Microglia depletion ameliorates neuroinflammation, anxiety-like behavior, and cognitive deficits in a sex-specific manner in Rev-erbα knockout mice.

The circadian system is an evolutionarily adaptive system that synchronizes biological and physiological activities within the body to the 24 h oscillations on Earth. At the molecular level, circadian clock proteins are transcriptional factors that regulate the rhythmic expression of genes involved in numerous physiological processes such as sleep, cognition, mood, and immune function. Environmental and genetic disruption of the circadian clock can lead to pathology. For example, global deletion of the circadian clock gene Rev-erbα (RKO) leads to hyperlocomotion, increased anxiety-like behaviors, and cognitive impairments in male mice; however, the mechanisms underlying behavioral changes remain unclear. Here we hypothesized that RKO alters microglia function leading to neuroinflammation and altered mood and cognition, and that microglia depletion can resolve neuroinflammation and restore behavior. We show that microglia depletion (CSF1R inhibitor, PLX5622) in 8-month-old RKO mice ameliorated hyperactivity, memory impairments, and anxiety/risky-like behaviors. RKO mice exhibited striking increases in expression of pro-inflammatory cytokines (e.g., IL-1ß and IL-6). Surprisingly, these increases were only fully reversed by microglia depletion in the male but not female RKO hippocampus. In contrast, male RKO mice showed greater alterations in microglial morphology and phagocytic activity than females. In both sexes, microglia depletion reduced microglial branching and decreased CD68 production without altering astrogliosis. Taken together, we show that male and female RKO mice exhibit unique perturbations to the neuroimmune system, but microglia depletion is effective at rescuing aspects of behavioral changes in both sexes. These results demonstrate that microglia are involved in Rev-erbα-mediated changes in behavior and neuroinflammation.

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.

Sex differences in microglia function in aged rats underlie vulnerability to cognitive decline.

Aging is associated with a significant shift in immune system reactivity ("inflammaging"), as basal inflammation increases but protective responses to infection are compromised. The immune system exhibits considerable sex differences, which may influence the process of inflammaging, including immune cell activation and behavioral consequences of immune signaling (i.e., impaired memory). Here, we test the hypothesis that sex differences in immune aging may mediate sex differences in cognitive decline. Aged male and female rats received peripheral immune stimulation using lipopolysaccharide (LPS), then molecular, cellular, and behavioral outcomes were assessed. We observed that LPS-treated aged male rats showed cognitive impairment and increased neuroinflammatory responses relative to adult males. In contrast, aged female rats did not display these aging-related deficits. Using transcriptomic and flow cytometry analyses, we further observed significant age- and sex- dependent changes in immune cell populations in the brain parenchyma and meninges, indicating a broad shift in the neuroinflammatory environment that may potentiate these behavioral effects. Ovariectomized aged female rats were also resistant to inflammation-induced memory deficits, indicating that ovarian hormones are not required for the attenuated neuroinflammation in aged females. Overall, our results indicate that males have amplified inflammatory priming with age, which contributes to age-associated cognitive decline. Our findings highlight sexual dimorphism in mechanisms of aging, and suggest that sex is a crucial consideration for identifying therapies for aging and neuroinflammation.

Distinct immune and transcriptomic profiles in dominant versus subordinate males in mouse social hierarchies.

Social status is a critical factor determining health outcomes in human and nonhuman social species. In social hierarchies with reproductive skew, individuals compete to monopolize resources and increase mating opportunities. This can come at a significant energetic cost leading to trade-offs between different physiological systems. In particular, changes in energetic investment in the immune system can have significant short and long-term effects on fitness and health. We have previously found that dominant alpha male mice living in social hierarchies have increased metabolic demands related to territorial defense. In this study, we tested the hypothesis that high-ranking male mice favor adaptive immunity, while subordinate mice show higher investment in innate immunity. We housed 12 groups of 10 outbred CD-1 male mice in a social housing system. All formed linear social hierarchies and subordinate mice had higher concentrations of plasma corticosterone (CORT) than alpha males. This difference was heightened in highly despotic hierarchies. Using flow cytometry, we found that dominant status was associated with a significant shift in immunophenotypes towards favoring adaptive versus innate immunity. Using Tag-Seq to profile hepatic and splenic transcriptomes of alpha and subordinate males, we identified genes that regulate metabolic and immune defense pathways that are associated with status and/or CORT concentration. In the liver, dominant animals showed a relatively higher expression of specific genes involved in major urinary production and catabolic processes, whereas subordinate animals showed relatively higher expression of genes promoting biosynthetic processes, wound healing, and proinflammatory responses. In spleen, subordinate mice showed relatively higher expression of genes facilitating oxidative phosphorylation and DNA repair and CORT was negatively associated with genes involved in lymphocyte proliferation and activation. Together, our findings suggest that dominant and subordinate animals adaptively shift immune profiles and peripheral gene expression to match their contextual needs.

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.

Aging and miR-155 in mice influence survival and neuropathic pain after spinal cord injury.

Spinal cord injury (SCI) elicits chronic pain in 65% of individuals. In addition, SCI afflicts an increasing number of aged individuals, and those with SCI are predisposed to shorter lifespan. Our group previously identified that deletion of the microRNA miR-155 reduced neuroinflammation and locomotor deficits after SCI. Here, we hypothesized that aged mice would be more susceptible to pain symptoms and death soon after SCI, and that miR-155 deletion would reduce pain symptoms in adult and aged mice and improve survival. Adult (2 month-old) and aged (20 month-old) female wildtype (WT) and miR-155 knockout (KO) mice received T9 contusion SCI. Aged WT mice displayed reduced survival and increased autotomy - a symptom of spontaneous pain. In contrast, aged miR-155 KO mice after SCI were less susceptible to death or spontaneous pain. Evoked pain symptoms were tested using heat (Hargreaves test) and mechanical (von Frey) stimuli. At baseline, aged mice showed heightened heat sensitivity. After SCI, adult and aged WT and miR-155 KO mice all exhibited heat and mechanical hypersensitivity at all timepoints. miR-155 deletion in adult (but not aged) mice reduced mechanical hypersensitivity at 7 and 14 d post-SCI. Therefore, aging predisposes mice to SCI-elicited spontaneous pain and expedited mortality. miR-155 deletion in adult mice reduces evoked pain symptoms, and miR-155 deletion in aged mice reduces spontaneous pain and expedited mortality post-SCI. This study highlights the importance of studying geriatric models of SCI, and that inflammatory mediators such as miR-155 are promising targets after SCI for improving pain relief and longevity.

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.

Circadian regulation of depression: A role for serotonin.

Synchronizing circadian (24 h) rhythms in physiology and behavior with the environmental light-dark cycle is critical for maintaining optimal health. Dysregulation of the circadian system increases susceptibility to numerous pathological conditions including major depressive disorder. Stress is a common etiological factor in the development of depression and the circadian system is highly interconnected to stress-sensitive neurotransmitter systems such as the serotonin (5-hydroxytryptamine, 5-HT) system. Thus, here we propose that stress-induced perturbation of the 5-HT system disrupts circadian processes and increases susceptibility to depression. In this review, we first provide an overview of the basic components of the circadian system. Next, we discuss evidence that circadian dysfunction is associated with changes in mood in humans and rodent models. Finally, we provide evidence that 5-HT is a critical factor linking dysregulation of the circadian system and mood. Determining how these two systems interact may provide novel therapeutic targets for depression.

Dim light at night exacerbates stroke outcome.

Circadian rhythms are endogenous biological cycles that synchronize physiology and behaviour to promote optimal function. These ~24-hr internal rhythms are set to precisely 24 hr daily by exposure to the sun. However, the prevalence of night-time lighting has the potential to dysregulate these biological functions. Hospital patients may be particularly vulnerable to the consequences of light at night because of their compromised physiological state. A mouse model of stroke (middle cerebral artery occlusion; MCAO) was used to test the hypothesis that exposure to dim light at night impairs responses to a major insult. Stroke lesion size was substantially larger among animals housed in dLAN after reperfusion than animals maintained in dark nights. Mice housed in dLAN for three days after the stroke displayed increased post-stroke anxiety-like behaviour. Overall, dLAN amplified pro-inflammatory pathways in the CNS, which may have exacerbated neuronal damage. Our results suggest that exposure to LAN is detrimental to stroke recovery.

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.

The behavioral and neurochemical effects of an inescapable stressor are time of day dependent.

Circadian rhythms are ∼24 h fluctuations in physiology and behavior that are synchronized with the light-dark cycle. The circadian system ensures homeostatic balance by regulating multiple systems that respond to environmental stimuli including stress systems. In rats, acute exposure to a series of uncontrollable tailshocks (inescapable stress, IS) produces an anxiety and depression-like phenotype. Anxiety- and fear-related behavioral changes produced by IS are driven by sensitization of serotonergic (5-hydroxytryptamine, 5-HT) neurons in the dorsal raphe nucleus (DRN). Because the circadian and serotonergic systems are closely linked, here we tested whether the DRN-dependent behavioral and neurochemical effects of IS are time of day dependent. Exposure to IS during the light (inactive) phase elicited the expected changes in mood related behaviors. In contrast, rats that underwent IS during the dark (active) phase were buffered against stress-induced changes in juvenile social exploration and shock-elicited freezing, both DRN-dependent outcomes. Interestingly, behavioral anhedonia, which is not a DRN-dependent behavior, was comparably reduced by stress at both times of day. Neurochemical changes complimented the behavioral results: IS-induced activation of DRN 5-HT neurons was greater during the light phase compared to the dark phase. Additionally, 5-HT1AR and 5-HTT, two genes that regulate 5-HT activity were up-regulated during the middle of the light cycle. These data suggest that DRN-dependent behavioral outcomes of IS are time of day dependent and may be mediated by circadian gating of the DRN response to stress.Lay summaryHere we show that the time of day at which a stressor occurs impacts the behavioral and neurochemical outcomes of the stressor. In particular, animals appear more vulnerable to a stressor that occurs during their rest phase. This work may have important implications for shift-workers and other populations that are more likely to encounter stressors during their rest phase.

Endocrine Effects of Circadian Disruption.

Disruption of circadian rhythms, provoked by artificial lighting at night, inconsistent sleep-wake schedules, and transmeridian air travel, is increasingly prevalent in modern society. Desynchrony of biological rhythms from environmental light cycles has dramatic consequences for human health. In particular, disrupting homeostatic oscillations in endocrine tissues and the hormones that these tissues regulate can have cascading effects on physiology and behavior. Accumulating evidence suggests that chronic disruption of circadian organization of endocrine function may lead to metabolic, reproductive, sleep, and mood disorders. This review discusses circadian control of endocrine systems and the consequences of distorting rhythmicity of these systems.

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.

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.

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.

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.

The redox state of the alarmin HMGB1 is a pivotal factor in neuroinflammatory and microglial priming: A role for the NLRP3 inflammasome.

The alarmin high mobility group box-1 (HMGB1) has been implicated as a key factor mediating neuroinflammatory processes. Recent findings suggest that the redox state of HMGB1 is a critical molecular feature of HMGB1 such that the reduced form (fr-HMGB1) is chemotactic, while the disulfide form (ds-HMGB1) is pro-inflammatory. The present study examined the neuroinflammatory effects of these molecular forms as well as the ability of these forms to prime the neuroinflammatory and microglial response to an immune challenge. To examine the neuroinflammatory effects of these molecular forms in vivo, animals were administered intra-cisterna magna (ICM) a single dose of fr-HMGB1 (10µg), ds-HMGB1 (10µg) or vehicle and basal pro-inflammatory effects were measured 2 and 24h post-injection in hippocampus. Results of this initial experiment demonstrated that ds-HMGB1 increased hippocampal pro-inflammatory mediators at 2h (NF-κBIα mRNA, NLRP3 mRNA and IL-1ß protein) and 24h (NF-κBIα mRNA, TNFα mRNA, and NLRP3 protein) after injection. fr-HMGB1 had no effect on these mediators. These neuroinflammatory effects of ds-HMGB1 suggested that ds-HMGB1 may function to prime the neuroinflammatory response to a subsequent immune challenge. To assess the neuroinflammatory priming effects of these molecular forms, animals were administered ICM a single dose of fr-HMGB1 (10µg), ds-HMGB1 (10µg) or vehicle and 24h after injection, animals were challenged with LPS (10µg/kg IP) or vehicle. Neuroinflammatory mediators and the sickness response (3, 8 and 24h after injection) were measured 2h after immune challenge. We found that ds-HMGB1 potentiated the neuroinflammatory (NF-κBIα mRNA, TNFα mRNA, IL-1ß mRNA, IL-6 mRNA, NLRP3 mRNA and IL-1ß protein) and sickness response (reduced social exploration) to LPS challenge. fr-HMGB1 failed to potentiate the neuroinflammatory response to LPS. To examine whether these molecular forms of HMGB1 directly induce neuroinflammatory effects in isolated microglia, whole brain microglia were isolated and treated with fr-HMGB1 (0, 1, 10, 100, or 1000ng/ml) or ds-HMGB1 (0, 1, 10, 100, or 1000ng/ml) for 4h and pro-inflammatory mediators measured. To assess the effects of these molecular forms on microglia priming, whole brain microglia were pre-exposed to these forms of HMGB1 (0, 1, 10, 100, or 1000ng/ml) and subsequently challenged with LPS (10ng/ml). We found that ds-HMGB1 increased expression of NF-κBIα mRNA and NLRP3 mRNA in isolated microglia, and potentiated the microglial pro-inflammatory response (TNFα mRNA, IL-1ß mRNA and IL-1ß protein) to LPS. fr-HMGB1 failed to potentiate the microglial pro-inflammatory response to LPS. Consistent with prior reports, the present findings demonstrate that the disulfide form of HMGB1 not only potentiates the neuroinflammatory response to a subsequent immune challenge in vivo, but also potentiates the sickness response to that challenge. Moreover, the present findings demonstrate for the first time that ds-HMGB1 directly potentiates the microglia pro-inflammatory response to an immune challenge, a finding that parallels the effects of ds-HMGB1 in vivo. In addition, ds-HMGB1 induced expression of NLRP3 and NF-κBIα in vivo and in vitro suggesting that the NLRP3 inflammasome may play role in the priming effects of ds-HMGB1. Taken together, the present results suggest that the redox state of HMGB1 is a critical determinant of the priming properties of HMGB1 such that the disulfide form of HMGB1 induces a primed immunophenotype in the CNS, which may result in an exacerbated neuroinflammatory response upon exposure to a subsequent pro-inflammatory stimulus.

Microglia inflammatory responses are controlled by an intrinsic circadian clock.

The circadian system regulates many physiological functions including inflammatory responses. For example, mortality caused by lipopolysaccharide (LPS) injection varies depending on the time of immunostimulation in mammals. The effects of more subtle challenges on the immune system and cellular mechanisms underlying circadian differences in neuroinflammatory responses are not well understood. Here we show that adult male Sprague-Dawley rats injected with a sub-septic dose of LPS during the light phase displayed elevated sickness behaviors and hippocampal cytokine production compared to rats injected during the dark phase. Microglia are the primary central nervous system (CNS) immune cell type and may mediate diurnal differences in sickness response, thus we explored whether microglia demonstrate temporal variations in inflammatory factors. Hippocampal microglia isolated from adult rats rhythmically expressed inflammatory factors and circadian clock genes. Microglia displayed robust rhythms of TNFα, IL1ß and IL6 mRNA, with peak cytokine gene expression occurring during the middle of the light phase. Microglia isolated during the light phase were also more reactive to immune stimulation; such that, ex vivo LPS treatment induced an exaggerated cytokine response in light phase-isolated microglia. Treating microglia with corticosterone ex vivo induced expression of the circadian clock gene Per1. However, microglia isolated from adrenalectomized rats maintained temporal differences in clock and inflammatory gene expression. This suggests circadian clock gene expression in microglia is entrained by, but oscillates in the absence of, glucocorticoids. Taken together, these findings demonstrate that microglia possess a circadian clock that influences inflammatory responses. These results indicate time-of-day is an important factor to consider when planning inflammatory interventions such as surgeries or immunotherapies.