Phospholipase Cγ-2 and intracellular calcium are required for lipopolysaccharide-induced Toll-like receptor 4 (TLR4) endocytosis and interferon regulatory factor 3 (IRF3) activation

Toll-like receptor 4 (TLR4) is unique among the TLRs in its use of multiple adaptor proteins leading to activation of both the interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) pathways. Previous work has demonstrated that TLR4 initiates NF-κB activation from the plasma membrane, but that subsequent TLR4 translocation to the endosomes is required for IRF3 activation. Here we have characterized several components of the signaling pathway that governs TLR4 translocation and subsequent IRF3 activation. We find that phospholipase C γ2 (PLCγ2) accounts for LPS-induced inositol 1,4,5-trisphosphate (IP(3)) production and subsequent calcium (Ca(2+)) release. Blockage of PLCγ2 function by inhibitors or knockdown of PLCγ2 expression by siRNAs in RAW 264.7 macrophages lead to reduced IRF3, but enhanced NF-κB activation. In addition, bone marrow-derived macrophages from PLCγ2-deficient mice showed impaired IRF3 phosphorylation and expression of IRF3-regulated genes after LPS stimulation. Using cell fractionation, we show that PLCγ2-IP(3)-Ca(2+) signaling cascade is required for TLR4 endocytosis following LPS stimulation. In conclusion, our results describe a novel role of the PLCγ2-IP(3)-Ca(2+) cascade in the LPS-induced innate immune response pathway where release of intracellular Ca(2+) mediates TLR4 trafficking and subsequent activation of IRF3.     

Eicosapentaenoic acid inhibits TNF-alpha-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

Eicosapentaenoic acid (EPA) is an omega-3 (ω-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-κB activation induced by tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-α-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-α induced MMP-9 expression by NF-κB-dependent pathway. Pretreatment of EPA inhibited TNF-α-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect IκB-α phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-κB. EPA inhibited TNF-α-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKKα-dependent event. Taken together, we demonstrate that EPA inhibits TNF-α-induced MMP-9 expression through inhibition of p38 and Akt activation.     

Conjugated linoleic acid can prevent tumor necrosis factor gene expression by inhibiting nuclear factor binding activity in peripheral blood mononuclear cells from weaned pigs challenged with lipopolysaccharide

Twenty-four weanling barrows were fed corn-soybean diets supplemented with 2% conjugated linolenic acid (CLA) or soybean oil. On day 14 and 21, pigs were injected intraperitoneally with lipopolysaccharide (LPS) or sterile saline. Plasma samples were collected 2h after injection. Peripheral blood mononuclear cells (PBMC) were also collected on day 21, 2 h after injection to determine tumor necrosis factor-alpha (TNF-alpha) production and its mRNA expression. The results indicate that dietary CLA inhibited the production of TNF-alpha by pig PBMC both at the protein and mRNA expression level. In a second experiment, PBMC, collected from a healthy pig, were incubated with either c9,t11-CLA or t10,c12-CLA, or without CLA and stimulated with LPS. Both CLA isomers inhibited LPS-stimulated TNF-alpha production and expression, which may be partially due to inhibition of the binding activity of nuclear factor-kappaB. The t10,c12 isomer was more effective than the c9,t11-CLA isomer in reducing TNF-alpha levels and nuclear factor-kappaB activation.     

Interleukin-10 activates Toll-like receptor 4 and requires MyD88 for cardiomyocyte survival

Toll-like receptors (TLRs) are important in a variety of inflammatory diseases including acute cardiac disorders. TLR4 innate signaling regulates the synthesis of anti-inflammatory cytokine, interleukin-10 (IL-10) upon TLR4 agonists’ re-stimulation. Anti-apoptotic action of IL-10 in cardiac dysfunction is generally accepted but its protective mechanism through TLR4 is not yet understood. We studied the effect of IL-10 in the activation of TLR4 downstream signals leading to cardiomyocytes survival. IL-10 caused a significant increase in the expression of CD14, MyD88 and TLR4. TLR4 activation led to the translocation of the interferon regulatory factor 3 (IRF3) into the nucleus. Phosphorylation of IRF3 enhanced mRNA synthesis for IL-1β but not TNF-α and was elevated even after removal of IL-10 stimulation. Furthermore, degradation of inhibitory kappa B (IκB) kinase (Ikk) suggested that IκBβ was the main activating kinase for IRF3-regulated NF-κB activation and phosphorylation of p65. Phosphorylated NF-κB p65 was translocated into the nucleus. Concomitantly, an increase in Bcl-xL activity inhibited Bax and the proteolytic activity of caspase 3 as well as a decrease in PARP cleavage. An inhibition of MyD88, modulated the above listed responses to IL-10 as there was a decrease in TLR4 and IRF3 and an increase in TNF-α mRNA. This was associated with a decrease in NF-κB p65, Bcl-xL mRNA and protein levels as well as there was an activation of Bax and PARP cleavage independent of caspase 3 activation. These data in cardiomyocytes suggest that IL-10 induced anti-apoptotic signaling involves upregulation of TLR4 through MyD88 activation.     

Glycyrrhizin inhibits lipopolysaccharide-induced inflammatory response by reducing TLR4 recruitment into lipid rafts in RAW264.7 cells

Background

The aim of this study was to investigate the effect of glycyrrhizin on LPS-induced endotoxemia in mice and clarify the possible mechanism.

Methods

An LPS-induced endotoxemia mouse model was used to confirm the anti-inflammatory activity of glycyrrhizin in vivo. In vitro, RAW264.7 cells were stimulated with LPS in the presence or absence of glycyrrhizin. The expression of cytokines was determined by ELISA. Toll-like receptor 4 (TLR4) was determined by Western blot analysis. Nuclear factor-kB (NF-κB) and Interferon regulatory factor 3 (IRF3) activation were detected by Western blotting and luciferase assay. Lipid raft staining was detected by immunocytochemistry.

Results

In vivo, the results showed that glycyrrhizin can improve survival during lethal endotoxemia. In vitro, glycyrrhizin dose-dependently inhibited the expression of TNF-α, IL-6, IL-1β and RANTES in LPS-stimulated RAW264.7 cells. Western blot analysis showed that glycyrrhizin suppressed LPS-induced NF-κB and IRF3 activation. However, glycyrrhizin did not inhibit NF-κB and IRF3 activation induced by MyD88-dependent (MyD88, IKKβ) or TRIF-dependent (TRIF, TBK1) downstream signaling components. Moreover, glycyrrhizin did not affect the expression of TLR4 and CD14 induced by LPS. Significantly, we found that glycyrrhizin decreased the levels of cholesterol of lipid rafts and inhibited translocation of TLR4 to lipid rafts. Moreover, glycyrrhizin activated ABCA1, which could induce cholesterol efflux from lipid rafts.

Conclusion

Glycyrrhizin exerts an anti-inflammatory property by disrupting lipid rafts and inhibiting translocation of TLR4 to lipid rafts, thereby attenuating LPS-mediated inflammatory response.

General significance

Learning the anti-inflammatory mechanism of glycyrrhizin is crucial for the anti-inflammatory drug development.     

Characterization of sparstolonin B, a Chinese herb-derived compound, as a selective Toll-like receptor antagonist with potent anti-inflammatory properties

Blockade of excessive Toll-like receptor (TLR) signaling is a therapeutic approach being actively pursued for many inflammatory diseases. Here we report a Chinese herb-derived compound, sparstolonin B (SsnB), which selectively blocks TLR2- and TLR4-mediated inflammatory signaling. SsnB was isolated from a Chinese herb, Spaganium stoloniferum; its structure was determined by NMR spectroscopy and x-ray crystallography. SsnB effectively inhibited inflammatory cytokine expression in mouse macrophages induced by lipopolysaccharide (LPS, a TLR4 ligand), Pam3CSK4 (a TLR1/TLR2 ligand), and Fsl-1 (a TLR2/TLR6 ligand) but not that by poly(I:C) (a TLR3 ligand) or ODN1668 (a TLR9 ligand). It suppressed LPS-induced cytokine secretion from macrophages and diminished phosphorylation of Erk1/2, p38a, IκBα, and JNK in these cells. In THP-1 cells expressing a chimeric receptor CD4-TLR4, which triggers constitutive NF-κB activation, SsnB effectively blunted the NF-κB activity. Co-immunoprecipitation showed that SsnB reduced the association of MyD88 with TLR4 and TLR2, but not that with TLR9, in HEK293T cells and THP-1 cells overexpressing MyD88 and TLRs. Furthermore, administration of SsnB suppressed splenocyte inflammatory cytokine expression in mice challenged with LPS. These results demonstrate that SsnB acts as a selective TLR2 and TLR4 antagonist by blocking the early intracellular events in the TLR2 and TLR4 signaling. Thus, SssB may serve as a promising lead for the development of selective TLR antagonistic agents for inflammatory diseases.     

The Role of p38 and CK2 Protein Kinases in the Response of RAW 264.7 Macrophages to Lipopolysaccharide

The role of protein kinases p38 and CK2 (casein kinase II) in the response of RAW 264.7 macrophages to the lipopolysaccharide (LPS) from gram-negative bacteria was studied. Using specific p38 and CK2 inhibitors (p38 MAP kinase Inhibitor XI and casein kinase II Inhibitor III, respectively), we investigated the effects of these protein kinases on (i) LPS-induced activation of signaling pathways involving nuclear factor κB (NF-κB), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and interferon regulatory factor 3 (IRF3); (ii) expression of Toll-like receptor 4 (TLR4) and inducible heat-shock proteins HSP72 and HSP90; and (iii) production of interleukins IL-1α, IL-1β, IL-6, tumor necrosis factor α, and IL-10. Activation of the proapoptotic signaling in the macrophages was evaluated from the ratio between the active and inactive caspase-3 forms and p53 phosphorylation. Six hours after LPS addition (2.5 μg/ml) to RAW 264.7 cells, activation of the TLR4 signaling pathways was observed that was accompanied by a significant increase in phosphorylation of IκB kinase α/β, NF-κB (at both Ser536 and Ser276), p38, JNK, and IRF3. Other effects of macrophage incubation with LPS were an increase in the contents of TLR4, inducible heat-shock proteins (HSPs), and pro- and anti-inflammatory cytokines, as well as slight activation of the pro-apoptotic signaling in the cells. Using inhibitor analysis, we found that during the early response of macrophages to the LPS, both CK2 and p38 modulate activation of MAP kinase and NF-κB signaling pathways and p65 phosphorylation at Ser276/Ser536 and cause accumulation of HSP72, HSP90 and the LPS-recognizing receptor TLR4. Suppression of the p38 MAP kinase and CK2 activities by specific inhibitors (Inhibitor XI and Inhibitor III, respectively) resulted in the impairment of the macrophage effector function manifested as a decrease in the production of the early-response proinflammatory cytokines and disbalance between the pro- and anti-apoptotic signaling pathways leading presumably to apoptosis development. Taken together, our data indicate the inefficiency of therapeutic application of p38 and CK2 inhibitors during the early stages of inflammatory response.     

Atorvastatin suppresses LPS-induced rapid upregulation of Toll-like receptor 4 and its signaling pathway in endothelial cells

In the present study, we tested our hypothesis that atorvastatin exerts its anti-inflammation effect via suppressing LPS-induced rapid upregulation of Toll-like receptor 4 (TLR4) mRNA and its downstream p38, ERK, and NF-κB signaling pathways in human umbilical-vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). TLR4 mRNA expression and its downstream kinase activities induced by LPS alone or atorvastatin + LPS in endothelial cells were quantified using quantitative real-time PCR and enzyme-linked immunosorbent assay. Preincubation of LPS-stimulated endothelial cells with TLR4 siRNA was conducted to identify the target of the anti-inflammatory effects of atorvastatin. Atorvastatin incubation resulted in the reduction of LPS-induced TLR4 mRNA expression, ERK1/2 and P38 MAPK phosphorylation, and NF-κB binding activity. Pretreatment with MEK/ERK1/2 inhibitor PD98059 attenuated atorvastatin + LPS-induced NF-κB activity but had no effect on P38 MAPK phosphorylation. In contrast, pretreatment with P38 MAPK inhibitor SB203580 resulted in upregulation of atorvastatin + LPS-induced ERK1/2 phosphorylation but had no significant effects on NF-κB activity. On the other hand, blocking NF-κB with SN50 produced no effects on atorvastatin + LPS-induced ERK1/2 and P38 MAPK phosphorylation. Moreover, TLR4 gene silencing produced the same effects as the atorvastatin treatment. In conclusion, atorvastatin downregulated TLR4 mRNA expression by two distinct signaling pathways. First, atorvastatin stabilized Iκ-Bα, which directly inhibited NF-κB activation. Second, atorvastatin inactivated ERK phosphorylation, which indirectly inhibited NF-κB activation. Suppression of p38 MAPK by atorvastatin upregulates ERK but exerts no effect on NF-κB.     

Lipopolysaccharide induces proliferation and osteogenic differentiation of adipose-derived mesenchymal stromal cells in vitro via TLR4 activation

Multipotent mesenchymal stromal cells (MSC) are capable of multi-lineage differentiation and support regenerative processes. In bacterial infections, resident MSC can come intocontact with and need to react to bacterial components. Lipopolysaccharide (LPS), a typical structure of Gram-negative bacteria, increases the proliferation and osteogenic differentiation of MSC. LPS is usually recognized by the toll-like receptor (TLR) 4 and induces pro-inflammatory reactions in numerous cell types. In this study, we quantified the protein expression of TLR4 and CD14 on adipose-derived MSC (adMSC) in osteogenic differentiation and investigated the effect of TLR4 activation by LPS on NF-κB activation, proliferation and osteogenic differentiation of adMSC. We found that TLR4 is expressed on adMSC whereas CD14 is not, and that osteogenic differentiation induced an increase of the amount of TLR4 protein whereas LPS stimulation did not. Moreover, we could show that NF-κB activation via TLR4 occurs upon LPS treatment. Furthermore, we were able to show that competitive inhibition of TLR4 completely abolished the stimulatory effect of LPS on the proliferation and osteogenic differentiation of adMSC. In addition, the inhibition of TLR4 leads to the complete absence of osteogenic differentiation of adMSC, even when osteogenically stimulated. Thus, we conclude that LPS induces proliferation and osteogenic differentiation of adMSC in vitro through the activation of TLR4 and that the TLR4 receptor seems to play a role during osteogenic differentiation of adMSC.