Microglial Priming Research Articles

Microglia maintain the normal structure and function of the hippocampal astrocyte network

Microglial control of activity‐dependent plasticity and synaptic remodeling in neuronal networks has been the subject of intense research in the past several years. Although microglia–neuron interactions have been extensively studied, less is known about how microglia influence astrocyte‐dependent control over neuronal structure and function. Here, we explored a role for microglia in regulating the structure and function of the astrocyte syncytium in mouse hippocampus. After depleting microglia using a CSF1R antagonist (PLX5622, Plexxikon), we observed severe disruption of astrocyte syncytial isopotentiality and dye coupling. A decrease in astrocyte‐specific gap junction connexin (Cx) 30 and 43 expression, at least partially accounts for these microglia‐dependent changes in astrocytes. Because neuronal function requires intact astrocyte coupling, we also evaluated the effects of microglia depletion on synaptic transmission in the hippocampus. Without microglia, the strength of synaptic transmission was reduced at baseline and after long‐term potentiation (LTP). Conversely, priming microglia with systemic injections of lipopolysaccharide enhanced CA3‐CA1 synaptic transmission. This microglia‐induced scaling of synaptic transmission was associated with increased expression of post‐synaptic scaffold proteins (Homer1) in CA1. However, astrocyte network function was not affected by microglia priming, indicating that microglia‐dependent effects on astrocytes and neurons vary across functional states. Through manipulation of microglia in the brain, our results reveal the importance of microglia in homeostatic regulation of the astrocyte syncytium and scaling of synaptic transmission. These novel mechanisms uncover a new direction for future studies interrogating microglia function in various physiological and pathological contexts.

Luteolin ameliorates depression-like behaviors by suppressing ER stress in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) is the most common cause of dementia in the elderly population. Inflammation plays an important role in AD, as microglia respond to several pathological insults, such as Aβ, and exert protective homeostatic functions (anti-inflammatory) and detrimental inflammatory functions (proinflammatory). During the development of AD, chronic inflammation that accompanies aging causes microglial priming, a state of hyperactivation in response to stimulation, indicating that suppressing microglial priming may be a therapeutic intervention for AD. Endoplasmic reticulum (ER) stress is crucial for inflammation through NF-kB and inflammasome activation. To identify natural flavonoids that regulate ER stress, a DNA microarray was performed using the brains of AD model mice after long-term intake of quercetin, after which the connectivity map (CMap) assay was carried out. We found that luteolin suppresses lipopolysaccharide (LPS)-induced interleukin 1β (IL1β) expression by inhibiting ER stress. Immunohistochemical analyses showed that CD68 levels were reduced in the brain after intraperitoneal injection of luteolin in a mouse model of AD, suppressing IL1β production. As shown by behavioral analyses using the tail suspension test (TST) and forced swimming test (FST), depression-like behaviors were ameliorated in luteolin-treated AD model mice. These findings indicate that luteolin prevents ER stress to suppress microglial activation in the brain, improving individual activity.

Biological and Psychological Factors Determining Neuropsychiatric Outcomes in COVID-19.

Purpose of ReviewWe present biological and psychological factors implicated in psychiatric manifestations of SARS-CoV-2, as well as its neuroinvasive capability and immune pathophysiology.Recent FindingsPreexisting mental illness leads to worse clinical outcomes in COVID-19. The presence of the virus was reported in the cerebrospinal fluid (CSF) and brain tissue post-mortem. Most common psychiatric manifestations include delirium, mood disorders, anxiety disorders, and posttraumatic stress disorder. “Long-COVID” non-syndromal presentations include “brain-fogginess,” autonomic instability, fatigue, and insomnia. SummarySARS-CoV-2 infection can trigger prior vulnerabilities based on the priming of microglia and other cells, induced or perpetuated by aging and mental and physical illnesses. COVID-19 could further induce priming of neuroimmunological substrates leading to exacerbated immune response and autoimmunity targeting structures in the central nervous system (CNS), in response to minor immune activating environmental exposures, including stress, minor infections, allergens, pollutants, and traumatic brain injury.

Lipopolysaccharide-induced maternal immune activation modulates microglial CX3CR1 protein expression and morphological phenotype in the hippocampus and dentate gyrus, resulting in cognitive inflexibility during late adolescence

Inflammation during pregnancy can disturb brain development and lead to disorders in the progeny, including autism spectrum disorder and schizophrenia. However, the mechanism by which a prenatal, short-lived increase of cytokines results in adverse neurodevelopmental outcomes remains largely unknown. Microglia—the brain’s resident immune-cells—stand as fundamental cellular mediators, being highly sensitive and responsive to immune signals, which also play key roles during normal development.The fractalkine signaling axis is a neuron-microglia communication mechanism used to regulate neurogenesis and network formation. Previously, we showed hippocampal reduction of fractalkine receptor (Cx3cr1) mRNA at postnatal day (P) 15 in male offspring exposed to maternal immune activation induced with lipopolysaccharide (LPS) during late gestation, which was concomitant to an increased dendritic spine density in the dentate gyrus, a neurogenic niche. The current study sought to evaluate the origin and impact of this reduced hippocampal Cx3cr1 mRNA expression on microglia and cognition. We found that microglial total cell number and density are not affected in the dorsal hippocampus and dentate gyrus, respectively, but that the microglial CX3CR1 protein is decreased in the hippocampus of LPS-male offspring at P15. Further characterization of microglial morphology in the dentate gyrus identified a more ameboid phenotype in LPS-exposed offspring, predominantly in males, at P15. We thus explored maternal plasma and fetal brain cytokines to understand the mechanism behind microglial priming, showing a robust immune activation in the mother at 2 and 4 hrs after LPS administration, while only IL-10 tended towards upregulation at 2 hrs after LPS in fetal brains. To evaluate the functional long-term consequences, we assessed learning and cognitive flexibility behavior during late adolescence, finding that LPS affects only the latter with a male predominance on perseveration. A CX3CR1 gene variant in humans that results in disrupted fractalkine signaling has been recently associated with an increased risk for neurodevelopmental disorders. We show that an acute immune insult during late gestation can alter fractalkine signaling by reducing the microglial CX3CR1 protein expression, highlighting neuron-microglial fractalkine signaling as a relevant target underlying the outcomes of environmental risk factors on neurodevelopmental disorders.

Microglia react to partner loss in a sex- and brain site-specific manner in prairie voles

Positive social relationships are paramount for the survival of mammals and beneficial for mental and physical health, buffer against stressors, and even promote appropriate immune system functioning. By contrast, impaired social relationships, social isolation, or the loss of a bonded partner lead to aggravated physical and mental health. For example, in humans partner loss is detrimental for the functioning of the immune system and heightens the susceptibility for the development of post-traumatic stress disorders, anxiety disorders, and major depressive disorders. To understand potential underlying mechanisms, the monogamous prairie vole can provide important insights. In the present study, we separated pair bonded male and female prairie voles after five days of co-housing, subjected them to the forced swim test on the fourth day following separation, and studied their microglia morphology and activation in specific brain regions. Partner loss increased passive stress-coping in male, but not female, prairie voles. Moreover, partner loss was associated with microglial priming within the parvocellular region of the paraventricular nucleus of the hypothalamus (PVN) in male prairie voles, whereas in female prairie voles the morphological activation within the whole PVN and the prelimbic cortex (PrL) was decreased, marked by a shift towards ramified microglial morphology. Expression of the immediate early protein c-Fos following partner loss was changed within the PrL of male, but not female, prairie voles. However, the loss of a partner did not affect the investigated aspects of the peripheral immune response. These data suggest a potential sex-dependent mechanism for the regulation of microglial activity following the loss of a partner, which might contribute to the observed differences in passive stress-coping. This study furthers our understanding of the effects of partner loss and its short-term impact on the CNS as well as the CNS immune system and the peripheral innate immune system in both male and female prairie voles.

Rapamycin Modulates the Proinflammatory Memory-Like Response of Microglia Induced by BAFF.

BackgroundRecently trained immunity of microglia provided an opportunity to study the chronic effect of microglial activation and its metabolic rewiring in neuroimmunological diseases. Since elevated levels of B cell-activating factor (BAFF) have been proved to be associated with some chronic neuroimmunological disorders. Here, we used the trained innate immunity model to analyze the effect of BAFF, a vital regulator of the adaptive immune system, on long-term microglial activation and metabolic reprogramming in vitro and in vivo.Methods and resultsIn vitro, BV2 cells and mouse primary microglial cells were incubated with BAFF for 24 h (BAFF priming). After 5 days of resting, microglia were restimulated with LPS (LPS restimulation) or BAFF (BAFF restimulation). BAFF priming induced a pro-inflammatory trained immunity-phenotype of both BV2 cells and primary microglial cells, which was indicated by morphological change, secretion of pro-inflammatory cytokine and chemokine upon LPS restimulation or BAFF restimulation. The production of lactate and NAD+/NADH ratio were elevated 5 days after BAFF priming. The activation of the Akt/mTOR/HIF-1α pathway was induced by BAFF priming and lasted for 5 days. Pretreating the BV2 cells or mouse primary microglial cells with rapamycin blocked mTOR/HIF-1α activation and cellular metabolic reprogramming induced by BAFF training. Consistently, rapamycin efficiently suppressed the trained immunity-like responses of microglia triggered by BAFF. In vivo, adult male mice were treated with BAFF by intracerebroventricular injection for priming and 7 days later with BAFF for restimulation. BAFF training activated microglia in the cortex and hippocampus. The production of proinflammatory cytokines and chemokines was elevated after BAFF training.ConclusionOur current data, for the first time, demonstrate that BAFF priming induces a proinflammatory memory-like response of microglia not only to LPS but also to BAFF itself. Rapamycin inhibits microglial priming triggered by BAFF through targeting the mTOR/HIF-1α signaling pathway. Our data reveal a novel role of BAFF in trained immunity and that rapamycin may be a potential therapeutic target of neuroimmunological diseases.

Papaverine Exerts Neuroprotective Effect by Inhibiting NLRP3 Inflammasome Activation in an MPTP-Induced Microglial Priming Mouse Model Challenged with LPS.

Microglial priming is the process of microglial proliferation and activation in response to neurodegeneration and abnormal protein accumulation. Priming makes microglia susceptible to secondary inflammatory stimuli and causes exaggerated inflammatory responses. In the present study, we established a microglial priming model in mice by administering a single injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 20 mg/kg). MPTP induced microglial activation without dopaminergic degeneration; however, subsequent treatment with a sub-toxic dose of lipopolysaccharides (LPS) induced an amplified inflammatory response and caused nigrostriatal dopaminergic degeneration. These pathological and inflammatory changes, including microglial activation and dopaminergic cell loss in the substantia nigra (SN) area were reversed by papaverine (PAP) administration. In addition, MPTP/LPS enhanced interleukin-1β (IL-1β) expression and processing via nod-like receptor protein 3 (NLRP3) inflammasome activation in the SN region of mice. However, PAP treatment suppressed inflammasome activation and subsequent IL-1β maturation. Moreover, PAP inhibited nuclear factor-κB (NF-κB) and enhanced cAMP-response element binding protein (CREB) activity in the SN of MPTP/LPS mice. These results suggest that PAP inhibits the activation of NLRP3 inflammasome by modulating NF-κB and CREB signaling pathways, which results in reduced microglial activation and neuronal cell death. Thus, PAP may be a potential candidate for the treatment of Parkinsons’s disease, which is aggravated by systemic inflammation.

Umbilical cord blood-derived microglia-like cells to model COVID-19 exposure

Microglia, the resident brain immune cells, play a critical role in normal brain development, and are impacted by the intrauterine environment, including maternal immune activation and inflammatory exposures. The COVID-19 pandemic presents a potential developmental immune challenge to the fetal brain, in the setting of maternal SARS-CoV-2 infection with its attendant potential for cytokine production and, in severe cases, cytokine storming. There is currently no biomarker or model for in utero microglial priming and function that might aid in identifying the neonates and children most vulnerable to neurodevelopmental morbidity, as microglia remain inaccessible in fetal life and after birth. This study aimed to generate patient-derived microglial-like cell models unique to each neonate from reprogrammed umbilical cord blood mononuclear cells, adapting and extending a novel methodology previously validated for adult peripheral blood mononuclear cells. We demonstrate that umbilical cord blood mononuclear cells can be used to create microglial-like cell models morphologically and functionally similar to microglia observed in vivo. We illustrate the application of this approach by generating microglia from cells exposed and unexposed to maternal SARS-CoV-2 infection. Our ability to create personalized neonatal models of fetal brain immune programming enables non-invasive insights into fetal brain development and potential childhood neurodevelopmental vulnerabilities for a range of maternal exposures, including COVID-19.

The Role of the Pathogen Dose and PI3Kγ in Immunometabolic Reprogramming of Microglia for Innate Immune Memory.

Microglia, the innate immune cells of the CNS, exhibit long-term response changes indicative of innate immune memory (IIM). Our previous studies revealed IIM patterns of microglia with opposing immune phenotypes: trained immunity after a low dose and immune tolerance after a high dose challenge with pathogen-associated molecular patterns (PAMP). Compelling evidence shows that innate immune cells adopt features of IIM via immunometabolic control. However, immunometabolic reprogramming involved in the regulation of IIM in microglia has not been fully addressed. Here, we evaluated the impact of dose-dependent microglial priming with ultra-low (ULP, 1 fg/mL) and high (HP, 100 ng/mL) lipopolysaccharide (LPS) doses on immunometabolic rewiring. Furthermore, we addressed the role of PI3Kγ on immunometabolic control using naïve primary microglia derived from newborn wild-type mice, PI3Kγ-deficient mice and mice carrying a targeted mutation causing loss of lipid kinase activity. We found that ULP-induced IIM triggered an enhancement of oxygen consumption and ATP production. In contrast, HP was followed by suppressed oxygen consumption and glycolytic activity indicative of immune tolerance. PI3Kγ inhibited glycolysis due to modulation of cAMP-dependent pathways. However, no impact of specific PI3Kγ signaling on immunometabolic rewiring due to dose-dependent LPS priming was detected. In conclusion, immunometabolic reprogramming of microglia is involved in IIM in a dose-dependent manner via the glycolytic pathway, oxygen consumption and ATP production: ULP (ultra-low-dose priming) increases it, while HP reduces it.

Microglial innate memory and epigenetic reprogramming in neurological disorders.

Microglia are myeloid-derived cells recognized as brain-resident macrophages. They act as the first and main line of immune defense in the central nervous system (CNS). Microglia have high phenotypic plasticity and are essential for regulating healthy brain homeostasis, and their dysregulation underlies the onset and progression of several CNS pathologies through impaired inflammatory responses. Aberrant microglial activation, following an inflammatory insult, is associated with epigenetic dysregulation in various CNS pathologies. Emerging data suggest that certain stimuli to myeloid cells determine enhanced or attenuated responses to subsequent stimuli. These phenomena, generally termed innate immune memory (IIM), are highly dependent on epigenetic reprogramming. Microglial priming has been reported in several neurological diseases and corresponds to a state of increased permissiveness or exacerbated response, promoted by continuous exposure to a chronic pro-inflammatory environment. In this article, we provide extensive evidence of these epigenetic-mediated phenomena under neurological conditions and discuss their contribution to pathogenesis and their clinical implications, including those concerning potential novel therapeutic approaches.

Lifestyle modifications with anti-neuroinflammatory benefits in the aging population

Aging-associated microglial priming results in the potential for an exaggerated neuroinflammatory response to a subsequent inflammatory challenge in regions of the brain known to support learning and memory. This excessive neuroinflammation in the aging brain is known to occur following a variety of peripheral insults, including infection and surgery, where it has been associated with precipitous declines in cognition and memory. As the average lifespan increases worldwide, identifying interventions to prevent and treat aging-associated excessive neuroinflammation and ensuing cognitive impairments is of critical importance. Lifestyle has emerged as a potential non-pharmacological target in this endeavor. Here, we review important and recent preclinical and clinical literature demonstrating the anti-inflammatory effects of lifestyle modifications such as exercise, diet, and environmental enrichment in the context of aging and memory. Importantly, we focus on research indicating that these lifestyle modifications do not need to be lifelong, suggesting that such interventions may be efficacious in the prevention and treatment of aging- and neuroinflammation-associated cognitive impairment, even when initiated in older age.

Microglial and peripheral immune priming is partially sexually dimorphic in adolescent mouse offspring exposed to maternal high-fat diet

BackgroundMaternal nutrition is critical for proper fetal development. While increased nutrient intake is essential during pregnancy, an excessive consumption of certain nutrients, like fat, can lead to long-lasting detrimental consequences on the offspring. Animal work investigating the consequences of maternal high-fat diet (mHFD) revealed in the offspring a maternal immune activation (MIA) phenotype associated with increased inflammatory signals. This inflammation was proposed as one of the mechanisms causing neuronal circuit dysfunction, notably in the hippocampus, by altering the brain-resident macrophages—microglia. However, the understanding of mechanisms linking inflammation and microglial activities to pathological brain development remains limited. We hypothesized that mHFD-induced inflammation could prime microglia by altering their specific gene expression signature, population density, and/or functions.MethodsWe used an integrative approach combining molecular (i.e., multiplex-ELISA, rt-qPCR) and cellular (i.e., histochemistry, electron microscopy) techniques to investigate the effects of mHFD (saturated and unsaturated fats) vs control diet on inflammatory priming, as well as microglial transcriptomic signature, density, distribution, morphology, and ultrastructure in mice. These analyses were performed on the mothers and/or their adolescent offspring at postnatal day 30.ResultsOur study revealed that mHFD results in MIA defined by increased circulating levels of interleukin (IL)-6 in the mothers. This phenotype was associated with an exacerbated inflammatory response to peripheral lipopolysaccharide in mHFD-exposed offspring of both sexes. Microglial morphology was also altered, and there were increased microglial interactions with astrocytes in the hippocampus CA1 of mHFD-exposed male offspring, as well as decreased microglia-associated extracellular space pockets in the same region of mHFD-exposed offspring of the two sexes. A decreased mRNA expression of the inflammatory-regulating cytokine Tgfb1 and microglial receptors Tmem119, Trem2, and Cx3cr1 was additionally measured in the hippocampus of mHFD-exposed offspring, especially in males.ConclusionsHere, we described how dietary habits during pregnancy and nurturing, particularly the consumption of an enriched fat diet, can influence peripheral immune priming in the offspring. We also found that microglia are affected in terms of gene expression signature, morphology, and interactions with the hippocampal parenchyma, in a partially sexually dimorphic manner, which may contribute to the adverse neurodevelopmental outcomes on the offspring.

Pharmacologically reversible zonation-dependent endothelial cell transcriptomic changes with neurodegenerative disease associations in the aged brain

The molecular signatures of cells in the brain have been revealed in unprecedented detail, yet the ageing-associated genome-wide expression changes that may contribute to neurovascular dysfunction in neurodegenerative diseases remain elusive. Here, we report zonation-dependent transcriptomic changes in aged mouse brain endothelial cells (ECs), which prominently implicate altered immune/cytokine signaling in ECs of all vascular segments, and functional changes impacting the blood–brain barrier (BBB) and glucose/energy metabolism especially in capillary ECs (capECs). An overrepresentation of Alzheimer disease (AD) GWAS genes is evident among the human orthologs of the differentially expressed genes of aged capECs, while comparative analysis revealed a subset of concordantly downregulated, functionally important genes in human AD brains. Treatment with exenatide, a glucagon-like peptide-1 receptor agonist, strongly reverses aged mouse brain EC transcriptomic changes and BBB leakage, with associated attenuation of microglial priming. We thus revealed transcriptomic alterations underlying brain EC ageing that are complex yet pharmacologically reversible.

Priming of microglia with IFN-γ impairs adult hippocampal neurogenesis and leads to depression-like behaviors and cognitive defects.

Neuroinflammation driven by interferon-gamma (IFN-γ) and microglial activation has been linked to neurological disease. However, the effects of IFN-γ-activated microglia on hippocampal neurogenesis and behavior are unclear. In the present study, IFN-γ was administered to mice via intracerebroventricular injection. Mice received intraperitoneal injection of ruxolitinib to inhibit the JAK/STAT1 pathway or injection of minocycline to inhibit microglial activation. During a 7-day period, mice were assessed for depressive-like behaviors and cognitive impairment based on a series of behavioral analyses. Effects of the activated microglia on neural stem/precursor cells (NSPCs) were examined, as was pro-inflammatory cytokine expression by activated microglia. We showed that IFN-γ-injected animals showed long-term adult hippocampal neurogenesis reduction, behavior despair, anhedonia, and cognitive impairment. Chronic activation with IFN-γ induces reactive phenotypes in microglia associated with morphological changes, population expansion, MHC II and CD68 up-regulation, and pro-inflammatory cytokine (IL-1β, TNF-α, IL-6) and nitric oxide (NO) release. Microglia isolated from the hippocampus of IFN-γ-injected mice suppressed NSPCs proliferation and stimulated apoptosis of immature neurons. Inhibiting of the JAK/STAT1 pathway in IFN-γ-injected animals to block microglial activation suppressed microglia-mediated neuroinflammation and neurogenic injury, and alleviated depressive-like behaviors and cognitive impairment. Collectively, these findings suggested that priming of microglia with IFN-γ impairs adult hippocampal neurogenesis and leads to depression-like behaviors and cognitive defects. Targeting microglia by modulating levels of IFN-γ the brain may be a therapeutic strategy for neurodegenerative diseases and psychiatric disorders.

Fatty food, fatty acids, and microglial priming in the adult and aged hippocampus and amygdala

Short-term (3-day) consumption of a high fat diet (HFD) rich in saturated fats is associated with a neuroinflammatory response and subsequent cognitive impairment in aged, but not young adult, male rats. This exaggerated effect in aged rats could be due to a “primed” microglial phenotype observed in the normal aging process in rodents in which aged microglia display a potentiated response to immune challenge. Here, we investigated the impact of HFD on microglial priming and lipid composition in the hippocampus and amygdala of young and aged rats. Furthermore, we investigated the microglial response to palmitate, the main saturated fatty acid (SFA) found in HFD that is proinflammatory. Our results indicate that HFD increased gene expression of microglial markers of activation indicative of microglial priming, including CD11b, MHCII, CX3CR1, and NLRP3, as well as the pro-inflammatory marker IL-1β in both hippocampus and amygdala-derived microglia. Furthermore, HFD increased the concentration of SFAs and decreased the concentration of polyunsaturated fatty acids (PUFAs) in the hippocampus. We also observed a specific decrease in the anti-inflammatory PUFA docosahexaenoic acid (DHA) in the hippocampus and amygdala of aged rats. In a separate cohort of young and aged animals, isolated microglia from the hippocampus and amygdala exposed to palmitate in vitro induced an inflammatory gene expression profile mimicking the effects of HFD in vivo. These data suggest that palmitate may be a critical nutritional signal from the HFD that is directly involved in hippocampal and amygdalar inflammation. Interestingly, microglial activation markers were increased in response to HFD or palmitate in an age-independent manner, suggesting that HFD sensitivity of microglia, under these experimental conditions, is not the sole mediator of the exaggerated inflammatory response observed in whole tissue extracts from aged HFD-fed rats.

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-1β, 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-1β, 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.

Progesterone Attenuates Stress-Induced NLRP3 Inflammasome Activation and Enhances Autophagy following Ischemic Brain Injury.

NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome inhibition and autophagy induction attenuate inflammation and improve outcome in rodent models of cerebral ischemia. However, the impact of chronic stress on NLRP3 inflammasome and autophagic response to ischemia remains unknown. Progesterone (PROG), a neuroprotective steroid, shows promise in reducing excessive inflammation associated with poor outcome in ischemic brain injury patients with comorbid conditions, including elevated stress. Stress primes microglia, mainly by the release of alarmins such as high-mobility group box-1 (HMGB1). HMGB1 activates the NLRP3 inflammasome, resulting in pro-inflammatory interleukin (IL)-1β production. In experiment 1, adult male Sprague-Dawley rats were exposed to social defeat stress for 8 days and then subjected to global ischemia by the 4-vessel occlusion model, a clinically relevant brain injury associated with cardiac arrest. PROG was administered 2 and 6 h after occlusion and then daily for 7 days. Animals were killed at 7 or 14 days post-ischemia. Here, we show that stress and global ischemia exert a synergistic effect in HMGB1 release, resulting in exacerbation of NLRP3 inflammasome activation and autophagy impairment in the hippocampus of ischemic animals. In experiment 2, an in vitro inflammasome assay, primary microglia isolated from neonatal brain tissue, were primed with lipopolysaccharide (LPS) and stimulated with adenosine triphosphate (ATP), displaying impaired autophagy and increased IL-1β production. In experiment 3, hippocampal microglia isolated from stressed and unstressed animals, were stimulated ex vivo with LPS, exhibiting similar changes than primary microglia. Treatment with PROG reduced HMGB1 release and NLRP3 inflammasome activation, and enhanced autophagy in stressed and unstressed ischemic animals. Pre-treatment with an autophagy inhibitor blocked Progesterone’s (PROG’s) beneficial effects in microglia. Our data suggest that modulation of microglial priming is one of the molecular mechanisms by which PROG ameliorates ischemic brain injury under stressful conditions.

Tgfb1‐mediated Regulation of Microglia Maturation and Activation

The pleiotropic cytokine transforming growth factor‐beta 1 (TGFβ1) has been shown to play pivotal roles in a variety of different cell types including immune cells such as T cells, monocytes/macrophages as well as microglia. As the resident immune cell population of the central nervous system (CNS), microglia are essential effector cells during physiological and pathological events. Microglia originate from mesodermal progenitors that arise from the yolk sac and colonize the embryonic CNS where they adopt a mature microglia‐specific phenotypic pattern including expression of P2ry12, Tmem119, Olfml3, Siglech, Gpr34, Hexb, and Fcrls. The differentiation and maturation of postnatal microglia, as well as the regulation of the complex functional repertoire of mature microglia, needs to be carefully orchestrated. Here, we demonstrate that the deletion of Tgfbr2 in early postnatal microglia results in a loss of microglia maturation whereas silencing of TGFβ signaling in adult microglia does neither result in impairment of the microglia‐specific gene expression signatures, nor is microglial survival and maintenance affected. Tgfbr2‐deficient microglia were characterized by distinct morphological changes and transcriptome analysis using RNAseq revealed that loss of TGFβ signaling results in the upregulation of microglia activation and priming markers. Taken together, recent studies employing sophisticated transgenic approaches to silence microglial TGFβ signaling have revealed that TGFβ1 is indispensable for microglia maturation and the establishment of the microglia‐specific gene expression pattern as well as the homeostasis of adult microglia and the control of excessive microglia activation.

Microglial depletion and repopulation in brain slice culture normalizes sensitized proinflammatory signaling

BackgroundMicroglia are critical mediators of neuroimmune pathology across multiple neurologic disorders. Microglia can be persistently activated or “primed” by Toll-like receptor (TLR) activation, ethanol, stress, and other insults. Thus, strategies to prevent or reverse microglial priming may be beneficial for conditions that involve progressively increasing microglial activation. Microglial depletion with repopulation is emerging as a potential therapy to normalize chronic immune activation. Primary organotypic hippocampal slice culture (OHSC) allows for the study of neuroimmune activation as well as microglial depletion and repopulation without involvement of peripheral immune activation. OHSC undergoes functional maturation and retains cytoarchitecture similar to in vivo.MethodsOHSC underwent microglial depletion with the CSF1R antagonist PLX3397 with or without repopulation after removal of PLX3397. Immune, trophic, and synaptic gene changes in response to agonists of TLRs 2, 3, 4, 7, and 9 as well as ethanol were assessed in the settings of microglial depletion and repopulation. Gi-DREADD inhibition of microglia was used to confirm select findings seen with depletion. The ability of microglial repopulation to prevent progressive proinflammatory gene induction by chronic ethanol was also investigated.ResultsMicroglia were depleted (> 90%) by PLX3397 in OHSC. Microglial depletion blunted proinflammatory responses to several TLR agonists as well as ethanol, which was mimicked by Gi-DREADD inhibition of OHSC microglia. Removal of PLX3397 was followed by complete repopulation of microglia. OHSCs with repopulated microglia showed increased baseline expression of anti-inflammatory cytokines (e.g., IL-10), microglial inhibitory signals (e.g., CX3CL1), and growth factors (e.g., BDNF). This was associated with blunted induction (~ 50%) of TNFα and IL-1β in response to agonists to TLR4 and TLR7. Further, chronic cycled ethanol from 4 days in vitro (DIV) to 16DIV caused immediate 2-fold inductions of TNFα and IL-1β that grew to ~4-fold of age-matched control slices by 40DIV. This persistent inflammatory gene expression was completely reversed by microglial depletion and repopulation after chronic ethanol.ConclusionsMicroglia in OHSCs mediate proinflammatory responses to TLR agonists and ethanol. Microglial repopulation promoted an anti-inflammatory, trophic neuroenvironment and normalized proinflammatory gene expression. This supports the possibility of microglial depletion with repopulation as a strategy to reverse chronic neuroimmune activation.

PRIMING OF MICROGLIA ACTIVITY INCREASES SUSCEPTIBILITY TO DEPRESSION-LIKE BEHAVIORS

This study investigates the role of microglia activity in stress-induced depression and anxiety and the mechanisms associated with the role of certain microbiome derived anti-inflammatory polyphenols in attenuating stress-induced microglia immune priming and symptoms of depression. We implemented a chronic unpredictable stress (CUS) paradigm to exhibit priming of microglia innate immunity in the context of the onset of depression and anxiety phenotypes. Mechanistic studies related to prophylactic treatment using dietary microbiome derived polyphenols were also investigated in this model. Depression and anxiety phenotypes, gene expression in microglia and protein expression in the cortex of mice were measured following a primary exposure to short-term unpredictable stress (US) followed by CUS. We examined the long-term, persistent CUS induced changes at 4-weeks of post-stress rest following a secondary US exposure. We found depression phenotypes resulted from US only following exposure to CUS. This was accompanied by an increase and persistent upregulation of toll-like receptor 4 (TLR4), RAGE, and HMGB1 gene expression in isolated cortical microglia. Priming by CUS also amplified gene expression of IL-1β in microglia and protein IL-1β in the cerebral cortex following US re-exposure. Increased activity of NF-kB was also noted in the period following CUS. Furthermore, polyphenol treatment prevented stress-induced phenotypes, upregulation of HMGB1, IL-1B, and TLR4 gene expression, as well as upregulation of IL-1β and NF-kB. The study suggests that latent activity of the TLR4-NFkB-IL1β pathway contributes to immune priming and increases susceptibility to depression-like behaviors. Anti-depressant effects of polyphenols may result from their ability to attenuate microglia priming.