Priming Microglia Research Articles

Fingolimod Alleviates Cognitive Deficit in Type 2 Diabetes by Promoting Microglial M2 Polarization via the pSTAT3-jmjd3 Axis.

Sphingosine receptors (S1PRs) are implicated in the progression of neurodegenerative diseases and metabolic disorders like obesity and type 2 diabetes (T2D). The link between S1PRs and cognition in type 2 diabetes, as well as the mechanisms that underpin it, are yet unknown. Neuroinflammation is the common pathology shared among T2D and cognitive impairment. However, the interplay between the M1 and M2 polarization state of microglia, a primary driver of neuroinflammation, could be the driving factor for impaired learning and memory in diabetes. In the present study, we investigated the effects of fingolimod (S1PR1 modulator) on cognition in high-fat diet and streptozotocin-induced diabetic mice. We further assessed the potential pathways linking microglial polarization and cognition in T2D. Fingolimod (0.5mg/kg and 1mg/kg) improved M2 polarization and synaptic plasticity while ameliorating cognitive decline and neuroinflammation. Sphingolipid dysregulation was mimicked in vitro using palmitate in BV2 cells, followed by conditioned media exposure to Neuro2A cells. Mechanistically, type 2 diabetes induced microglial activation, priming microglia towards the M1 phenotype. In the hippocampus and cortex of type 2 diabetic mice, there was a substantial drop in pSTAT3, which was reversed by fingolimod. This protective effect of fingolimod on microglial M2 polarization was primarily suppressed by selective jmjd3 blockade in vitro using GSK-J4, revealing that jmjd3 was involved downstream of STAT3 in the fingolimod-enabled shift of microglia from M1 to M2 polarization state. This study suggested that fingolimod might effectively improve cognition in type 2 diabetes by promoting M2 polarization.

Homotypic Neonatal and Adult Inflammation Primes Adult Microglia Without Impairing Adult Breathing

A population of adults are predisposed to breathing deficits during subsequent illness and disease, yet it is unknown what factor(s) contribute to this adult vulnerability. Since neonatal inflammation primes adult microglia in non‐respiratory control regions and augments peripheral immune responses, we hypothesized that neonatal and adult homotypic inflammation (same inflammatory stimulus at both ages) primes male and female microglia in respiratory control regions, contributing to impairments in adult breathing during subsequent inflammatory challenges. Using lipopolysaccharide (LPS) to induce homotypic inflammation in neonates (1mg/kg LPS, P4) and adults (24µg/kg LPS, i.p.), medullary microglia were primed in adult males (neonatal LPS + adult LPS: 25±4% microglia, n=9; neonatal LPS + adult saline: 18±2% microglia, n=9; neonatal saline + adult saline: 10±1% microglia, n=9; p<0.01) and adult females (neonatal LPS + adult LPS: 25±3% microglia, n=9; neonatal LPS + adult saline: 14±2% microglia, n=6; neonatal saline + adult saline: 10±2% microglia, n=8; p<0.0001). However, spinal cord microglia were only primed in adult males (neonatal LPS + adult LPS: 13±2% microglia, n=9; neonatal LPS + adult saline: 9±2% microglia, n=9; neonatal saline + adult saline: 10±2% microglia, n=9; p<0.01) and not adult females (neonatal LPS + adult LPS: 13±2% microglia, n=9; neonatal LPS + adult saline: 9±1% microglia, n=6; neonatal saline + adult saline: 9±1% microglia, n=8; p<0.01). Thus, neonatal and adult homotypic inflammation region‐ and sex‐dependently prime microglia in respiratory control regions. To test whether primed microglia in respiratory control regions contribute to deficits in adult breathing, breathing was assessed by plethysmography in adults at two time points: during the peak inflammatory response (3hrs) and peak microglial migration and proliferation (24hrs). Neither adult eupneic breathing nor chemoreflexes (hypercapnic ventilatory response, HCVR, and hypoxic ventilatory response, HVR) were primed in males or females by neonatal and adult homotypic inflammation at either time point. Interestingly, adult female chemoreflexes were time‐dependently increased by neonatal and adult homotypic inflammation. In adult females, neonatal and adult homotypic inflammation increased the peak HCVR at 3hrs (neonatal LPS + adult LPS: 298±112% ∆VE from baseline, n=6) compared to 24hrs (neonatal LPS + adult LPS: 169±41% ∆VE from baseline, n=6, p=0.04), and increased the peak HVR at 3hrs (neonatal LPS + adult LPS: 159±37% ∆VE from baseline) compared to 24hrs (neonatal LPS + adult LPS: 62±23% ∆VE from baseline, n=6, p=0.02). Thus, adult female chemoreflexes were time‐dependently impacted by the combination of neonatal and adult homotypic inflammation, suggesting time‐specific effects of adult inflammation. Despite no significant impact of primed microglia on baseline breathing and chemoreflexes, primed respiratory control microglia have the potential to significantly impair respiratory control during more severe inflammatory challenges associated with illness and disease. Further, sex differences in primed respiratory control microglia suggest sex‐specific vulnerability to adult ventilatory control disorders.

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.

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.

Toll-like receptors 2, -3 and -4 prime microglia but not astrocytes across central nervous system regions for ATP-dependent interleukin-1β release.

Interleukin-1β (IL-1β) is a crucial mediator in the pathogenesis of inflammatory diseases at the periphery and in the central nervous system (CNS). Produced as an unprocessed and inactive pro-form which accumulates intracellularly, release of the processed cytokine is strongly promoted by ATP acting at the purinergic P2X7 receptor (P2X7R) in cells primed with lipopolysaccharide (LPS), a Toll-like receptor (TLR) 4 ligand. Microglia are central to the inflammatory process and a major source of IL-1β when activated. Here we show that purified (>99%) microglia cultured from rat cortex, spinal cord and cerebellum respond robustly to ATP-dependent IL-1β release, upon priming with a number of TLR isoform ligands (zymosan and Pam3CSK4 for TLR2, poly(I:C) for TLR3). Cytokine release was prevented by a P2X7R antagonist and inhibitors of stress-activated protein kinases. Enriched astrocytes (≤5% microglia) from these CNS regions displayed responses qualitatively similar to microglia but became unresponsive upon eradication of residual microglia with the lysosomotropic agent Leu-Leu-OMe. Activation of multiple TLR isoforms in nervous system pathology, coupled with elevated extracellular ATP levels and subsequent P2X7R activation may represent an important route for microglia-derived IL-1β. This phenomenon may have important consequences for neuroinflammation and its position to the common pathology of CNS diseases.

Neonatal immune challenge exacerbates seizure-induced hippocampus-dependent memory impairment in adult rats

Our aim was to examine whether neonatal lipopolysaccharide (LPS) exposure is associated with changes in microglia and whether these alternations could influence later seizure-induced neurobehavioral outcomes. Male pups were first injected intraperitoneally with either LPS or saline on postnatal day 3 (P3) and postnatal day 5 (P5). Immunohistochemical analysis showed that LPS-treated animals exhibited increased microglia activation that persisted into adolescence. At P45, seizures were induced in rats by intraperitoneal injection of kainic acid (KA). Rats treated with LPS neonatally showed significantly greater proinflammatory responses and performed significantly worse in the Y-maze, Morris water maze, and inhibitory avoidance tasks after KA insult. Treatment with minocycline at the time of neonatal LPS exposure to block LPS-induced microglia alternation attenuated the exaggerated neuroinflammatory responses and alleviated memory impairment associated with the KA insult. Our findings suggest that neonatal immune activation can predispose the brain to exacerbated behavioral deficits following seizures in adulthood, possibly by priming microglia.

170. Characterization of the neuroendocrine system in healthy aged rats

Healthy aged individuals are more likely to suffer profound memory impairments following a challenging life event such as a severe bacterial infection, surgery, or an intense psychological stressor, than are younger adults. Importantly, these peripheral challenges are capable of producing a neuroinflammatory response (e.g., increased pro-inflammatory cytokines) and in the healthy aged brain this response is exaggerated and prolonged. Normal aging primes or sensitizes microglia and this appears to be the source of this amplified response. Here, we present data suggesting that a dysregulated neuroendocrine response in the aged (24 mos) F344xBN rat is skewed toward higher brain corticosterone (CORT) levels and greater hippocampal glucocorticoid receptor activation compared to young adult rats (3 mos). In addition, we confirm others’ findings that 11beta-hydroxysteroid dehydrogenase (11beta-HSD1), the enzyme that transforms cortisone (an inactive metabolite of CORT) to the active hormone CORT, is elevated in the aged hippocampus. In concert, these factors may play a critical role in priming microglia in the aged rat brain, resulting in exaggerated and longer lasting cytokine responses in the brain and greater memory deficits following a challenge.

Abstract SY31-02: Behavioral consequences of peripheral tumors: Neuroimmune mechanisms

Abstract SY31-02: Behavioral consequences of peripheral tumors: Neuroimmune mechanisms

Glial Activation Links Early‐Life Seizures and Long‐Term Neurologic Dysfunction: Evidence Using a Small Molecule Inhibitor of Proinflammatory Cytokine Upregulation

Early-life seizures increase vulnerability to subsequent neurologic insult. We tested the hypothesis that early-life seizures increase susceptibility to later neurologic injury by causing chronic glial activation. To determine the mechanisms by which glial activation may modulate neurologic injury, we examined both acute changes in proinflammatory cytokines and long-term changes in astrocyte and microglial activation and astrocyte glutamate transporters in a "two-hit" model of kainic acid (KA)-induced seizures. Postnatal day (P) 15 male rats were administered KA or phosphate buffered saline (PBS). On P45 animals either received a second treatment of KA or PBS. On P55, control (PBS-PBS), early-life seizure (KA-PBS), adult seizure (PBS-KA), and "two-hit" (KA-KA) groups were examined for astrocyte and microglial activation, alteration in glutamate transporters, and expression of the glial protein, clusterin. P15 seizures resulted in an acute increase in hippocampal levels of IL-1beta and S100B, followed by behavioral impairment and long-term increases in GFAP and S100B. Animals in the "two-hit" group showed greater microglial activation, neurologic injury, and susceptibility to seizures compared to the adult seizure group. Glutamate transporters increased following seizures but did not differ between these two groups. Treatment with Minozac, a small molecule inhibitor of proinflammatory cytokine upregulation, following early-life seizures prevented both the long-term increase in activated glia and the associated behavioral impairment. These data suggest that glial activation following early-life seizures results in increased susceptibility to seizures in adulthood, in part through priming microglia and enhanced microglial activation. Glial activation may be a novel therapeutic target in pediatric epilepsy.

κ-Opioid Modulation of Human Microglial Cell Superoxide Anion Generation

Opioids have been postulated to play an immunomodulatory role in the CNS. Recently, we found that priming microglia with interferon (IFN)-γ or tumor necrosis factor (TNF)-α resulted in an enhanced production of superoxide anion, a reactive oxygen intermediate that may be pathogenic during brain inflammation. In the present study, we investigated the effects of trans-3,4-dichloro- N-methyl- N[2-(1-pyrolidinyl)cyclohexyl]benzeneaceamide methanesulfonate (U50,488), a selective κ-opioid ligand, on microglial cell superoxide production when cells were primed with cytokines or stimulated with phorbol myristate acetate. While treatment of microglial cells with U50,488 had little effect on nonstimulated or stimulated superoxide production, this opioid inhibited (by >70%) the priming effects of cytokines. Maximal inhibition of microglial cell superoxide generation by U50,488 was observed at 10 nM for the priming effect of interferon-γ and at 1 μM for tumor necrosis factor-α. Pretreatment of microglial cell cultures for 30 min with an equal concentration of the selective κ-opioid receptor antagonist nor-binaltorphimine (nor-BNI) completely blocked the inhibitory effect of U50,488. The results of this study suggest that κ-opioids may have therapeutic potential in inflammatory diseases of the CNS involving reactive oxygen intermediates produced by activated microglia.