Peripheral innate immunophenotype in neurodegenerative disease: blood-based profiles and links to survival.

The innate immune system plays an integral role in the progression of many neurodegenerative diseases. In addition to central innate immune cells (e.g., microglia), peripheral innate immune cells (e.g., blood monocytes, natural killer cells, and dendritic cells) may also differ in these conditions. However, the characterization of peripheral innate immune cell types across different neurodegenerative diseases remains incomplete. This study aimed to characterize peripheral innate immune profiles using flow cytometry for immunophenotyping of peripheral blood mononuclear cells in n = 148 people with Alzheimer's disease (AD), frontotemporal dementia (FTD), corticobasal syndrome (CBS), progressive supranuclear palsy (PSP), Lewy body dementia (LBD) as compared to n = 37 healthy controls. To compare groups, we used multivariate dissimilarity analysis and principal component analysis across 19 innate immune cell types. We identified pro-inflammatory profiles that significantly differ between patients with all-cause dementia and healthy controls, with some significant differences between patient groups. Regression analysis confirmed that time to death following the blood test correlated with the individuals' immune profile weighting, positively to TREM2+ and non-classical monocytes and negatively to classical monocytes. Taken together, these results describe transdiagnostic peripheral immune profiles and highlight the link between prognosis and the monocyte cellular subdivision and function (as measured by surface protein expression). The results suggest that blood-derived innate immune profiles can inform sub-populations of cells relevant for specific neurodegenerative diseases that are significantly linked to accelerated disease progression and worse survival outcomes across diagnoses. Blood-based innate immune profiles may contribute to enhanced precision medicine approaches in dementia, helping to identify and monitor therapeutic targets and stratify patients for candidate immunotherapies.

CCR2 monocytes repair cerebrovascular damage caused by chronic social defeat stress.

Immune surveillance of the brain plays an important role in health and disease. Peripheral leukocytes patrol blood-brain barrier interfaces, and after injury, monocytes cross the cerebrovasculature and follow a pattern of pro- and anti-inflammatory activity leading to tissue repair. We have shown that chronic social defeat (CSD) causes scattered vasculature disruptions. Here, we assessed CCR2+ monocyte trafficking to the vascular injury sites in Ccr2wt/rfp reporter mice both during CSD and one week following CSD cessation. We found that CSD for 14 days induced microhemorrhages where plasma fibrinogen leaked into perivascular spaces, but it did not affect the distribution or density of CCR2rfp+ monocytes in the brain. However, after recovery from CSD, many vascularly adhered CCR2+ cells were detected, and gene expression of the CCR2 chemokine receptor ligands CCL7 and CCL12, but not CCL2, was elevated in endothelial cells. Adhered CCR2+ cells were mostly the non-classical, anti-inflammatory Ly6Clo type, and they phagocytosed fibrinogen in perivascular spaces. In CCR2-deficient Ccr2rfp/rfp mice, fibrinogen levels remained elevated in recovery. Fibrinogen infused intracerebroventricularly induced CCR2+ cells to adhere to the vasculature and phagocytose perivascular fibrinogen in Ccr2wt/rfp but not Ccr2rfp/rfp mice. Depletion of monocytes with clodronate liposomes during CSD recovery prevented fibrinogen clearance and blocked behavioral recovery. We hypothesize that peripheral CCR2+ monocytes are not elevated in the brain on day 14 at the end of CSD and do not contribute to its behavioral effects at that time, but in recovery following cessation of stress, they enter the brain and exert restorative functions mediating vascular repair and normalization of behavior.

B-cells are abnormal in psychosocial stress and regulate meningeal myeloid cell activation.

There is increasing interest in how immune cells, including those within the meninges at the blood-brain interface, influence brain function and mood disorders, but little data on humoral immunity in this context. Here, we show that in mice exposed to psychosocial stress, there is increased splenic B cell activation and secretion of the immunoregulatory cytokine interleukin (IL)-10. Meningeal B cells were prevalent in homeostasis but substantially decreased following stress, whereas Ly6Chi monocytes increased, and meningeal myeloid cells showed augmented expression of activation markers. Single-cell RNA sequencing of meningeal B cells demonstrated the induction of innate immune transcriptional programmes following stress, including genes encoding antimicrobial peptides that are known to alter myeloid cell activation. Cd19-/- mice, that have reduced B cells, showed baseline meningeal myeloid cell activation and decreased exploratory behaviour. Together, these data suggest that B cells may influence behaviour by regulating meningeal myeloid cell activation.

Gadd45b is an epigenetic regulator of juvenile social behavior and alters local pro-inflammatory cytokine production in the rodent amygdala.

Precise regulation of the epigenome during perinatal development is critical to the formation of species-typical behavior later in life. Recent data suggests that Gadd45b facilitates active DNA demethylation by recruiting proteins involved in base excision repair (BER), which will catalyze substitution of 5-methyl-cytosine (5mC) for an unmodified cytosine. While a role for Gadd45b has been implicated in both hippocampal and amygdalar learning tasks, to the best of our knowledge, no study has been done investigating the involvement of Gadd45b in neurodevelopmental programming of social behavior. To address this, we used a targeted siRNA delivery approach to transiently knock down Gadd45b expression in the neonatal rat amygdala. We chose to examine social behavior in the juvenile period, as social deficits associated with neurodevelopmental disorders tend to emerge in humans at an equivalent age. We find that neonatal Gadd45b knock-down results in altered juvenile social behavior and reduced expression of several genes implicated in psychiatric disorders, including methyl-CpG-binding protein 2 (MeCP2), Reelin, and brain derived neurotrophic factor (BDNF). We furthermore report a novel role for Gadd45b in the programmed expression of α2-adrenoceptor (Adra2a). Consistent with Gadd45b's role in the periphery, we also observed changes in the expression of pro-inflammatory cytokines interleukin-6 (Il-6) and interleukin-1beta (Il-1beta) in the amygdala, which could potentially mediate or exacerbate effects of Gadd45b knockdown on the organization of social behavior. These data suggest a prominent role for Gadd45b in the epigenetic programming of complex juvenile social interactions, and may provide insight into the etiology of juvenile behavioral disorders such as ADHD, autism, and/or schizophrenia.

Brain, behavior, and physiological changes associated with predator stress-An animal model for trauma exposure in adult and neonatal rats.

The use of predators and predator odor as stressors is an important and ecologically relevant model for studying the impact of behavioral responses to threat. Here we summarize neural substrates and behavioral changes in rats resulting from predator exposure. We briefly define the impact predator exposure has on neural targets throughout development (neonatal, juvenile, and adulthood). These findings allow us to conceptualize the impact of predator exposure in the brain, which in turn may have broader implications for human disorders such as PTSD. Importantly, inclusion of sex as a biological variable yields distinct results that may indicate neural substrates impacted by predator exposure differ based on sex.

Hot metacognition: poorer metacognitive efficiency following acute but not traumatic stress.

Aberrations to metacognition-the ability to reflect on and evaluate self-performance-are a feature of poor mental health. Theoretical models of post-traumatic stress disorder propose that following severe stress or trauma, maladaptive metacognitive evaluations and appraisals of the event drive the development of symptoms. Empirical research is required in order to reveal whether disruptions to metacognition cause or contribute to symptom development in line with theoretical accounts, or are simply a consequence of ongoing psychopathology. In two experiments, using hierarchical Bayesian modelling of metacognition measured in a memory recognition task, we assessed whether distortions to metacognition occur at a state-level after an acute stress induction, and/or at a trait-level in a sample of individuals experiencing intrusive memories following traumatic stress. Results from experiment 1, an in-person laboratory-based experiment, demonstrated that heightened psychological responses to the stress induction were associated with poorer metacognitive efficiency, despite there being no overall change in metacognitive efficiency from pre- to post-stress (N = 27). Conversely, in experiment 2, an online experiment using the same metamemory task, we did not find evidence of metacognitive alterations in a transdiagnostic sample of patients with intrusive memory symptomatology following traumatic stress (N = 36, compared to 44 matched controls). Our results indicate a relationship between state-level psychological responses to stress and metacognitive alterations. The lack of evidence for pre- to post-stress differences in metamemory illustrates the importance for future studies to reveal the direction of this relationship, and consequently the duration of stress-associated metacognitive impairments and their impact on mental health.

Chronic social defeat stress induces meningeal neutrophilia via type I interferon signaling.

Animal models of stress and stress-related disorders are also associated with blood neutrophilia. The mechanistic relevance of this to symptoms or behavior is unclear. We used cytometry, immunohistochemistry, whole tissue clearing, and single-cell sequencing to characterize the meningeal immune response to chronic social defeat (CSD) stress in mice. We find that chronic, but not acute, stress causes meningeal neutrophil accumulation, and CSD increases neutrophil trafficking in vascular channels emanating from skull bone marrow (BM). Transcriptional analysis suggested CSD increases type I interferon (IFN-I) signaling in meningeal neutrophils. Blocking this pathway via the IFN-I receptor (IFNAR) protected against the anhedonic and anxiogenic effects of CSD stress, potentially through reduced infiltration of IFNAR+ neutrophils into the meninges from skull BM. Our identification of IFN-I signaling as a putative mediator of meningeal neutrophil recruitment may facilitate development of new therapies for stress-related disorders.

DNA topoisomerase Top3ß is impacted by early life stress in the developing female and male rat brain.

DNA topoisomerases are essential for preserving genomic integrity. DNA topoisomerases induce breakage of DNA to facilitate replication and transcription by relaxing DNA and relieving supercoiling. Aberrant expression and deletions of topoisomerases are associated with psychiatric disorders such as schizophrenia and autism. Our study investigated the effects of early life stress (ELS) on three topoisomerases, Top1, Top3α, and Top3ß in the developing rat brain. Newborn rats were exposed to a predator odor stress on postnatal days 1, 2, and 3; brain tissue was collected either 30 min after the last stressor on postnatal day 3 or during the juvenile period. We found that exposure to predator odor resulted in a decrease in Top3ß expression levels in the neonatal male amygdala and in the juvenile prefrontal cortex of males and females. These data suggest that developing males and females respond differently to predator odor-induced stress. As ELS results in lower Top3ß levels, these data suggest that ELS experienced during development may have consequences for genomic structural integrity and increased mental health risk.

Early life stress during the neonatal period alters social play and Line1 during the juvenile stage of development.

Early life stress (ELS) has been shown to have a significant impact on typical brain development and the manifestation of psychological disorders through epigenetic modifications that alter gene expression. Line1, a retrotransposon associated with genetic diversity, has been linked with various psychological disorders that are associated with ELS. Our previous work demonstrated altered Line1 DNA copy number in the neonatal period following stressful experiences; we therefore chose to investigate whether early life stress altered Line1 retrotransposition persists into the juvenile period of development. Our study uses a neonatal predator odor exposure (POE) paradigm to model ELS in rats. We examined Line1 using qPCR to assess Line1 expression levels and DNA copy number in the male and female juvenile amygdala, hippocampus and prefrontal cortex-areas chosen for their association with affective disorders and stress. We report a sex difference in Line1 levels within the juvenile amygdala. We also find that ELS significantly increases Line1 DNA copy number within the juvenile amygdala which correlates with reduced juvenile social play levels, suggesting the possibility that Line1 may influence juvenile social development.

Early life stress increases Line1 within the developing brain in a sex-dependent manner.

Long-interspersing element 1 (Line1)-a retrotransposon that comprises ~17% of the human genome and ~24% of the rat genome -is aberrantly expressed in psychiatric disorders such as schizophrenia, bipolar disorder, and Rett syndrome, suggesting it may play an important role in neurodevelopment. Retrotransposons such as Line1 have the ability to self-replicate via reverse transcription and can subsequently be reinserted throughout the genome, potentially increasing genetic diversity. We sought to understand whether early life stress (ELS), a known risk factor for the development of later psychiatric disorders in humans, would affect Line1 expression and DNA copy number. Our study uses a neonatal predator odor exposure (POE) paradigm to model ELS in rats. We found sex- and region-specific increases in both Line1 Open Reading Frame 1 (ORF1) and ORF2 mRNA following POE-induced stress. Interestingly, ELS increased Line1 DNA copy number within the male hippocampus. These data suggest the possibility that early life stress can mobilize Line1 in a sex- and region-specific manner, resulting in genomic heterogeneity between cells in the brain suggesting that some cells may have a different genetic makeup than others resulting in genomic heterogeneity.

Early life stress alters opioid receptor mRNA levels within the nucleus accumbens in a sex-dependent manner.

Early life stress (ELS) strongly impacts mental health, but little is known about its interaction with biological sex and postnatal development to influence risk and resilience to psychopathologies. A number of psychiatric disorders, such as social anhedonia and drug addiction, involve dysfunctional opioid signaling; moreover, there is evidence for differential central opioid function in males vs. females. The present study examined opioid receptor gene expression in the nucleus accumbens (NAc) and amygdala of male and female rats subjected to a neonatal predator odor exposure (POE) paradigm to model ELS. Brain tissue was collected at two developmental time points: neonatal and juvenile. Results showed that, following the neonatal POE experience, opioid receptor mRNA levels in the NAc were differentially regulated at the neonatal and juvenile time points. POE downregulated neonatal mu- and kappa-opioid receptor mRNA levels in neonatal females, but upregulated mu- and delta-opioid receptor mRNA levels in juvenile females. Intriguingly, POE had no significant effect on NAc opioid receptor mRNA levels in males at either time point, indicating that the impact of POE on opioid system development is sex-dependent. Finally, POE failed to alter amygdalar opioid receptor gene expression in either sex at either time-point. The spatiotemporally- and sex-specific impact of ELS within the developing brain may confer differential risk or resilience for males and females to develop atypical opioid-regulated behaviors associated with conditions such as depression and addiction.

Decoding microglia responses to psychosocial stress reveals blood-brain barrier breakdown that may drive stress susceptibility.

An animal's ability to cope with or succumb to deleterious effects of chronic psychological stress may be rooted in the brain's immune responses manifested in microglial activity. Mice subjected to chronic social defeat (CSD) were categorized as susceptible (CSD-S) or resilient (CSD-R) based on behavioral phenotyping, and their microglia were isolated and analyzed by microarray. Microglia transcriptomes from CSD-S mice were enriched for pathways associated with inflammation, phagocytosis, oxidative stress, and extracellular matrix remodeling. Histochemical experiments confirmed the array predictions: CSD-S microglia showed elevated phagocytosis and oxidative stress, and the brains of CSD-S but not CSD-R or non-stressed control mice showed vascular leakage of intravenously injected fluorescent tracers. The results suggest that the inflammatory profile of CSD-S microglia may be precipitated by extracellular matrix degradation, oxidative stress, microbleeds, and entry and phagocytosis of blood-borne substances into brain parenchyma. We hypothesize that these CNS-centric responses contribute to the stress-susceptible behavioral phenotype.

Contributions of the adaptive immune system to mood regulation: Mechanisms and pathways of neuroimmune interactions.

Clinical and basic studies of functional interactions between adaptive immunity, affective states, and brain function are reviewed, and the neural, humoral, and cellular routes of bidirectional communication between the brain and the adaptive immune system are evaluated. In clinical studies of depressed populations, lymphocytes-the principal cells of the adaptive immune system-exhibit altered T cell subtype ratios and CD4+ helper T cell polarization profiles. In basic studies using psychological stress to model depression, T cell profiles are altered as well, consistent with stress effects conveyed by the hypothalamic-pituitary-adrenal axis and sympathetic nervous system. Lymphocytes in turn have effects on behavior and CNS structure and function. CD4+ T cells in particular appear to modify affective behavior and rates of hippocampal dentate gyrus neurogenesis. These observations force the question of how such actions are carried out. CNS effects may occur via cellular and molecular mechanisms whereby effector memory T cells and the cytokine profiles they produce in the blood interact with the blood-brain barrier in ways that remain to be clarified. Understanding the mechanisms by which T cells polarize and interact with the brain to alter mood states is key to advances in the field, and may permit development of therapies that target cells in the periphery, thus bypassing problems associated with bioavailability of drugs within the brain.

N6-methyladenine is an epigenetic marker of mammalian early life stress.

Recent evidence described 6-methyladenine (6 mA) as a novel epigenetic regulator in a variety of multicellular species, including rodents; however, its capacity to influence gene expression in the mammalian brain remains unknown. We examined if 6 mA is present and regulated by early life stress associated with predator odor exposure (POE) within the developing rat amygdala. Our results provide evidence that 6 mA is present in the mammalian brain, is altered within the Htr2a gene promoter by early life stress and biological sex, and increased 6 mA is associated with gene repression. These data suggest that methylation of adenosine within mammalian DNA may be used as an additional epigenetic biomarker for investigating the development of stress-induced neuropathology.

Sex differences in Gadd45b expression and methylation in the developing rodent amygdala.

Precise spatiotemporal epigenetic regulation of the genome facilitates species-typical development; sexual differentiation of the brain by gonadal hormones and sex chromosomes causes extensive epigenetic reprogramming of many cells in the body, including the brain, and may indirectly predispose males and females to different psychiatric conditions. We and others have demonstrated sex differences in DNA methylation, as well as in the enzymes that form, or 'write', this epigenetic modification. However, while a growing body of evidence suggests that DNA methylation undergoes rapid turnover and is dynamically regulated in vivo, to our knowledge no studies have been done investigating whether sex differences exist in the epigenetic 'erasers' during postnatal development. Here we report sex differences in the expression of growth arrest and DNA damage inducible factor ß (Gadd45b), but not family members α (a) or γ (g), in the neonatal and juvenile rodent amygdala.