Modelling Neuroinflammation In Vitro: A Tool to Test the Potential Neuroprotective Effect of Anti-Inflammatory Agents

Neuron-microglia co-cultures treated with pro-inflammatory agents are a useful tool to study neuroinflammation in vitro, where to test the potential neuroprotective effect of anti-inflammatory compounds. However, a great diversity of experimental conditions can be found in the literature, making difficult to select the working conditions when considering this approach for the first time. We compared the use of neuron-primary microglia and neuron-BV2 cells (a microglial cell line) co-cultures, using different neuron:microglia ratios, treatments and time post-treatment to induce glial activation and derived neurotoxicity. We show that each model requires different experimental conditions, but that both neuron-BV2 and neuron-primary microglia LPS/IFN-γ-treated co-cultures are good to study the potential neuroprotective effect of anti-inflammatory agents. The contribution of different pro-inflammatory parameters in the neurotoxicity induced by reactive microglial cells was determined. IL-10 pre-treatment completely inhibited LPS/IFN-γ-induced TNF-α and IL-6 release, and COX-2 expression both in BV2 and primary microglial cultures, but not NO production and iNOS expression. However, LPS/IFN-γ induced neurotoxicity was not inhibited in IL-10 pre-treated co-cultures. The inhibition of NO production using the specific iNOS inhibitor 1400 W totally abolished the neurotoxic effect of LPS/IFN-γ, suggesting a major role for NO in the neurotoxic effect of activated microglia. Consequently, among the anti-inflammatory agents, special attention should be paid to compounds that inhibit NO production.     

Expression of Inducible Nitric Oxide Synthase (iNOS) in Microglia of the Developing Quail Retina

Inducible nitric oxide synthase (iNOS), which produce large amounts of nitric oxide (NO), is induced in macrophages and microglia in response to inflammatory mediators such as LPS and cytokines. Although iNOS is mainly expressed by microglia that become activated in different pathological and experimental situations, it was recently reported that undifferentiated amoeboid microglia can also express iNOS during normal development. The aim of this study was to investigate the pattern of iNOS expression in microglial cells during normal development and after their activation with LPS by using the quail retina as model. iNOS expression was analyzed by iNOS immunolabeling, western-blot, and RT-PCR. NO production was determined by using DAR-4M AM, a reliable fluorescent indicator of subcellular NO production by iNOS. Embryonic, postnatal, and adult in situ quail retinas were used to analyze the pattern of iNOS expression in microglial cells during normal development. iNOS expression and NO production in LPS-treated microglial cells were investigated by an in vitro approach based on organotypic cultures of E8 retinas, in which microglial cell behavior is similar to that of the in situ retina, as previously demonstrated in our laboratory. We show here that amoeboid microglia in the quail retina express iNOS during normal development. This expression is stronger in microglial cells migrating tangentially in the vitreal part of the retina and is downregulated, albeit maintained, when microglia differentiate and become ramified. LPS treatment of retina explants also induces changes in the morphology of amoeboid microglia compatible with their activation, increasing their lysosomal compartment and upregulating iNOS expression with a concomitant production of NO. Taken together, our findings demonstrate that immature microglial cells express iNOS during normal development, suggesting a certain degree of activation. Furthermore, LPS treatment induces overactivation of amoeboid microglia, resulting in a significant iNOS upregulation.     

A self-propelling cycle mediated by reactive oxide species and nitric oxide exists in LPS-activated microglia

It has been widely accepted that microglia, the innate immune cells in the brain, can be chronically activated in response to neuron death, fuelling a self-renewing cycle of microglial activation followed by further neuron damage (reactive microgliosis), which has been considered as the main reason responsible for the progressive nature of neurodegenerative diseases. In the present study, it was found that LPS (lipopolysaccharide) significantly induced the activation of N9 microglia, and the increase of NO level induced by pretreatment of LPS could last after the removal of LPS. The culture medium of activated microglia significantly decreased the viability of rat primary cortical neuron. These results can be blocked by the antioxidant N-acetylcysteine (NAC) and nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase inhibitor diphenyleneiodonium sulfate (DPI), suggesting that intracellular reactive oxide species (iROS) released from the activated microglial cells may continue to further activate microglia. Next, it was shown that the iROS level increased rapidly after the LPS treatment in microglia cells followed by the NO production through the regulation of iNOS (inducible nitric oxide synthase) expression. The increase of iROS could be reversed by gp91phox (the critical and catalytic subunit of NADPH oxidase) siRNA. Moreover, NO released from sodium nitroprusside (SNP) was able to increase the iROS production of N9 microglia by regulating of the activity and the expression of NADPH oxidase. In conclusion, our research suggests for the first time that there may exist a self-propelling cycle in microglial cells possibly mediated by iROS and NO when they become activated by LPS. It may be responsible partially for the ongoing microglial activation and the progressive nature of neurodegenerative diseases.     

Nitric oxide regulates antagonistically phagocytic and neurite outgrowth inhibiting capacities of microglia

Traumatic injury or the pathogenesis of some neurological disorders is accompanied by inflammatory cellular mechanisms, mainly resulting from the activation of central nervous system (CNS) resident microglia. Under inflammatory conditions, microglia up‐regulate the inducible isoform of NOS (iNOS), leading to the production of high concentrations of the radical molecule nitric oxide (NO). At the onset of inflammation, high levels of microglial‐derived NO may serve as a cellular defense mechanism helping to clear the damaged tissue and combat infection of the CNS by invading pathogens. However, the excessive overproduction of NO by activated microglia has been suggested to govern the inflammation‐mediated neuronal loss causing eventually complete neurodegeneration. Here, we investigated how NO influences phagocytosis of neuronal debris by BV‐2 microglia, and how neurite outgrowth of human NT2 model neurons is affected by microglial‐derived NO. The presence of NO greatly increased microglial phagocytic capacity in a model of acute inflammation comprising lipopolysaccharide (LPS)‐activated microglia and apoptotic neurons. Chemical manipulations suggested that NO up‐regulates phagocytosis independently of the sGC/cGMP pathway. Using a transwell system, we showed that reactive microglia inhibit neurite outgrowth of human neurons via the generation of large amounts of NO over effective distances in the millimeter range. Application of a NOS blocker prevented the LPS‐induced NO production, totally reversed the inhibitory effect of microglia on neurite outgrowth, but reduced the engulfment of neuronal debris. Our results indicate that a rather simple notion of treating excessive inflammation in the CNS by NO synthesis blocking agents has to consider functionally antagonistic microglial cell responses during pharmaceutic therapy. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 566–584, 2016     

Dalesconols B inhibits lipopolysaccharide induced inflammation and suppresses NF-κB and p38/JNK activation in microglial cells

Therapeutic strategies designed to inhibit the activation of microglia may lead to significant advancement in the treatment of most neurodegenerative diseases. Dalesconols B, also termed as TL2, is a newly found polyketide from a mantis-associated fungus and has been reported to exert potent immunosuppressive effects. In the present study, the anti-inflammatory effects of TL2 was investigated in lipopolysaccharide (LPS)-treated BV2 microglia and primary microglia cells. Our observations indicated that pretreatment with TL2 significantly inhibited the production of NO and PGE2 and suppressed the expression of pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), COX-2, TNF-α, IL-1β, IL-6, MCP-1 and MIP-1α in LPS-stimulated BV2 microglia. The nuclear translocation of NF-κB and the phosphorylation level of Akt, p38 and JNK MAP kinase pathways were also inhibited by TL2 in LPS-treated BV2 microglia. Moreover, TL2 also decreased Aβ-induced production of TNF-α, IL-1β and IL-6 in BV2 microglia. Additionally, TL2 protected primary cortical neurons against microglia-mediated neurotoxicity. Overall, our findings suggested that TL2 might be a promising therapeutic agent for alleviating the progress of neurodegenerative diseases associated with microglia activation.     

Distinct signaling pathways for induction of type II NOS by IFNgamma and LPS in BV-2 microglial cells

Nitric oxide (NO) release upon microglial cell activation has been implicated in the tissue injury and cell death in many neurodegenerative diseases. Recent studies have indicated the ability of interferon-gamma (IFNgamma) and lipopolysaccharides (LPS) to independently induce type II nitric oxide synthase (iNOS) expression and NO production in BV-2 microglial cells. However, a detailed comparison between the signaling pathways activating iNOS by these two agents has not been accomplished. Analysis of PKC isoforms revealed mainly the presence of PKCdelta, iota and lambda in BV-2 cells. Although both IFNgamma and LPS could specifically enhance the tyrosine phosphorylation of PKCdelta, treatment with IFNgamma induced a steady increase of phospho-PKCdelta for up to 1h, whereas treatment with LPS elevated phospho-PKCdelta levels only transiently, with peak activity at 5 min. Rottlerin, a specific inhibitor for PKCdelta, dose-dependently inhibited IFNgamma- and LPS-induced NO production. Despite the common involvement of PKCdelta, IFNgamma- but not LPS-induced NO production involved extracellular signal-regulated kinases (ERK1/2) cascade and IFNgamma-induced phosphorylation of ERK1/2 was mediated through PKC. On the other hand, LPS- but not IFNgamma-induced NO production was through stimulation of NF-kappaB activation and nuclear translocation to interact with DNA. These results demonstrated distinct signaling pathways for induction of iNOS by IFNgamma and LPS in BV-2 microglial cells.     

Inducible nitric oxide synthase is key to peroxynitrite-mediated,LPS-induced protein radical formation in murine microglial BV2 cells

Microglia are the resident immune cells in the brain. Microglial activation is characteristic of several inflammatory and neurodegenerative diseases including Alzheimer's disease, multiple sclerosis, and Parkinson's disease. Though lipopolysaccharide (LPS)-induced microglial activation in models of Parkinson's disease is well documented, the free radical-mediated protein radical formation and its underlying mechanism during LPS-induced microglial activation are not known. Here we have used immuno-spin trapping and RNA interference to investigate the role of inducible nitric oxide synthase (iNOS) in peroxynitrite-mediated protein radical formation in murine microglial BV2 cells treated with LPS. Treatment of BV2 cells with LPS resulted in morphological changes, induction of iNOS, and increased protein radical formation. Pretreatments with FeTPPS (a peroxynitrite decomposition catalyst), L-NAME (total NOS inhibitor), 1400W (iNOS inhibitor), and apocynin significantly attenuated LPS-induced protein radical formation and tyrosine nitration. Results obtained with coumarin-7-boronic acid, a highly specific probe for peroxynitrite detection, correlated with LPS-induced tyrosine nitration, which demonstrated involvement of peroxynitrite in protein radical formation. A similar degree of protection conferred by 1400W and L-NAME led us to conclude that only iNOS, and no other forms of NOS, is involved in LPS-induced peroxynitrite formation. Subsequently, siRNA for iNOS, the iNOS-specific inhibitor 1400W, the NF-κB inhibitor PDTC, and the p38 MAPK inhibitor SB202190 was used to inhibit iNOS directly or indirectly. Inhibition of iNOS precisely correlated with decreased protein radical formation in LPS-treated BV2 cells. The time course of protein radical formation also matched the time course of iNOS expression. Taken together, these results prove the role of iNOS in peroxynitrite-mediated protein radical formation in LPS-treated microglial BV2 cells.     

Attenuation of pro-inflammatory mediators in LPS-stimulated BV2 microglia by chitooligosaccharides via the MAPK signaling pathway

Chitooligosaccharides (COS), depolymerized products of chitosan, has received considerable attention as bioactive material due to their biocompatible, biodegradable, non-toxic and non-allergenic natures. In this study, COS of four different molecular weight ranges (<1, 1-3, 3-5 and 5-10 kDa) were investigated for their abilities to modulate inflammatory mediators in lipopolysaccharides (LPS)-stimulated BV2 microglia. At the concentration of 500 μg/ml, COS attenuate the productions of nitric oxide (NO) and prostaglandin E₂ (PGE₂) by inhibiting inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions. Furthermore, the release and expression levels of inflammatory cytokines; including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were also attenuated by COS. Notably, the inhibitory activity of COS depends significantly on its molecular weight, with lower molecular weight showed higher activity. In addition, the suppressive effects on the phosphorylation of JNK and p38 mitogen-activated protein kinase (MAPK) by COS were confirmed. These results indicate that COS could be used as an inhibitor in regulating microglial inflammatory responses. Moreover, COS may assist therapeutic treatment of neurodegenerative diseases which accompanied with microglial activation.     

Activated microglia mediate neuronal cell injury via a nitric oxide mechanism.

Activated microglial have been proposed to play a pathogenetic role in immune-mediated neurodegenerative diseases. To test this hypothesis, purified murine neonatal microglial were cocultured with neuronal cells derived from fetal brain. Activation with IFN-gamma and LPS of these cocultures brought about a sharp decrease in uptake of gamma-amino butyric acid and a marked reduction in neuronal cell survival. These effects varied with the density of microglia, the concentrations of the activation signals (IFN-gamma and LPS), and the duration of coculture. Inasmuch as addition of NG-monomethyl-L-arginine blocked these effects, a L-arginine-dependent neurocytotoxic mechanism was implicated. Abundant nitrite, a metabolite of the free radical nitric oxide (NO) derived from L-arginine, was detected in activated microglial/neuronal cell cocultures and in purified microglial cell cultures but not in purified astrocyte or neuronal cell cultures, suggesting that microglial were the principal source of the NO. These findings support the hypothesis that microglia are the source of a neurocytotoxic-free radical, and shed light on an additional mechanism of immune-mediated brain injury.     

Antagonizing peroxisome proliferator‐activated receptor γ facilitates M1‐to‐M2 shift of microglia by enhancing autophagy via the LKB1–AMPK signaling pathway

Microglia‐mediated neuroinflammation plays a dual role in various brain diseases due to distinct microglial phenotypes, including deleterious M1 and neuroprotective M2. There is growing evidence that the peroxisome proliferator‐activated receptor γ (PPARγ) agonist rosiglitazone prevents lipopolysaccharide (LPS)‐induced microglial activation. Here, we observed that antagonizing PPARγ promoted LPS‐stimulated changes in polarization from the M1 to the M2 phenotype in primary microglia. PPARγ antagonist T0070907 increased the expression of M2 markers, including CD206, IL‐4, IGF‐1, TGF‐β1, TGF‐β2, TGF‐β3, G‐CSF, and GM‐CSF, and reduced the expression of M1 markers, such as CD86, Cox‐2, iNOS, IL‐1β, IL‐6, TNF‐α, IFN‐γ, and CCL2, thereby inhibiting NFκB–IKKβ activation. Moreover, antagonizing PPARγ promoted microglial autophagy, as indicated by the downregulation of P62 and the upregulation of Beclin1, Atg5, and LC3‐II/LC3‐I, thereby enhancing the formation of autophagosomes and their degradation by lysosomes in microglia. Furthermore, we found that an increase in LKB1–STRAD–MO25 complex formation enhances autophagy. The LKB1 inhibitor radicicol or knocking down LKB1 prevented autophagy improvement and the M1‐to‐M2 phenotype shift by T0070907. Simultaneously, we found that knocking down PPARγ in BV2 microglial cells also activated LKB1–AMPK signaling and inhibited NFκB–IKKβ activation, which are similar to the effects of antagonizing PPARγ. Taken together, our findings demonstrate that antagonizing PPARγ promotes the M1‐to‐M2 phenotypic shift in LPS‐induced microglia, which might be due to improved autophagy via the activation of the LKB1–AMPK signaling pathway.     

Effects of resveratrol and its derivatives on lipopolysaccharide-induced microglial activation and their structure-activity relationships

The inhibitory effects of 21 resveratrol derivatives on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in microglia and their structure-activity relationships were studied. It was found, for the first time, that certain resveratrol derivatives that have 3,5-dimethoxyl groups in the A-ring, such as (E)-4-(3,5-dimethoxystyryl)phenol (pterostilbene, compound 2), or have substituted the B-ring of resveratrol with quinolyl, such as (E)-5-[2-(quinolin-4-yl)vinyl]benzene-1,3-diol (compound 18) and (E)-4-(3,5-dimethoxystyryl)quinoline (compound 19), strongly inhibited NO production. Compounds 2, 18, and 19 reduced LPS-induced protein and mRNA expression of inducible NO synthase (iNOS), but did not display direct NO-scavenging activity up to 30 microM in sodium nitroprusside (SNP) solution. Moreover, compounds 2, 18, and 19 could also significantly inhibit the production of TNF-alpha by LPS-activated microglia. Further studies revealed that compounds 2, 18, and 19 inhibited LPS-induced NO and TNF-alpha production in microglia by blocking IkappaBalpha phosphorylation and degradation. The potent inhibitory effects of compounds 2, 18, and 19 on microglial activation suggest their potential for treatment of neurodegenerative diseases accompanied by microglial activation.     

4-O-carboxymethylascochlorin protected against microglial-mediated neurotoxicity in SH-SY5Y and BV2 cocultured cells from LPS–induced neuroinflammation and death by inhibiting MAPK,NF-κB,and Akt pathways

In our previous studies, structurally similar compounds of ascochlorin and ascofuranone exhibited anti-inflammatory activity. Neural inflammation plays a significant role in the commence and advancement of neurodegenerative diseases. It is not known whether 4-O-carboxymethylascochlorin (AS-6) regulates the initial stage of inflammatory responses at the cellular level in BV2 microglia cells. We here investigated the anti-inflammatory effects of AS-6 treatment in microglia cells with the microglial protection in neurons. We found that the lipopolysaccharide (LPS)-stimulated production of nitric oxide, a main regulator of inflammation, is suppressed by AS-6 in BV2 microglial cells. In addition, AS-6 dose-dependently suppressed the increase in COX-2 protein and messenger RNA levels in LPS-stimulated BV2 cells. Moreover, AS-6 inhibited the expression and secretion of proinflammatory cytokines in BV2 microglial cells. At the intracellular level, AS-6 inhibited LPS-activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in BV2 microglial cells. AS-6 negatively affected mitogen-activated protein kinases (MAPK) and Akt phosphorylation: Phosphorylated forms of ERK, JNK, p38, and Akt decreased. To check whether AS-6 protects against inflammatory inducer-mediated neurotoxicity, neuronal SH-SY5Y cells were coincubated with BV2 cells in conditioned medium. AS-6 exerted a neuroprotective effect by suppressing microglial activation by LPS or amyloid-β peptide. AS-6 is a promising suppressor of inflammatory responses in LPS-induced BV2 cells by attenuating NF-κB and MAPKs signaling. AS-6 protected against microglial-mediated neurotoxicity in SH-SY5Y and BV2 cocultured cells from LPS–induced neuroinflammation and death via inhibiting MAPK, NF-κB, and Akt pathways.     

TGF-beta1 represses activation and resultant death of microglia via inhibition of phosphatidylinositol 3-kinase activity

Overactivation of microglial cells may cause severe brain tissue damage in various neurodegenerative diseases. Therefore, the overactivation of microglia should be repressed by any means. The present study investigated the potential mechanism and signaling pathway for the repressive effect of TGF-beta1, a major anti-inflammatory cytokine, on overactivation and resultant death of microglial cells. A bacterial endotoxin LPS stimulated expression of inducible NO synthase (iNOS) and caused death in cultured microglial cells. TGF-beta1 markedly blocked these LPS effects. However, the LPS-evoked death of microglial cells was not solely attributed to excess production of NO. Because phosphatidylinositol 3-kinase (PI3K) was previously shown to play a crucial role in iNOS expression and cell survival signals, we further studied whether PI3K signaling was associated with the suppressive effect of TGF-beta1. Like TGF-beta1, the PI3K inhibitor LY294002 blocked iNOS expression and death in cultured microglial cells. Both TGF-beta1 and LY294002 decreased the activation of caspases 3 and 11 and the mRNA expression of various kinds of inflammatory molecules caused by LPS. TGF-beta1 was further found to decrease LPS-induced activation of PI3K and Akt. TGF-beta1 and LY294002 suppressed LPS-induced p38 mitogen-activated kinase and c-Jun N-terminal kinase activity. In contrast, TGF-beta1 and LY294002 enhanced LPS-induced NF-kappaB activity. Our data indicate that TGF-beta1 protect normal or damaged brain tissue by repressing overactivation of microglial cells via inhibition of PI3K and its downstream signaling molecules.     

Inhibitory effect of curcumin on nitric oxide production from lipopolysaccharide-activated primary microglia

Curcumin has been shown to exhibit anti-inflammatory, antimutagenic, and anticarcinogenic activities. However, the modulatory effect of curcumin on the functional activation of primary microglial cells, brain mononuclear phagocytes causing the neuronal damage, largely remains unknown. The current study examined whether curcumin influenced NO production in rat primary microglia and investigated its underlying signaling pathways. Curcumin decreased NO production in LPS-stimulated microglial cells in a dose-dependent manner, with an IC(50) value of 3.7 microM. It also suppressed both mRNA and protein levels of inducible nitric oxide synthase (iNOS), indicating that this drug may affect iNOS gene expression process. Indeed, curcumin altered biochemical patterns induced by LPS such as phosphorylation of all mitogen-activated protein kinases (MAPKs), and DNA binding activities of nuclear factor-kappaB (NF-kappaB) and activator protein (AP)-1, assessed by reporter gene assay. By analysis of inhibitory features of specific MAPK inhibitors, a series of signaling cascades including c-Jun N-terminal kinase (JNK), p38 and NF-kappaB was found to play a critical role in curcumin-mediated NO inhibition in microglial cells. The current results suggest that curcumin is a promising agent for the prevention and treatment of both NO and microglial cell-mediated neurodegenerative disorders.     

Bone Marrow-Derived Mesenchymal Stem Cells Maintain the Resting Phenotype of Microglia and Inhibit Microglial Activation

Many studies have shown that microglia in the activated state may be neurotoxic. It has been proven that uncontrolled or over-activated microglia play an important role in many neurodegenerative disorders. Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown in many animal models to have a therapeutic effect on neural damage. Such a therapeutic effect is attributed to the fact that BMSCs have the ability to differentiate into neurons and to produce trophic factors, but there is little information available in the literature concerning whether BMSCs play a therapeutic role by affecting microglial activity. In this study, we triggered an inflammatory response situation in vitro by stimulating microglia with the bacterial endotoxin lipopolysaccharide (LPS), and then culturing these microglia with BMSC-conditioned medium (BMSC-CM). We found that BMSC-CM significantly inhibited proliferation and secretion of pro-inflammatory factors by activated microglia. Furthermore, we found that the phagocytic capacity of microglia was also inhibited by BMSC-CM. Finally, we investigated whether the induction of apoptosis and the production of nitric oxide (NO) were involved in the inhibition of microglial activation. We found that BMSC-CM significantly induced apoptosis of microglia, while no apoptosis was apparent in the LPS-stimulated microglia. Our study also provides evidence that NO participates in the inhibitory effect of BMSCs. Our experimental results provide evidence that BMSCs have the ability to maintain the resting phenotype of microglia or to control microglial activation through their production of several factors, indicating that BMSCs could be a promising therapeutic tool for treatment of diseases associated with microglial activation.