typically developing children (ASDGI:1.93% â€‹± â€‹0.75% and ASDNoGI:1.85% â€‹± â€‹0.89 compared to 2.93% â€‹± â€‹1.16%). Children with ASD may have deficits in immune regulation that lead to differential inflammatory T cell subsets that could be linked to associated co-morbidities.

Variability in PolyIC induced immune response: Implications for preclinical maternal immune activation models.

Maternal infection during pregnancy may increase the risk of offspring neurodevelopmental disorders. The preclinical Polyinosinic-polycytidylic acid (PolyIC) model has become one of the most widely used approaches in maternal immune activation (MIA) research. However, variability in molecular weight may impact the immune activating potential of PolyIC. Nulliparous rats injected with high molecular weight PolyIC exhibit pronounced cytokine response and sickness behavior that was not observed in rats injected low molecular weight PolyIC. Although an essential next step is to extend these studies to pregnant animals, the preliminary results suggest that PolyIC molecular weight is an important experimental design consideration.

Cytokine alterations in first-episode schizophrenia and bipolar disorder: relationships to brain structure and symptoms.

BACKGROUND: Over the past 30 years, evidence has been accumulating for an immunological component to schizophrenia etiology, including genetic links to the major histocompatibility complex, microglia activation, and dysregulated cytokine profiles. However, the degree of similarity in cytokine profiles for schizophrenia and bipolar disorder, as well as the relationship between cytokine levels and brain structure, is less well understood. METHODS: To address this, we recruited 69 first-episode schizophrenia-spectrum patients, 16 first-episode bipolar patients with psychotic features, and 53 healthy controls, from the UC Davis EDAPT clinic. Blood plasma was collected and analyzed for all participants with a subset of participants that also underwent structural MRI on a 1.5T GE scanner. RESULTS: Plasma levels of interleukin (IL)-1ß, IL-2, IL-6, and interferon (IFN)-γ were elevated in schizophrenia patients compared to those in controls. Patients with bipolar disorder had elevated plasma IL-10 levels compared to controls, and the two patient groups did not differ significantly on any immunological measure. Percent whole-brain gray matter was inversely correlated with IFN-γ and IL-12 levels in patients with schizophrenia, with a trend relationship between IFN-γ and IL-12 and prefrontal cortical thickness. Furthermore, psychotic symptoms were positively related to IL-1ß levels in individuals with schizophrenia. CONCLUSIONS: These data suggest a partially overlapping pattern of elevated blood cytokine levels in patients with first-episode schizophrenia and bipolar disorder with psychotic features. Furthermore, our findings suggest that elevated pro-inflammatory cytokines may be particularly involved in schizophrenia etiology, given evidence of cytokine-related decreases in total gray matter.

Differential immune responses and microbiota profiles in children with autism spectrum disorders and co-morbid gastrointestinal symptoms.

OBJECTIVES: Many studies have reported the increased presence of gastrointestinal (GI) symptoms in children with autism spectrum disorders (ASD). Altered microbiome profiles, pro-inflammatory responses and impaired intestinal permeability have been observed in children with ASD and co-morbid GI symptoms, yet few studies have compared these findings to ASD children without GI issues or similarly aged typical developing children. The aim of this study was to determine whether there are biological signatures in terms of immune dysfunction and microbiota composition in children with ASD with GI symptoms. METHODS: Children were enrolled in one of four groups: ASD and GI symptoms of irregular bowel habits (ASDGI), children with ASD but without current or previous GI symptoms (ASDNoGI), typically developing children with GI symptoms (TDGI) and typically developing children without current or previous GI symptoms (TDNoGI). Peripheral blood mononuclear cells (PBMC) were isolated from the blood, stimulated and assessed for cytokine production, while stool samples were analyzed for microbial composition. RESULTS: Following Toll-Like receptor (TLR)-4 stimulation, the ASDGI group produced increased levels of mucosa-relevant cytokines including IL-5, IL-15 and IL-17 compared to ASDNoGI. The production of the regulatory cytokine TGFß1 was decreased in the ASDGI group compared with both the ASDNoGI and TDNoGI groups. Analysis of the microbiome at the family level revealed differences in microbiome composition between ASD and TD children with GI symptoms; furthermore, a predictive metagenome functional content analysis revealed that pathways were differentially represented between ASD and TD subjects, independently of the presence of GI symptoms. The ASDGI also showed an over-representation of the gene encoding zonulin, a molecule regulating gut permeability, compared to the other groups. CONCLUSIONS: Overall our findings suggest that children with ASD who experience GI symptoms have an imbalance in their immune response, possibly influenced by or influencing metagenomic changes, and may have a propensity to impaired gut barrier function which may contribute to their symptoms and clinical outcome.

Developmental social communication deficits in the Shank3 rat model of phelan-mcdermid syndrome and autism spectrum disorder.

Mutations in the SHANK3 gene have been discovered in autism spectrum disorder (ASD), and the intellectual disability, Phelan-McDermid Syndrome. This study leveraged a new rat model of Shank3 deficiency to assess complex behavioral phenomena, unique to rats, which display a richer social behavior repertoire than mice. Uniquely detectable emissions of ultrasonic vocalizations (USV) in rats serve as situation-dependent affective signals and accomplish important communicative functions. We report, for the first time, a call and response acoustic playback assay of bidirectional social communication in juvenile Shank3 rats. Interestingly, we found that Shank3-deficient null males did not demonstrate the enhanced social approach behavior typically exhibited following playback of pro-social USV. Concomitantly, we discovered that emission of USV in response to playback was not genotype-dependent and emitted response calls were divergent in meaning. This is the first report of these socially relevant responses using a genetic model of ASD. A comprehensive and empirical analysis of vigorous play during juvenile reciprocal social interactions further revealed fewer bouts and reduced durations of time spent playing by multiple key parameters, including reduced anogenital sniffing and allogrooming. We further discovered that male null Shank3-deficient pups emitted fewer isolation-induced USV than Shank3 wildtype controls. Postnatal whole brain anatomical phenotyping was applied to visualize anatomical substrates that underlie developmental phenotypes. The data presented here lend support for the important role of Shank3 in social communication, the core symptom domain of ASD. By increasing the number of in vivo functional outcome measures, we improved the likelihood for identifying and moving forward with medical interventions. Autism Res 2018, 11: 587-601. © 2018 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Clinically relevant outcomes are required to demonstrate the utility of therapeutics. We introduce findings in a rat model, and assess the impact of mutations in Shank3, an autism risk gene. We found that males with deficient expression of Shank3 did not demonstrate typical responses in a bi-directional social communication test and that social interaction was lower on key parameters. Outcome measures reported herein extend earlier results in mice and capture responses to acoustic calls, which is analogous to measuring receptive and expressive communication.

Microglia from offspring of dams with allergic asthma exhibit epigenomic alterations in genes dysregulated in autism.

Dysregulation in immune responses during pregnancy increases the risk of a having a child with an autism spectrum disorder (ASD). Asthma is one of the most common chronic diseases among pregnant women, and symptoms often worsen during pregnancy. We recently developed a mouse model of maternal allergic asthma (MAA) that induces changes in sociability, repetitive, and perseverative behaviors in the offspring. Since epigenetic changes help a static genome adapt to the maternal environment, activation of the immune system may epigenetically alter fetal microglia, the brain's resident immune cells. We therefore tested the hypothesis that epigenomic alterations to microglia may be involved in behavioral abnormalities observed in MAA offspring. We used the genome-wide approaches of whole genome bisulfite sequencing to examine DNA methylation and RNA sequencing to examine gene expression in microglia from juvenile MAA offspring. Differentially methylated regions were enriched for immune signaling pathways and important microglial developmental transcription factor binding motifs. Differential expression analysis identified genes involved in controlling microglial sensitivity to the environment and shaping neuronal connections in the developing brain. Differentially expressed genes significantly overlapped genes with altered expression in human ASD cortex, supporting a role for microglia in the pathogenesis of ASD.

Maternal Immune Activation and Autism Spectrum Disorder: From Rodents to Nonhuman and Human Primates.

A subset of women who are exposed to infection during pregnancy have an increased risk of giving birth to a child who will later be diagnosed with a neurodevelopmental or neuropsychiatric disorder. Although epidemiology studies have primarily focused on the association between maternal infection and an increased risk of offspring schizophrenia, mounting evidence indicates that maternal infection may also increase the risk of autism spectrum disorder. A number of factors, including genetic susceptibility, the intensity and timing of the infection, and exposure to additional aversive postnatal events, may influence the extent to which maternal infection alters fetal brain development and which disease phenotype (autism spectrum disorder, schizophrenia, other neurodevelopmental disorders) is expressed. Preclinical animal models provide a test bed to systematically evaluate the effects of maternal infection on fetal brain development, determine the relevance to human central nervous system disorders, and to evaluate novel preventive and therapeutic strategies. Maternal immune activation models in mice, rats, and nonhuman primates suggest that the maternal immune response is the critical link between exposure to infection during pregnancy and subsequent changes in brain and behavioral development of offspring. However, differences in the type, severity, and timing of prenatal immune challenge paired with inconsistencies in behavioral phenotyping approaches have hindered the translation of preclinical results to human studies. Here we highlight the promises and limitations of the maternal immune activation model as a preclinical tool to study prenatal risk factors for autism spectrum disorder, and suggest specific changes to improve reproducibility and maximize translational potential.

Immune Endophenotypes in Children With Autism Spectrum Disorder.

BACKGROUND: Autism spectrum disorder (ASD) is characterized by social communication deficits and restricted, repetitive patterns of behavior. Varied immunological findings have been reported in children with ASD. To address the question of heterogeneity in immune responses, we sought to examine the diversity of immune profiles within a representative cohort of boys with ASD. METHODS: Peripheral blood mononuclear cells from male children with ASD (n = 50) and from typically developing age-matched male control subjects (n = 16) were stimulated with either lipopolysaccharide or phytohemagglutinin. Cytokine production was assessed after stimulation. The ASD study population was clustered into subgroups based on immune responses and assessed for behavioral outcomes. RESULTS: Children with ASD who had a proinflammatory profile based on lipopolysaccharide stimulation were more developmentally impaired as assessed by the Mullen Scales of Early Learning. They also had greater impairments in social affect as measured by the Autism Diagnostic Observation Schedule. These children also displayed more frequent sleep disturbances and episodes of aggression. Similarly, children with ASD and a more activated T cell cytokine profile after phytohemagglutinin stimulation were more developmentally impaired as measured by the Mullen Scales of Early Learning. CONCLUSIONS: Children with ASD may be phenotypically characterized based upon their immune profile. Those showing either an innate proinflammatory response or increased T cell activation/skewing display a more impaired behavioral profile than children with noninflamed or non-T cell activated immune profiles. These data suggest that there may be several possible immune subphenotypes within the ASD population that correlate with more severe behavioral impairments.

Long-term altered immune responses following fetal priming in a non-human primate model of maternal immune activation.

Infection during pregnancy can lead to activation of the maternal immune system and has been associated with an increased risk of having an offspring later diagnosed with a neurodevelopmental disorders (NDD) such as autism spectrum disorder (ASD) or schizophrenia (SZ). Most maternal immune activation (MIA) studies to date have been in rodents and usually involve the use of lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C). However, since NDD are based on behavioral changes, a model of MIA in non-human primates could potentially provide data that helps illuminate complex behavioral and immune outputs in human NDD. In this study twenty-one pregnant rhesus macaques were either given three injections over 72 hours of poly I:C-LC, a double stranded RNA analog (viral mimic), or saline as a control. Injections were given near the end of the first trimester or near the end of the second trimester to determine if there were differences in immune output due to the timing of MIA.An additional three non-treated animals were used as controls. The offspring were followed until 4 years of age, with blood collected at the end of their first (year 1) and fourth (year 4) years to assess dynamic cellular immune function. Induced responses from peripheral immune cells were measured using multiplex assays.At one year of age, MIA exposed offspring displayed elevated production of innate inflammatory cytokines including: interleukin (IL)-1ß, IL-6, IL-12p40, and tumor necrosis factor (TNF)α at baseline and following stimulation. At four years of age, the MIA exposed offspring continued to display elevated IL-1ß, and there was also a pattern of an increased production of T-cell helper type (TH)-2 cytokines, IL-4 and IL-13. Throughout this time period, the offspring of MIA treated dams exhibited altered behavioral phenotypes including increased stereotyped behaviors. During the first two years, stereotyped behaviors were associated with innate cytokine production. Self-directed behaviors were associated with TH2 cytokine production at year 4. Data from this study suggests long-term behavioral and immune activation was present in offspring following MIA. This novel non-human primate model of MIA may provide a relevant clinically translational model to help further elucidate the role between immune dysfunction and complex behavioral outputs following MIA.

Behavioral impact of maternal allergic-asthma in two genetically distinct mouse strains.

Recent population-based studies of expecting mothers identified a unique profile of immune markers that are associated with an increased risk of having a child diagnosed with autism spectrum disorder (ASD). This immune profile, including increased levels of maternal and placental interleukin (IL)-4 and IL-5, is consistent with an immune response found in an allergic-asthma phenotype. Allergies and asthma reflect an imbalance in immune responses including polarization towards T-helper type 2 (TH2) responses, with both genetic susceptibility and environmental factors affecting this T-cell polarization. Mouse strains provide a known and controlled source of genetic diversity to explore the role of genetic predisposition on environmental factors. In particular, the FVB background exhibits a skew towards TH2-mediated allergic-asthma response in traditional models of asthma whereas the C57 strain exhibits a more blunted TH2 polarized phenotype resulting in an attenuated allergic-asthma response. C57BL/6J (C57) and the sighted FVB.129P2-Pde6b(+) Tyr(c-ch)/Ant (FVB/Ant) lines were selected based on their characteristic high sociability and differing sensitivity to TH2-mediated stimuli. Based on the distinct allergy-sensitive immune responses of these two strains, we hypothesized that unique developmental consequences would occur in offspring following maternal allergy-asthma exposure. Female C57 and FVB/Ant dams were primed/sensitized with an exposure to ovalbumin (OVA) before pregnancy, then exposed to either aerosolized OVA or PBS-vehicle throughout gestation. Sera from pregnant dams were analyzed for changes in cytokine profiles using multiplex-arrays and offspring were assessed for changes in autism-like behavioral responses. Analysis of maternal sera revealed elevated IL-4 and IL-5 in OVA-treated dams of both strains but only C57 mice expressed increased levels of IL-1ß, IL-6, TNFα, and IL-17. Behavioral assessments revealed strain-dependent changes in juvenile reciprocal social interaction in offspring of maternal allergic asthma dams. Moreover, mice of both strains showed decreased repetitive grooming and increased marble burying behavior when born to OVA-exposed dams. Together, these findings support the important role genetic predisposition plays in the effects of maternal immune activation and underscore differences in ASD-like behavioral outcomes across mouse strains.

What has been learned from mouse models of the Fragile X Premutation and Fragile X-associated tremor/ataxia syndrome?

OBJECTIVE: To describe in this review how research using mouse models developed to study the Fragile X premutation (PM) and Fragile X-associated tremor/ataxia syndrome (FXTAS) have contributed to understanding these disorders. PM carriers bear an expanded CGG trinucleotide repeat on the Fragile X Mental Retardation 1 (FMR1) gene, and are at risk for developing the late onset neurodegenerative disorder FXTAS. CONCLUSIONS: Much has been learned about these genetic disorders from the development and study of mouse models. This includes new insights into the early cellular and molecular events that occur in PM carriers and in FXTAS, the presence of multiorgan pathology beyond the CNS, immunological dysregulation, unexpected synthesis of a potentially toxic peptide in FXTAS (i.e., FMRpolyG), and evidence that the disease process may be halted or reversed by appropriate molecular therapies given early in the course of disease.

Inflammatory profiles in the BTBR mouse: how relevant are they to autism spectrum disorders?

Autism spectrum disorders (ASD) are a group of disorders characterized by core behavioral features including stereotyped interests, repetitive behaviors and impairments in communication and social interaction. In addition, widespread changes in the immune systems of individuals with ASD have been identified, in particular increased evidence of inflammation in the periphery and central nervous system. While the etiology of these disorders remains unclear, it appears that multiple gene and environmental factors are involved. The need for animal models paralleling the behavioral and immunological features of ASD is paramount to better understand the link between immune system dysregulation and behavioral deficits observed in these disorders. As such, the asocial BTBR mouse strain displays both ASD relevant behaviors and persistent immune dysregulation, providing a model system that has and continues to be instructive in understanding the complex nature of ASD.

Group I metabotropic glutamate receptor mediated dynamic immune dysfunction in children with fragile X syndrome.

BACKGROUND: Fragile X syndrome (FXS) is the leading cause of inheritable intellectual disability in male children, and is predominantly caused by a single gene mutation resulting in expanded trinucleotide CGG-repeats within the 5' untranslated region of the fragile X mental retardation (FMR1) gene. Reports have suggested the presence of immune dysregulation in FXS with evidence of altered plasma cytokine levels; however, no studies have directly assessed functional cellular immune responses in children with FXS. In order to ascertain if immune dysregulation is present in children with FXS, dynamic cellular responses to immune stimulation were examined. METHODS: Peripheral blood mononuclear cells (PBMC) were from male children with FXS (n=27) and from male aged-matched typically developing (TD) controls (n=8). PBMC were cultured for 48 hours in media alone or with lipopolysaccharides (LPS; 1 µg/mL) to stimulate the innate immune response or with phytohemagglutinin (PHA; 8 µg/mL) to stimulate the adaptive T-cell response. Additionally, the group I mGluR agonist, DHPG, was added to cultures to ascertain the role of mGluR signaling in the immune response in subject with FXS. Supernatants were harvested and cytokine levels were assessed using Luminex multiplexing technology. RESULTS: Children with FXS displayed similar innate immune response following challenge with LPS alone when compared with TD controls; however, when LPS was added in the presence of a group I mGluR agonist, DHPG, increased immune response were observed in children with FXS for a number of pro-inflammatory cytokines including IL-6 (P=0.02), and IL-12p40 (P<0.01). Following PHA stimulation, with or without DHPG, no significant differences between subjects with FXS and TD were seen. CONCLUSIONS: In unstimulated cultures, subjects with FXS did not display altered dynamic immune response to LPS or PHA alone; however, subjects with FXS showed an altered response to co-current stimulation of LPS and DHPG, such that subjects with FXS failed to inhibit production of pro-inflammatory cytokines, suggesting a role of group I mGluR signaling in innate immune responses in FXS.

Change in plasma cytokine levels during risperidone treatment in children with autism.

BACKGROUND: Atypical antipsychotics decrease irritability in autism. They also affect the cytokine network. Psychological stress, depression, and, possibly, autism spectrum disorder (ASD) are associated with the production of pro-inflammatory cytokines. We sought to determine if risperidone treatment led to changes in plasma cytokine levels. METHODS: Forty-five subjects from an open-label study of risperidone treatment of children and adolescents with ASD, ages 4-18 years, had an analysis of 27 different cytokines at baseline and after 8 weeks of treatment using multiplex assays (Millipore) and read on the Luminex 100(™) platform. We examined changes in each of the cytokine levels in the entire group, and also compared changes in cytokines in responders versus nonresponders. RESULTS: After 8 weeks of risperidone treatment, 2 of the 27 plasma cytokines showed statistically significant decreases in median levels: Eotaxin (p=0.0003) and monocyte chemoattractant protein-1 (MCP-1) (p=0.0024). Six of the 48 subjects met two criteria for responders to risperidone, and the median values of interleukin (IL)-5 were significantly higher (p=0.005) in the overall responder group than in nonresponders. CONCLUSIONS: Two cytokines, eotaxin and MCP-1, which have previously been identified as abnormally elevated in children with autism, decreased during treatment with risperidone. This suggests a possible mechanism of action of risperidone treatment and a balancing of the immune system in affected subjects in this very preliminary study.

Immune dysregulation as a cause of autoinflammation in fragile X premutation carriers: link between FMRI CGG repeat number and decreased cytokine responses.

BACKGROUND: Increased rates of autoinflammatory and autoimmune disorders have been observed in female premutation carriers of CGG repeat expansion alleles of between 55-200 repeats in the fragile X mental retardation 1 (FMR1) gene. To determine whether an abnormal immune profile was present at a cellular level that may predispose female carriers to autoinflammatory conditions, we investigated dynamic cytokine production following stimulation of blood cells. In addition, splenocyte responses were examined in an FMR1 CGG knock-in mouse model of the fragile X premutation. METHODS: Human monocyte and peripheral blood leukocytes (PBLs) were isolated from the blood of 36 female FMR1 premutation carriers and 15 age-matched controls. Cells were cultured with media alone, LPS or PHA. In the animal model, splenocytes were isolated from 32 CGG knock-in mice and 32 wild type littermates. Splenocytes were cultured with media alone or LPS or PMA/Ionomycin. Concentrations of cytokines (GM-CSF, IL-1ß, IL-6, IL-10, IL-13, IL-17, IFNγ, TNFα, and MCP-1) were determined from the supernatants of cellular cultures via Luminex multiplex assay. Additionally, phenotypic cellular markers were assessed on cells isolated from human subjects via flow cytometry. RESULTS: We found decreases in cytokine production in human premutation carriers as well as in the FMR1 knock-in mice when compared with controls. Levels of cytokines were found to be associated with CGG repeat length in both human and mouse. Furthermore, T cells from human premutation carriers showed decreases in cell surface markers of activation when compared with controls. CONCLUSIONS: In this study, FMR1 CGG repeat expansions are associated with decreased immune responses and immune dysregulation in both humans and mice. Deficits in immune responses in female premutation carriers may lead to increased susceptibility to autoimmunity and further research is warranted to determine the link between FMR1 CGG repeat lengths and onset of autoinflammatory conditions.

Maternal immune activation leads to activated inflammatory macrophages in offspring.

Several epidemiological studies have shown an association between infection or inflammation during pregnancy and increased risk of autism in the child. In addition, animal models have illustrated that maternal inflammation during gestation can cause autism-relevant behaviors in the offspring; so called maternal immune activation (MIA) models. More recently, permanent changes in T cell cytokine responses were reported in children with autism and in offspring of MIA mice; however, the cytokine responses of other immune cell populations have not been thoroughly investigated in these MIA models. Similar to changes in T cell function, we hypothesized that following MIA, offspring will have long-term changes in macrophage function. To test this theory, we utilized the poly (I:C) MIA mouse model in C57BL/6J mice and examined macrophage cytokine production in adult offspring. Pregnant dams were given either a single injection of 20mg/kg polyinosinic-polycytidylic acid, poly (I:C), or saline delivered intraperitoneally on gestational day 12.5. When offspring of poly (I:C) treated dams reached 10weeks of age, femurs were collected and bone marrow-derived macrophages were generated. Cytokine production was measured in bone marrow-derived macrophages incubated for 24h in either growth media alone, LPS, IL-4/LPS, or IFN-γ/LPS. Following stimulation with LPS alone, or the combination of IFN-γ/LPS, macrophages from offspring of poly (I:C) treated dams produced higher levels of IL-12(p40) (p<0.04) suggesting an increased M1 polarization. In addition, even without the presence of a polarizing cytokine or LPS stimulus, macrophages from offspring of poly (I:C) treated dams exhibited a higher production of CCL3 (p=0.05). Moreover, CCL3 levels were further increased when stimulated with LPS, or polarized with either IL-4/LPS or IFN-γ/LPS (p<0.05) suggesting a general increase in production of this chemokine. Collectively, these data suggest that MIA can produce lasting changes in macrophage function that are sustained into adulthood.