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Chronic exposure to exogenous glucocorticoids primes microglia to pro-inflammatory stimuli and induces NLRP3 mRNA in the hippocampus.

ABSTRACT: Chronic stress as well as chronic treatment with glucocorticoids (GCs) primes the neuroinflammatory response to a subsequent pro-inflammatory challenge. However, it remains unclear whether chronic GCs sensitize the response of key CNS immune substrates (i.e. microglia) to pro-inflammatory stimuli. In the present set of studies, male Sprague-Dawley rats underwent sham surgery or were adrenalectomized and then treated with varying concentrations of corticosterone (CORT; 0, 25, 50, and 75 ?g/ml) administered in their drinking water. After 10 days of CORT exposure, whole hippocampus was collected and expression of glial activation markers measured or hippocampal microglia were isolated and challenged with LPS to probe for CORT-induced sensitization of pro-inflammatory responses. Chronic CORT exposure increased the gene expression of NLRP3, Iba-1, MHCII, and NF-?BI? in a concentration dependent manner. Chronic CORT (75 ?g/ml) exposure potentiated the microglial proinflammatory response (TNF?, IL-1?, IL-6 and NLRP3) to LPS compared to the microglial response of sham surgery animals treated with vehicle. The present set of results demonstrate that chronic exposure to GCs primes microglia to pro-inflammatory stimuli and add to a growing body of evidence suggesting that a permissive function of GCs is that of an endogenous danger signal or alarmin.

SUBMITTER: Frank MG

PROVIDER: S-EPMC3912460 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Chronic exposure to exogenous glucocorticoids primes microglia to pro-inflammatory stimuli and induces NLRP3 mRNA in the hippocampus.

Frank Matthew G MG Hershman Sarah A SA Weber Michael D MD Watkins Linda R LR Maier Steven F SF

Psychoneuroendocrinology 20131127

Chronic stress as well as chronic treatment with glucocorticoids (GCs) primes the neuroinflammatory response to a subsequent pro-inflammatory challenge. However, it remains unclear whether chronic GCs sensitize the response of key CNS immune substrates (i.e. microglia) to pro-inflammatory stimuli. In the present set of studies, male Sprague-Dawley rats underwent sham surgery or were adrenalectomized and then treated with varying concentrations of corticosterone (CORT; 0, 25, 50, and 75 μg/ml) ad ...[more]

PMID: 24485491

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Project description:Microglia respond to CNS injuries and diseases with complex reactions, often called "activation." A pro-inflammatory phenotype (also called classical or M1 activation) lies at one extreme of the reactivity spectrum. There were several motivations for this study. First, bacterial endotoxin (lipopolysaccharide, LPS) is the most commonly used pro-inflammatory stimulus for microglia, both in vitro and in vivo; however, pro-inflammatory cytokines (e.g., IFNγ, TNFα) rather than LPS will be encountered with sterile CNS damage and disease. We lack direct comparisons of responses between LPS and such cytokines. Second, while transcriptional profiling is providing substantial data on microglial responses to LPS, these studies mainly use mouse cells and models, and there is increasing evidence that responses of rat microglia can differ. Third, the cytokine milieu is dynamic after acute CNS damage, and an important question in microglial biology is: How malleable are their responses? There are very few studies of effects of resolving cytokines, particularly for rat microglia, and much of the work has focused on pro-inflammatory outcomes. Here, we first exposed primary rat microglia to LPS or to IFNγ+TNFα (I+T) and compared hallmark functional (nitric oxide production, migration) and molecular responses (almost 100 genes), including surface receptors that can be considered part of the sensome. Protein changes for exemplary molecules were also quantified: ARG1, CD206/MRC1, COX-2, iNOS, and PYK2. Despite some similarities, there were notable differences in responses to LPS and I+T. For instance, LPS often evoked higher pro-inflammatory gene expression and also increased several anti-inflammatory genes. Second, we compared the ability of two anti-inflammatory, resolving cytokines (IL-4, IL-10), to counteract responses to LPS and I+T. IL-4 was more effective after I+T than after LPS, and IL-10 was surprisingly ineffective after either stimulus. These results should prove useful in modeling microglial reactivity in vitro; and comparing transcriptional responses to sterile CNS inflammation in vivo.

Project description:Neuroinflammation associated with HIV-1 infection is a problem affecting ∼50% of HIV-infected individuals. NLR family pyrin domain containing 3 (NLRP3) inflammasome has been implicated in HIV-induced microglial activation, but the mechanism(s) remain unclear. Because HIV-1 Transactivator of Transcription (Tat) protein continues to be present despite antiretroviral therapy and activates NF-kB, we hypothesized that Tat could prime the NLRP3 inflammasome. We found a dose- and time-dependent induction of NLRP3 expression in microglia exposed to Tat compared with control. Tat exposure also time-dependently increased the mature caspase-1 and IL-1β levels and enhanced the IL-1β secretion. These in vitro findings were validated in archival brain tissues from Simian Immunodeficiency Virus (SIV)-infected and uninfected rhesus macaques. Further validation of NLRP3 priming in vivo involved administration of lipopolysaccharide (LPS) to HIV transgenic (Tg) rats followed by assessment of IL-1β mRNA expression and inflammasome activation (ASC oligomers and mature IL-1β). Intriguingly, LPS potentiated upregulation of IL-1β mRNA and inflammasome activation in HIV-Tg rats compared with the wild-type controls. Interestingly, we found an inverse relationship in the expression of NLRP3 and its negative regulator, miR-223, suggesting a miR-223-mediated mechanism for Tat-induced NLRP3 priming. Furthermore, blockade of NLRP3 resulted in decreased IL-1β secretion. Collectively, these findings suggest a novel role of Tat in priming and activating the NLRP3 inflammasome. Therefore, NLRP3 can be envisioned as a therapeutic target for ameliorating Tat-mediated neuroinflammation.SIGNIFICANCE STATEMENT Despite successful suppression of viremia with increased longevity in the era of combined antiretroviral therapy, chronic inflammation with underlying neurocognitive impairment continues to afflict almost 50% of infected individuals. Viral, bacterial, and cellular products have all been implicated in promoting the chronic inflammation found in these individuals. Understanding the molecular mechanism(s) by which viral proteins such as HIV-1 Transactivator of Transcription (Tat) protein can activate microglia is thus of paramount importance. Herein, we demonstrate a novel role of Tat in priming and activating NLR family pyrin domain containing 3 (NLRP3) inflammasomes in microglial cells and in HIV-Tg rats administered lipopolysaccharide. Targeting NLRP3 inflammasome pathway mediators could thus be developed as therapeutic interventions to alleviate or prevent neuroinflammation and subsequent cognitive impairment in HIV-positive patients.

Project description:BACKGROUND:Acute-phase response is a systemic reaction to environmental/inflammatory insults and involves production of acute-phase proteins, including serum amyloid A (SAA). Interleukin-1? (IL-1?), a master regulator of neuroinflammation produced by activated inflammatory cells of the myeloid lineage, in particular microglia, plays a key role in the pathogenesis of acute and chronic diseases of the peripheral nervous system and CNS. IL-1? release is promoted by ATP acting at the purinergic P2X7 receptor (P2X7R) in cells primed with toll-like receptor (TLR) ligands. METHODS:Purified (>?99%) microglia cultured from neonatal rat cortex and cerebellum were first primed with the putative TLR4/TLR2 agonist SAA (recombinant human Apo-SAA) or the established TLR4 agonist lipopolysaccharide (LPS) followed by addition of ATP. Expression of genes for the NLRP3 inflammasome, IL-1?, tumor necrosis factor-? (TNF-?), and SAA1 was measured by quantitative real-time polymerase chain reaction (q-PCR). Intracellular and extracellular amounts of IL-1? were determined by ELISA. RESULTS:Apo-SAA stimulated, in a time-dependent manner, the expression of NLRP3, IL-1?, and TNF-? in cortical microglia, and produced a concentration-dependent increase in the intracellular content of IL-1? in these cells. A 2-h 'priming' of the microglia with Apo-SAA followed by addition of ATP for 1 h, resulting in a robust release of IL-1? into the culture medium, with a concomitant reduction in its intracellular content. The selective P2X7R antagonist A740003 blocked ATP-dependent release of IL-1?. Microglia prepared from rat cerebellum displayed similar behaviors. As with LPS, Apo-SAA upregulated SAA1 and TLR2 mRNA, and downregulated that of TLR4. LPS was less efficacious than Apo-SAA, perhaps reflecting an action of the latter at TLR4 and TLR2. The TLR4 antagonist CLI-095 fully blocked the action of LPS, but only partially that of Apo-SAA. Although the TLR2 antagonist CU-CPT22 was inactive against Apo-SAA, it also failed to block the TLR2 agonist Pam3CSK4. CONCLUSIONS:Microglia are central to the inflammatory process and a major source of IL-1? when activated. P2X7R-triggered IL-1? maturation and export is thus likely to represent an important contributor to this cytokine pool. Given that SAA is detected in Alzheimer disease and multiple sclerosis brain, together with IL-1?-immunopositive microglia, these findings propose a link between P2X7R, SAA, and IL-1? in CNS pathophysiology.

Project description:BackgroundAcute CNS damage is commonly studied using rat and mouse models, but increasingly, molecular analysis is finding species differences that might affect the ability to translate findings to humans. Microglia can undergo complex molecular and functional changes, often studied by in vitro responses to discrete activating stimuli. There is considerable evidence that pro-inflammatory (M1) activation can exacerbate tissue damage, while anti-inflammatory (M2) states help resolve inflammation and promote tissue repair. However, in assessing potential therapeutic targets for controlling inflammation, it is crucial to determine whether rat and mouse microglia respond the same.MethodsPrimary microglia from Sprague-Dawley rats and C57BL/6 mice were cultured, then stimulated with interferon-γ + tumor necrosis factor-α (I + T; M1 activation), interleukin (IL)-4 (M2a, alternative activation), or IL-10 (M2c, acquired deactivation). To profile their activation responses, NanoString was used to monitor messenger RNA (mRNA) expression of numerous pro- and anti-inflammatory mediators, microglial markers, immunomodulators, and other molecules. Western analysis was used to measure selected proteins. Two potential targets for controlling inflammation-inward- and outward-rectifier K+ channels (Kir2.1, Kv1.3)-were examined (mRNA, currents) and specific channel blockers were applied to determine their contributions to microglial migration in the different activation states.ResultsPro-inflammatory molecules increased after I + T treatment but there were several qualitative and quantitative differences between the species (e.g., iNOS and nitric oxide, COX-2). Several molecules commonly associated with an M2a state differed between species or they were induced in additional activation states (e.g., CD206, ARG1). Resting levels and/or responses of several microglial markers (Iba1, CD11b, CD68) differed with the activation state, species, or both. Transcripts for several Kir2 and Kv1 family members were detected in both species. However, the current amplitudes (mainly Kir2.1 and Kv1.3) depended on activation state and species. Treatment-induced changes in morphology and migratory capacity were similar between the species (migration reduced by I + T, increased by IL-4 or IL-10). In both species, Kir2.1 block reduced migration and Kv1.3 block increased it, regardless of activation state; thus, these channels might affect microglial migration to damage sites.ConclusionsCaution is recommended in generalizing molecular and functional responses of microglia to activating stimuli between species.

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Chronic exposure to exogenous glucocorticoids primes microglia to pro-inflammatory stimuli and induces NLRP3 mRNA in the hippocampus.

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Chronic exposure to exogenous glucocorticoids primes microglia to pro-inflammatory stimuli and induces NLRP3 mRNA in the hippocampus.

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