Inhibition Of Toll-like Receptor Research Articles

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.

Bisphenol A: A potential Toll-like receptor 4/myeloid differentiation factor 2 complex agonist.

In addition to endocrine disruption, bisphenol A (BPA) is known to induce inflammation through the activation of nuclear factor-κB (NF-κB). However, detailed studies on the mechanism of NF-κB activation by BPA have not been sufficiently conducted. In the present study, we observed that low concentrations of BPA (≤1μM) upregulated the release of proinflammatory mediators, including nitric oxide (NO) and prostaglandin E2 (PGE2), as well as proinflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-12, and IL-6. Molecular modeling predicted that BPA docked with the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) complex activates downstream molecules including myeloid differentiation primary response 88 (MyD88) and IL-1 receptor-associated kinase 4 (IRAK-4) and results in the upregulation of the NF-κB signaling pathway. Additionally, BPA increased morphological abnormalities and mortality in zebrafish larvae and enhanced the dispersal of macrophages and neutrophils in the whole body, thereby causing an endotoxemia-like disorder. However, a specific TLR4 inhibitor, TLR4-IN-C34, mitigated BPA-induced mortality and morphological abnormalities, which indicates that the TLR4/MD2 complex is a molecular target of BPA-induced immunotoxicity. Collectively, our results indicate that low concentrations of BPA, which is a potential agonist of the TLR4/MD2 complex, can intensify the immune response and eventually cause an endotoxemia-like disorder.

Porphyromonas gingivalis lipopolysaccharide induces interleukin-6 and c-c motif chemokine ligand 2 expression in cultured hCMEC/D3 human brain microvascular endothelial cells.

This paper describes the effect of Porphyromonas gingivalis (Pgingivalis) lipopolysaccharide (LPS) on the expression of interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2) in cultured hCMEC/D3 human brain microvascular endothelial cells. Pgingivalis is one of the important pathogens in periodontitis, and periodontitis is a risk factor for brain disorders including cerebrovascular diseases and Alzheimer's disease. However, the mechanisms underlying the pathogenesis of Pgingivalis-mediated brain diseases are incompletely understood. Effects of Pgingivalis LPS on brain endothelial cells are not known well. The hCMEC/D3 human brain microvascular endothelial cells were cultured and treated with Pgingivalis LPS. The expression of IL-6 and CCL2 mRNA and protein was examined using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Effect of inhibitors of Toll-like receptor (TLR) 2, TLR4, nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) was also investigated. Phosphorylation of NF-κB p65, p38 MAPK and JNK was examined using Western blotting. Pgingivalis LPS-induced mRNA and protein expression of IL-6 and CCL2 in hCMEC/D3 cells in a concentration-dependent manner at the concentration of 0.5-50µg/mL. Maximal mRNA expression of IL-6 and CCL2 was found 2 and 4hours after stimulation, respectively. Induction of IL-6 and CCL2 by Pgingivalis LPS was almost completely inhibited by pretreatment of cells with TLR4 inhibitor but not by TLR2 inhibitor. Treatment of cells with Pgingivalis LPS for up to 2hours induced phosphorylation of NF-κB p65, p38 MAPK and JNK. IL-6 induction was decreased by pretreatment of cells with NF-κB inhibitor SN50 or p38 MAPK inhibitor SB203580, while CCL2 induction was reduced by SN50 or JNK inhibitor SP600125. IL-6 and CCL2 produced upon Pgingivalis LPS stimulation may contribute to the inflammatory reactions in brain endothelial cells and subsequent neurological disorders such as cerebrovascular and Alzheimer's diseases.

TLR4/IL-6/IRF1 signaling regulates androgen receptor expression: A potential therapeutic target to overcome taxol resistance in ovarian cancer

Ovarian cancer is poorly treatable due, at least in part, to induced drug resistance to taxol- and cisplatin-based chemotherapy. Recent studies showed that ectopic overexpression of toll-like receptor 4 (TLR4) in ovarian cancer cells leads to upregulation of the androgen receptor (AR) and transactivation of taxol resistance genes, thereby causing chemoresistance. In the present study, we examined the signaling pathways involving TLR4 and interleukin 6 (IL-6) that enhance AR expression. Based on transcriptomic analysis, we show that IL-6 functions as a hub gene among the upregulated genes in taxol-treated TLR4-overexpressing ovarian cancer cells. Both the TLR4 activator taxol and IL-6 can induce AKT phosphorylation, whereas TLR4 knockdown or inhibition of the IL-6 signal transducer GP130 abrogates AKT activation. Furthermore, expression of AR and IL-6 is downregulated in TLR4-knockdown, taxol-resistant cells. In addition, TLR4 knockdown inhibits GP130 and IL-6 receptor alpha (IL6Rα) activities, indicating that TLR4 plays a critical role in IL-6 signaling. On the other hand, nuclear translocation of AR is induced by IL-6 treatment, whereas knockdown of endogenous IL-6 reduces AR and TLR4 expression in taxol-resistant ovarian cancer cells. These results indicate that TLR4 and IL-6 play a crucial role in AR gene regulation and function. We also identify interferon regulatory factor 1 (IRF1) as a downstream target of IL-6 signaling and as a regulator of AR expression. Moreover, analysis of clinical samples indicates that high IL-6 expression correlates with poor progression-free survival in ovarian cancer patients treated with taxol. Overall, our findings indicate that the TLR4/IL-6/IRF1 signaling axis represents a potential therapeutic target to overcome AR-based taxol resistance in ovarian cancer.

S100A8 Promotes Inflammation via Toll-Like Receptor 4 After Experimental Traumatic Brain Injury.

IntroductionS100 calcium-binding protein A8 (S100A8) is also known as macrophage-related protein 8, which is involved in various pathological processes in the central nervous system post-traumatic brain injury (TBI), and plays a critical role in inducing inflammatory cytokines. Accumulating evidences have indicated that toll-like receptor 4 (TLR4) is considered to be involved in inflammatory responses post TBI. The present study was designed to analyze the hypothesis that S100A8 is the key molecule that induces inflammation via TLR4 in TBI.MethodsThe weight-drop TBI model was used and randomly implemented on mice that were categorized into six groups: Sham, NS, S100A8, S100A8+TAK-242, TBI, and TBI+TAK-242 groups. In the S100A8+TAK-242 and TBI+TAK-242 groups, at half an hour prior to the intracerebroventricular administration of S100A8 or TBI, mice were intraperitoneally treated with TAK-242 that acts as a selective antagonist and inhibitor of TLR4. Furthermore, the protein recombinant of S100A8 was injected into the lateral ventricle of the brain of mice in the S100A8 and S100A8+TAK-242 groups. Sterile normal saline was injected into the lateral ventricle in the NS group. To evaluate the association between S100A8 and TLR4, Western blot, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and Nissl staining were employed. Simultaneously, the neurological score and brain water content were assessed. In the in vitro analysis, BV-2 microglial cells were stimulated with lipopolysaccharide LPS or S100A8 recombinant protein, with or without TAK-242. The expression of the related proteins was subsequently detected by Western blot or enzyme-linked immunosorbent assay.ResultsThe levels of S100A8 protein and pro-inflammatory cytokines were significantly elevated after TBI. There was a reduction in the neurological scores of non-TBI animals with remarkable severe brain edema after the intracerebroventricular administration of S100A8. Furthermore, the TLR4, p-p65, and myeloid differentiation factor 88 (MyD88) levels were elevated after the administration of S100A8 or TBI, which could be restored by TAK-242. Meanwhile, in the in vitro analysis, due to the stimulation of S100A8 or LPS, there was an upregulation of p-p65 and MyD88, which could also be suppressed by TAK-242.ConclusionThe present study demonstrated that the TLR4-MyD88 pathway was activated by S100A8, which is essential for the development of inflammation in the brain after TBI.

Toll-like receptor 4 (TLR4) antagonists as potential therapeutics for intestinal inflammation.

Gastrointestinal inflammation is a hallmark of highly prevalent disorders, including cancer treatment–induced mucositis and ulcerative colitis. These disorders cause debilitating symptoms, have a significant impact on quality of life, and are poorly managed. The activation of toll-like receptor 4 (TLR4) has been proposed to have a major influence on the inflammatory signalling pathways of the intestinal tract. Inhibition of TLR4 has been postulated as an effective way to treat intestinal inflammation. However, there are a limited number of studies looking into the potential of TLR4 antagonism as a therapeutic approach for intestinal inflammation. This review surveyed available literature and reported on the in vitro, ex vivo and in vivo effects of TLR4 antagonism on different models of intestinal inflammation. Of the studies reviewed, evidence suggests that there is indeed potential for TLR4 antagonists to treat inflammation, although only a limited number of studies have investigated treating intestinal inflammation with TLR4 antagonists directly. These results warrant further research into the effect of TLR4 antagonists in the intestinal tract.

Specific TLR4 Blocking Effect of a Novel 3,4-Dihydropyrimidinone Derivative.

Background: Toll-like receptor 4 (TLR4) initiates both innate and adaptive immune responses, which plays an important protective role in self-defense mechanisms. Excessive or inappropriate TLR4 activation causes the development of many autoimmune diseases. Dihydropyrimidinone derivatives are medicinally important molecules with diverse pharmacological activities, including anti-inflammatory activity. The present study focused on novel synthesized 3,4-dihydropyrimidinone derivatives and evaluated their inhibitory effects on TLR4. Methods: A series of 3,4-dihydropyrimidinone derivatives were recently synthesized and evaluated for their TLR4 inhibition activities and cytotoxic on HEK-BlueTM hTLR4 cells with the help of QUANTI-Blue assay and MTS assay. Selected compound 3 was analyzed for its molecular docking with TLR4 by using Autodock vina 1.1.2. Its effect on the TLR4 pathway related cytokines was also evaluated in THP-1 cells and human peripheral blood mononuclear cells by using real-time PCR, ELISA and western blot. Results: Five compounds were synthesized and characterized for effectiveness based on 3,4-dihydropyrimidinone. Compound 3 was found to be the potent hybrid among the synthesized compounds, with high TLR4 inhibition activities and low cytotoxic activities against HEK-BlueTM hTLR4 cells. Molecular docking analysis showed that two hydrogen bonds between compound 3 and residues Asp209(TLR4) and Asp99(MD-2) mainly contribute to the TLR4 inhibition. In addition, compound 3 suppressed LPS-induced of the mRNA expression of TLR4, IP-10, TNF-α, IL-6, IL-12A, and IL-12B, the protein expression of pIRF3 and pNFκB and the secretion of IP-10, TNF-α in THP-1 cell line. Compound 3 also inhibited LPS-induced expression of TNF-α, IL-6, and IL-1β but increased IP-10 at mRNA levels in human peripheral blood mononuclear cells. Conclusion: Our study reveals compound 3, a novel 3,4-dihydropyrimidinone derivative, is a potential TLR4 antagonist, which opens up new research avenues for the development of promising therapeutic agents for inflammatory and autoimmune diseases.

Toll-like receptor 4 signaling activates ERG function in prostate cancer and provides a therapeutic target.

The TMPRSS2–ERG gene fusion and subsequent overexpression of the ERG transcription factor occurs in ∼50% of prostate tumors, making it the most common abnormality of the prostate cancer genome. While ERG has been shown to drive tumor progression and cancer-related phenotypes, as a transcription factor it is difficult to target therapeutically. Using a genetic screen, we identified the toll-like receptor 4 (TLR4) signaling pathway as important for ERG function in prostate cells. Our data confirm previous reports that ERG can transcriptionally activate TLR4 gene expression; however, using a constitutively active ERG mutant, we demonstrate that the critical function of TLR4 signaling is upstream, promoting ERG phosphorylation at serine 96 and ERG transcriptional activation. The TLR4 inhibitor, TAK-242, attenuated ERG-mediated migration, clonogenic survival, target gene activation and tumor growth. Together these data indicate a mechanistic basis for inhibition of TLR4 signaling as a treatment for ERG-positive prostate cancer.

Sleep deprivation aggravates brain injury after experimental subarachnoid hemorrhage via TLR4-MyD88 pathway.

Subarachnoid hemorrhage (SAH) is a life-threatening cerebrovascular disease, and most of the SAH patients experience sleep deprivation during their hospital stay. It is well-known that sleep deprivation is one of the key components of developing several neurological disorders, but its effect on brain damage after SAH has not been determined. Therefore, this study was designed to evaluate the effect of sleep deprivation using an experimental SAH model in rats. Induction of sleep deprivation for 24 h aggravated the SAH-induced brain damage, as evidenced by brain edema, neuronal apoptosis and activation of caspase-3. Sleep deprivation also worsened the neurological impairment and cognitive deficits after SAH. The results of immunostaining and western blot showed that sleep deprivation increased the activation of microglial cells. In addition, sleep deprivation differently regulated the expression of anti-inflammatory and pro-inflammatory cytokines. The results of immunofluorescence staining and western blot showed that sleep deprivation markedly increased the activation of Toll-like receptor 4 (TLR4) and myeloid differentiation primary response protein 88 (MyD88). Mechanically, treatment with the TLR4 inhibitor TAK-242 or the MyD88 inhibitor ST2825 significantly attenuated the brain damage and neuroinflammation induced by sleep deprivation after SAH. In conclusion, our results indicate that sleep deprivation aggravates brain damage and neurological dysfunction following experimental SAH in rats. These effects were mediated by the activation of the TLR4-MyD88 cascades and regulation of neuroinflammation.

Endothelial TLR4 Expression Mediates Vaso-Occlusive Crisis in Sickle Cell Disease.

Heme, released from red blood cells in sickle cell disease (SCD), interacts with toll-like receptor 4 (TLR4) to activate NF-κB leading to the production of cytokines and adhesion molecules which promote inflammation, pain, and vaso-occlusion. In SCD, TLR4 inhibition has been shown to modulate heme-induced microvascular stasis and lung injury. We sought to delineate the role of endothelial verses hematopoietic TLR4 in SCD by developing a TLR4 null transgenic sickle mouse. We bred a global Tlr4-/- deficiency state into Townes-AA mice expressing normal human adult hemoglobin A and Townes-SS mice expressing sickle hemoglobin S. SS-Tlr4-/- had similar complete blood counts and serum chemistries as SS-Tlr4+/+ mice. However, SS-Tlr4-/- mice developed significantly less microvascular stasis in dorsal skin fold chambers than SS-Tlr4+/+ mice in response to challenges with heme, lipopolysaccharide (LPS), and hypoxia/reoxygenation (H/R). To define a potential mechanism for decreased microvascular stasis in SS-Tlr4-/- mice, we measured pro-inflammatory NF-κB and adhesion molecules in livers post-heme challenge. Compared to heme-challenged SS-Tlr4+/+ livers, SS-Tlr4-/- livers had lower adhesion molecule and cytokine mRNAs, NF-κB phospho-p65, and adhesion molecule protein expression. Furthermore, lung P-selectin and von Willebrand factor immunostaining was reduced. Next, to establish if endothelial or hematopoietic cell TLR4 signaling is critical to vaso-occlusive physiology, we created chimeric mice by transplanting SS-Tlr4-/- or SS-Tlr4+/+ bone marrow into AA-Tlr4-/- or AA-Tlr4+/+ recipients. Hemin-stimulated microvascular stasis was significantly decreased when the recipient was AA-Tlr4-/-. These data demonstrate that endothelial, but not hematopoietic, TLR4 expression is necessary to initiate vaso-occlusive physiology in SS mice.

Attenuation of the Induction of TLRs 2 and 4 Mitigates Inflammation and Promotes Neurological Recovery After Focal Cerebral Ischemia.

The intense inflammatory response triggered in the brain after focal cerebral ischemia is detrimental. Recently, we showed that the suppression of toll-like receptors (TLRs) 2 and 4 attenuates infarct size and reduces the expression of pro-inflammatory cytokines in the ischemic brain. In this study, we further examined the effect of unsuppressed induction of TLRs 2 and 4 on the expression of its downstream signaling molecules and pro-inflammatory cytokines 1week after reperfusion. The primary purpose of this study was to investigate the effect of simultaneous knockdown of TLRs 2 and 4 on M1/M2 microglial polarization dynamics and post-stroke neurological deficits and the recovery. Transient focal cerebral ischemia was induced in young adult male Sprague-Dawley rats by the middle cerebral artery occlusion (MCAO) procedure using a monofilament suture. Appropriate cohorts of rats were treated with a nanoparticle formulation of TLR2shRNA and TLR4shRNA (T2sh+T4sh) expressing plasmids (1mg/kg each of T2sh and T4sh) or scrambled sequence inserted vector (vehicle control) expressing plasmids (2mg/kg) intravenously via tail vein immediately after reperfusion. Animals from various cohorts were euthanized during reperfusion, and the ischemic brain tissue was isolated and utilized for PCR followed by agarose gel electrophoresis, real-time PCR, immunoblot, and immunofluorescence analysis. Appropriate groups were subjected to a battery of standard neurological tests at regular intervals until 14days after reperfusion. The increased expression of both TLRs 2 and 4 and their downstream signaling molecules including the pro-inflammatory cytokines was observed even at 1-week after reperfusion. T2sh+T4sh treatment immediately after reperfusion attenuated the post-ischemic inflammation, preserved the motor function, and promoted recovery of the sensory and motor functions. We conclude that the post-ischemic induction of TLRs 2 and 4 persists for at least 7days after reperfusion, contributes to the severity of acute inflammation, and impedes neurological recovery. Unlike previous studies in TLRs 2 or 4 knockout models, results of this study in a pharmacologically relevant preclinical rodent stroke model have translational significance.

Resveratrol attenuates inflammation by regulating macrophage polarization via inhibition of toll-like receptor 4/MyD88 signaling pathway

Background: Resveratrol (RES) can induce macrophage polarization to achieve the immune response. Objectives: In this study, we aimed to determine whether RES attenuates inflammation by regulating macrophage polarization through inhibition of toll-like receptor 4 (TLR4)/MyD88 signaling. Materials and Methods: We measured the effects of different concentrations of RES on cellular activity of RAW264.7 and measured it using the methyl thiazolyl blue tetrazolium bromide method. The immunomodulatory effects of RES on lipopolysaccharide (LPS)-induced RAW264.7 cells were detected by measuring the levels of nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α. The quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the markers of M1 and M2 polarization of macrophages. The changes in the expression of both mRNA and proteins related to the TLR4/ myeloid differentiation factor 88 (MyD88) receptor pathway detected by Western blot (WB) and RT-qPCR analyses. Results: According to our results, 2, 4, and 8 μmol/L RES decreased the levels of NO, IL-6, and TNF-α in LPS-induced RAW264.7 cells, thereby reducing inflammation and increasing immunity. IL-1 and inducible NO synthase, which are the markers of M1-type macrophages, were increased by LPS, and arginase-1, CD206, which are the markers of M2-type macrophages, were decreased. However, in LPS-induced RAW264.7 cells incubated with RES, we observed the opposite results for both M1-and M2-type macrophage markers. Proteins and mRNA related to the TLR4 pathway were detected by WB and RT-qPCR analysis and TLR4, P65, MyD88, interleukin receptor-associated kinases 1, tumor necrosis factor receptor associated factor 6, activated kinase 1, and IKKβ were significantly increased by LPS. In contrast, when the cells were incubated with RES, the TLR4 pathway-related proteins and mRNA were significantly decreased and showed a volume-response relationship. Conclusion: RES can polarize M1-type macrophages to M2-type macrophages and regulate them through the TLR4/MyD88 receptor pathway. The polarization of macrophages can reduce the level of inflammation and regulate the immune system.

Recombinant High-Mobility Group Box 1 (rHMGB1) Promotes NRF2-Independent Mitochondrial Fusion through CXCR4/PSMB5-Mediated Drp1 Degradation in Endothelial Cells.

Mitochondrial dynamics plays an important role in maintaining normal endothelial cell function and in the pathogenesis of cardiovascular disease. It is not identified whether high-mobility group box 1 (HMGB1), a representative damage-associated molecular pattern (DAMP) molecule, could influence mitochondrial dynamics in endothelial cells. The objective of this study is to clarify the effect of HMGB1 on mitochondrial dynamics in endothelial cells and the underlying mechanism. EA.hy926 human endothelial cells were incubated with recombinant HMGB1 (rHMGB1); mitochondrial morphology was observed with a confocal microscope and transmission electron microscope (TEM). The expression of dynamin-related protein 1 (Drp1), Mitofusin 1 (Mfn1), Mitofusin 2 (Mfn2), Optic atrophy 1 (Opa1), phosphatase and tensin homolog- (PTEN-) induced kinase 1 (PINK1), NOD-like receptor 3 (NLRP3), caspase 1, cleaved caspase 1, 20S proteasome subunit beta 5 (PSMB5), and antioxidative master nuclear factor E2-related factor 2 (NRF2) and the concentration of interleukin 1β (IL-1β) were determined. Specific inhibitors C29, TAK-242, FPS-ZM1, AMD3100, and epoxomicin were used to block toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), receptor for advanced glycation end products (RAGE), C-X-C-chemokine receptor 4 (CXCR4), and PSMB5, respectively. siRNAs were used to silence the expression of NRF2. rHMGB1 promoted mitochondrial fusion in endothelial cells, while no significant proinflammatory effects were found. The expression of mitochondrial fission protein Drp1 and phosphorylated subtypes p-Drp1-S616 and p-Drp1-S637 were all downregulated; no significant expression changes of PINK1 and Mfn1, Mfn2, and Opa1 were found. Inhibition of CXCR4 but not TLR4, RAGE, or TLR2 reversed rHMGB1-induced Drp1 downregulation and mitochondrial fusion. Interestingly, inhibition of TLR4 with TAK-242 promoted Drp1 downregulation and mitochondrial fusion. rHMGB1 increased the expression of NRF2 and PSMB5; inhibition of PSMB5 but not silencing NRF2 abolished rHMGB1-induced Drp1 downregulation and mitochondrial fusion. These results indicate that rHMGB1 promotes NRF2 independent mitochondrial fusion via CXCR4/PSMB5 pathway-mediated Drp1 proteolysis. rHMGB1 may influence mitochondrial and endothelial function through this effect on mitochondrial dynamics.

In vitro macrophage activation by Sageretia thea fruits through TLR2/TLR4-dependent activation of MAPK, NF-κB and PI3K/AKT signalling in RAW264.7 cells

ABSTRACTSageretia thea fruits (STF) are known to have a higher content of functional ingredients and nutrients than blueberries, but studies related to its pharmacological activity are insufficient. Thus, in this study, we investigated whether STF exhibits immune-enhancing activity through macrophage activation. STF increased the production of immunomodulators, cell viability and phagocytosis in RAW264.7 cells. The inhibition of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) blocked STF-mediated production of immunomodulators in RAW264.7 cells. Inhibition of mitogen-activated protein kinases (MAPKs), nuclear factor-kappaB (NF-κB) and phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) signalling pathway reduced STF-mediated production of immunomodulators and the activation of MAPK, NF-κB and PI3K/AKT signalling pathway by STF suppressed the inhibition of TLR2/TLR4. However, STF blocked the overproduction of immunomodulators in LPS-stimulated RAW264.7 cells. Taken together, STF induced macrophage activation through TLR2/TLR4-dependent activation of MAPK, NF-κB and PI3K/AKT signalling.

Rice sprouts exert immunostimulatory activity in mouse macrophages, RAW264.7 cells

ABSTRACTRice sprouts are reported to have potential as a functional food agent for health promotion, but studies on the biological activity of rice sprouts are insufficient. Thus, in this study, we investigated whether WERS exhibits immunostimulatory activity in macrophages. WERS increased the production of immunomodulatory factors such as nitric oxide (NO), inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) in RAW264.7 cells. In addition, WERS activated the phagocytosis of RAW264.7 cells. The inhibition of toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) blocked WERS-mediated production of immunomodulatory factors in RAW264.7 cells. In addition, the inhibition of p38 and JNK suppressed the production of immunomodulatory factors by WERS, and the activation of p38 and JNK was blocked by the inhibition of TLR2 and TLR4. Taken together, WERS induced macrophage activation through TLR2/4-dependent activation of p38 and JNK.

Immuno-enhancing effects of fruit of Actinidia polygama in macrophages

ABSTRACTIn this study, we evaluated the functionality of water extracts from the fruit of Actinidia polygama (APF) in macrophages. The APF is a medicinal plant belonging to Actinidiaceae, it has been reported to exert anti-inflammatory, analgesic and hypouricemic activities. However, the potential mechanism for the immune activation of the APF is still insufficient. So, we evaluated whether APF exerts immune activation activities and elucidated its potential mechanism in macrophages. The APF dose-dependently increased the production of immunomodulators in macrophages. The inhibition of Toll-like receptor 4 (TLR4) blocked APF-mediated production of immunomodulators in RAW264.7 cells. In addition, APF-mediated production of immunomodulators was attenuated by MAPKs and NF-κB inhibition in RAW264.7 cells. Also, we analyzed the sugars content of the APF. The contents of glucose, galactose and fructose were 3597, 904, 7582 mg/L, respectively. These results suggest that the APF may have great potential for the development of immunomodulatory drugs.

Mechanism of lipopolysaccharide-mediated induction of epithelial-mesenchymal transition of alveolar type II epithelial cells in absence of other inflammatory cells

Several studies have shown that gram-negative bacilli infection can cause acute lung injury, and that consequent pulmonary fibrosis is caused when alveolar type-II epithelial cells undergo epithelial-mesenchymal transition (EMT). However, the mechanism underlying this change remains unclear. This study aimed to elucidate whether the main toxin of gram-negative bacteria, lipopolysaccharide (LPS), can induce EMT in human alveolar epithelial cells, and the underlying molecular mechanisms. Human alveolar type-II epithelial cells (A549) were used in EMT induction experiments. Cells were collected after LPS exposure, and changes in the expression levels of epithelial and mesenchymal cell markers were determined. Further, the effect of LPS exposure on the expression of Toll-like Receptor 4 (TLR4), Transforming Growth Factor-beta 1 (TGF-β1) and Smad2/3 was assessed. The expression level of a mesenchymal cell marker was also assessed after pharmacological inhibition of TLR4 and TGF-β1 prior to addition of LPS, to identify downstream pathways involved in EMT induction. Results showed that LPS exposure caused significant downregulation of epithelial marker E-cadherin, and upregulation of mesenchymal marker vimentin, together with increased expression of TGF-β1 and activation of the TGF-β1/Smad2/3 pathway. Furthermore, pretreatment with TGF-β1 and TLR4 inhibitors suppressed EMT, and treatment with the latter also reduced the expression level of TGF-β1. Overall, we conclude that LPS directly induces EMT in A549 cells through upregulation of TLR4 and activation of the TGF-β1/Smad2/3 signalling pathway. Our results suggest that LPS-mediated pulmonary fibrosis may occur in ALI patients even if the LPS-induced inflammatory response is inhibited.

FSTL1 aggravates sepsis-induced acute kidney injury through regulating TLR4/MyD88/NF-κB pathway in newborn rats

Background: The aim of this work was to investigate whether Follistatin like 1 (FSTL1) exerted an effect on acute kidney injury (AKI) induced by sepsis, and to explore the molecular mechanism. Methods: A cecal ligation and puncture (CLP) model was established and adenoviral solution was administrated to newborn rats to achieve FSTL1 knockdown. Colorimetric assays measured concentrations of serum creatinine and blood urea nitrogen (BUN) and ELISA assays were performed to examine TNF-α, IL-1β and IL-6 levels in serum and kidney tissues. Kidney histological analysis was performed using hematoxylin-eosin (HE) staining. Protein levels of toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), phosphorylated p65 (p-p65) and total p65 (p65) were determined by western blotting. Results: FSTL1 was significantly up-regulated in kidneys following CLP, but subsequent FSTL1 inhibition alleviated AKI. FSTL1 knockdown following CLP inhibited the production of TNF-α, IL-1β, IL-6 and inactivated the TLR4/MyD88/NF-κB pathway. Furthermore, FSTL1 overexpression activated the TLR4/MyD88/NF-κB pathway following CLP, but TLR4 inhibitor TAK242 abolished this effect. Conclusions: FSTL1 aggravates sepsis-induced acute kidney injury through regulating the TLR4/MyD88/NF-κB pathway.

Electronic Cigarettes Induce Mitochondrial DNA Damage and Trigger TLR9 (Toll-Like Receptor 9)-Mediated Atherosclerosis.

Electronic cigarette (e-cig) use has recently been implicated in promoting atherosclerosis. In this study, we aimed to investigate the mechanism of e-cig exposure accelerated atherosclerotic lesion development. Approach and Results: Eight-week-old ApoE-/- mice fed normal laboratory diet were exposed to e-cig vapor (ECV) for 2 hours/day, 5 days/week for 16 weeks. We found that ECV exposure significantly induced atherosclerotic lesions as examined by Oil Red O staining and greatly upregulated TLR9 (toll-like receptor 9) expression in classical monocytes and in the atherosclerotic plaques, which the latter was corroborated by enhanced TLR9 expression in human femoral artery atherosclerotic plaques from e-cig smokers. Intriguingly, we found a significant increase of oxidative mitochondria DNA lesion in the plasma of ECV-exposed mice. Administration of TLR9 antagonist before ECV exposure not only alleviated atherosclerosis and the upregulation of TLR9 in plaques but also attenuated the increase of plasma levels of inflammatory cytokines, reduced the plaque accumulation of lipid and macrophages, and decreased the frequency of blood CCR2+ (C-C chemokine receptor type 2) classical monocytes. Surprisingly, we found that cytoplasmic mitochondrial DNA isolated from ECV extract-treated macrophages can enhance TLR9 activation in reporter cells and the induction of inflammatory cytokine could be suppressed by TLR9 inhibitor in macrophages. E-cig increases level of damaged mitochondrial DNA in circulating blood and induces the expression of TLR9, which elevate the expression of proinflammatory cytokines in monocyte/macrophage and consequently lead to atherosclerosis. Our results raise the possibility that intervention of TLR9 activation is a potential pharmacological target of ECV-related inflammation and cardiovascular diseases.

N-Formylated Peptide Induces Increased Expression of Both Formyl Peptide Receptor 2 (Fpr2) and Toll-Like Receptor 9 (TLR9) in Schwannoma Cells-An In Vitro Model for Early Inflammatory Profiling of Schwann Cells.

Following nerve injury, disintegrated axonal mitochondria distal to the injury site release mitochondrial formylated peptides and DNA that can induce activation and inflammatory profiling of Schwann cells via formyl peptide receptor 2 (Fpr2) and toll-like receptor 9 (TLR9), respectively. We studied RT4 schwannoma cells to investigate the regulation of Fpr2 and TLR9 after stimulation with fMLF as a prototypical formylated peptide. RT4 cells were treated with fMLF at various concentrations and times with and without pretreatment with inhibitors (chloroquine for activated TLR9, PBP10 for Fpr2). Western blots of Fpr2, TLR9, p-p38, p-NFκB, and IL-6 were compared in relation to inflammatory profiling of RT4 cells and chemokine receptors (CCR2, CXCR4) as potential co-receptors of Fpr2. fMLF stimulation upregulated Fpr2 in RT4 cells at low concentrations (10 nM and 100 nM) but higher concentrations were required (10 µM and 50 µM) when the cells were pretreated with an activated TLR9 inhibitor. Moreover, the higher concentrations of fMLF could modulate TLR9 and inflammatory markers. Upregulation of Fpr2 triggered by 10 nM and 100 nM fMLF coincided with higher levels of chemokine receptors (CCR2, CXCR4) and PKCβ. Treating RT4 cells with fMLF, as an in vitro model of Schwann cells, uncovered Schwann cells’ complex responses to molecular patterns of release from injured axonal mitochondria.