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1.  The phosphoproteome of toll-like receptor-activated macrophages 
First global and quantitative analysis of phosphorylation cascades induced by toll-like receptor (TLR) stimulation in macrophages identifies nearly 7000 phosphorylation sites and shows extensive and dynamic up-regulation and down-regulation after lipopolysaccharide (LPS).In addition to the canonical TLR-associated pathways, mining of the phosphorylation data suggests an involvement of ATM/ATR kinases in signalling and shows that the cytoskeleton is a hotspot of TLR-induced phosphorylation.Intersecting transcription factor phosphorylation with bioinformatic promoter analysis of genes induced by LPS identified several candidate transcriptional regulators that were previously not implicated in TLR-induced transcriptional control.
Toll-like receptors (TLR) are a family of pattern recognition receptors that enable innate immune cells to sense infectious danger. Recognition of microbial structures, like lipopolysaccharide (LPS) of Gram-negative bacteria by TLR4, causes within hours substantial re-programming of macrophage gene expression, including up-regulation of chemokines driving inflammation, anti-microbial effector molecules and cytokines directing adaptive immune responses. TLR signalling is initiated by the adapter protein Myd88 and leads to the activation of kinase cascades that result in activation of the MAPK and NFkB pathways. Phosphorylation has an essential role in these early steps of TLR signalling, and in addition regulates critical transcription factors (TFs). Although TLR signalling has been extensively studied, a comprehensive analysis of phosphorylation events in TLR-activated macrophages is lacking. It is therefore unknown whether the canonical MAPK and NFkB pathways comprise the main phosphorylation events and which other molecular functions and processes are regulated by phosphorylation after stimulation with LPS.
Recent progress in mass spectrometry-based proteomics has opened the possibility to quantitatively investigate global changes in protein abundance and post-translational modifications. Stable isotope labelling with amino acids in cell culture (SILAC) allows highly accurate quantification, and has proved especially useful for direct comparison of phosphopeptide abundance in time-course or treatment analyses.
Here, we adapted SILAC to primary mouse macrophages, and performed a global, quantitative and kinetic analysis of the macrophage phosphoproteome after LPS stimulation. Bioinformatic analyses were used to identify kinases, pathways and biological processes enriched in the LPS-regulated phosphoproteome. To connect TF phosphorylation with transcription, we generated a parallel dataset of nascent RNA and used in silico promoter analysis to identify transcriptional regulators with binding site enrichment among the LPS-regulated gene set.
After establishing SILAC conditions for efficient labelling of primary bone marrow-derived macrophages in two independent experiments 1850 phosphoproteins with a total of 6956 phosphorylation sites were reproducibly identified. Phosphoproteins were detected from all cellular compartments, with a clear enrichment for nuclear and cytoskeleton-associated proteins. LPS caused major regulation of a large fraction of phosphopeptides, with 24% of all sites up-regulated and 9% down-regulated after stimulation (Figure 3A and B). These changes were highly dynamic, as the majority of the regulated phosphopeptides were up-regulated or down-regulated transiently or in a delayed manner (Figure 3C). Overall, the extent of changes in the phosphoproteome was comparable to the transcriptional re-programming, underscoring the importance of phosphorylation cascades in TLR signalling. Our parallel transcriptome data also showed that widespread phosphorylation precedes massive transcriptional changes.
To obtain footprints of kinase activation in response to TLR ligation, we searched phosphopeptide sequences for known linear sequence motifs of 33 kinases and identified kinase motifs enriched among LPS-regulated phosphorylation sites (compared to non-regulated phosphorylation sites) (Table I). Motif ERK/MAPK was highly enriched, in accordance with the essential role of the MAPK module in TLR signalling. Other kinases with motif enrichment have also recently been linked to TLR signalling (e.g. PKD; AKT and its targets GSK3 and mTOR). However, the DNA damage-actviated kinases ATM/ATR and the cell cycle-associated kinases AURORA and CHK1/2 have not been associated with the macrophage response to TLR activation yet. These finding shed new light on older data on the effect of TLR on macrophage proliferation in response to macrophage colony stimulating factor. Of interest, in follow-up experiments using pharmacological inhibitors of the kinases with motif enrichment, we observed that inhibition of ATM kinase activity caused increased LPS-induced expression of several cytokines and chemokines, suggesting that this pathway regulates inflammatory responses.
In further bioinformatic analyses, the Gene Ontology and signalling pathway annotations of phosphoproteins were used to identify signalling pathways and cellular processes targeted by TLR4-controlled phosphorylation (Table II). Among the expected hits, based on the known TLR pathways, were TLR signalling, MAPK and AKT as well as mTOR signalling. Of interest, the annotation terms ‘Rho GTPase cycle' and ‘cytoskeleton' were significantly enriched among LPS-regulated phosphoproteins, indicating a more prominent role for cytoskeletal proteins in the transduction of TLR signals or in the biological response to it.
We were especially interested in the phosphorylation of TFs and its regulation by LPS (Figure 6A). We hypothesised that functionally important TFs should have an increased frequency of binding sites in the promoters of LPS-regulated genes (Figure 6B). To identify transcriptionally regulated genes with high sensitivity, we isolated nascent RNA after metabolic labelling (Figure 6C–E). In silico promoter scanning using Genomatix software for binding sites for all 50 TF families with phosphorylated members was used to test for enrichment in transciptionally induced genes (Figure 6F). At the early time point, binding site enrichment for the canonical TLR-associated TF NFkB was detected, and in addition we found that several other TF families with an established role in the transcription of individual LPS-target genes showed binding site enrichment (CEBP, MEF2, NFAT and HEAT). In addition, enrichment for OCT and HOXC binding sites at the early time point and SORY matrices later after stimulation indicated an involvement of the phosphorylated members of the respective TF families in the execution of TLR-induced transcriptional responses. An initial test of the function for a few of these candidate transcriptional regulators was performed using siRNA knockdown in primary macrophages. These experiments suggested that knock down of the SORY binding phosphoprotein Capicua homolog (Cic) and to a lesser extent of the CREB family member Atf7 selectively attenuates LPS-induced expression of Il1a and Il1b.
In summary, this study provides a novel and global perspective on innate immune activation by TLR signalling (Figure 5). We quantitatively detected a large number of previously unknown site-specific phosphorylation events, which are now publicly available through the Phosida database. By combining different data mining approaches, we consistently identified canonical and newly implicated TLR-activated signalling modules. In particular, the PI3K/AKT and the related mTOR pathway were highlighted; furthermore, DNA damage–response associated ATM/ATR kinases and the cytoskeleton emerged as unexpected hotspots for phosphorylation. Finally, weaving together corresponding phophoproteome and nascent transcriptome datasets through the loom of in silico promoter analysis we identified TFs with a likely role in mediating TLR-induced gene expression programmes.
Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome of primary macrophages using stable isotope labelling with amino acids in cell culture, phosphopeptide enrichment and high-resolution mass spectrometry. In parallel, nascent RNA was profiled to link transcription factor (TF) phosphorylation to TLR4-induced transcriptional activation. We reproducibly identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier. LPS caused major dynamic changes in the phosphoproteome (24% up-regulation and 9% down-regulation). Functional bioinformatic analyses confirmed canonical players of the TLR pathway and highlighted other signalling modules (e.g. mTOR, ATM/ATR kinases) and the cytoskeleton as hotspots of LPS-regulated phosphorylation. Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression.
PMCID: PMC2913394  PMID: 20531401
macrophage; nascent RNA; phosphoproteome; SILAC; toll-like receptors
2.  Roles of a Novel Molecule ‘Shati’ in the Development of Methamphetamine-Induced Dependence 
Current Neuropharmacology  2011;9(1):104-108.
The ability of drugs of abuse to cause dependence can be viewed as a form of neural plasticity. Recently, we have demonstrated that tumor necrosis factor-α (TNF-α) increases dopamine uptake and inhibits methamphetamine-induced dependence. Moreover, we have identified a novel molecule ‘shati’ in the nucleus accumbens of mice treated with methamphetamine using the PCR-select cDNA subtraction method and clarified that it is involved in the development of methamphetamine dependence: Treatment with the shati antisense oligonucleotide (shati-AS), which inhibits the expression of shati mRNA, enhanced the methamphetamine-induced hyperlocomotion, sensitization, and conditioned place preference. Further, blockage of shati mRNA by shati-AS potentiated the methamphetamine-induced increase of dopamine overflow and the methamphetamine-induced decrease in dopamine uptake in the nucleus accumbens. Interestingly, treatment with shati-AS also inhibited expression of TNF-α. Transfection of the vector containing shati cDNA into PC12 cells, dramatically induced the expression of shati and TNF-α mRNA, accelerated dopamine uptake, and inhibited the methamphetamine-induced decrease in dopamine uptake. These effects were blocked by neutralizing TNF-α. These results suggest that the functional roles of shati in methamphetamine-induced behavioral changes are mediated through the induction of TNF-α expression which inhibits the methamphetamine-induced increase of dopamine overflow and decrease in dopamine uptake.
PMCID: PMC3137161  PMID: 21886572
Shati; methamphetamine; dependence; tumor necrosis factor-α; dopamine; uptake; nucleus accumbens; anti-addictive.
3.  Induction of cytokine granulocyte-macrophage colony-stimulating factor and chemokine macrophage inflammatory protein 2 mRNAs in macrophages by Legionella pneumophila or Salmonella typhimurium attachment requires different ligand-receptor systems. 
Infection and Immunity  1996;64(8):3062-3068.
The attachment of bacteria to macrophages is mediated by different ligands and receptors and induces various intracellular molecular responses. In the present study, induction of cytokines and chemokines, especially granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage inflammatory protein 2 (MIP-2), was examined, following bacterial attachment, with regard to the ligand-receptor systems involved. Attachment of Legionella pneumophila or Salmonella typhimurium to cultured mouse peritoneal macrophages increased the steady-state levels of cellular mRNAs for the cytokines interleukin 1beta (IL-1beta), IL-6, and GM-CSF as well as the chemokines MIP-1beta, MIP-2, and KC. However, when macrophages were treated with alpha-methyl-D-mannoside (alphaMM), a competitor of glycopeptide ligands, induction of cytokine mRNAs was inhibited, but the levels of chemokine mRNAs were not. Pretreatment of the bacteria with fresh mouse serum enhanced the level of GM-CSF mRNA but not the level of MIP-2 mRNA. In addition, serum treatment reduced the inhibitory effect of alphaMM on GM-CSF mRNA. These results indicate that bacterial attachment increases the steady-state levels of the cytokine and chemokine mRNAs tested by at least two distinct receptor-ligand systems, namely, one linked to cytokine induction and involving mannose or other sugar residues and the other linked to chemokine induction and relatively alphaMM insensitive. Furthermore, opsonization with serum engages other pathways in the cytokine response which are relatively independent of the alphaMM-sensitive system. Regarding bacterial surface ligands involved in cytokine mRNA induction, evidence is presented that the flagellum may be important in stimulating cytokine GM-CSF message but not chemokine MIP-2 message. Analysis of cytokine GM-CSF and chemokine MIP-2 signaling pathways with protein kinase inhibitors revealed the involvement of calmodulin and myosin light-chain kinase in GM-CSF but not MIP-2 mRNA induction, adding further evidence that several distinct receptor systems are engaged during the process of bacterial attachment and induction of cytokines and chemokines, such as GM-CSF and MIP-2, respectively.
PMCID: PMC174188  PMID: 8757834
4.  Involvement of metabotropic glutamate receptor 5, AKT/PI3K Signaling and NF-κB pathway in methamphetamine-mediated increase in IL-6 and IL-8 expression in astrocytes 
Methamphetamine (MA) is one of the commonly used illicit drugs and the central nervous system toxicity of MA is well documented. The mechanisms contributing to this toxicity have not been fully elucidated. In this study, we investigated the effect of MA on the expression levels of the proinflammatory cytokines/chemokines, IL-6 and IL-8 in an astrocytic cell line. The IL-6 and IL-8 RNA levels were found to increase by 4.6 ± 0.2 fold and 3.5 ± 0.2 fold, respectively, after exposure to MA for three days. Exposure of astrocytes to MA for 24 hours also caused increased expression of IL-6 and IL-8 at the level of both RNA and protein. The potential involvement of the nuclear factor-Kappa B (NF-κB) pathway was explored as one of the possible mechanism(s) responsible for the increased induction of IL-6 and IL-8 by MA. The MA-mediated increases in IL-6 and IL-8 were significantly abrogated by SC514. We also found that exposure of astrocytes to MA results in activation of NF-κB through the phosphorylation of IκB-α, followed by translocation of active NF-κB from the cytoplasm to the nucleus. In addition, treatment of cells with a specific inhibitor of metabotropic glutamate receptor-5 (mGluR5) revealed that MA-mediated expression levels of IL-6 and IL-8 were abrogated by this treatment by 42.6 ± 5.8% and 65.5 ± 3.5%, respectively. Also, LY294002, an inhibitor of the Akt/PI3K pathway, abrogated the MA-mediated induction of IL-6 and IL-8 by 77.9 ± 6.6% and 81.4 ± 2.6%, respectively. Thus, our study demonstrates the involvement of an NF-κB-mediated signaling mechanism in the induction of IL-6 and IL-8 by MA. Furthermore, we showed that blockade of mGluR5 can protect astrocytes from MA-mediated increases of proinflammatory cytokines/chemokines suggesting mGluR5 as a potential therapeutic target in treating MA-mediated neurotoxicity.
PMCID: PMC3338363  PMID: 22420994
gp120; IL-8; Astrocytes; NF-kB; siRNA
5.  A Francisella Mutant in Lipid A Carbohydrate Modification Elicits Protective Immunity 
PLoS Pathogens  2008;4(2):e24.
Francisella tularensis (Ft) is a highly infectious Gram-negative bacterium and the causative agent of the human disease tularemia. Ft is designated a class A select agent by the Centers for Disease Control and Prevention. Human clinical isolates of Ft produce lipid A of similar structure to Ft subspecies novicida (Fn), a pathogen of mice. We identified three enzymes required for Fn lipid A carbohydrate modifications, specifically the presence of mannose (flmF1), galactosamine (flmF2), or both carbohydrates (flmK). Mutants lacking either galactosamine (flmF2) or galactosamine/mannose (flmK) addition to their lipid A were attenuated in mice by both pulmonary and subcutaneous routes of infection. In addition, aerosolization of the mutants (flmF2 and flmK) provided protection against challenge with wild-type (WT) Fn, whereas subcutaneous administration of only the flmK mutant provided protection from challenge with WT Fn. Furthermore, infection of an alveolar macrophage cell line by the flmK mutant induced higher levels of tumor necrosis factor-α (TNF-α) and macrophage inhibitory protein-2 (MIP-2) when compared to infection with WT Fn. Bone marrow–derived macrophages (BMMø) from Toll-like receptor 4 (TLR4) and TLR2/4 knockout mice infected with the flmK mutant also produced significantly higher amounts of interleukin-6 (IL-6) and MIP-2 than BMMø infected with WT Fn. However, production of IL-6 and MIP-2 was undetectable in BMMø from MyD88−/− mice infected with either strain. MyD88−/− mice were also susceptible to flmK mutant infection. We hypothesize that the ability of the flmK mutant to activate pro-inflammatory cytokine/chemokine production and innate immune responses mediated by the MyD88 signaling pathway may be responsible for its attenuation, leading to the induction of protective immunity by this mutant.
Author Summary
Bacterial pathogens modify outer membrane components, such as lipid A or endotoxin, the lipid anchor of lipopolysaccharide, to enhance the ability to colonize, spread to different tissues, and/or avoid the host's immune defenses. Lipopolysaccharide also plays an essential role in maintaining membrane integrity and is a key factor in host innate immune recognition of Gram-negative bacterial infections. Francisella tularensis is the causative agent of the human disease tularemia and is classified as a category A select agent. Francisella novicida (Fn) is the murine counterpart of F. tularensis. The structure of Francisella spp. lipid A is unique in that it is modified by various carbohydrates that play a role in virulence and altered endotoxicity. In our study, we identified and defined the role of three genes involved in the carbohydrate modification of the base Fn lipid A structure. We showed that the lack of specific modification(s) of the Fn lipid A molecule lead to bacterial attenuation and activation of a protective immune response against a lethal wild-type infection. Therefore, alteration of Francisella lipid A structure may represent a pathogenesis strategy common to the Francisella species, and specific lipid A mutant strains may be candidates for inclusion in future vaccine studies.
PMCID: PMC2233673  PMID: 18266468
6.  Lipopolysaccharide signals activation of tumor necrosis factor biosynthesis through the ras/raf-1/MEK/MAPK pathway. 
Molecular Medicine  1994;1(1):93-103.
BACKGROUND: Lipopolysaccharide (LPS) is known to activate macrophages, causing the release of toxic cytokines that may provoke inflammation and shock. One of the most important and best studied of these cytokines is tumor necrosis factor (TNF). Details of the signaling pathway leading to TNF biosynthesis remain unclear. The pathway is branched in the sense that TNF gene transcription and TNF mRNA translation are both strongly stimulated by LPS. Recent evidence has indicated that MAP kinase homologs become phosphorylated in LPS-stimulated cells, suggesting their possible involvement in signal transduction. We sought to test this hypothesis. MATERIALS AND METHODS: Measurements of LPS-induced MEK and ERK2 activity were undertaken in LPS-sensitive and LPS-insensitive cells. Transfection studies, in which dominant inhibitors of ras and raf-1 were used to block signaling to the level of MAP kinase, were carried out in order to judge whether the TNF gene transcription and TNF mRNA translation are modulated through this pathway. RESULTS: In RAW 264.7 mouse macrophages, both ERK2 and MEK1 activity are induced by LPS treatment. In the same cell line, dominant negative inhibitors of ras and raf-1 block LPS-induced activation of the TNF promoter, as well as derepression of the translational blockade normally imposed by the TNF 3'-untranslated region. A constitutively active form of raf-1 (raf-BXB) was found to augment, but not replace, the LPS signal. In LPS-insensitive cells (RAW 264.7 x NIH 3T3 fusion hybrid cells and primary macrophages derived from C3H/HeJ mice), ERK2 activity was found to be refractory to induction by LPS. CONCLUSIONS: The ras/raf-1/MEK/MAPK pathway is chiefly responsible for transduction of the LPS signal to the level of the TNF gene and mRNA. raf and raf-1 lie upstream from (or actually represent) the physical branchpoints of the transcriptional and translation activation signals generated by LPS. The lesions that prevent LPS signaling in macrophages from C3H/HeJ mice, or in RAW 264.7 x NIH 3T3 fusion hybrid cells, occupy a proximal position in the signaling pathway.
PMCID: PMC2229930  PMID: 8790605
7.  Expression of cytokine and chemokine mRNA and secretion of tumor necrosis factor-α by gallbladder epithelial cells: Response to bacterial lipopolysaccharides 
BMC Gastroenterology  2002;2:23.
In addition to immune cells, many other cell types are known to produce cytokines. Cultured normal mouse gallbladder epithelial cells, used as a model system for gallbladder epithelium, were examined for their ability to express the mRNA of various cytokines and chemokines in response to bacterial lipopolysaccharide. The synthesis and secretion of the tumor necrosis factor-α (TNF-α) protein by these cells was also measured.
Untreated mouse gallbladder cells expressed mRNA for TNF-α, RANTES, and macrophage inflammatory protein-2 (MIP-2). Upon treatment with lipopolysaccharide, these cells now produced mRNA for Interleukin-1β (IL-1β), IL-6, monocyte chemoattractant protein-1 (MCP-1), and showed increased expression of TNF-α and MIP-2 mRNA. Untreated mouse gallbladder cells did not synthesize TNF-α protein; however, they did synthesize and secrete TNF-α upon treatment with lipopolysaccharide.
Cells were treated with lipopolysaccharides from 3 strains of bacteria. Qualitative and semi-quantitative RT-PCR, using cytokine or chemokine-specific primers, was used to measure mRNA levels of TNFα, IL-1β, IL-6, IL-10, KC, RANTES, MCP-1, and MIP-2. TNF-α protein was measured by immunoassays.
This research demonstrates that gallbladder epithelial cells in response to lipopolysaccharide exposure can alter their cytokine and chemokine RNA expression pattern and can synthesize and secrete TNFα protein. This suggests a mechanism whereby gallbladder epithelial cells in vivo may mediate gallbladder secretory function, inflammation and diseases in an autocrine/paracrine fashion by producing and secreting cytokines and/or chemokines during sepsis.
PMCID: PMC130965  PMID: 12377103
8.  JNK and ERK MAP kinases mediate induction of IL-1β, TNF-α and IL-8 following hyperosmolar stress in human limbal epithelial cells⋆ 
Experimental eye research  2005;82(4):588-596.
Hyperosmolarity has been recognized to be a pro-inflammatory stress to the corneal epithelium. The cell signalling pathways linking hyperosmolar stress and inflammation have not been well elucidated. This study investigated whether exposure of human limbal epithelial cells to hyperosmotic stress activates the mitogen-activated protein kinase (MAPK) pathways and induces production of pro-inflammatory cytokines, interleukin (IL) -1β, tumor necrosis factor (TNF) α, and the C-X-C chemokine IL-8. Primary human limbal epithelial cultures in normal osmolar media (312 mOsM) were exposed to media with higher osmolarity (400–500 mOsM) by adding 50–90 mM NaCl, with or without SB202190, an inhibitor of c-Jun N-terminal kinases (JNK) pathway, PD 98059, an inhibitor of extracellular-regulated kinase (ERK) pathway, dexamethasone or doxycycline for different lengths of time. The conditioned media were collected after 24 hr of treatment for ELISA. Total RNA was extracted from cultures treated for 6 hr for semi-quantitative RT-PCR. Cells treated for 15–60 min were lysed in RIPA buffer and subjected to Western blot with phospho (p)-specific antibodies against p-JNK and p-ERK. The concentrations of IL-1β, TNF-α and IL-8 proteins in 24 hr conditioned media of limbal epithelial cells progressively increased as the media osmolarity increased from 312 to 500 mOsM. Active p-JNK-1/p-JNK-2 and p-ERK-1/p-ERK-2 were detected by Western blot and peaked at 60 min in cells exposed to hyperosmolar media. The levels of p-JNK-1/p-JNK-2 and p-ERK1/p-ERK2 were positively correlated with the medium osmolarity. SB202190, PD98059 and doxycycline markedly suppressed the levels of p-JNK-1/p-JNK-2 and/or p-ERK1/p-ERK2, as well as IL-1β, TNF-α and IL-8 mRNAs and proteins stimulated by hyperosmolar media. These findings provide direct evidence that hyperosmolarity induces inflammation in human limbal epithelial cells by increasing expression and production of pro-inflammatory cytokines and chemokines, a process that appears to be mediated through activation of the JNK and ERK MAPK signalling pathways. The efficacy of doxycycline in treating ocular surface diseases may be due to its ability to suppress JNK and ERK signalling activation and inflammatory mediator production in the limbal epithelium.
PMCID: PMC2198933  PMID: 16202406
cornea; epithelium; hyperosmolarity; inflammatory cytokine; chemokine; JNK; ERK; MAPK
9.  Synergistic Inhibition of Tumor Necrosis Factor-Alpha-Stimulated Pro-Inflammatory Cytokine Expression in HaCaT Cells by a Combination of Rapamycin and Mycophenolic Acid 
Annals of Dermatology  2015;27(1):32-39.
Keratinocytes release various pro-inflammatory cytokines, chemokines, and adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) in response to cytokines such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ. Rapamycin and mycophenolic acid (MPA) have potent immunosuppressive activity because they inhibit lymphocyte proliferation.
We investigated the effects of rapamycin and MPA on the expression of inflammation-related factors such as ICAM-1 and inducible nitric oxide synthase (iNOS), pro-inflammatory cytokines and chemokines, and related signaling pathways in TNF-α-stimulated HaCaT cells.
The viability of HaCaT cells treated with rapamycin and MPA was confirmed using MTT assay. The expression of various cytokines such as interleukin (IL)-1β, IL-6, and IL-8; inflammation-related factors such as ICAM-1 and iNOS; and the activation of mitogen activated protein kinase (MAPK) signaling pathways mediated by extracellular signal-related kinases (ERK), p38, and c-Jun N-terminal kinases (JNK) in TNF-α-stimulated HaCaT cells were confirmed using reverse transcription-polymerase chain reaction and western blotting.
Combined treatment of TNF-α-induced HaCaT cells with rapamycin and MPA decreased ICAM-1 and iNOS expression and ERK and p38 activation more than treatment with either drug alone. The most significant decrease was observed with a combination of rapamycin (80 nM) and MPA (20 nM). These results show that co-treatment with these agents has a synergistic anti-inflammatory effect by blocking the activation of the ERK/p38 MAPK signaling pathway and thus suppressing the TNF-α-induced expression of ICAM-1 and iNOS.
The combination of rapamycin and MPA could potentially be used as a therapeutic approach in inflammatory skin diseases.
PMCID: PMC4323600
Anti-inflammation; Mycophenolic acid; Sirolimus; Tumor necrosis factor-alpha
10.  Role of anti-inflammatory compounds in human immunodeficiency virus-1 glycoprotein120-mediated brain inflammation 
Neuroinflammation is a common immune response associated with brain human immunodeficiency virus-1 (HIV-1) infection. Identifying therapeutic compounds that exhibit better brain permeability and can target signaling pathways involved in inflammation may benefit treatment of HIV-associated neurological complications. The objective of this study was to implement an in vivo model of brain inflammation by intracerebroventricular administration of the HIV-1 viral coat protein gp120 in rats and to examine anti-inflammatory properties of HIV adjuvant therapies such as minocycline, chloroquine and simvastatin.
Male Wistar rats were administered a single dose of gp120ADA (500 ng) daily for seven consecutive days, intracerebroventricularly, with or without prior intraperitoneal administration of minocycline, chloroquine or simvastatin. Maraviroc, a CCR5 antagonist, was administered intracerebroventricularly prior to gp120 administration for seven days as control. Real-time qPCR was used to assess gene expression of inflammatory markers in the frontal cortex, hippocampus and striatum. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) secretion in cerebrospinal fluid (CSF) was measured applying ELISA. Protein expression of mitogen-activated protein kinases (MAPKs) (extracellular signal-related kinase 1/2 (ERK1/2), c-Jun N-terminal kinases (JNKs) and P38 kinases (P38Ks)) was detected using immunoblot analysis. Student’s t-test and ANOVA were applied to determine statistical significance.
In gp120ADA-injected rats, mRNA transcripts of interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) were significantly elevated in the frontal cortex, striatum and hippocampus compared to saline or heat-inactivated gp120-injected controls. In CSF, a significant increase in TNF-α and IL-1β was detected. Maraviroc reduced upregulation of these markers suggesting that the interaction of R5-tropic gp120 to CCR5 chemokine receptor is critical for induction of an inflammatory response. Minocycline, chloroquine or simvastatin attenuated upregulation of IL-1β and iNOS transcripts in different brain regions. In CSF, minocycline suppressed TNF-α and IL-1β secretion, whereas chloroquine attenuated IL-1β secretion. In gp120-injected animals, activation of ERK1/2 and JNKs was observed in the hippocampus and ERK1/2 activation was significantly reduced by the anti-inflammatory agents.
Our data demonstrate that anti-inflammatory compounds can completely or partially reverse gp120-associated brain inflammation through an interaction with MAPK signaling pathways and suggest their potential role in contributing towards the prevention and treatment of HIV-associated neurological complications.
PMCID: PMC4046047  PMID: 24884548
Chloroquine; HIV-1 brain inflammation; HIV-1 gp120; Inducible nitric oxide synthase; Maraviroc; Minocycline; Mitogen-activated protein kinase (MAPK); Pro-inflammatory cytokines; Simvastatin
11.  Spred-2 Deficiency Exacerbates Lipopolysaccharide-Induced Acute Lung Inflammation in Mice 
PLoS ONE  2014;9(10):e108914.
Acute respiratory distress syndrome (ARDS) is a severe and life-threatening acute lung injury (ALI) that is caused by noxious stimuli and pathogens. ALI is characterized by marked acute inflammation with elevated alveolar cytokine levels. Mitogen-activated protein kinase (MAPK) pathways are involved in cytokine production, but the mechanisms that regulate these pathways remain poorly characterized. Here, we focused on the role of Sprouty-related EVH1-domain-containing protein (Spred)-2, a negative regulator of the Ras-Raf-extracellular signal-regulated kinase (ERK)-MAPK pathway, in lipopolysaccharide (LPS)-induced acute lung inflammation.
Wild-type (WT) mice and Spred-2−/− mice were exposed to intratracheal LPS (50 µg in 50 µL PBS) to induce pulmonary inflammation. After LPS-injection, the lungs were harvested to assess leukocyte infiltration, cytokine and chemokine production, ERK-MAPK activation and immunopathology. For ex vivo experiments, alveolar macrophages were harvested from untreated WT and Spred-2−/− mice and stimulated with LPS. In in vitro experiments, specific knock down of Spred-2 by siRNA or overexpression of Spred-2 by transfection with a plasmid encoding the Spred-2 sense sequence was introduced into murine RAW264.7 macrophage cells or MLE-12 lung epithelial cells.
LPS-induced acute lung inflammation was significantly exacerbated in Spred-2−/− mice compared with WT mice, as indicated by the numbers of infiltrating leukocytes, levels of alveolar TNF-α, CXCL2 and CCL2 in a later phase, and lung pathology. U0126, a selective MEK/ERK inhibitor, reduced the augmented LPS-induced inflammation in Spred-2−/− mice. Specific knock down of Spred-2 augmented LPS-induced cytokine and chemokine responses in RAW264.7 cells and MLE-12 cells, whereas Spred-2 overexpression decreased this response in RAW264.7 cells.
The ERK-MAPK pathway is involved in LPS-induced acute lung inflammation. Spred-2 controls the development of LPS-induced lung inflammation by negatively regulating the ERK-MAPK pathway. Thus, Spred-2 may represent a therapeutic target for the treatment of ALI.
PMCID: PMC4183529  PMID: 25275324
12.  Simultaneous Induction of Apoptotic and Survival Signaling Pathways in Macrophage-Like THP-1 Cells by Shiga Toxin 1▿  
Infection and Immunity  2006;75(3):1291-1302.
Shiga toxins have been shown to induce apoptosis in many cell types. However, Shiga toxin 1 (Stx1) induced only limited apoptosis of macrophage-like THP-1 cells in vitro. The mechanisms regulating macrophage death or survival following toxin challenge are unknown. Differentiated THP-1 cells expressed tumor necrosis factor receptors and membrane-associated tumor necrosis factor alpha (TNF-α) and produced soluble TNF-α after exposure to Stx1. However, the cells were refractory to apoptosis induced by TNF-α, although the cytokine modestly increased apoptosis in the presence of Stx1. Despite the partial resistance of macrophage-like THP-1 cells to Stx1-mediated killing, treatment of these cells with Stx1 activated a broad array of caspases, disrupted the mitochondrial membrane potential (ΔΨm), and released cytochrome c into the cytoplasm. The ΔΨm values were greatest in cells that had detached from plastic surfaces. Specific caspase inhibitors revealed that caspase-3, caspase-6, caspase-8, and caspase-9 were primarily involved in apoptosis induction. The antiapoptotic factors involved in macrophage survival following toxin challenge include inhibitors of apoptosis proteins and X-linked inhibitor of apoptosis protein. NF-κB and JNK mitogen-activated protein kinases (MAPKs) appeared to activate survival pathways, while p38 MAPK was involved in proapoptotic signaling. The JNK and p38 MAPKs were shown to be upstream signaling pathways which may regulate caspase activation. Finally, the protein synthesis inhibitors Stx1 and anisomycin triggered limited apoptosis and prolonged JNK and p38 MAPK activation, while macrophage-like cells treated with cycloheximide remained viable and showed transient activation of MAPKs. Collectively, these data suggest that Stx1 activates both apoptotic and cell survival signaling pathways in macrophage-like THP-1 cells.
PMCID: PMC1828570  PMID: 17194804
13.  Identification of Proteins Differentially Expressed in Human Monocytes Exposed to Porphyromonas gingivalis and Its Purified Components by High-Throughput Immunoblotting  
Infection and Immunity  2006;74(2):1204-1214.
To characterize the roles of Porphyromonas gingivalis and its components in disease processes, we investigated the cytokine profiles induced by live P. gingivalis, its lipopolysaccharide (LPS), and its major fimbrial protein, fimbrillin (FimA). A cytokine antibody array revealed that human monocyte-derived macrophages were induced to produce chemokines (e.g., monocyte chemoattractant protein 1, macrophage inflammatory protein 1β [MIP-1β], and MIP-3α) as early as 1 h after exposure to P. gingivalis, with production declining after 24 h of exposure. As expected, an extensive repertoire of inflammatory mediators increased subsequent to infection, most predominantly tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor. The induction of cytokines by P. gingivalis was not triggered simply by bacterial cell surface components, since purified P. gingivalis LPS and FimA induced similar patterns of cytokines, while the pattern of cytokines induced by live P. gingivalis was significantly different, indicating that the host defense system senses live bacteria differently than it does the cell surface components LPS and FimA. To further understand the mechanisms by which live P. gingivalis and its components exert their effects, we used a high-throughput immunoblot screening approach (Becton-Dickinson PowerBlot) to analyze intracellular proteins involved in P. gingivalis infection in human macrophages. Exposure of human macrophages to either live P. gingivalis, its LPS, or its FimA protein led to the up-regulation of 12, 8, and 10 proteins and the down-regulation of 15, 8, and 17 proteins, respectively. The expression of proteins involved in gene transcription (e.g., monocyte enhancer factor 2D [MEF2D], signal transducer and activator of transcription 1 [STAT1], STAT3, STAT6, and IL enhancer binding factors [ILF3]), of protein kinases (e.g., mitogen-activated protein kinase 3 [MAPK3], MAP3K8, double-stranded RNA-activated protein kinase [PRKR], and MAP2K4), and of proteins involved in immune responses (e.g., TNF super family member 6 [TNFSF6] and interferon-induced protein with tetratricopeptide repeat 4 [IFIT4]), apoptosis (e.g., genes associated with retinoid interferon-induced mortality 19 [GRIM19]), and other fundamental cellular processes (e.g., clathrin heavy-chain polypeptide, culreticulin, and Ras-associated protein RAB27A) was found to be modulated differentially by P. gingivalis, LPS, and FimA. These differential changes are interpreted as preferential signal pathway activation in host immune/inflammatory responses to P. gingivalis infection.
PMCID: PMC1360359  PMID: 16428770
14.  Effects of alpha-mangostin on the expression of anti-inflammatory genes in U937 cells 
Chinese Medicine  2012;7:19.
α-Mangostin (α-MG) is a main constituent of the fruit hull of the mangosteen. Previous studies have shown that α-MG has pharmacological activities such as antioxidant, antitumor, anti-inflammatory, antiallergic, antibacterial, antifungal and antiviral effects. This study aims to investigate the anti-inflammatory molecular action of α-MG on gene expression profiles.
U937 and EL4 cells were treated with different concentrations of α-MG in the presence of 0.1 ng/mL lipopolysaccharide (LPS) for 4 h. The anti-inflammatory effects of α-MG were measured by the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-4 in cell culture media, which were determined with enzyme-linked immunosorbent assay kits. The gene expression profiles of all samples were analyzed with a whole human genome microarray, Illumina BeadChip WG-6 version 3, containing 48804 probes. The protein levels were determined by Western blotting analyses.
α-MG decreased the LPS induction of the inflammatory cytokines TNF-α (P = 0.038) and IL-4 (P = 0.04). α-MG decreased the gene expressions in oncostatin M signaling via mitogen-activated protein kinase (MAPK) pathways, including extracellular signal-regulated kinases (P = 0.016), c-Jun N-terminal kinase (P = 0.01) , and p38 (P = 0.008). α-MG treatment of U937 cells reduced the phosphorylation of MAPK kinase 3 / MAPK kinase 6 (P = 0.0441), MAPK-activated protein kinase-2 (P = 0.0453), signal transducers and activators of transcription-1 (STAT1) (P = 0.0012), c-Fos (P = 0.04), c-Jun (P = 0.019) and Ets-like molecule 1 (Elk-1) (P = 0.038).
This study demonstrates that α-MG attenuates LPS-mediated activation of MAPK, STAT1, c-Fos, c-Jun and EIK-1, inhibiting TNF-α and IL-4 production in U937 cells.
PMCID: PMC3476971  PMID: 22920833
15.  Human intestinal epithelial and smooth muscle cells are potent producers of IL-6. 
Mediators of Inflammation  2003;12(1):3-8.
BACKGROUND: Interleukin-6 (IL-6), a pluripotent cytokine, has traditionally been considered the product of proinflammatory cells. However, many other cell types have been shown to produce IL-6. Since intestinal inflammation is commonly associated with a vigorous systemic inflammatory response, we hypothesized that intestinal epithelial and smooth muscle cells might contribute to that response by producing IL-6. We therefore studied the capacity of differentiated human intestinal epithelial and smooth muscle cell lines to produce IL-6 in response to various proinflammatory stimuli. MATERIALS AND METHODS: CCL-241, a human intestinal epithelial cell line, and HISM, a human intestinal muscle cell line, were grown to confluency and then treated for 24 h with various concentrations of lipopolysaccharide, Clostridium difficile culture extract containing both toxin A and toxin B, recombinant human tumor necrosis factor-alpha (TNF-alpha), or recombinant human interleukin-1 beta (IL-1beta). Supernatants were then collected for IL-6 determination using an enzyme-linked immunosorbent assay. Cell numbers were determined using a Coulter counter. For comparison, parallel studies were performed using phorbol ester-primed U-937 and THP-1 human macrophage cell lines. RESULTS: Both human intestinal epithelial and smooth muscle cells produced IL-6 under basal conditions. In HISM cells, but not in CCL-241 cells, IL-6 release was increased slightly by treatment with C. difficile culture extract containing both toxin A and toxin B and with lipopolysaccharide. In both cell lines, IL-6 production was profoundly stimulated by treatment with IL-1beta and less so with TNF-alpha. Combinations of high-dose TNF-alpha and IL-1beta may have a slightly additive, but not synergistic, effect on IL-6 release. The amount of IL-6 produced by IL-1-stimulated intestinal cell lines was 70-fold higher than that produced by stimulated macrophage cell lines. CONCLUSIONS; Both intestinal epithelial and smooth muscle cells demonstrate the ability to release significant amounts of IL-6. The profound response to IL-1beta and TNF-alpha stimulation by both cell lines suggests that human intestinal parenchymal cells, influenced by paracrine mediators liberated from proinflammatory cells, might significantly contribute to the overall systemic inflammatory response by producing IL-6.
PMCID: PMC1781593  PMID: 12745542
16.  The role of sphingosine 1-phosphate in the TNF-α induction of IL-8 gene expression in lung epithelial cells 
Gene  2006;391(1-2):150-160.
Tumor necrosis factor-α (TNF-α) is an important cytokine involved in the pathogenesis of inflammatory diseases of the lung. Inteleukin-8 (IL-8), a C-X-C chemokine, is induced by TNF-α and initiates injury by acting as a chemoattractant for neutrophils and other immune cells. Although sphingolipids such as ceramide and sphingosine 1-phosphate (S1-P) have been shown to serve as signaling molecules in the TNF-α inflammatory response, their role in the TNF-α induction of IL-8 gene expression in lung epithelial cells is not known. We investigated the role of sphingolipids in the TNF-α induction of IL-8 gene expression in H441 lung epithelial cells. We found that TNF-α induced IL-8 mRNA levels by increasing gene transcription, and the stability of IL-8 mRNA was not affected. Exogenous S1-P but not ceramide or sphingosine increased IL-8 mRNA levels and IL-8 secretion. Dimethylsphingosine, an inhibitor of sphingosine kinase, partially inhibited TNF-α induction of IL-8 mRNA levels indicating the importance of intracellular increases in S1-P in the IL-8 induction. S1-P induction of IL-8 mRNA was due to an increase in gene transcription, and the stability of IL-8 mRNA was not affected. S1P induction of IL-8 mRNA was associated with an increase in the binding activity of AP-1 but the activities of NF-κB and NF IL-6 were unchanged. S1-P induced the phosphorylation of ERK, p38 and JNK MAPKs. Pharmacological inhibitors of ERK and p38 but not JNK partly inhibited S1-P induction of IL-8 mRNA levels. These data show that increases in the intracellular S1-P partly mediate TNF-α induction of IL-8 gene expression in H441 lung epithelial cells via ERK and p38 MAPK signaling pathways and increased AP-1 DNA binding.
PMCID: PMC1892234  PMID: 17306937
sphingolipids; transcription; lung injury; inflammation
17.  Phosphorylation of Akt Mediates Anti-Inflammatory Activity of 1-p-Coumaroyl β-D-Glucoside Against Lipopolysaccharide-Induced Inflammation in RAW264.7 Cells 
Hydroxycinnamic acids have been reported to possess numerous pharmacological activities such as antioxidant, anti-inflammatory, and anti-tumor properties. However, the biological activity of 1-p-coumaroyl β-D-glucoside (CG), a glucose ester derivative of p-coumaric acid, has not been clearly examined. The objective of this study is to elucidate the anti-inflammatory action of CG in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. In the present study, CG significantly suppressed LPS-induced excessive production of pro-inflammatory mediators such as nitric oxide (NO) and PGE2 and the protein expression of iNOS and COX-2. CG also inhibited LPS-induced secretion of pro-inflammatory cytokines, IL-1β and TNF-α. In addition, CG significantly suppressed LPS-induced degradation of IκB. To elucidate the underlying mechanism by which CG exerts its anti-inflammatory action, involvement of various signaling pathways were examined. CG exhibited significantly increased Akt phosphorylation in a concentration-dependent manner, although MAPKs such as Erk, JNK, and p38 appeared not to be involved. Furthermore, inhibition of Akt/PI3K signaling pathway with wortmannin significantly, albeit not completely, abolished CG-induced Akt phosphorylation and anti-inflammatory actions. Taken together, the present study demonstrates that Akt signaling pathway might play a major role in CG-mediated anti-inflammatory activity in LPS-stimulated RAW264.7 macrophage cells.
PMCID: PMC3951828  PMID: 24634601
1-p-coumaroyl β-D-glucoside (CG); Akt; COX-2; iNOS; Lipopolysaccharide; NF-κB; RAW264.7 cells
18.  Chronic alcohol exposure exacerbates inflammation and triggers pancreatic acinar-to-ductal metaplasia through PI3K/Akt/IKK 
Pancreatic acinar-to-ductal metaplasia (ADM) has been identified as an initiating event that can progress to pancreatic intraepithelial neoplasia (PanIN) or pancreatic ductal adenocarcinoma (PDAC). Acini transdifferentiation can be induced by persistent inflammation. Notably, compelling evidence has emerged that chronic alcohol exposure may trigger an inflammatory response of macrophages/monocytes stimulated by endotoxins. In the present study, we aimed to evaluate the role of inflammation induced by chronic alcohol and lipopolysaccharide (LPS) exposure in the progression of pancreatic ADM, as well as to elucidate the possible mechanisms involved. For this purpose, cultured macrophages were exposed to varying doses of alcohol for 1 week prior to stimulation with LPS. Tumor necrosis factor-α (TNF-α) and regulated upon activation, normal T cell expression and secreted (RANTES) expression were upregulated in the intoxicated macrophages with activated nuclear factor-κB (NF-κB). Following treatment with the supernatant of intoxicated macrophages, ADM of primary acinar cells was induced. Furthermore, the expression of TNF-α and RANTES, as well as the phosphatidylinositol-3-kinase (PI3K)/protein kinase B(Akt)/inhibitory κB kinase (IKK) signaling pathway have been proven to be involved in the ADM of acinar cells. Moreover, Sprague-Dawley (SD) rats were employed to further explore the induction of pancreatic ADM by chronic alcohol and LPS exposure in vivo. At the end of the treatment period, a number of physiological parameters, such as body weight, liver weight and pancreatic weight were reduced in the exposed rats. Plasma alcohol concentrations and oxidative stress levels in the serum, as well as TNF-α and RANTES expression in monocytes were also induced following chronic alcohol and LPS exposure. In addition, pancreatic ADM was induced through the PI3K/Akt/IKK signaling pathway by the augmented TNF-α and RANTES expression levels in the exposed rats. Overall, we characterized the link between inflammation induced by chronic alcohol and LPS exposure and pancreatic ADM. However, the mechanisms behind the induction of pancreatic ADM warrant further investigation.
PMCID: PMC4314411  PMID: 25573338
chronic alcohol, lipopolysaccharide; acinar-to-ductal metaplasia; PI3K/Akt/IKK
19.  Chemokine Receptor CCR8 Is Required for Lipopolysaccharide-Triggered Cytokine Production in Mouse Peritoneal Macrophages 
PLoS ONE  2014;9(4):e94445.
Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8-/-) and wild-type (WT) mice. We found that CCR8-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca2+ flux and CCL1-driven PMφ aggregation. Similar to CCR8-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.
PMCID: PMC3979852  PMID: 24714157
20.  Genistein inhibits TNF-α-induced endothelial inflammation through the protein kinase pathway A and improves vascular inflammation in C57BL/6 mice 
International journal of cardiology  2013;168(3):2637-2645.
Genistein, a soy isoflavone, has received wide attention for its potential to improve vascular function, but the mechanism of this effect is unclear. Here, we report that genistein at physiological concentrations (0.1 µM–5 µM) significantly inhibited TNF-α-induced adhesion of monocytes to human umbilical vein endothelial cells (HUVECs), a key event in the pathogenesis of atherosclerosis. Genistein also significantly suppressed TNF-α-induced production of adhesion molecules and chemokines such as sICAM-1, sVCAM-1,sE-selectin, MCP-1 and IL-8, which play key role in the firm adhesion of monocytes to activated endothelial cells (ECs). Genistein at physiologically relevant concentrations didn’t significantly induce antioxidant enzyme activities or scavenge free radicals. Further, blocking the estrogen receptors (ERs) in ECs didn’t alter the preventive effect of genistein on endothelial inflammation. However, inhibition of protein kinase A (PKA) significantly attenuated the inhibitory effects of genistein on TNF-α-induced monocyte adhesion to ECs as well as the production of MCP-1 and IL-8. In animal study, dietary genistein (0.1% genistein in the diet) significantly suppressed TNF-α-induced increase in circulating chemokines and adhesion molecules in C57BL/6 mice. Genistein treatment also reduced VCAM-1 and monocytes-derived F4/80-positive macrophages in the aorta of TNF-α treated mice. In conclusion, genistein protects against TNF-α induced vascular endothelial inflammation both in vitro and in vivo models. This anti-inflammatory effect of genistein is independent of the ER-mediated signaling machinery or antioxidant activity, but mediated via the PKA signaling pathway.
PMCID: PMC3758913  PMID: 23587398
genistein; vascular inflammation; TNF-α; protein kinase A; endothelial cells
21.  Anti-inflammatory effects of Lacto-Wolfberry in a mouse model of experimental colitis 
AIM: To investigate the anti-inflammatory properties of Lacto-Wolfberry (LWB), both in vitro and using a mouse model of experimental colitis.
METHODS: The effects of LWB on lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) and interleukin (IL)-6 secretion were assessed in a murine macrophage cell line. in vitro assessment also included characterizing the effects of LWB on the activation of NF-E2 related 2 pathway and inhibition of tumor necrosis factor-α (TNF-α)-induced nuclear factor-κB (NF-κB) activation, utilizing reporter cell lines. Following the in vitro assessment, the anti-inflammatory efficacy of an oral intervention with LWB was tested in vivo using a preclinical model of intestinal inflammation. Multiple outcomes including body weight, intestinal histology, colonic cytokine levels and anti-oxidative measures were investigated.
RESULTS: LWB reduced the LPS-mediated induction of ROS production [+LPS vs 1% LWB + LPS, 1590 ± 188.5 relative luminescence units (RLU) vs 389 ± 5.9 RLU, P < 0.001]. LWB was more effective than wolfberry alone in reducing LPS-induced IL-6 secretion in vitro (wolfberry vs 0.5% LWB, 15% ± 7.8% vs 64% ± 5%, P < 0.001). In addition, LWB increased reporter gene expression via the anti-oxidant response element activation (wolfberry vs LWB, 73% ± 6.9% vs 148% ± 28.3%, P < 0.001) and inhibited the TNF-α-induced activation of the NF-κB pathway (milk vs LWB, 10% ± 6.7% vs 35% ± 3.3%, P < 0.05). Furthermore, oral supplementation with LWB resulted in a reduction of macroscopic (-LWB vs +LWB, 5.39 ± 0.61 vs 3.66 ± 0.59, P = 0.0445) and histological scores (-LWB vs +LWB, 5.44 ± 0.32 vs 3.66 ± 0.59, P = 0.0087) in colitic mice. These effects were associated with a significant decrease in levels of inflammatory cytokines such as IL-1β (-LWB vs +LWB, 570 ± 245 μg/L vs 89 ± 38 μg/L, P = 0.0106), keratinocyte-derived chemokine/growth regulated protein-α (-LWB vs +LWB, 184 ± 49 μg/L vs 75 ± 20 μg/L, P = 0.0244), IL-6 (-LWB vs +LWB, 318 ± 99 μg/L vs 117 ± 18 μg/L, P = 0.0315) and other pro-inflammatory proteins such as cyclooxygenase-2 (-LWB vs +LWB, 0.95 ± 0.12 AU vs 0.36 ± 0.11 AU, P = 0.0036) and phosphorylated signal transducer and activator of transcription-3 (-LWB vs +LWB, 0.51 ± 0.15 AU vs 0.1 ± 0.04 AU, P = 0.057). Moreover, antioxidant biomarkers, including expression of gene encoding for the glutathione peroxidase, in the colon and the plasma anti-oxidant capacity were significantly increased by supplementation with LWB (-LWB vs +LWB, 1.2 ± 0.21 mmol/L vs 2.1 ± 0.19 mmol/L, P = 0.0095).
CONCLUSION: These results demonstrate the anti-inflammatory properties of LWB and suggest that the underlying mechanism is at least in part due to NF-κB inhibition and improved anti-oxidative capacity.
PMCID: PMC3471103  PMID: 23082051
Lacto-Wolfberry; Colitis; Nutrition; Inflammation; Wolfberry; Inflammatory bowel disease; Crohn’s disease
22.  CD45RB Is a Novel Molecular Therapeutic Target to Inhibit Aβ Peptide-Induced Microglial MAPK Activation 
PLoS ONE  2008;3(5):e2135.
Microglial activation, characterized by p38 MAPK or p44/42 MAPK pathway signal transduction, occurs in Alzheimer's disease (AD). Our previous studies demonstrated CD45, a membrane-bound protein tyrosine phosphatase (PTP), opposed β-amyloid (Aβ) peptide-induced microglial activation via inhibition of p44/42 MAPK. Additionally we have shown agonism of the RB isoform of CD45 (CD45RB) abrogates lipopolysaccharide (LPS)-induced microglial activation.
Methodology and Results
In this study, CD45RB modulation of Aβ peptide or LPS-activated primary cultured microglial cells was further investigated. Microglial cells were co-treated with “aged” FITC-Aβ1–42 and multiple CD45 isoform agonist antibodies. Data revealed cross-linking of CD45, particularly the CD45RB isoform, enhances microglial phagocytosis of Aβ1–42 peptide and inhibits LPS-induced activation of p44/42 and p38 pathways. Co-treatment of microglial cells with agonist CD45 antibodies results in significant inhibition of LPS-induced microglial TNF-α and IL-6 release through p44/42 and/or p38 pathways. Moreover, inhibition of either of these pathways augmented CD45RB cross-linking induced microglial phagocytosis of Aβ1–42 peptide. To investigate the mechanism(s) involved, microglial cells were co-treated with a PTP inhibitor (potassium bisperoxo [1,10-phenanthroline oxovanadate; Phen]) and Aβ1–42 peptides. Data showed synergistic induction of microglial activation as evidenced by TNF-α and IL-6 release; both of which are demonstrated to be dependent on increased p44/42 and/or p38 activation. Finally, it was observed that cross-linking of CD45RB in the presence of Aβ1–42 peptide, inhibits co-localization of microglial MHC class II and Aβ peptide; suggesting CD45 activation inhibits the antigen presenting phenotype of microglial cells.
In summary, p38 MAPK is another novel signaling pathway, besides p44/42, in which CD45RB cross-linking negatively regulates microglial Aβ phagocytosis while increasing potentially neurotoxic inflammation. Therefore, agonism of CD45RB PTP activity may be an effective therapeutic target for novel agents to treat AD due to its Aβ lowering, and inflammation reducing, properties that are particularly targeted at microglial cells. Such treatments may be more effective with less potential to produce systemic side-effects than therapeutics which induce non-specific, systemic down-regulation of inflammation.
PMCID: PMC2366070  PMID: 18478117
23.  Protection against cartilage and bone destruction by systemic interleukin-4 treatment in established murine type II collagen-induced arthritis 
Arthritis Research  1999;1(1):81-91.
Destruction of cartilage and bone are hallmarks of human rheumatoid arthritis (RA), and controlling these erosive processes is the most challenging objective in the treatment of RA. Systemic interleukin-4 treatment of established murine collagen-induced arthritis suppressed disease activity and protected against cartilage and bone destruction. Reduced cartilage pathology was confirmed by both decreased serum cartilage oligomeric matrix protein (COMP) and histological examination. In addition, radiological analysis revealed that bone destruction was also partially prevented. Improved suppression of joint swelling was achieved when interleukin-4 treatment was combined with low-dose prednisolone treatment. Interestingly, synergistic reduction of both serum COMP and inflammatory parameters was noted when low-dose interleukin-4 was combined with prednisolone. Systemic treatment with interleukin-4 appeared to be a protective therapy for cartilage and bone in arthritis, and in combination with prednisolone at low dosages may offer an alternative therapy in RA.
Rheumatoid arthritis (RA) is associated with an increased production of a range of cytokines including tumour necrosis factor (TNF)-α and interleukin (IL)-1, which display potent proinflammatory actions that are thought to contribute to the pathogenesis of the disease. Although TNF-α seems to be the major cytokine in the inflammatory process, IL-1 is the key mediator with regard to cartilage and bone destruction. Apart from direct blockade of IL-1/TNF, regulation can be exerted at the level of modulatory cytokines such as IL-4 and IL-10. IL-4 is a pleiotropic T-cell derived cytokine that can exert either suppressive or stimulatory effects on different cell types, and was originally identified as a B-cell growth factor and regulator of humoral immune pathways. IL-4 is produced by activated CD4+ T cells and it promotes the maturation of Th2 cells. IL-4 stimulates proliferation, differentiation and activation of several cell types, including fibroblasts, endothelial cells and epithelial cells. IL-4 is also known to be a potent anti-inflammatory cytokine that acts by inhibiting the synthesis of proinflammatory cytokines such as IL-1, TNF-α, IL-6, IL-8 and IL-12 by macrophages and monocytes. Moreover, IL-4 stimulates the synthesis of several cytokine inhibitors such as interleukin-1 receptor antagonist (IL-1Ra), soluble IL-1-receptor type II and TNF receptors IL-4 suppresses metalloproteinase production and stimulates tissue inhibitor of metalloproteinase-1 production in human mononuclear phagocytes and cartilage explants, indicating a protective effect of IL-4 towards extracellular matrix degradation. Furthermore, IL-4 inhibits both osteoclast activity and survival, and thereby blocks bone resorption in vitro. Of great importance is that IL-4 could not be detected in synovial fluid or in tissues. This absence of IL-4 in the joint probably contributes to the disturbance in the Th1/Th2 balance in chronic RA.
Collagen-induced arthritis (CIA) is a widely used model of arthritis that displays several features of human RA. Recently it was demonstrated that the onset of CIA is under stringent control of IL-4 and IL-10. Furthermore, it was demonstrated that exposure to IL-4 during the immunization stage reduced onset and severity of CIA. However, after cessation of IL-4 treatment disease expression increased to control values.
Because it was reported that IL-4 suppresses several proinflammatory cytokines and matrix degrading enzymes and upregulates inhibitors of both cytokines and catabolic enzymes, we investigated the tissue protective effect of systemic IL-4 treatment using established murine CIA as a model. Potential synergy of low dosages of anti-inflammatory glucocorticosteroids and IL-4 was also evaluated.
DBA-1J/Bom mice were immunized with bovine type II collagen and boosted at day 21. Mice with established CIA were selected at day 28 after immunization and treated for days with IL-4, prednisolone, or combinations of prednisolone and IL-4. Arthritis score was monitored visually. Joint pathology was evaluated by histology, radiology and serum cartilage oligomeric matrix protein (COMP). In addition, serum levels of IL-1Ra and anticollagen antibodies were determined.
Treatment of established CIA with IL-4 (1 μg/day) resulted in suppression of disease activity as depicted in Figure 1. Of great interest is that, although 1 μg/day IL-4 had only a moderate effect on the inflammatory component of the disease activity, it strongly reduced cartilage pathology, as determined by histological examination (Fig. 1). Moreover, serum COMP levels were significantly reduced, confirming decreased cartilage involvement. In addition, both histological and radiological analysis showed that bone destruction was prevented (Fig. 1). Systemic IL-4 administration increased serum IL-1Ra levels and reduced anticollagen type II antibody levels. Treatment with low-dose IL-4 (0.1 μg/day) was ineffective in suppressing disease score, serum COMP or joint destruction. Synergistic suppression of both arthritis severity and COMP levels was noted when low-dose IL-4 was combined with prednisolone (0.05 mg/kg/day), however, which in itself was not effective.
In the present study, we demonstrate that systemic IL-4 treatment ameliorates disease progression of established CIA. Although clinical disease progression was only arrested and not reversed, clear protection against cartilage and bone destruction was noted. This is in accord with findings in both human RA and animal models of RA that show that inflammation and tissue destruction sometimes are uncoupled processes. Of great importance is that, although inflammation was still present, strong reduction in serum COMP was found after exposure to IL-4. This indicated that serum COMP levels reflected cartilage damage, although a limited contribution of the inflamed synovium cannot be excluded.
Increased serum IL-1Ra level (twofold) was found after systemic treatment with IL-4, but it is not likely that this could explain the suppression of CIA. We and others have reported that high dosages of IL-1Ra are needed for marked suppression of CIA. As reported previously, lower dosages of IL-4 did not reduce clinical disease severity of established CIA. Of importance is that combined treatment of low dosages of IL-4 and IL-10 appeared to have more potent anti-inflammatory effects, and markedly protected against cartilage destruction. Improved anti-inflammatory effect was achieved with IL-4/prednisolone treatment. In addition, synergistic effects were found for the reduction of cartilage and bone destruction. This indicates that systemic IL-4/prednisolone treatment may provide a cartilage and bone protective therapy for human RA.
Effects in mice of treatment with interleukin-4 or control on disease activity, cartilage damage and bone destruction. Mice were treated intraperitoneally for 7 days with either vehicle (control) or 1 μg/day interleukin-4 (IL-4). CIA, collagen-induced arthritis. *P < 0.05, versus control, by Mann-Whitney U test.
PMCID: PMC17779  PMID: 11056663
bone destruction; cartilage oligomeric matrix protein levels; collagen-induced arthritis; interleukin-4; prednisolone
24.  Terameprocol, a methylated derivative of nordihydroguaiaretic acid, inhibits production of prostaglandins and several key inflammatory cytokines and chemokines 
Extracts of the creosote bush, Larrea tridentata, have been used for centuries by natives of western American and Mexican deserts to treat a variety of infectious diseases and inflammatory disorders. The beneficial activity of this plant has been linked to the compound nordihydroguaiaretic acid (NDGA) and its various substituted derivatives. Recently, tetra-O-methyl NDGA or terameprocol (TMP) has been shown to inhibit the growth of certain tumor-derived cell lines and is now in clinical trials for the treatment of human cancer. In this report, we ask whether TMP also displays anti-inflammatory activity. TMP was tested for its ability to inhibit the LPS-induced production of inflammatory lipids and cytokines in vitro. We also examined the effects of TMP on production of TNF-α in C57BL6/J mice following a sublethal challenge with LPS. Finally, we examined the molecular mechanisms underlying the effects we observed.
RAW 264.7 cells and resident peritoneal macrophages from C57BL6/J mice, stimulated with 1 μg/ml LPS, were used in experiments designed to measure the effects of TMP on the production of prostaglandins, cytokines and chemokines. Prostaglandin production was determined by ELISA. Cytokine and chemokine production were determined by antibody array and ELISA.
Western blots, q-RT-PCR, and enzyme assays were used to assess the effects of TMP on expression and activity of COX-2.
q-RT-PCR was used to assess the effects of TMP on levels of cytokine and chemokine mRNA.
C57BL6/J mice injected i.p. with LPS were used in experiments designed to measure the effects of TMP in vivo. Serum levels of TNF-α were determined by ELISA.
TMP strongly inhibited the production of prostaglandins from RAW 264.7 cells and normal peritoneal macrophages. This effect correlated with a TMP-dependent reduction in levels of COX-2 mRNA and protein, and inhibition of the enzymatic activity of COX-2.
TMP inhibited, to varying degrees, the production of several cytokines, and chemokines from RAW 264.7 macrophages and normal peritoneal macrophages. Affected molecules included TNF-α and MCP-1. Levels of cytokine mRNA were affected similarly, suggesting that TMP is acting to prevent gene expression.
TMP partially blocked the production of TNF-α and MCP-1 in vivo in the serum of C57BL6/J mice that were challenged i.p. with LPS.
TMP inhibited the LPS-induced production of lipid mediators and several key inflammatory cytokines and chemokines, both in vitro and in vivo, raising the possibility that TMP might be useful as a treatment for a variety of inflammatory disorders.
PMCID: PMC2631502  PMID: 19133137
25.  TNF-α Involvement in Insulin Resistance Induced by Experimental Scorpion Envenomation 
Scorpion venom induces systemic inflammation characterized by an increase in cytokine release and chemokine production. There have been few experimental studies assessing the effects of scorpion venom on adipose tissue function in vivo.
Methodology/Principal Findings
To study the adipose tissue inflammation (ATI) induced by Androctonus australis hector (Aah) venom and to assess possible mechanisms of ATI, mice (n = 6, aged 1 month) were injected with Aah (0.45 mg/kg), toxic fraction of Aah (FTox-G50; 0.2 mg/kg) or saline solution (control). Inflammatory responses were evaluated by ELISA and cell sorting analyses in adipose tissue 45 minutes and 24 hours after injection. Quantitative real-time PCR was used to assess the regulation of genes implicated in glucose uptake. The titers of selected inflammatory cytokines (IL-1β, IL-6 and TNF-α) were also determined in sera and in insulin target tissues. The serum concentration of IL-1β rose 45 minutes after envenomation and returned to basal level after 24 hours. The pathophysiological effects of the venom after 24 hours mainly involved M1-proinflammatory macrophage infiltration in adipose tissue combined with high titers of IL-1β, IL-6 and TNF-α. Indeed, TNF-α was strongly induced in both adipose tissue and skeletal muscle. We studied the effects of Aah venom on genes implicated in insulin-stimulated glucose uptake. Insulin induced a significant increase in the expression of the mRNAs for hexokinase 2 and phosphatidylinositol 3-kinase in both skeletal muscle and adipose tissue in control mice; this upregulation was completely abolished after 24 hours in mice envenomed with Aah or FTox-G50.
Our findings suggest that Aah venom induces insulin resistance by mechanisms involving TNF-α-dependent Map4k4 kinase activation in the adipose tissue.
Author Summary
Androctonus australis hector (Aah) is the scorpion most frequently causing serious human envenomation. In Algeria, Aah is responsible for approximately 50,000 cases of scorpion envenomation per year. The Aah sting causes multi-system failure that may be fatal; the manifestations include cardiopulmonary abnormalities, lung edema and inflammation. In addition, hyperglycemia and hyperinsulinemia have been described in scorpion-envenomed animals. The mechanisms causing systemic and local inflammation are poorly understood. Here, we report that Aah venom causes pronounced upregulation of TNF-α, IL1-β and IL-6 expression in the adipose tissue, exacerbating inflammation. As the inflammatory state intensifies, 24 hours after envenomation, TNF-α and other factors are upregulated, and Map4k4 expression increases, blunting the insulin response in adipocytes by decreasing Hexokinase 2 expression. Administration of TNF-α inhibitor following the envenomation reduces Map4k4 expression and restores glucose uptake in adipose tissue. These findings provide coherent evidence linking Aah venom-induced adipose tissue inflammation to insulin resistance. The value of TNF-α inhibitors as a treatment complementary to anti-scorpion venom immunotherapy should be evaluated clinically.
PMCID: PMC3398957  PMID: 22816003

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