Use of adeno-associated viruses for transgenic modulation of microglia structure and function: A review of technical considerations and challenges.

Microglia play a central role in the etiology of many neuropathologies. Transgenic tools are a powerful experiment approach to gain reliable and specific control over microglia function. Adeno-associated virus (AAVs) vectors are already an indispensable tool in neuroscience research. Despite ubiquitous use of AAVs and substantial interest in the role of microglia in the study of central nervous system (CNS) function and disease, transduction of microglia using AAVs is seldom reported. This review explores the challenges and advancements made in using AAVs for expressing transgenes in microglia. First, we will examine the functional anatomy of the AAV capsid, which will serve as a basis for subsequent discussions of studies exploring the relationship between capsid mutations and microglia transduction efficacy. After outlining the functional anatomy of AAVs, we will consider the experimental evidence demonstrating AAV-mediated transduction of microglia and microglia-like cell lines followed by an examination of the most promising experimental approaches identified in the literature. Finally, technical limitations will be considered in future applications of AAV experimental approaches.

Interleukin-1beta and inflammasome expression in spinal cord following chronic constriction injury in male and female rats.

Females represent a majority of chronic pain patients and show greater inflammatory immune responses in human chronic pain patient populations as well as in animal models of neuropathic pain. Recent discoveries in chronic pain research have revealed sex differences in inflammatory signaling, a key component of sensory pathology in chronic neuropathic pain, inviting more research into the nuances of these sex differences. Here we use the chronic constriction injury (CCI) model to explore similarities and differences in expression and production of Inflammatory cytokine IL-1beta in the lumbar spinal cord, as well as its role in chronic pain. We have discovered that intrathecal IL-1 receptor antagonist reverses established pain in both sexes, and increased gene expression of inflammasome NLRP3 is specific to microglia and astrocytes rather than neurons, while IL-1beta is specific to microglia in both sexes. We report several sex differences in the expression level of the genes coding for IL-1beta, as well as the four inflammasomes responsible for IL-1beta release: NLRP3, AIM2, NLRP1, and NLRC4 in the spinal cord. Total mRNA, but not protein expression of IL-1beta is greater in females than males after CCI. Also, while CCI increases all four inflammasomes in both sexes, there are sex differences in relative levels of inflammasome expression. NLRP3 and AIM2 are more highly expressed in females, whereas NLRP1 expression is greater in males.

SARS-CoV-2 S1 subunit produces a protracted priming of the neuroinflammatory, physiological, and behavioral responses to a remote immune challenge: A role for corticosteroids.

Long COVID is a major public health consequence of COVID-19 and is characterized by multiple neurological and neuropsychatric symptoms. SARS-CoV-2 antigens (e.g., spike S1 subunit) are found in the circulation of Long COVID patients, have been detected in post-mortem brain of COVID patients, and exhibit neuroinflammatory properties. Considering recent observations of chronic neuroinflammation in Long COVID patients, the present study explores the idea that antigens derived from SARS-CoV-2 might produce a long-term priming or sensitization of neuroinflammatory processes, thereby potentiating the magnitude and/or duration of the neuroinflammatory response to future inflammatory insults. Rats were administered S1 or vehicle intra-cisterna magna and 7d later challenged with vehicle or LPS. The neuroinflammatory, physiological, and behavioral responses to LPS were measured at various time points post-LPS. We found that prior S1 treatment potentiated many of these responses to LPS suggesting that S1 produces a protracted priming of these processes. Further, S1 produced a protracted reduction in basal brain corticosteroids. Considering the anti-inflammatory properties of corticosteroids, these findings suggest that S1 might disinhibit innate immune processes in brain by reducing anti-inflammatory drive, thereby priming neuroinflammatory processes. Given that hypocortisolism is observed in Long COVID, we propose that similar S1-induced innate immune priming processes might play role in the pathophysiology of Long COVID.

Exploring the immunogenic properties of SARS-CoV-2 structural proteins: PAMP:TLR signaling in the mediation of the neuroinflammatory and neurologic sequelae of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) produces an array of neurologic and neuropsychiatric symptoms in the acute and post-acute phase of infection (PASC; post-acute sequelae of SARS-CoV-2 infection). Neuroinflammatory processes are considered key factors in the etiology of these symptoms. Several mechanisms underpinning the development of inflammatory events in the brain have been proposed including SARS-CoV-2 neurotropism and peripheral inflammatory responses (i.e., cytokine storm) to infection, which might produce neuroinflammation via immune-to-brain signaling pathways. In this review, we explore evidence in support of an alternate mechanism whereby structural proteins (e.g., spike and spike S1 subunit) derived from SARS-CoV-2 virions function as pathogen-associated molecular patterns (PAMPs) to elicit proinflammatory immune responses in the periphery and/or brain via classical Toll-Like Receptor (TLR) inflammatory pathways. We propose that SARS-CoV-2 structural proteins might directly produce inflammatory processes in brain independent of and/or in addition to peripheral proinflammatory effects, which might converge to play a causal role in the development of neurologic/neuropsychiatric symptoms in COVID-19.

Immunization with a heat-killed preparation of Mycobacterium vaccae NCTC 11659 enhances auditory-cued fear extinction in a stress-dependent manner.

Stress-related psychiatric disorders including anxiety disorders, mood disorders, and trauma and stressor-related disorders, such as posttraumatic stress disorder (PTSD), affect millions of people world-wide each year. Individuals with stress-related psychiatric disorders have been found to have poor immunoregulation, increased proinflammatory markers, and dysregulation of fear memory. The "Old Friends" hypothesis proposes that a lack of immunoregulatory inputs has led to a higher prevalence of inflammatory disorders and stress-related psychiatric disorders, in which inappropriate inflammation is thought to be a risk factor. Immunization with a soil-derived saprophytic bacterium with anti-inflammatory and immunoregulatory properties, Mycobacterium vaccae NCTC 11659, can lower proinflammatory biomarkers, increase stress resilience, and, when given prior to or after fear conditioning in a rat model of fear-potentiated startle, enhance fear extinction. In this study, we investigated whether immunization with heat-killed M. vaccae NCTC 11659 would enhance fear extinction in contextual or auditory-cued fear conditioning paradigms and whether M. vaccae NCTC 11659 would prevent stress-induced exaggeration of fear expression or stress-induced resistance to extinction learning. Adult male Sprague Dawley rats were immunized with M. vaccae NCTC 11659 (subcutaneous injections once a week for three weeks), and underwent either: Experiment 1) one-trial contextual fear conditioning; Experiment 2) two-trial contextual fear conditioning; Experiment 3) stress-induced enhancement of contextual fear conditioning; Experiment 4) stress-induced enhancement of auditory-cued fear conditioning; or Experiment 5) stress-induced enhancement of auditory-cued fear conditioning exploring short-term memory. Immunizations with M. vaccae NCTC 11659 had no effect on one- or two-trial contextual fear conditioning or contextual fear extinction, with or without exposure to inescapable stress. However, inescapable stress increased resistance to auditory-cued fear extinction. Immunization with M. vaccae NCTC 11659 prevented the stress-induced increase in resistance to auditory-cued fear extinction learning. Finally, in an auditory-cued fear conditioning paradigm exploring short-term memory and fear acquisition, immunization with M. vaccae did not prevent fear acquisition, either with or without exposure to inescapable stress, consistent with the hypothesis that M. vaccae NCTC 11659 has no effect on fear acquisition but enhances fear extinction. These data are consistent with the hypothesis that increased immunoregulation following immunization with M. vaccae NCTC 11659 promotes stress resilience, in particular by preventing stress-induced resistance to fear extinction, and may be a potential therapeutic intervention for trauma- and stressor-related disorders such as PTSD.

Mycobacterium vaccae NCTC 11659, a Soil-Derived Bacterium with Stress Resilience Properties, Modulates the Proinflammatory Effects of LPS in Macrophages.

Inflammatory conditions, including allergic asthma and conditions in which chronic low-grade inflammation is a risk factor, such as stress-related psychiatric disorders, are prevalent and are a significant cause of disability worldwide. Novel approaches for the prevention and treatment of these disorders are needed. One approach is the use of immunoregulatory microorganisms, such as Mycobacterium vaccae NCTC 11659, which have anti-inflammatory, immunoregulatory, and stress-resilience properties. However, little is known about how M. vaccae NCTC 11659 affects specific immune cell targets, including monocytes, which can traffic to peripheral organs and the central nervous system and differentiate into monocyte-derived macrophages that, in turn, can drive inflammation and neuroinflammation. In this study, we investigated the effects of M. vaccae NCTC 11659 and subsequent lipopolysaccharide (LPS) challenge on gene expression in human monocyte-derived macrophages. THP-1 monocytes were differentiated into macrophages, exposed to M. vaccae NCTC 11659 (0, 10, 30, 100, 300 µg/mL), then, 24 h later, challenged with LPS (0, 0.5, 2.5, 250 ng/mL), and assessed for gene expression 24 h following challenge with LPS. Exposure to M. vaccae NCTC 11659 prior to challenge with higher concentrations of LPS (250 ng/mL) polarized human monocyte-derived macrophages with decreased IL12A, IL12B, and IL23A expression relative to IL10 and TGFB1 mRNA expression. These data identify human monocyte-derived macrophages as a direct target of M. vaccae NCTC 11659 and support the development of M. vaccae NCTC 11659 as a potential intervention to prevent stress-induced inflammation and neuroinflammation implicated in the etiology and pathophysiology of inflammatory conditions and stress-related psychiatric disorders.

Effects of Mycobacterium vaccae NCTC 11659 and Lipopolysaccharide Challenge on Polarization of Murine BV-2 Microglial Cells.

Previous studies have shown that the in vivo administration of soil-derived bacteria with anti-inflammatory and immunoregulatory properties, such as Mycobacterium vaccae NCTC 11659, can prevent a stress-induced shift toward an inflammatory M1 microglial immunophenotype and microglial priming in the central nervous system (CNS). It remains unclear whether M. vaccae NCTC 11659 can act directly on microglia to mediate these effects. This study was designed to determine the effects of M. vaccae NCTC 11659 on the polarization of naïve BV-2 cells, a murine microglial cell line, and BV-2 cells subsequently challenged with lipopolysaccharide (LPS). Briefly, murine BV-2 cells were exposed to 100 µg/mL whole-cell, heat-killed M. vaccae NCTC 11659 or sterile borate-buffered saline (BBS) vehicle, followed, 24 h later, by exposure to 0.250 µg/mL LPS (Escherichia coli 0111: B4; n = 3) in cell culture media vehicle (CMV) or a CMV control condition. Twenty-four hours after the LPS or CMV challenge, cells were harvested to isolate total RNA. An analysis using the NanoString platform revealed that, by itself, M. vaccae NCTC 11659 had an "adjuvant-like" effect, while exposure to LPS increased the expression of mRNAs encoding proinflammatory cytokines, chemokine ligands, the C3 component of complement, and components of inflammasome signaling such as Nlrp3. Among LPS-challenged cells, M. vaccae NCTC 11659 had limited effects on differential gene expression using a threshold of 1.5-fold change. A subset of genes was assessed using real-time reverse transcription polymerase chain reaction (real-time RT-PCR), including Arg1, Ccl2, Il1b, Il6, Nlrp3, and Tnf. Based on the analysis using real-time RT-PCR, M. vaccae NCTC 11659 by itself again induced "adjuvant-like" effects, increasing the expression of Il1b, Il6, and Tnf while decreasing the expression of Arg1. LPS by itself increased the expression of Ccl2, Il1b, Il6, Nlrp3, and Tnf while decreasing the expression of Arg1. Among LPS-challenged cells, M. vaccae NCTC 11659 enhanced LPS-induced increases in the expression of Nlrp3 and Tnf, consistent with microglial priming. In contrast, among LPS-challenged cells, although M. vaccae NCTC 11659 did not fully prevent the effects of LPS relative to vehicle-treated control conditions, it increased Arg1 mRNA expression, suggesting that M. vaccae NCTC 11659 induces an atypical microglial phenotype. Thus, M. vaccae NCTC 11659 acutely (within 48 h) induced immune-activating and microglial-priming effects when applied directly to murine BV-2 microglial cells, in contrast to its long-term anti-inflammatory and immunoregulatory effects observed on the CNS when whole-cell, heat-killed preparations of M. vaccae NCTC 11659 were given peripherally in vivo.

SARS-CoV-2 spike S1 subunit induces neuroinflammatory, microglial and behavioral sickness responses: Evidence of PAMP-like properties.

SARS-CoV-2 infection produces neuroinflammation as well as neurological, cognitive (i.e., brain fog), and neuropsychiatric symptoms (e.g., depression, anxiety), which can persist for an extended period (6 months) after resolution of the infection. The neuroimmune mechanism(s) that produces SARS-CoV-2-induced neuroinflammation has not been characterized. Proposed mechanisms include peripheral cytokine signaling to the brain and/or direct viral infection of the CNS. Here, we explore the novel hypothesis that a structural protein (S1) derived from SARS-CoV-2 functions as a pathogen-associated molecular pattern (PAMP) to induce neuroinflammatory processes independent of viral infection. Prior evidence suggests that the S1 subunit of the SARS-CoV-2 spike protein is inflammatory in vitro and signals through the pattern recognition receptor TLR4. Therefore, we examined whether the S1 subunit is sufficient to drive 1) a behavioral sickness response, 2) a neuroinflammatory response, 3) direct activation of microglia in vitro, and 4) activation of transgenic human TLR2 and TLR4 HEK293 cells. Adult male Sprague-Dawley rats were injected intra-cisterna magna (ICM) with vehicle or S1. In-cage behavioral monitoring (8 h post-ICM) demonstrated that S1 reduced several behaviors, including total activity, self-grooming, and wall-rearing. S1 also increased social avoidance in the juvenile social exploration test (24 h post-ICM). S1 increased and/or modulated neuroimmune gene expression (Iba1, Cd11b, MhcIIα, Cd200r1, Gfap, Tlr2, Tlr4, Nlrp3, Il1b, Hmgb1) and protein levels (IFNγ, IL-1ß, TNF, CXCL1, IL-2, IL-10), which varied across brain regions (hypothalamus, hippocampus, and frontal cortex) and time (24 h and 7d) post-S1 treatment. Direct exposure of microglia to S1 resulted in increased gene expression (Il1b, Il6, Tnf, Nlrp3) and protein levels (IL-1ß, IL-6, TNF, CXCL1, IL-10). S1 also activated TLR2 and TLR4 receptor signaling in HEK293 transgenic cells. Taken together, these findings suggest that structural proteins derived from SARS-CoV-2 might function independently as PAMPs to induce neuroinflammatory processes via pattern recognition receptor engagement.

Microglia: Neuroimmune-sensors of stress.

Exposure to stressors disrupts homeostasis and results in the release of stress hormones including glucocorticoids, epinepherine and norepinepherine. Interestingly, stress also has profound affects on microglia, which are tissue-resident macrophages in the brain parenchyma. Microglia express a diverse array of receptors, which also allows them to respond to stress hormones derived from peripheral as well as central sources. Here, we review studies of how exposure to acute and chronic stressors alters the immunophenotype and function of microglia. Further, we examine a causal for stress hormones in these effects of stress on microglia. We propose that microglia serve as immunosensors of the stress response, which puts them in the unique position to sense and respond rapidly to alterations in homeostasis and integrate the neural response to threats.

Comparing the effects of two different strains of mycobacteria, Mycobacterium vaccae NCTC 11659 and M. vaccae ATCC 15483, on stress-resilient behaviors and lipid-immune signaling in rats.

Stress-related disorders, such as posttraumatic stress disorder (PTSD), are highly prevalent and often difficult to treat. In rodents, stress-related, anxiety-like defensive behavioral responses may be characterized by social avoidance, exacerbated inflammation, and altered metabolic states. We have previously shown that, in rodents, subcutaneous injections of a heat-killed preparation of the soil-derived bacterium Mycobacterium vaccae NCTC 11659 promotes stress resilience effects that are associated with immunoregulatory signaling in the periphery and the brain. In the current study, we sought to determine whether treatment with a heat-killed preparation of the closely related M. vaccae type strain, M. vaccae ATCC 15483, would also promote stress-resilience in adult male rats, likely due to biologically similar characteristics of the two strains. Here we show that immunization with either M. vaccae NCTC 11659 or M. vaccae ATCC 15483 prevents stress-induced increases in hippocampal interleukin 6 mRNA expression, consistent with previous studies showing that M. vaccae NCTC 11659 prevents stress-induced increases in peripheral IL-6 secretion, and prevents exaggeration of anxiety-like defensive behavioral responses assessed 24 h after exposure to inescapable tail shock stress (IS) in adult male rats. Analysis of mRNA expression, protein abundance, and flow cytometry data demonstrate overlapping but also unique effects of treatment with the two M. vaccae strains on immunological and metabolic signaling in the host. These data support the hypothesis that treatment with different M. vaccae strains may immunize the host against stress-induced dysregulation of physiology and behavior.

Acute stress induces chronic neuroinflammatory, microglial and behavioral priming: A role for potentiated NLRP3 inflammasome activation.

Prior exposure to acute and chronic stressors potentiates the neuroinflammatory and microglial pro-inflammatory response to subsequent immune challenges suggesting that stressors sensitize or prime microglia. Stress-induced priming of the NLRP3 inflammasome has been implicated in this priming phenomenon, however the duration/persistence of these effects has not been investigated. In the present study, we examined whether exposure to a single acute stressor (inescapable tailshock) induced a protracted priming of the NLRP3 inflammasome as well as the neuroinflammatory, behavioral and microglial proinflammatory response to a subsequent immune challenge in hippocampus. In male Sprague-Dawley rats, acute stress potentiated the neuroinflammatory response (IL-1ß, IL-6, and NFκBIα) to an immune challenge (lipopolysaccharide; LPS) administered 8 days after stressor exposure. Acute stress also potentiated the proinflammatory cytokine response (IL-1ß, IL-6, TNF and NFκBIα) to LPS ex vivo. This stress-induced priming of microglia also was observed 28 days post-stress. Furthermore, challenge with LPS reduced juvenile social exploration, but not sucrose preference, in animals exposed to stress 8 days prior to immune challenge. Exposure to acute stress also increased basal mRNA levels of NLRP3 and potentiated LPS-induction of caspase-1 mRNA and protein activity 8 days after stress. The present findings suggest that acute stress produces a protracted vulnerability to the neuroinflammatory effects of subsequent immune challenges, thereby increasing risk for stress-related psychiatric disorders with an etiological inflammatory component. Further, these findings suggest the unique possibility that acute stress might induce innate immune memory in microglia.

Acute stress induces the rapid and transient induction of caspase-1, gasdermin D and release of constitutive IL-1ß protein in dorsal hippocampus.

The proinflammatory cytokine interleukin (IL)-1ß plays a pivotal role in the behavioral manifestations (i.e., sickness) of the stress response. Indeed, exposure to acute and chronic stressors induces the expression of IL-1ß in stress-sensitive brain regions. Thus, it is typically presumed that exposure to stressors induces the extra-cellular release of IL-1ß in the brain parenchyma. However, this stress-evoked neuroimmune phenomenon has not been directly demonstrated nor has the cellular process of IL-1ß release into the extracellular milieu been characterized in brain. This cellular process involves a form of inflammatory cell death, termed pyroptosis, which involves: 1) activation of caspase-1, 2) caspase-1 maturation of IL-1ß, 3) caspase-1 cleavage of gasdermin D (GSDMD), and 4) GSDMD-induced permeability of the cell membrane through which IL-1ß is released into the extracellular space. Thus, the present study examined whether stress induces the extra-cellular release of IL-1ß and engages the above cellular process in mediating IL-1ß release in the brain. Male Sprague-Dawley rats were exposed to inescapable tailshock (IS). IL-1ß extra-cellular release, caspase-1 activity and cleavage of GSDMD were measured in dorsal hippocampus. We found that exposure to IS induced a transient increase in the release of IL-1ß into the extracellular space immediately after termination of the stressor. IS also induced a transient increase in caspase-1 activity prior to IL-1ß release, while activation of GSDMD was observed immediately after termination of the stressor. IS also increased mRNA and protein expression of the ESCRTIII protein CHMP4B, which is involved in cellular repair. The present results suggest that exposure to an acute stressor induces the hallmarks of pyroptosis in brain, which might serve as a key cellular process involved in the release of IL-1ß into the extracellular milieu of the brain parenchyma.

Glucocorticoids mediate stress induction of the alarmin HMGB1 and reduction of the microglia checkpoint receptor CD200R1 in limbic brain structures.

Exposure to stressors primes neuroinflammatory responses to subsequent immune challenges and stress-induced glucocorticoids (GCs) play a mediating role in this phenomenon of neuroinflammatory priming. Recent evidence also suggests that the alarmin high-mobility group box-1 (HMGB1) and the microglial checkpoint receptor CD200R1 serve as proximal mechanisms of stress-induced neuroinflammatory priming. However, it is unclear whether stress-induced GCs play a causal role in these proximal mechanisms of neuroinflammatory priming; this forms the focus of the present investigation. Here, we found that exposure to a severe acute stressor (inescapable tailshock) induced HMGB1 and reduced CD200R1 expression in limbic brain regions and pharmacological blockade of GC signaling (RU486) mitigated these effects of stress. To confirm these effects of RU486, adrenalectomy (ADX) with basal corticosterone (CORT) replacement was used to block the stress-induced increase in GCs as well as effects on HMGB1 and CD200R1. As with RU486, ADX mitigated the effects of stress on HMGB1 and CD200R1. Subsequently, exogenous CORT was administered to determine whether GCs are sufficient to recapitulate the effects of stress. Indeed, exogenous CORT induced expression of HMGB1 and reduced expression of CD200R1. In addition, exposure of primary microglia to CORT also recapitulated the effects of stress on CD200R1 suggesting that CORT acts directly on microglia to reduce expression of CD200R1. Taken together, these findings suggest that GCs mediate the effects of stress on these proximal mechanisms of neuroinflammatory priming.

Pattern recognition receptors mediate pro-inflammatory effects of extracellular mitochondrial transcription factor A (TFAM).

Neuroinflammation is a common pathogenic mechanism for a number of neurodegenerative disorders including Alzheimer's and Parkinson's diseases. Microglia, the immune cells of the brain, contribute to the onset and progression of the neuroinflammation observed in these diseases. Microglia become activated and initiate an inflammatory response by interacting with a diverse set of molecules, including the group of endogenous proteins released upon cell damage, termed damage-associated molecular patterns (DAMPs). One of these molecules, mitochondrial transcription factor A (TFAM), has been shown to induce pro-inflammatory and cytotoxic responses of microglia in vitro. Here, we demonstrate that TFAM injected into the cisterna magna of male Sprague-Dawley rats upregulates (i) the expression of monocyte chemotactic protein (MCP)-1, interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α and nuclear factor-kappa B inhibitor alpha (NF-κBIA) in the hippocampus; (ii) the expression of MCP-1, IL-1ß and TNF-α in the frontal cortex; and (iii) IL-1ß protein concentration in both these brain regions. These same inflammatory mediators are upregulated in isolated rat microglia following their in vitro exposure to extracellular TFAM. Blocking the receptor for advanced glycation endproducts (RAGE) and the macrophage antigen complex (Mac)-1 by specific antibodies inhibited the TFAM-induced secretion of MCP-1 by THP-1 monocytic cells, which were used to model human microglia. Our data support the hypothesis that extracellular TFAM can interact with RAGE and Mac-1 to function as a DAMP that causes pro-inflammatory microglial activation. Blocking this interaction may represent a potential target for attenuating the neuroinflammation observed in neurodegenerative diseases.

Stress and aging act through common mechanisms to elicit neuroinflammatory priming.

Over the course of an animal's lifespan, there is a protracted breakdown in basic homeostatic functions. Stressors (both psychological and physiological) can accelerate this process and compromise multiple homeostatic mechanisms. For example, both stress and aging can modulate neuroinflammatory function and cause a primed phenotype resulting in a heightened neuroinflammatory profile upon immune activation. Microglia, the brain's resident myeloid cell, produce "silent" immune machinery in response to stress and aging that does not cause immediate immune activation; rather, these changes prime the cell for a subsequent immune insult. Primed microglia exhibit a hyperinflammatory response upon immune activation that can exacerbate pathology. In this review, we will explore parallels between stress- and aging-induced neuroinflammatory priming. First, we will provide a background on the basic principles of neuroimmunology. Next, we will discuss evidence that neuroinflammatory responses become primed in the context of both stress and aging. We will also describe cell-specific contributions to neuroinflammatory priming with a focus on microglia. Finally, common mechanisms underlying priming in the context of stress and aging will be discussed: these mechanisms include glucocorticoid signaling; accumulation of danger signals; dis-inhibition of microglia; and breakdown of circadian rhythms. Overall, there are multifarious parallels between stress- and aging-elicited neuroinflammatory priming, suggesting that stress may promote a form of premature aging. Further unravelling mechanisms underlying priming could lead to improved treatments for buffering against stress- and aging-elicited behavioral pathologies.

Stress disinhibits microglia via down-regulation of CD200R: A mechanism of neuroinflammatory priming.

Exposure to stressors primes the neuroinflammatory and microglial proinflammatory response to subsequent immune challenges, suggesting that stress might attenuate immunoregulatory mechanisms in the CNS microenvironment. CD200:CD200R is a key immunoregulatory signaling dyad that constrains microglial activation, and disruption of CD200:CD200R signaling primes microglia to subsequent immune challenges. Therefore, the present study examined the mediating role of CD200:CD200R signaling in stress-induced microglial priming. Here, we found that exposure to an acute stressor reduced CD200R expression across sub-regions of the hippocampus, amygdala as well as in isolated hippocampal microglia. A transcriptional suppressor of CD200R, CAAT/Enhancer Binding Proteinß, was induced by stress and inversely associated with CD200R expression. To examine whether disrupted CD200:CD200R signaling plays a mediating role in stress-induced microglial priming, a soluble fragment of CD200 (mCD200Fc) was administered intra-cisterna magna prior to stressor exposure and stress-induced microglia priming assessed ex vivo 24 h later. Treatment with mCD200Fc blocked the stress-induced priming of the microglial pro-inflammatory response. Further, treatment with mCD200R1Fc recapitulated the effects of stress on microglial priming. We previously found that stress increases the alarmin high mobility group box-1 (HMGB1) in hippocampus, and that HMGB1 mediates stress-induced priming of microglia. Thus, we examined whether stress-induced increases in hippocampal HMGB1 are a consequence of disrupted CD200:CD200R signaling. Indeed, treatment with mCD200Fc prior to stress exposure blocked the stress-induced increase in hippocampal HMGB1. The present study suggests that stress exposure disrupts immunoregulatory mechanisms in the brain, which typically constrain the immune response of CNS innate immune cells. This attenuation of immunoregulatory mechanisms may thus permit a primed activation state of microglia to manifest.

A novel platform for in vivo detection of cytokine release within discrete brain regions.

Mounting evidence indicates that cytokines secreted by innate immune cells in the brain play a central role in regulating neural circuits that subserve mood, cognition, and sickness responses. A major impediment to the study of neuroimmune signaling in healthy and disease states is the absence of tools for in vivo detection of cytokine release in the brain. Here we describe the design and application of a cytokine detection device capable of serial monitoring of local cytokine release in discrete brain regions. The immunocapture device consisted of a modified optical fiber labeled with a capture antibody specific for the pro-inflammatory cytokine interleukin-1 beta (IL-1ß). Using a sandwich immunoassay method, in vitro data demonstrate that the sensing interface of the modified optical fiber has a linear detection range of 3.9 pg mL-1-500 pg mL-1 and spatial resolution on the order of 200-450 µm. Finally, we show that the immunocapture device can be introduced into a perforated guide cannula for repeated analyte measurements in vivo. An increase in fluorescence detection of spatially localized intrahippocampal IL-1ß release was observed following a peripheral lipopolysaccharide challenge in Sprague-Dawley rats. This novel immunosensing technology represents an opportunity for unlocking the function of neuroimmune signaling.

Immunization with Mycobacterium vaccae induces an anti-inflammatory milieu in the CNS: Attenuation of stress-induced microglial priming, alarmins and anxiety-like behavior.

Exposure to stressors induces anxiety- and depressive-like behaviors, which are mediated, in part, by neuroinflammatory processes. Recent findings demonstrate that treatment with the immunoregulatory and anti-inflammatory bacterium, Mycobacterium vaccae (M. vaccae), attenuates stress-induced exaggeration of peripheral inflammation and stress-induced anxiety-like behavioral responses. However, the effects of M. vaccae on neuroimmune processes have largely been unexplored. In the present study, we examined the effect of M. vaccae NCTC11659 on neuroimmune regulation, stress-induced neuroinflammatory processes and anxiety-like behavior. Adult male rats were immunized 3× with a heat-killed preparation of M. vaccae (0.1 mg, s.c.) or vehicle. M. vaccae induced an anti-inflammatory immunophenotype in hippocampus (increased interleukin (Il)4, Cd200r1, and Mrc1 mRNA expression) and increased IL4 protein 8 d after the last immunization. Central administration of recombinant IL4 recapitulated the effects of M. vaccae on Cd200r1 and Mrc1 mRNA expression. M. vaccae reduced basal levels of genes (Nlrp3 and Nfkbia) involved in microglial priming; thus, we explored the effects of M. vaccae on stress-induced hippocampal microglial priming and HMGB1, which mediates priming. We found that M. vaccae blocked stress-induced decreases in Cd200r1, increases in the alarmin HMGB1, and priming of the microglial response to immune challenge. Furthermore, M. vaccae prevented stress-induced increases in anxiety-like behavior. The present findings suggest that M. vaccae enhances immunomodulation in the CNS and mitigates the neuroinflammatory and behavioral effects of stress, which may underpin its capacity to impart a stress resilient phenotype.

Neuroinflammatory priming to stress is differentially regulated in male and female rats.

Exposure to stressors can enhance neuroinflammatory responses, and both stress and neuroinflammation are predisposing factors in the development of psychiatric disorders. Females suffer disproportionately more from several psychiatric disorders, yet stress-induced changes in neuroinflammation have primarily been studied in males. Here we tested whether exposure to inescapable tail shock sensitizes or 'primes' neuroinflammatory responses in male and female rats. At 24 h post-stress, male and female rats exposed to a peripheral immune challenge enhanced neuroinflammatory responses and exacerbated anxiety- and depressive-like behaviors. These changes are likely glucocorticoid dependent, as administering exogenous CORT, caused a similar primed inflammatory response in the hippocampus of male and female rats. Further, stress disinhibited anti-inflammatory signaling mechanisms (such as CD200R) in the hippocampus of male and female rats. In males, microglia are considered the likely cellular source mediating neuroinflammatory priming; stress increased cytokine expression in ex vivo male microglia. Conversely, microglia isolated from stressed or CORT treated females did not exhibit elevated cytokine responses. Microglia isolated from both stressed male and female rats reduced phagocytic activity; however, suggesting that microglia from both sexes experience stress-induced functional impairments. Finally, an immune challenge following either stress or CORT in females, but not males, increased peripheral inflammation (serum IL-1ß). These novel data suggest that although males and females both enhance stress-induced neuroinflammatory and behavioral responses to an immune challenge, this priming may occur through distinct, sex-specific mechanisms.

The Alarmin HMGB1 Mediates Age-Induced Neuroinflammatory Priming.

UNLABELLED: Amplified neuroinflammatory responses following an immune challenge occur with normal aging and can elicit or exacerbate neuropathology. The mechanisms mediating this sensitized or "primed" immune response in the aged brain are not fully understood. The alarmin high mobility group box 1 (HMGB1) can be released under chronic pathological conditions and initiate inflammatory cascades. This led us to investigate whether HMGB1 regulates age-related priming of the neuroinflammatory response. Here, we show that HMGB1 protein and mRNA were elevated in the hippocampus of unmanipulated aged rats (24-month-old F344XBN rats). Furthermore, aged rats had increased HMGB1 in the CSF, suggesting increased HMGB1 release. We demonstrate that blocking HMGB1 signaling with an intracisterna magna (ICM) injection of the competitive antagonist to HMGB1, Box-A, downregulates basal expression of several inflammatory pathway genes in the hippocampus of aged rats. This indicates that blocking the actions of HMGB1 might reduce age-associated inflammatory priming. To test this hypothesis, we evaluated whether HMGB1 antagonism blocks the protracted neuroinflammatory and sickness response to peripheral Escherichia coli (E. coli) infection in aged rats. ICM pretreatment of aged rats with Box-A 24 h before E. coli infection prevented the extended hippocampal cytokine response and associated cognitive and affective behavioral changes. ICM pretreatment with Box-A also inhibited aging-induced potentiation of the microglial proinflammatory response to lipopolysaccharide ex vivo Together, these results suggest that HMGB1 mediates neuroinflammatory priming in the aged brain. Blocking the actions of HMGB1 appears to "desensitize" aged microglia to an immune challenge, thereby preventing exaggerated behavioral and neuroinflammatory responses following infection. SIGNIFICANCE STATEMENT: The world's population is aging, highlighting a need to develop treatments that promote quality of life in aged individuals. Normal aging is associated with precipitous drops in cognition, typically following events that induce peripheral inflammation (e.g., infection, surgery, heart attack). Peripheral immune stimuli cause exaggerated immune responses in the aged brain, which likely underlie these behavioral deficits. Here, we investigated whether the alarmin high mobility group box 1 (HMGB1) mediates age-associated "priming" of the neuroinflammatory response. HMGB1 is elevated in aged rodent brain and CSF. Blocking HMGB1 signaling downregulated expression of inflammatory pathway genes in aged rat brain. Further, HMGB1 antagonism prevented prolonged infection-induced neuroinflammatory and sickness responses in aged rats. Overall, blocking HMGB1 "desensitized" microglia in the aged brain, thereby preventing pathological infection-elicited neuroinflammatory responses.