Toll-like Receptor 4 Knockdown Research Articles

Paeoniflorin abrogates DSS-induced colitis via a TLR4-dependent pathway

Paeonia lactiflora Pall is one of the most well-known herbs in China, Korea, and Japan for more than 1,200 years. Paeoniflorin, the major bioactive component of peony root, has recently been reported to have anticolitic activity. However, the underlying molecular mechanism is unclear. The present study was to explore the possible mechanism of paeoniflorin in attenuating dextran sulfate sodium (DSS)-induced colitis. Pre- and coadministration of paeoniflorin significantly reduced the severity of colitis and resulted in downregulation of several inflammatory parameters in the colon, including the activity of myeloperoxidase (MPO), the levels of TNF-α and IL-6, and the mRNA expression of proinflammatory mediators (MCP-1, Cox2, IFN-γ, TNF-α, IL-6, and IL-17). The decline in the activation of NF-κB p65, ERK, JNK, and p38 MAPK correlated with a decrease in mucosal Toll-like receptor 4 (TLR4) but not TLR2 or TLR5 expression. In accordance with the in vivo results, paeoniflorin downregulated TLR4 expression, blocked nuclear translocation of NF-κB p65, and reduced the production of IL-6 in LPS-stimulated mouse macrophage RAW264.7 cells. Transient transfection assay performed in LPS-stimulated human colon cancer HT-29 cells indicated that paeoniflorin inhibits NF-κB transcriptional activity in a dose-dependent manner. TLR4 knockdown and overexpression experiments demonstrated a requirement for TLR4 in paeoniflorin-mediated downregulation of inflammatory cytokines. Thus, for the first time, the present study indicates that paeoniflorin abrogates DSS-induced colitis via decreasing the expression of TLR4 and suppressing the activation of NF-κB and MAPK pathways.

Toll-like receptor 4 signaling is required for induction of gluconeogenic gene expression by palmitate in human hepatic carcinoma cells

Saturated free fatty acids (FFA) can activate inflammatory cascades including the toll-like receptor 4 (TLR4) pathway. TLR4 is expressed by hepatocytes and may help link FFA to altered hepatic gluconeogenesis in type 2 diabetes mellitus. This study examined the role of TLR4 in mediating palmitate effects on the expression of phosphoenolpyruvate carboxykinase (PCK1) and the catalytic subunit of glucose-6-phosphatase (G6PC), rate-determining gluconeogenic enzymes. Human hepatocellular carcinoma cells (HepG2 and HuH7) were incubated in media including 2% bovine serum albumin and 250 to 1000 μM palmitate for 24 h. Signaling mediated by TLR4 was blocked by a TLR4 decoy peptide or small interfering RNA knockdown of TLR4. Palmitate induced dose-dependent increases in PCK1 and G6PC mRNA abundance, which were prevented by the TLR4 decoy peptide. Palmitate doubled PCK1 promoter activity, and TLR4 knockdown ablated this response. Lipopolysaccharide and monophosphoryl lipid A also up-regulated G6PC and PCK1 transcript abundance in a TLR4-dependent manner. Addition of oleate attenuated palmitate-induced increases in G6PC and PCK1 mRNA abundance. Palmitate increased nuclear factor κ-light-chain-enhancer of activated B cells reporter gene activity, which was unaffected by TLR4 blockade, but increased mRNA abundance of hepatocyte-specific cyclic AMP response element binding protein, a transcriptional regulator of PCK1, in a TLR4-dependent manner. Finally, TLR4 activation by palmitate increased subsequent cellular uptake of palmitate, and inhibiting ceramide synthesis ablated palmitate effects on PCK1 mRNA abundance and promoter activity. These results suggest that TLR4 signaling could play a critical role in linking elevated saturated FFA to increased transcription of gluconeogenic genes.

TLR4 signaling induces retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5 in mesangial cells

It is known that recognition of bacterial lipopolysaccharide (LPS) and various endogenous ligands by Toll-like receptor 4 (TLR4) induces inflammatory reactions. However, the role of TLR4 activation in mesangial inflammation remains to be elucidated. Retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are putative RNA helicases and are involved in immune and inflammatory reactions. The purpose of the present study was to investigate the implication of RIG-I and MDA5 in TLR4 signaling in mesangial cells. Normal human mesangial cells in culture were treated with LPS. Expression of RIG-I, MDA5, interferon-β (IFN-β), CXCL10 and CXCL8 was examined using real-time RT-PCR, Western blotting and ELISA. The cells were also subjected to RNA interference against TLR4, IFN-β, RIG-I or MDA5. LPS induced the expression of IFN-β, RIG-I, MDA5, CXCL8 and CXCL10 in human mesangial cells. RNA interference against either TLR4 or IFN-β inhibited LPS-induced RIG-I and MDA5 expression. Knockdown of TLR4, IFN-β, RIG-I or MDA5 resulted in decreased induction of CXCL10, while only TLR4 knockdown inhibited CXCL8 induction. TLR4 signaling induces the expression of RIG-I and MDA5 in mesangial cells. RIG-I and MDA5 may be involved in inflammatory reactions by regulating CXCL10 expression in the downstream of TLR4 signaling in human mesangial cells.

Toll‐like receptor 4 is involved in bacterial endotoxin‐induced endothelial cell injury and SOC‐mediated calcium regulation

Bacterial endotoxins may lead to vascular endothelial cell injury. Our study explored the role of TLR4 (Toll-like receptor 4) and STIM1 (stromal interaction molecule 1) in bacterial endotoxin-induced calcium overload and inflammatory reactions in HUVECs (human umbilical vein endothelial cells). It showed that under LPS (lipopolysaccharide) stimulation, LBP (LPS-binding protein) mRNA levels peaked at 24 h, TLR4 levels at 12 h and NF-κB (nuclear factor κB) levels at 6 h (all P<0.01). LBP levels increased gradually and peaked at 24 h of LPS treatment. TLR4 protein levels increased significantly at 1 h and peaked at 12 h. NF-κB protein levels markedly increased at 1 h and peaked at 6 h. Knockdown of STIM1 alone, TLR4 alone or both STIM1 and TLR4 together, markedly abolished LPS-induced increase in calcium influx into cells (P<0.05, P<0.01 and P<0.01 respectively). LBP-TLR4 and STIM-NF-κB interactions were detected without LPS treatment, enhanced by LPS stimulation, and markedly reduced by knocking down TLR4 and STIM respectively. Both the NF-κB inhibitor, PDTC (pyrrolidine dithiocarbamate) and TLR4 knockdown could block LPS induction of NF-κB, STIM, TNFα (tumour necrosis factor α) and IL-6 (interleukin 6). The data indicate LPS-LBP may activate TLR4 signalling and downstream transcription factor NF-κB, which further can activate STIM1 and eventually lead to calcium influx and injury of HUVECs. Inhibition of TLR4 effectively reverses LPS induction of inflammatory mediator generation and extracellular calcium influx mediated by STIM1.

Lipopolysaccharide Induces Lung Fibroblast Proliferation through Toll-Like Receptor 4 Signaling and the Phosphoinositide3-Kinase-Akt Pathway

Pulmonary fibrosis is characterized by lung fibroblast proliferation and collagen secretion. In lipopolysaccharide (LPS)-induced acute lung injury (ALI), aberrant proliferation of lung fibroblasts is initiated in early disease stages, but the underlying mechanism remains unknown. In this study, we knocked down Toll-like receptor 4 (TLR4) expression in cultured mouse lung fibroblasts using TLR4-siRNA-lentivirus in order to investigate the effects of LPS challenge on lung fibroblast proliferation, phosphoinositide3-kinase (PI3K)-Akt pathway activation, and phosphatase and tensin homolog (PTEN) expression. Lung fibroblast proliferation, detected by BrdU assay, was unaffected by 1 mug/mL LPS challenge up to 24 hours, but at 72 hours, cell proliferation increased significantly. This proliferation was inhibited by siRNA-mediated TLR4 knockdown or treatment with the PI3K inhibitor, Ly294002. In addition, siRNA-mediated knockdown of TLR4 inhibited the LPS-induced up-regulation of TLR4, down-regulation of PTEN, and activation of the PI3K-Akt pathway (overexpression of phospho-Akt) at 72 hours, as detected by real-time PCR and Western blot analysis. Treatment with the PTEN inhibitor, bpV(phen), led to activation of the PI3K-Akt pathway. Neither the baseline expression nor LPS-induced down-regulation of PTEN in lung fibroblasts was influenced by PI3K activation state. PTEN inhibition was sufficient to exert the LPS effect on lung fibroblast proliferation, and PI3K-Akt pathway inhibition could reverse this process. Collectively, these results indicate that LPS can promote lung fibroblast proliferation via a TLR4 signaling mechanism that involves PTEN expression down-regulation and PI3K-Akt pathway activation. Moreover, PI3K-Akt pathway activation is a downstream effect of PTEN inhibition and plays a critical role in lung fibroblast proliferation. This mechanism could contribute to, and possibly accelerate, pulmonary fibrosis in the early stages of ALI/ARDS.

Abstract 5585: Generation of novel cell lines for analyzing the role of TLR4 in breast cancer

Abstract Background: Toll like receptor 4 (TLR4) is a transmembrane receptor expressed in macrophages that plays a major role in pathogen recognition and activation of innate immunity. TLR4 signaling has been linked to tumorigenesis and promotion of metastasis in ovarian, lung, prostate and head-neck cancers through up-regulation of the expression of inflammatory cytokines (e.g., IL-6, IL-8 and TNF-alpha). These factors increase cancer cell survival and resistance to chemotherapy through up-regulation of anti-apoptotic and down-regulation of apoptotic proteins, respectively. The goal of this study was to generate new isogenic cell lines that would allow analysis of the TLR4 role in paclitaxel resistance in breast cancer. To this end, we used TLR4-positive and negative lines (MDA-MB-231 and HCC1806, respectively) to generate stable TLR4 knockdown and over-expressing line derivatives. Methods: To generate a TLR4 knockdown line, MDA-MB-231 cells were transfected with pisiRNA-TLR4 plasmid and selected with zeocin (50μg/ml). To generate a TLR4 over-expressing line, HCC1806 cells were transfected with pKT2-TLR4-PURO plasmid and selected with puromycin (1μg/ml). Ten to 20 monoclonal colonies were selected from each line and characterized for TLR4 expression and TLR4-dependent cell activities by qRT-PCR and western blot. Results: TLR4 mRNA expression was reduced by &amp;gt;90% as compared with parental line in five out of 8 MDA-MB-231TLR4− clones examined. One clone had 60% reduction and two clones had no effect on TLR4 mRNA determined by qRT-PCR. TLR4 mRNA was up-regulated 1,000-12,000 fold in five (out of 11) clones derived from HCC1806 line. Two clones overexpressed TLR4 mRNA by 300-500 fold and four clones had increased TLR4 expression by 1-6 fold. All selected clones were analyzed for TLR4 functional signaling by exposure to LPS (1μg/ml), which was reduced by 50-70% in MDA-MB-231TLR4− clones and increased by 15-25 folds in HCC1806TLR4+ sub-lines, as compared with cells transfected with empty vectors. Conclusions: These data show that we successfully created sub-lines of MDA-MB-231 with stable knockdown of TLR4 and sub-lines of breast carcinoma HCC1806 line with stable over-expression of TLR4. Ectopically expressed human TLR4 as well as TLR4 downregulating sequence had significant functional effects on the TLR4 signaling as demonstrated by increase and decrease of LPS-activated targets (IL-6, IL-8 and TNF-alpha) in MDA-MB-231TLR4− and HCC1806TLR4+ sub-lines, respectively. These new sub-lines will serve as excellent tools for analyzing the role of the TLR4 pathway signaling in breast cancer progression, metastasis and resistance to therapy. * These authors contributed equally in this work. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5585. doi:10.1158/1538-7445.AM2011-5585

Rosiglitazone Regulates C-reactive Protein–induced Inflammatory Responses Via Glucocorticoid Receptor–mediated Inhibition of p38 Mitogen-activated Protein Kinase–Toll-like Receptor 4 Signal Pathway in Vascular Smooth Muscle Cells

C-reactive protein (CRP) activates toll-like receptor 4 (TLR4) to initiate inflammatory response involved in the pathogenesis of atherosclerosis through mitogen-activated protein kinase (MAPK) signal pathways. Rosiglitazone, a synthetic peroxisome proliferator-activated receptor γ (PPARγ) agonist, is considered to be an important inhibitor of the inflammatory response. The present study was to explore the effect of rosiglitazone on the CRP-induced inflammatory responses and the related signal pathway in vascular smooth muscle cells (VSMCs). The results showed that rosiglitazone reduced the expressions of proinflammatory cytokines, such as vascular endothelial growth factor-A and inducible nitric oxide synthase, and enhanced the expression or activation of anti-inflammatory transcription factors including PPARγ and glucocorticoid receptor (GR) in VSMCs in response to CRP. The further investigations indicated that rosiglitazone inhibited CRP-induced TLR4 expression and p38 MAPK phosphorylation in VSMCs, and TLR4 knockdown potentiated the inhibitory effects of rosiglitazone on vascular endothelial growth factor-A and inducible nitric oxide synthase expressions. In addition, GR antagonist RU486 but not PPARγ inhibitor GW9662 remarkably weakened the inhibitory effects of rosiglitazone on CRP-induced TLR4 expression and p38 phosphorylation in VSMCs. But GW9662 did not affect rosiglitazone-induced GR phosphorylation. These suggest that rosiglitazone exerts its anti-inflammatory effect through activating GR and subsequently inhibiting p38 MAPK-TLR4 signaling pathway in CRP-stimulated VSMCs.

C-Reactive Protein Induces TNF-α Secretion by p38 MAPK–TLR4 Signal Pathway in Rat Vascular Smooth Muscle Cells

Atherosclerosis is a chronic inflammatory disease. C-reactive protein (CRP) not only is an inflammatory marker but also regulates the expressions of other inflammatory cytokines associated with the pathogenesis of atherosclerosis. Toll-like receptor 4 (TLR4) also contributes to atherogenesis via transducting inflammatory signals. Herein, our studies focused on characterizing the effect of CRP on tumor necrosis factor α (TNF-α) production and TLR4-related molecular mechanisms in rat vascular smooth muscle cells (VSMCs). The results showed that CRP stimulated VSMCs to secrete TNF-α and enhanced TLR4 expression in a time-concentration-dependent manner. TLR4 knockdown significantly inhibited CRP-induced TNF-α generation, and p38 mitogen-activated protein kinase (MAPK) blocker SB203580 depressed TLR4 expression and TNF-α production initiated by CRP in VSMCs. The data demonstrate that CRP triggers an inflammatory response in rat VSMCs by inducing TNF-α secretion, which is mediated by p38 MAPK-TLR4 signaling pathway.

C-Reactive Protein and Reendothelialization

See related article, pages 1452–1459 Disruption of the endothelium and its subsequent dysfunction appears to set the stage for atherogenesis.1 The maintenance of vascular homeostasis depends in part on a balance between endothelium-derived relaxing and contracting factors. Alterations in this balance lead to inflammation and the formation of fatty streaks and fibrous plaques. The diminished production or availability of nitric oxide (NO) appears paramount to setting the stage for inflammation and vascular injury. In addition to its vasodilatory effect on the vasculature, endothelium-derived NO promotes endothelial cell growth, survival and migration,2 stimulates angiogenesis and neovascularization in part by endothelial progenitor cell (EPC) recruitment,3 inhibits leukocyte adhesion to the endothelium,4 maintains vascular smooth muscle in a nonproliferative state,5 and limits platelet aggregation and thrombosis.6 Maintenance of an intact and functional endothelium protects against vascular disease while its disruption is detrimental. The study by Schwartz et al,7 in this issue of Circulation Research , adds to our understanding of how C-reactive protein (CRP) works to promote vascular pathology by inhibiting NO synthesis. CRP, long regarded as an acute phase protein and an active participant in the innate immune system, has more recently been linked to the development of cardiovascular disease.8 On the basis of several prospective epidemiologic studies, circulating high sensitivity CRP has emerged as an independent predictor of cardiovascular disease risk in various subgroups of patients, including those without overt cardiovascular disease, patients with stable angina or acute coronary syndromes, and in those with the metabolic syndrome.9–12 In primary prevention, CRP confers additional prognostic value at all levels of Framingham risk score and blood pressure.13,14 However, whether or not CRP plays a direct biological role in the initiation and progression of atherosclerosis is controversial.15,16 …

The biological effects of CRP are not attributable to endotoxin contamination: evidence from TLR4 knockdown human aortic endothelial cells

C-reactive protein (CRP) is the prototypic marker of inflammation and a strong predictor of cardiovascular events in humans. There are questions regarding the validity of the biological effects reported for CRP, in spite of adherence to rigorous control measures minimizing endotoxin [lipopolysaccharide (LPS)] contamination in these in vitro studies. In this study, we addressed the key question of endotoxin contamination in CRP preparations using Toll-like receptor 4 (TLR4) knockdown endothelial cells. Human aortic endothelial cells (HAECs) transfected with prevalidated TLR4 small interfering RNA (siRNA) and scrambled siRNA controls were challenged with pleural fluid-derived CRP or LPS for 12-16 h. Secreted interleukin-6 (IL-6), IL-1beta, IL-8, and plasminogen activator inhibitor-1 (PAI-1) levels and endothelial Nitric oxide synthase (eNOS) activity were determined. TLR4 knockdown in HAECs significantly decreased LPS-induced IL-1beta, IL-6, and IL-8, whereas the stimulatory effects of CRP were similar in both scrambled control and TLR4 knockdown cells. Furthermore, CRP significantly stimulated PAI-1 levels in both control and TLR4-transfected cells and inhibited eNOS activity, whereas LPS effects were negated in TLR4-transfected cells. The data presented cogently demonstrate and further confirm that the biological effects of CRP on HAECs are independent of LPS and thus are attributable to native protein per se. This is the first study to positively authenticate the significance of earlier in vitro reports on CRP biological effects.