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CX3CR1 deficiency suppresses activation and neurotoxicity of microglia/macrophage in experimental ischemic stroke.

ABSTRACT: Chemokine (C-X3-C motif) ligand 1 (CX3CL1)/ CX3C chemokine receptor 1 (CX3CR1) signaling is important in modulating the communication between neurons and resident microglia/migrated macrophages in the central nervous system (CNS). Although CX3CR1 deficiency is associated with an improved outcome following ischemic brain injury, the mechanism of this observation is largely unknown. The aim of this study was to investigate how CX3CR1 deficiency influences microglia/macrophage functions in the context of its protection following brain ischemia.Wild-type (WT) and CX3CR1-deficient (CX3CR1?/?) mice were subjected to transient middle cerebral artery occlusion (MCAO) and reperfusion. The ischemic brain damage was monitored by rodent high-field magnetic resonance imaging. Neurological deficit was assessed daily. Neuronal apoptotic death and reactive oxygen species (ROS) production were analyzed by immunostaining and live imaging. Activation/inflammatory response of microglia/macrophage were assessed using immunohistochemistry, flow cytometry, 5-bromo-2-deoxyuridine labeling, cytokine ELISA, and real-time PCR.CX3CR1?/? mice displayed significantly smaller infarcts and less severe neurological deficits compared to WT controls, following MCAO. In addition, CX3CR1?/? MCAO mice displayed fewer apoptotic neurons and reduced ROS levels. Impaired CX3CR1 signaling abrogated the recruitment of monocyte-derived macrophages from the periphery, suppressed the proliferation of CNS microglia and infiltrated macrophage, facilitated the alternative activation (M2 state) of microglia/macrophages, and attenuated their ability to synthesize and release inflammatory cytokines.Our results suggest that inhibition of CX3CR1 signaling could function as a therapeutic modality in ischemic brain injury, by reducing recruitment of peripheral macrophages and expansion/activation of CNS microglia and macrophages, resulting in protection of neurological function.

SUBMITTER: Tang Z

PROVIDER: S-EPMC3942808 | biostudies-other | 2014 Feb

REPOSITORIES: biostudies-other

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CX3CR1 deficiency suppresses activation and neurotoxicity of microglia/macrophage in experimental ischemic stroke.

Tang Zhiwei Z Gan Yan Y Liu Qiang Q Yin Jun-Xiang JX Liu Qingwei Q Shi Jiong J Shi Fu-Dong FD

Journal of neuroinflammation 20140203

<h4>Background</h4>Chemokine (C-X3-C motif) ligand 1 (CX3CL1)/ CX3C chemokine receptor 1 (CX3CR1) signaling is important in modulating the communication between neurons and resident microglia/migrated macrophages in the central nervous system (CNS). Although CX3CR1 deficiency is associated with an improved outcome following ischemic brain injury, the mechanism of this observation is largely unknown. The aim of this study was to investigate how CX3CR1 deficiency influences microglia/macrophage fu ...[more]

PMID: 24490760

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Project description:Stroke is the most common type of cerebrovascular disease and is a leading cause of disability and death. Ischemic stroke accounts for approximately 80% of all strokes. The remaining 20% of strokes are hemorrhagic in nature. To date, therapeutic options for acute ischemic stroke are very limited. Recent research suggests that shifting microglial phenotype from the pro-inflammatory M1 state toward the anti-inflammatory and tissue-reparative M2 phenotype may be an effective therapeutic strategy for ischemic stroke. The dietary phytochemical curcumin has shown promise in experimental stroke models, but its effects on microglial polarization and long-term recovery after stroke are unknown. Here we address these gaps by subjecting mice to distal middle cerebral artery occlusion (dMCAO) and administering curcumin intraperitoneally (150 mg/kg) immediately after ischemia and 24 h later. Histological studies revealed that curcumin post-treatment significantly reduced cerebral ischemic damage 3 days after dMCAO. Sensorimotor functions-as measured by the adhesive removal test and modified Garcia scores-were superior in curcumin-treated mice at 3, 5, 7 and 10 days after stroke. RT-PCR measurements revealed an elevation of M2 microglia/macrophage phenotypic markers and a reduction in M1 markers in curcumin-treated brains 3 days after dMCAO. Immunofluorescent staining further showed that curcumin treatment significantly increased the number of CD206+Iba1+ M2 microglia/macrophages and reduced the number of CD16+Iba1+ M1 cells 10 days after stroke. In vitro studies using the BV2 microglial cell line confirmed that curcumin inhibited lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-induced M1 polarization. Curcumin treatment concentration-dependently reduced the expression of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-12p70, in the absence of any toxic effect on microglial cell survival. In conclusion, we demonstrate that curcumin has a profound regulatory effect on microglial responses, promoting M2 microglial polarization and inhibiting microglia-mediated pro-inflammatory responses. Curcumin post-treatment reduces ischemic stroke-induced brain damage and improves functional outcomes, providing new evidence that curcumin might be a promising therapeutic strategy for stroke.

Project description:Background: Innate immune response to neuronal death is one of the key events of the pathogenesis of ischemic brain injury. Interleukin-1 receptor-associated kinase (IRAK)-M, encoded by gene Irak3, negatively regulates toll-like receptor signaling by interacting with the MyD88-IRAK-4-IRAK-1 complex and blocking the phosphorylation and dissociation of IRAK-1. Its function in the ischemic stroke is unknown. Objective: This study aims to investigate whether IRAK-M deficiency could exacerbate neuroinflammation and neurovascular injuries during cerebral ischemia and reperfusion. Methods: Male C57BL/6 mice and Irak3 knockout mice were subjected to 45 min of middle cerebral artery occlusion and 4 or 24 h of reperfusion. Transcription of Irak3 gene was evaluated by quantitative real-time PCR (qRT-PCR). Then, infarct volume, neurological score, brain water content, and Evans blue leakage were compared between knock-out and wild-type mice after reperfusion. Through the observation of gross brain specimen after cerebral ischemia, the incidence of hemorrhage transformation was compared between KO and WT mice. To explore underlying signaling pathways involved in IRAK-M deficiency, major proinflammatory cytokines and NF-κB signaling were measured by qRT-PCR and Western blot. Results: The expression of IRAK-M peaked at 1 h after reperfusion, and then gradually decreased within the first 24 h, which was abolished by blocking the expression of hypoxia induced factor 1α. IRAK-M deficiency increased infarct volume, brain edema, the incidence of hemorrhage transformation, and the permeability of blood-brain barrier. In addition, the NF-κB-mediated expressions of proinflammatory cytokines and the activation of microglia in the ipsilateral brain from knock-out mice were much higher than those in wild-type littermates. Conclusion: IRAK-M deletion exacerbates neurovascular damages which are related to the pronounced activation of NF-κB signaling and neuroinflammatory responses during cerebral ischemia-reperfusion in mice. Our study indicates that IRAK-M has neuroprotective effect and has potential to facilitate the development of new pharmaceuticals that reduce neurovascular complications.

Project description:Rationale: Stroke is a leading causes of human death worldwide. Ischemic damage induces the sterile neuroinflammation, which directly determines the recovery of patients. Lipids, a major component of the brain, significantly altered after stroke. Cholesterol sulfate, a naturally occurring analog of cholesterol, can directly regulate immune cell activation, indicating the possible involvement of cholesterol metabolites in neuroinflammation. Sulfotransferase family 2b member 1 (Sult2b1) is the key enzyme that catalyzes the synthesis of cholesterol sulfate. This study aimed to investigate the function of Sult2b1 and cholesterol sulfate in the neuroinflammation after ischemic stroke. Methods and Results: Sult2b1 -/- and wild-type mice were subjected to transient middle cerebral artery occlusion. Our data showed that Sult2b1 -/- mice had larger infarction and worse neurological scores. To determine whether immune cells were involved in the worsening stroke outcome in Sult2b1 -/- mice, bone marrow transplantation, immune cell depletion, and adoptive monocyte transfer were performed. Combined with CyTOF and immunofluorescence techniques, we demonstrated that after stroke, the peripheral monocyte-derived macrophages were the dominant cell type promoting the pro-inflammatory status in Sult2b1 -/-mice. Using primary bone marrow-derived macrophages, we showed that cholesterol sulfate could attenuate the pro-inflammatory polarization of macrophages under both normal and oxygen-glucose deprivation conditions by regulating the levels of nicotinamide adenine dinucleotide phosphate (NADPH), reactive oxygen species (ROS), and activating the AMP-activated protein kinase (AMPK) - cAMP responsive element-binding protein (CREB) signaling pathway. Conclusions: Sult2b1 -/- promoted the polarization of macrophages into pro-inflammatory status. This trend could be attenuated by adding cholesterol sulfate, which promotes the polarization of macrophages into anti-inflammatory status by metabolic regulation. In this study, we established an inflammation-metabolism axis during the macrophage polarization after ischemic stroke.

Project description:BACKGROUND:As the primary immune response cell in the central nervous system, microglia constantly monitor the microenvironment and respond rapidly to stress, infection, and injury, making them important modulators of neuroinflammatory responses. In diseases such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, and human immunodeficiency virus-induced dementia, activation of microglia precedes astrogliosis and overt neuronal loss. Although microgliosis is implicated in manganese (Mn) neurotoxicity, the role of microglia and glial crosstalk in Mn-induced neurodegeneration is poorly understood. METHODS:Experiments utilized immunopurified murine microglia and astrocytes using column-free magnetic separation. The effect of Mn on microglia was investigated using gene expression analysis, Mn uptake measurements, protein production, and changes in morphology. Additionally, gene expression analysis was used to determine the effect Mn-treated microglia had on inflammatory responses in Mn-exposed astrocytes. RESULTS:Immunofluorescence and flow cytometric analysis of immunopurified microglia and astrocytes indicated cultures were 97 and 90% pure, respectively. Mn treatment in microglia resulted in a dose-dependent increase in pro-inflammatory gene expression, transition to a mixed M1/M2 phenotype, and a de-ramified morphology. Conditioned media from Mn-exposed microglia (MCM) dramatically enhanced expression of mRNA for Tnf, Il-1?, Il-6, Ccl2, and Ccl5 in astrocytes, as did exposure to Mn in the presence of co-cultured microglia. MCM had increased levels of cytokines and chemokines including IL-6, TNF, CCL2, and CCL5. Pharmacological inhibition of NF-?B in microglia using Bay 11-7082 completely blocked microglial-induced astrocyte activation, whereas siRNA knockdown of Tnf in primary microglia only partially inhibited neuroinflammatory responses in astrocytes. CONCLUSIONS:These results provide evidence that NF-?B signaling in microglia plays an essential role in inflammatory responses in Mn toxicity by regulating cytokines and chemokines that amplify the activation of astrocytes.

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CX3CR1 deficiency suppresses activation and neurotoxicity of microglia/macrophage in experimental ischemic stroke.

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CX3CR1 deficiency suppresses activation and neurotoxicity of microglia/macrophage in experimental ischemic stroke.

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