Response Of Microglial Cells Research Articles

Regulation of genes involved in the metabolic adaptation of murine microglial cells in response to elevated HIF-1α mediated activation.

Microglia cells are activated in response to different stress signals. Several metabolic adaptations underlie microglia activation in the brain. Among these, in conditions like ischemic strokeand, hypoxic stress stimuli activate microglia cells. Hypoxic stress is mediated by HIF-1α. Although HIF-1α has been implicated in the alteration of metabolic pathways, changes in microglia lipid metabolism during M1 activation of microglia induced by elevated HIF-1α levels are yet to be understood. This can also merit interest in the development of novel targets to mitigate chronic inflammation. Our study aims to elucidate the transcriptional regulation of metabolic pathways in microglia cells during HIF-1α mediated activation. To study the adaptations in the metabolic pathways we induced microglia activation, by activating HIF-1α. Here, we show that microglia cells activated in response to elevated HIF-1α require ongoing lipogenesis and fatty acid breakdown. Notably, autophagy is activated during the initial stages of microglia activation. Inhibition of autophagy in activated microglia affects their viability and phagocytic activity. Collectively, our study expands the understanding of the molecular link between autophagy, lipid metabolism, and inflammation during HIF-1α mediated microglial activation that can lead to the development of promising strategies for controlling maladaptive activation states of microglia responsible for neuroinflammation. Together, our findings suggest that the role of HIF-1α in regulating metabolic pathways during hypoxia in microglia is beyond optimization of glucose utilization and distinctly regulates lipid metabolism during pro-inflammatory activation.

Sortilin inhibition in microglial cells cannot alleviate ischemia and hypoxia-induced neuronal injury in co-culture.

Sortilin is a single-pass type I transmembrane protein which can bind to various cargo proteins, regulating their surface location, secretion, or degradation in lysosomes. In our previous study, we found that sortilin can regulate progranulin expression by transporting it to lysosomes and reduce neuronal cell injury in hypoxia-ischemia, but the expression and function of sortilin in microglial cells during hypoxia-ischemia are unknown. The purpose of this study was to further investigate the function of sortilin in microglial cells and its effect on neuron cells. In rat BV2 microglial cells, sortilin was knocked down by lentivirus. After oxygen-glucose deprivation/reperfusion (OGD/R), expression of sortilin, progranulin (PGRN) and JNK pathway was detected by western blot, immunofluorescence was used to show the localization of PGRN, secretion of TNFα/IL-6 was measured by Elisa. Then co-culture microglial cells with neuron cells during hypoxia-ischemia and detected the neuron injury by CCK-8 and TUNEL. The expression of sortilin, mature and cleaved PGRN were all increased after OGD/R in microglial cells. Furthermore, sortilin inhibition accompany with less PGRN localization in lysosomes and more mature and less cleaved PGRN expression in microglial cells. Sortilin inhibition also can reduce the inflammatory response in microglial cells, but it does not alleviate neuronal injury in co-culture. This study demonstrated that sortilin can regulate the expression of PGRN and reduce the inflammatory response in microglial cells. However, only inhibiting sortilin in microglial cells did not have an impact on the survival of neurons during ischemia-hypoxia.

Amlexanox attenuates LPS-induced neuroinflammatory responses in microglial cells via inhibition of NF–κB and STAT3 signaling pathways

Amlexanox is an anti-inflammatory and anti-allergic agent used clinically for the treatment of aphthous ulcers, allergic rhinitis, and asthma. Recent studies have demonstrated that amlexanox, a selective inhibitor of IkB kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1), suppresses a range of diseases or inflammatory conditions, such as obesity-related metabolic dysfunction and type 2 diabetes. However, the effects of amlexanox on neuroinflammatory responses to amlexanox have not yet been comprehensively studied. In this study, we investigated the novel therapeutic effect of amlexanox on LPS-induced neuroinflammation in vivo, and intraperitoneal injection of amlexanox markedly reduced LPS-induced IKKε levels, proinflammatory cytokines, and microglial activation, as evidenced by ionized calcium-binding adapter molecule 1 (Iba1) immunostaining. Furthermore, amlexanox significantly reduced proinflammatory cytokines and chemokines in LPS-induced bone marrow-derived macrophages (BMDM), murine BV2, and human HMC3 microglial cells. This data provided considerable evidence that amlexanox can be used as a preventive and curative therapy for neuroinflammatory and neurodegenerative diseases. In terms of mechanism aspects, our results demonstrated that the anti-inflammatory action of amlexanox in BV2 microglial cells was through the downregulation of NF-κB and STAT3 signaling pathways. In addition, the combination of amlexanox and SPI (a STAT3 selective inhibitor) showed high efficiency in inhibiting the production of neurotoxic and pro-inflammatory mediators. Overall, our data provide rational insights into the mechanisms of amlexanox as a potential therapeutic strategy for neuroinflammation-related diseases.

Simulated microgravity induces DNA damage concurrent with impairment of DNA repair and activation of cell-type specific DNA damage response in microglial and glioblastoma cells

Long-term spaceflights affect the structural changes in brain, alter motor or cognitive function and associated development of neuro-optic syndrome in astronauts. Studies addressing the impact of microgravity on brain cells are very limited. Herein, we employed microglial (CHME3) and glioblastoma (U87MG and A172) cells to study their molecular and functional adaptations under simulated microgravity (SMG) exposure. A reduction in cell viability and proliferation with decreased levels of PCNA were observed in these cells. SMG caused extensive DNA damage with an increase in γH2A.X (ser139) phosphorylation and differential activation/expression of DNA damage response (DDR) proteins including ATM, ATR, Chk1, Chk2 and p53 in all the three cell lines. Unlike CHME3, the ATM/Chk2-dependent DDR pathway was activated in glioblastoma cells suggesting a marked difference in the adaptation between normal and cancer cells to SMG. Five different classes of DNA repair pathways including BER, NER, MMR, NHEJ and HR were suppressed in both cell lines with the notable exception of NHEJ (Ku70/80 and DNA-PK) activation in U87MG cells. SMG induced mitochondrial apoptosis with increased expression of Bax, cleaved caspase-3 and cleaved poly-(ADP-ribose) polymerase, and reduced Bcl-2 level. SMG triggered apoptosis simultaneously via ERK1/2 and AKT activation, and inhibition of GSK3β activity which was reversed by MEK1 and PI3K inhibitors. Taken together, our study revealed that microgravity is a strong stressor to trigger DNA damage and apoptosis through activation of ERK1/2 and AKT, and impairment of DNA repair capacity, albeit with a cell-type difference in DDR and NHEJ regulation, in microglial and glioblastoma cells.

Multi-Omics Profiling Identifies Microglial Annexin A2 as a Key Mediator of NF-κB Pro-inflammatory Signaling in Ischemic Reperfusion Injury

Cerebral stroke is one of the leading causes of mortality and disability worldwide. Restoring the cerebral circulation following a period of occlusion and subsequent tissue oxygenation leads to reperfusion injury. Cerebral ischemic reperfusion (I/R) injury triggers immune and inflammatory responses, apoptosis, neuronal damage, and even death. However, the cellular function and molecular mechanisms underlying cerebral I/R-induced neuronal injury are incompletely understood. By integrating proteomic, phosphoproteomic, and transcriptomic profiling in mouse hippocampi after cerebral I/R, we revealed that the differentially expressed genes and proteins mainly fall into several immune inflammatory response-related pathways. We identified that Annexin 2 (Anxa2) was exclusively upregulated in microglial cells in response to cerebral I/R invivo and oxygen-glucose deprivation and reoxygenation (OGD/R) invitro. RNA-seq analysis revealed a critical role of Anxa2 in the expression of inflammation-related genes in microglia via the NF-κB signaling. Mechanistically, microglial Anxa2 is required for nuclear translocation of the p65 subunit of NF-κB and its transcriptional activity upon OGD/R in BV2 microglial cells. Anxa2 knockdown inhibited the OGD/R-induced microglia activation and markedly reduced the expression of pro-inflammatory factors, including TNF-α, IL-1β, and IL-6. Interestingly, conditional medium derived from Anxa2-depleted BV2 cell cultures with OGD/R treatment alleviated neuronal death invitro. Altogether, our findings revealed that microglia Anxa2 plays a critical role in I/R injury by regulating NF-κB inflammatory responses in a non-cell-autonomous manner, which might be a potential target for the neuroprotection against cerebral I/R injury.

New Dihydropyridine Derivative Attenuates NF-κB Activation via Suppression of Calcium Influx in a Mouse BV-2 Microglial Cell Line.

Activated microglia contribute to many neuroinflammatory diseases in the central nervous system. In this study, we attempted to identify an anti-inflammatory compound that could suppress microglial activation. We performed high-throughput screening with a chemical library developed at our institute. We performed a luciferase assay of nuclear factor-kappa B (NF-κB) reporter stable HT22 cells and identified a compound that was confirmed to inhibit the anti-inflammatory response in BV2 microglial cells. The selected dihydropyridine derivative can suppress the expression response of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor (TNF), as well as NF-κB phosphorylation and nuclear translocation, and reduce the intracellular calcium level. Thus, our identified compound has a potential role in suppressing microglial activation and may contribute to the development of a new therapeutic molecule against neuroinflammatory diseases.

Foeniculum vulgare Mill. inhibits lipopolysaccharide-induced microglia activation and ameliorates neuroinflammation-mediated behavioral deficits in mice

Objective: To investigate the effect of Foeniculum vulgare extract against lipopolysaccharide (LPS)-induced microglial activation in vitro as well as cognitive behavioral deficits in mice. Methods: LPS-activated BV-2 cell viability was measured using MTT assay and reactive oxygen species (ROS) was studied using DCF-DA assay. The antioxidative enzymes and pro-inflammatory mediators were analyzed using respective ELISA kits and Western blotting. For in vivo testing, LPS (1 mg/kg, i.p.) was given daily for five days in male Swiss albino mice to produce chronic neuroinflammation. Cognitive and behavioral tests were performed using open-field, passive avoidance, and rotarod experiments in LPS-induced mice. Results: Foeniculum vulgare extract (25, 50 and 100 μg/mL) significantly attenuated the LPS-activated increase in nitric oxide (NO), ROS, cyclooxygenase-2, inducible NO synthase, IL-6, and TNF-alpha (P < 0.05). Moreover, LPS-induced oxidative stress and reduced antioxidative enzyme levels were significantly improved by Foeniculum vulgare extract (P < 0.05). The extract also regulated the NF-κB/MAPK signaling in BV-2 cells. In an in vivo study, Foeniculum vulgare extract (50, 100, and 200 mg/kg) markedly mitigated the LPS-induced cognitive and locomotor impairments in mice. The fingerprinting analysis showed distinctive peaks with rutin, kaempferol-3-O-glucoside, and anethole as identifiable compounds. Conclusions: Foeniculum vulgare extract can ameliorate LPS-stimulated neuroinflammatory responses in BV-2 microglial cells and improve cognitive and locomotor performance in LPS-administered mice.

Insulin Mediates Lipopolysaccharide-Induced Inflammatory Responses and Oxidative Stress in BV2 Microglia.

Insulin, the key hormone for glucose regulation, has garnered attention for its role as an immune modulator. Impaired insulin signaling in the central nervous system is linked to neuroinflammation and neurodegenerative diseases. Microglia, the resident macrophage-like immune cells in the brain, are key regulators of neuroinflammation. However, the mechanisms by which insulin influences microglial immune responses remain relatively unknown. This study aimed to assess the effects of post-treatment with insulin [30minutes after lipopolysaccharide (LPS) exposure] on LPS-induced inflammatory responses in BV2 microglial cells. Post-treatment with insulin potentiated LPS-induced production of nitric oxide and pro-inflammatory cytokines, such as TNF and IL-6, through activation of the Akt/NF-κB pathway. Insulin also enhanced the ability of BV2 cells to phagocytose bacteria particles and β-amyloid fibrils. Conversely, insulin inhibited activation of NADPH oxidase and reduced intracellular levels of reactive oxygen species in LPS-treated BV2 cells. Insulin enhances microglial immune competence when challenged by endotoxins but mitigates oxidative stress in these cells.

Potential neuroprotective effects of anti‐cytokine antibodies against lipopolysaccharide‐induced microglial inflammation

Aims/Purpose: Microglia‐mediated neuroinflammation is involved in many neurodegenerative diseases, including glaucoma and diabetic retinopathy. This study examined the effects of anti‐cytokine antibodies on LPS‐induced inflammatory response of microglial cells, and explored the underlying molecular mechanisms by proteomic analysis.Methods: A lipopolysaccharide (LPS)‐induced neuroinflammation model was established in murine BV‐2 microglial cells in vitro. The expression of microglial cell markers (CD68 and CD206) were detected by immunocytochemistry. BV‐2 was stimulated with LPS (1 μg/mL) with or without anti‐tumour necrosis factor‐alpha (anti‐TNF‐α, 2 μg /ml) or anti‐interleukin‐1 beta (anti‐IL‐1β, 4 μg /ml) for 24 h. LPS‐induced inflammation level was analysed using the Griess assay to determine nitric oxide (NO) concentration. Proteome alterations were characterized employing the mass spectrometry‐based proteomics and bioinformatics analyses.Results: Immunocytochemistry confirmed the expression of microglial markers in BV‐2 cells. LPS stimulation induced excessive activation of BV‐2, with significant increase in NO generation (p < 0.0001). Anti‐IL‐1β significantly attenuated 34% of the LPS‐induced NO generation (p < 0.0001). Interestingly, anti‐TNF‐α showed a stronger anti‐inflammatory effect by reducing 57% of LPS‐induced NO generation (p < 0.0001). Bioinformatics analyses revealed that differentially expressed proteins involved in inflammation (p = 3.8 × 10−5) and apoptotic (p = 3.2 × 10−11) signalling pathways were significantly activated by LPS. Particularly, LPS‐mediated upregulation of STAT1 (p = 8.6 × 10−6) was restored by anti‐IL‐1β and anti‐TNF‐α (p = 3.1 × 10−4 and p = 2.7 × 10−3, respectively). Anti‐IL‐1β induced proteome alterations involved in anti‐inflammatory signalling pathways and promote glial cell polarization towards neuroprotective M2 phenotype (p = 5.3 × 10−6), while anti‐TNF‐α treatment significantly regulated proteins involved in immune response of macrophages (p = 2.7 × 10−8).Conclusions: In summary, anti‐TNF‐α and anti‐IL‐1β antibodies alleviated LPS‐induced neuroinflammation and altered proteins involved in neuroprotective pathways. Hence, anti‐cytokine antibodies have therapeutic potential as neuroprotective agents.

CGAS Mediates the Inflammatory Responses of Human Microglial Cells to Genotoxic DNA Damage.

Genomic instability is a key driving force for the development and progression of many age-related neurodegenerative diseases and central nervous system (CNS) cancers. Recently, the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), has been shown to detect and respond to self-DNA accumulation resulting from DNA damaging insults in peripheral cell types. cGAS has been shown to be important in the responses of microglia to DNA viruses and amyloid beta, and we have reported that it underlies the responses of human microglia to exogenous DNA. However, the role of this cytosolic sensor in the detection of self-DNA by glia is poorly understood and its ability to mediate the cellular responses of human microglia to genotoxic DNA damage has not been established. Here, we describe the ability of ionizing radiation and oxidative stress to elicit genomic DNA damage in human microglial cells and to stimulate the production of key inflammatory mediators by these cells in an NF-kB dependent manner. Importantly, we have utilized CRISPR/Cas9 and siRNA-mediated knockdown approaches and a pharmacological inhibitor of the cGAS adaptor protein stimulator of interferon genes (STING) to demonstrate that the cGAS-STING pathway plays a critical role in the generation of these microglial immune responses to such genotoxic insults. Together, these studies support the notion that cGAS mediates the detection of cytosolic self-DNA by microglia, providing a potential mechanism linking genomic instability to the development of CNS cancers and neurodegenerative disorders.

Immune response of BV-2 microglial cells is impacted by peroxisomal beta-oxidation.

Microglia are crucial for brain homeostasis, and dysfunction of these cells is a key driver in most neurodegenerative diseases, including peroxisomal leukodystrophies. In X-linked adrenoleukodystrophy (X-ALD), a neuroinflammatory disorder, very long-chain fatty acid (VLCFA) accumulation due to impaired degradation within peroxisomes results in microglial defects, but the underlying mechanisms remain unclear. Using CRISPR/Cas9 gene editing of key genes in peroxisomal VLCFA breakdown (Abcd1, Abcd2, and Acox1), we recently established easily accessible microglial BV-2 cell models to study the impact of dysfunctional peroxisomal β-oxidation and revealed a disease-associated microglial-like signature in these cell lines. Transcriptomic analysis suggested consequences on the immune response. To clarify how impaired lipid degradation impacts the immune function of microglia, we here used RNA-sequencing and functional assays related to the immune response to compare wild-type and mutant BV-2 cell lines under basal conditions and upon pro-inflammatory lipopolysaccharide (LPS) activation. A majority of genes encoding proinflammatory cytokines, as well as genes involved in phagocytosis, antigen presentation, and co-stimulation of T lymphocytes, were found differentially overexpressed. The transcriptomic alterations were reflected by altered phagocytic capacity, inflammasome activation, increased release of inflammatory cytokines, including TNF, and upregulated response of T lymphocytes primed by mutant BV-2 cells presenting peptides. Together, the present study shows that peroxisomal β-oxidation defects resulting in lipid alterations, including VLCFA accumulation, directly reprogram the main cellular functions of microglia. The elucidation of this link between lipid metabolism and the immune response of microglia will help to better understand the pathogenesis of peroxisomal leukodystrophies.

The Influence of Nucleoside Reverse Transcriptase Inhibitors on Mitochondrial Activity, Lipid Content, and Fatty-Acid-Binding Protein Levels in Microglial HMC3 Cells.

Despite the availability of a wide range of preventive measures and comprehensive treatment options following infection, the development of acquired immunodeficiency syndrome (AIDS) remains a persistent challenge. Nucleoside reverse transcriptase inhibitors (NRTIs) represent the most commonly utilized therapeutic approach, despite being on the pharmaceutical market for nearly four decades. During this time, a spectrum of side effects ranging from mild discomfort and hypersensitivity reactions to the more prevalent nephrotoxicity and hepatotoxicity has been documented. In light of these considerations, our study aimed to investigate the impacts of two NRTIs, lamivudine and zidovudine, on lipid metabolism in HMC3 microglial cells. Our findings revealed statistically significant reductions in the ATP levels (nearly 8%) and increased mitochondrial superoxide levels (around 10%) after 24 h of treatment with the maximum therapeutic concentration of zidovudine compared to the untreated microglial cells. Furthermore, the concentrations of fatty-acid-binding proteins 4 and 5 were significantly lower (approximately 40%) in the microglial cells that were exposed to NRTIs than in the untreated cells. Notably, the total lipid concentration within the microglial cells markedly increased following NRTI administration with a 13% rise after treatment with 10 µM lamivudine and a remarkable 70% surge following the administration of 6 µM zidovudine. These results suggest that the prolonged administration of NRTIs may potentially lead to lipid accumulation, posing a significant risk to the delicate homeostasis of the neuronal system and potentially triggering a pro-inflammatory response in microglial cells.

Naegleria fowleri Extracellular Vesicles Induce Proinflammatory Immune Responses in BV-2 Microglial Cells.

Extracellular vesicles (EVs) of protozoan parasites have diverse biological functions that are essential for parasite survival and host-parasite interactions. In this study, we characterized the functional properties of EVs from Naegleria fowleri, a pathogenic amoeba that causes a fatal brain infection called primary amoebic meningoencephalitis (PAM). N. fowleri EVs (NfEVs) have been shown to be internalized by host cells such as C6 glial cells and BV-2 microglial cells without causing direct cell death, indicating their potential roles in modulating host cell functions. NfEVs induced increased expression of proinflammatory cytokines and chemokines such as TNF-α, IL-1α, IL-1β, IL-6, IL-17, IFN-γ, MIP-1α, and MIP-2 in BV-2 microglial cells; these increases were initiated via MyD88-dependent TLR-2/TLR-4. The production levels of proinflammatory cytokines and chemokines in NfEVs-stimulated BV-2 microglial cells were effectively downregulated by inhibitors of MAPK, NF-κB, or JAK-STAT. Phosphorylation levels of JNK, p38, ERK, p65, JAK-1, and STAT3 were increased in NfEVs-stimulated BV-2 microglial cells but were effectively suppressed by each corresponding inhibitor. These results suggest that NfEVs could induce proinflammatory immune responses in BV-2 microglial cells via the NF-κB-dependent MAPK and JAK-STAT signaling pathways. Taken together, these findings suggest that NfEVs are pathogenic factors involved in the contact-independent pathogenic mechanisms of N. fowleri by inducing proinflammatory immune responses in BV-2 microglial cells, further contributing to deleterious inflammation in infected foci by activating subsequent inflammation cascades in other brain cells.

The STING inhibitor C-176 attenuates MPTP-induced neuroinflammation and neurodegeneration in mouse parkinsonian models

Recent emerging evidence reveals that cGAS-STING-mediated Type I interferon (IFN) signaling axis takes part in the microglial-associated neuroinflammation. However, the potential role of pharmacological inhibition of STING on neuroinflammation and dopaminergic neurodegeneration remains unknown. In the present study, we investigated whether pharmacological inhibition of STING attenuates neuroinflammation and neurodegeneration in experimental models of Parkinson’s disease. We report that therapeutic inhibition of STING with C-176 significantly inhibited the activation of downstream signaling pathway, suppressed neuroinflammation, and ameliorated MPTP-induced dopaminergic neurotoxicity and motor deficit. Furthermore, pharmacological inhibition of STING with C-176 attenuated proinflammatory response in BV2 microglial cells exposed to LPS/MPP+. More importantly, C-176 also reduced NLRP3 inflammasome activation both in vitro and in vivo. The results of our study suggest that pharmacologic inhibition of STING protects against dopaminergic neurodegeneration and neuroinflammation that may act at least in part through suppressing NLRP3 inflammasome activation. STING signaling may hold great promise for the development of new treatment strategy for PD.

Ferulic Acid reduces amyloid beta mediated neuroinflammation through modulation of Nurr1 expression in microglial cells.

Microglial cells (MGCs) serve as the resident macrophages in the brain and spinal cord, acting as the first line of immune defense against pathological changes. With various phenotypes, they can shift from a homeostatic state to a reactive state or transit from a reactive to a non-inflammatory reactive state (alternative homeostatic). A well-timed transit is crucial in limiting excessive microglial reaction and promoting the healing process. Studies indicate that increased Nurr1 expression promotes anti-neuroinflammatory responses in the brain. In this study, we investigated the possible role of ferulic acid (FA) in facilitating microglia transition due to its anti-inflammatory and Nurr1-inducing effects. MGCs were extracted from the brains of male NMRI mice at postnatal day 2 (P2) and cultured with or without FA and beta-amyloid (Aβ). Real-time qRT-PCR was conducted to measure the expressions of Nurr1, IL-1β, and IL-10 genes. Immunostaining was performed to determine the number of NURR1-positive cells, and the ramification index (RI) of MGCs was calculated using Image J software. Treating MGCs with FA (50 μg/ml) induced Nurr1 and IL-10 expressions, while reducing the level of IL-1β in the absence of Aβ-stress. Further assessments on cells under Aβ-stress showed that FA treatment restored the IL-10 and Nurr1 levels, increased the RI of cells, and the number of NURR1-positive cells. Morphological assessments and measurements of the RI revealed that FA treatment reversed amoeboid and rod-like cells to a ramified state, which is specific morphology for non-inflammatory reactive microglia. To conclude, FA can provide potential alternative homeostatic transition in Aβ-reactive microglia by recruiting the NURR1 dependent anti-inflammatory responses. This makes it a promising therapeutic candidate for suppressing Aβ-induced neuroinflammatory responses in MGCs. Furthermore, given that FA has the ability to increase NURR1 levels in homeostatic microglia, it could be utilized as a preventative medication.

Palmitic Acid Modulates Microglial Cell Response to Metabolic Endotoxemia in an In Vitro Study.

Metabolic endotoxemia (ME) is characterized by a 2-3-fold increase in blood endotoxin levels and low-grade systemic inflammation without apparent infection. ME is usually accompanied by metabolic syndrome, characterized by central obesity and hyperlipidemia. According to numerous studies, ME may lead to functional brain disorders, including cognitive decline, depression, and dementia. In the current in vitro study, we aimed to determine the direct and indirect impact of endotoxin (LPS) and palmitic acid (PA), representing saturated fatty acids, on the inflammatory and oxidative stress response in the human microglial HMC3 cells unstimulated and stimulated with IFNγ. The study's results revealed that direct HMC3 cell exposition to endotoxin and PA increased inflammatory response measured as levels of IL-6 and MCP-1 released into the medium and PGE2 levels in cell lysates. Moreover, direct HMC3 cell treatment with PA and LPS induced oxidative stress, i.e., ROS and COX-2 production and lipid peroxidation. On the contrary, an indirect effect of LPS and PA on microglial cells, assessed as the impact of macrophage metabolites, was much lower regarding the inflammatory response, although still associated with oxidative stress. Interestingly, IFNγ had a protective effect on microglial cells, reducing the production of pro-inflammatory mediators and oxidative stress in HMC3 cells treated directly and indirectly with LPS and PA.

T1AM/TAAR1 System Reduces Inflammatory Response and β-Amyloid Toxicity in Human Microglial HMC3 Cell Line.

Microglial dysfunction is one of the hallmarks and leading causes of common neurodegenerative diseases (NDDs), including Alzheimer's disease (AD) and Parkinson's disease (PD). All these pathologies are characterized by aberrant aggregation of disease-causing proteins in the brain, which can directly activate microglia, trigger microglia-mediated neuroinflammation, and increase oxidative stress. Inhibition of glial activation may represent a therapeutic target to alleviate neurodegeneration. Recently, 3-iodothyronamine (T1AM), an endogenous derivative of thyroid hormone (TH) able to interact directly with a specific GPCR known as trace amine-associated receptor 1 (TAAR1), gained interest for its ability to promote neuroprotection in several models. Nevertheless, T1AM's effects on microglial disfunction remain still elusive. In the present work we investigated whether T1AM could inhibit the inflammatory response of human HMC3 microglial cells to LPS/TNFα or β-amyloid peptide 25-35 (Aβ25-35) stimuli. The results of ELISA and qPCR assays revealed that T1AM was able to reduce microglia-mediated inflammatory response by inhibiting the release of proinflammatory factors, including IL-6, TNFα, NF-kB, MCP1, and MIP1, while promoting the release of anti-inflammatory mediators, such as IL-10. Notably, T1AM anti-inflammatory action in HMC3 cells turned out to be a TAAR1-mediated response, further increasing the relevance of the T1AM/TAAR1 system in the management of NDDs.

3',4'-Dihydroxyflavonol Attenuates Lipopolysaccharide-Induced Neuroinflammatory Responses of Microglial Cells by Suppressing AKT-mTOR and NF-κB Pathways.

Microglia-related neuroinflammation contributes to the pathogenesis of a variety of neurodegenerative disorders such as Alzheimer's disease. The synthetic flavonoid, 3',4'-dihydroxyflavonol (3,3',4'-trihydroxyflavone), has been shown to protect brain or myocardial ischemia reperfusion-induced cell death and prevent the aggregation of amyloid-β protein, a process that causes progressive neurodegeneration in Alzheimer's disease. Here, we explored the anti-neuroinflammatory ability of 3',4'-dihydroxyflavonol in lipopolysaccharide (LPS)-activated MG6 microglial cells. 3',4'-Dihydroxyflavonol attenuated LPS-induced tumor necrosis factor-α and nitric oxide secretion in MG6 cells. LPS-induced phosphorylation of mammalian target of rapamycin (mTOR), nuclear factor-κB (NF-κB), and protein kinase B (AKT) (which are all associated with the neuroinflammatory response in microglia) were attenuated by 3',4'-dihydroxyflavonol treatment. Treatment with the mTOR inhibitor, rapamycin, NF-κB inhibitor, caffeic acid phenethyl ester, or AKT inhibitor, LY294002, also attenuated LPS-induced tumor necrosis factor-α and nitric oxide secretion in MG6 cells. LY294002 treatment attenuated LPS-induced phosphorylation of mTOR and NF-κB in MG6 cells. Hence, our study suggests that 3',4'-dihydroxyflavonol can attenuate the neuroinflammatory response of microglial cells by suppressing the AKT-mTOR and NF-κB pathways.

Lomerizine inhibits LPS-mediated neuroinflammation and tau hyperphosphorylation by modulating NLRP3, DYRK1A, and GSK3α/β.

Lomerizine is a calcium channel blocker that crosses the blood-brain barrier and is used clinically in the treatment of migraines. However, whether lomerizine is beneficial in modulating neuroinflammatory responses has not been tested yet. To assess the potential of lomerizine for repurposing as a treatment for neuroinflammation, we investigated the effects of lomerizine on LPS-induced proinflammatory responses in BV2 microglial cells, Alzheimer's disease (AD) excitatory neurons differentiated from induced pluripotent stem cells (iPSCs), and in LPS-treated wild type mice. In BV2 microglial cells, lomerizine pretreatment significantly reduced LPS-evoked proinflammatory cytokine and NLRP3 mRNA levels. Similarly, lomerizine pretreatment significantly suppressed the increases in Iba-1, GFAP, proinflammatory cytokine and NLRP3 expression induced by LPS in wild-type mice. In addition, lomerizine posttreatment significantly decreased LPS-stimulated proinflammatory cytokine and SOD2 mRNA levels in BV2 microglial cells and/or wild-type mice. In LPS-treated wild-type mice and AD excitatory neurons differentiated from iPSCs, lomerizine pretreatment ameliorated tau hyperphosphorylation. Finally, lomerizine abolished the LPS-mediated activation of GSK3α/β and upregulation of DYRK1A, which is responsible for tau hyperphosphorylation, in wild-type mice. These data suggest that lomerizine attenuates LPS-mediated neuroinflammatory responses and tau hyperphosphorylation and is a potential drug for neuroinflammation- or tauopathy-associated diseases.

Pharmacologic inhibition of NLRP3 reduces the levels of α-synuclein and protects dopaminergic neurons in a model of Parkinson’s disease

BackgroundParkinson’s disease (PD) is characterized by a progressive degeneration of dopaminergic neurons, which leads to irreversible loss of peripheral motor functions. Death of dopaminergic neurons induces an inflammatory response in microglial cells, which further exacerbates neuronal loss. Reducing inflammation is expected to ameliorate neuronal loss and arrest motor dysfunctions. Because of the contribution of the NLRP3 inflammasome to the inflammatory response in PD, we targeted NLRP3 using the specific inhibitor OLT1177®.MethodsWe evaluated the effectiveness of OLT1177® in reducing the inflammatory response in an MPTP neurotoxic model of PD. Using a combination of in vitro and in vivo studies, we analyzed the effects of NLRP3 inhibition on pro-inflammatory markers in the brain, α-synuclein aggregation, and dopaminergic neuron survival. We also determined the effects of OLT1177® on locomotor deficits associated with MPTP and brain penetrance.ResultsTreatment with OLT1177® prevented the loss of motor function, reduced the levels of α-synuclein, modulated pro-inflammatory markers in the nigrostriatal areas of the brain, and protected dopaminergic neurons from degeneration in the MPTP model of PD. We also demonstrated that OLT1177® crosses the blood–brain barrier and reaches therapeutic concentrations in the brain.ConclusionsThese data suggest that targeting the NLRP3 inflammasome by OLT1177® may be a safe and novel therapeutic approach to arrest neuroinflammation and protect against neurological deficits of Parkinson’s disease in humans.