TLR4 Dimerization Research Articles

Ginsenoside Rh2 Alleviates LPS-Induced Inflammatory Responses by Binding to TLR4/MD-2 and Blocking TLR4 Dimerization.

Lipopolysaccharide (LPS) triggers a severe systemic inflammatory reaction in mammals, with the dimerization of TLR4/MD-2 upon LPS stimulation serving as the pivotal mechanism in the transmission of inflammatory signals. Ginsenoside Rh2 (G-Rh2), one of the active constituents of red ginseng, exerts potent anti-inflammatory activity. However, whether G-Rh2 can block the TLR4 dimerization to exert anti-inflammatory effects remains unclear. Here, we first investigated the non-cytotoxic concentration of G-Rh2 on RAW 264.7 cells, and detected the releases of pro-inflammatory cytokines in LPS-treated RAW 264.7 cells, and then uncovered the mechanisms involved in the anti-inflammatory activity of G-Rh2 through flow cytometry, fluorescent membrane localization, Western blotting, co-immunoprecipitation (Co-IP), molecular docking and surface plasmon resonance (SPR) analysis in LPS-stimulated macrophages. Our results show that G-Rh2 stimulation markedly inhibited the secretion of LPS-induced interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and nitric oxide (NO). Additionally, G-Rh2 blocked the binding of LPS with the membrane of RAW 264.7 cells through direct interaction with TLR4 and MD-2 proteins, leading to the disruption of the dimerization of TLR4 and MD-2, followed by suppression of the TLR4/NF-κB signaling pathway. Our results suggest that G-Rh2 acts as a new inhibitor of TLR4 dimerization and may serve as a promising therapeutic agent against inflammation.

In Silico Analyses Indicate a Lower Potency for Dimerization of TLR4/MD-2 as the Reason for the Lower Pathogenicity of Omicron Compared to Wild-Type Virus and Earlier SARS-CoV-2 Variants.

The SARS-CoV-2 Omicron variants have replaced all earlier variants, due to increased infectivity and effective evasion from infection- and vaccination-induced neutralizing antibodies. Compared to earlier variants of concern (VoCs), the Omicron variants show high TMPRSS2-independent replication in the upper airway organs, but lower replication in the lungs and lower mortality rates. The shift in cellular tropism and towards lower pathogenicity of Omicron was hypothesized to correlate with a lower toll-like receptor (TLR) activation, although the underlying molecular mechanisms remained undefined. In silico analyses presented here indicate that the Omicron spike protein has a lower potency to induce dimerization of TLR4/MD-2 compared to wild type virus despite a comparable binding activity to TLR4. A model illustrating the molecular consequences of the different potencies of the Omicron spike protein vs. wild-type spike protein for TLR4 activation is presented. Further analyses indicate a clear tendency for decreasing TLR4 dimerization potential during SARS-CoV-2 evolution via Alpha to Gamma to Delta to Omicron variants.

Pterostilbene participates in TLR4- mediated inflammatory response and autophagy-dependent Aβ1–42 endocytosis in Alzheimer's disease

BackgroundAlzheimer's disease (AD), the most prevalent form of dementia, remains untreatable. One of the factors that contributes to its progression is microglia-mediated inflammation. Pterostilbene, a compound isolated from Chinese dragon's blood, can reduce inflammation caused by overactive microglia. However, its effects on AD transgenic animals and the possible underlying mechanism remain unknown. MethodsWe evaluated the effect of pterostilbene on learning and memory difficulties in transgenic APP/PS1 mice. We used immunofluorescence to detect microglial activation and Aβ aggregation. We explored the cellular mechanism of pterostilbene by establishing LPS- stimulated BV2 cells and oAβ1–42- exposed HEK 293T cells that overexpress TLR4 and/or MD2 via lentivirus. We applied flow cytometry and immunoprecipitation to examine how pterostilbene regulates TLR4 signaling. ResultsPterostilbene enhanced the learning and memory abilities of APP/PS1 mice and reduced microglial activation and Aβ aggregation in their hippocampus. Pterostilbene alleviated oAβ1–42-induced inflammation, which required the involvement of MD2. Pterostilbene disrupted the binding between TLR4 and MD2, which may further prevent TLR4 dimerization and subsequent inflammatory response. Moreover, pterostilbene restored the impaired endocytosis of oAβ1–42 through an autophagy-dependent mechanism. ConclusionThis is the first demonstration that pterostilbene can potentially treat AD by blocking the interaction of TLR4 and MD2, thereby suppressing TLR4-mediated inflammation.

Piperlongumine mitigates LPS-induced inflammation and lung injury via targeting MD2/TLR4

PurposeAcute lung injury (ALI) is a fatal acute inflammatory illness with restricted therapeutic choices clinically. Piperlongumine (PL) is recognized as an alkaloid separated from Piper longum L, which was suggested to exhibit multiple pharmacological activities (e.g., anti-inflammatory activity). However, the effects of PL on LPS-triggered ALI and its anti-inflammatory target remain unclear. This paper intended to assess the roles of PL in LPS-triggered ALI, as well as its underlying mechanism and target. MethodsIn vivo, ALI was induced by intratracheal injection of LPS to evaluate protective effects of PL and assessed by the changes of histopathological. In vitro, the anti-inflammatory activity and mechanism of PL were investigated by ELISA, RT-qPCR, transcription factor enrichment analysis, Western blotting and Immunofluorescence assay. The binding affinity of PL to MD2 was analyzed using computer docking, surface plasmon resonance, ELISA and immunoprecipitation assay. ResultsIt was reported here that PL treatment alleviated LPS-induced pulmonary damage, inflammatory cells infiltration and inflammatory response in mice. In culture cells, PCR array showed that PL significantly inhibited LPS-induced inflammatory cytokines, chemokines, and type I IFNs genetic expression, along with the inhibition of TAK1 and TBK1 pathway. It is noteworthy that PL is capable of straightly binding to MD2 and inhibiting MD2/TLR4 complex formation and TLR4 dimerization. ConclusionsAs revealed from this study, PL directly binding to MD2 to block cytokines expression by inhibiting the activation of TAK1 and TBK1 pathway, which then exerted its pulmonary protective activity. Accordingly, PL may act as an underlying candidate for treating LPS-triggered ALI.

Hederacoside C ameliorates colitis via restoring impaired intestinal barrier through moderating S100A9/MAPK and neutrophil recruitment inactivation.

Hederacoside C (HSC) has attracted much attention as a novel modulator of inflammation, but its anti-inflammatory mechanism remains elusive. In the present study, we investigated how HSC attenuated intestinal inflammation in vivo and in vitro. HSC injection significantly alleviated TNBS-induced colitis by inhibiting pro-inflammatory cytokine production and colonic epithelial cell apoptosis, and partially restored colonic epithelial cell proliferation. The therapeutic effect of HSC injection was comparable to that of oral administration of mesalazine (200 mg·kg−1·d−1, i.g.). In LPS-stimulated human intestinal epithelial Caco-2 cells, pretreatment with HSC (0.1, 1, 10 μM) significantly inhibited activation of MAPK/NF-κB and its downstream signaling pathways. Pretreatment with HSC prevented LPS-induced TLR4 dimerization and MyD88 recruitment in vitro. Quantitative proteomic analysis revealed that HSC injection regulated 18 proteins in the colon samples, mainly clustered in neutrophil degranulation. Among them, S100A9 involved in the degranulation of neutrophils was one of the most significantly down-regulated proteins. HSC suppressed the expression of S100A9 and its downstream genes including TLR4, MAPK, and NF-κB axes in colon. In Caco-2 cells, recombinant S100A9 protein activated the MAPK/NF-κB signaling pathway and induced inflammation, which were ameliorated by pretreatment with HSC. Notably, HSC attenuated neutrophil recruitment and degranulation as well as S100A9 release in vitro and in vivo. In addition, HSC promoted the expression of tight junction proteins and repaired the epithelial barrier via inhibiting S100A9. Our results verify that HSC ameliorates colitis via restoring impaired intestinal barrier through moderating S100A9/MAPK and neutrophil recruitment inactivation, suggesting that HSC is a promising therapeutic candidate for colitis.

Rotundic acid reduces LPS-induced acute lung injury in vitro and in vivo through regulating TLR4 dimer.

Acute lung injury (ALI) is a serious clinical disease. Rotundic acid (RA), a natural ingredient isolated from Ilex rotunda Thunb, exhibits multiple pharmacological activities. However, RA's therapeutic effect and mechanism on ALI remain to be elucidated. The present study aimed to further clarify its regulating effects on inflammation in vitro and in vivo. Our results indicated that RA significantly inhibited the overproduction of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). RA decreased ROS production and calcium influx. In addition, RA inhibited the activation of PI3K, MAPK, and NF-κB pathways and enhanced the activity of nuclear factor E2-related factor 2 (Nrf2) signaling. The cellular thermal shift assay and docking results indicated that RA bind to TLR4 to block TLR4 dimerization. Furthermore, RA pretreatment effectively inhibited ear edema induced by xylene and LPS-induced endotoxin death and had a protective effect on LPS-induced ALI. Our findings collectively indicated that RA has anti-inflammatory effects, which may serve as a potential therapeutic option for pulmonary inflammation.

Oleuropein Attenuates Lipopolysaccharide-Induced Acute Kidney Injury In Vitro and In Vivo by Regulating Toll-Like Receptor 4 Dimerization.

Acute kidney injury (AKI) is a common critical illness that involves multiple systems and multiple organs with a rapid decline in kidney function over short period. It has a high mortality rate and presents a great treatment challenge for physicians. Oleuropein, the main active constituent of Ilex pubescens Hook. et Arn. var. kwangsiensis Hand.-Mazz. displays significant anti-inflammatory activity, although oleuropein’s therapeutic effect and mechanism of action in AKI remain to be elucidated. The present study aimed to further clarify the mechanism by which oleuropein exerts effects on inflammation in vitro and in vivo. In vitro, the inflammatory effect and mechanism were investigated through ELISA, Western blotting, the thermal shift assay, co-immunoprecipitation, and immunofluorescence staining. Lipopolysaccharide (LPS) induced acute kidney injury was employed in an animal model to investigate oleuropein’s therapeutic effect on AKI and mechanism in vivo. The underlying mechanisms were investigated by Western blot analysis of kidney tissue. In LPS-stimulated macrophages, our data demonstrated that oleuropein significantly reduced the expression of inflammatory mediators like NO, IL-6, TNF-α, iNOS, and COX-2. Moreover, oleuropein inhibited NF-κB/p65 translocation, and had a negative regulatory effect on key proteins in the NF-κB and MAPK pathways. In addition, the thermal shift and co-immunoprecipitation assays revealed that oleuropein played an essential role in binding to the active sites of TLR4, as well as inhibiting TLR4 dimerization and suppressing the binding of TLR4 to MyD88. Oleuropein markedly alleviated LPS induced acute kidney injury, decreased serum creatinine and blood urea nitrogen (BUN) levels and proinflammatory cytokines. More importantly, the TLR4-MyD88-NF-κB/MAPK pathways were confirmed to play an important role in the oleuropein treatment of AKI. In this study, oleuropein exhibited excellent anti-inflammatory effects by regulating TLR4-MyD88-NF-κB/MAPK axis in vitro and in vivo, suggesting oleuropein as a candidate molecule for treating AKI.

Artemisinin inhibits TLR4 signaling by targeting co-receptor MD2 in microglial BV-2 cells and prevents lipopolysaccharide-induced blood-brain barrier leakage in mice.

Artemisinin and its derivatives have been the frontline drugs for treating malaria. In addition to the antiparasitic effect, accumulating evidence shows that artemisinins can alleviate neuroinflammatory responses in the central nervous system (CNS). However, the precise mechanisms underlying their anti-neuroinflammatory effects are unclear. Herein we attempted to delineate the molecule target of artemisinin in microglia. In vitro protein intrinsic fluorescence titrations and saturation transfer difference (STD)-NMR showed the direct binding of artemisinin to Toll-like receptor TLR4 co-receptor MD2. Cellular thermal shift assay (CETSA) showed that artemisinin binding increased MD2 stability, which implies that artemisinin directly binds to MD2 in the cellular context. Artemisinin bound MD2 showed much less collapse during the molecular dynamic simulations, which supports the increased stability of MD2 upon artemisinin binding. Flow cytometry analysis showed artemisinin inhibited LPS-induced TLR4 dimerization and endocytosis in microglial BV-2 cells. Therefore, artemisinin was found to inhibit the TLR4-JNK signaling axis and block LPS-induced pro-inflammatory factors nitric oxide, IL-1β and TNF-α in BV-2 cells. Furthermore, artemisinin restored LPS-induced decrease of junction proteins ZO-1, Occludin and Claudin-5 in primary brain microvessel endothelial cells, and attenuated LPS-induced blood-brain barrier disruption in mice as assessed by Evans blue. In all, this study unambiguously adds MD2 as a direct binding target of artemisinin in its anti-neuroinflammatory function. The results also suggest that artemisinin could be repurposed as a potential therapeutic intervention for inflammatory CNS diseases.

Anemoside B4 Protects against Acute Lung Injury by Attenuating Inflammation through Blocking NLRP3 Inflammasome Activation and TLR4 Dimerization.

Acute lung injury (ALI) is an acute inflammatory process in the lung parenchyma. Anemoside B4 (B4) was isolated from Pulsatilla, a plant-based drug against inflammation and commonly applied in traditional Chinese medicine. However, the anti-inflammatory effect and the mechanisms of B4 are not clear. In this study, we explored the potential mechanisms and anti-inflammatory activity of B4 both in vitro and in vivo. The results indicated that B4 suppressed the expression of iNOS, COX-2, NLRP3, caspase-1, and IL-1β. The ELISA assay results showed that B4 significantly restrained the release of inflammatory cytokines like TNF-α, IL-6, and IL-1β in macrophage cells. In addition, B4 rescued mitochondrial membrane potential (MMP) loss in (lipopolysaccharide) LPS plus ATP stimulated macrophage cells. Co-IP and molecular docking results illustrated that B4 disrupted the dimerization of TLR4. For in vivo results, B4 exhibited a protective effect on LPS and bleomycin- (BLM-) induced ALI in mice through suppressing the lesions of lung tissues, the release of inflammatory cytokines, and the levels of white blood cells, neutrophils, and lymphoid cells in the blood. Collectively, B4 has a protective effect on ALI via blocking TLR4 dimerization and NLRP3 inflammasome activation, suggesting that B4 is a potential agent for the treatment of ALI.

Abstract P129: Angiotensin II Does Not Directly Stimulate The TLR4-MD2-MyD88 Inflammatory Pathway

Current evidence suggests that the deleterious actions of Angiotensin II (Ang II) reflect activation of both innate and adaptive inflammatory pathways. Indeed, recent studies find that Ang II stimulates the TLR-4 signaling cascade to promote cytokine release and renal injury by directly binding to the TLR4 accessory protein MD2 to induce TLR-4 dimerization and internalization that is independent of the AT 1 receptor (AT 1 R). Moreover, knockdown of TLR4 or MD2 attenuates renal injury and expression of pro-inflammatory cytokines in a chronic Ang II infusion model and in rat proximal tubule NRK-52e cells treated with Ang II. In the present study, we evaluated the proposed Ang II-TLR4-MD2 pathway in the NRK-52e cells regarding the stimulation of the chemokine MCP-1 (CCL2) and activation of the TLR4-MyD88 complex. NRK-52e cells, maintained in antibiotic-free media, were transferred to serum-free media prior to stimulation with the TLR4 agonists LPS and palmitate or Ang II for 24 hrs; reported data are the means ± SEM, n=4. The NRK-52e cells were sensitive to a low dose of LPS (1 ng/ml) stimulating CCL2 release 20-fold [Basal: 24.3 ± 1.0 pg/ml vs. 504 ± 30.4 pg/ml, p<0.001]. The LPS induced release of CCL2 was abolished by the specific TLR4 inhibitor Tak-242 [TAK: 23.9 ± 1.3 pg/ml] and was significantly reduced by the MD2 inhibitor L48H37 [135 ± 24.2 pg/ml]. Palmitate [100 μM] also stimulated CCL2 release [482 ± 21.0 pg/ml; p<0.001] that was abolished by TAK [32.3 ± 6.3 pg/ml]. The overall effects are consistent with previously reported responses to LPS and palmitate in these cells, as well as TLR4 protein expression and TAK inhibition. However, Ang II treatment over a dose range of 0.1 to 10 μM for 24 hrs failed to elicit CCL2 release [24.3 ± 1.0, 22.6 ± 0.5, and 27.6 ± 2.3 pg/ml, respectively; P>0.1 vs. Basal]. Ang II [1 μM] also failed to augment the CCL2 response to LPS [496 ± 6.5 pg/ml] or palmitate [511 ± 35.0 pg/ml]. Finally, pre-treatment of the NRK-52e cells with the AT 1 R antagonist candesartan [5 μM] failed to attenuate CCL2 release to LPS [512 ± 21 pg/ml]. We conclude that Ang II does not directly stimulate the TLR4-MD2 complex to induce CCL2 and that the inflammatory events associated with Ang II in vivo may reflect the release of other factors (LPS, AGEs, HMGB1) that serve as TLR-4 agonists.

1,7-Dihydroxy-3,4-Dimethoxyxanthone Inhibits Lipopolysaccharide-Induced Inflammation in RAW264.7 Macrophages by Suppressing TLR4/NF-κB Signaling Cascades.

Securidaca inappendiculata Hassk. is a traditional Chinese anti-rheumatic herbal medicine native to southern China. In this study, we identified a possible TLR4 inhibitor from this plant. General effects of its xanthone-rich fraction (XRF) on inflammation in vitro were investigated by immunoblotting experiments performed on lipopolysaccharides (LPS)-treated RAW264.7 cells, and the possible ligand of TLR4 within was screened out by analyzing chemical composition differences of the XRF containing cell culture medium under different inflammatory circumstances. The interaction between ligand and TLR4 was validated by cellular thermal shift assay (CETSA) and molecular docking simulation, and TLR4/NF-κB pathway status was investigated by immunoprecipitation, ELISA, immunofluorescence, dual-luciferase reporter, and immunoblotting experiments. Treatment with XRF resulted in significant decrease in p-p65 and p-JNK, and the signal accounting for 1,7-dihydroxy-3,4-dimethoxyxanthone (XAN) at 12.5min with mass of 289.29 was greatly decreased in XRF containing medium after LPS stimulus because of enhanced interaction with increased TLR4. CETSA and molecular docking simulation demonstrated that XAN could bind to TLR4 directly on a smooth region adjacent to its contact interface with MD-2. XAN treatment inhibited the dimerization of TLR4 and transcriptional activity of NF-κB in HEK293T cells and decreased p65 accumulation in nucleus and pro-inflammatory cytokines production in RAW264.7 cells receiving LPS treatment. Overall evidences suggest that XAN could be a selective TLR4 inhibitor with potent anti-inflammatory effects. Also, it indicated that xanthone derivatives could have promising clinical application in many immune-mediated inflammations by acting as TLR4 inhibitors.

Anthocyanins isolated from Hibiscus syriacus L. attenuate lipopolysaccharide-induced inflammation and endotoxic shock by inhibiting the TLR4/MD2-mediated NF-κB signaling pathway

BackgroundHibiscus syriacus L. has been used as a medicinal plant in many Asian countries. However, anti-inflammatory activity of H. syriacus L. remains unknown. PurposeThis study was aimed to investigating the anti-inflammatory effect of anthocyanin fractions from the H. syriacus L. variety Pulsae (PS) on the lipopolysaccharide (LPS)-induced inflammation and endotoxic shock. Study design and methodsMTT assay and flow cytometry analysis were performed to determine cytotoxicity of PS. RT-PCR, western blotting, and ELISA were conducted to evaluate the expression of proinflammatory mediators and cytokines. Molecular docking study predicted the binding scores and sites of PS to TLR4/MD2 complex. Immunohistochemical assay was conducted to evaluate the binding capability of PS to TLR4/MD2 and nuclear translocation of NF-κB p65. A zebrafish endotoxic shock model was used to evaluate anti-inflammatory activity of PS in vivo. ResultsPS suppressed LPS-induced nitric oxide and prostaglandin E2 secretion concomitant with the downregulation of inducible nitric oxide synthase and cyclooxygenase-2 expression. Furthermore, PS inhibited the production of proinflammatory cytokines such as TNF-α, IL-6, and IL-12 in LPS-stimulated RAW 264.7 macrophages. Additionally, molecular docking data showed that PS mostly fit into the hydrophobic pocket of MD2 and bound to TLR4. In particular, apigenin-7-O-glucoside powerfully bound to MD2 and TLR4 via hydrogen bonding. Additionally, immunohistochemistry assay revealed that PS inhibited LPS-induced TLR4 dimerization or expression on the cell surface, which consequently decreased MyD88 recruitment and IRAK4 phosphorylation, resulting in the inhibition of NF-κB activity. PS also attenuated LPS-mediated mortality and abnormality in zebrafish larvae and diminished the recruitment of neutrophils and macrophages at the inflammatory site accompanied by the low levels of proinflammatory mediators and cytokines. ConclusionPS might be a novel immunomodulator for the effective treatment of LPS-mediated inflammatory diseases.

Ginsenoside Rb1 exerts anti-inflammatory effects in vitro and in vivo by modulating toll-like receptor 4 dimerization and NF-kB/MAPKs signaling pathways

BackgoundGinsenoside Rb1, the main active constituent of Panax ginseng, displays significant anti-inflammatory activity, although the mechanism has not been clearly unraveled. In this study, Rb1’s mechanism of anti-inflammatory effects were investigated. MethodsThe flow cytometry and enzyme-linked immunosorbent assay (ELISA) were empolyed to detect pro-inflammatory cytokines release. The related protein and gene expression was investigated by western blotting and qRT-PCR. The dimerization of TLR4 was measured by co-immunoprecipitation and molecular docking assays. Cellular thermal shift assay was used for the determination of the binding of Rb1 and TLR4. For animal moldels, LPS- or cantharidin-induced acute kidney injury, LPS-induced septic death, and dimethyl benzene-induced ear edema were employed to investigate Rb1’s anti-inflammatory activity in vivo. ResultsRb1 significantly decreased inflammatory cytokines release in LPS-stimulated RAW264.7 cells and BMDMs, as well as COX-2 and iNOS amounts. Rb1 reduced LPS-associated calcium influx, ROS production, and NO generation. The NF-κB and MAPK axes participated in Rb1’s anti-inflammatory effects. Molecular docking simulation indicated Rb1 bound to TLR4 to prevent TLR4 dimerization, as confirmed by co-immunoprecipitation and cellular thermal shift assay. Furthermore, MyD88 recruitment and TAK1 expression were altered by reduced TLR4 dimerization, indicating the TLR4-MyD88-NF-κB/MAPK pathways contributed to Rb1’s anti-inflammatory process. In animal models, Rb1 markedly alleviated LPS- or cantharidin-induced acute kidney injury, rescued LPS-induced septic mice from death, and inhibited dimethyl benzene-induced mouse ear edema. ConclusionOverall, these findings demonstrate Rb1 exhibits marked anti-inflammatory effects, suggesting Rb1 represents an optimal molecule for treating inflammatory diseases.

Conjugate of Enkephalin and Temporin Peptides as a Novel Therapeutic Agent for Sepsis.

Antimicrobial peptides (AMPs) exhibit a wide spectrum of actions, ranging from a direct bactericidal effect to multifunctional activities as immune effector molecules. The aim of this study was to examine the anti-inflammatory properties of a DAL-PEG-DK5 conjugate composed of a lysine-rich derivative of amphibian temporin-1CEb (DK5) and dalargin (DAL), the synthetic Leu-enkephalin analogue. Detailed study of the endotoxin-neutralizing activity of the peptide revealed that DAL-PEG-DK5 interacts with LPS and the LPS binding protein (LBP). Moreover, DAL-PEG-DK5 prevented dimerization of TLR4 at the macrophage surface upon LPS stimulation. This inhibited activation of the NF-κB signaling pathway and markedly reduced pro-inflammatory cytokine production. Finally, we showed that aggregation of DAL-PEG-DK5 into amyloid-like structures induced by LPS neutralized the endotoxin proinflammatory activity. Consequently, DAL-PEG-DK5 reduced morbidity and mortality in vivo, in a mouse model of endotoxin-induced septic shock. Collectively, the data suggest that DAL-PEG-DK5 is a promising therapeutic compound for sepsis.

P204 Is Required for Canonical Lipopolysaccharide-induced TLR4 Signaling in Mice

p204, a murine member of an interferon-inducible p200 family, was reported to recognize intracellular viral and bacterial DNAs, however, its role in the innate immunity in vivo remains unknown due to the lack of p204-deficient animal models. In this study we first generated the p204−/− mice. Unexpectedly, p204 deficiency led to significant defect in extracellular LPS signaling in macrophages, as demonstrated by dramatic reductions of LPS-mediated IFN-β and pro-inflammatory cytokines. The serum levels of IFN-β and pro-inflammatory cytokines were also significantly reduced in p204−/− mice following LPS challenge. In addition, p204−/− mice were resistant to LPS-induced shock. LPS-activated NF-ĸB and IRF-3 pathways were all defective in p204-deficient macrophages. p204 binds to TLR4 through its Pyrin domain, and it is required for the dimerization of TLR4 following LPS-challenge. Collectively, p204 is a critical component of canonical LPS-TLR4 signaling pathway, and these studies also suggest that p204 could be a potential target to prevent and treat inflammatory and infectious diseases.

Isoacteoside, a dihydroxyphenylethyl glycoside, exhibits anti-inflammatory effects through blocking toll-like receptor 4 dimerization.

Isoacteoside (is a phenylethanoid isolated from Monochasma savatieri Franch. ex Maxim., which is an anti-inflammatory herb widely used in traditional Chinese medicine. However, the exact mechanism of the anti-inflammatory activity of isoacteoside is not completely understood. In this study, its anti-inflammatory mechanism was elucidated in mouse macrophages. The expression of the NF-κB pathway, MAPK pathway, iNOS, TNF-α, IL-6 and IL-1β was evaluated using Western blotting, quantitative real-time PCR or ELISA. TLR4 dimerization was determined by transfecting HEK293T cells with TLR4 plasmids. The in vivo anti-inflammatory effect of isoacteoside was determined using mouse models of xylene-induced ear oedema, LPS-induced endotoxic shock and LPS-induced endotoxaemia-associated acute kidney injury (AKI). Isoacteoside suppressed COX-2, iNOS, TNF-α, IL-6 and IL-1β expression. Furthermore, isoacteoside attenuated the LPS-induced transcriptional activity of NF-κB by decreasing the levels of phosphorylated IκB-α and IKK and NF-κB/p65 nuclear translocation. In addition, isoacteoside inhibited LPS-induced transcriptional activity of AP-1 by reducing the levels of phosphorylated JNK1/2 and p38MAPK. Isoacteoside blocked LPS-induced TLR4 dimerization, resulting in a reduction in the recruitment of MyD88 and TIR-domain-containing adapter-inducing interferon-β (TRIF) and the phosphorylation of TGF-β-activated kinase-1 (TAK1). Pretreatment of mice with isoacteoside effectively inhibited xylene-induced ear oedema and LPS-induced endotoxic death and protected against LPS-induced AKI. Isoacteoside blocked TLR4 dimerization, which activates the MyD88-TAK1-NF-κB/MAPK signalling cascades and TRIF pathway. Our data indicate that isoacteoside is a potential lead compound for the treatment of inflammatory diseases.

Tanshinones and diethyl blechnics with anti-inflammatory and anti-cancer activities from Salvia miltiorrhiza Bunge (Danshen).

Four novel compounds (1–4) as well as fourteen reported compounds (5–18) were isolated and purified from Salvia miltiorrhiza Bunge (Danshen). The structures of novel compounds were determined by 1D and 2D NMR, HRESIMS data, etc. The anti-inflammatory properties of all the compounds on RAW264.7 macrophages and their cytotoxicity on H1299 and Bel-7402 cell lines coupled with a structure-activity relationship (SAR) were investigated. Compound 4 demonstrated the best anti-inflammatory activity and was chosen for further research. Compound 4 greatly suppressed secretion of nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) in the RAW264.7 macrophages stimulated by LPS. Additionally, the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was decreased and the nuclear translocation of NF-κB was attenuated after treatment with compound 4 in vitro. Compound 4 was able to dramatically inhibit LPS-induced activation of JNK1/2 and ERK1/2 and remarkably disrupted the TLR4 dimerization in LPS-induced RAW264.7 macrophages. Thus, the new compound 4 suppressed LPS-induced inflammation partially is due to the blocking TLR4 dimerization. In addition, the anti-cancer activity investigation indicated that most of isolated compounds exhibited cytotoxicity and the SAR analysis showed that the intact D ring was indispensable and unsaturated D ring played vital role.

Mechanisms for the activation of Toll-like receptor 2/4 by saturated fatty acids and inhibition by docosahexaenoic acid

Saturated fatty acids can activate Toll-like receptor 2 (TLR2) and TLR4 but polyunsaturated fatty acids, particularly docosahexaenoic acid (DHA) inhibit the activation. Lipopolysaccharides (LPS) and lipopetides, ligands for TLR4 and TLR2, respectively, are acylated by saturated fatty acids. Removal of these fatty acids results in loss of their ligand activity suggesting that the saturated fatty acyl moieties are required for the receptor activation. X-ray crystallographic studies revealed that these saturated fatty acyl groups of the ligands directly occupy hydrophobic lipid binding domains of the receptors (or co-receptor) and induce the dimerization which is prerequisite for the receptor activation. Saturated fatty acids also induce the dimerization and translocation of TLR4 and TLR2 into lipid rafts in plasma membrane and this process is inhibited by DHA. Whether saturated fatty acids induce the dimerization of the receptors by interacting with these lipid binding domains is not known. Many experimental results suggest that saturated fatty acids promote the formation of lipid rafts and recruitment of TLRs into lipid rafts leading to ligand independent dimerization of the receptors. Such a mode of ligand independent receptor activation defies the conventional concept of ligand induced receptor activation; however, this may enable diverse non-microbial molecules with endogenous and dietary origins to modulate TLR-mediated immune responses. Emerging experimental evidence reveals that TLRs play a key role in bridging diet-induced endocrine and metabolic changes to immune responses.

The Thrombin-Derived Host Defense Peptide GKY25 Inhibits Endotoxin-Induced Responses through Interactions with Lipopolysaccharide and Macrophages/Monocytes.

Host defense peptides have recently gained much interest as novel anti-infectives owing to their ability to kill bacteria and simultaneously modulate host cell responses. The cationic host defense peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), derived from the C terminus of human thrombin, inhibits proinflammatory responses in vitro and in vivo, but the mode of action is unclear. In this study, we show that GKY25, apart from binding bacterial LPS, also interacts directly with monocytes and macrophages in vitro, ex vivo, and in vivo. Moreover, GKY25 inhibits TLR4- and TLR2-induced NF-κB activation in response to several microbe-derived agonists. Furthermore, GKY25 reduces LPS-induced phosphorylation of MAPKs p38α and JNK1/2/3. FACS and electron microscopy analyses showed that GKY25 interferes with TLR4/myeloid differentiation protein-2 dimerization. The results demonstrate a previously undisclosed activity of the host defense peptide GKY25, based on combined LPS and cell interactions leading to inhibition of TLR4 dimerization and subsequent reduction of NF-κB activity and proinflammatory cytokine production in monocytes and macrophages.

Polyoxygenated Cholesterol Ester Hydroperoxide Activates TLR4 and SYK Dependent Signaling in Macrophages

Oxidation of low-density lipoprotein (LDL) is one of the major causative mechanisms in the development of atherosclerosis. In previous studies, we showed that minimally oxidized LDL (mmLDL) induced inflammatory responses in macrophages, macropinocytosis and intracellular lipid accumulation and that oxidized cholesterol esters (OxCEs) were biologically active components of mmLDL. Here we identified a specific OxCE molecule responsible for the biological activity of mmLDL and characterized signaling pathways in macrophages in response to this OxCE. Using liquid chromatography – tandem mass spectrometry and biological assays, we identified an oxidized cholesteryl arachidonate with bicyclic endoperoxide and hydroperoxide groups (BEP-CE) as a specific OxCE that activates macrophages in a TLR4/MD-2-dependent manner. BEP-CE induced TLR4/MD-2 binding and TLR4 dimerization, phosphorylation of SYK, ERK1/2, JNK and c-Jun, cell spreading and uptake of dextran and native LDL by macrophages. The enhanced macropinocytosis resulted in intracellular lipid accumulation and macrophage foam cell formation. Bone marrow-derived macrophages isolated from TLR4 and SYK knockout mice did not respond to BEP-CE. The presence of BEP-CE was demonstrated in human plasma and in the human plaque material captured in distal protection devices during percutaneous intervention. Our results suggest that BEP-CE is an endogenous ligand that activates the TLR4/SYK signaling pathway. Because BEP-CE is present in human plasma and human atherosclerotic lesions, BEP-CE-induced and TLR4/SYK-mediated macrophage responses may contribute to chronic inflammation in human atherosclerosis.