To Toll-like Receptor 4 Expression Research Articles

205-LB: ERK Is Required for LPS-Induced TLR4 Internalization in Macrophages

Insulin resistance is associated with circulating levels of lipopolysaccharide (LPS) released from the gut. Animal and human studies demonstrate that obesity increases gut permeability leading to elevated plasma LPS levels resulting in inflammation and metabolic dysfunction. Binding of LPS to Toll like receptor 4 (TLR4) leads to the internalization and trafficking of TLR4, inducing activation of downstream signaling pathways. TLR4 internalization in macrophages leads to activation of pro-inflammatory signaling pathways and production of factors linked to the development of insulin resistance. The extracellular signal-regulated kinase 1 and 2 (ERK1 and 2) are activated downstream of TLR4 and this activation is associated with insulin resistance. We hypothesized that ERK1 and ERK2 regulate TLR4 internalization in macrophages that are exposed to LPS. We examined whether inhibition of ERK activity blocks LPS-mediated internalization of TLR4 in bone marrow derived macrophages (BMDM) .We used siRNA to knockdown ERK1, ERK2, or both. BMDM were treated with LPS (100 ng/ml, 6hr) . Loss of cell surface TLR4 expression was measured by flow cytometry as a readout for TLR4 internalization. LPS decreased TLR4 surface expression by 31.3%, but knockdown of ERK1, ERK2, or both prevented LPS-induced decrease of TLR4 surface expression (5.6%, no decrease, 0.9%, respectively) . In addition, knockdown of either ERK1, ERK2, or both in RAW264.7 cells prevented LPS-induced activity of Rab5, the early endosomal protein associated with TLR4 translocation. In summary, ERK regulates TLR4 endocytosis and trafficking in macrophages. We propose that ERK positively regulates LPS-mediated TLR4 internalization and inhibition of ERK signaling will protect against insulin resistance. Disclosure P. F. Chang: n/a. S. M. Reyna: n/a. Funding National Institutes of Health (SC2GM127272)

Phlorotannins from Ecklonia cava Attenuates Palmitate-Induced Endoplasmic Reticulum Stress and Leptin Resistance in Hypothalamic Neurons.

Leptin resistance in the hypothalamus has an essential role in obesity. Saturated fatty acids such as palmitate bind to Toll-like receptor 4 (TLR4) and lead to endoplasmic reticulum (ER) stress and leptin resistance. In this study, we evaluated whether extracts of Ecklonia cava would attenuate the ER stress induced by palmitate and reduce leptin resistance in hypothalamic neurons and microglia. We added palmitate to these cells to mimic the environment induced by high-fat diet in the hypothalamus and evaluated which of the E. cava phlorotannins—dieckol (DK), 2,7-phloroglucinol-6,6-bieckol (PHB), pyrogallol-phloroglucinol-6,6-bieckol (PPB), or phlorofucofuroeckol-A (PFFA)—had the most potent effect on attenuating leptin resistance. TLR4 and NF-κB expression induced by palmitate was attenuated most effectively by PPB in both hypothalamic neurons and microglia. ER stress markers were increased by palmitate and were attenuated by PPB in both hypothalamic neurons and microglia. Leptin resistance, which was evaluated as an increase in SOCS3 and a decrease in STAT3 with leptin receptor expression, was increased by palmitate and was decreased by PPB in hypothalamic neurons. The culture medium from palmitate-treated microglia increased leptin resistance in hypothalamic neurons and this resistance was attenuated by PPB. In conclusion, PPB attenuated leptin resistance by decreasing ER stress in both hypothalamic neurons and microglia.

Baicalin ameliorates neuroinflammation-induced depressive-like behavior through inhibition of toll-like receptor 4 expression via the PI3K/AKT/FoxO1 pathway

BackgroundBaicalin, which is isolated from Radix Scutellariae, possesses strong biological activities including an anti-inflammation property. Recent studies have shown that the anti-inflammatory effect of baicalin is linked to toll-like receptor 4 (TLR4), which participates in pathological changes of central nervous system diseases such as depression. In this study, we explored whether baicalin could produce antidepressant effects via regulation of TLR4 signaling in mice and attempted to elucidate the underlying mechanisms.MethodsA chronic unpredictable mild stress (CUMS) mice model was performed to explore whether baicalin could produce antidepressant effects via the inhibition of neuroinflammation. To clarify the role of TLR4 in the anti-neuroinflammatory efficacy of baicalin, a lipopolysaccharide (LPS) was employed in mice to specially activate TLR4 and the behavioral changes were determined. Furthermore, we used LY294002 to examine the molecular mechanisms of baicalin in regulating the expression of TLR4 in vivo and in vitro using western blot, ELISA kits, and immunostaining. In the in vitro tests, the BV2 microglia cell lines and primary microglia cultures were pretreated with baicalin and LY292002 for 1 h and then stimulated 24 h with LPS. The primary microglial cells were transfected with the forkhead transcription factor forkhead box protein O 1 (FoxO1)-specific siRNA for 5 h and then co-stimulated with baicalin and LPS to investigate whether FoxO1 participated in the effect of baicalin on TLR4 expression.ResultsThe administration of baicalin (especially 60 mg/kg) dramatically ameliorated CUMS-induced depressive-like symptoms; substantially decreased the levels of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) in the hippocampus; and significantly decreased the expression of TLR4. The activation of TLR4 by the LPS triggered neuroinflammation and evoked depressive-like behaviors in mice, which were also alleviated by the treatment with baicalin (60 mg/kg). Furthermore, the application of baicalin significantly increased the phosphorylation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and FoxO1. The application of baicalin also promoted FoxO1 nuclear exclusion and contributed to the inhibition of the FoxO1 transactivation potential, which led to the downregulation of the expression of TLR4 in CUMS mice or LPS-treated BV2 cells and primary microglia cells. However, prophylactic treatment of LY294002 abolished the above effects of baicalin. In addition, we found that FoxO1 played a vital role in baicalin by regulating the TLR4 and TLR4-mediating neuroinflammation triggered by the LPS via knocking down the expression of FoxO1 in the primary microglia.ConclusionCollectively, these results demonstrate that baicalin ameliorated neuroinflammation-induced depressive-like behaviors through the inhibition of TLR4 expression via the PI3K/AKT/FoxO1 pathway.

Inhibition of Neuroinflammation by AIBP: Spinal Effects upon Facilitated Pain States

Apolipoprotein A-I binding protein (AIBP) reduces lipid raft abundance by augmenting the removal of excess cholesterol from the plasma membrane. Here, we report that AIBP prevents and reverses processes associated with neuroinflammatory-mediated spinal nociceptive processing. The mechanism involves AIBP binding to Toll-like receptor-4 (TLR4) and increased binding of AIBP to activated microglia, which mediates selective regulation of lipid rafts in inflammatory cells. AIBP-mediated lipid raft reductions downregulate LPS-induced TLR4 dimerization, inflammatory signaling, and expression of cytokines in microglia. In mice, intrathecal injections of AIBP reduce spinal myeloid cell lipid rafts, TLR4 dimerization, neuroinflammation, and glial activation. Intrathecal AIBP reverses established allodynia in mice in which pain states were induced by the chemotherapeutic cisplatin, intraplantar formalin, or intrathecal LPS, all of which are pro-nociceptive interventions known to be regulated by TLR4 signaling. These findings demonstrate a mechanism by which AIBP regulates neuroinflammation and suggest the therapeutic potential of AIBP in treating preexisting pain states.

Maximal Exercise Alters the Inflammatory Phenotype and Response of Mononuclear Cells.

Monocytes express the CD14 receptor that facilitates lipopolysaccharide (LPS) ligation to toll-like receptor 4 (TLR4) to elicit production of interleukin (IL)-6, IL-10, and tumor necrosis factor alpha (TNF-α). However, proinflammatory conditions, such as strenuous exercise, increase the percentage of monocytes expressing CD16, a receptor that enhances LPS stimulated TNF-α production. Therefore, we examined whether maximal treadmill exercise would alter the inflammatory phenotype of classical (CD14/CD16) and proinflammatory monocytes (intermediate [CD14/CD16] and nonclassical [CD14/CD16]), evidenced by changes in TLR4, CD14, and CD16 receptor expression, and their inflammatory response to ex vivo LPS stimulation. Human mononuclear cells from 25 male participants (age, 24.2 ± 4.0 yr) were isolated before and after exercise to assess TLR4, CD14, and CD16 expression by flow cytometry and ex vivo production of LPS-stimulated inflammatory cytokines (IL-6, IL-10, and TNF-α). Exercise reduced the percentage of classical monocytes and increased the percentage of intermediate and nonclassical monocytes. In addition, TLR4 expression decreased on classical and intermediate monocytes, but not the nonclassical monocyte subset. Furthermore, although CD14 expression decreased on all monocyte subsets, CD16 expression increased on intermediate monocytes only. In parallel with these phenotypic changes, the inflammatory milieu shifted toward a proinflammatory response after LPS stimulation (decreased IL-6 and IL-10 and increased IL-6 to IL-10 ratio and TNF-α production). These findings demonstrate that acute maximal exercise elicits a proinflammatory phenotype of isolated monocytes exposed to LPS and highlight potential mechanisms that will help elucidate the role of acute and chronic exercise on the innate immune response of circulating monocytes.

Endotoxin, Toll-like Receptor-4, and Atherosclerotic Heart Disease

Background:Endotoxin is a lipopolysaccharide (LPS) constituent of the outer membrane of most gram negative bacteria. Ubiquitous in the environment, it has been implicated as a cause or con-tributing factor in several disparate disorders from sepsis to heatstroke and Type II diabetes mellitus. Starting at birth, the innate immune system develops cellular defense mechanisms against environmen-tal microbes that are in part modulated through a series of receptors known as toll-like receptors. Endo-toxin, often referred to as LPS, binds to toll-like receptor 4 (TLR4)/ myeloid differentiation protein 2 (MD2) complexes on various tissues including cells of the innate immune system, smooth muscle and endothelial cells of blood vessels including coronary arteries, and adipose tissue. Entry of LPS into the systemic circulation ultimately leads to intracellular transcription of several inflammatory mediators. The subsequent inflammation has been implicated in the development and progression atherosclerosis and subsequent coronary artery disease and heart failure.Objective:The potential roles of endotoxin and TLR4 are reviewed regarding their role in the pathogen-esis of atherosclerotic heart disease.Conclusion:Atherosclerosis is initiated by inflammation in arterial endothelial and subendothelial cells, and inflammatory processes are implicated in its progression to clinical heart disease. Endotoxin and TLR4 play a central role in the inflammatory process, and represent potential targets for therapeutic intervention. Therapy with HMG-CoA inhibitors may reduce the expression of TLR4 on monocytes. Other therapeutic interventions targeting TLR4 expression or function may prove beneficial in athero-sclerotic disease prevention and treatment.

Effect of LPS on the Viability and Proliferation of Human Oral and Esophageal Cancer Cell Lines

The esophagus and mouth tumors are very frequent malignancies worldwide. Lipopolysaccharides (LPS) are capable of regulating gene expression of pro-inflammatory cytokines by binding to toll-like receptor 4 (TLR4). Recent studies show that LPS can increase the migration ability of human esophageal cancer cell line HKESC-2 by increasing its adhesion properties. However, the effect of LPS has not been tested on viability of human esophageal and oral cancer cells. This study aimed to determine the action of LPS on the cell proliferation and viability in OE19 (adenocarcinoma) and OE21 (squamous carcinoma) cell lines, representative of human esophageal cancer, and HN30 cell line, representative of human oral carcinoma. LPS was used as treatment to OE19 and OE21 cells, and PgLPS (Porphyromonasgingivalis lipopolysaccharide) to HN30 cells. Viability was assessed by MTT assay and proliferation by cell counting. TLR4 expression was evaluated by real-time PCR. LPS at higher concentrations decreased significantly cell viability in both cell lines, adenocarcinoma (OE19) and squamous esophageal carcinoma (OE21) at different times of treatment. In addition, both cell lines, OE19 and OE21, expressed TLR4 receptor. Taken together, our data demonstrated that LPS at high concentrations might contribute to tumor death, in agreement with previously data.

Low shear stress induced HMGB1 translocation and release via PECAM-1/PARP-1 pathway to induce inflammation response.

Low shear stress (LSS) plays a critical role in the site predilection of atherosclerosis through activation of cellular mechanosensors, such as platelet endothelial cell adhesion molecule 1 (PECAM-1). Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that regulates the expression of various inflammatory cytokines. The nuclear enzyme high mobility group box 1 (HMGB1) can induce inflammation response by binding to toll-like receptor 4 (TLR4). In the present study, we aimed to investigate the role and mechanism of HMGB1 in LSS induced inflammation in human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated by undisturbed shear stress (USS, 1 Pa) and LSS (0.4 Pa) in our experiments. Gene expression was inhibited by small interfering RNA (siRNA). ICAM-1 expression was regulated by LSS in a time dependent manner. LSS can induce HMGB1 translocation from nucleus to cytoplasm and release. Compared with the USS, LSS could increase the protein expression of PECAM-1 and PARP-1 as well as the secretion of TNF-α and IL-1β. LSS induced the translocation of HMGB1 from nucleus to cytoplasm. Inhibition of HGMB1 reduced LSS-induced inflammatory response. Inhibition of PARP-1 suppressed inflammatory response through inhibiting TLR4 expression and HMGB1 translocation. PECAM-1 inhibition reduced LSS-induced ICAM-1 expression, TNF-α and IL-1β secretion, and monocytes adhesion. LSS can induce inflammatory response via PECAM-1/PARP-1/HMGB1 pathway. PARP-1 plays a fundamental role in HMGB1 translocation and TLR4 expression. Inhibition of PARP-1 may shed light on the treatment of HMGB1 involved inflammation during atherosclerosis.

Association between toll-like receptor 4 expression and neural stem cell proliferation in the hippocampus following traumatic brain injury in mice.

Whether or how neural stem cells (NSCs) respond to toll-like receptor 4 (TLR4) in an inflammatory environment caused by traumatic brain injury (TBI) has not been understood. In the present study, association between TLR4 expression and NSCs proliferation in the hippocampus was investigated in a mouse model of TBI using controlled cortical impact (CCI). Hippocampal proliferating cells were labeled with the thymidine analog 5-bromo-2-deoxyuridine (BrdU). In order to identify NSCs, the proliferating cells were further co-labeled with BrdU/sex determination region of Y chromosome related high mobility group box gene 2 (SOX2). Morphological observation on the expression of BrdU, SOX2, and TLR4 in the hippocampus was performed by inmmunofluorescence (IF). Relative quantification of TLR4 expression at the protein and mRNA level was performed using Western blotting and real-time polymerase chain reaction (PCR). It was observed that BrdU+/SOX2+ cells accounted for 95.80% ± 7.91% among BrdU+ cells; several BrdU+ cells and SOX2+ cells in the hippocampus were also TLR4-positive post injury, and that BrdU+ cell numbers, together with TLR4 expression at either protein or mRNA level, increased significantly in TBI mice over 1, 3, 7, 14, and 21 days survivals and changed in a similar temporal pattern with a peak at 3 day post-injury. These results indicate that hippocampal proliferating cells (suggestive of NSCs) expressed TLR4, and that there was a potential association between increased expression of TLR4 and the proliferation of NSCs post TBI. It is concluded that hippocampal TLR4 may play a potential role in endogenous neurogenesis after TBI.

Toll-Like Receptor 4 Mediates Acute Lung Injury Induced by High Mobility Group Box-1

BackgroundAcute lung injury (ALI) is considered to be the major cause of respiratory failure in critically ill patients. Clinical studies have found that in patients with sepsis and after hemorrhage, the elevated level of high mobility group box-1(HMGB-1) in their circulation is highly associated with ALI, but the underlying mechanism remains unclear. Extracellular HMGB-1 has cytokine-like properties and can bind to Toll-like Receptor-4 (TLR4), which was reported to play an important role in the pathogenesis of ALI. The aim of this study was to determine whether HMGB-1 directly contributes to ALI and whether TLR4 signaling pathway is involved in this process.MethodsRecombinant human HMGB-1 (rhHMGB-1) was used to induce ALI in male Sprague-Dawley rats. Lung specimens were collected 2 h after HMGB-1 treatment. The levels of TNF-α, IL-1β, TLR4 protein, and TLR4 mRNA in lungs as well as pathological changes of lung tissue were assessed. In cell studies, the alveolar macrophage cell line, NR8383, was collected 24 h after rhHMGB-1 treatment and the levels of TNF-α and IL-1β in cultured medium as well as TLR4 protein and mRNA levels in the cell were examined. TLR4-shRNA-lentivirus was used to inhibit TLR4 expression, and a neutralizing anti-HMGB1 antibody was used to neutralize rhHMGB-1 both in vitro and in vivo.ResultsFeatures of lung injury and significant elevation of IL-1β and TNF-α levels were found in lungs of rhHMGB-1-treated animals. Cultured NR8383 cells were activated by rhHMGB-1 treatment and resulted in the release of IL-1β and TNF-α. TLR4 expression was greatly up-regulated by rhHMGB-1. Inhibition of TLR4 or neutralization of HMGB1 with a specific antibody also attenuated the inflammatory response induced by HMGB-1 both in vivo and in vitro.ConclusionHMGB-1 can activate alveolar macrophages to produce proinflammatory cytokines and induce ALI through a mechanism that relies on TLR-4.

Down-Regulation of MicroRNA-146a in the Early Stage of Liver Ischemia-Reperfusion Injury

BackgroundMicroRNAs (miRNAs), 21-23-nucleotide noncoding RNAs, act as regulators of gene expression transcriptionally. MicroRNA-146a(miR-146a) has been demonstrated to be one of the key molecules in oncogenesis and inflammatory responses. Few data describe the expression of miR-146a in liver ischemia-reperfusion (IR) injury. The present study sought to explore the relationship of miR-146a to Toll-like receptor 4 (TLR4) signaling pathways in a rat model of warm IR injury. MethodsThe expression of miR-146a was detected by real-time reverse-transcriptase polymerase chain reaction using a partial warm hepatic IR injury model. The expression of TLR4, tumor necrosis factor receptor-associated factor 6 (TRAF6), and interleukin-1 receptor-associated kinase (IRAK 1) protein was assessed by Western blotting as well as the signaling pathways induced by TLR4. ResultsThe expression of hepatic miR-146a was down-regulated in IR injury during the 24 hours after reperfusion, reaching the lowest level at 6 hours after reperfusion. Increases in TLR4, TRAF6, and IRAK1 were accompanied by decreased miR-146a during the 24 hours after reperfusion, peaking at 6 hours. Immunohistochemistry showed cytoplasmic expression of cells positive for TLR4, and nuclear expression of cells positive for nuclear factor κB p65 and c-jun to be increased among IR groups after reperfusion. ConclusionmiR-146a was down-regulated in the early stage of liver IR injury.

Moderate temperature alterations affect Gram-negative immune signalling in ex vivo whole blood

Background. Alterations in body temperature may influence immune system function and consequently affect the risk of infection and inflammatory diseases. Lipopolysaccharide (LPS) from gram-negative bacteria induces production of inflammatory cytokines after ligand binding to Toll-like receptor 4 (TLR4) on immune cells (especially monocytes/ macrophages). Our aim was to explore how clinically relevant hypo- and hyperthermia affect this signalling in an ex vivo whole blood model, and investigate if the cytokine response was correlated with monocyte TLR4 expression level. Methods. Blood from 11 healthy volunteers was incubated with LPS 10 ng/ml for 6 h at 33, 37 or 40°C. The concentrations of selected pro-inflammatory (tumour necrosis factor-α (TNF-α) and interleukin (IL)-1β) and anti-inflammatory (IL-10) cytokines were measured in plasma, and the surface expression of TLR4 was quantified on CD14 + monocytes. Results. Monocyte TLR4 expression and plasma IL-1β were inversely related to temperature. The TNF-α production was unaffected by hypothermia but increased significantly during hyperthermia, whereas plasma IL-10 was significantly reduced during both hypo- and hyperthermic incubation. No correlation was found between TLR4 expression and cytokine concentrations. During hypothermia, the TNF-α/IL-10 and IL-1β/IL-10 ratios increased seven and nine times, respectively. Hyperthermia increased the TNF-α/IL-10 ratio, but to a lesser extent (doubling), whereas the IL-1β/IL-10 ratio remained unchanged. Conclusion. Hypothermia significantly changed the cytokine ratios in the pro-inflammatory direction. In comparison, the effect of hyperthermia was sparse, with a modest increase in the TNF-α/IL-10 ratio only. No association was found between LPS-stimulated cytokine production and TLR4 expression on CD14 + monocytes.

Alteration of CXCR7 Expression Mediated by TLR4 Promotes Tumor Cell Proliferation and Migration in Human Colorectal Carcinoma

The link between inflammation and colorectal carcinoma has been acknowledged. However, the impact of bacterial lipopolysaccharide (LPS) binding to Toll-like receptor 4 (TLR4) on chemokine receptors in human colorectal carcinoma cells still remains to be elucidated. The present study shows that exposure to LPS elevated CXC chemokine receptor 7 (CXCR7) expression in colorectal carcinoma SW480 and Colo 205 cell lines expressing TLR4/myeloid differential protein (MD-2). CXCR7 is associated with SW480 cell proliferation and migration. However, exposure of SW480 and Colo 205 cells to LPS had no effect on CXCR4 expression. To further support the above results, the expression of TLR4, MD-2, and CXCR7 was analyzed in human colorectal carcinoma tissues. Higher rates of TLR4 (53%), MD-2 (70%), and CXCR7 (29%) expression were found in colorectal carcinoma tissues than in normal tissues. We demonstrated that the recombination of TLR4, MD-2 and CXCR7 strongly correlated with tumor size, lymph node metastasis and distant metastasis in colorectal carcinoma tissue samples (p = 0.037, p = 0.002, p = 0.042, resp.). Accordingly, simultaneous examination of the expression of TLR4, MD-2 and CXCR7 in cancer tissues of colorectal carcinoma may provide valuable prognostic diagnosis of carcinoma growth and metastasis. Interplay of TLR4, MD-2 and CXCR7 may be of interest in the context of novel immunomodulatory therapies for colorectal carcinoma.

Constitutive activation of epithelial TLR4 augments inflammatory responses to mucosal injury and drives colitis-associated tumorigenesis

Chronic intestinal inflammation culminates in cancer and a link to Toll-like receptor-4 (TLR4) has been suggested by our observation that TLR4 deficiency prevents colitis-associated neoplasia. In the current study we address the effect of the aberrant activation of epithelial TLR4 on induction of colitis and colitis-associated tumor development. We take a translational approach to address the consequences of increased TLR signaling in the intestinal mucosa. Mice transgenic for a constitutively active TLR4 under the intestine-specific villin promoter (villin-TLR4 mice) were treated with dextran sodium sulfate (DSS) for acute colitis and azoxymethane (AOM)-DSS TLR4 expression was analyzed by immunohistochemistry in colonic tissue from patients with ulcerative colitis (UC) and UC-associated cancer. The effect of an antagonist TLR4 antibody (Ab) was tested in prevention of colitis-associated neoplasia in the AOM-DSS model. Villin-TLR4 mice were highly susceptible to both acute colitis and colitis-associated neoplasia. Villin-TLR4 mice had increased epithelial expression of COX-2 and mucosal PGE₂ production at baseline. Increased severity of colitis in villin-TLR4 mice was characterized by enhanced expression of inflammatory mediators and increased neutrophilic infiltration. In human UC samples, TLR4 expression was upregulated in almost all colitis-associated cancer and progressively increased with grade of dysplasia. As a proof of principle, a TLR4/MD-2 antagonist antibody inhibited colitis-associated neoplasia in the mouse model. Our results show that regulation of TLRs can affect the outcome of both acute colitis and its consequences, cancer. Targeting TLR4 and other TLRs may ultimately play a role in prevention or treatment of colitis-associated cancer.

Role of TLR4 for paclitaxel chemotherapy in human epithelial ovarian cancer cells

Paclitaxel has been reported to be a ligand to Toll like receptor 4 (TLR4). Myeloid differentiation factor 88(MyD88) was described as a myeloid differentiation primary response gene. TLR4 signalling owns two pathways: MyD88-dependent and MyD88-independent pathways. XIAP is a key member of the inhibitor of apoptosis protein family. Akt is a major downstream target of growth factor receptor tyrosine kinases, which negatively regulates apoptotic pathways through phosphorylation (pAkt). The aim of the present study is to investigate the role of TLR4 in paclitaxel resistance of ovarian cancer cells. We reconstructed the RNA interference expression vector, pGenesil-1-U6 specifically targeting TLR4 mRNA, which was stable transfected into the human ovarian cancer cell line SKOV3 (MyD88-positive expression) and A2780 (MyD88-negative expression). Cell proliferation, cell cycle distribution and cell apoptosis were assessed in the cells transfected with scramble control shRNA (SKOV3/shControl, A2780/shControl) and TLR4 shRNA (SKOV3/shTLR4, A2780/shTLR4) to explore the possible functions of TLR4 in ovarian cancer cells growth. The expression of TLR4, MyD88, XIAP, Akt and pAkt was analysed by Western blot analysis. A knockdown of TLR4 levels down-regulated the expression of XIAP and pAkt. And it restored the inhibitory effect of paclitaxel on cell proliferation and impeding cell cycle progression in SKOV3 cells. It suggests that TLR4 negatively regulates paclitaxel chemotherapy and MyD88 is an essential downstream factor to TLR4 signalling for this resistance. Knockdown of TLR4 induces paclitaxel chemosensitivity which might depress the Akt pathway. The TLR4-MyD88 signalling represents an important source to promote tumour growth.

Wnt5a is expressed in murine and human atherosclerotic lesions

Atherosclerosis is an inflammatory disease involving the accumulation of macrophages in the intima. Wnt5a is a noncanonical member of the Wnt family of secreted glycoproteins. Recently, human macrophages have been shown to express Wnt5a upon stimulation with bacterial pathogens in vitro and in granulomatous lesions in the lung of Mycobacterium tuberculosis-infected patients. Wnt5a expression has also been liked to Toll-like receptor-4 (TLR-4), an innate immune receptor implicated in atherosclerosis. These observations, along with the fact that Wnt5a is involved in cell migration and proliferation, led us to postulate that Wnt5a plays a role in atherosclerosis. To investigate this hypothesis, we characterized Wnt5a expression in murine and human atherosclerotic lesions. Tissue sections derived from the aortic sinus to the aortic arch of apolipoprotein E-deficient mice and sections derived from the carotid arteries of patients undergoing endarterectomy were subjected to immunohistochemical analysis. All samples were found to be positive for Wnt5a with predominant staining in the areas of macrophage accumulation within the intima. In parallel, we probed for the presence of TLR-4 and found coincident TLR-4 and Wnt5a expression. For both the Wnt5a and TLR-4 staining, consecutive tissue sections treated with an isotype- and species-matched Ig served as a negative control and exhibited little, if any, reactivity. Quantitative RT-PCR revealed that Wnt5a mRNA expression in RAW264.7 murine macrophages can be induced by stimulation with LPS, a known ligand for TLR-4. Combined, these findings demonstrate for the first time Wnt5a expression in human and murine atherosclerotic lesions and suggest that cross talk between TLR-4 and Wnt5a is operative in atherosclerosis.

Mutational Analysis of Membrane and Soluble Forms of Human MD-2

Toll-like receptor 4 and MD-2 form a receptor for lipopolysaccharide (LPS), a major constituent of Gram-negative bacteria. MD-2 is a 20-25-kDa extracellular glycoprotein that binds to Tolllike receptor 4 (TLR4) and LPS and is a critical part of the LPS receptor. Here we have shown that the level of MD-2 expression regulates TLR4 activation by LPS. Using site-directed mutagenesis, we have found that glycosylation has no effect on MD-2 function as a membrane receptor for LPS. We used alanine-scanning mutagenesis to identify regions of human MD-2 that are important for TLR4 and LPS binding. We found that mutation in the N-terminal 46 amino acids of MD-2 did not substantially diminish LPS activation of Chinese hamster ovary (CHO) cells co-transfected with TLR4 and mutant MD-2. The residues 46-50 were important for LPS activation but not LPS binding. The residues 79-83, 121-124, and 125-129 are identified as important in LPS activation but not surface expression of membrane MD-2. The function of soluble MD-2 is somewhat more sensitive to mutation than membrane MD-2. Our results suggest that the 46-50 and 127-131 regions of soluble MD-2 bind to TLR4. The region 79-120 is not involved in LPS binding but affects monomerization of soluble MD-2 as well as TLR4 binding. We define the LPS binding region of monomeric soluble MD-2 as a cluster of basic residues 125-131. Studies on both membrane and soluble MD-2 suggest that domains of MD-2 for TLR4 and LPS binding are separate as well as overlapping. By mapping these regions on a three-dimensional model, we show the likely binding regions of MD-2 to TLR4 and LPS.

Heterogeneous Expression of Toll-like Receptor 4 and Downregulation of Toll-like Receptor 4 Expression on Human Gingival Fibroblasts by Porphyromonas gingivalis Lipopolysaccharide

Porphyromonas gingivalis (P. gingivalis) is implicated in the initiation and progression of periodontitis. Human gingival fibroblasts (HGFs) are the major constituent of gingival connective tissue. P. gingivalis or its components such as lipopolysaccharide (LPS) upregulate the production of various inflammatory cytokines including interleukin (IL)-1 and IL-6 in HGFs. Recently, we demonstrated that the binding of P. gingivalis LPS to Toll-like receptor 4 (TLR4) on HGFs activates various second messenger systems (Biochem. Biophys. Res. Commun. 273, 1161–1167, 2000). In the present study, we examined the level of TLR4 expression on HGFs by flow cytometric analysis (FACS), and studied the levels of IL-1 and IL-6 in the culture medium upon LPS stimulation of HGFs by enzyme-linked immunosorbent assay (ELISA). Upon stimulation by P. gingivalis LPS for 24 h, HGFs that expressed a high level of TLR4 secreted significantly higher levels of IL-1 and IL-6 than HGFs that expressed a low level of TLR4. On the other hand, after stimulation with P. gingivalis LPS for 24 h, the level of TLR4 on the surface of HGFs decreased. These results suggest that the level of TLR4 expression on HGFs reflects the extent of inflammation in the gingival tissue, and that P. gingivalis LPS downregulates TLR4 expression on HGFs. These findings may be used to control inflammatory and immune responses in periodontal disease.