Floridoside suppresses pro-inflammatory responses by blocking MAPK signaling in activated microglia

Inflammatory conditions mediated by activated microglia lead to chronic neuro-degenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. This study was conducted to determine the effect of floridoside isolated from marine red algae Laurencia undulata on LPS (100 ng/ml) activated inflammatory responses in BV-2 microglia cells. The results show that floridoside has the ability to suppress pro-inflammatory responses in microglia by markedly inhibiting the production of nitric oxide (NO) and reactive oxygen species (ROS). Moreover, floridoside down-regulated the protein and gene expression levels of iNOS and COX-2 by significantly blocking the phosphorylation of p38 and ERK in BV-2 cells. Collectively, these results indicate that floridoside has the potential to be developed as an active agent for the treatment of neuro-inflammation. [BMB Reports 2013; 46(8): 398-403]     

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.     

Differential protein acetylation induced by novel histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors induce the hyperacetylation of nucleosomal histones in carcinoma cells resulting in the expression of repressed genes that cause growth arrest, terminal differentiation, and/or apoptosis. In vitro selectivity of several novel hydroxamate HDAC inhibitors including succinimide macrocyclic hydroxamates and the non-hydroxamate alpha-ketoamide inhibitors was investigated using isolated enzyme preparations and cellular assays. In vitro selectivity for the HDAC isozymes (HDAC1/2, 3, 4/3, and 6) was not observed for these HDAC inhibitors or the reference HDAC inhibitors, MS-275 and SAHA. In T24 and HCT116 cells these compounds caused the accumulation of acetylated histones H3 and H4; however, the succinimide macrocyclic hydroxamates and the alpha-ketoamides did not cause the accumulation of acetylated alpha-tubulin. These data suggest "selectivity" can be observed at the cellular level with HDAC inhibitors and that the nature of the zinc-chelating moiety is an important determinant of activity against tubulin deacetylase.     

Follistatin-like protein 1 suppressed pro-inflammatory cytokines expression during neuroinflammation induced by lipopolysaccharide

Follistain-like protein 1 (FSTL1), has been recently demonstrated to be involved in the embryo development of nervous system and glioblastoma. However, the role of FSTL1 in neuroinflammation remains unexplored. In this study, the expression of FSTL1 in astrocytes was verified and its role was studied in neuroinflammation induced by in vivo intracerebroventricular (ICV) injection of lipopolysaccharide (LPS) or LPS treatment to astrocytes in vitro. FSTL1 was significantly induced after ICV LPS injection or LPS treatment. FSTL1 suppressed upregulation of pro-inflammatory cytokines in astrocytes after LPS treatment. Moreover, FSTL1 downregulated expression of pro-inflammatory cytokines through suppressing MAPK/p-ERK1/2 pathway in astrocytes. Our results suggest that FSTL1 may play an anti-inflammatory role in neuroinflammation mediated by astrocytes.     

Characterization of the protein ubiquitination response induced by Doxorubicin

Doxorubicin is commonly considered to exert its anti-tumor activity by triggering apoptosis of cancer cells through DNA damage. Recent reports have shown that Doxorubicin elicits a marked heat shock response, and that either inhibition or silencing of heat shock proteins enhance the Doxorubicin apoptotic effect in neuroblastoma cells. In order to investigate whether Doxorubicin may also act through protein modification, we performed a proteomic analysis of ubiquitinated proteins. Here we show that nanomolar Doxorubicin treatment of neuroblastoma cells caused: (a) dose-dependent over-ubiquitination of a specific set of proteins in the absence of measurable inhibition of proteasome, (b) protein ubiquination patterns similar to those with Bortezomib, a proteasome inhibitor, (c) depletion and loss of activity of ubiquitinated enzymes such as lactate dehydrogenase and α-enolase, and (d) a decrease in HSP27 solubility, probably a consequence of its binding to denatured proteins. These data strongly reinforce the hypothesis that Doxorubicin may also exert its effect by damaging proteins.     

CCL5 induces a pro-inflammatory profile in microglia in vitro

The chemokine receptors CCR1, CCR2, CCR3, CCR5, and CXCR2 have been found to be expressed on microglia in many neurodegenerative diseases, such as multiple sclerosis and Alzheimer’s disease. There is emerging evidence that chemokines, besides chemoattraction, might directly modulate reactive profiles of microglia. To address this hypothesis we have investigated the effects of CCL2, CCL3, CCL5, and CXCL1 on cytokine and growth factor production, NO synthesis, and phagocytosis in non-stimulated and lipopolysaccharide-stimulated primary rat microglia. The respective receptors CCR1, CCR5, and CXCR2 were shown to be functionally expressed on microglia. All tested chemokines stimulated chemotaxis whereas only CCL5 increased NO secretion and attenuated IL-10 as well as IGF-1 production in activated microglia. Based on these findings we propose that besides its chemoattractant function CCL5 has a modulatory effect on activated microglia.     

NIMA-related kinase 7 amplifies NLRP3 inflammasome pro-inflammatory signaling in microglia/macrophages and mice models of spinal cord injury

BackgroundNIMA-related kinase-7 (NEK7) is a serine/threonine kinase that drives cell-cycle dynamics by modulating mitotic spindle formation and cytokinesis. It is also a crucial modulator of the pro-inflammatory effects of NOD-like receptor 3 (NLRP3) inflammasome. However, the role of NEK7 in microglia/macrophages post-spinal cord injury (SCI) is not well defined.MethodsIn this study, we performed both in vivo and in vitro experiments. Using an in vivo mouse SCI model, NEK7 siRNAs were administered intraspinally. For in vitro analysis, BV-2 microglia cells with NEK7-siRNA were stimulated with 1 μg/ml lipopolysaccharide (LPS) and 2 mM Adenosine triphosphate (ATP).ResultsHere, we found that the mRNA and protein levels of NEK7 and NLRP3 inflammasomes were upregulated in spinal cord tissues of injured mice and BV-2 microglia cells exposed to Lipopolysaccharide (LPS) and Adenosine triphosphate (ATP). Further experiments established that NEK7 and NLRP3 interacted in BV-2 microglia cells, an effect that was eliminated following NEK7 ablation. Moreover, NEK7 ablation suppressed the activation of NLRP3 inflammasomes. Although NEK7 inhibition did not significantly improve motor function post-SCI in mice, it was found to attenuate local inflammatory response and inhibit the activation of NLRP3 inflammasome in microglia/macrophages of the injured spinal cord.ConclusionNEK7 amplifies NLRP3 inflammasome pro-inflammatory signaling in BV-2 microglia cells and mice models of SCI. Therefore, agents targeting the NEK7/NLRP3 signaling offers great promise in the treatment of inflammatory response post-SCI.     

Prion protein induced signaling cascades in monocytes

Prion proteins play a central role in transmission and pathogenesis of transmissible spongiform encephalopathies. The cellular prion protein (PrP(C)), whose physiological function remains elusive, is anchored to the surface of a variety of cell types including neurons and cells of the lymphoreticular system. In this study, we investigated the response of a mouse monocyte/macrophage cell line to exposure with PrP(C) fusion proteins synthesized with a human Fc-tag. PrP(C) fusion proteins showed an attachment to the surface of monocyte/macrophages in nanomolar concentrations. This was accompanied by an increase of cellular tyrosine phosphorylation as a result of activated signaling pathways. Detailed investigations exhibited activation of downstream pathways through a stimulation with PrP fusion proteins, which include phosphorylation of ERK(1,2) and Akt kinase. Macrophages opsonize and present antigenic structures, contact lymphocytes, and deliver cytokines. The findings reported here may become the basis of understanding the molecular function of PrP(C) in monocytes and macrophages.     

Characterization of the protein unfolding processes induced by urea and temperature

Correct folding is critical for the biological activities of proteins. As a contribution to a better understanding of the protein (un)folding problem, we studied the effect of temperature and of urea on peptostreptococcal Protein L destructuration. We performed standard molecular dynamics simulations at 300 K, 350 K, 400 K, and 480 K, both in 10 M urea and in water. Protein L followed at least two alternative unfolding pathways. Urea caused the loss of secondary structure acting preferentially on the β-sheets, while leaving the α-helices almost intact; on the contrary, high temperature preserved the β-sheets and led to a complete loss of the α-helices. These data suggest that urea and high temperature act through different unfolding mechanisms, and protein secondary motives reveal a differential sensitivity to various denaturant treatments. As further validation of our results, replica-exchange molecular dynamics simulations of the temperature-induced unfolding process in the presence of urea were performed. This set of simulations allowed us to compute the thermodynamical parameters of the process and confirmed that, in the configurational space of Protein L unfolding, both of the above pathways are accessible, although to a different relative extent.     

Acetate reduces microglia inflammatory signaling in vitro

Acetate supplementation increases brain acetyl‐CoA and histone acetylation and reduces lipopolysaccharide (LPS)‐induced neuroglial activation and interleukin (IL)‐1β expression in vivo. To determine how acetate imparts these properties, we tested the hypothesis that acetate metabolism reduces inflammatory signaling in microglia. To test this, we measured the effect acetate treatment had on cytokine expression, mitogen‐activated protein kinase (MAPK) signaling, histone H3 at lysine 9 acetylation, and alterations of nuclear factor‐kappa B (NF‐κB) in primary and BV‐2 cultured microglia. We found that treatment induced H3K9 hyperacetylation and reversed LPS‐induced H3K9 hypoacetylation similar to that found in vivo. LPS also increased IL‐1β, IL‐6, and tumor necrosis factor‐alpha (TNF‐α) mRNA and protein, whereas treatment returned the protein to control levels and only partially attenuated IL‐6 mRNA. In contrast, treatment increased mRNA levels of transforming growth factor‐β1 (TGF‐β1) and both IL‐4 mRNA and protein. LPS increased p38 MAPK and JNK phosphorylation at 4 and 2–4 h, respectively, whereas treatment reduced p38 MAPK and JNK phosphorylation only at 2 h. In addition, treatment reversed the LPS‐induced elevation of NF‐κB p65 protein and phosphorylation at serine 468 and induced acetylation at lysine 310. These data suggest that acetate metabolism reduces inflammatory signaling and alters histone and non‐histone protein acetylation.     

Regulation of caspase-3 processing by cIAP2 controls the switch between pro-inflammatory activation and cell death in microglia

The activation of microglia, resident immune cells of the central nervous system, and inflammation-mediated neurotoxicity are typical features of neurodegenerative diseases, for example, Alzheimer''s and Parkinson''s diseases. An unexpected role of caspase-3, commonly known to have executioner role for apoptosis, was uncovered in the microglia activation process. A central question emerging from this finding is what prevents caspase-3 during the microglia activation from killing those cells? Caspase-3 activation occurs as a two-step process, where the zymogen is first cleaved by upstream caspases, such as caspase-8, to form intermediate, yet still active, p19/p12 complex; thereafter, autocatalytic processing generates the fully mature p17/p12 form of the enzyme. Here, we show that the induction of cellular inhibitor of apoptosis protein 2 (cIAP2) expression upon microglia activation prevents the conversion of caspase-3 p19 subunit to p17 subunit and is responsible for restraining caspase-3 in terms of activity and subcellular localization. We demonstrate that counteracting the repressive effect of cIAP2 on caspase-3 activation, using small interfering RNA targeting cIAP2 or a SMAC mimetic such as the BV6 compound, reduced the pro-inflammatory activation of microglia cells and promoted their death. We propose that the different caspase-3 functions in microglia, and potentially other cell types, reside in the active caspase-3 complexes formed. These results also could indicate cIAP2 as a possible therapeutic target to modulate microglia pro-inflammatory activation and associated neurotoxicity observed in neurodegenerative disorders.     

Atypical antiinflammatory activation of microglia induced by apoptotic neurons

In the central nervous system (CNS), apoptosis plays an important role during development and is a primary pathogenic mechanism in several adult neurodegenerative diseases. A main feature of apoptotic cell death is the efficient and fast removal of dying cells by macrophages and nonprofessional phagocytes, without eliciting inflammation in the surrounding tissue. Apoptotic cells undergo several membrane changes, including the externalization of so-called "eat me" signals whose cognate receptors are present on professional phagocytes. Among these signals, the aminophospholipid phosphatidylserine (PS) appears to have a crucial and unique role in preventing the classical pro-inflammatory activation of macrophages, thus ensuring the silent and safe removal of apoptotic cells. Although extensively studied in the peripheral organs, the process of recognition and removal of apoptotic cells in the brain has only recently begun to be unraveled. Here, we summarize the evidence suggesting that upon interaction with PS-expressing apoptotic neurons, microglia may no longer promote the inflammatory cascade, but rather facilitate the elimination of damaged neurons through antiinflammatory and neuroprotective functions. We propose that the anti-inflammatory microglial phenotype induced through the activation of the specific PS receptor (PtdSerR), expressed by resting and activated microglial cells, could be relevant to the final outcome of neurodegenerative diseases, in which apoptosis seems to play a crucial role.     

The viral control of cellular acetylation signaling

It is becoming clear that the post-translational modification of histone and non-histone proteins by acetylation is part of an important cellular signaling process controlling a wide variety of functions in both the nucleus and the cytoplasm. Recent investigations designate this signaling pathway as one of the primary targets of viral proteins after infection. Indeed, specific viral proteins have acquired the capacity to interact with cellular acetyltransferases (HATs) and deacetylases (HDACs) and consequently to disrupt normal acetylation signaling pathways, thereby affecting viral and cellular gene expression. Here we review the targeting of cellular HATs and HDACs by viral proteins and highlight different strategies adopted by viruses to control cellular acetylation signaling and to accomplish their life cycle.     

A TLR4/MD2 fusion protein inhibits LPS-induced pro-inflammatory signaling in hepatic stellate cells

Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis. In injured liver they are the main extracellular matrix protein producing cell type and further perpetuate hepatic injury by secretion of pro-inflammatory mediators. Since LPS-mediated signaling through toll-like receptor 4 (TLR4) has been identified as key fibrogenic signal in HSCs we aimed to test TLR4 as potential target of therapy via ligand-binding soluble receptors. Incubation of human HSCs with a fusion protein between the extracellular domain of TLR4 and MD2 which binds LPS inhibited LPS-induced NFκB and JNK activation. TLR4/MD2 abolished LPS-induced secretion of IL-6, IL-8, MCP1, and RANTES in HSCs. In addition, TLR4/MD2 fused to human IgG-Fc neutralized LPS activity. Since TLR4 mutant mice are resistant to liver fibrosis, the TLR4/MD2 soluble receptor might represent a new therapeutic molecule for liver fibrogenesis in vivo.