HMO6 Cells Research Articles

Molecular Docking, Synthesis, and Characterization of Furanyl-Pyrazolyl Acetamide and 2,4-Thiazolidinyl-Furan-3-Carboxamide Derivatives as Neuroinflammatory Protective Agents.

Microglia are key immune cells in the brain that maintain homeostasis and defend against immune threats. Targeting the dysfunctional microglia is one of the most promising approaches to inhibit neuroinflammation. In the current study, a diverse series of molecular hybrids were designed and screened through molecular docking against two neuroinflammatory targets, namely HMGB1 (2LY4) and HMGB1 Box A (4QR9) proteins. Based on the outcomes of docking scores fifteen compounds; ten furanyl-pyrazolyl acetamides 11(a-j), and five 2,4-thiazolidinyl-furan-3-carboxamide 15(a-e) derivatives were selected for further synthesis, followed by biological evaluation. The selected compounds, 11(a-j) and 15(a-e) were successfully synthesized with moderate to good yields, and structures were confirmed by IR, NMR, and mass spectra. The in-vitro cytotoxicity was evaluated on microglial cells namely BV-2, N-9, HMO6, leukemic HAP1, and human fibroblast cells. Further western-blot analysis revealed that 11h, 11f, 11c, 11j, 15d, 15c, 15e, and 15b compounds significantly suppressed anti-inflammatory markers such as TNF-α, IL-1, IL-6, and Bcl-2. All derivatives were moderate in potency compared to reference doxorubicin and could potentially act as novel anti-neuroinflammatory agents. This study can act as a beacon for further research in the application of furan-pyrazole and furan-2,4-thiazolidinediones as lead moieties for anti-neuroinflammatory and related diseases.

Exosomes secreted from induced pluripotent stem cell ameliorate the lipopolysaccharide induced neuroinflammatory response via lncRNA-0949.

To study the effect of exosomes derived from the induced pluripotent stem cells (iPSCs) in the neuroinflammatory response of microglia caused by lipopolysaccharide (LPS) and reveal the potential underlying mechanism. A permanent microglia cell line HMO6 was activated by LPS. The features of exosomes were analyzed by nano flow cytometry, Western blot and transmission electron microscope. The RNA-seq was used to analyze the difference of noncoding RNA profiles between iPSC-Exos and HMO6 derived exosomes and proved that long no-coding RNA (lncRNA-0949) was highly expressed in the iPSC-Exos. Activated HMO6 cells were cocultured with iPSC-Exos in which lncRNA-0949 was overexpressed, knocked down or normally expressed. Quantitative real-time polymerase chain reaction (RT-qPCR), Enzyme-Linked Immunosorbent Assay and Western blot assay were adopted to analyze RNA and protein expression of inflammatory factors in HMO6 cells. The oxidative stress and inflammatory response of microglia were significantly attenuated with the iPSC derived exosomes treatment. LncRNA-0949 was effectively delivered into the HMO6 cells through the iPSC-Exos, which largely alleviated the production of malondialdehyde, IL-6, IL-1β and TNF-α in HMO6 cells. Overexpression of lncRNA-0949 could enhance the anti-inflammatory effect of the iPSC-Exos, and knock-down of lncRNA-0949 impaired this availability. According to our results, lncRNA-0949 enriched exosomes from iPSC could potentially be used as a therapeutic strategy to prevent/treat neuroinflammatory diseases.

Effectiveness of Combinational Treatments for Alzheimer's Disease with Human Neural Stem Cells and Microglial Cells Over-Expressing Functional Genes.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. In AD patients, amyloid-β (Aβ) peptide-mediated degeneration of the cholinergic system utilizing acetylcholine (ACh) for memory acquisition is observed. Since AD therapy using acetylcholinesterase (AChE) inhibitors are only palliative for memory deficits without reversing disease progress, there is a need for effective therapies, and cell-based therapeutic approaches should fulfil this requirement. We established F3.ChAT human neural stem cells (NSCs) encoding the choline acetyltransferase (ChAT) gene, an ACh-synthesizing enzyme, HMO6.NEP human microglial cells encoding the neprilysin (NEP) gene, an Aβ-degrading enzyme, and HMO6.SRA cells encoding the scavenger receptor A (SRA) gene, an Aβ-uptaking receptor. For the efficacy evaluation of the cells, first, we established an appropriate animal model based on Aβ accumulation and cognitive dysfunction. Among various AD models, intracerebroventricular (ICV) injection of ethylcholine mustard azirinium ion (AF64A) induced the most severe Aβ accumulation and memory dysfunction. Established NSCs and HMO6 cells were transplanted ICV to mice showing memory loss induced by AF64A challenge, and brain Aβ accumulation, ACh concentration and cognitive function were analyzed. All the transplanted F3.ChAT, HMO6.NEP and HMO6.SRA cells were found to survive up to 4 weeks in the mouse brain and expressed their functional genes. Combinational treatment with the NSCs (F3.ChAT) and microglial cells encoding each functional gene (HMO6.NEP or HMO6.SRA) synergistically restored the learning and memory function of AF64A-challenged mice by eliminating Aβ deposits and recovering ACh level. The cells also attenuated inflammatory astrocytic (glial fibrillary acidic protein) response by reducing Aβ accumulation. Taken together, it is expected that NSCs and microglial cells over-expressing ChAT, NEP or SRA genes could be strategies for replacement cell therapy of AD.

1,25-Dihydroxy Vitamin D3 Facilitates the M2 Polarization and β-Amyloid Uptake by Human Microglia in a TREM2-Dependent Manner.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by dementia as the primary clinical symptom. The production and accumulation of aggregated β-amyloid (Aβ) in patient brain tissues is one of the hallmarks of AD pathogenesis. Microglia, brain-resident macrophages, produce inflammatory cytokines in response to Aβ oligomers or fibrils exacerbating Aβ pathology in AD. HMO6 cells were treated with Aβ42 in the presence or absence of 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) to determine its potential immunomodulatory effects, and the expression of pro-/anti-inflammatory cytokines, M1/M2-associated markers, Toll-like receptors (TLRs), and triggering receptor expressed on myeloid cells 2 (TREM2) was examined. 1,25(OH)2D3 was found to suppress Aβ-induced expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6), M1 markers (CD86 and iNOS), and TLR2/4, whilst increasing the expression of anti-inflammatory cytokines (IL-4, IL-10, and CCL17) and M2 markers (CD206 and Arg-1). Furthermore, 1,25(OH)2D3 promoted TREM2 expression and Aβ uptake by HMO6 cells, and the enhancement of Aβ uptake and M2 polarization was revealed to be TREM2-dependent. The findings of this study suggest that 1,25(OH)2D3 facilitates M2 polarization and Aβ uptake in a TREM2-dependent manner.

Anti-inflammatory effects of 9-cis-retinoic acid on β-amyloid treated human microglial cells

Objective: Alzheimer’s disease (AD) is a neurodegenerative disease that abolishes cognitive and analytical abilities to perform basic day-to-day tasks. Microglia are involved in AD-associated neuroinflammation in response to amyloid β‐peptide (Aβ). This study focused on observing the immunomodulatory effects of 9-cis-retinoic acid (9-Cis-RA) the active metabolite of vitamin A, on Aβ-treated human microglial HMO6 cells. Methods: HMO6 cells were treated with Aβ42 in the absence or presence of 9-cis-RA, and the expression of M1-and M2-associated molecules, Toll like receptors (TLRs), and triggering receptor expressed on myeloid cells 2 (TREM2) were examined. Results: The levels of M1-markers [cluster of differentiation (CD86) and inducible nitric oxide synthase (iNOS)] and -cytokines [tumor necrosis factor (TNF-α), interleukin (IL)-6, and IL-1β], inflammatory receptors (TLR2 and TLR4), and reactive oxygen species increased significantly in Aβ-treated HMO6 cells. In contrast, the levels of M2-markers (CD206 and arginase-1) and -cytokines (IL-10, IL-4, and C-C motif chemokine ligand 17) the anti-inflammatory receptor TLR10 was significantly suppressed. However, 9-cis-RA treatment reversed the Aβ-induced upregulation of expression of M1-associated molecules and upregulated the expression of M2-associated molecules. Moreover, 9-cis-RA treatment augmented Aβ uptake by HMO6 cells, possibly by increasing the cell surface protein levels of TREM2, which is a receptor of Aβ that promotes Aβ phagocytosis by microglia. Conclusion: Our results suggest that 9-cis-RA is a potential therapeutic agent for AD.

Transforming growth factor‑β1 functions as a competitive endogenous RNA that ameliorates intracranial hemorrhage injury by sponging microRNA‑93‑5p.

Intracerebral hemorrhage (ICH) has the highest mortality rate of all stroke subtypes but an effective treatment has yet to be clinically implemented. Transforming growth factor-β1 (TGF-β1) has been reported to modulate microglia-mediated neuroinflammation after ICH and promote functional recovery; however, the underlying mechanisms remain unclear. Non-coding RNAs such as microRNAs (miRNAs) and competitive endogenous RNAs (ceRNAs) have surfaced as critical regulators in human disease. A known miR-93 target, nuclear factor erythroid 2-related factor 2 (Nrf2), has been shown to be neuroprotective after ICH. It was hypothesized that TGF-β1 functions as a ceRNA that sponges miR-93-5p and thereby ameliorates ICH injury in the brain. Short interfering RNA (siRNA) was used to knock down TGF-β1 and miR-93 expression was also pharmacologically manipulated to elucidate the mechanistic association between miR-93-5p, Nrf2, and TGF-β1 in an in vitro model of ICH (thrombin-treated human microglial HMO6 cells). Bioinformatics predictive analyses showed that miR-93-5p could bind to both TGF-β1 and Nrf2. It was found that neuronal miR-93-5p was dramatically decreased in these HMO6 cells, and similar changes were observed in fresh brain tissue from patients with ICH. Most importantly, luciferase reporter assays were used to demonstrate that miR-93-5p directly targeted Nrf2 to inhibit its expression and the addition of the TGF-β1 untranslated region restored the levels of Nrf2. Moreover, an miR-93-5p inhibitor increased the expression of TGF-β1 and Nrf2 and decreased apoptosis. Collectively, these results identified a novel function of TGF-β1 as a ceRNA that sponges miR-93-5p to increase the expression of neuroprotective Nrf2 and decrease cell death after ICH. The present findings provided evidence to support miR-93-5p as a potential therapeutic target for the treatment of ICH.

Tp47 induces cell death involving autophagy and mTOR in human microglial HMO6 cells

BackgroundTp47 can induce immune cells to produce numerous inflammatory factors, some of which can trigger autophagy. Increased autophagy has a dual effect on cell survival. However, whether Tp47 induces autophagy in microglia is unknown. ObjectiveTo evaluate the potential role of Tp47 in microglia. MethodsAfter treatment with Tp47, autophagy-related proteins were assessed in HMO6 human microglial cells by flow cytometry, Western blotting and immunofluorescence. Cell death was assessed by flow cytometry and trypan blue staining. Changes in mTOR pathway proteins were explored by using Western blotting. ResultsAfter treatment with Tp47, a gradual increase in total LC3 expression was observed as a dose- and time-dependent accumulation of its active form, LC3-II (P < 0.05), but P62 expression was downregulated (P < 0.05). Moreover, microglial mortality gradually increased in a dose- and time-dependent manner. 3-Methyladenine (3-MA), a specific inhibitor of PI3KC3, reversed autophagy and cell death. The mortality rate of HMO6 microglial cells treated with Tp47 was approximately 13.7 ± 2%, and the basal expression of p-mTOR, p-p70s6k and p-S6 in these cells was significantly downregulated by Tp47. Moreover, the mortality rate of microglia was significantly reduced after mTOR agonist intervention. ConclusionIn human microglial HMO6 cells, Tp47 induces autophagy- and mTOR pathway-dependent cell death.

Ecklonia cava -derived Polysaccharide Prevent Hydro Peroxide-induced Oxidative Stress and Neurotoxicity in Human Microglial HMO6 Cells

In the present study, we examined the beneficial effect of Ecklonia cava-derived polysaccharide (ECAP), which is residual product after bioactive polyphenol isolation form Ecklonia cava, on neurotoxicity and oxidative stress in hydro peroxide-treated human microglia HMO6 cells. We sought that ECAP inhibited the expression of p53 and Bcl-2 in hydro peroxide-induced HMO6 cells. ECAP dramatically suppressed the cleaved form of caspase-3 in hydro peroxide-treated HMO6 cells. Moreover, we observed that hydro peroxide stimulated the expression of glucose-6-phosphate dehydrogenase (G6PDH) and its downstream target NADPH oxidase 4 (NOX4) in HMO6 cells. In contrast, ECAP suppressed the expression of G6PDH and NOX4 in hydro peroxide-treated HMO6 cells. We also observed that anti-oxidant enzymes including superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), and catalase were stimulated by ECAP to protect the neurotoxicity in hydro peroxide-treated HMO6 cells. Taken together, we demonstrated that ECAP may inhibit hydro peroxide-mediated neurotoxicity through the regulation of p53 and Bcl-2 genes in HMO6 cells and ameliorate oxidative stress in hydro peroxide-treated HMO6 cells. Therefore we suggest that ECAP may hold the potential to prevent the incidence of neuronal damage-mediated neurodegenerative diseases.

1,25-Dihydroxyvitamin D3 Facilitates M2 Polarization and Upregulates TLR10 Expression on Human Microglial Cells

Objective: To explore the effects of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], the active metabolite of vitamin D, on M1/M2 polarization of human microglia and the expression of Toll-like receptor 10 (TLR10) on these cells, which has been suggested to play an inhibitory role in inflammation previously. Methods: Microglial HMO6 cells were treated with 1,25(OH)₂D₃, and mRNA or protein levels of M1 and M2 cytokines and TLR10 were examined. Results: 1,25(OH)₂D₃ upregulated TLR10 in HMO6 cells at both mRNA and protein level. 1,25(OH)₂D₃ enhanced basal mRNA expression of M2 cytokines, such as IL-10 and CCL17, but did not affect the expression of M1 cytokines, including IL-12 and TNF-α. 1,25(OH)₂D₃ downregulated the lipopolysaccharide (LPS)-induced mRNA expression of M1 cytokines IL-12 and TNF-α. Concomitantly, it upregulated not only the M2 cytokines IL-10 and CCL17, but also TLR10 in microglial cells treated with LPS, in a concentration-dependent manner. Conclusions: Our results suggest that 1,25(OH)₂D₃ may exert anti-inflammatory action by facilitating the M2 polarization of human microglial cells.

Exposure to electromagnetic field attenuates oxygen-glucose deprivation-induced microglial cell death by reducing intracellular Ca2+ and ROS

Purpose The aim of this research was to demonstrate the protective effects of electromagnetic field (EMF) exposure on the human microglial cell line, HMO6, against ischemic cell death induced by in vitro oxygen-glucose deprivation (OGD).Materials and methods HMO6 cells were cultured for 4 h under OGD with or without exposure to EMF with different combinations of frequencies and intensities (10, 50, or 100 Hz/1 mT and 50 Hz/0.01, 0.1, or 1 mT). Cell survival, intracellular calcium and reactive oxygen species (ROS) levels were measured.Results OGD caused significant HMO6 cell death as well as elevation of intracellular Ca2+ and ROS levels. Among different combinations of EMF frequencies and intensities, 50 Hz/1 mT EMF was the most potent to attenuate OGD-induced cell death and intracellular Ca2+ and ROS levels. A significant but less potent protective effect was also found at 10 Hz/1 mT, whereas no protective effect was found at other combinations of EMF. A xanthine oxidase inhibitor reversed OGD-induced ROS production and cell death, while NADPH oxidase and mitochondrial respiration chain complex II inhibitors did not affect cell death.Conclusions 50 Hz/1 mT EMF protects human microglial cells from OGD-induced cell death by interfering with OGD-induced elevation of intracellular Ca2+ and ROS levels, and xanthine oxidase is one of the main mediators involved in OGD-induced HMO6 cell death. Non-invasive treatment of EMF radiation may be clinically useful to attenuate hypoxic-ischemic brain injury.

Glucosamine suppresses platelet-activating factor-induced activation of microglia through inhibition of store-operated calcium influx.

Microglia activation and subsequent release of inflammatory mediators are implicated in the pathophysiology of neurodegenerative diseases. Platelet-activating factor (PAF), a potent lipid mediator synthesized by microglia, is known to stimulate microglia functional responses. In this study, we determined that endogenous PAF exert autocrine effects on microglia activation, as well as the underlying mechanism involved. We also investigated the effect of D-glucosamine (GlcN) on PAF-induced cellular activation in human HMO6 microglial cells. PAF induced sustained intracellular Ca(2+) ([Ca(2+)]i) increase through store-operated Ca(2+) channels (SOC) and reactive oxygen species (ROS) generation. PAF also induced pro-inflammatory markers through NFκB/COX-2 signaling. GlcN significantly inhibited PAF-induced Ca(2+) influx and ROS generation without significant cytotoxicity. GlcN downregulated excessive expression of pro-inflammatory markers and promoted filopodia formation through NFκB/COX-2 inhibition in PAF-stimulated HMO6 cells. Taken together, these data suggest that GlcN may offer substantial therapeutic potential for treating inflammatory and neurodegenerative diseases accompanied by microglial activation.

An Aβ42 uptake and degradation via Rg3 requires an activation of caveolin, clathrin and Aβ-degrading enzymes in microglia

We demonstrated previously that ginsenoside Rg3 enhances the expression of macrophage scavenger receptor class A (SRA) and amyloid β peptide 1–42 (Aβ42) uptake in BV2 cells. In this study, we investigated the biochemical and mechanistic roles of Rg3 in human microglia and animal models to identify the determinants that participate in restoring memory and learning in brains disrupted by the Aβ42 peptide. SRA was expressed highly in Rg3-treated rats, and learning and memory functions were maintained at a normal level after the infusion of Aβ42. SRA-transfected HMO6 human microglial cells (HMO6.hSRA) overexpressed SRA and took up a remarkable amount of Aβ42. Rg3-treated HMO6 cells showed highly enhanced SRA expression and dramatically promoted Aβ42 uptake. Moreover, high levels of clathrin and caveolin1 supported the roles of Rg3 in endocytic biogenesis by activating p38 and extracellular signal-regulated protein kinase signaling. Notably, both neprilysin (NEP) and insulin-degrading enzyme (IDE) were significantly expressed by Rg3, suggesting independent and compensatory hydrolytic activity for the Aβ peptide. In conclusion, Rg3 successfully triggered Aβ42 uptake via SRA and clathrin-/caveolae-mediated endocytic mechanisms and further contributed to accelerate the degradation of Aβ peptide via the increase of intracellular NEP and IDE, which may be a promising Alzheimer׳s disease therapy.

Non-phosphorylated FTY720 induces apoptosis of human microglia by activating SREBP2.

A synthetic analog of sphingosine named FTY720 (Fingolimod), phosphorylated by sphingosine kinase-2, interacts with sphingosine-1-phosphate (S1P) receptors expressed on various cells. FTY720 suppresses the disease activity of multiple sclerosis (MS) chiefly by inhibiting S1P-dependent egress of autoreactive T lymphocytes from secondary lymphoid organs, and possibly by exerting anti-inflammatory and neuroprotective effects directly on brain cells. However, at present, biological effects of FTY720 on human microglia are largely unknown. We studied FTY720-mediated apoptosis of a human microglia cell line HMO6. The exposure of HMO6 cells to non-phosphorylated FTY720 (FTY720-non-P) induced apoptosis in a dose-dependent manner with IC50 of 10.6 ± 2.0 μM, accompanied by the cleavage of caspase-7 and caspase-3 but not of caspase-9. The apoptosis was inhibited by Z-DQMD-FMK, a caspase-3 inhibitor, but not by Pertussis toxin, a Gi protein inhibitor, suramin, a S1P3/S1P5 inhibitor, or W123, a S1P1 competitive antagonist, although HMO6 expressed S1P1, S1P2, and S1P3. Furthermore, both phosphorylated FTY720 (FTY720-P) and SEW2871, S1P1 selective agonists, did not induce apoptosis of HMO6. Genome-wide gene expression profiling and molecular network analysis indicated activation of transcriptional regulation by sterol regulatory element-binding protein (SREBP) in FTY720-non-P-treated HMO6 cells. Western blot verified activation of SREBP2 in these cells, and apoptosis was enhanced by pretreatment with simvastatin, an activator of SREBP2, and by overexpression of the N-terminal fragment of SREBP2. These observations suggest that FTY720-non-P-induced apoptosis of HMO6 human microglia is independent of S1P receptor binding, and positively regulated by the SREBP2-dependent proapoptotic signaling pathway.

Human Microglia Transplanted in Rat Focal Ischemia Brain Induce Neuroprotection and Behavioral Improvement

Background and PurposeMicroglia are resident immunocompenent and phagocytic cells of central nervous system (CNS), which produce various cytokines and growth factors in response to injury and thereby regulate disease pathology. The purpose of this study is to investigate the effects of microglial transplantation on focal cerebral ischemia model in rat.MethodsTransient middle cerebral artery occlusion (MCAO) in rats was induced by the intraluminal filament technique. HMO6 cells, human microglial cell line, were transplanted intravenously at 48 hours after MCAO. Functional tests were performed and the infarct volume was measured at 7 and 14 days after MCAO. Migration and cell survival of transplanted microglial cells and host glial reaction in the brain were studied by immunohistochemistry. Gene expression of neurotrophic factors, cytokines and chemokines in transplanted cells and host rat glial cells was determined by laser capture microdissection (LCM) and quantitative real time-PCR.ResultsHMO6 human microglial cells transplantion group demonstrated significant functional recovery compared with control group. At 7 and 14 days after MCAO, infarct volume was significantly reduced in the HMO group. In the HMO6 group, number of apoptotic cells was time-dependently reduced in the infarct core and penumbra. In addition, number of host rat microglia/macrophages and reactive astrocytes was significantly decreased at 7 and 14 days after MCAO in the penumbra. Gene expression of various neurotrophic factors (GDNF, BDNF, VEGF and BMP7) and anti-inflammatory cytokines (IL4 and IL5) was up-regulated in transplanted HMO6 cells of brain tissue compared with those in culture. The expression of GDNF and VEGF in astrocytes in penumbra was significantly up-regulated in the HMO6 group.ConclusionsOur results indicate that transplantation of HMO6 human microglial cells reduces ischemic deficits and apoptotic events in stroke animals. The results were mediated by modulation of gliosis and neuroinflammation, and neuroprotection provided by neurotrophic factors of endogenous and transplanted cells-origin.

Microglia transplantation attenuates white matter injury in rat chronic ischemia model via matrix metalloproteinase-2 inhibition

Chronic cerebral ischemia is thought to induce white matter lesions (WMLs), which contribute to subcortical vascular dementia. Although glial activation and protease upregulation are believed to modify WML pathology, effective therapy remains elusive. Here, we compare the efficacy of microglial cell transplantation and mesenchymal stem cell (MSC) transplantation in protecting against WML development in a chronic cerebral hypoperfusion rat model. A microglial cell line (HMO6), MSC cell line (B10) or vehicle (phosphate-buffered saline; PBS) was intravenously injected, and the appearance and severity of WMLs were evaluated. Transplanted HMO6 and B10 cells migrated to sites of WMLs, including the corpus callosum (CC) and caudoputamen (CP), reduced the severity of WMLs, and inhibited the accumulation and activation of microglia and astrocytes. Transplantation of both cell types reduced the level of matrix metalloproteinase (MMP)-2 mRNA in microglia of the CC. MMP-2 protein level and activity were also both greatly reduced in the same region. Our results indicate that transplantation of either microglial cells or mesenchymal stem cells could inhibit chronic cerebral ischemia-induced WML formation by decreasing MMP-2 expression in microglia and decreasing MMP-2 activity in the CC region.

Lysophosphatidylcholine induces glial cell activation: Role of rho kinase

Lysophosphatidylcholine (LPC), a major phospholipid component of atherogenic oxidized LDL, is implicated in atherosclerosis and, recently, in neurodegenerative diseases. We investigated the immunomodulatory functions of LPC in the central nervous system (CNS) using both an in vivo rat model, and in vitro culture systems of human primary astrocytes and a microglia cell line, HMO6. Compared with PBS injection, 20 nmol LPC-injection into the rat striatum increased astrocyte and microglial accumulation and elevated iNOS expression; concomitantly a time-dependent decrease in number of neurons was exhibited. In vitro studies on astrocytes and HMO6 cells showed that LPC increased the gene expression of proinflammatory factors IL-1beta, COX-2, and GM-CSF. LPC also induced chemotactic responses in HMO6 cells. Inhibition of rho kinase by fasudil, Y27632, or expressing a dominant negative form of rho kinase inhibited the LPC-induced IL-1beta mRNA expression in both astrocytes and HMO6. Moreover, intraperitoneal fasudil injection inhibited the LPC-induced microglial accumulation and iNOS expression and also was effective in protecting against neuronal loss. Silencing G2A, a specific receptor for LPC, inhibited proinflammatory gene expression and HMO6 migration. Overall, our results indicate that LPC induced considerable neuroinflammatory reactivity in glia mediated by rho kinase-dependent pathways with inhibition of these pathways conferring significant extents of neuroprotection.

Microglial activation and cell death induced by the mitochondrial toxin 3-nitropropionic acid: in vitro and in vivo studies

Metabolic impairment of neurons has been implicated in several neurological disorders, but it is not at present known whether such metabolic impairment has deleterious effects on microglia, the phagocytic cells of the central nervous system (CNS). In the present study, we examined whether metabolic impairment induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, affects the function and viability of microglia in vitro and in vivo. Treatment of HMO6 human microglia cell line with 3-NP induced the elevation of intracellular Ca 2+ concentration ([Ca 2+] i) and activation of microglia with production of reactive oxygen species (ROS). Exposure of HMO6 cells to 3-NP also induced cell death as indicated by nuclear fragmentation in a dose- and time-dependent manner. Trolox, an antioxidant agent, was effective in reduction in ROS production and cell death caused by 3-NP. Consistent with in vitro findings, intrastriatal injection of 3-NP in adult rats resulted in an increase in ROS production in microglia in vivo, as evidenced by the oxidation of the reduced MitoTracker probe. ROS production induced by 3-NP was inhibited when trolox was coinjected with 3-NP. Caspase-3 immunoreactivity was demonstrated in OX-42+ microglia in the core and penumbra area of the 3-NP-injected striatum. Apoptotic cell death of microglia was also demonstrated by terminal deoxynucleotidyl- transferase-mediated biotin-dUTP nick end labeling reaction in the 3-NP-induced lesion area. The present results indicate that metabolic impairment in the CNS could involve both activation and cell death of microglia and contribute to pathology in neurodegenerative diseases.

Generation and Characterization of Immortalized Human Microglial Cell Lines: Expression of Cytokines and Chemokines

Microglia are a major glial component of the central nervous system (CNS), play a critical role as resident immunocompetent and phagocytic cells in the CNS, and serve as scavenger cells in the event of infection, inflammation, trauma, ischemia, and neurodegeneration in the CNS. Studies of human microglia have been hampered by the difficulty of obtaining sufficient numbers of human microglia. One way to circumvent this difficulty is to establish permanent cell lines of human microglia. In the present study we report the generation of immortalized human microglial cell line, HMO6, from human embryonic telencephalon tissue using a retroviral vector encoding myc oncogene. The HMO6 cells exhibited cell type-specific antigens for microglia-macrophage lineage cells including CD11b (Mac-1), CD68, CD86 (B7-2), HLA-ABC, HLA-DR, and ricinus communis aggulutinin lectin-1 (RCA), and actively phagocytosed latex beads. In addition, HMO6 cells showed ATP-induced responses similar to human primary microglia in Ca2+ influx spectroscopy. Both human primary microglia and HMO6 cells showed the similar cytokine gene expression in IL-1β, IL-6, IL-8, IL-10, IL-12, IL-15, and TNF-α. Using HMO6 cells, we investigated whether activation was induced by Amyloid-β fragments or lipopolysaccharide (LPS). Treatment of HMO6 cells with Amyloid-β 25–35 fragment (Aβ25–35) or Amyloid-β 1–42 fragment (Aβ1–42) led to increased expression of mRNA levels of cytokine/chemokine IL-8, IL-10, IL-12, MIP-1β MIP-1, and MCP-1, and treatment with LPS produced same results. Expression of TNF-α and MIP1-α was not detected in unstimulated HMO6 cells, but their expression was later induced by long-term exposure to Aβ25–35 or Aβ1–42. ELISA assays of spent culture media showed increased protein levels of TNF-α and IL-8 in HMO6 cells following treatment with Aβ25–35 or LPS. Taken together, our results demonstrate that treatment of human primary microglia and HMO6 immortalized human microglia cell line with Aβ25–35, Aβ1–42 and LPS upregulate gene expression and protein production of proinflammatory cytokines and chemokines in these cells. The human microglial cell line HMO6 exhibits similar properties to those documented in human microglia and should have considerable utility as an in vitro model for the studies of human microglia in health and disease.