Lipopolysaccharide Activation Research Articles

Antibiotic-induced imbalances in gut microbiota aggravates cholesterol accumulation and liver injuries in rats fed a high-cholesterol diet.

Increasing evidence suggests that maintenance of homeostasis between gut microbiota and host plays an important role in human health. Many diseases, such as those affecting the liver, have been linked to imbalances in gut microbial communities. However, it is not clear whether an imbalance in gut microbiota promotes the onset of liver injury or if the imbalance results from the pathological state. In the current study, antibiotics were used to disturb the gut microbiota of both rats fed a high-cholesterol diet and rats fed a normal diet (controls). The prevalence of Bacteroidetes and Firmicutes were reduced, and Proteobacteria was greatly increased in the guts of rats after antibiotic treatment. The antibiotic-induced perturbation of gut microbiota aggravated cholesterol accumulation and liver injury in rats fed a high-cholesterol diet. This may have been due to an increase in intestinal permeability and plasma lipopolysaccharide (LPS), which lead to an increase in LPS absorption and activation of TLR4 signaling, resulting in the synthesis of pro-inflammatory cytokines and chemokines in liver tissues. This study suggests that imbalances in gut microbiota may be a predisposing factor for the onset of metabolic diseases and liver injuries related to cholesterol and high-cholesterol diets. Modulation of gut microbiota could be a novel target for preventing cholesterol-related metabolic disorders.

Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay

Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo.

Evaluation of the proliferative activity of immunocompetent cells in the jejunal and iliac lymph nodes of prepubertal female wild boars diagnosed with mixed mycotoxicosis

Abstract The study evaluated the proliferative activity of immunocompetent cells in the jejunal and iliac lymph nodes of prepubertal female wild boars exposed to deoxynivalenol and zearalenone in naturally contaminated feed. The evaluation was performed with the use of the MTT assay and 2 mitogens: lipopolysaccharide (LPS) and concanavalin A. Intensified proliferative processes in T and B lymphocytes were revealed. The mitogenic activity of LPS was more expressed in the lymphocytes of both iliac and jejunal lymph nodes in comparison with the control group. Proliferative activity was higher in iliac lymph nodes than in jejunal lymph nodes. A reverse trend was observed in the percentage of live cells, which was higher in jejunal lymph nodes during the evaluation of lymphocyte proliferation.

Podocyte apoptosis is prevented by blocking the Toll-like receptor pathway.

High serum lipopolysaccharide (LPS) activity in normoalbuminuric patients with type 1 diabetes (T1D) predicts the progression of diabetic nephropathy (DN), but the mechanisms behind this remain unclear. We observed that treatment of cultured human podocytes with sera from normoalbuminuric T1D patients with high LPS activity downregulated 3-phosphoinositide-dependent kinase-1 (PDK1), an activator of the Akt cell survival pathway, and induced apoptosis. Knockdown of PDK1 in cultured human podocytes inhibited antiapoptotic Akt pathway, stimulated proapoptotic p38 MAPK pathway, and increased apoptosis demonstrating an antiapoptotic role for PDK1 in podocytes. Interestingly, PDK1 was downregulated in the glomeruli of diabetic rats and patients with type 2 diabetes before the onset of proteinuria, further suggesting that reduced expression of PDK1 associates with podocyte injury and development of DN. Treatment of podocytes in vitro and mice in vivo with LPS reduced PDK1 expression and induced apoptosis, which were prevented by inhibiting the Toll-like receptor (TLR) signaling pathway with the immunomodulatory agent GIT27. Our data show that LPS downregulates the cell survival factor PDK1 and induces podocyte apoptosis, and that blocking the TLR pathway with GIT27 may provide a non-nephrotoxic means to prevent the progression of DN.

Scavenger receptor SREC-I mediated entry of TLR4 into lipid microdomains and triggered inflammatory cytokine release in macrophages upon LPS activation (IRC4P.454)

Abstract Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation. As our previous studies indicated that SREC-I required intact Toll-like receptor 4 (TLR4) expression for its immune functions in tumor immunity, we examined potential interactions between these two receptors. We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS). The receptors then became internalized together in intracellular endosomes. SREC-I then promoted TLR4-induced signal transduction through the NFĸB and MAP kinase pathways, leading to enhanced inflammatory cytokine release. Activation of inflammatory signaling through SREC-I / TLR4 complexes appeared to involve recruitment of the receptors into detergent-insoluble, cholesterol-rich lipid microdomains that contain the small GTPase Cdc42 and the non-receptor tyrosine kinase c-Src. Under conditions of SREC-I activation by LPS, TLR4 activity required Cdc42 as well as cholesterol and actin polymerization for signaling through NFkB and MAP kinase pathways. SREC-I appeared to respond differently to another ligand, the molecular chaperone Hsp90 that, while triggering SREC-I-TLR4 binding caused only faint activation of the NFkB pathway. Our experiments indicated that SREC-I could bind LPS and might be involved in innate/inflammatory immune responses to extracellular danger signals.

The Science of Fatty Acids and Inflammation

AbstractInflammation is believed to play a central role in many of the chronic diseases that characterize modern society. In the past decade, our understanding of how dietary fats affect our immune system and subsequently our inflammatory status has grown considerably. There are compelling data showing that high-fat meals promote endotoxin [e.g., lipopolysaccharide (LPS)] translocation into the bloodstream, stimulating innate immune cells and leading to a transient postprandial inflammatory response. The nature of this effect is influenced by the amount and type of fat consumed. The role of various dietary constituents, including fats, on gut microflora and subsequent health outcomes in the host is another exciting and novel area of inquiry. The impact of specific fatty acids on inflammation may be central to how dietary fats affect health. Three key fatty acid–inflammation interactions are briefly described. First, the evidence suggests that saturated fatty acids induce inflammation in part by mimicking the actions of LPS. Second, the often-repeated claim that dietary linoleic acid promotes inflammation was not supported in a recent systematic review of the evidence. Third, an explanation is offered for why omega-3 (n–3) polyunsaturated fatty acids are so much less anti-inflammatory in humans than in mice. The article closes with a cautionary tale from the genomic literature that illustrates why extrapolating the results from inflammation studies in mice to humans is problematic.

LPS up-regulates FasL expression in Breg population in mice (IRM12P.643)

Abstract Regulatory B cell plays important role in negative immune regulation, it can exert the inhibitory functions partly by the secretion of IL-10. FasL is also considered to be associated with the immune regulatory function of B cells. Here, we first observed the expression of FasL in CD5+CD1dhi Breg population as well as other B cell subsets after LPS stimulation.In this study, splenocytes were isolated from C57/BL6 wild-type mice and cultured with LPS for 24-48 hours to activate Breg. Results showed that the expression of FasL in CD5+CD1dhi B cell subset is much higher than CD19+CD5+CD1dlow and CD19+CD5neg B cell subset. The lowest FasL expression was seen in CD19+CD5neg B cell subset. This is consistent with the expression of IL-10 in corresponding B cell subsets. In addition, studies showed that the expression of CD25 molecules in CD19+CD5+CD1dhi Breg was significantly higher than other two B cell subsets after stimulating by LPS for 24 hours. CD86 molecule was highly expressed in all these three B cell subsets, and it is higher in CD19+CD5+CD1dhi Breg compared with other B subsets. In contact hypersensitivity model, the expression of FasL in CD19+CD5+CD1dhi Breg after LPS activation showed significant difference in comparison with control group.These results suggested that FasL might also contribute to the inhibitory function of CD5+CD1dhi Breg population after LPS stimulation and thus participate in the regulation of some inflammatory immune diseases.

Scavenger receptor SREC-I mediated entry of TLR4 into lipid microdomains and triggered inflammatory cytokine release in RAW 264.7 cells upon LPS activation.

Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation. As our previous studies indicated that SREC-I required intact Toll-like receptor 4 (TLR4) expression for its functions in tumor immunity, we examined potential interactions between these two receptors. We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS) in RAW 264.7 and HEK 293 cells overexpressing these two receptors. The receptors then became internalized together in intracellular endosomes. SREC-I promoted TLR4-induced signal transduction through the NF-kB and MAP kinase pathways, leading to enhanced inflammatory cytokine release. Activation of inflammatory signaling through SREC-I/TLR4 complexes appeared to involve recruitment of the receptors into detergent-insoluble, cholesterol-rich lipid microdomains that contained the small GTPase Cdc42 and the non-receptor tyrosine kinase c-src. Under conditions of SREC-I activation by LPS, TLR4 activity required Cdc42 as well as cholesterol and actin polymerization for signaling through NF-kB and MAP kinase pathways in RAW 264.7 cells. SREC-I appeared to respond differently to another ligand, the molecular chaperone Hsp90 that, while triggering SREC-I-TLR4 binding caused only faint activation of the NF-kB pathway. Our experiments therefore indicated that SREC-I could bind LPS and might be involved in innate inflammatory immune responses to extracellular danger signals in RAW 264.7 cells or bone marrow-derived macrophages.

Antibacterial and membrane-damaging activities of mannosylated bovine serum albumin

The aim of this study was to test whether mannosylated BSA (Man-BSA) exerts antibacterial activity on Escherichia coli (gram-negative bacteria) and Staphylococcus aureus (gram-positive bacteria) via its membrane-damaging effect. Man-BSA caused inhibition of growth of E. coli and S. aureus. Moreover, bactericidal action of Man-BSA on E. coli and S. aureus positively correlated with the increase in membrane permeability of the bacterial cells. Morphological examination showed that Man-BSA disrupted bacterial membrane integrity. Destabilization of the lipopolysaccharide (LPS) layer and inhibition of lipoteichoic acid (LTA) biosynthesis in the cell wall increased the bactericidal effect of Man-BSA on E. coli and S. aureus. Man-BSA also induced leakage and fusion of membrane-mimicking liposomes in E. coli and S. aureus. Man-BSA showed similar binding affinity for LPS and LTA. LPS and LTA strongly suppressed the membrane-damaging activity of Man-BSA, whereas an increase in the Man-BSA concentration attenuated the inhibitory action of LPS and LTA. Taken together, our data indicate that Man-BSA’s bactericidal activity depends strongly on its ability to induce membrane permeability. Moreover, the bactericidal action of Man-BSA proven in this study suggests that Man-BSA may serve as a prototype for the development of anti-infective agents targeting E. coli and S. aureus.

Beneficial effect of honokiol on lipopolysaccharide induced anxiety-like behavior and liver damage in mice

Anxiety disorders are commonly occurring co-morbid neuropsychiatric disorders with chronic inflammatory conditions such as live damage. Numerous studies revealed that peripheral inflammation, oxidative stress and brain derived neurotrophic factor (BDNF) play important roles in the pathophysiology of anxiety disorders. Honokiol (HNK) is a polyphenol, possessing multiple biological activities including antioxidant, anti-inflammatory, anxiolytic, antidepressant and hepatoprotection. The present study was designed to investigate the effect of HNK, in lipopolysaccharide (LPS)-induced anxiety-like behavior and liver damage in mice. Mice (n=6–10/group) were pre-treated with different doses of HNK (2.5 and 5mg/kg; i.p.) for two days, and challenged with saline or LPS (0.83mg/kg; i.p.) on third day. Anxiety-like behavior was monitored using elevated plus maze (EPM) and open field test (OFT). Animals were sacrificed to evaluate various biochemical parameters in plasma and liver. HNK pre-treatment provided significant (P<0.01) protection against LPS-induced reduction in body weight, food and water intake in mice. HNK at higher dose significantly (P<0.05) attenuated LPS-induced anxiety-like behavior by increasing the number of entries and time spent in open arm in EPM test, and by increasing the frequency in central zone in OFT. HNK pre-treatment ameliorated LPS-induced peripheral inflammation by reducing plasma IL-1β, IL-6, TNF-α level, and also improved the plasma BDNF level, prevented liver damage via attenuating transaminases (AST, ALT), liver oxidative stress and TNF-α activity in LPS challenged mice. In conclusion, the current investigation suggests that HNK provided beneficial effect against LPS-induced anxiety-like behavior and liver damage which may be governed by inhibition of cytokines production, oxidative stress and depletion of plasma BDNF level. Our result suggests that HNK could be a therapeutic approach for the treatment of anxiety and other neuropsychiatric disorders associated with inflammation and oxidative stress.

Supercritical fluid extraction as an alternative process to obtain essential oils with anti-inflammatory properties from marjoram and sweet basil

The anti-inflammatory potential of marjoram and sweet basil essential oils obtained by supercritical fluid extraction (SFE) was tested in vitro using THP-1 human macrophage cells. THP-1 cells were activated by lipopolysaccharide or human ox-LDL and the cytokine secretion and gene expression of, respectively, TNF-α, IL-1β, IL-6 and IL-10 along with COX-2 and NFκB gene expression was evaluated. Results indicated that both marjoram and sweet basil essential oils at a concentration of 10μg/mL markedly suppressed the production of pro-inflammatory cytokines and gene expression in LPS and ox-LDL THP-1 activation. The chemical composition of marjoram and basil extracts was evaluated and the activity of the main compounds was also tested for cytokine production and gene expression. We concluded that the anti-inflammatory activity of both oils is dedicated to their main compounds, respectively, sabinene hydrate and terpineol for marjoram and linalool and eugenol for sweet basil extracts. Furthermore, these results confirmed their application as anti-atherosclerotic agents.Main compounds presented in supercritical basil (linalool and eugenol) and marjoram (sabinene hydrate and terpineol) essential oils also presented an important anti-inflammatory activity, which allowed us to propose these compounds as responsible for the anti-inflammatory activity found in essential oils.In conclusion, SFE allowed to obtain sage and marjoram essential oils with significant anti-inflammatory properties. Furthermore, these supercritical essential oils have also shown an important anti-inflammatory effect in an atherosclerotic environment and presented a highly potential application in the prevention of atherosclerosis.

Paradise revealed II: Top science, sunny beach, tropical fruits and caipirinhas….

Some people say that lightning never strikes twice in the same place. Gratefully, sunbeam does! In February 2010, Brazilian scientists organized an outstanding multidisciplinary meeting at a beach resort in Angra dos Reis, Rio de Janeiro, Brazil, that brought together leaders of the cell death, tumor biology and immunology fields. Along with couple of hundred participants, speakers shared their latest work, experience and enthusiasm in an excited atmosphere rocked by the sound of waves.1 Keystone Symposia on Molecular and Cellular Biology and Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), the Sao Paulo State Research Foundation, decided to surf this idea and worked together to implement the first Keystone meeting in the State of Sao Paulo—the second organized in Brazil. This time the venue was the delightful Enseada beach, in Guaruja, a coastal paradisiacal town located in the south of the State of Sao Paulo. The sophisticated and yet casual hotel, the amazing fine sand beach, the fresh breeze, the tasty fruits and coconut water and, of course, the famous Brazilian caipirinhas provided an incredibly relaxed and friendly atmosphere where top scientists were overwhelmed by the quality and depth of students' interrogations. Organized by Gustavo Amarante-Mendes, Douglas Green and Kim Newton, the event started with a pre-meeting course on Cell Death modalities. The course brought together 60 PhD students or postdocs fully supported by FAPESP, half coming from Brazil and half from abroad, creating a dynamic and highly interactive environment that lasted throughout the meeting. Vishva Dixit (Genentech, Inc., USA) presented the 'Keynote Address' on inflammasome structure and signaling events, describing the activation of non-canonical inflammasome by cytosolic LPS and its role in septic shock. In line with this, Petr Broz (University of Basel, Switzerland) demonstrated that guanylate-binding proteins (GBPs) targeted the bacteria and promoted disruption of bacteria-containing vacuoles, allowing cytosolic sensing of LPS and caspase-11 activation. Guy Salvesen (Sanford-Burnham Medical Research Institute, USA) introduced us to novel specific probes highly specific for each caspase active site, enabling unambiguous discrimination of caspase activation. Mohanish Deshmukh (University of North Carolina at Chapel Hill, USA) demonstrated that cytosolic cytochrome c is not able to engage in the apoptosome formation in neurons because of very low levels of Apaf-1 in these cells, and that free cytosolic cytochrome c is ubiquitinated and degraded by the proteasome. Marion MacFarlane (MRC Toxicologic Unit, UK), demonstrated that caspase-8 and c-FLIP were able to form chains, and that the respective ratio of caspase-8/c-FLIPS/L determines whether the cell would engage the apoptotic pathway or rather continue to live. Necroptotic pathways were extensively discussed by Peter Vandenabeele (VIB, Ghent University, Belgium), Francis Chan (University of Massachussets Medical School, USA) and Kim Newton (Genentech, Inc., USA). Peter Vandenabeele described that RIPK1 protects intestinal epithelial cells (IECs) from apoptosis. RIPK1IEC-KO mice presented increased apoptosis, barrier disruption, bacterial translocation and inflammatory infiltrates, resulting in significant weight loss and lethality. Importantly, antibiotic treatment of RIPK1IEC-KO mice decreased the inflammation and improved survival. Francis Chan revealed that, in dendritic cells, RIPK3 is necessary for TLR4-mediated activation of caspases-8 (requirement for FADD and RIPK1) and -1 (requirement for ROS) and IL-1β processing, and that RIPK3-mediated maturation of IL-1β is critical to injury-induced tissue repair. Kim Newton described that necroptosis-related inflammation is dependent on RIPK1 and RIPK3, but not on MLKL. Douglas Green (St Jude Children's Research Hospital, USA) delivered a captivating lecture in which he approached the question of non-genetic control of cell survival. He presented new results obtained by single-cell mass spectrometry to investigate the cell population that consistently survives TRAIL-induced apoptosis.

Photoactivated Polycationic Bioactive Chitosan Nanoparticles Inactivate Bacterial Endotoxins

IntroductionThe current root canal disinfection protocols fail to markedly inactivate bacterial endotoxins from infected root dentin. This study aimed to evaluate the ability of antibacterial photodynamic therapy with chitosan-conjugated rose bengal nanoparticles (CSRBnps) to selectively inactivate endotoxins/lipopolysaccharides (LPSs). MethodsAntimicrobial agents such as calcium hydroxide (Ca[OH]2), chitosan nanoparticles (CSnps), CSRBnps, and methylene blue (MB) were assessed for their ability to neutralize LPSs obtained from Pseudomonas aeruginosa in a time-dependent interaction with/without photoactivation (20 and 40 J/cm2). The inflammatory potential of the treated/untreated LPSs was assessed on macrophage cells (RAW 267.4) using nitric oxide– and enzyme-linked immunosorbent assay (tumor necrosis factor α and interleukin-6 expression)–based analysis. These antimicrobials were tested directly on macrophage cells for cytotoxicity using the mitochondrial activity assay and light microscopy. The data were analyzed using 1-way analysis of variance and the Tukey test. ResultsCSnps were least effective in LPS inactivation. Interluekin-6 expression was reduced only with CSRBnp treatment. CSnps and CSRBnps were completely nontoxic, and MB showed slight toxicity to macrophage cells. Ca(OH)2 was highly cytotoxic (P < .005) even at 30 minutes of exposure. CSRBnps and MB with/without photoactivation significantly inactivated LPSs with reduced nitric oxide and tumor necrosis factor α expression (P < .05). Cell death and detachment after Ca(OH)2 treatment resulted in complete absence of all 3 inflammatory markers. ConclusionsPhotodynamically activated CSRBnps caused significant inactivation of endotoxins and the subsequent reduction of all tested inflammatory markers from activated macrophages. Antimicrobial CSRBnps in combination with photodynamic therapy showed the potential to effectively inactivate bacterial endotoxins.

Scutellarin regulates the Notch pathway and affects the migration and morphological transformation of activated microglia in experimentally induced cerebral ischemia in rats and in activated BV-2 microglia.

BackgroundActivated microglial cells release an excess of inflammatory mediators after an ischemic stroke. We reported previously that scutellarin effectively suppressed the inflammatory response induced by activated microglia in rats subjected to middle cerebral artery occlusion (MCAO); however, the mechanism via which scutellarin exerts its effects on microglial activation has not been explored. This study aimed to elucidate if scutellarin can regulate the Notch pathway that is linked to microglia activation in MCAO rat, and in lipopolysaccharide (LPS)-induced BV-2 microglia. Along with this, we also investigated some characteristic behavioral responses of activated microglia.MethodsExpression of various members of the Notch pathway, including Notch-1, intracellular Notch receptor domain (NICD), recombining binding protein suppressor of hairless (RBP-JK) and transcription factor hairy and enhancer of split-1 (Hes-1) in activated microglia was assessed by immunofluorescence staining and western blot after experimental MCAO and in vitro LPS activation. The effect of scutellarin on migration of microglia was determined by the transwell chamber assay as well as expression of monocyte chemoattractant protein-1 (MCP-1). The morphological change of microglia induced by scutellarin was detected by F-actin staining and electron microscopy.ResultsScutellarin markedly attenuated the expression of NF-κB, Notch-1, NICD, RBP-JK and Hes-1 both in vivo and in activated microglia. It decreased the expression of MCP-1 and microglial migration, but increased the ability of microglia adhesion. Scutellarin also altered the phenotype of microglia by causing rearrangement or reorganization of its cytoskeleton.ConclusionsThe results suggest that scutellarin regulates the activation of microglia via the Notch pathway and concurrently induces morphological and functional changes in activated microglia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0226-z) contains supplementary material, which is available to authorized users.

Endotoxemia Analysis by the Limulus Amoebocyte Lysate Assay in Different Mammal Species Used in Metabolic Studies

Introduction: Lipopolysaccharides (LPS) or so-called endotoxins are potent pro-inflammatory compounds. LPS can be present in the bloodstream in case of septic conditions, leading to measure endotoxemia that is the activity of LPS in plasma. Recent research also reveals a low-grade or so-called metabolic endotoxemia associated with metabolic diseases (e.g. obesity, type 2 diabetes, cardiovascular diseases). In this context, research studies use different experimental models, mostly in humans and rodents. Pig is now emerging as a new animal model in nutritional and metabolic studies. However, information is lacking to date about optimal dilution to be used for sample preparation for the Limulus Amebocyte Lysate test, according to species, especially for pig. Methods: strong>Endotoxemia was measured using the LAL standard reference method. We describe the method of sample preparation and the LAL technique to measure endotoxemia in 4 mammal species: human, mouse, rat and pig. Results: Plasma dilution is necessary to overcome interferences leading to erroneously low or high results. Optimal dilution to avoid interferences in most samples, while maintaining a satisfactory sensitivity, was found to be at least 1/10 for human vs 1/40 for mice, while much higher dilution was mandatory for pigs, namely 1/200. Altogether, mean plasma endotoxemia in all tested samples using the optimal dilution for each species was 0.73 ± 0.05 EU/mL in humans (n=903), 0.9 ± 0.2 EU/mL in rodents (n=295) and 8.5 ±1.3 EU/mL in pigs (n=186), regardless of fasting or postprandial state and/or type of dietary intervention. Conclusion: We show that the LAL assay can be used to determine endotoxemia in fasting and postprandial blood samples from different mammal species including pig. Because its detection is made difficult by interference from other plasma constituents, an essential parameter to overcome this difficulty is the dilution factor that depends on the studied species.

GSTO1-1 modulates metabolism in macrophages activated through the LPS and TLR4 pathway.

Macrophages mediate innate immune responses that recognise foreign pathogens, and bacterial lipopolysaccharide (LPS) recruits a signalling pathway through Toll-like receptor 4 (TLR4) to induce pro-inflammatory cytokines and reactive oxygen species (ROS). LPS activation also skews the metabolism of macrophages towards a glycolytic phenotype. Here, we demonstrate that the LPS-triggered glycolytic switch is significantly attenuated in macrophages deficient for glutathione transferase omega-1 (GSTO1, note that GSTO1-1 refers to the dimeric molecule with identical type 1 subunits). In response to LPS, GSTO1-1-deficient macrophages do not produce excess lactate, or dephosphorylate AMPK, a key metabolic stress regulator. In addition, GSTO1-1-deficient cells do not induce HIF1α, which plays a key role in maintaining the pro-inflammatory state of activated macrophages. The accumulation of the TCA cycle intermediates succinate and fumarate that occurs in LPS-treated macrophages was also blocked in GSTO1-1-deficient cells. These data indicate that GSTO1-1 is required for LPS-mediated signalling in macrophages and that it acts early in the LPS-TLR4 pro-inflammatory pathway.

Contrast-enhanced micro-computed tomography using ExiTron nano6000 for assessment of liver injury.

To explore the potential of contrast-enhanced computed tomography (CECT) using ExiTron nano6000 for assessment of liver lesions in mouse models. Three mouse models of liver lesions were used: bile duct ligation (BDL), lipopolysaccharide (LPS)/D-galactosamine (D-GalN), and alcohol. After injection with the contrast agent ExiTron nano6000, the mice were scanned with micro-CT. Liver lesions were evaluated using CECT images, hematoxylin and eosin staining, and serum aminotransferase levels. Macrophage distribution in the injury models was shown by immunohistochemical staining of CD68. The in vitro studies measured the densities of RAW264.7 under different conditions by CECT. In the in vitro studies, CECT provided specific and strong contrast enhancement of liver in mice. CECT could present heterogeneous images and densities of injured livers induced by BDL, LPS/D-GalN, and alcohol. The liver histology and immunochemistry of CD68 demonstrated that both dilated biliary tracts and necrosis in the injured livers could lead to the heterogeneous distribution of macrophages. The in vitro study showed that the RAW264.7 cell masses had higher densities after LPS activation. Micro-CT with the contrast agent ExiTron nano6000 is feasible for detecting various liver lesions by emphasizing the heterogeneous textures and densities of CECT images.

Foeniculum vulgare Mill. Protects against Lipopolysaccharide-induced Acute Lung Injury in Mice through ERK-dependent NF-κB Activation.

Foeniculum vulgare Mill. (fennel) is used to flavor food, in cosmetics, as an antioxidant, and to treat microbial, diabetic and common inflammation. No study to date, however, has assessed the anti-inflammatory effects of fennel in experimental models of inflammation. The aims of this study were to investigate the anti-inflammatory effects of fennel in model of lipopolysaccharide (LPS)-induced acute lung injury. Mice were randomly assigned to seven groups (n=7~10). In five groups, the mice were intraperitoneally injected with 1% Tween 80-saline (vehicle), fennel (125, 250, 500µl/kg), or dexamethasone (1 mg/kg), followed 1 h later by intratracheal instillation of LPS (1.5 mg/kg). In two groups, the mice were intraperitoneally injected with vehicle or fennel (250µl/kg), followed 1 h later by intratracheal instillation of sterile saline. Mice were sacrificed 4 h later, and bronchoalveolar lavage fluid (BALF) and lung tissues were obtained. Fennel significantly and dose-dependently reduced LDH activity and immune cell numbers in LPS treated mice. In addition fennel effectively suppressed the LPS-induced increases in the production of the inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha, with 500µl/kg fennel showing maximal reduction. Fennel also significantly and dose-dependently reduced the activity of the proinflammatory mediator matrix metalloproteinase 9 and the immune modulator nitric oxide (NO). Assessments of the involvement of the MAPK signaling pathway showed that fennel significantly decreased the LPS-induced phosphorylation of ERK. Fennel effectively blocked the inflammatory processes induced by LPS, by regulating pro-inflammatory cytokine production, transcription factors, and NO.

Time-dependent changes in inhibitory action of lipopolysaccharide on intestinal motility in rat.

Endotoxin causes gastrointestinal motility disorder. Aim of this study is to clarify inhibitory mechanisms of lipopolysaccharide (LPS) on smooth muscle contraction in rat ileum. Ileal tissues were isolated from control rat or from LPS-induced peritonitis model rat. Treatment with LPS inhibited carbachol (CCh)-mediated contraction in a time-dependent manner. Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) genes were also upregulated, but iNOS expression was preceded by a rising of COX-2. All subtypes of prostaglandin E2 (PGE2) receptors (EP1-EP4) were expressed in ileum, and PGE2 and selective EP2 or EP4 agonist inhibited CCh-mediated contraction. Selective iNOS inhibitor did not reverse LPS-induced inhibition of contraction by CCh at 1 and 2 hr, but reduced the inhibitory action at 4 hr after the LPS treatment. COX-2 inhibitor reversed the inhibitory action by LPS in all exposure time. Finally, in ileal tissues isolated from peritonitis model rat, iNOS expression was upregulated only at 4 hr after LPS administration, resulting in enhanced inhibitory action of LPS against CCh-induced contraction. In conclusion, LPS induces COX-2 to produce PGE2, which initially activates EP2 and/or EP4 on smooth muscle cells to inhibit the contractility in early phase of LPS exposure. Moreover, in late phase of LPS treatment, iNOS is expressed to produce NO, which in turn inhibited the contraction by CCh. The inhibitory cascade is similar in the ileum isolated from peritonitis model rat, indicating time-dependent changes of inhibitory action by LPS on intestinal motility in peritonitis.

Inhibition of Lipopolysaccharide-Induced Expression of Fractalkine by Methylprednisolone via NF-Κb in Human Renal Tubular Epithelial Cells

Objective: to study the effect of the glucocorticoid, methylprednisolone (MP), in lipopolysaccharide (LPS)-induced fractalkine (FKN) expression in HK-2 cells and to determine the role of NF-κB in this signaling pathway. Methods: HK-2 cells were stimulated by LPS to set up an in vitro inflammation model. The concentration of FKN in cell culture supernatant was measured by ELISA. FKN and p65 mRNA expression were detected by RT-PCR. FKN, p65 protein expression and the activity of the NF-κB were detected by immunofluorescence staining and western blotting. The effect of MP and SC-514 (a selective and reversible inhibitor of IKK beta) in FKN expression and NF-κB activation induced by LPS were evaluated. Results: LPS induced FKN expression and secretion in HK-2 cells occurred in a time- and dose-dependent manner and correlated with the activation of NF-κB. MP was able to inhibit FKN expression and secretion as well as the NF-κB induced activation of LPS, whereas SC-514 abolished this effect. Conclusions: MP inhibited FKN expression induced by LPS through the NF-κB pathway in human renal tubular epithelial cells in vitro. Use of HK-2 cells to study the renal inflammatory process will allow the further elucidation of the pathways involved in kidney disease.