Activate Toll-like Receptor 4 Signaling Research Articles

Inhibition of TLR4 attenuates vascular dysfunction and oxidative stress in diabetic rats.

Hyperglycemia-induced reactive oxygen species (ROS) production plays a major role in the pathogenesis of diabetic vascular dysfunction. However, the underlying mechanisms remain unclear. Toll-like receptor 4 (TLR4), a key component of innate immunity, is known to be activated during diabetes. Therefore, we hypothesize that hyperglycemia activates TLR4 signaling in vascular smooth muscle cells (VSMCs) that triggers ROS production and causes vascular dysfunction. Rat mesenteric VSMCs exposed to high glucose (25 mmol/l) increased TLR4 expression and activated TLR4 signaling via upregulation of myeloid differentiation factor 88 (MyD88). TLR4 inhibitor CLI-095 significantly attenuated elevated levels of ROS and nuclear factor-kappa B (NF-κB) activity in VSMCs exposed to high glucose. Mesenteric arteries from streptozotocin-induced diabetic rats treated with CLI-095 (2 mg/kg/day) intraperitoneally for 2 weeks exhibited reduced ROS generation and attenuated noradrenaline-induced contraction. These results suggest that hyperglycemia-induced ROS generation and NF-κB activation in VSMCs are at least, in part, mediated by TLR4 signaling. Therefore, strategies to block TLR4 signaling pathways pose a promising avenue to alleviate diabetic-induced vascular complications. High glucose-induced TLR4 activation in vascular smooth muscle cells. Inhibition of TLR4 attenuated high glucose-induced ROS production and NF-κB activity in VSMC. Suppression of TLR4 signaling attenuated mesenteric contraction in diabetic rat.

The common food additive carrageenan is not a ligand for Toll-Like- Receptor 4 (TLR4) in an HEK293-TLR4 reporter cell-line model

Carrageenan (CGN) is widely used in the food manufacturing industry as an additive that stabilizes and thickens food products. Standard animal safety studies in which CGN was administered in diet showed no adverse effects. However, several in vitro studies have reported that intestinal inflammation is caused by CGN and that this effect is mediated through Toll-Like-Receptor 4 (TLR4). The purpose of this study was to evaluate the ability of different types of CGN to bind and activate TLR4 signaling. To accomplish this a TLR4/MD-2/CD14/NFκB/SEAP reporter construct in a HEK293 cell line was used. The reporter molecule, secretable alkaline phosphatase (SEAP), was measured as an indicator of TLR4 activation. The test compounds were exposed to this system at concentrations of 0.1, 1, 10, 50, 100, 500, 1000, and 5000 ng/mL for 24 h. Cytotoxicity was evaluated following the 24 h exposure period by LDH leakage and ATP. CGN binding to serum proteins was characterized by Toluidine Blue. The results show that CGN does not bind to TLR4 and is not cytotoxic to the HEK293 cells at the concentrations and experimental conditions tested and that CGN binds tightly to serum proteins.

TLR4 elimination prevents synaptic and myelin alterations and long-term cognitive dysfunctions in adolescent mice with intermittent ethanol treatment.

The adolescent brain undergoes important dynamic and plastic cell changes, including overproduction of axons and synapses, followed by rapid pruning along with ongoing axon myelination. These developmental changes make the adolescent brain particularly vulnerable to neurotoxic and behavioral effects of alcohol. Although the mechanisms of these effects are largely unknown, we demonstrated that ethanol by activating innate immune receptors toll-like receptor 4 (TLR4), induces neuroinflammation and brain damage in adult mice. The present study aims to evaluate whether intermittent ethanol treatment in adolescence promotes TLR4-dependent pro-inflammatory processes, leading to myelin and synaptic dysfunctions, and long-term cognitive impairments. Using wild-type (WT) and TLR4-deficient (TLR4-KO) adolescent mice treated intermittently with ethanol (3.0g/kg) for 2weeks, we show that binge-like ethanol treatment activates TLR4 signaling pathways (MAPK, NFκB) leading to the up-regulation of cytokines and pro-inflammatory mediators (COX-2, iNOS, HMGB1), impairing synaptic and myelin protein levels and causing ultrastructural alterations. These changes were associated with long-lasting cognitive dysfunctions in young adult mice, as demonstrated with the object recognition, passive avoidance and olfactory behavior tests. Notably, elimination of TLR4 receptors prevented neuroinflammation along with synaptic and myelin derangements, as well as long-term cognitive alterations. These results support the role of the neuroimmune response and TLR4 signaling in the neurotoxic and behavioral effects of ethanol in adolescence.

Abstract 4794: NANOG represses mitochondrial energy production and promotes fatty acid synthesis to promote self-renewal in tumor-initiating cells

Abstract Stem cell markers such as Nanog have been implicated in various cancer, but whether they are functionally contributing to cancer pathogenesis has remained unclear. We previously reported that Toll-like receptor 4 (TLR4) signaling regulated NANOG expression in liver cancers (Chen et al., J. Clin. Inv., 2013). Here, we determined novel targets of NANOG in CD133+CD49f+ tumor-initiating cells (TICs) from patient and mouse models of hepatocellular carcinoma (HCC) using genome-wide NANOG-binding site analysis (ChIP-seq). Silencing NANOG inhibits tumor development in HCC mouse models and genesis of TICs as alcohol/obesity-associated increase of serum bacterial products activates TLR4 signaling. To determine novel targets of NANOG in TICs isolated from several HCC models, ChIP-seq in TICs was performed. NANOG binds genes of oxidative phosphorylation and β-oxidation in mitochondria. Silencing NANOG promotes oxidative phosphorylation and β-oxidation, indicating that NANOG is a suppressor of mitochondria-mediated energy production. We identified the TLR4-NANOG oncogenic pathway in the genesis of TICs and liver tumor in HCC mouse models, including alcohol/high-fat-diet-fed hepatitis C virus (HCV) transgenic mice, DEN/phenobarbtal-treated and Spnb2+/- mice. We identified the stimulation of TLR4 transactivates Nanog through E2F1 phosphorylation at serine residues 332/336 and generates TICs, leading to development of liver tumors. Metabolomic analysis demonstrates metabolic reprogramming in TICs through NANOG. Reduction of NANOG expression resulted in an improved cellular energy balance, including reduction of the glycolysis pathway, increased fatty acid consumption and greater uptake of amino acids. The improved energy status is linked to a shift away from glycolysis and a greater reliance on fatty acid β-oxidation and amino acid anaplerosis. Taken together, these results suggest that NANOG-mediated metabolic reprogramming through suppression of mitochondria function in both experimental and clinical HCC downstream of TLR4/NANOG generates TICs and drives liver tumorigenesis. Citation Format: Chia-Lin Chen, Vasu Punj, Linda Sher, Lydia Petrovic, Keigo Machida. NANOG represses mitochondrial energy production and promotes fatty acid synthesis to promote self-renewal in tumor-initiating cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4794. doi:10.1158/1538-7445.AM2014-4794

Lipopolysaccharide-Induced Toll-Like Receptor 4 Signaling in Cancer Cells Promotes Cell Survival and Proliferation in Hepatocellular Carcinoma

Recent studies have shown that toll-like receptor 4 (TLR4) is involved in hepatocarcinogenesis. However, the significance of TLR4 signaling in cancer development and progression remains unclear. The purpose of this study was to investigate the role of TLR4 in cancer cell survival and proliferation in hepatocellular carcinoma (HCC). Fifty-three HCC and ten normal liver specimens were analyzed by immunohistochemistry, and three cell lines (HL-7702, PLC/PRF/5 and HepG2) were used for in vitro studies. Lipopolysaccharide (LPS), a specific ligand of TLR4, was used to activate TLR4 signaling. The effects of LPS-TLR4 signaling on cell survival, proliferation and invasion were examined. Specific inhibitors of NF-κB and MAPK (JNK, ERK and p38) signaling pathways were used to explore the role of each pathway in LPS-TLR4 signaling. TLR4 was overexpressed in HCC cell lines and in human HCC tissues, where it correlated with Ki-67 expression. LPS-induced activation of TLR4 signaling promoted cancer cell survival and proliferation. LPS-TLR4 signaling was associated with regulation on the activation of NF-κB and MAPK signaling pathways. LPS-TLR4-induced activation of ERK and JNK signaling promotes cell proliferation through regulating Bax translocation to mitochondria. Activation of NF-κB and p38 mediates cytotoxicity of LPS, and inhibition on these two pathways promotes cell proliferation in HCC cells. Our results indicate that TLR4 signaling in cancer cells promotes cell survival and proliferation in HCC.

HMGB1-mediated activation of TLR4 signaling in hepatic stellate cells

To determine the potential of the high mobility group box-1 protein 1 (HMGB1) to activate Toll-like receptor 4 (TLR4) signaling in hepatic stellate cells (HSCs) and investigate the subsequent transition of HSC towards the inflammatory phenotype. Three immortalized mouse HSC cell lines, wild-type (JS1), TLR4-/- (JS2) and MyD88-/- (JS3), were subcultured in plates and divided into groups of normal control (untreated), postive control (lipopolysaccaride, LPS treatment), and experimental (HMGB1 treatment). All groups were transfected with luciferase reporter plasmids carrying responsive elements for either the nuclear factor-kappa B (NF-kB) or activator protein-1 AP-1 transcription factors. Following stimulation with normal saline, LPS (100 ng/mL) or HMGB1 (100 ng/mL), the activation of NF-kB or AP-1 was detected by a dual-luciferase reporter assay system. The induction of monocyte chemotactic protein-1 (MCP-1) transcription was determined by measuring the mRNA levels using real time quantitative reverse transcription PCR (qRT-PCR). The secreted protein levels of MCP-1 were determined by enzyme-linked immunosorbent assay (ELISA) of the culture supernatants. Activation of NF-kB- and AP-1-responsive reporters was significantly up-regulated in JS1 cells treated with HMGB1 or LPS, and the activation was coincident with markedly up-regulated transcription and secretion of MCP-1. However, HMGB1 and LPS treatment produced no responsive of the NF-kB and AP-1 reporters, and no increase in expression or secretion of MCP-1, in JS2 or JS3 cells. As an endogeous ligand of TLR4, HMGB1 may activate TLR4 signaling and the TLR4-mediated inflammatory response of HSC.

TNFα and IL-1β are mediated by both TLR4 and Nod1 pathways in the cultured HAPI cells stimulated by LPS

A growing body of evidence recently suggests that glial cell activation plays an important role in several neurodegenerative diseases and neuropathic pain. Microglia in the central nervous system express toll-like receptor 4 (TLR4) that is traditionally accepted as the primary receptor of lipopolysaccharide (LPS). LPS activates TLR4 signaling pathways to induce the production of proinflammatory molecules. In the present studies, we verified the LPS signaling pathways using cultured highly aggressively proliferating immortalized (HAPI) microglial cells. We found that HAPI cells treated with LPS upregulated the expression of TLR4, phospho-JNK (pJNK) and phospho-NF-κB (pNF-κB), TNFα and IL-1β. Silencing TLR4 with siRNA reduced the expression of pJNK, TNFα and IL-1β, but not pNF-κB in the cells. Inhibition of JNK with SP600125 (a JNK inhibitor) decreased the expression of TNFα and IL-1β. Unexpectedly, we found that inhibition of Nod1 with ML130 significantly reduced the expression of pNF-κB. Inhibition of NF-κB also reduced the expression of TNFα and IL-1β. Nod1 ligand, DAP induced the upregulation of pNF-κB which was blocked by Nod1 inhibitor. These data indicate that LPS-induced pJNK is TLR4-dependent, and that pNF-κB is Nod1-dependent in HAPI cells treated with LPS. Either TLR4–JNK or Nod1–NF-κB pathways is involved in the expression of TNFα and IL-1β.

Follistatin-related protein/follistatin-like 1 evokes an innate immune response via CD14 and toll-like receptor 4

Follistatin-related protein (FRP)/follistatin-like 1 (FSTL1) has multi-specific binding nature especially with TGF-β superfamily proteins, and thereby modulates organ development. However, its function in immune systems remains unclear. Previously, we reported FRP interacts with CD14, which is known to mediate toll-like receptor 4 (TLR4) signaling. Here, we investigated whether FRP activates TLR4 signaling. Recombinant FRP induced interleukin 6 or interleukin 8 production from target cells in a CD14- and TLR4-dependent manner. Moreover, similar to lipopolysaccharide (LPS), FRP induced tolerance to the second LPS stimulation. FRP has the function of evoking innate immune responses as one of the endogenous TLR4 agonists. Structured summary of protein interactions:FRP physically interacts with CD14 by fluorescence-activated cell sorting (View interaction)

TLR4 Activity Is Required in the Resolution of Pulmonary Inflammation and Fibrosis after Acute and Chronic Lung Injury

Pulmonary fibrosis is an inflammation-driven lung disease with a poor prognosis and no cure. Here we report that basal toll-like receptor 4 (TLR4) activity is critical for the resolution of acute and chronic inflammation and pulmonary fibrosis in mouse models of lung injury. We found that genetic or pharmacologic inhibition of TLR4 exacerbates bleomycin-induced pulmonary inflammation, fibrosis, dysfunction, and animal death through promoting formation of an immunosuppressive tissue microenvironment and attenuating autophagy-associated degradation of collagen and cell death in the fibrotic lung tissues. In contrast, pharmacologic activation of TLR4 resulted in a quick resolution of acute inflammation, reversed the established pulmonary fibrosis, improved lung function, and rescued mice from death. Similarly, blocking TLR4 impaired the resolution of silica-induced chronic inflammation and fibrosis. Importantly, altering autophagic activity could reverse the TLR4-regulated lung inflammation, fibrosis, dysfunction, and animal death. Rapamycin, an autophagy activator, reversed the effects of TLR4 antagonism. In contrast, inhibition of autophagy by 3-methyladenine reversed the proresolving and antifibrotic roles of TLR4 agonists and increased animal death. These results not only highlight a pivotal role for TLR4-mediated basal immunity, particularly autophagic activity, in the proresolution of inflammation and fibrosis after chemical-induced lung injury but also provide proof for the concept for activating TLR4 signaling, particularly TLR4-mediated autophagy, as a novel therapeutic strategy against chronic fibroproliferative diseases that are unresponsive to current therapy.

Hypoxia Activates Toll-like Receptor 4 Signaling in Primary Mouse Hepatocytes Through the Receptor Clustering within Lipid Rafts

Purpose: Transient hypoxia is an initial event that accentuates ischemia/reperfusion (I/R) injury in the liver. Hepatic ischemia/reperfusion (I/R) injury is largely related to innate immunity via Toll-like receptor 4 (TLR4) signaling. However, the mechanism by which hypoxia could lead to activate TLR4 signaling remains unclear. Therefore, the aim of this experimental study investigates how TLR4 signalling is activated by hypoxia. Methods: Hepatocytes were isolated from male wild-type (C57BL/6) mice (8∼12 weeks old) by an in situ collagenase (Type Ⅳ, Sigma-Aldrich) perfusion technique. In this study, using primary mouse hepatocytes in culture to 1% oxygen, detection of TLR4 translocation to the lipid rafts on the cell membrane by immunofluorescence staining and immunoblotting was saught. Results: Hypoxia caused TLR4/MD2 and β2-Integrin (CD11b/CD18) translocation to lipid rafts associated with CD14 in hepatocytes. The cholesterol sequestering agent, Nystatin and Filipin prevented hypoxia-induced TLR4/MD2 translocation to lipid rafts. Consistent with a role for oxidative stress in this effect, in vitro H2O2 treatment of hepatocytes similarly caused TLR4/MD2 translocation to lipid rafts. In addition, translocation of hypoxia-induced TLR4 complex was inhibited by N-acetylcysteine (NAC) demonstrating that the activation of TLR4 signaling is dependent on ROS. Further, the cholesterol sequestering agent, nystatin, prevented hypoxia-induced high mobility group box 1 (HMGB1) release in hepatocytes. Conclusion: These results suggest that ROS dependent TLR4 signaling is achieved following receptor translocation to the lipid raft in hepatocytes. We hypothesized that this mechanism is required for the release of HMGB1, an early mediator of injury and inflammation in hepatic I/R injury.

Evidence that opioids may have toll-like receptor 4 and MD-2 effects

Opioid-induced proinflammatory glial activation modulates wide-ranging aspects of opioid pharmacology including: opposition of acute and chronic opioid analgesia, opioid analgesic tolerance, opioid-induced hyperalgesia, development of opioid dependence, opioid reward, and opioid respiratory depression. However, the mechanism(s) contributing to opioid-induced proinflammatory actions remains unresolved. The potential involvement of toll-like receptor 4 (TLR4) was examined using in vitro, in vivo, and in silico techniques. Morphine non-stereoselectively induced TLR4 signaling in vitro, blocked by a classical TLR4 antagonist and non-stereoselectively by naloxone. Pharmacological blockade of TLR4 signaling in vivo potentiated acute intrathecal morphine analgesia, attenuated development of analgesic tolerance, hyperalgesia, and opioid withdrawal behaviors. TLR4 opposition to opioid actions was supported by morphine treatment of TLR4 knockout mice, which revealed a significant threefold leftward shift in the analgesia dose response function, versus wildtype mice. A range of structurally diverse clinically-employed opioid analgesics was found to be capable of activating TLR4 signaling in vitro. Selectivity in the response was identified since morphine-3-glucuronide, a morphine metabolite with no opioid receptor activity, displayed significant TLR4 activity, whilst the opioid receptor active metabolite, morphine-6-glucuronide, was devoid of such properties. In silico docking simulations revealed ligands bound preferentially to the LPS binding pocket of MD-2 rather than TLR4. An in silico to in vitro prediction model was built and tested with substantial accuracy. These data provide evidence that select opioids may non-stereoselectively influence TLR4 signaling and have behavioral consequences resulting, in part, via TLR4 signaling.

Lactobacilli inhibit interleukin-8 production induced by Helicobacter pylori lipopolysaccharide-activated Toll-like receptor 4

To investigate the effect of Lactobacillus bulgaricus (LBG) on the Toll-like receptor 4 (TLR4) pathway and interleukin-8 (IL-8) production in SGC-7901 cells treated with Helicobacter pyloriSydney strain 1 lipopolysaccharide (H pyloriSS1-LPS). SGC-7901 cells were treated with H pyloriSS1-LPS in the presence or absence of pretreatment for 1 h with viable LBG or supernatant recovered from LBG culture MRS broth (LBG-(s)). Cellular lysates were prepared for Western blot with anti-TLR4, anti-transforming growth factor beta-activated kinase 1 (TAK1), anti-phospho-TAK1, anti-nuclear factor kappaB (NF-kappaB), anti-p38 mitogen-activated protein kinase (p38MAPK), and anti-phospho-p38MAPK antibodies. The amount of IL-8 in cell culture medium was measured by ELISA. H pyloriSS1-LPS up-regulated the expression of TLR4, stimulated the phosphorylation of TAK1, subsequently enhanced the activation of NF-kappaB and the phosphorylation of p38MAPK in a time-dependent manner, leading to augmentation of IL-8 production in SGC-7901 cells. Viable LBG or LBG-(s) pretreatment attenuated the expression of TLR4, inhibited the phosphorylation of TAK1 and p38MAPK, prevented the activation of NF-kappaB, and consequently blocked IL-8 production. H pyloriSS1-LPS induces IL-8 production through activating TLR4 signaling in SGC-7901 cells and viable LBG or LBG-(s) prevents H pyloriSS1-LPS-mediated IL-8 production via inhibition of the TLR4 pathway.

Activation of Toll-like Receptor 4 on Tumor CellsIn vitroInhibits Subsequent Tumor GrowthIn vivo

Although an eruption of information on the role of Toll-like receptor 4 (TLR4), the main receptor for bacterial lipopolysaccharide, in activating macrophages and dendritic cells has emerged, very little is known about the role of TLR4 present on epithelial cells from sterile environments like tumors. The main goal of this work was to investigate the consequences of TLR4 activation present on tumor cells in two different animal models of cancer: the Dunning rat prostate cancer and the B16 murine melanoma models. We show that (a) activating TLR4 signaling in two different tumor cell lines in vitro modifies the tumor outgrowth in vivo; (b) this effect is not due to a direct consequence of TLR4 signaling on the proliferation/apoptosis balance of the tumor cells; (c) the T-cell compartment is somehow involved in the described phenomenon because the inhibitory effect observed is not seen in athymic nude mice; and (d) tumor-infiltrating lymphocytes purified from tumors induced by TLR4-activated cells show strong induction of IFN gamma transcript in detriment of interleukin-10 transcript, suggesting a change in their functionality. We hypothesize that TLR4 signaling in tumor cells in vitro induces the expression of proinflammatory mediators, which could dramatically alter the maturation state of dendritic cells present at the site of inoculation, switching the type of immune response elicited against the tumor. These results open up new avenues for understanding the role of TLR4 in tumor cells and for identifying potential new therapy strategies for cancer.

A synthetic analog of α-galactosylceramide induces macrophage activation via the TLR4-signaling pathways

α-Galactosylceramide (α-GalCer), a bioactive glycolipid isolated from the marine sponge Agelas mauritianus, is a potent immunomodulator with therapeutic potential for the treatment of autoimmune diseases and cancer. The Toll-like receptor 4 (TLR4), one of the promising molecular targets for immune-modulating drugs, is commonly expressed in innate immune cells especially macrophages and dendritic cells. Currently, whether α-GalCer can activate TLR4 signaling pathways remains unreported. In this study, we examined the effects of α-GalCer and its various structural analogs, CCL-1∼47, on TLR4 activation. We found that one α-GalCer analog (CCL-34), but not α-GalCer itself, strongly stimulated NF-κB activity in RAW 264.7 cells. CCL-34 activated NF-κB in a TLR4-dependent manner and stimulated TNF-α production in bone marrow cells of TLR4-functional C3H/HeN mice but not in those of TLR4-defective C3H/HeJ mice. Furthermore, CCL-34 treatment stimulated NF-κB activation and IL-8 production in a 293 cell line constitutively expressing human TLR4, MD-2 and CD14. Treatment of RAW 264.7 cells with CCL-34 also activated TLR4-downstream mitogen-activated protein kinases (ERK, JNK and p38), induced expression of TLR4-downstream genes (TNF-α, IL-6, IL-1β and iNOS) and promoted production of cytokines characteristic of activated macrophages. CCL-34-treated RAW 264.7 cells acquired a distinct morphology similar to that of LPS-activated macrophages and exhibited higher phagocytotic activity. Moreover, treatment with a TLR4-neutalizing antibody inhibited the CCL-34-induced morphological alteration. In summary, we identify a novel synthetic compound CCL-34 that can activate macrophages via TLR4-dependent signaling pathways. Our results suggest that CCL-34 is an immune modulator and may serve as a potential drug lead for immunotherapy.