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Side-Chain Immune Oxysterols Induce Neuroinflammation by Activating Microglia.

ABSTRACT: In individuals with Alzheimer's disease, the brain exhibits elevated levels of IL-1β and oxygenated cholesterol molecules (oxysterols). This study aimed to investigate the effects of side-chain oxysterols on IL-1β expression using HMC3 microglial cells and ApoE-deficient mice. Treatment of HMC3 cells with 25-hydroxycholesterol (25OHChol) and 27-hydroxycholesterol (27OHChol) led to increased IL-1β expression at the transcript and protein levels. Additionally, these oxysterols upregulated the surface expression of MHC II, a marker of activated microglia. Immunohistochemistry performed on the mice showed increased microglial expression of IL-1β and MHC II when fed a high-cholesterol diet. However, cholesterol and 24s-hydroxycholesterol did not increase IL-1β transcript levels or MHC II expression. The extent of IL-1β increase induced by 25OHChol and 27OHChol was comparable to that caused by oligomeric β-amyloid, and the IL-1β expression induced by the oxysterols was not impaired by polymyxin B, which inhibited lipopolysaccharide-induced IL-1β expression. Both oxysterols enhanced the phosphorylation of Akt, ERK, and Src, and inhibition of these kinase pathways with pharmacological inhibitors suppressed the expression of IL-1β and MHC II. The pharmacological agents chlorpromazine and cyclosporin A also impaired the oxysterol-induced expression of IL-1β and upregulation of MHC II. Overall, these findings suggest that dysregulated cholesterol metabolism leading to elevated levels of side-chain oxysterols, such as 25OHChol and 27OHChol, can activate microglia to secrete IL-1β through a mechanism amenable to pharmacologic intervention. The activation of microglia and subsequent neuroinflammation elicited by the immune oxysterols can contribute to the development of neurodegenerative diseases.

SUBMITTER: Son Y

PROVIDER: S-EPMC10607006 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Side-Chain Immune Oxysterols Induce Neuroinflammation by Activating Microglia.

Son Yonghae Y Yeo In-Jun IJ Hong Jin-Tae JT Eo Seong-Kug SK Lee Dongjun D Kim Koanhoi K

International journal of molecular sciences 20231018 20

In individuals with Alzheimer's disease, the brain exhibits elevated levels of IL-1β and oxygenated cholesterol molecules (oxysterols). This study aimed to investigate the effects of side-chain oxysterols on IL-1β expression using HMC3 microglial cells and ApoE-deficient mice. Treatment of HMC3 cells with 25-hydroxycholesterol (25OHChol) and 27-hydroxycholesterol (27OHChol) led to increased IL-1β expression at the transcript and protein levels. Additionally, these oxysterols upregulated the surf ...[more]

PMID: 37894967

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Project description:Among Alzheimer's disease (AD) brain hallmarks, the presence of reactive astrocytes was demonstrated to correlate with neuronal loss and cognitive deficits. Evidence indeed supports the role of reactive astrocytes as mediators of changes in neurons, including synapses. However, the complexity and the outcomes of astrocyte reactivity are far from being completely elucidated. Another key role in AD pathogenesis is played by alterations in brain cholesterol metabolism. Oxysterols (cholesterol oxidation products) are crucial for brain cholesterol homeostasis, and we previously demonstrated that changes in the brain levels of various oxysterols correlate with AD progression. Moreover, oxysterols have been shown to contribute to various pathological mechanisms involved in AD pathogenesis. In order to deepen the role of oxysterols in AD, we investigated whether they could contribute to astrocyte reactivity, and consequently impact on neuronal health. Results showed that oxysterols present in mild or severe AD brains induce a clear morphological change in mouse primary astrocytes, accompanied by the upregulation of some reactive astrocyte markers, including lipocalin-2 (Lcn2). Moreover, astrocyte conditioned media analysis revealed a significant increase in the release of Lcn2, cytokines, and chemokines in response to oxysterols. A significant reduction of postsynaptic density protein 95 (PSD95) and a concurrent increase in cleaved caspase-3 protein levels have been demonstrated in neurons co-cultured with oxysterol-treated astrocytes, pointing out that mediators released by astrocytes have an impact on neurons. Among these mediators, Lcn2 has been demonstrated to play a major role on synapses, affecting neurite morphology and decreasing dendritic spine density. These data demonstrated that oxysterols present in the AD brain promote astrocyte reactivity, determining the release of several mediators that affect neuronal health and synapses. Lcn2 has been shown to exert a key role in mediating the synaptotoxic effect of oxysterol-treated astrocytes.

Project description:BackgroundMicroglial activation is a prominent feature of neuroinflammation, which is present in almost all neurodegenerative diseases. While an initial inflammatory response mediated by microglia is considered to be protective, excessive pro-inflammatory response of microglia contributes to the pathogenesis of neurodegeneration. Although autophagy is involved in the suppression of inflammation, its role and mechanism in microglia are unclear.MethodsIn the present study, we studied the mechanism by which lipopolysaccharide (LPS) affects microglial autophagy and the effects of autophagy on the production of pro-inflammatory factors in microglial cells by western blotting, immunocytochemistry, transfection, transmission electron microscopy (TEM), and real-time PCR. In a mouse model of neuroinflammation, generated by intraventricular injection of LPS (5 μg/animal), we induced autophagy by rapamycin injection and investigated the effects of enhanced autophagy on microglial activation by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry.ResultsWe found that autophagic flux was suppressed in LPS-stimulated N9 microglial cells, as evidenced by decreased expression of the autophagy marker LC3-II (lipidated form of MAP1LC3), as well as increased levels of the autophagy adaptor protein SQSTM1. LPS significantly decreased Vps34 expression in N9 microglial cells by activating the PI3KI/AKT/MTOR pathway without affecting the levels of lysosome-associated proteins and enzymes. More importantly, overexpression of Vps34 significantly enhanced the autophagic flux and decreased the accumulation of SQSTM1 in LPS-stimulated N9 microglial cells. Moreover, our results revealed that an LPS-induced reduction in the level of Vps34 prevented the maturation of omegasomes to phagophores. Furthermore, LPS-induced neuroinflammation was significantly ameliorated by treatment with the autophagy inducer rapamycin both in vitro and in vivo.ConclusionsThese data reveal that LPS-induced neuroinflammation in N9 microglial cells is associated with the inhibition of autophagic flux through the activation of the PI3KI/AKT/MTOR pathway, while enhanced microglial autophagy downregulates LPS-induced neuroinflammation. Thus, this study suggests that promoting the early stages of autophagy might be a potential therapeutic approach for neuroinflammation-associated diseases.

Project description:BackgroundNeuroinflammation-induced injury is intimately associated with poor prognosis in patients with cerebral venous sinus thrombosis (CVST). The cyclic GMP-AMP synthase-stimulator of interferon gene (cGAS-STING) axis is a cytoplasmic double-stranded DNA (dsDNA) sensing pathway has recently emerged as a crucial mediator of neuroinflammation in ischemic stroke. However, the role of the cGAS-STING pathway in modulating post-CVST inflammation and the underlying mechanisms involved remain unclear.MethodsA CVST model was induced by ferric chloride in male C57BL/6J mice. The selective cGAS inhibitor RU.521, STING agonist 2'3'-cGAMP, and STING siRNA were delivered by intranasal administration or intraventricular injection. Post-CVST assessments included rotarod test, TUNEL staining, Fluoro-Jade C staining, dihydroethidium staining, western blotting, qPCR, immunofluorescence, immunohistochemistry, ELISA and flow cytometry.ResultscGAS, STING, NLRP3 and GSDMD were significantly upregulated after CVST and mostly in the microglia of the mouse brain. CVST triggered the release of dsDNA into the cytoplasm and elicited an inflammatory response via activating the cGAS-STING axis. RU.521 decreased the levels of 2'3'-cGAMP, STING and downstream inflammatory cytokines, and suppressed the expressions of NLRP3 inflammasome and pyroptosis-pertinent components containing cleaved caspase-1, GSDMD, GSDMD-C, pro- and cleaved IL-1β, and cleaved IL-1β/pro-IL-1β. Besides, RU.521 treatment also reduced oxidative stress, lessened the numbers of microglia and neutrophils, and ameliorated neuronal apoptosis, degeneration along with neurological deficits post-CVST. 2'3'-cGAMP delivery enhanced the expressions of STING and related inflammatory mediators, NLRP3 inflammasome and pyroptosis-relevant proteins, whereas these alterations were significantly abrogated by the silencing of STING by siRNA.ConclusionsOur data demonstrate that repression of the cGAS-STING pathway diminishes the neuroinflammatory burden of CVST and highlight this approach as a potential therapeutic tactic in CVST-mediated pathologies.

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Side-Chain Immune Oxysterols Induce Neuroinflammation by Activating Microglia.

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Side-Chain Immune Oxysterols Induce Neuroinflammation by Activating Microglia.

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