Showing papers on "Lipopolysaccharide published in 2013"

Succinate is an inflammatory signal that induces IL-1β through HIF-1α

Abstract: Macrophages activated by the Gram-negative bacterial product lipopolysaccharide switch their core metabolism from oxidative phosphorylation to glycolysis. Here we show that inhibition of glycolysis with 2-deoxyglucose suppresses lipopolysaccharide-induced interleukin-1β but not tumour-necrosis factor-α in mouse macrophages. A comprehensive metabolic map of lipopolysaccharide-activated macrophages shows upregulation of glycolytic and downregulation of mitochondrial genes, which correlates directly with the expression profiles of altered metabolites. Lipopolysaccharide strongly increases the levels of the tricarboxylic-acid cycle intermediate succinate. Glutamine-dependent anerplerosis is the principal source of succinate, although the 'GABA (γ-aminobutyric acid) shunt' pathway also has a role. Lipopolysaccharide-induced succinate stabilizes hypoxia-inducible factor-1α, an effect that is inhibited by 2-deoxyglucose, with interleukin-1β as an important target. Lipopolysaccharide also increases succinylation of several proteins. We therefore identify succinate as a metabolite in innate immune signalling, which enhances interleukin-1β production during inflammation.

Noncanonical Inflammasome Activation by Intracellular LPS Independent of TLR4

Abstract: Gram-negative bacteria including Escherichia coli, Citrobacter rodentium, Salmonella typhimurium, and Shigella flexneri are sensed in an ill-defined manner by an intracellular inflammasome complex that activates caspase-11. We show that macrophages loaded with synthetic lipid A, E. coli lipopolysaccharide (LPS), or S. typhimurium LPS activate caspase-11 independently of the LPS receptor Toll-like receptor 4 (TLR4). Consistent with lipid A triggering the noncanonical inflammasome, LPS containing a divergent lipid A structure antagonized caspase-11 activation in response to E. coli LPS or Gram-negative bacteria. Moreover, LPS-mutant E. coli failed to activate caspase-11. Tlr4(-/-) mice primed with TLR3 agonist polyinosinic:polycytidylic acid [poly(I:C)] to induce pro-caspase-11 expression were as susceptible as wild-type mice were to sepsis induced by E. coli LPS. These data unveil a TLR4-independent mechanism for innate immune recognition of LPS.

Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock

Abstract: Inflammatory caspases, such as caspase-1 and -11, mediate innate immune detection of pathogens. Caspase-11 induces pyroptosis, a form of programmed cell death, and specifically defends against bacterial pathogens that invade the cytosol. During endotoxemia, however, excessive caspase-11 activation causes shock. We report that contamination of the cytoplasm by lipopolysaccharide (LPS) is the signal that triggers caspase-11 activation in mice. Specifically, caspase-11 responds to penta- and hexa-acylated lipid A, whereas tetra-acylated lipid A is not detected, providing a mechanism of evasion for cytosol-invasive Francisella. Priming the caspase-11 pathway in vivo resulted in extreme sensitivity to subsequent LPS challenge in both wild-type and Tlr4-deficient mice, whereas Casp11-deficient mice were relatively resistant. Together, our data reveal a new pathway for detecting cytoplasmic LPS.

Recognition of lipopolysaccharide pattern by TLR4 complexes

Abstract: Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. Minute amounts of LPS released from infecting pathogens can initiate potent innate immune responses that prime the immune system against further infection. However, when the LPS response is not properly controlled it can lead to fatal septic shock syndrome. The common structural pattern of LPS in diverse bacterial species is recognized by a cascade of LPS receptors and accessory proteins, LPS binding protein (LBP), CD14 and the Toll-like receptor4 (TLR4)–MD-2 complex. The structures of these proteins account for how our immune system differentiates LPS molecules from structurally similar host molecules. They also provide insights useful for discovery of anti-sepsis drugs. In this review, we summarize these structures and describe the structural basis of LPS recognition by LPS receptors and accessory proteins.

Lipopolysaccharide Causes an Increase in Intestinal Tight Junction Permeability in Vitro and in Vivo by Inducing Enterocyte Membrane Expression and Localization of TLR-4 and CD14

Abstract: Bacterial-derived lipopolysaccharides (LPS) play an essential role in the inflammatory process of inflammatory bowel disease. A defective intestinal tight junction (TJ) barrier is an important pathogenic factor of inflammatory bowel disease and other inflammatory conditions of the gut. Despite its importance in mediating intestinal inflammation, the physiological effects of LPS on the intestinal epithelial barrier remain unclear. The major aims of this study were to determine the effects of physiologically relevant concentrations of LPS (0 to 1 ng/mL) on intestinal barrier function using an in vitro (filter-grown Caco-2 monolayers) and an in vivo (mouse intestinal perfusion) intestinal epithelial model system. LPS, at physiologically relevant concentrations (0 to 1 ng/mL), in the basolateral compartment produced a time-dependent increase in Caco-2 TJ permeability without inducing cell death. Intraperitoneal injection of LPS (0.1 mg/kg), leading to clinically relevant plasma concentrations, also caused a time-dependent increase in intestinal permeability in vivo . The LPS-induced increase in intestinal TJ permeability was mediated by an increase in enterocyte membrane TLR-4 expression and a TLR-4–dependent increase in membrane colocalization of membrane-associated protein CD14. In conclusion, these studies show for the first time that LPS causes an increase in intestinal permeability via an intracellular mechanism involving TLR-4–dependent up-regulation of CD14 membrane expression.

HMGB1 promotes neutrophil extracellular trap formation through interactions with Toll-like receptor 4

Abstract: Although neutrophil extracellular traps (NETs) form to prevent dissemination of pathogenic microorganisms, excessive release of DNA and DNA-associated proteins can also perpetuate sterile inflammation. In this study, we found that the danger-associated molecular pattern protein high-mobility group box 1 (HMGB1) can induce NET formation. NET formation was found after exposure of wild-type and receptor for advanced glycation end products-deficient neutrophil to HMGB1, whereas deficiency of Toll-like receptor (TLR)4 diminished the ability of neutrophils to produce NETs. Incubation of neutrophils with HMGB1 significantly increased the amount of DNA and histone 3 released as well as intracellular histone 3 citrullination, a signaling event that precedes chromatin decondensation. In vivo, neutrophils isolated from bronchoalveolar lavages of mice exposed to LPS and HMGB1 showed consistently greater ability to produce NETs compared with pulmonary neutrophils from mice that received LPS alone. In contrast, mice treated with LPS and neutralizing antibody to HMGB1 had decreased amounts of the inflammatory cytokines TNF-α and macrophage inflammatory protein 2, as well as of free DNA and histone 3 in bronchoalveolar lavage fluids. Airway neutrophils from LPS-exposed mice that had been treated with anti-HMGB1 antibodies showed decreased citrullination of histone 3. These results demonstrate that interactions between HMGB1 and TLR4 enhance the formation of NETs and provide a novel mechanism through which HMGB1 may contribute to the severity of neutrophil-associated inflammatory conditions.

Monocytes, peripheral blood mononuclear cells, and THP-1 cells exhibit different cytokine expression patterns following stimulation with lipopolysaccharide.

Abstract: THP-1 cells are widely applied to mimic monocytes in cell culture models. In this study, we compared the cytokine release from THP-1, peripheral blood mononuclear cells (PBMC), monocytes, or whole blood after stimulation with lipopolysaccharide (LPS) and investigated the consequences of different cytokine profiles on human umbilical vein endothelial cell (HUVEC) activation. While Pseudomonas aeruginosa-stimulated (10 ng/mL) THP-1 secreted similar amounts of tumor necrosis factor alpha (TNF-α) as monocytes and PBMC, they produced lower amounts of interleukin(IL)-8 and no IL-6 and IL-10. Whole blood required a higher concentration of Pseudomonas aeruginosa (1000 ng/mL) to induce cytokine release than isolated monocytes or PBMC (10 ng/mL). HUVEC secreted more IL-6 and IL-8 after stimulation with conditioned medium derived from whole blood than from THP-1, despite equal concentrations of TNF-α in both media. Specific adsorption of TNF-α or selective cytokine adsorption from the conditioned media prior to HUVEC stimulation significantly reduced HUVEC activation. Our findings show that THP-1 differ from monocytes, PBMC, and whole blood with respect to cytokine release after stimulation with LPS. Additionally, we could demonstrate that adsorption of inflammatory mediators results in reduced endothelial activation, which supports the concept of extracorporeal mediator modulation as supportive therapy for sepsis.

Recognition of lipid A variants by the TLR4-MD-2 receptor complex.

Abstract: Lipopolysaccharide (LPS) is a component of the outer membrane of almost all Gram-negative bacteria and consists of lipid A, core sugars, and O-antigen. LPS is recognized by Toll-like receptor 4 (TLR4) and MD-2 on host innate immune cells and can signal to activate the transcription factor NFκB, leading to the production of pro-inflammatory cytokines that initiate and shape the adaptive immune response. Most of what is known about how LPS is recognized by the TLR4-MD-2 receptor complex on animal cells has been studied using Escherichia coli lipid A, which is a strong agonist of TLR4 signaling. Recent work from several groups, including our own, has shown that several important pathogenic bacteria can modify their LPS or lipid A molecules in ways that significantly alter TLR4 signaling to NFκB. Thus, it has been hypothesized that expression of lipid A variants is one mechanism by which pathogens modulate or evade the host immune response. Additionally, several key differences in the amino acid sequences of human and mouse TLR4-MD-2 receptors have been shown to alter the ability to recognize these variations in lipid A, suggesting a host-specific effect on the immune response to these pathogens. In this review, we provide an overview of lipid A variants from several human pathogens, how the basic structure of lipid A is recognized by mouse and human TLR4-MD-2 receptor complexes, as well as how alteration of this pattern affects its recognition by TLR4 and impacts the downstream immune response.

In depression, bacterial translocation may drive inflammatory responses, oxidative and nitrosative stress (O&NS), and autoimmune responses directed against O&NS-damaged neoepitopes

Abstract: OBJECTIVE: Depression is accompanied by activation of immuno-inflammatory and oxidative and nitrosative stress (IO&NS) pathways, and increased IgM/IgA responses to lipopolysaccharide (LPS) of gram-negative commensal bacteria. The latter suggests that bacterial translocation has caused IgM/IgA responses directed against LPS. Bacterial translocation may drive IO&NS responses. METHOD: To examine the associations between IgM/IgA responses to LPS and IO&NS measurements, including plasma/serum interleukin-1 (IL-1), tumor necrosis factor (TNF)α, neopterin, lysozyme, oxidized LDL (oxLDL) antibodies, peroxides, and IgM (auto)immune responses against malondialdehyde (MDA), azelaic acid, phophatidyl inositol (Pi), NO-tryptophan and NO-tyrosine in depressed patients and controls. RESULTS: We found significant positive associations between IgM/IgA responses to LPS and oxLDL antibodies, IgM responses against MDA, azelaic acid, Pi, NO-tryptophan, and NO-tyrosine. The IgA responses to LPS were correlated with lysozyme. There were no significant positive correlations between the IgM/IgA responses to LPS and IL-1 and neopterin. CONCLUSION: The findings show that in depression there is an association between increased bacterial translocation and lysozyme production, an antibacterial compound, O&NS processes, and autoimmune responses directed against O&NS generated neoantigenic determinants. It is suggested that bacterial translocation may drive IO&NS pathways in depression and thus play a role in its pathophysiology.

Lipopolysaccharide Clearance, Bacterial Clearance, and Systemic Inflammatory Responses Are Regulated by Cell Type–Specific Functions of TLR4 during Sepsis

Abstract: The morbidity associated with bacterial sepsis is the result of host immune responses to pathogens, which are dependent on pathogen recognition by pattern recognition receptors, such as TLR4. TLR4 is expressed on a range of cell types, yet the mechanisms by which cell-specific functions of TLR4 lead to an integrated sepsis response are poorly understood. To address this, we generated mice in which TLR4 was specifically deleted from myeloid cells (LysMTLR4KO) or hepatocytes (HCTLR4KO) and then determined survival, bacterial counts, host inflammatory responses, and organ injury in a model of cecal ligation and puncture (CLP), with or without antibiotics. LysM-TLR4 was required for phagocytosis and efficient bacterial clearance in the absence of antibiotics. Survival, the magnitude of the systemic and local inflammatory responses, and liver damage were associated with bacterial levels. HCTLR4 was required for efficient LPS clearance from the circulation, and deletion of HCTLR4 was associated with enhanced macrophage phagocytosis, lower bacterial levels, and improved survival in CLP without antibiotics. Antibiotic administration during CLP revealed an important role for hepatocyte LPS clearance in limiting sepsis-induced inflammation and organ injury. Our work defines cell type–selective roles for TLR4 in coordinating complex immune responses to bacterial sepsis and suggests that future strategies for modulating microbial molecule recognition should account for varying roles of pattern recognition receptors in multiple cell populations.

Structural modifications of bacterial lipopolysaccharide that facilitate Gram-negative bacteria evasion of host innate immunity

Abstract: Bacterial lipopolysaccharide (LPS), a cell wall component characteristic of Gram-negative bacteria, is a representative pathogen-associated molecular pattern that allows mammalian cells to recognize bacterial invasion and trigger innate immune responses. The polysaccharide moiety of LPS primary plays protective roles for bacteria such as prevention from complement attacks or camouflage with common host carbohydrate residues. The lipid moiety, termed lipid A, is recognized by the Toll-like receptor 4 (TLR4)/MD-2 complex, which transduces signals for activation of host innate immunity. The basic structure of lipid A is a glucosamine disaccharide substituted by phosphate groups and acyl groups. Lipid A with 6 acyl groups (hexa-acylated form) has been indicated to be a strong stimulator of the TLR4/MD-2 complex. This type of lipid A is conserved among a wide variety of Gram-negative bacteria, and those bacteria are easily recognized by host cells for activation of defensive innate immune responses. Modifications of the lipid A structure to less-acylated forms have been observed in some bacterial species, and those forms are poor stimulators of the TLR4/MD-2 complex. Such modifications are thought to facilitate bacterial evasion of host innate immunity, thereby enhancing pathogenicity. This hypothesis is supported by studies of Yersinia pestis LPS, which contains hexa-acylated lipid A when the bacterium grows at 27oC (the temperature of the vector flea), and shifts to contain less-acylated forms when grown at the human body temperature of 37oC. This alteration of lipid A forms following transmission of Y. pestis from fleas to humans contributes predominantly to the virulence of this bacterium over other virulence factors. A similar role for less-acylated lipid A forms has been indicated in some other bacterial species, such as Francisella tularensis, Helicobacter pylori, and Porphyromonas gingivalis, and further studies to explore this concept are expected.

Ethanol induces TLR4/TLR2 association, triggering an inflammatory response in microglial cells.

Abstract: Alcohol consumption can induce brain damage, demyelination, and neuronal death, although the mechanisms are poorly understood. Toll-like receptors are sensors of the innate immune system and their activation induces inflammatory processes. We have reported that ethanol activates and recruits Toll-like receptor (TLR)4 receptors within the lipid rafts of glial cells, triggering the production of inflammatory mediators and causing neuroinflammation. Since TLR2 can also participate in the glial response and in the neuroinflammation, we investigate the effects of ethanol on TLR4/TLR2 responses. Here, we demonstrate that ethanol up-regulates TLR4 and TLR2 expression in microglial cells, inducing the production of inflammatory mediators which triggers reactive oxygen species generation and neuronal apoptosis. Ethanol also promotes TLR4/TLR2 recruitment into lipid rafts-caveolae, mimicking their activation by their ligands, lipopolysaccharide, and lipoteichoic acid (LTA). Immunoprecipitation and confocal microscopy studies reveal that ethanol induces a physical association between TLR2 and TLR4 receptors, suggesting the formation of heterodimers. Using microglia from either TLR2 or TLR4 knockout mice, we show that TLR2 potentiates the effects of ethanol on the TLR4 response reflected by the activation of MAPKs and inducible NO synthase. In summary, we provide evidence for a mechanism by which ethanol triggers TLR4/TLR2 association contributing to the neuroinflammation and neurodegeneration associated with alcohol abuse.

TGR5 signalling inhibits the production of pro‐inflammatory cytokines by in vitro differentiated inflammatory and intestinal macrophages in Crohn's disease

Abstract: Bile acids (BAs) play important roles not only in lipid metabolism, but also in signal transduction. TGR5, a transmembrane receptor of BAs, is an immunomodulative factor, but its detailed mechanism remains unclear. Here, we aimed to delineate how BAs operate in immunological responses via the TGR5 pathway in human mononuclear cell lineages. We examined TGR5 expression in human peripheral blood monocytes, several types of in vitro differentiated macrophages (Mϕs) and dendritic cells. Mϕs differentiated with macrophage colony-stimulating factor and interferon-γ (Mγ-Mϕs), which are similar to the human intestinal lamina propria CD14(+) Mϕs that contribute to Crohn's disease (CD) pathogenesis by production of pro-inflammatory cytokines, highly expressed TGR5 compared with any other type of differentiated Mϕ and dendritic cells. We also showed that a TGR5 agonist and two types of BAs, deoxycholic acid and lithocholic acid, could inhibit tumour necrosis factor-α production in Mγ-Mϕs stimulated by commensal bacterial antigen or lipopolysaccharide. This inhibitory effect was mediated by the TGR5-cAMP pathway to induce phosphorylation of c-Fos that regulated nuclear factor-κB p65 activation. Next, we analysed TGR5 levels in lamina propria mononuclear cells (LPMCs) obtained from the intestinal mucosa of patients with CD. Compared with non-inflammatory bowel disease, inflamed CD LPMCs contained more TGR5 transcripts. Among LPMCs, isolated CD14(+) intestinal Mϕs from patients with CD expressed TGR5. In isolated intestinal CD14(+) Mϕs, a TGR5 agonist could inhibit tumour necrosis factor-α production. These results indicate that TGR5 signalling may have the potential to modulate immune responses in inflammatory bowel disease.

G protein-coupled receptor 43 moderates gut inflammation through cytokine regulation from mononuclear cells.

Abstract: BACKGROUND Short-chain fatty acids (SCFAs), which are produced by the fermentation of dietary fiber by intestinal microbiota, may positively influence immune responses and protect against gut inflammation. SCFAs bind to G protein-coupled receptor 43 (GPR43). Here, we show that SCFA-GPR43 interactions profoundly affect the gut inflammatory response. METHODS Colitis was induced by adding dextran sulfate sodium to the drinking water of GPR43 knockout (-/-) and wild-type mice. RESULTS Dextran sulfate sodium-treated GPR43 mice exhibited weight loss, increased disease activity index (a combined measure of weight loss, rectal bleeding, and stool consistency), decreased hematocrit, and colon shortening, resulting in significantly worse colonic inflammation than in wild-type mice. Tumor necrosis factor alpha and interleukin 17 protein levels in the colonic mucosa of GPR43 mice were significantly higher than in wild-type mice. Treatment of wild-type mice with 150 mM acetate in their drinking water markedly improved these disease indices, with an increase in colon length and decrease in the disease activity index; however, it had no effect on GPR43 mice. Mononuclear cell production of tumor necrosis factor alpha after lipopolysaccharide stimulation was suppressed by acetate. This effect was inhibited by anti-GPR43 antibody. CONCLUSIONS SCFA-GPR43 interactions modulate colitis by regulating inflammatory cytokine production in mononuclear cells.

Upregulation of miR-146a contributes to the suppression of inflammatory responses in LPS-induced acute lung injury

Abstract: Despite the critical role of microRNA in inflammatory response, little is known about its function in inflammation-induced Acute Lung Injury (ALI)/Acute Respiratory Distress Syndrome (ARDS). To investigate the potential role of microRNA146a (miR-146a) in ALI, we used lipopolysaccharide (LPS)-induced ALI rat model. Our data revealed that LPS-induced lung injury in rats resulted in significant upregulation of proinflammatory cytokine tumor necrosis factor-alpha (TNF-α), IL-6, IL-1β, and miR-146a expression. LPS treatment also leads to higher expression of miR-146a as well as increase in secretion of TNF-α, IL-6, and IL-1β in alveolar macrophage (AM) NR8383 cells in a time-dependent manner. Manipulation with miR146a mimic significantly suppressed LPS-mediated TNF-α, IL-6, and IL-1β induction in NR8383 cells by repressing expression of IRAK-1 and TRAF-6. These data clearly indicate that the upregulation of miR146a suppresses inflammatory mediators in LPS induced-ALI model. Therefore, miR-146a may be therapeutically targeted as a mean to repress inflammatory response following ALI.

PGE2 suppression of innate immunity during mucosal bacterial infection

Abstract: Prostaglandin E2 (PGE2) is an important lipid mediator in inflammatory and immune responses during acute and chronic infections. Upon stimulation by various proinflammatory stimuli such as lipopolysaccharide (LPS), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α, PGE2 synthesis is upregulated by the expression of cyclooxygenases. Biologically active PGE2 is then able to signal through four primary receptors to elicit a response. PGE2 is a critical molecule that regulates the activation, maturation, migration, and cytokine secretion of several immune cells, particularly those involved in innate immunity such as macrophages, neutrophils, natural killer cells, and dendritic cells. Both Gram-negative and Gram-positive bacteria can induce PGE2 synthesis to regulate immune responses during bacterial pathogenesis. This review will focus on PGE2 in innate immunity and how bacterial pathogens influence PGE2 production during enteric and pulmonary infections. The conserved ability of many bacterial pathogens to promote PGE2 responses during infection suggests a common signaling mechanism to deter protective pro-inflammatory immune responses. Inhibition of PGE2 production and signaling during infection may represent a therapeutic alternative to treat bacterial infections. Further study of the immunosuppressive effects of PGE2 on innate immunity will lead to a better understanding of potential therapeutic targets within the PGE2 pathway.

The immunobiology of toll-like receptor 4 agonists: from endotoxin tolerance to immunoadjuvants.

Abstract: Lipopolysaccharide (LPS, endotoxin) is a structural component of the Gram negative outer membrane. The lipid A moiety of LPS binds to the LPS receptor complex expressed by leukocytes, endothelial cells and parenchymal cells and is the primary component of Gram negative bacteria that is recognized by the immune system. Activation of the LPS receptor complex by native lipid A induces robust cytokine production, leukocyte activation and inflammation, which is beneficial for clearing bacterial infections at the local level but can cause severe systemic inflammation and shock at higher challenge doses. Interestingly, prior exposure to LPS renders the host resistant to shock caused by subsequent LPS challenge, a phenomenon known as endotoxin tolerance. Treatment with lipid A has also been shown to augment the host response to infection and to serve as a potent vaccine adjuvant. However, the side effects associated with the pronounced inflammatory response limits the use of native lipid A as a clinical immunomodulator. More recently, analogs of lipid A have been developed that possess attenuated pro-inflammatory activity but retain attractive immunomodulatory properties. The lipid A analog monophosphoryl lipid A (MPLA) exhibits approximately 1/1000th of the toxicity of native lipid A but retains potent immunoadjuvant activity. As such, MPLA is currently employed as an adjuvant in several human vaccine preparations. Due to the potency of lipid A analogs as immunoadjuvants, numerous laboratories are actively working to identify and develop new lipid A mimetics and to optimize their efficacy and safety. Based on those characteristics, lipid A analogs represent an attractive family of immunomodulators.

Autocrine Regulation of Macrophage Activation via Exocytosis of ATP and Activation of P2Y11 Receptor

Abstract: It is important to understand the mechanisms that regulate macrophage activation to establish novel therapies for inflammatory diseases, such as sepsis; a systemic inflammatory response syndrome generally caused by bacterial lipopolysaccharide (LPS). In this study, we investigated the involvement of extracellular ATP-mediated autocrine signaling in LPS-induced macrophage activation. We show here that ATP release via exocytosis, followed by activation of P2Y11 receptor, is a major pathway of the macrophage activation, leading to release of cytokines. Treatment of human monocyte THP-1 cells with LPS induced rapid ATP release from cells, and this release was blocked by knockdown of SLC17A9 (vesicular nucleotide transporter, VNUT), which is responsible for exocytosis of ATP. ATP-enriched vesicles were found in cytosol of THP-1 cells. These data suggest the involvement of vesicular exocytosis in the release of ATP. Knockdown of SLC17A9, the P2Y11 antagonist NF157 or knockdown of P2Y11 receptor significantly suppressed both M1-type polarization and IL-6 production in THP-1 cells, indicating an important role of activation of P2Y11 receptor by released ATP in macrophage activation. Next, the effect of NF157 on LPS-induced immune activation was examined in vivo. Administration of LPS to mice caused increase of serum IL-1s, IL-6, IL-12 and TNF-alpha levels at 3–24 h after the administration. Pre-treatment of LPS-treated mice with NF157 suppressed both elevation of proinflammatory cytokines in serum and M1 polarization of peritoneal/spleen macrophages. Moreover, post-treatment with NF157 at 30 min after administration of LPS also suppressed the elevation of serum cytokines levels. We conclude that vesicular exocytosis of ATP and autocrine, positive feedback through P2Y11 receptors is required for the effective activation of macrophages. Consequently, P2Y11 receptor antagonists may be drug candidates for treatment of inflammatory diseases such as sepsis.

Lactobacillus brevis G‐101 ameliorates colitis in mice by inhibiting NF‐κB, MAPK and AKT pathways and by polarizing M1 macrophages to M2‐like macrophages

Abstract: Aim: We isolated Lactobacillus brevis G-101 from kimchi lactic acid bacteria (LAB) strains, which induced IL-10 expression in lipopolysaccharide (LPS)-stimulated peritoneal macrophages. To evaluate the inflammatory effect of G-101, we examined its inhibitory effect in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice. Materials and Results: The colitic mice were prepared by intrarectal injection of TNBS. We measured intestinal mucosal cytokines by enzyme-linked immunosorbent assay; activation of transcription factors, by immunoblotting; and macrophage polarization markers, by real-time polymerase chain reaction. Of 200 LAB strains tested, Lact.brevis G-101 showed most potent activity for induction of IL-10 expression in LPS-stimulated peritoneal macrophages. However, it significantly inhibited the expression of TNF-alpha, IL-1 beta and IL-6 and the phosphorylation of IRAK1 and AKT, and activated NF-kappa B and MAPKs. Treatment with TNBS caused colon shortening; increased myeloperoxidase activity; and increased IL-1 beta, IL-6 and TNF-alpha expression in mice. Oral administration of Lact. brevis G-101 significantly inhibited these activities. Lactobacillus brevis G-101 inhibited TNBS-induced IRAK-1 phosphorylation and NF-kappa B activation, as well as the expression of COX-2 and iNOS. Lactobacillus brevis G-101 inhibited the expression of M1 macrophage markers, but increased the expression of M2 macrophages in the colons of TNBS-treated mice. Conclusions: Lactobacillus brevis G-101 may improve colitis by inhibiting the IRAK1/NF-kappa B, MAPK and AKT pathways and by polarizing M1 macrophages to M2-like macrophages. Significance and Impact of the Study: These results suggest that IL-10 expression-inducing LAB can ameliorate colitis by inhibiting NF-kappa B activation and macrophage polarization.

The uremic toxin indoxyl sulphate enhances macrophage response to LPS.

Abstract: Indoxyl sulphate (IS) is a protein-bound uremic toxin that results from the metabolism of dietary tryptophan normally excreted by kidney through the proximal tubules. Thus the toxin accumulates in the blood of patients with impaired renal function such as in chronic kidney disease (CKD). High IS serum levels in patients with CKD suggest its involvement in CKD progression and in the onset of complications. Its presence in plasma is also a powerful predictor of overall and cardiovascular morbidity/mortality. IS is a well known nephrovascular toxin but very little is known regarding its effects on the immune system and in particular during inflammation. In this study we examined the effect of IS on macrophage activation in response to lipopolysaccharide from E. coli (LPS), a gram negative bacterial endotoxin associated with inflammation and septic shock. To simulate the uremic condition, J774A.1 macrophages were incubated with IS at concentrations observed in uremic patients (1000–62.5 µM) both alone and during LPS challenge. IS alone induced release of reactive oxygen species (ROS), through a mechanism involving pro- and anti-oxidant systems, and alteration in intracellular calcium homeostasis. When added to J774A.1 macrophages in presence of LPS, IS significantly increased the nitric oxide (NO) release, inducible nitric oxide synthase (iNOS) and cycloxygenase-2 (COX-2) expression. IS pre-treatment was also associated with an increase in tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production by macrophages stimulated with LPS. Mechanistic studies revealed that IS increased LPS-induced NF-kB nuclear translocation, ROS release and altered calcium concentrations, mainly because of mitochondrial calcium overloading. Moreover also in primary mouse peritoneal macrophages IS enhances the inflammatory response to LPS increasing ROS, NO, iNOS, COX-2, TNF-α, IL-6 and NF-kB levels. This study provides evidences that IS stimulates macrophage function and enhances inflammatory reasponse associated with LPS, thus contributing to altered immune response dysfunctions observed in CKD.

Risperidone normalizes increased inflammatory parameters and restores anti-inflammatory pathways in a model of neuroinflammation.

Abstract: UNLABELLED Inflammation, caused by both external and endogenous factors, has been implicated as a main pathophysiological feature of chronic mental illnesses, including schizophrenia. An increase in pro-inflammatory cytokines has been described both in experimental models and in schizophrenia patients. However, not much is known about the effects that antipsychotic drugs have on intra- and intercellular mechanisms controlling inflammation. The aim of the present study was to investigate the possible anti-inflammatory effect of a standard schizophrenia treatment not only at the level of soluble mediators, but also at intra- and intercellular inflammatory pathways. The present study was conducted in a model of mild neuroinflammation using a lipopolysaccharide (LPS) challenge that was not an endotoxaemic dose (0.5 mg/kg i.p.) in young adult rats. MAIN RESULTS single doses of risperidone (0.3-3.0 mg/kg i.p.) prevented increased inflammatory parameters induced by LPS in brain cortex [expression of inflammatory cytokines, interleukin (IL)-1β and tumour necrosis factor (TNF)-α, activity of the inducible inflammatory enzymes nitric oxide synthase and cyclooxygenase, p38 mitogen-activated protein kinase (MAPK) and inflammatory nuclear transcription factor κB] and restored anti-inflammatory pathways decreased by LPS challenge (deoxyprostaglandins and peroxisome proliferator activated receptor γ). This is the first study demonstrating that risperidone elicits a preventive effect on the anti-inflammatory arm of the homeostatic mechanism controlling inflammation in a model of mild encephalitis in rats. Our findings suggest a possible protective effect of risperidone on brain cells.

Anti-inflammatory Effects of Oleanolic Acid on LPS-Induced Inflammation In Vitro and In Vivo

Abstract: Oleanolic acid (OA) is a triterpenoid known for its anti-inflammatory and anti-cancer properties; however, the anti-inflammatory effects of OA on lipopolysaccharide (LPS)-mediated pro-inflammatory responses have not been studied. Here, we first investigated the possible anti-inflammatory effects of OA against pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) induced by LPS and the associated signaling pathways. We found that OA inhibited LPS-induced barrier disruption, expression of cell adhesion molecules (CAMs), and adhesion/transendothelial migration of monocytes to HUVECs. OA also suppressed acetic acid-induced hyperpermeability and carboxymethylcellulose-induced leukocyte migration in vivo. Further studies revealed that OA suppressed the production of tumor necrosis factor-α and activation of nuclear factor-κB by LPS. Collectively, these results suggest that OA has anti-inflammatory effects by inhibiting hyperpermeability, the expression of CAMs, and the adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapeutic agent for vascular inflammatory diseases.

Macrophage glucose-6-phosphate dehydrogenase stimulates proinflammatory responses with oxidative stress

Abstract: Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme that regulates cellular redox potential. In this study, we demonstrate that macrophage G6PD plays an important role in the modulation of proinflammatory responses and oxidative stress. The G6PD levels in macrophages in the adipose tissue of obese animals were elevated, and G6PD mRNA levels positively correlated with those of proinflammatory genes. Lipopolysaccharide (LPS) and free fatty acids, which initiate proinflammatory signals, stimulated macrophage G6PD. Overexpression of macrophage G6PD potentiated the expression of proinflammatory and pro-oxidative genes responsible for the aggravation of insulin sensitivity in adipocytes. In contrast, when macrophage G6PD was inhibited or suppressed via chemical inhibitors or small interfering RNA (siRNA), respectively, basal and LPS-induced proinflammatory gene expression was attenuated. Furthermore, macrophage G6PD increased activation of the p38 mitogen-activated protein kinase (MAPK) and NF-κB pathways, which may lead to a vicious cycle of oxidative stress and proinflammatory cascade. Together, these data suggest that an abnormal increase of G6PD in macrophages promotes oxidative stress and inflammatory responses in the adipose tissue of obese animals.

Intestinal anti‐inflammatory activity of luteolin: Role of the aglycone in NF‐κB inactivation in macrophages co‐cultured with intestinal epithelial cells

Abstract: The flavonoid luteolin is reported to exert anti-inflammatory properties. In this study, we investigated whether luteolin inhibits gut inflammation, using in vivo and in vitro inflammation models. In a dextran sulfate sodium (DSS)-induced colitis mouse model, luteolin (20 and 50 mg/kg) significantly ameliorated shortening of colon length and histological score. Immunohistochemical analysis showed that luteolin also significantly inhibited infiltration of macrophages and interferon (IFN)-γ-producing CD4⁺ T cells into the colonic mucosa. Treatment with luteolin also improved IFN-γ mRNA expression in the colon. At the cellular level, a co-culture consisting of intestinal epithelial Caco-2 and macrophage RAW264.7 cells, stimulated with lipopolysaccharide, the addition of luteolin (100 μM) suppressed interleukin (IL)-8 mRNA expression in Caco-2 cells without epithelial monolayer disruption. Expression of tumor necrosis factor (TNF)-α protein and proinflammatory cytokines mRNA (TNF-α, IL-6, and IL-1β) in RAW264.7 cells were also suppressed. HPLC analysis and subsequent cellular assay revealed that aglycone of luteolin was present in the basolateral supernatant of this system at a sufficient concentration to suppress TNF-α production and nuclear factor (NF)-κB activation of RAW264.7 cells. These results suggest that the luteolin aglycones released from the Caco-2 epithelium inhibits NF-κB nuclear translocation in RAW264.7 cells, followed by reduction of TNF-α mRNA expression, which results in downregulation of IL-8 mRNA expression in Caco-2 cells. The mechanism by which aglycone inhibits inflammation is important for understanding the roles of luteolin in diet.