Lipopolysaccharide-treated Cells Research Articles

Human Infrapatellar Fat Pad Mesenchymal Stem Cell-derived Extracellular Vesicles Purified by Anion Exchange Chromatography Suppress Osteoarthritis Progression in a Mouse Model.

Human Infrapatellar Fat Pad Mesenchymal Stem Cell-derived Extracellular Vesicles Purified by Anion Exchange Chromatography Suppress Osteoarthritis Progression in a Mouse Model.

Novel alkyl-substituted 4-methoxy benzaldehyde thiosemicarbazones: Multi-target directed ligands for the treatment of Alzheimer's disease

Alzheimer's disease (AD) is a devastating neurodegenerative disorder affecting mental ability and interrupts neurocognitive functions. Treating multifactorial conditions of AD with a single-target-directed drug is highly difficult. Thus, a multi-target-directed ligand (MTDL) development strategy has been developed as a promising approach for the treatment of AD.Herein, we have synthesized two novel thiosemicarbazones as MTDLs and reported their bioactivities against diverse neuropathological events involved in AD. In vitro studies revealed that both compounds exhibited promising anticholinesterase activity (AChE, IC50 = 15.98 μM, MZET and IC50 = 30.23 μM, MZMT), well supported by a detailed computational study. Both analogs have shown good thermodynamic behaviour and stability through interactions with characteristic amino acid residues throughout simulation of 100 ns against acetylcholinesterase enzyme. In an electrophysiology assay, these analogs have shown a characteristic inhibitory response against the GluN1-1a + GluN2B subunit of N-methyl-D-aspartate receptors. Pre-treatment of BV-2 microglial cells with MZET effectively decreased nitrite production compared to nitrite produced by lipopolysaccharide-treated cells alone. Further, the effect of MZMT and MZET on autophagy regulation was determined using stably transfected SH-SY5Y neuroblastoma cells. MZET significantly enhanced the autophagy flux in neuroblastoma cells. A significant decrease in copper-catalysed oxidation of amyloid-β in presence of synthesized thiosemicarbazones was also observed.Collectively, our findings indicated that these analogs have potential as effective anti-AD candidates and can be used as a prototype to develop more safer multi-targeted anti-AD drugs.

Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages.

Here, we elucidated the structural characteristics of a polysaccharide isolated from Gardenia jasminoides Ellis (labeled as GP2a) and its immunomodulatory activity. GP2a is an acidic polysaccharide with a molecular weight of 44.8 kDa, mostly comprising galacturonic acid. Methylation analysis revealed 4-GalpA (74.8%) to be the major sugar residue in GP2a. Nuclear magnetic resonance analysis indicated that its main chain comprised →4)-α-D-GalpA-6-OMe-(1→4)-α-D-GalpA-(1→ and →4)-α-D-GalpA-6-OMe-(1→2)-α-L-Rhap-(1→, with galactan and arabinans linked to the C-4 position of →2)-α-L-Rhap-(1→ residue as branched chains. Furthermore, GP2a showed no obvious toxicity to macrophages (RAW 264.7) while enhancing cell viability in a dose- and time-dependent manner. Compared with untreated cells, nitric oxide production and secretion of cytokines, such as tumor necrosis factor-α, interferon-γ, interleukin (IL)-1β, IL-6, and granulocyte macrophage colony stimulating factor, in GP2a-treated cells significantly increased after 48 h. At 300 µg/mL GP2a concentration, there was no significant difference in the cytokine levels in GP2a- and lipopolysaccharide-treated cells (the positive control). In summary, GP2a is a pectic polysaccharide with homogalacturonan and rhamnogalacturonan-I structural regions in the main chain. Based on its immunomodulatory effects in vitro, GP2a may have potential uses in functional food and medicine.

Polyphyllin I alleviates lipopolysaccharide-induced inflammation reduces pyroptosis in BEAS-2B and HPAEC cells by inhibiting NF-κB signaling.

Polyphyllin I is an active steroidal saponin isolated from Paris polyphylla with anti-cancer and anti-inflammatory properties. The present study investigates the role of polyphyllin I in acute lung injury. Firstly, the human bronchial epithelial cells (BEAS-2B) and human pulmonary artery endothelial cells (HPAEC) were stimulated with increasing concentrations of lipopolysaccharide at 2, 5, and 10 μg/mL. The treatment with lipopolysaccharide reduced the cell viabilities of BEAS-2B and HPAEC, downregulated superoxide dismutase (SOD) and glutathione (GSH), and up-regulated myeloperoxidase (MPO) and malondialdehyde (MDA). Moreover, the levels of TNF-α, IL-1β, and IL-6 were also up-regulated in lipopolysaccharide-treated BEAS-2B/HPAEC cells. Secondly, the lipopolysaccharide-treated cells were then incubated with different concentrations of polyphyllin I. Incubation with polyphyllin I enhanced the cell viabilities of lipopolysaccharide--treated BEAS-2B/HPAEC, up-regulated levels of SOD and GSH, and reduced MPO and MDA. Moreover, polyphyllin I reduced TNF-α, IL-1β, and IL-6 in lipopolysaccharide-treated BEAS-2B/HPAEC cells. Thirdly, the up-regulation of GSDMD-N, pro-caspase-1, and cleaved caspase-1 proteins in lipopolysaccharide-treated BEAS-2B/HPAEC cells were decreased by polyphyllin I. Polyphyllin I increased the protein expression of GSDMD-D in the lipopolysaccharide-treated BEAS-2B/HPAEC cells, and inhibited the translocation of GSDMD from cytoplasm to plasma membrane. Lastly, polyphyllin I reduced the expression of p-p65 in lipopolysaccharide-treated BEAS-2B/HPAEC cells. The over-expression of p65 counteracted with the inhibitory effects of polyphyllin I on oxidative stress and inflammation in lipopolysaccharide-treated BEAS-2B. In conclusion, polyphyllin I repressed the lipopolysaccharide-induced oxidative stress and inflammation in BEAS-2B and HPAEC, and reduced pyroptosis through inhibition of NF-κB signaling.

Skullcapflavone II Alleviates Lipopolysaccharide-Induced Apoptosis and Inflammation in WI-38 Cells

The role of skullcapflavone II in pneumonia was investigated using lipopolysaccharide treated human lung fibroblasts WI-38 cells as a model. Lipopolysaccharide treatment led to decreased cell viability, increased cell apoptosis and inflammation. Exposure of these lipopolysaccharide-treated cells to skullcapflavone II resulted in amelioration of cytotoxic effects of the lipopolysaccharide. The reduced IκBα expression and enhanced p-IκBα and p-p65 in WI-38 cells caused by the lipopolysaccharide treatment were reversed by skullcapflavone II. In conclusion, skullcapflavone II exerts antiapoptotic and anti-inflammatory effects on lipopolysaccharide-induced WI-38 through inhibition of NF-κB pathway.

Long non-coding RNA LINC01194 promotes the inflammatory response and apoptosis of lipopolysaccharide-treated MLE-12 cells through the miR-203a-3p/MIP-2 axis.

Acute lung injury (ALI) induced by bacteria lipopolysaccharide (LPS) is characterized by the upregulation of the apoptosis rate of tissue cells and aggravation of inflammatory response. Although many studies have focused on the pathogenesis of this disease, its mechanism remains unknown. This study examined the regulatory role of long non-coding RNA (lncRNA) LINC01194 in the progression of ALI through various bioinformatics analyses and experimental work, including ELISA assay, dual-luciferase reporter assay, biotinylated RNA pull-down assay, and Western blot analysis. The result showed that the LINC01194 was overexpressed in the ALI-induced mice model. We observed a significant upregulation of LINC01194 in LPS-treated mouse lung epithelial type II cells (MLE-12 cells) after 24 h of induction. Bioinformatics analysis, ELISA assay, quantitative reverse transcription polymerase chain reaction analysis, biotinylated RNA pull-down assay, apoptosis test, and Western blot analysis demonstrated that the LINC01194 could act as a microRNA (miR) miR-203a-3p sponge to activate the inflammatory response in LPS-induced ALI model through post-transcriptional upregulation of macrophage inflammatory protein (MIP-2). We showed that LINC01194 regulates the inflammatory response and apoptosis of LPS-induced mice and MLE-12 cells via the miR-203a-3p/MIP-2 axis. LINC01194 could be a potential biomarker for early diagnosis and the treatment of ALI.

Anacardic acid-mediated regulation of osteoblast differentiation involves mitigation of inflammasome activation pathways.

Disruption of the finely tuned osteoblast-osteoclast balance is the underlying basis of several inflammatory bone diseases, such as osteomyelitis, osteoporosis, and septic arthritis. Prolonged and unrestrained exposure to inflammatory environment results in reduction of bone mineral density by downregulating osteoblast differentiation. Earlier studies from our laboratory have identified that Anacardic acid (AA), a constituent of Cashew nut shell liquid that is used widely in traditional medicine, has potential inhibitory effect on gelatinases (MMP2 and MMP9) which are over-expressed in numerous inflammatory conditions (Omanakuttan et al. in Mol Pharmacol, 2012 and Nambiar et al. in Exp Cell Res, 2016). The study demonstrated for the first time that AA promotes osteoblast differentiation in lipopolysaccharide-treated osteosarcoma cells (MG63) by upregulating specific markers, like osteocalcin, receptor activator of NF-κB ligand, and alkaline phosphatase. Furthermore, expression of the negative regulators, such as nuclear factor-κB, matrix metalloproteinases (MMPs), namely MMP13, and MMP1, along with several inflammatory markers, such as Interleukin-1β and Nod-like receptor protein 3 were downregulated by AA. Taken together, AA expounds as a novel template for development of potential pharmacological therapeutics for inflammatory bone diseases.

Altered paracellular permeability in intestinal cell monolayer challenged with lipopolysaccharide: Modulatory effects of pterostilbene metabolites

Epithelial barrier alteration is a central event in the pathogenesis of inflammatory bowel diseases. Lipopolysaccharide, correlated to the pathogenesis of such pathologies, has been demonstrated to cause altered membrane permeability, through the disruption and/or relocation of tight junction proteins, following redox-sensitive mitogen-activated protein kinases (MAPKs) modulation. Pterostilbene and its metabolite pinostilbene are natural stilbenoids which may reach relevant concentrations at intestinal level, together with their glucuronide and sulfate metabolites. The aim of our study was to evaluate the ability of these compounds to inhibit lipopolysaccharide-induced toxic effects on intestinal cell monolayer integrity and to explore the mechanism of action. Caco-2 cells, differentiated as enterocytes, were treated with lipopolysaccharide following pretreatment with the phenolic compounds at 1 μM physiological concentration. Caco-2 monolayer's permeability was monitored with time, measuring the transepithelial electrical resistance. Tight junction proteins were assessed by western blotting and immunofluorescence in lipopolysaccharide-treated cells, in relation to MAPK p38 and ERK1/2 activation. Pretreatment with all the phenolic compounds significantly slowed lipopolysaccharide-induced transepithelial electrical resistance decrease, preserved tight junction proteins levels and reduced MAPKs phosphorylation. The reported findings indicate that pterostilbene and its metabolites may counteract lipopolysaccharide-induced alteration of epithelial permeability, one of the initial events in the intestinal inflammatory process.

Natural Polyketides Isolated from the Endophytic Fungus Phomopsis sp. CAM212 with a Semisynthetic Derivative Downregulating the ERK/IκBα Signaling Pathways

Three previously undescribed natural products, phomopsinin A - C (1: - 3: ), together with three known compounds, namely, cis-hydroxymellein (4: ), phomoxanthone A (5: ) and cytochalasin L-696,474 (6: ), were isolated from the solid culture of Phomopsis sp. CAM212, an endophytic fungus obtained from Garcinia xanthochymus. Their structures were determined on the basis of spectroscopic data, including IR, NMR, and MS. The absolute configurations of 1: and 2: were assigned by comparing their experimental and calculated ECD spectra. Acetylation of compound 1: yielded 1A: , a new natural product derivative that was tested together with other isolated compounds on lipopolysaccharide-stimulated RAW 264.7 cells. Cytochalasin L-696,474 (6: ) was found to significantly inhibit nitric oxide production, but was highly cytotoxic to the treated cells, whereas compound 1: slightly inhibited nitric oxide production, which was not significantly different compared to lipopolysaccharide-treated cells. Remarkably, the acetylated derivative of 1: , compound 1A: , significantly inhibited nitric oxide production with an IC50 value of 14.8 µM and no cytotoxic effect on treated cells, thereby showing the importance of the acetyl group in the anti-inflammatory activity of 1A: . The study of the mechanism of action revealed that 1A: decreases the expression of inducible nitric oxide synthase, cyclooxygenase 2, and proinflammatory cytokine IL-6 without an effect on IL-1β expression. Moreover, it was found that 1A: exerts its anti-inflammatory activity in lipopolysaccharide-stimulated RAW 264.7 macrophage cells by downregulating the activation of ERK1/2 and by preventing the translocation of nuclear factor κB. Thus, derivatives of phomopsinin A (1: ), such as compound 1A: , could provide new anti-inflammatory leads.

The immunostimulatory effect of indole-6-carboxaldehyde isolated from Sargassum thunbergii (Mertens) Kuntze in RAW 264.7 macrophages

ABSTRACT Indole-6-carboxaldehyde (I6CA), an indole derivative isolated from the marine brown algae Sargassum thunbergii, is known to have several beneficial effects, but no studies on immune regulation have been conducted. In this study, the immunomodulatory properties of I6CA on murine RAW 264.7 monocyte/macrophage cells were evaluated. As the concentration of I6CA increased, the morphology of RAW 264.7 cells changed to a typical active macrophage shape, and the phagocytic activity increased significantly. I6CA effectively enhanced the production and secretion of immunomodulatory mediators and cytokines due to increased expression of their respective genes. Additionally, I6CA markedly stimulated the expression of Toll-like receptor 4 (TLR4) and its adapter molecule, myeloid differentiation factor 88 (Myd88), and increased TLR4 complexed with Myd88. Furthermore, I6CA promoted the nuclear translocation of nuclear factor-kappa B (NF-κB) by increasing the degradation of the inhibitor of NF-κB-α. Meanwhile, similar trends were also found in lipopolysaccharide-treated cells as a positive control. Furthermore, molecular docking simulation showed that I6CA interacted with TLR4-myeloid differentiation 2 complex. Taken together, the results support the concept that I6CA may increase the activity of the TLR4/NF-κB signaling pathway in order to enhance the immunomodulatory activity of RAW 264.7 cells.

Exosome-derived uterine miR-218 isolated from cows with endometritis regulates the release of cytokines and chemokines.

SummaryAs an inflammation of the endometrium, endometritis can affect fertility and lead to serious economic losses in the dairy industry. Widely found in various tissues and body fluids, exosomes and exosome micro (mi)RNAs have been shown to play an important regulatory role in the immune responses. As one of differentially expressed exosome miRNAs, miR‐218 is involved in the pathogenesis of bovine endometritis. The mechanisms of miR‐218 in regulating the release of cytokines and chemokines in endometritis, however, are poorly understood. Exosomes were isolated from bovine uterine cavity fluid and verified by transmission electron microscopy. An in vitro lipopolysaccharide‐treated cell model for bovine endometritis was then established to evaluate the correlation between exosome‐derived miR‐218 and the immune responses. We demonstrated that exosomes could be used to deliver miR‐218 from endometrial epithelial cells (EECs) into the uterine microenvironment and adjacent recipient cells to modulate local immune responses. miR‐218 packaged in the exosomes secreted from EECs acts as an inhibitor by blocking immune factors such as interleukin (IL)‐6, IL‐1β, tumour necrosis factor‐α, the chemokines macrophage inflammatory genes (MIP)‐1α and MIP‐1β to maintain the immune balance in the uterus. However, uterine inflammation altered the immunoregulatory mechanism of exosome miR‐218. MiR‐218 is a potential biomarker for the detection of endometritis. Our findings also revealed a new mechanism for the development of endometritis in cows.

Assessment of adipogenic, antioxidant, and anti-inflammatory properties of whole and whey bovine colostrum

Bovine colostrum (BC) has been used for nutraceutical purposes for animals and humans. Bovine colostrum is a complex heterogeneous product and its antimicrobial activity, antioxidant potential, and growth factors can vary depending on age and species of the cow as well as their environment. Bovine colostrum preparation in skimmed or whey fractions can also alter properties of BC. Our goal was to compare cumulative anti-inflammatory, antioxidant, and adipogenic properties of natural (whole) versus whey BC. We compared properties of whole and whey BC in 3T3-L1 preadipocytes permanently transfected with reporters responding to changes in inflammatory (NfκbRE/green fluorescent protein), anti-inflammatory (Nrf2/YFP), and adipogenic (Fabp4/cyan fluorescent protein) status in cells. Interleukin-6 secretion in these cells was measured by ELISA. Whole and whey BC induce IL-6 secretion from 3T3-L1 fibroblasts; however, whey preparation stimulated less IL-6 secretion. Cumulative inflammatory nuclear factor (NF)κB activation in the presence of lipopolysaccharide was reduced by both whole (-27%) and whey BC (-22%) compared with lipopolysaccharide-treated cells (100%). Treatment with whole BC was more effective in the reduction of NFκB activation compared with whey BC and occurred in a dose-dependent manner. In consonance with decreased NFκB activation, the Nrf2 promoter activity was also reduced in response to whole (-27%) and whey (-13%) treatments compared with nontreated cells (100%). Whole and whey BC suppressed adipogenesis, measured as induction of Fabp4, by -27 and -13%, respectively, compared with nontreated 3T3-L1 fibroblasts (100%). Our results showed distinct differences in properties of whey and whole BC that could be used to attain reduced adipogenic or cumulative inflammatory responses.

Strawberry extracts efficiently counteract inflammatory stress induced by the endotoxin lipopolysaccharide in Human Dermal Fibroblast

A protracted pro-inflammatory state is the common denominator in the development, progression and complication of the common chronic diseases. Dietary antioxidants represent an efficient tool to counteract this inflammatory state. The aim of the present work was to evaluate the effects of strawberry extracts on inflammation evoked by E. Coli lipopolysaccharide in Human Dermal Fibroblast, by measuring reactive oxygen species production, apoptosis rate, antioxidant enzymes activity, mitochondria functionality and also investigating the molecular pathway involved in inflammatory and antioxidant response. The results demonstrated that strawberry pre-treatment reduced intracellular reactive oxygen species levels, apoptotic rate, improved antioxidant defences and mitochondria functionality in lipopolysaccharide -treated cells. Strawberry exerted these protective activities through the inhibition of the NF-kB signalling pathway and the stimulation of the Nrf2 pathway, with a mechanism AMPK-dependent. These results confirm the health benefits of strawberry in the prevention of inflammation and oxidative stress condition in lipopolysaccharide-treated cells.

In vitro treatment of lipopolysaccharide increases invasion of Pasteurella multocida serotype B:2 into bovine aortic endothelial cells.

Pasteurella multocida serotype B:2 causes hemorrhagic septicemia in cattle and buffalo. The invasion mechanism of the bacterium when invading the bloodstream is unclear. This study aimed to characterize the effects of immunomodulatory molecules, namely dexamethasone and lipopolysaccharide, on the invasion efficiency of P. multocida serotype B:2 toward bovine aortic endothelial cells (BAECs) and the involvement of actin microfilaments in the invasion mechanism. The results imply that treatment of BAECs with lipopolysaccharide at 100 ng/mL for 24 h significantly increases the intracellular bacteria number per cell (p < 0.01) compared with those in untreated and dexamethasone-treated cells. The lipopolysaccharide-treated cells showed a significant decrease in F-actin expression and an increase in G-actin expression (p < 0.001), indicating actin depolymerization of BAECs. However, no significant differences were detected in the invasion efficiency and actin filament reorganization between the dexamethasone-treated and untreated cells. Transmission electron microscopy showed that P. multocida B:2 resided in a vacuolar compartment of dexamethasone-treated and untreated cells, whereas the bacteria resided in cellular membrane of lipopolysaccharide-treated cells. The results suggest that lipopolysaccharide destabilizes the actin filaments of BAECs, which could facilitate the invasion of P. multocida B:2 into BAECs.

Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses

Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi’s sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman’s disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase-3 processing of LANA. This indicates that these are the principal sites that are susceptible to caspase cleavage. Using peptides spanning the identified LANA cleavage sites, we show that caspase activity can be inhibited in vitro and that a cell-permeable peptide spanning the C-terminal cleavage site could inhibit cleavage of poly (ADP-ribose) polymerase and increase viability in cells undergoing etoposide-induced apoptosis. The C-terminal peptide of LANA also inhibited interleukin-1beta (IL-1β) production from lipopolysaccharide-treated THP-1 cells by more than 50%. Furthermore, mutation of the two cleavage sites in LANA led to a significant increase in IL-1β production in transfected THP-1 cells; this provides evidence that these sites function to blunt the inflammasome, which is known to be activated in latently infected PEL cells. These results suggest that specific caspase cleavage sites in KSHV LANA function to blunt apoptosis as well as interfere with the caspase-1-mediated inflammasome, thus thwarting key cellular defense mechanisms.

Utocrine role of endogenous interleukin‐18 on inflammatory cytokine generation by human neutrophils

Neutrophils are key players of innate immunity and influence inflammatory and immune reactions through the production of numerous cytokines. Interleukin-18 (IL-18) is known to stimulate several neutrophil responses, and recent evidence suggests that neutrophils might represent a source of IL-18. Here, we show that neutrophils constitutively produce both IL-18 and its antagonist, IL-18BP. Cell activation does not affect IL-18BP release but leads to an increased gene expression and secretion of IL-18, a process that depends on NF-kappaB activation. Moreover, endogenous IL-18 feeds back on the neutrophils to augment cytokine generation in lipopolysaccharide-treated cells. Accordingly, exogenous IL-18 can induce the gene expression and release of several inflammatory cytokines in neutrophils, including its own expression. We finally report that IL-18 activates the p38 MAPK, MEK/ERK, and PI3K/Akt pathways in neutrophils. The IKK cascade is also activated by IL-18, resulting in IkappaB-alpha degradation, NF-kappaB activation, and RelA phosphorylation. Accordingly, these pathways contribute to the generation of inflammatory cytokines in IL-18-stimulated neutrophils. By contrast, the phosphorylation and DNA-binding activity of various STAT proteins were not induced by IL-18. Collectively, our results unveil new interactions between IL-18 and neutrophils and further support a role for these cells in influencing both innate and adaptive immunity.

Inhibitory effect of somatostatin on prostaglandin E 2 synthesis by primary neonatal rat glial cells

Glial inflammation plays an integral role in the development of neurodegenerative disease. Although somatostatin is known to be a local anti-inflammatory factor in the periphery, evidence of a similar function in the brain is scarce. The aim of the present study was to investigate the effect of somatostatin on prostaglandin E 2 synthesis in primary neonatal rat glial cells. The data shows that high concentrations of somatostatin (10 − 5 –10 − 4 ) significantly increased prostaglandin synthesis. By contrast, when used at physiologically relevant concentrations (10 − 9 –10 − 7 M), somatostatin and somatostatin receptor agonists decreased prostaglandin E 2 synthesis in non-stimulated glial cells as well as in lipopolysaccharide-induced prostaglandin synthesis. The inhibitory effect of somatostatin in lipopolysaccharide-treated cells could be mimicked by protein kinase A inhibitor and was prevented by forskolin. These observations suggest the presence of a novel neuro-immune feedback pathway through which somatostatin inhibits glial prostaglandin synthesis, and thus may prove to play a role in brain inflammation. This action of somatostatin may have a therapeutic potential in pathological conditions of the brain, where an inflammatory response is involved .

CEP-11004, an inhibitor of the SAPK/JNK pathway, reduces TNF-α release from lipopolysaccharide-treated cells and mice

CEP-11004, a mixed lineage kinase (MLK) inhibitor, was examined for its effects on tumor necrosis factor-alpha (TNF-α) production in human THP-1 monocytes, mouse BV-2 microglia, and C57Bl/6 mice. CEP-11004 inhibited TNF-α secretion up to 90% in THP-1 cells incubated with 3 μg/ml lipopolysaccharide, with an IC50 of 137±14 nM. CEP-11004 also inhibited TNF-α production in lipopolysaccharide-stimulated microglial cells, but did not inhibit the initial increase in TNF-α mRNA expression as measured by real-time polymerase chain reaction (PCR). The mitogen-activated protein kinases (MAPKs) phospho-c-jun N-terminal kinase (JNK), phospho-p38, and phospho-MAPK kinase 4 (MKK4) levels were increased in THP-1 cells following lipopolysaccharide treatment, and were reduced by CEP-11004 treatment. For in vivo studies, CEP-11004 was injected 2 h prior to lipopolysaccharide (20 mg/kg) administration. CEP-11004 significantly inhibited TNF-α production at doses of 1–10 mg/kg as measured by enzyme-linked immunosorbent assay (ELISA). These results suggest that MLK blockade may be useful in inhibiting pro-inflammatory cytokine production in a wide range of diseases.

Argininosuccinate Synthetase is Reversibly Inactivated by S-Nitrosylation in Vitro and in Vivo

Prior studies have demonstrated that the substrate for NO synthesis, l-arginine, can be regenerated from the NOS co-product l-citrulline. This requires the sequential action of two enzymes, argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). AS activity has been shown to be rate-limiting for high output NO synthesis by immunostimulant-activated cells and represents a potential site for metabolic control of NO synthesis. We now demonstrate that NO mediates reversible S-nitrosylation and inactivation of AS in vitro and in lipopolysaccharide-treated cells and mice. Using a novel mass spectrometry-based method, we show that Cys-132 in human AS is the sole target for S-nitrosylation among five Cys residues. Mutagenesis studies confirm that S-nitrosylation of Cys-132 is both necessary and sufficient for the inhibition of AS by NO donors. S-nitroso-AS content is regulated by cellular glutathione levels and selectively influences NO production when citrulline is provided to cells as a protosubstrate of NOS but not when l-arginine is provided. A phylogenetic comparison of AS sequences suggests that Cys-132 evolved as a site for post-translational regulation of activity in the AS in NOS-expressing species, endowing NO with the capacity to limit its own synthesis by restricting arginine availability.

Identification of the Minimal Phosphoacceptor Site Required for in Vivo Activation of Interferon Regulatory Factor 3 in Response to Virus and Double-stranded RNA

The ubiquitously expressed latent interferon regulatory factor (IRF) 3 transcription factor is activated in response to virus infection by phosphorylation events that target a cluster of Ser/Thr residues, (382)GGASSLENTVDLHISNSHPLSLTSDQY(408) at the C-terminal end of the protein. To delineate the minimal phosphoacceptor sites required for IRF-3 activation, several point mutations were generated and tested for transactivation potential and cAMP-response element-binding protein-binding protein/p300 coactivator association. Expression of the IRF-3 S396D mutant alone was sufficient to induce type I IFN beta, IFNalpha1, RANTES, and the interferon-stimulated gene 561 promoters. Using SDS-PAGE and immunoblotting with a novel phosphospecific antibody, we show for the first time that, in vivo, IRF-3 is phosphorylated on Ser(396) following Sendai virus infection, expression of viral nucleocapsid, and double-stranded RNA treatment. These results demonstrate that Ser(396) within the C-terminal Ser/Thr cluster is targeted in vivo for phosphorylation following virus infection and plays an essential role in IRF-3 activation. The inability of the phosphospecific antibody to detect Ser(396) phosphorylation in lipopolysaccharide-treated cells suggests that other major pathways may be involved in IRF-3 activation following Toll-like receptor 4 stimulation.