Cell-Type-Specific Interleukin 1 Receptor 1 Signaling in the Brain Regulates Distinct Neuroimmune Activities.

Interleukin-1 (IL-1) signaling is important for multiple potentially pathogenic processes in the central nervous system (CNS), but the cell-type-specific roles of IL-1 signaling are unclear. We used a genetic knockin reporter system in mice to track and reciprocally delete or express IL-1 receptor 1 (IL-1R1) in specific cell types, including endothelial cells, ventricular cells, peripheral myeloid cells, microglia, astrocytes, and neurons. We found that endothelial IL-1R1 was necessary and sufficient for mediating sickness behavior and drove leukocyte recruitment to the CNS and impaired neurogenesis, whereas ventricular IL-1R1 was critical for monocyte recruitment to the CNS. Although microglia did not express IL-1R1, IL-1 stimulation of endothelial cells led to the induction of IL-1 in microglia. Together, these findings describe the structure and functions of the brain's IL-1R1-expressing system and lay a foundation for the dissection and identification of IL-1R1 signaling pathways in the pathogenesis of CNS diseases.

Gonadal hormones impart male-biased behavioral vulnerabilities to immune activation via microglial mitochondrial function.

There is a strong male bias in the prevalence of many neurodevelopmental disorders such as autism spectrum disorder. However, the mechanisms underlying this sex bias remain elusive. Infection during the perinatal period is associated with an increased risk of neurodevelopmental disorder development. Here, we used a mouse model of early-life immune activation that reliably induces deficits in social behaviors only in males. We demonstrate that male-biased alterations in social behavior are dependent upon microglial immune signaling and are coupled to alterations in mitochondrial morphology, gene expression, and function specifically within microglia, the innate immune cells of the brain. Additionally, we show that this behavioral and microglial mitochondrial vulnerability to early-life immune activation is programmed by the male-typical perinatal gonadal hormone surge. These findings demonstrate that social behavior in males over the lifespan are regulated by microglia-specific mechanisms that are shaped by events that occur in early development.

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.

Stressed-Out T Cells Fragment the Mind.

The immune system is increasingly recognized to play an integral role in regulating stress responses. In a recent article in Cell, Fan et al. demonstrate a novel mechanism through which stress drives mitochondrial fragmentation-induced xanthine accumulation in mouse CD4+ T cells, subsequently acting on oligodendrocytes to induce anxiety-like behaviors.

Sickness and the Social Brain: How the Immune System Regulates Behavior across Species.

Many instances of sickness critically involve the immune system. The immune system talks to the brain in a bidirectional loop. This discourse affords the immune system immense control, such that it can influence behavior and optimize recovery from illness. These behavioral responses to infection are called sickness behaviors and can manifest in many ways, including changes in mood, motivation, or energy. Fascinatingly, most of these changes are conserved across species, and most organisms demonstrate some form of sickness behaviors. One of the most interesting sickness behaviors, and not immediately obvious, is altered sociability. Here, we discuss how the immune system impacts social behavior, by examining the brain regions and immune mediators involved in this process. We first outline how social behavior changes in response to infection in various species. Next, we explore which brain regions control social behavior and their evolutionary origins. Finally, we describe which immune mediators establish the link between illness and social behavior, in the context of both normal development and infection. Overall, we hope to make clear the striking similarities between the mechanisms that facilitate changes in sociability in derived and ancestral vertebrate, as well as invertebrate, species.

Sex-dependent neurobiological features of prenatal immune activation via TLR7.

Immune activation during pregnancy via infection or autoimmune disease is a risk factor for neuropsychiatric illness. Mouse models of prenatal immune activation often involve maternal administration of agents that activate toll-like receptors (TLRs), a class of pattern recognition receptors that initiate innate immune responses. Such studies have focused primarily on activating the TLR3 or TLR4 subtypes, to mimic immune responses to viral or bacterial infections, respectively. Here, we characterize the effects of prenatal activation of TLR7, which is implicated in the pathogenesis of autoimmune disease. Prenatal TLR7 activation via administration of the selective agonist imiquimod (5.0 mg/kg) induces a phenotype in offspring characterized by reduced anxiety-like behavior, fragmented social behavior, and altered ultrasonic vocalization patterns at 6-12 weeks of age. The characteristics of this phenotype are readily distinguishable from-and in some ways opposite to-those seen following prenatal activation of TLR3 and/or TLR4. Prenatal TLR7-activated mice have normal baseline locomotor activity, but are hyperresponsive to stimuli including social partners, circadian cues, and gonadal hormone fluctuations. These alterations are accompanied by decreases in microglia density but increases in ramifications. RNA-sequencing of dorsal striatum, a region showing profound changes in microglial markers, indicates that prenatal TLR7 activation induces differential expression of hundreds of genes at 13 weeks of age, with virtually no overlap in differentially expressed genes between males and females. Our findings demonstrate that prenatal immune activation can promote a wide range of developmental trajectories, depending on the type and/or pattern of TLR activation and the sex of the offspring.

Microglia and sexual differentiation of the developing brain: A focus on ontogeny and intrinsic factors.

Sexual differentiation of the brain during early development likely underlies the strong sex biases prevalent in many neurological conditions. Mounting evidence indicates that microglia, the innate immune cells of the central nervous system, are intricately involved in these sex-specific processes of differentiation. In this review, we synthesize literature demonstrating sex differences in microglial number, morphology, transcriptional state, and functionality throughout spatiotemporal development as well as highlight current literature regarding ontogeny of microglia. Along with vanRyzin et al. in this issue, we explore the idea that differences in microglia imparted by chromosomal or ontogeny-related programming can influence microglial-driven sexual differentiation of the brain, as well as the idea that extrinsic differences in the male and female brain microenvironment may in turn impart sex differences in microglia.

The inflammatory event of birth: How oxytocin signaling may guide the development of the brain and gastrointestinal system.

The role of oxytocin (OT) as a neuropeptide that modulates social behavior has been extensively studied and reviewed, but beyond these functions, OT's adaptive functions at birth are quite numerous, as OT coordinates many physiological processes in the mother and fetus to ensure a successful delivery. In this review we explore in detail the potential adaptive roles of oxytocin as an anti-inflammatory, protective molecule at birth for the developing fetal brain and gastrointestinal system based on evidence that birth is a potent inflammatory/immune event. We discuss data with relevance for a number of neurodevelopmental disorders, as well as the emerging role of the gut-brain axis for health and disease. Finally, we discuss the potential relevance of sex differences in OT signaling present at birth in the increased male vulnerability to neurodevelopmental disabilities.

Neonatal immune challenge induces female-specific changes in social behavior and somatostatin cell number.

Decreases in social behavior are a hallmark aspect of acute "sickness behavior" in response to infection. However, immune insults that occur during the perinatal period may have long-lasting consequences for adult social behavior by impacting the developmental organization of underlying neural circuits. Microglia, the resident immune cells of the central nervous system, are sensitive to immune stimulation and play a critical role in the developmental sculpting of neural circuits, making them likely mediators of this process. Here, we investigated the impact of a postnatal day (PND) 4 lipopolysaccharide (LPS) challenge on social behavior in adult mice. Somewhat surprisingly, neonatal LPS treatment decreased sociability in adult female, but not male mice. LPS-treated females also displayed reduced social interaction and social memory in a social discrimination task as compared to saline-treated females. Somatostatin (SST) interneurons within the anterior cingulate cortex (ACC) have recently been suggested to modulate a variety of social behaviors. Interestingly, the female-specific changes in social behavior observed here were accompanied by an increase in SST interneuron number in the ACC. Finally, these changes in social behavior and SST cell number do not appear to depend on microglial inflammatory signaling, because microglia-specific genetic knock-down of myeloid differentiation response protein 88 (MyD88; the removal of which prevents LPS from increasing proinflammatory cytokines such as TNFα and IL-1ß) did not prevent these LPS-induced changes. This study provides novel evidence for enduring effects of neonatal immune activation on social behavior and SST interneurons in females, largely independent of microglial inflammatory signaling.

Removal of microglial-specific MyD88 signaling alters dentate gyrus doublecortin and enhances opioid addiction-like behaviors.

Drugs of abuse promote a potent immune response in central nervous system (CNS) via the activation of microglia and astrocytes. However, the molecular mechanisms underlying microglial activation during addiction are not well known. We developed and functionally characterized a novel transgenic mouse (Cx3cr1-CreBTtg/0:MyD88f/f [Cretg/0]) wherein the immune signaling adaptor gene, MyD88, was specifically deleted in microglia. To test the downstream effects of loss of microglia-specific MyD88 signaling in morphine addiction, Cretg/0 and Cre0/0 mice were tested for reward learning, extinction, and reinstatement using a conditioned place preference (CPP) paradigm. There were no differences in drug acquisition, but Cretg/0 mice had prolonged extinction and enhanced reinstatement compared to Cre0/0 controls. Furthermore, morphine-treated Cretg/0 mice showed increased doublecortin (DCX) signal relative to Cre0/0 control mice in the hippocampus, indicative of increased number of immature neurons. Additionally, there was an increase in colocalization of microglial lysosomal marker CD68 with DCX+cells in morphine-treated Cretg/0 mice but not in Cre0/0 or drug-naїve mice, suggesting a specific role for microglial MyD88 signaling in neuronal phagocytosis in the hippocampus. Our results show that MyD88 deletion in microglia may negatively impact maturing neurons within the adult hippocampus and thus reward memories, suggesting a novel protective role for microglia in opioid addiction.

Associations between maternal cytokine levels during gestation and measures of child cognitive abilities and executive functioning.

Preclinical studies demonstrate that environmentally-induced alterations in inflammatory cytokines generated by the maternal and fetal immune system can significantly impact fetal brain development. Yet, the relationship between maternal cytokines during gestation and later cognitive ability and executive function remains understudied. Children (n = 246) were born of mothers enrolled in the Newborn Epigenetic Study - a prospective pre-birth cohort in the Southeastern US. We characterized seven cytokines [IL-1ß, IL-4,IL-6, IL-12p70, IL-17A, tumor necrosis factor-α (TNFα), and interferon-γ (IFNγ)] and one chemokine (IL-8) from maternal plasma collected during pregnancy. We assessed children's cognitive abilities and executive functioning at a mean age of 4.5 (SD = 1.1) years. Children's DAS-II and NIH toolbox scores were regressed on cytokines and the chemokine, controlling for maternal age, race, education, body mass index, IQ, parity, smoking status, delivery type, gestational weeks, and child birth weight and sex. Higher IL-12p70 (ßIL-12p70 = 4.26, p = 0.023) and IL-17A (ßIL-17A = 3.70, p = 0.042) levels were related to higher DAS-II GCA score, whereas higher IL-1ß (ßIL-1B = -6.07, p = 0.003) was related to lower GCA score. Higher IL-12p70 was related to higher performance on NIH toolbox measures of executive functions related to inhibitory control and attention (ßIL-12p70 = 5.20, p = 0.046) and cognitive flexibility (ßIL-12p70 = 5.10, p = 0.047). Results suggest that dysregulation in gestational immune activity are associated with child cognitive ability and executive functioning.

Generation of a microglial developmental index in mice and in humans reveals a sex difference in maturation and immune reactivity.

Evidence suggests many neurological disorders emerge when normal neurodevelopmental trajectories are disrupted, i.e., when circuits or cells do not reach their fully mature state. Microglia play a critical role in normal neurodevelopment and are hypothesized to contribute to brain disease. We used whole transcriptome profiling with Next Generation sequencing of purified developing microglia to identify a microglial developmental gene expression program involving thousands of genes whose expression levels change monotonically (up or down) across development. Importantly, the gene expression program was delayed in males relative to females and exposure of adult male mice to LPS, a potent immune activator, accelerated microglial development in males. Next, a microglial developmental index (MDI) generated from gene expression patterns obtained from purified mouse microglia, was applied to human brain transcriptome datasets to test the hypothesis that variability in microglial development is associated with human diseases such as Alzheimer's and autism where microglia have been suggested to play a role. MDI was significantly increased in both Alzheimer's Disease and in autism, suggesting that accelerated microglial development may contribute to neuropathology. In conclusion, we identified a microglia-specific gene expression program in mice that was used to create a microglia developmental index, which was applied to human datasets containing heterogeneous cell types to reveal differences between healthy and diseased brain samples, and between males and females. This powerful tool has wide ranging applicability to examine microglial development within the context of disease and in response to other variables such as stress and pharmacological treatments.

Got worms? Perinatal exposure to helminths prevents persistent immune sensitization and cognitive dysfunction induced by early-life infection.

The incidence of autoimmune and inflammatory diseases has risen dramatically in post-industrial societies. "Biome depletion" - loss of commensal microbial and multicellular organisms such as helminths (intestinal worms) that profoundly modulate the immune system - may contribute to these increases. Hyperimmune-associated disorders also affect the brain, especially neurodevelopment, and increasing evidence links early-life infection to cognitive and neurodevelopmental disorders. We have demonstrated previously that rats infected with bacteria as newborns display life-long vulnerabilities to cognitive dysfunction, a vulnerability that is specifically linked to long-term hypersensitivity of microglial cell function, the resident immune cells of the brain. Here, we demonstrate that helminth colonization of pregnant dams attenuated the exaggerated brain cytokine response of their offspring to bacterial infection, and that combined with post-weaning colonization of offspring with helminths (consistent with their mothers treatment) completely prevented enduring microglial sensitization and cognitive dysfunction in adulthood. Importantly, helminths had no overt impact on adaptive immune cell subsets, whereas exaggerated innate inflammatory responses in splenic macrophages were prevented. Finally, helminths altered the effect of neonatal infection on the gut microbiome; neonatal infection with Escherichia coli caused a shift from genera within the Actinobacteria and Tenericutes phyla to genera in the Bacteroidetes phylum in rats not colonized with helminths, but helminths attenuated this effect. In sum, these data point toward an inter-relatedness of various components of the biome, and suggest potential mechanisms by which this helminth might exert therapeutic benefits in the treatment of neuroinflammatory and cognitive disorders.

A model for the induction of autism in the ecosystem of the human body: the anatomy of a modern pandemic?

BACKGROUND: The field of autism research is currently divided based on a fundamental question regarding the nature of autism: Some are convinced that autism is a pandemic of modern culture, with environmental factors at the roots. Others are convinced that the disease is not pandemic in nature, but rather that it has been with humanity for millennia, with its biological and neurological underpinnings just now being understood. OBJECTIVE: In this review, two lines of reasoning are examined which suggest that autism is indeed a pandemic of modern culture. First, given the widely appreciated derailment of immune function by modern culture, evidence that autism is strongly associated with aberrant immune function is examined. Second, evidence is reviewed indicating that autism is associated with 'triggers' that are, for the most part, a construct of modern culture. In light of this reasoning, current epidemiological evidence regarding the incidence of autism, including the role of changing awareness and diagnostic criteria, is examined. Finally, the potential role of the microbial flora (the microbiome) in the pathogenesis of autism is discussed, with the view that the microbial flora is a subset of the life associated with the human body, and that the entire human biome, including both the microbial flora and the fauna, has been radically destabilized by modern culture. CONCLUSIONS: It is suggested that the unequivocal way to resolve the debate regarding the pandemic nature of autism is to perform an experiment: monitor the prevalence of autism after normalizing immune function in a Western population using readily available approaches that address the well-known factors underlying the immune dysfunction in that population.

Adolescent morphine exposure affects long-term microglial function and later-life relapse liability in a model of addiction.

Adolescence in humans represents a unique developmental time point associated with increased risk-taking behavior and experimentation with drugs of abuse. We hypothesized that exposure to drugs of abuse during adolescence may increase the risk of addiction in adulthood. To test this, rats were treated with a subchronic regimen of morphine or saline in adolescence, and their preference for morphine was examined using conditioned place preference (CPP) and drug-induced reinstatement in adulthood. The initial preference for morphine did not differ between groups; however, rats treated with morphine during adolescence showed robust reinstatement of morphine CPP after drug re-exposure in adulthood. This effect was not seen in rats pretreated with a subchronic regimen of morphine as adults, suggesting that exposure to morphine specifically during adolescence increases the risk of relapse to drug-seeking behavior in adulthood. We have previously established a role for microglia, the immune cells of the brain, and immune molecules in the risk of drug-induced reinstatement of morphine CPP. Thus, we examined the role of microglia within the nucleus accumbens of these rats and determined that rats exposed to morphine during adolescence had a significant increase in Toll-like receptor 4 (TLR4) mRNA and protein expression specifically on microglia. Morphine binds to TLR4 directly, and this increase in TLR4 was associated with exaggerated morphine-induced TLR4 signaling and microglial activation in rats previously exposed to morphine during adolescence. These data suggest that long-term changes in microglial function, caused by adolescent morphine exposure, alter the risk of drug-induced reinstatement in adulthood.

Neonatal E. coli infection causes neuro-behavioral deficits associated with hypomyelination and neuronal sequestration of iron.

Recent evidence indicates that inflammatory insults in neonates significantly influenced white matter development and caused behavioral deficits that manifest in young adulthood. The mechanisms underlying these developmental and behavioral complications, however, are not well understood. We hypothesize that acute brain inflammation caused by neonatal infection reduces the bioavailability of iron required for oligodendrocyte maturation and white matter development. Here, we confirm that peripheral Escherichia coli infection in neonates at postnatal day 3 (P3) caused acute brain inflammation that was resolved within 72 h. Nonetheless, transient early life infection (ELI) profoundly influenced behavior, white matter development, and iron homeostasis in the brain. For instance, mice exposed to E. coli as neonates had increased locomotor activity and impaired motor coordination as juveniles (P35) and young adults (P60). In addition, these behavioral deficits were associated with marked hypomyelination and a reduction of oligodendrocytes in subcortical white matter and motor cortex. Moreover, ELI altered transcripts related to cellular sequestration of iron in the brain including hepcidin, ferroportin, and L-ferritin. For example, ELI increased hepcidin mRNA and decreased ferroportin mRNA and protein in the brain at P4, which preceded increased L-ferritin mRNA at P12. Consistent with the mRNA results, L-ferritin protein was robustly increased at P12 specifically in neurons of E. coli infected mice. We interpret these data to indicate that neonatal infection causes significant neuronal sequestration of iron at a time point before myelination. Together, these data indicate a possible role for aberrant neuronal iron storage in neonatal infection-induced disturbances in myelination and behavior.

Prenatal air pollution exposure induces sexually dimorphic fetal programming of metabolic and neuroinflammatory outcomes in adult offspring.

Environmental chemical exposures during critical windows of development may contribute to the escalating prevalence of obesity. We tested the hypothesis that prenatal exposure to diesel exhaust particles (DEP), a primary component of air pollution, would prime microglia long-term, resulting in exacerbated metabolic and affective outcomes following exposure to a high-fat diet in adulthood. Time-mated mouse dams were intermittently exposed to respiratory instillations of either vehicle (VEH) or DEP throughout gestation. Adult male and female offspring were then fed either a low-fat diet (LFD) or high-fat diet (HFD) for 9 weeks. The male offspring of DEP-exposed dams exhibited exaggerated weight gain, insulin resistance, and anxiety-like behavior on HFD compared to the male offspring of VEH-exposed dams, whereas female offspring did not differ according to prenatal treatment. Furthermore, HFD induced evidence of macrophage infiltration of both adipose tissue and the brain in both sexes, but these cells were more activated specifically in DEP/HFD males. DEP/HFD males also expressed markedly higher levels of microglial/macrophage, but not astrocyte, activation markers in the hippocampus, whereas females exhibited only a suppression of astrocyte activation markers due to HFD. In a second experiment, DEP male offspring mounted an exaggerated peripheral IL-1ß response to an LPS challenge at postnatal day (P)30, whereas their central IL-1ß response did not differ from VEH male offspring, which is suggestive of macrophage priming due to prenatal DEP exposure. In sum, prenatal air pollution exposure "programs" offspring for increased susceptibility to diet-induced metabolic, behavioral, and neuroinflammatory changes in adulthood in a sexually dimorphic manner.

Microglial MyD88-dependent pathways are regulated in a sex-specific manner in the context of HMGB1-induced anxiety.

Chronic stress is a significant risk factor for the development and recurrence of anxiety disorders. Chronic stress impacts the immune system, causing microglial functional alterations in the medial prefrontal cortex (mPFC), a brain region involved in the pathogenesis of anxiety. High mobility group box 1 protein (HMGB1) is an established modulator of neuronal firing and a potent pro-inflammatory stimulus released from neuronal and non-neuronal cells following stress. HMGB1, in the context of stress, acts as a danger-associated molecular pattern (DAMP), instigating robust proinflammatory responses throughout the brain, so much so that localized drug delivery of HMGB1 alters behavior in the absence of any other forms of stress, i.e., social isolation, or behavioral stress models. Few studies have investigated the molecular mechanisms that underlie HMGB1-associated behavioral effects in a cell-specific manner. The aim of this study is to investigate cellular and molecular mechanisms underlying HMGB1-induced behavioral dysfunction with regard to cell-type specificity and potential sex differences. Here, we report that both male and female mice exhibited anxiety-like behavior following increased HMGB1 in the mPFC as well as changes in microglial morphology. Interestingly, our results demonstrate that HMGB1-induced anxiety may be mediated by distinct microglial MyD88-dependent mechanisms in females compared to males. This study supports the hypothesis that MyD88 signaling in microglia may be a crucial mediator of the stress response in adult female mice.

Neuron Derived Cytokine Interleukin-34 Controls Developmental Microglia Function.

Neuron-microglia interactions dictate the development of neuronal circuits in the brain. However, the factors that support and broadly regulate these processes across developmental stages are largely unknown. Here, we find that IL34, a neuron-derived cytokine, is upregulated in development and plays a critical role in supporting and maintaining neuroprotective, mature microglia in the anterior cingulate cortex (ACC) of mice. We show that IL34 mRNA and protein is upregulated in neurons in the second week of postnatal life and that this increase coincides with increases in microglia number and expression of mature, homeostatic markers, e.g., TMEM119. We also found that IL34 mRNA is higher in more active neurons, and higher in excitatory (compared to inhibitory) neurons. Genetic KO of IL34 prevents the functional maturation of microglia and results in an anxiolytic phenotype in these mice by adulthood. Acute, low dose blocking of IL34 at postnatal day (P)15 in mice decreased microglial TMEM119 expression and increased aberrant microglial phagocytosis of thalamocortical synapses within the ACC. In contrast, viral overexpression of IL34 early in life (P1-P8) caused early maturation of microglia and prevented microglial phagocytosis of thalamocortical synapses during the appropriate neurodevelopmental refinement window. Taken together, these findings establish IL34 as a key regulator of neuron-microglia crosstalk in postnatal brain development, controlling both microglial maturation and synapse engulfment.