Lipoteichoic acids as a major virulence factor causing inflammatory responses via Toll-like receptor 2

Lipoteichoic acid (LTA), a major cell wall component of Gram-positive bacteria, is associated with various inflammatory diseases ranging from minor skin diseases to severe sepsis. It is known that LTA is recognized by Toll-like receptor 2 (TLR2), leading to the initiation of innate immune responses and further development of adaptive immunity. However, excessive immune responses may result in the inflammatory sequelae that are involved in severe diseases such as sepsis. Although numerous studies have tried to identify the molecular basis for the pathophysiology of Gram-positive bacterial infection, the exact role of LTA during the infection has not been clearly elucidated. This review provides an overview of LTA structure and host recognition by TLR2 that leads to the activation of innate immune responses. Emphasis is placed on differential immunostimulating activities of LTAs of various Gram-positive bacteria at the molecular level.     

CQMUH-011, a novel adamantane sulfonamide compound,inhibits lipopolysaccharide- and D-galactosamine-induced fulminant hepatic failure in mice

CQMUH-011, a novel adamantane sulfonamide compound, was shown to suppress macrophage activation and proliferation in our previous study. However, it is unknown whether CQMUH-011 has anti-inflammatory and hepatoprotective properties. In this study, we investigated the potential effects and mechanisms of CQMUH-011 on lipopolysaccharide (LPS)-induced RAW264.7 cell activation in vitro and LPS- and D-galactosamine (D-GalN)-induced fulminant hepatic failure (FHF) in vivo. The results showed that in RAW264.7 cells challenged by LPS, CQMUH-011 inhibited cell proliferation and induced cell cycle arrest and apoptosis. Furthermore, CQMUH-011 reduced tumor necrosis factor (TNF)-α and interleukin (IL)-1β production and down-regulated the overexpression of toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB induced by LPS in RAW264.7 cells. In vivo, CQMUH-011 reduced serum levels of aspartic aminotransferase and alanine transaminase and improved the mortality and hepatic pathological damage induced by LPS/D-GalN in mice. Moreover, CQMUH-011 significantly inhibited the serum levels of proinflammatory mediators, including TNF-α, IL-6, IL-1β, nitric oxide (NO), and prostaglandin E2 (PGE₂), and down-regulated the protein expression of TLR4, p38 mitogen-activated protein kinases, NF-κB, NF-κB inhibitor α (IκBα), IκB kinase β (IKKβ), cyclooxygenase-2 (COX-2) and inducible NO synthases (iNOS) induced by LPS/D-GalN in mice. In conclusion, these results demonstrated that CQMUH-011 has a notable anti-inflammatory effect and protects mice from LPS/D-GalN-induced FHF and that the molecular mechanisms might be related to the inhibition of the TLR4/NF-κB signaling pathway activation, the subsequent decrease in proinflammatory mediator production, and the inhibition of macrophage activation.     

Vitisin A suppresses LPS-induced NO production by inhibiting ERK,p38, and NF-κB activation in RAW 264.7 cells

Vitisin A, a resveratrol tetramer isolated from Vitis vinifera roots, exhibits antioxidative, anticancer, antiapoptotic, and anti-inflammatory effects. It also inhibits nitric oxide (NO) production. Here, we examined the mechanism by which vitisin A inhibits NO production in lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells. Vitisin A dose dependently inhibited LPS-induced NO production and inducible NO synthase (iNOS) expression. In contrast, the production of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) was not altered by vitisin A. To investigate the signaling pathway for NO inhibition by vitisin A, we examined nuclear factor-κB (NF-κB) activation in the mitogen-activated protein kinase (MAPK) pathway, an inflammation-induced signal pathway in RAW 264.7 cells. Vitisin A inhibited LPS-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 phosphorylation and suppressed LPS-induced NF-κB activation in RAW 264.7 cells. This suggests that vitisin A decreased NO production via downregulation of ERK1/2 and p38 and the NF-κB signal pathway in RAW 264.7 cells.     

Lipoteichoic acid fractions from pathogenic and apathogenic Listeria species and Staphylococcus aureus induce similar amounts of macrophage-derived cytokines

Lipoteichoic acids (LTAs) of pathogenic and apathogenic Listeria species and of Staphylococcus aureus were fractionated and tested for their ability to stimulate production of cytokines (IL-1α, IL-6, TNF-α) in resident peritoneal macrophages (Mϕ) of endotoxin-resistant C3H/HeJ mice using a serum-free medium. For IL-1α and IL-6 there were no detectable differences in the ability of LTA fractions of pathogenic and apathogenic Listeria species and of Staphylococcus aureus. However, LTA-2 fractions of Staphylococcus aureus, which might be less hydrophobic than the LTA-2 fractions of the listeriae-induced lower amounts of TNF-α. Furthermore, the more lipophilic LTA-2 fractions of all LTAs employed were more potent inducers of cytokines than the less lipophilic LTA-1 fractions. The biologic effect of LTAs appears, therefore, to depend mainly on their hydrophobicity.     

Activation of macrophages by polysaccharide isolated from Paecilomyces cicadae through toll-like receptor 4

Paecilomyces cicadae have been reported to have immunomodulatory properties. In this study, we investigated the effect of polysaccharide (PCP) isolated from P. cicadae on the macrophages. PCP increased the production of nitric oxide (NO) and the gene expression of IL-1β, IL-6, and TNF-α in RAW 264.7 cells. To investigate the membrane receptor, we examined the effect of PCP on primary macrophages isolated from wild type C3H/HeN and C3H/HeJ mice having mutant-TLR4. PCP induced NO production and cytokine gene expression in macrophages from C3H/HeN, but not from tlr4-mutated C3H/HeJ mice, which suggests that TLR4 is the membrane receptor for PCP. PCP induced the phosphorylation of ERK, JNK, and p38, and the nuclear translocation of NF-κB p50/p65, which are the main signaling molecules downstream from TLR4. Among them, p38 and NF-κB signaling played a crucial role in PCP-induced NO production by macrophages. These results indicate that PCP activates macrophages through the TLR4 signaling pathway.     

8,8′-Bieckol,isolated from edible brown algae,exerts its anti-inflammatory effects through inhibition of NF-κB signaling and ROS production in LPS-stimulated macrophages

Ecklonia cava (E. cava) is an abundant brown alga that contains high levels of phlorotannins, which are unique marine polyphenolic compounds. It has been suggested that E. cava phlorotannins exert anti-inflammatory effects. However, the anti-inflammatory effects and underlying molecular mechanism exerted by 8,8′-bieckol isolated from E. cava have not been reported. Thus, in this study, we examined the anti-inflammatory effects of 8,8′-bieckol on lipopolysaccharide (LPS)-stimulated primary macrophages and RAW 264.7 macrophages. We found that 8,8′-bieckol suppressed key inflammatory mediator [i.e., nitric oxide (NO) and prostaglandin E₂ (PGE₂)] production in both primary and RAW 264.7 macrophages. 8,8′-Bieckol inhibited NO by suppressing LPS-induced expression of inducible nitric oxide synthase (iNOS) at the mRNA and protein levels in primary macrophages and RAW 264.7 cells. In addition, 8,8′-bieckol decreased the production and mRNA expression of the inflammatory cytokine interleukin-6 (IL-6), but not tumor necrosis factor (TNF)-α, in RAW 264.7 cells. Moreover, 8,8′-bieckol treatment diminished transactivation of nuclear factor-kappa B (NF-κB) and nuclear translocation of the NF-κB p65 subunit and suppressed LPS-induced intracellular reactive oxygen species (ROS) production in macrophages. Furthermore, 8,8′-bieckol markedly reduced mortality in LPS-induced septic mice. Taken together, these data indicate that the anti-inflammatory properties of 8,8′-bieckol are associated with the suppression of NO, PGE₂, and IL-6 via negative regulation of the NF-κB pathway and ROS production in LPS-stimulated RAW 264.7 cells. Moreover, 8,8′-bieckol protects mice from endotoxin shock.     

Fulvic acid promotes extracellular anti-cancer mediators from RAW 264.7 cells,causing to cancer cell death in vitro

Fulvic acid (FA) is known to promote electrochemical balance as a donor or a receptor possessing many biomedical functions. Nevertheless, the effect of FA on the anti-cancer activity has not been elucidated. In the current study, we first isolated FA from humus and investigated whether FA regulates immune-stimulating functions, such as production of nitric oxide (NO), in RAW 264.7 cells. Our data showed that FA slightly enhances cell viability in a dose-dependent manner and secretion of NO from RAW 264.7 cells. It upregulated the protein and mRNA expression of inducible NO synthesis (iNOS). In addition, FA enhanced the DNA-binding activity of nuclear factor-κB (NF-κB) in RAW 264.7 cells; the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) effectively attenuated the expression of FA-stimulated iNOS, suggesting that FA stimulates NF-κB to promote iNOS and NO production. Finally, FA-stimulated culture media (FA-CM) from RAW 264.7 cells were collected and MCA-102 fibrosarcoma cells were cultured in this media. The FA-CM augmented MCA-102 fibrosarcoma cell apoptosis; however, an NO inhibitor N^G-monomethyl-l-arginine (NMMA) slightly inhibited the FA-CM-mediated MCA-102 fibrosarcoma cell apoptosis, which was accompanied by low levels of NO. In the present study, we found that FA induces the generation of NO and iNOS in RAW 264.7 cells by inducing NF-κB activation; however, NO did not significantly stimulate MCA-102 fibrosarcoma cell apoptosis in the current study. In addition, FA-CM enhanced cell death in various human cancer cells such as Hep3B, LNCaP, and HL60. Taken together, FA most likely stimulates immune-modulating molecules such as NO and induces cancer cell apoptosis.     

3‑Bromo‑5‑(ethoxymethyl)‑1,2‑benzenediol inhibits LPS-induced pro-inflammatory responses by preventing ROS production and downregulating NF-κB in vitro and in a zebrafish model

The anti-inflammatory effects of 3‑bromo‑5‑(ethoxymethyl)‑1,2‑benzenediol (BEMB) from Polysiphonia morrowii were evaluated in lipopolysaccharide (LPS)-induced RAW264.7 cells and zebrafish embryo. BEMB showed anti-inflammatory effects by inhibiting the production of nitric oxide (NO) and reactive oxygen species (ROS), and the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in the LPS-activated RAW264.7 cells and zebrafish embryo without cytotoxicity. Moreover, BEMB suppressed the protein and mRNA expression levels of nuclear factor (NF)-κB (p65 and inhibitor of NF-κB [IκB]-A) in RAW264.7 cells and zebrafish embryo, respectively. Collectively, the results of this study indicate that BEMB suppressed the production of pro-inflammatory mediators such as NO, iNOS, and COX-2 as well as their regulation in LPS-induced RAW264.7 cells and zebrafish embryos by inhibiting ROS production and NF-κB expression. Therefore, this study suggests that BEMB could be a potential anti-inflammatory agent for the treatment of inflammatory diseases.     

Okra polysaccharide-2 plays a vital role on the activation of RAW264.7 cells by TLR2/4-mediated signal transduction pathways

Polysaccharide is the main active component of okra (Abelmoschus esculentus L.) and it can effectively stimulate the activation of macrophages. However, the immune regulatory mechanism is still not clear. Therefore, the present study aimed to reveal the possible mechanism by investigating the effect of okra polysaccharide-2 (RPS-2) on Toll-like receptor (TLR) 2/4-mediated signal transduction pathways in RAW264.7 murine macrophage cells. In order to confirm whether RPS-2 stimulated macrophages activation via TLR2 or TLR4, RAW264.7 murine macrophage cells were pretreated with TLR2/4 inhibitors for 1 h before RPS-2 treatment, and then the NO, IL-10, TNF-α levels were tested. The results indicated that both TLR2 and TLR4 were the keys of immune regulatory effect of RPS-2. Afterwards, the effect of RPS-2 on NF-κB and MAPKs signaling pathways were studied by western blot analysis. It showed RPS-2 induced the phosphorylation of p65, IκBα, p38, ERK1/2 and JNK. At the same time, the specific inhibitors reduced these phosphorylation levels as well as NO, IL-10 and TNF-α amounts. In a word, RPS-2 activated macrophages by NF-κB and MAPKs signal transduction pathways.     

Purification and characterization of a nitric oxide inhibitory peptide from Ruditapes philippinarum

Ruditapes philippinarum (R. philippinarum) were hydrolyzed using 8 proteases to produce an anti-inflammatory peptide of the various hydrolysates produced, the Alcalase hydrolysate exhibited the highest nitric oxide (NO) inhibitory activity. The derived peptide was purified using high performance liquid chromatography (HPLC) and NO-inhibitory activity of the purified compound was evaluated. The sequence of the NO-inhibitory peptide obtained was composed of 10 amino acid residues, Gln-Cys-Gln-Gln-Ala-Val-Gln-Ser-Ala-Val at N-terminal position. In addition, we investigated the inhibitory effect of the purified peptide from R. philippinarum on NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In this analysis the purified peptide from R. philippinarum was shown to inhibit LPS-induced NO production in RAW264.7 cells. The present results indicate that the purified peptide displayed potent anti-inflammation activity in RAW264.7 cells.     

Anti-Inflammatory Effect of Mangostenone F in Lipopolysaccharide-Stimulated RAW264.7 Macrophages by Suppressing NF-κB and MAPK Activation

Mangostenone F (MF) is a natural xanthone isolated from Garcinia mangostana. However, little is known about the biological activities of MF. This study was designed to investigate the anti-inflammatory effect and underlying molecular mechanisms of MF in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. MF dose-dependently inhibited the production of NO, iNOS, and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in LPS-stimulated RAW264.7 macrophages. Moreover, MF decreased the NF-κB luciferase activity and NF-κB DNA binding capacity in LPS-stimulated RAW264.7 macrophages. Furthermore, MF suppressed the NF-κB activation by inhibiting the degradation of IκBα and nuclear translocation of p65 subunit of NF-κB. In addition, MF attenuated the AP-1 luciferase activity and phosphorylation of ERK, JNK, and p38 MAP kinases. Taken together, these results suggest that the anti-inflammatory effect of MF is associated with the suppression of NO production and iNOS expression through the down-regulation of NF-κB activation and MAPK signaling pathway in LPS-stimulated RAW264.7 macrophages.     

Anti-inflammatory effect of berkeleyacetal C through the inhibition of interleukin-1 receptor-associated kinase-4 activity

Berkeleyacetal C (BAC) isolated from Penicillium sp. which had isolated from a soil sample collected in Fukushima, inhibited NO production and induction of iNOS protein in RAW264.7 cells stimulated by the Toll-like receptor (TLR) 2 ligand, peptidoglycan (PGN) or TLR4 ligand, lipopolysaccharide (LPS). The other inflammatory mediator production by these stimulators was also suppressed by BAC in a concentration-dependent manner. BAC inhibited LPS- or PGN-activated nuclear translocation of nuclear factor (NF)-κB and MyD88-dependent signaling molecules. However, it showed no effect on LPS-induced nuclear translocation of interferon regulatory factor (IRF)-3, a MyD88-independent signaling molecule. To clarify the mechanistic basis for BAC ability to inhibit translocation of NF-κB and activated MyD88-dependent signaling molecules, we examined interleukin-1 receptor-associated kinase (IRAK)-4, existing to the most upstream on MyD88-dependent signaling molecules, in vitro kinase assay. BAC suppressed IRAK-4 kinase activity in a concentration-dependent manner. These findings suggest that BAC inhibits LPS- and PGN- induced NO production and iNOS expression by decreasing the level of the translocating of NF-κB in nuclear through inhibiting the kinase activity of IRAK-4 in inflammatory cells.     

Immune response mechanism of mouse monocytes/macrophages treated with κ-carrageenan polysaccharide

This study investigated the function of κ-carrageenan polysaccharide in immune regulation. The immune response of RAW 264.7 cells treated with κ-carrageenan polysaccharide was explored by MTT assay, general morphological observation, neutral red phagocytosis assay, Griess method, fluorescence method, and enzyme-linked immunosorbent assay. In addition, TLR4 blocking experiment and double-fluorescence immunostaining were performed on cells to demonstrate their immune response mechanism. Results show that κ-carrageenan polysaccharide not only promotes cell proliferation but also activates RAW 264.7 cells, thereby improving the cells’ phagocytic capability, NO production, and tumor necrosis factor-α (TNF-α) secretion. In addition, the use of TLR4-specific inhibitors can significantly mediate the increased TNF-α secretion induced by κ-carrageenan polysaccharide. The RAW 264.7 cells treated with κ-carrageenan polysaccharide show upregulated TLR4 expression, and the main subunit of NF-κB (p65) is translocated. These results support the immunomodulatory function of κ-carrageenan polysaccharide in RAW 264.7 cells.     

Membrane-damaging activity of Taiwan cobra cardiotoxin 3 is responsible for its bactericidal activity

This study investigates the causal relationship between membrane-damaging activity and bactericidal activity of Naja naja atra (Taiwan cobra) cardiotoxin 3 (CTX3). CTX3 showed greater inhibitory activity for the growth of Staphylococcus aureus (Gram-positive bacteria) relative to that of Escherichia coli (Gram-negative bacteria). The CTX3 antibacterial activity is positively correlated with the increase in membrane permeability of bacterial cells. Morphological examination showed that CTX3 disrupted bacterial membrane integrity.CTX3 showed similar binding capability with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and destabilization of LPS layer and inhibition of LTA biosynthesis on cell wall increased the CTX3 bactericidal effect on E. coli. and S. aureus, respectively. Compared with that of E. coli, CTX3 notably permeabilized model membrane of S. aureus. CTX3 membrane-damaging activity was inhibited by LPS and LTA, while increasing the CTX3 concentration counteracted the inhibitory action of LPS and LTA. Oxidation of Met residues on loop II of CTX3 simultaneously reduced the membrane-permeabilizing activity and bactericidal effect of CTX3. Taken together, our data indicate that CTX3 bactericidal activity depends highly on its ability to induce membrane permeability.     

Artemisia fukudo essential oil attenuates LPS-induced inflammation by suppressing NF-κB and MAPK activation in RAW 264.7 macrophages

In the present study, the chemical constituents of Artemisia fukudo essential oil (AFE) were investigated using GC–MS. The major constituents were α-thujone (48.28%), β-thujone (12.69%), camphor (6.95%) and caryophyllene (6.01%). We also examined the effects of AFE on the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Western blotting and RT-PCR tests indicated that AFE has potent dose-dependent inhibitory effects on pro-inflammatory cytokines and mediators. We investigated the mechanism by which AFE inhibits NO and PGE₂ by examining the level of nuclear factor-κB (NF-κB) activation within the mitogen-activated protein kinase (MAPK) pathway, which is an inflammation-induced signal pathway in RAW 264.7 cells. AFE inhibited LPS-induced ERK, JNK, and p38 phosphorylation. Furthermore, AFE inhibited the LPS-induced phosphorylation and degradation of Iκ-B-α, which is required for the nuclear translocations of the p50 and p65 NF-κB subunits in RAW 264.7 cells. Our results suggest that AFE might exert an anti-inflammatory effect by inhibiting the expression of pro-inflammatory cytokines. Such an effect is mediated by a blocking of NF-κB activation which consequently inhibits the generation of inflammatory mediators in RAW264.7 cells. AFE may be useful for treating inflammatory diseases.     

Water extract of processed Hydrangea macrophylla (Thunb.) Ser. leaf attenuates the expression of pro-inflammatory mediators by suppressing Akt-mediated NF-κB activation

Although Hydrangea macrophylla is native to Northeast Asia and widely cultivated in many parts of the world, no studies on its anti-inflammatory effects have been reported. In this study, we evaluated the anti-inflammatory effect of a water extract of processed H. macrophylla leaf (WH) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. WH inhibited the expression of LPS-stimulated pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E₂ (PGE₂), and tumor necrosis factor-α (TNF-α), as well as their regulatory genes inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α without any accompanying cytotoxicity. Moreover, WH significantly suppressed the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB), as well as the nuclear translocation of the NF-κB subunits, p65 and p50 by suppressing of IκBα phosphorylation and degradation. WH also increased Akt dephosphorylation, leading to the suppression of the DNA-binding activity of NF-κB in LPS-stimulated RAW264.7 macrophage cells. Our results indicate that WH downregulates the expression of pro-inflammatory mediators such as NO, PGE₂, and TNF-α by suppressing the Akt-mediated NF-κB activity in LPS-stimulated RAW264.7 macrophage cells.