Escherichia coli Maltose-Binding Protein Induces M1 Polarity of RAW264.7 Macrophage Cells via a TLR2- and TLR4-Dependent Manner

Maltose-binding protein (MBP) is a critical player of the maltose/maltodextrin transport system in Escherichia coli. Our previous studies have revealed that MBP nonspecifically induces T helper type 1 (Th1) cell activation and activates peritoneal macrophages obtained from mouse. In the present study, we reported a direct stimulatory effect of MBP on RAW264.7 cells, a murine macrophage cell line. When stimulated with MBP, the production of nitric oxide (NO), IL-1β, IL-6 and IL-12p70, and the expressions of CD80, MHC class II and inducible nitric oxide synthase (iNOS) were all increased in RAW264.7 cells, indicating the activation and polarization of RAW264.7 cells into M1 macrophages induced by MBP. Further study showed that MBP stimulation upregulated the expression of TLR2 and TLR4 on RAW264.7 cells, which was accompanied by subsequent phosphorylation of IκB-α and p38 MAPK. Pretreatment with anti-TLR2 or anti-TLR4 antibodies largely inhibited the phosphorylation of IκB-α and p38 MAPK, and greatly reduced MBP-induced NO and IL-12p70 production, suggesting that the MBP-induced macrophage activation and polarization were mediated by TLR2 and TLR4 signaling pathways. The observed results were independent of lipopolysaccharide contamination. Our study provides a new insight into a mechanism by which MBP enhances immune responses and warrants the potential application of MBP as an immune adjuvant in immune therapies.     

Anti-Inflammatory Effect of Auranofin on Palmitic Acid and LPS-Induced Inflammatory Response by Modulating TLR4 and NOX4-Mediated NF-κB Signaling Pathway in RAW264.7 Macrophages

Chronic inflammation, which is promoted by the production and secretion of inflammatory mediators and cytokines in activated macrophages, is responsible for the development of many diseases. Auranofin is a Food and Drug Administration-approved gold-based compound for the treatment of rheumatoid arthritis, and evidence suggests that auranofin could be a potential therapeutic agent for inflammation. In this study, to demonstrate the inhibitory effect of auranofin on chronic inflammation, a saturated fatty acid, palmitic acid (PA), and a low concentration of lipopolysaccharide (LPS) were used to activate RAW264.7 macrophages. The results show that PA amplified LPS signals to produce nitric oxide (NO) and various cytokines. However, auranofin significantly inhibited the levels of NO, monocyte chemoattractant protein-1, and pro-inflammatory cytokines, such as interleukin (IL)-1β, tumor necrosis factor-α, and IL-6, which had been increased by co-treatment with PA and LPS. Moreover, the expression of inducible NO synthase, IL-1β, and IL-6 mRNA and protein levels increased by PA and LPS were reduced by auranofin. In particular, the upregulation of NADPH oxidase (NOX) 4 and the translocation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) induced by PA and LPS were suppressed by auranofin. The binding between the toll-like receptor (TLR) 4 and auranofin was also predicted, and the release of NO and cytokines was reduced more by simultaneous treatment with auranofin and TLR4 inhibitor than by auranofin alone. In conclusion, all these findings suggested that auranofin had anti-inflammatory effects in PA and LPS-induced macrophages by interacting with TLR4 and downregulating the NOX4-mediated NF-κB signaling pathway.     

Computational-Driven Epitope Verification and Affinity Maturation of TLR4-Targeting Antibodies

Toll-like receptor (TLR) signaling plays a critical role in the induction and progression of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematous, experimental autoimmune encephalitis, type 1 diabetes mellitus and neurodegenerative diseases. Deciphering antigen recognition by antibodies provides insights and defines the mechanism of action into the progression of immune responses. Multiple strategies, including phage display and hybridoma technologies, have been used to enhance the affinity of antibodies for their respective epitopes. Here, we investigate the TLR4 antibody-binding epitope by computational-driven approach. We demonstrate that three important residues, i.e., Y328, N329, and K349 of TLR4 antibody binding epitope identified upon in silico mutagenesis, affect not only the interaction and binding affinity of antibody but also influence the structural integrity of TLR4. Furthermore, we predict a novel epitope at the TLR4-MD2 interface which can be targeted and explored for therapeutic antibodies and small molecules. This technique provides an in-depth insight into antibody–antigen interactions at the resolution and will be beneficial for the development of new monoclonal antibodies. Computational techniques, if coupled with experimental methods, will shorten the duration of rational design and development of antibody therapeutics.     

The effect of toll-like receptor 4 on macrophage cytokines during endotoxin induced uveitis

Toll-like receptor 4 (TLR4) signal activation of macrophages can lead to endotoxin-induced uveitis (EIU). Previously, our research group has demonstrated a higher expression of TLR4 in vivo during EIU than normal. In this study, we analyzed levels of peritoneal macrophage cytokines from C3H/HeN mice with LPS stimulation in vitro to elucidate the effect of TLR4 on cytokines during EIU.     

Pharmacological Effects of Polyphenol Phytochemicals on the Intestinal Inflammation via Targeting TLR4/NF-κB Signaling Pathway

TLR4/NF-κB is a key inflammatory signaling transduction pathway, closely involved in cell differentiation, proliferation, apoptosis, and pro-inflammatory response. Toll like receptor 4 (TLR4), the first mammalian TLR to be characterized, is the innate immune receptor that plays a key role in inflammatory signal transductions. Nuclear factor kappa B (NF-κB), the TLR4 downstream, is the key to accounting for the expression of multiple genes involved in inflammatory responses, such as pro-inflammatory cytokines. Inflammatory bowel disease (IBD) in humans is a chronic inflammatory disease with high incidence and prevalence worldwide. Targeting the TLR4/NF-κB signaling pathway might be an effective strategy to alleviate intestinal inflammation. Polyphenol phytochemicals have shown noticeable alleviative effects by acting on the TLR4/NF-κB signaling pathway in intestinal inflammation. This review summarizes the pharmacological effects of more than 20 kinds of polyphenols on intestinal inflammation via targeting the TLR4/NF-κB signaling pathway. We expected that polyphenol phytochemicals targeting the TLR4/NF-κB signaling pathway might be an effective approach to treat IBD in future clinical research applications.     

Hybrid Cells Derived from Human Breast Cancer Cells and Human Breast Epithelial Cells Exhibit Differential TLR4 and TLR9 Signaling

TLRs are important receptors of cells of the innate immune system since they recognize various structurally conserved molecular patterns of different pathogens as well as endogenous ligands. In cancer, the role of TLRs is still controversial due to findings that both regression and progression of tumors could depend on TLR signaling. In the present study, M13SV1-EGFP-Neo human breast epithelial cells, MDA-MB-435-Hyg human breast cancer cells and two hybrids M13MDA435-1 and -3 were investigated for TLR4 and TLR9 expression and signaling. RT-PCR data revealed that LPS and CpG-ODN induced the expression of pro-inflammatory cytokines, like IFN-β, TNF-α, IL-1β and IL-6 in hybrid cells, but not parental cells. Interestingly, validation of RT-PCR data by Western blot showed detectable protein levels solely after LPS stimulation, suggesting that regulatory mechanisms are also controlled by TLR signaling. Analysis of pAKT and pERK1/2 levels upon LPS and CpG-ODN stimulation revealed a differential phosphorylation pattern in all cells. Finally, the migratory behavior of the cells was investigated showing that both LPS and CpG-ODN potently blocked the locomotory activity of the hybrid cells in a dose-dependent manner. In summary, hybrid cells exhibit differential TLR4 and TLR9 signaling.     

PCSK9 Induces Tissue Factor Expression by Activation of TLR4/NFkB Signaling

Proprotein convertase subtilisin kexin 9 (PCSK9) increases LDL cholesterol (C) concentration by accelerating the hepatic degradation of the LDL receptor (R) thus promoting atherogenesis. The molecule, however, also exerts proinflammatory effects independent of circulating LDL-C by enhancing local cytokine production and activation of NFkB, a process that might involve Toll-like receptor 4 (TLR4), a crucial component of the innate immunity system. Tissue factor (TF), a glycoprotein which plays an essential role in coagulation and inflammation, is rapidly induced by circulating monocytes stimulated by proinflammatory agents through NFkB-dependent mechanisms. The aims of our study were (1) to assess whether PCSK9 may induce monocytic TF expression and (2) to evaluate whether the TLR4/NFkB signaling pathway may contribute to that effect. Experiments were carried out in peripheral blood mononuclear cells (PBMCs), THP-1 cells, and HEK293 cells transfected with plasmids encoding the human TLR4 complex. PCSK9 increased procoagulant activity (PCA), mRNA and TF protein expression in both PBMCs and THP-1 cultures. Pre-treatment with inhibitors of TLR4/NFkB signaling such as LPS-RS, CLI-095, and BAY 11-7082, downregulated PCSK9-induced TF expression. A similar effect was obtained by incubating cell cultures with anti-PCSK9 human monoclonal antibody. In TLR4-HEK293 cells, PCSK9 activated the TLR4/NFkB signaling pathway to an extent comparable to LPS, the specific agonist of TLR4s and quantitative confocal microscopy documented the colocalization of PCSK9 and TLR4s. In conclusion, PCSK9 induces TF expression through activation of TLR4/NFkB signaling.     

Oxy210, a Semi-Synthetic Oxysterol,Exerts Anti-Inflammatory Effects in Macrophages via Inhibition of Toll-like Receptor (TLR) 4 and TLR2 Signaling and Modulation of Macrophage Polarization

Inflammatory responses by the innate and adaptive immune systems protect against infections and are essential to health and survival. Many diseases including atherosclerosis, osteoarthritis, rheumatoid arthritis, psoriasis, and obesity involve persistent chronic inflammation. Currently available anti-inflammatory agents, including non-steroidal anti-inflammatory drugs, steroids, and biologics, are often unsafe for chronic use due to adverse effects. The development of effective non-toxic anti-inflammatory agents for chronic use remains an important research arena. We previously reported that oral administration of Oxy210, a semi-synthetic oxysterol, ameliorates non-alcoholic steatohepatitis (NASH) induced by a high-fat diet in APOE*3-Leiden.CETP humanized mouse model of NASH and inhibits expression of hepatic and circulating levels of inflammatory cytokines. Here, we show that Oxy210 also inhibits diet-induced white adipose tissue inflammation in APOE*3-Leiden.CETP mice, evidenced by the inhibition of adipose tissue expression of IL-6, MCP-1, and CD68 macrophage marker. Oxy210 and related analogs exhibit anti-inflammatory effects in macrophages treated with lipopolysaccharide in vitro, mediated through inhibition of toll-like receptor 4 (TLR4), TLR2, and AP-1 signaling, independent of cyclooxygenase enzymes or steroid receptors. The anti-inflammatory effects of Oxy210 are correlated with the inhibition of macrophage polarization. We propose that Oxy210 and its structural analogs may be attractive candidates for future therapeutic development for targeting inflammatory diseases.     

Maternal Neutrophil Depletion Fails to Avert Systemic Lipopolysaccharide-Induced Early Pregnancy Defects in Mice

Maternal infection-induced early pregnancy complications arise from perturbation of the immune environment at the uterine early blastocyst implantation site (EBIS), yet the underlying mechanisms remain unclear. Here, we demonstrated in a mouse model that the progression of normal pregnancy from days 4 to 6 induced steady migration of leukocytes away from the uterine decidual stromal zone (DSZ) that surrounds the implanted blastocyst. Uterine macrophages were found to be CD206⁺ M2-polarized. While monocytes were nearly absent in the DSZ, DSZ cells were found to express monocyte marker protein Ly6C. Systemic endotoxic lipopolysaccharide (LPS) exposure on day 5 of pregnancy led to: (1) rapid (at 2 h) induction of neutrophil chemoattractants that promoted huge neutrophil infiltrations at the EBISs by 24 h; (2) rapid (at 2 h) elevation of mRNA levels of MyD88, but not Trif, modulated cytokines at the EBISs; and (3) dose-dependent EBIS defects by day 7 of pregnancy. Yet, elimination of maternal neutrophils using anti-Ly6G antibody prior to LPS exposure failed to avert LPS-induced EBIS defects allowing us to suggest that activation of Tlr4-MyD88 dependent inflammatory pathway is involved in LPS-induced defects at EBISs. Thus, blocking the activation of the Tlr4-MyD88 signaling pathway may be an interesting approach to prevent infection-induced pathology at EBISs.     

Resveratrol Attenuates Acute Inflammatory Injury in Experimental Subarachnoid Hemorrhage in Rats via Inhibition of TLR4 Pathway

Toll-like receptor 4 (TLR4) has been proven to play a critical role in neuroinflammation and to represent an important therapeutic target following subarachnoid hemorrhage (SAH). Resveratrol (RSV), a natural occurring polyphenolic compound, has a powerful anti-inflammatory property. However, the underlying molecular mechanisms of RSV in protecting against early brain injury (EBI) after SAH remain obscure. The purpose of this study was to investigate the effects of RSV on the TLR4-related inflammatory signaling pathway and EBI in rats after SAH. A prechiasmatic cistern SAH model was used in our experiment. The expressions of TLR4, high-mobility group box 1 (HMGB1), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) were evaluated by Western blot and immunohistochemistry. The expressions of Iba-1 and pro-inflammatory cytokines in brain cortex were determined by Western blot, immunofluorescence staining, or enzyme-linked immunosorbent assay. Neural apoptosis, brain edema, and neurological function were further evaluated to investigate the development of EBI. We found that post-SAH treatment with RSV could markedly inhibit the expressions of TLR4, HMGB1, MyD88, and NF-κB. Meanwhile, RSV significantly reduced microglia activation, as well as inflammatory cytokines leading to the amelioration of neural apoptosis, brain edema, and neurological behavior impairment at 24 h after SAH. However, RSV treatment failed to alleviate brain edema and neurological deficits at 72 h after SAH. These results indicated that RSV treatment could alleviate EBI after SAH, at least in part, via inhibition of TLR4-mediated inflammatory signaling pathway.     

A Novel 1,8-Naphthyridine-2-Carboxamide Derivative Attenuates Inflammatory Responses and Cell Migration in LPS-Treated BV2 Cells via the Suppression of ROS Generation and TLR4/Myd88/NF-κB Signaling Pathway

Novel 1,8-naphthyridine-2-carboxamide derivatives with various substituents (HSR2101-HSR2113) were synthesized and evaluated for their effects on the production of pro-inflammatory mediators and cell migration in lipopolysaccharide (LPS)-treated BV2 microglial cells. Among the tested compounds, HSR2104 exhibited the most potent inhibitory effects on the LPS-stimulated production of inflammatory mediators, including nitric oxide (NO), tumor necrosis factor-α, and interleukin-6. Therefore, this compound was chosen for further investigation. We found that HSR2104 attenuated levels of inducible NO synthase and cyclooxygenase 2 in LPS-treated BV2 cells. In addition, it markedly suppressed LPS-induced cell migration as well as the generation of intracellular reactive oxygen species (ROS). Moreover, HSR2104 abated the LPS-triggered nuclear translocation of nuclear factor-κB (NF-κB) through inhibition of inhibitor kappa Bα phosphorylation. Furthermore, it reduced the expressions of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) in LPS-treated BV2 cells. Similar results were observed with TAK242, a specific inhibitor of TLR4, suggesting that TLR4 is an upstream regulator of NF-κB signaling in BV2 cells. Collectively, our findings demonstrate that HSR2104 exhibits anti-inflammatory and anti-migratory activities in LPS-treated BV2 cells via the suppression of ROS and TLR4/MyD88/NF-κB signaling pathway. Based on our observations, HSR2104 may have a beneficial impact on inflammatory responses and microglial cell migration involved in the pathogenesis of various neurodegenerative disorders.     

Berteroin Present in Cruciferous Vegetables Exerts Potent Anti-Inflammatory Properties in Murine Macrophages and Mouse Skin

Berteroin (5-methylthiopentyl isothiocyanate) is a sulforaphane analog present in cruciferous vegetables, including Chinese cabbage, rucola salad leaves, and mustard oil. We examined whether berteroin exerts anti-inflammatory activities using lipopolysaccharide (LPS)-stimulated Raw 264.7 macrophages and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin inflammation models. Berteroin decreased LPS-induced release of inflammatory mediators and pro-inflammatory cytokines in Raw 264.7 macrophages. Berteroin inhibited LPS-induced degradation of inhibitor of κBα (IκBα) and nuclear factor-κB p65 translocation to the nucleus and DNA binding activity. Furthermore, berteroin suppressed degradation of IL-1 receptor-associated kinase and phosphorylation of transforming growth factor β activated kinase-1. Berteroin also inhibited LPS-induced phosphorylation of p38 MAPK, ERK1/2, and AKT. In the mouse ear, berteroin effectively suppressed TPA-induced edema formation and down-regulated iNOS and COX-2 expression as well as phosphorylation of AKT and ERK1/2. These results demonstrate that berteroin exhibits potent anti-inflammatory properties and suggest that berteroin can be developed as a skin anti-inflammatory agent.     

TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function

Migration of vascular smooth muscle cells (VSMCs) into the intima is considered to be a vital event in the pathophysiology of atherosclerosis. Despite substantial evidence supporting the pathogenic role of Toll-like receptor 4 (TLR4) in the progression of atherogenesis, its function in the regulation of VSMC migration remains unclear. The goal of the present study was to elucidate the mechanism by which TLR4 regulates VSMC migration. Inhibitor experiments revealed that TLR4-induced IL-6 secretion and VSMC migration were mediated via the concerted actions of MyD88 and TRIF on the activation of p38 MAPK and ERK1/2 signaling. Neutralizing anti-IL-6 antibodies abrogated TLR4-driven VSMC migration and F-actin polymerization. Blockade of p38 MAPK or ERK1/2 signaling cascade inhibited TLR4 agonist-mediated activation of cAMP response element binding protein (CREB). Moreover, siRNA-mediated suppression of CREB production repressed TLR4-induced IL-6 production and VSMC migration. Rac-1 inhibitor suppressed TLR4-driven VSMC migration but not IL-6 production. Importantly, the serum level of IL-6 and TLR4 endogenous ligand HMGB1 was significantly higher in patients with coronary artery diseases (CAD) than in healthy subjects. Serum HMGB1 level was positively correlated with serum IL-6 level in CAD patients. The expression of both HMGB1 and IL-6 was clearly detected in the atherosclerotic tissue of the CAD patients. Additionally, there was a positive association between p-CREB and HMGB1 in mouse atherosclerotic tissue. Based on our findings, we concluded that, upon ligand binding, TLR4 activates p38 MAPK and ERK1/2 signaling through MyD88 and TRIF in VSMCs. These signaling pathways subsequently coordinate an additive augmentation of CREB-driven IL-6 production, which in turn triggers Rac-1-mediated actin cytoskeleton to promote VSMC migration.     

Oleacein Attenuates Lipopolysaccharide-Induced Inflammation in THP-1-Derived Macrophages by the Inhibition of TLR4/MyD88/NF-κB Pathway †

It is known that plant phenolic compounds exert anti-inflammatory activity through both anti-oxidant effects and modulation of pivotal pro-inflammatory factors. Recently, Olea europaea has been studied as a natural source of bioactive molecules; however, few studies have focused on the biological effect of oleacein (OLC), the most abundant secoiridoid. Therefore, the aim of this study was to investigate the potential anti-oxidant activity of OLC, as well as to study its anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated THP-1-derived macrophages. LPS brought a dramatic increase of both release and gene expression of pro-inflammatory cytokines (IL-6, IL-1β and TNF-α), as well as a decrease of anti-inflammatory ones (IL-10), the effects of which are reverted by OLC. Moreover, it reduced the levels of COX-2, NO and PGE₂ elicited by LPS exposure in THP-1 macrophages. Interestingly, OLC modulated inflammatory signaling pathways through the inhibition of CD14/TLR4/CD14/MyD88 axis and the activation of NF-κB. Finally, OLC showed relevant anti-oxidant capability, assessed by abiotic assays, and reduced the intracellular amount of ROS generated by LPS exposure in THP-1 macrophages. Overall, these results suggest that the anti-oxidant activity and anti-inflammatory effect of OLC may cooperate in its protective effect against inflammatory stressors, thus being a possible alternative pharmacological strategy aimed at reducing the inflammatory process.     

云芝糖肽对乳腺癌患者外周血单个核细胞Toll样受体4的作用

目的探讨云芝糖肽(polysaccharopeptide,PSP)对乳腺癌患者外周血单个核细胞(peripheral blood mononuclearcell,PBMC)Toll样受体4(Toll-like receptor 4,TLR4)的作用。方法体外分离乳腺癌患者或健康人PBMC,并将其分别分为PSP组(分别加入终浓度为25μg/ml的PSP和终浓度为100μg/ml的PHA)和对照组(只加入终浓度为100μg/ml的PHA),采用TLR4单克隆抗体对各组细胞进行免疫荧光染色。将乳腺癌患者PBMC分为空白对照组、TLR4抗体组、PSP组和PSP+TLR4抗体组,采用Q-PCR检测各组PBMC IL-12、IL-6和TNF-α的表达水平。结果健康志愿者对照组PBMC荧光强度较强,而健康志愿者PSP组PBMC荧光强度较弱;乳腺癌患者PSP组与乳腺癌患者对照组相比,细胞荧光强度更弱。与空白对照组比较,TLR4抗体组PBMC IL-12和组TNF-α的表达水平均显著降低(P<0.05),PSP组PBMC IL-12、IL-6和TNF-α的表达水平均显著上调(P<0.05或<0.01);与PSP和TLR4抗体组比较,PSP+TLR4抗体组PBMC IL-12、IL-6和TNF-α的表达水平均显著上调(P<0.05或<0.01)。结论 TLR4可能是PSP的作用受体之一。     

激活素A对巨噬细胞系RAW264.7细胞吞饮活性及TLR4表达的双向调节作用

目的探讨激活素A(ActivinA)对巨噬细胞系RAW264.7细胞活性的调节作用及其可能的机制。方法取对数生长期的小鼠巨噬细胞系RAW264.7细胞,加入1μg/ml脂多糖(LPS),继续培养8h,采用ELISA法检测细胞分泌ActivinA水平;分别加入ActivinA、LPS和ActivinA+LPS,中性红染料法检测细胞吞饮活性;流式细胞术分析细胞表面分子MHCⅠ、MHCⅡ及Toll样受体4(TLR4)的表达水平。结果LPS呈时间依赖性刺激RAW264.7细胞分泌ActivinA;ActivinA可明显促进静息RAW264.7细胞的吞饮活性,而对MHCⅠ、MHCⅡ及TLR4的表达水平无明显影响;ActivinA和LPS共同作用,ActivinA明显抑制了LPS活化的RAW264.7细胞的吞饮活性,并下调TLR4的表达。结论ActivinA可能以自分泌/旁分泌形式参与巨噬细胞活性调节,其抑制LPS作用与TLR4表达有关。     

Cassiaside C Inhibits M1 Polarization of Macrophages by Downregulating Glycolysis

Classically activated M1 macrophages reprogram their metabolism towards enhanced glycolysis to obtain energy and produce pro-inflammatory cytokines after activation by mammalian target of rapamycin complex 1 (mTORC1) and hypoxia-inducible factor (HIF)-1α. Thus, a strategy that constrains M1 polarization of macrophages via downregulation of glycolysis is essential for treating chronic inflammatory diseases. Cassiae semen has pharmacological activity against various inflammatory diseases. However, it is unclear whether specific compounds within Cassia seeds affect M1 polarization of macrophages. Here, we investigated whether Cassiaside C napthopyrone from Cassiae semen inhibits M1 polarization by downregulating glycolysis. We found that Cassiaside C reduced expression of inducible nitric oxide synthase and cyclooxygenase-2 and the phosphorylation of nuclear factor kappa B, all of which are upregulated in lipopolysaccharide (LPS)/interferon (IFN)-γ-treated Raw264.7 cells and peritoneal macrophages. Moreover, Cassiaside C-treated macrophages showed marked suppression of LPS/IFN-γ-induced HIF-1α, pyruvate dehydrogenase kinase 1, and lactate dehydrogenase A expression, along with downregulation of the phosphoinositide 3-kinases (PI3K)/AKT/mTORC1 signaling pathway. Consequently, Cassiaside C attenuated enhanced glycolysis and lactate production, but rescued diminished oxidative phosphorylation, in M1 polarized macrophages. Thus, Cassiaside C dampens M1 polarization of macrophages by downregulating glycolysis, which could be exploited as a therapeutic strategy for chronic inflammatory conditions.     

Exopolysaccharide Isolated from Lactobacillus plantarum L-14 Has Anti-Inflammatory Effects via the Toll-Like Receptor 4 Pathway in LPS-Induced RAW 264.7 Cells

Inflammation is a biological response of the immune system to defend the body from negative stimulation. However, the excessive inflammatory response can damage host tissues and pose serious threats. Exopolysaccharide (EPS), one of the postbiotics, is secreted from lactic acid bacteria. Although many studies have described the beneficial effects of EPS, such as its anti-inflammatory and anti-oxidant effects, its underlying mechanisms have remained to be poorly understood. Thus, we identified that EPS obtained from Lactobacillus plantarum L-14 was a homogeneous polysaccharide primarily comprised of glucose. To examine these anti-inflammatory effects, an inflammatory response was induced by lipopolysaccharide (LPS) administration to mouse macrophage RAW 264.7 cells that were pretreated with EPS. The anti-inflammatory effects of EPS were identified by analyzing the changes within inflammatory markers at the molecular level. We demonstrate here that EPS suppressed proinflammatory mediators, such as cyclooxygenase-2, interleukin-6, tumor necrosis factor-α, and interleukin-1β, and downregulated the expression of an inducible nitric oxide synthase known to lead to oxidative stress. It was also confirmed that EPS had anti-inflammatory effects by blocking the interaction of LPS with Toll-like receptor 4 (TLR4), as demonstrated by using the known TLR4 inhibitor TAK-242. In addition, we found that EPS itself could suppress the expression of TLR4. Consequently, our data suggest that EPS can be a potential target for the development of natural product-derived medicine for treating inflammatory diseases related to TLR4.