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HMGB1/RAGE axis mediates stress-induced RVLM neuroinflammation in mice via impairing mitophagy flux in microglia.

ABSTRACT: BACKGROUND:Microglial mediated neuroinflammation in the rostral ventrolateral medulla (RVLM) plays roles in the etiology of stress-induced hypertension (SIH). It was reported that autophagy influenced inflammation via immunophenotypic switching of microglia. High-mobility group box 1 (HMGB1) acts as a regulator of autophagy and initiates the production of proinflammatory cytokines (PICs), but the underlying mechanisms remain unclear. METHODS:The stressed mice were subjected to intermittent electric foot shocks plus noises administered for 2?h twice daily for 15 consecutive days. In mice, blood pressure (BP) and renal sympathetic nerve activity (RSNA) were monitored by noninvasive tail-cuff method and platinum-iridium electrodes placed respectively. Microinjection of siRNA-HMGB1 (siHMGB1) into the RVLM of mice to study the effect of HMGB1 on microglia M1 activation was done. mRFP-GFP-tandem fluorescent LC3 (tf-LC3) vectors were transfected into the RVLM to evaluate the process of autolysosome formation/autophagy flux. The expression of RAB7, lysosomal-associated membrane protein 1 (LAMP1), and lysosomal pH change were used to evaluate lysosomal function in microglia. Mitophagy was identified by transmission electron microscopic observation or by checking LC3 and MitoTracker colocalization under a confocal microscope. RESULTS:We showed chronic stress increased cytoplasmic translocations of HMGB1 and upregulation of its receptor RAGE expression in microglia. The mitochondria injury, oxidative stress, and M1 polarization were attenuated in the RVLM of stressed Cre-CX3CR1/RAGEfl/fl mice. The HMGB1/RAGE axis increased at the early stage of stress-induced mitophagy flux while impairing the late stages of mitophagy flux in microglia, as revealed by decreased GFP fluorescence quenching of GFP-RFP-LC3-II puncta and decreased colocalization of lysosomes with mitochondria. The expressions of RAB7 and LAMP1 were decreased in the stressed microglia, while knockout of RAGE reversed these effects and caused an increase in acidity of lysosomes. siHMGB1 in the RVLM resulted in BP lowering and RSNA decreasing in SIH mice. When the autophagy inducer, rapamycin, is used to facilitate the mitophagy flux, this treatment results in attenuated NF-?B activation and reduced PIC release in exogenous disulfide HMGB1 (ds-HMGB1)-stimulated microglia. CONCLUSIONS:Collectively, we demonstrated that inhibition of the HMGB1/RAGE axis activation led to increased stress-induced mitophagy flux, hence reducing the activity of microglia-mediated neuroinflammation and consequently reduced the sympathetic vasoconstriction drive in the RVLM.

SUBMITTER: Zhang S

PROVIDER: S-EPMC6953162 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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HMGB1/RAGE axis mediates stress-induced RVLM neuroinflammation in mice via impairing mitophagy flux in microglia.

Zhang Shutian S Hu Li L Jiang Jialun J Li Hongji H Wu Qin Q Ooi Kokwin K Wang Jijiang J Feng Yi Y Zhu Danian D Xia Chunmei C

Journal of neuroinflammation 20200110 1

<h4>Background</h4>Microglial mediated neuroinflammation in the rostral ventrolateral medulla (RVLM) plays roles in the etiology of stress-induced hypertension (SIH). It was reported that autophagy influenced inflammation via immunophenotypic switching of microglia. High-mobility group box 1 (HMGB1) acts as a regulator of autophagy and initiates the production of proinflammatory cytokines (PICs), but the underlying mechanisms remain unclear.<h4>Methods</h4>The stressed mice were subjected to int ...[more]

PMID: 31924219

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Project description:BACKGROUND:Spinal cord injury (SCI) favors a persistent pro-inflammatory macrophages/microglia-mediated response with only a transient appearance of anti-inflammatory phenotype of immune cells. However, the mechanisms controlling this special sterile inflammation after SCI are still not fully elucidated. It is known that damage-associated molecular patterns (DAMPs) released from necrotic cells after injury can trigger severe inflammation. High mobility group box 1(HMGB1), a ubiquitously expressed DNA binding protein, is an identified DAMP, and our previous study demonstrated that reactive astrocytes could undergo necroptosis and release HMGB1 after SCI in mice. The present study aimed to explore the effects and the possible mechanism of HMGB1on macrophages/microglia polarization, as well as the neuroprotective effects by HMGB1 inhibition after SCI. METHODS:In this study, the expression and the concentration of HMGB1 was determined by qRT-PCR, ELISA, and immunohistochemistry. Glycyrrhizin was applied to inhibit HMGB1, while FPS-ZM1 to suppress receptor for advanced glycation end products (RAGE). The polarization of macrophages/microglia in vitro and in vivo was detected by qRT-PCR, immunostaining, and western blot. The lesion area was detected by GFAP staining, while neuronal survival was examined by Nissl staining. Luxol fast blue (LFB) staining, DAB staining, and western blot were adopted to evaluate the myelin loss. Basso-Beattie-Bresnahan (BBB) scoring and rump-height Index (RHI) assay was applied to evaluate locomotor functional recovery. RESULTS:Our data showed that HMGB1 can be elevated and released from necroptotic astrocytes and HMGB1 could induce pro-inflammatory microglia through the RAGE-nuclear factor-kappa B (NF-?B) pathway. We further demonstrated that inhibiting HMGB1 or RAGE effectively decreased the numbers of detrimental pro-inflammatory macrophages/microglia while increased anti-inflammatory cells after SCI. Furthermore, our data showed that inhibiting HMGB1 or RAGE significantly decreased neuronal loss and demyelination, and improved functional recovery after SCI. CONCLUSIONS:The data implicated that HMGB1-RAGE axis contributed to the dominant pro-inflammatory macrophages/microglia-mediated pro-inflammatory response, and inhibiting this pathway afforded neuroprotection for SCI. Thus, therapies designed to modulate immune microenvironment based on this cascade might be a prospective treatment for SCI.

Project description:AIM: High mobility group box protein 1 (HMGB1) and receptor for the advanced glycation end product (RAGE) play pivotal roles in vascular inflammation and atherosclerosis. The aim of this study was to determine whether the HMGB1-RAGE axis was involved in the actions of simvastatin on vascular inflammation and atherosclerosis in ApoE(-/-) mice. METHODS: Five-week old ApoE(-/-) mice and wild-type C57BL/6 mice were fed a Western diet. At 8 weeks of age, ApoE(-/-) mice were administered simvastatin (50 mg·kg(-1)·d(-1)) or vehicle by gavage, and the wild-type mice were treated with vehicle. The mice were sacrificed at 11 weeks of age, and the atherosclerotic lesions in aortic sinus were assessed with Oil Red O staining. Macrophage migration was determined with scanning EM and immunohistochemistry. Human umbilical vein endothelial cells (HUVECs) were used for in vitro study. Western blots were used to quantify the protein expression of HMGB1, RAGE, vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1). RESULTS: Vehicle-treated ApoE(-/-) mice exhibited significant increases in aortic inflammation and atherosclerosis as well as enhanced expression of HMGB1, RAGE, VCAM-1, and MCP-1 in aortic tissues as compared to the wild-type mice. Furthermore, serum total cholesterol, triglyceride and LDL levels were markedly increased, while serum HDL level was decreased in vehicle-treated ApoE(-/-) mice. Administration with simvastatin in ApoE(-/-) mice markedly attenuated the vascular inflammation and atherosclerotic lesion area, and decreased the aortic expression of HMGB1, RAGE, VCAM-1, and MCP-1. However, simvastatin did not affect the abnormal levels of serum total cholesterol, triglyceride, LDL and HDL in ApoE(-/-) mice. Exposure of HUVECs to HMGB1 (100 ng/mL) markedly increased the expression of HMGB1, RAGE and VCAM-1, whereas pretreatment of the cells with simvastatin (10 μmol/L) blocked the HMGB1-caused changes. CONCLUSION: Simvastatin inhibits vascular inflammation and atherosclerosis in ApoE(-/-) mice, which may be mediated through downregulation of the HMGB1-RAGE axis.

Dataset Information

HMGB1/RAGE axis mediates stress-induced RVLM neuroinflammation in mice via impairing mitophagy flux in microglia.

Publications

HMGB1/RAGE axis mediates stress-induced RVLM neuroinflammation in mice via impairing mitophagy flux in microglia.

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