Reduced Toll-like Receptor 4 Expression Research Articles

Yeast extract improves the inflammatory response of HepG2 cells induced by ethyl alcohol and lipopolysaccharide

To explore the ameliorative effect of yeast extract(YE) on the inflammatory response of human hepatoma cells(HepG2) induced by ethyl alcohol(EtOH) and lipopolysaccharide(LPS), and further explore the potential molecular mechanism based on Toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB) signaling pathway. HepG2 cells were induced by 50 mmol/L EtOH and 1 μg/mL LPS combined with YE intervention. The expression level of inflammatory cytokines was detected by ELISA. The expression level of TLR4 and the nuclear translocation of NF-κB were detected by immunofluorescence staining. The expression levels of TLR4, NF-κB, phospho-NF-κB-P65(P-NF-κB-p65), nucleus-phospho-NF-κB-p65(N-P-NF-κB-p65), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1β(IL-1β) were detected by Western blot. Compared with the control group, the cells in EtOH+LPS group produced a large number of inflammatory factors and had a significant inflammatory response. YE intervention significantly alleviated EtOH+LPS induced hepatocyte inflammatory response. Further molecular mechanism studies showed that YE significantly reduced TLR4 expression level and inhibited NF-κB nuclear translocation in hepatocytes. YE can effectively inhibit the inflammatory response of HepG2 cells induced by EtOH and LPS, and its molecular mechanism may be related to the down-regulation of TLR4/NF-κB pathway.

Cannabidiol reduces LPS-induced nociception via endocannabinoid system activation.

Bacterial infections are often accompanied by fever and generalized muscle pain. However, the treatment of pain with an infectious etiology has been overlooked. Thus, we investigated the impact of cannabidiol (CBD) in bacterial lipopolysaccharide (LPS)-induced nociception. Male Swiss mice received intrathecal (i.t.) LPS injection, and the nociceptive threshold was measured by the von Frey filaments test. Spinal involvement of the cannabinoid CB2 receptor, toll-like receptor 4 (TLR4), microglia and astrocytes were evaluated by i.t. administration of their respectively antagonists or inhibitors. Western blot, immunofluorescence, ELISA and liquid chromatography-mass spectrometry were used to assess Cannabinoid CB2 receptors and TLR4 spinal expression, proinflammatory cytokines and endocannabinoid levels. CBD was administered intraperitoneally at 10 mg/kg. The pharmacological assay demonstrated TLR4 participation in LPS-induced nociception. In addition, spinal TLR4 expression and proinflammatory cytokine levels were increased in this process. CBD treatment prevented LPS-induced nociception and TLR4 expression. AM630 reversed antinociception and reduced CBD-induced endocannabinoids upregulation. Increased spinal expression of the cannabinoid CB2 receptor was also found in animals receiving LPS, which was accompanied by reduced TLR4 expression in CBD-treated mice. Taken together, our findings indicated that CBD is a potential treatment strategy to control LPS-induced pain by attenuating TLR4 activation via the endocannabinoid system.

Protective effect of Lactobacillus-containing probiotics on intestinal mucosa of rats experiencing traumatic hemorrhagic shock.

This study was conducted to assess whether Lactobacillus-containing probiotics could protect intestinal mucosa in rats during traumatic hemorrhagic shock and to determine its underlying mechanisms. Healthy male Sprague–Dawley rats (300 ± 20 g) were randomly divided into four groups. During the study, reverse transcription polymerase chain reaction, western blotting, and hematoxylin and eosin methods were used. There was a significant increase in the expression of toll-like receptor 4 (TLR4) in the rats that experienced traumatic hemorrhagic shock, along with increased mRNA of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6. Pretreatment with Lactobacillus-containing probiotics reduced TLR4 expression, decreased phosphorylation (Ser536) and acetylation (Lys310) of p65, and decreased TNF-α and IL-6 mRNA. The probiotics combined acetate Ringer’s group showed a less severe pathological manifestation compared to the other experimental groups. Lactobacillus-containing probiotics inhibited nuclear factor-kappa B signaling via the downregulation of TLR4, resulting in inflammatory homeostasis, which might be the mechanism whereby Lactobacillus protects the intestinal mucosa from damage caused by the traumatic hemorrhagic shock.

Glial A2B Adenosine Receptors Modulate Abnormal Tachykininergic Responses and Prevent Enteric Inflammation Associated with High Fat Diet-Induced Obesity.

The role played by adenosine A2B receptors (A2BRs) in the regulation of enteric glial cell (EGC) functions remains unclear. This study was aimed at investigating the involvement of A2BRs in the control of EGC functions in a model of obesity. C57BL/6 mice were fed with standard diet (SD) or high fat diet (HFD) for eight weeks. Colonic tachykininergic contractions were recorded in the presence of BAY60-6583 (A2BRs agonist), MRS1754 (A2BRs antagonist), and the gliotoxin fluorocitrate. Immunofluorescence distribution of HuC/D, S100β, and A2BRs was assessed in whole mount preparations of colonic myenteric plexus. To mimic HFD, EGCs were incubated in vitro with palmitate (PA) and lipopolysaccharide (LPS), in the absence or in the presence of A2BR ligands. Toll-like receptor 4 (TLR4) expression was assessed by Western blot analysis. Interleukin-1β (IL-1β), substance P (SP), and glial cell derived neurotrophic factor (GDNF) release were determined by enzyme-linked immunosorbent assay (ELISA) assays. MRS1754 enhanced electrically evoked tachykininergic contractions of colonic preparations from HFD mice. BAY60-6583 decreased the evoked tachykininergic contractions, with higher efficacy in HFD mice. Such effects were blunted upon incubation with fluorocitrate. In in vitro experiments on EGCs, PA and LPS increased TLR4 expression as well as IL-1β, GDNF, and SP release. Incubation with BAY60-6583 reduced TLR4 expression as well as IL-1β, GDNF, and SP release. Such effects were blunted by MRS1754. The present results suggest that A2BRs, expressed on EGCs, participate in the modulation of enteric inflammation and altered tachykininergic responses associated with obesity, thus representing a potential therapeutic target.

Cold-inducible RNA-binding protein contributes to intracerebral hemorrhage-induced brain injury via TLR4 signaling.

IntroductionExcessive neuroinflammation aggravates the brain injury caused by intracerebral hemorrhage (ICH), while the upstream mechanisms that initiate neuroinflammation remain unclear. Toll‐like receptor 4 (TLR4) signaling is important to trigger inflammatory responses in ICH, and cold‐inducible RNA‐binding protein (CIRP) has been shown as a novel ligand of TLR4 by recent studies. However, whether the CIRP could trigger the neuroinflammation via activating TLR4 signaling in ICH still needs to be investigated.MethodsHuman serum CIRP levels were measured using the ELISA kits. Western blot, FJB staining, brain water content, and neurological deficit scores were used to investigate the roles of CIRP in brain injury caused by ICH.ResultFirst, we found increased CIRP levels in the blood of patients with ICH when compared to the control individuals, and the ICH patients with mRS > 2 have higher serum CIRP levels in contrast to those with mRS ≤ 2. In the ICH mice, we also found that brain CIRP protein and mRNA levels were also increased after ICH. Furthermore, using the CIRP−/− mice, we found that CIRP−/− mice had less brain damages showing in less FJB+ cells, reduced brain water content (BWC) and lower neurological deficit scores (NDS) compared to that in WT mice after ICH. Cytokines including IL‐6, TNF‐α, and IL‐1β from CIRP−/− mice were attenuated after ICH. CIRP−/− mice also exhibited reduced TLR4 expression which was accompanied by the decreased activity of NF‐κB. This suggests that TLR4 signaling might be involved in CIRP‐mediated inflammatory injury possibly via NF‐κB activation after ICH.ConclusionOur findings suggest that CIRP may activate TLR4 signaling, and further inducing NF‐κB activation to increase the expression levels of cytokines and aggravate inflammatory injury in ICH. Targeting CIRP may be a promising strategy for ICH treatment.

Inhibition of glycogen synthase kinase 3β improves cognitive function in aged mice by upregulating claudin presences in cerebral endothelial cells.

Glycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase, is widely distributed in mammalian brains. Since GSK-3β plays a vital role in the development of neurodegenerative disorders, the present study was designed to investigate the role of GSK-3β in the blood-brain barrier (BBB) permeability in aged mice. Morris water maze test was used to examine mouse cognitive function. BBB permeability was examined by the leakage of fluorescence signals of low-molecular weight dextran. GSK-3β inhibitor, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), was administrated in aged mice and in cultured mouse brain microvascular endothelial cells (bEnd.3). Compared with young mice, aged mice had increased leftover signals of dextran in the hippocampus and a lower score in the maze test, suggesting that aged mice have abnormal leakage of BBB and cognitive dysfunction. The protein expression of Toll-like receptor 4 (TLR4) was increased, whereas the protein expressions of junction proteins (claudin1 and claudin5) were reduced in endothelial cells of BBB in aged mice. Phosphorylated level of serine 9, an inhibitory residue in GSK-3β protein, was decreased. TDZD-8 treatment downregulated TLR4 protein expression, upregulated claudin1 and claudin5 protein expressions, and significantly improved cognitive function in aged mice. In bEnd.3 cells, TDZD-8 treatment reduced TLR4 expression and increased claudin5 expression in cells stimulated with lipopolysaccharides. In conclusion, the inhibition of GSK-3β activity downregulates aging-induced TLR4 expression and restores the BBB integrity, resulting in the improvement of cognitive function in aged mice.

Dexmedetomidine-mediated protection against septic liver injury depends on TLR4/MyD88/NF-κB signaling downregulation partly via cholinergic anti-inflammatory mechanisms

BackgroundUncontrolled inflammatory responses exacerbate the pathogenesis of septic acute liver injury (ALI), posing a lethal threat to the host. Dexmedetomidine (DEX) has been reported to possess protective properties in inflammatory conditions. This study aimed to investigate whether DEX pretreatment exhibits hepatoprotection against ALI induced by lipopolysaccharide (LPS) in rats and determine its possible molecular mechanism. MethodsSeptic ALI was induced by intravenous injection of LPS. The rats received DEX intraperitoneally 30 min before LPS administration. α-Bungarotoxin (α-BGT), a specific α7 nicotinic acetylcholine receptor (α7nAChR) antagonist, was administered intraperitoneally 1 h before LPS exposure. The role of the vagus nerve was verified by performing unilateral cervical vagotomy or sham surgery before sepsis. ResultsThe expression of α7nAChR, toll-like receptor 4 (TLR4), high mobility group box 1 (HMGB1), and cleaved caspase-3 increased, peaking 24 h during sepsis. DEX enhanced α7nAChR activation and reduced TLR4 expression upon challenge with LPS. DEX significantly prevented LPS-induced ALI, which was associated with increased survival, the mitigation of pathological changes, the attenuation of inflammatory cytokine expression and apoptosis, and the downregulation of TLR4/MyD88/NF-κB pathway. Moreover, the hepatoprotective effect of DEX was abolished by α-BGT. Further investigation established that vagotomy, compared to sham surgery, triggered more severe pathogenic manifestations and higher proinflammatory cytokine levels. The inhibitory effects of DEX were shown in sham-operated rats but not in vagotomized rats. ConclusionsOur data highlight the pivotal function of α7nAChR and intact vagus nerves in protecting against LPS-induced ALI through inhibiting the TLR4/MyD88/NF-κB signaling pathway upon pretreatment with DEX.

Toll‑like receptor 4 plays a tumor‑suppressive role in cutaneous squamous cell carcinoma.

Toll‑like receptor4 (TLR4), a key regulator of the innate immune system, is expressed not only in immune cells, but also in a number of cancer cells. A biological role for TLR4 in cutaneous squamous cell carcinoma (SCC), however, is unclear. In this study, we first examined TLR4 expression and localization in cases of SCC, actinic keratosis (AK) and Bowen's disease (BD) by immunohistochemistry. TLR4 expression was significantly higher in the SCC than in the AK or BD tissues. We then determined the TLR4 expression level invivo, in 3histological subtypes of SCC. TLR4 expression in poorly differentiated SCC was significantly lower compared with that of the moderately and well‑differentiated type. In addition, the CD44 immunoreactivity tended to be high in the cell membrane of poorly differentiated SCC. Of note, poorly differentiated SCC is a risk factor of unfavorable outcomes in affected patients. We then assessed the biological role of TLR4 in HSC‑1 and HSC‑5 SCC cells and HaCaT human keratinocytes. TLR4 knockdown by transfection with siRNA accelerated HSC‑1 and HaCaT cell migration and invasion compared to the control siRNA‑transfected cells. TLR4 knockdown resulted in an increased CD44 expression and in an enhanced filopodia protrusion formation, particularly in HSC‑1. On the whole, these results suggest that a reduced TLR4 expression enhances the malignant features in SCC cases and cultured SCC cell lines. TLR4 may thus play an anti‑tumor role in cutaneous SCC.

Downregulation of glucose-6-phosphate dehydrogenase contributes to diabetic neuropathic pain through upregulation of toll-like receptor 4 in rats.

Background and aimDiabetic neuropathic pain is a refractory and disabling complication of diabetes mellitus. The pathogenesis of the diabetic neuropathic pain is still unclear, and treatment is insufficient. The aim of this study is to investigate the roles of glucose-6-phosphate dehydrogenase (G6PD) and toll-like receptor 4 (TLR4) in neuropathic pain in rats with diabetes.MethodsType 1 diabetes model was induced by intraperitoneal injection of streptozotocin (STZ, 75 mg/kg) in adult female Sprague-Dawley rats. Paw withdrawal threshold and paw withdrawal latency of rats were measured by von Frey filaments and thermal radiation, respectively. The expressions of G6PD and TLR4 in L4-L6 dorsal root ganglions (DRGs) were measured by western blotting and quantitative real-time polymerase chain reaction analysis. Fluorescent immunohistochemistry was employed to detect expressions of G6PD and TLR4 and co-location of G6PD with TLR4.ResultsThe mRNA and protein expression levels of G6PD in DRGs were significantly decreased in diabetic rats when compared with age-matched control rats. Upregulation of G6PD by intrathecal injection of G6PD overexpression adenovirus markedly attenuated hindpaw pain hypersensitivity of diabetic rats. The mRNA and protein expression levels of TLR4 in DRGs of diabetic rats were significantly increased when compared with control rats. Intrathecal injection of TLR4-selective inhibitor CLI-095 attenuated diabetic pain in dose- and time-dependent manners. Furthermore, G6PD and TLR4 were co-localized in DRG neurons. Intrathecal injection of G6PD overexpression adenovirus greatly reduced TLR4 expression, while intrathecal injection of CLI-095 had no significant effect on G6PD expression in diabetic rats.ConclusionsOur results suggest that decrease in G6PD expression was involved in diabetic peripheral neuropathic pain, which was most likely through upregulation of TLR4 expression in the DRGs of rats.

Curcumin Protects against Atherosclerosis in Apolipoprotein E-Knockout Mice by Inhibiting Toll-like Receptor 4 Expression.

Toll-like receptor 4 (TLR4) has been reported to play a critical role in the pathogenesis of atherosclerosis, the current study aimed to investigate whether curcumin suppresses atherosclerosis development in ApoE-knockout (ApoE-/-) mice by inhibiting TLR4 expression. ApoE-/- mice were fed a high-fat diet supplemented with or without curcumin (0.1% w/w) for 16 weeks. Curcumin supplementation significantly reduced TLR4 expression and macrophage infiltration in atherosclerotic plaques. Curcumin also reduced aortic interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression, nuclear factor-κB (NF-κB) activity, and plasma IL-1β, TNF-α, soluble VCAM-1 and ICAM-1 levels. In addition, aortic sinus sections revealed that curcumin treatment reduced the extent of atherosclerotic lesions and inhibited atherosclerosis development. In vitro, curcumin inhibited NF-κB activation in macrophages and reduced TLR4 expression induced by lipopolysaccharide. Our results indicate that curcumin protects against atherosclerosis at least partially by inhibiting TLR4 expression and its related inflammatory reaction.

MicroRNA-1906, a Novel Regulator of Toll-Like Receptor 4, Ameliorates Ischemic Injury after Experimental Stroke in Mice.

Toll-like receptor 4 (TLR4) is a proinflammatory cascade initiator in poststroke inflammation. In this study, miR-1906, a novel regulator of TLR4, was identified via in silico analysis and microRNA profiling in male adult mice and its expression was then quantitated in the ischemic hemisphere. We found miR-1906 to be significantly brain enriched in the ischemic hemisphere and even more drastically enriched in the peri-infarct regions. Furthermore, in vitro experiments demonstrated that, during oxygen-glucose deprivation, miR-1906 expression was increased in glial cells but decreased in neurons. Surprisingly, despite the augmentation of intracellular abundance, miR-1906 expression in extracellular vesicles was decreased in astrocyte cell culture supernatants, suggesting reduced sources of miR-1906 from glia to neurons. When exogenous miR-1906 was administered, decreased TLR4 protein expression was observed both in vitro and in vivo Using Cy3 labeling, exogenous miR-1906 uptake by astrocytes, microglia, and neurons was visualized directly in vivo Reduced infarct volumes and improved functional outcomes were observed in middle cerebral artery occlusion mice receiving miR-1906. However, the protective effects of miR-1906 disappeared with the genetic knock-out of TLR4, suggesting that TLR4 is a major target of miR-1906 through which the microRNA exerts its therapeutic effects.SIGNIFICANCE STATEMENT The current study identified miR-1906 as a novel specific regulator of Toll-like receptor 4 (TLR4) and depicted its distinct expression patterns in different cerebral regions and cell types during ischemic attack. Therefore, the therapeutic supplementation of miR-1906 can be beneficial in the modulation of poststroke inflammation. Using Cy3 labeling, exogenous miR-1906 expression was visualized and shown to enter astrocytes, microglia, and neurons successfully in vivo Supplemental therapeutic miR-1906 resulted in reduced TLR4 expression and improved outcomes after middle cerebral artery occlusion in a mouse model, but its neuroprotective function was TLR4 dependent, suggesting that TLR4 is a major target of miR-1906.

Short-Term Versus Long-Term Effects of Adipocyte Toll-Like Receptor 4 Activation on Insulin Resistance in Male Mice.

Chronic exposure to high-saturated fat diets (HFDs) increases the prevalence of obesity and contributes to the development of low-grade inflammation and insulin resistance. A possible mediator accounting for obesity-associated inflammation and insulin resistance is Toll-like receptor 4 (TLR4). We investigated the role of adipocyte TLR4 in lipid and glucose homeostasis through an inducible, adipocyte-specific deletion of TLR4 in a mouse model that is referred to as the "Tadipo" mouse. Consistent with a critical role for inflammation as a positive force for healthy adipose tissue expansion, chronic HFD exposure results in exacerbated whole-body and muscle insulin resistance in the absence of TLR4 in the adipocyte. Elimination of TLR4 in adipocytes affects TLR4 expression in other tissues, with reduced TLR4 expression in peritoneal macrophages and in the liver. In contrast, TLR4 deletion from adipocytes protects whole-body insulin sensitivity after an acute lipid challenge during a hyperinsulinemic euglycemic clamp. Our results therefore demonstrate dichotomous effects of TLR4 on adipose tissue functionality, with an important positive role of TLR4 during a chronic HFD challenge due to the lack of adipose tissue remodeling and a negative role of TLR4 as a mediator of insulin resistance in the adipocyte during an acute challenge with saturated fatty acids.

LPS preconditioning ameliorates intestinal injury in a rat model of hemorrhagic shock.

Previous studies indicate that endotoxin preconditioning may decrease the inflammatory response and alleviate intestinal mucosal damage caused by sepsis. However, it is not known whether preconditioning with endotoxin might protect the intestinal mucosa after hemorrhagic shock. In this study, we investigated the effect of lipopolysaccharide (LPS) preconditioning on the intestinal mucosa following hemorrhagic shock in a rat model. Given that intestinal toll-like receptor 4 (TLR4) signaling is exaggerated in response to LPS, we further investigated the role of TLR4 signaling in endotoxin tolerance. Animals were pre-treated with intra-peritoneal Escherichia coli LPS for 5days prior to hemorrhagic shock. Animals were bled to achieve a mean arterial pressure (MAP) of 35-40mmHg, then resuscitated with Ringer solution and the heparinized shed blood to maintain MAP between 90 and 100mmHg. The distal ileum was harvested after resuscitation and graded for mucosal damage. TNF-α, TLR4, cleaved caspase-3, and intestinal trefoil factor 3 (TFF3) levels were measured at different time points. Pretreatment with LPS significantly reduced intestinal mucosal damage and protein levels of cleaved caspase-3. Furthermore, animals pre-treated with LPS experienced reduction of TNF-α and increased mucosal expression of TFF3. LPS tolerance was associated with reduced TLR4 expression. Endotoxin preconditioning can lessen the effects of ischemia and reperfusion injury in intestinal mucosa of a rat model with hemorrhagic shock. It is hypothesized that this effect is mediated via inhibition of TLR4 over-expression.

Genomic analysis of between-cow variation in dermal fibroblast response to lipopolysaccharide

The innate immune response plays a major role in defense against mastitis-causing pathogens. Identification of existing variation in innate immune signaling among cows and the underlying molecular causes for the variation may help in design of new mastitis control strategies. The dermal fibroblast has been used as a model cell type to explore between-cow variation in the ability of cells to produce IL-8 in response to lipopolysaccharide (LPS) treatment, and this response appears related to an animal's ability to respond to in vivo challenge with LPS or Escherichia coli mastitis. In this study, primary dermal fibroblast cultures of cows and microarray-based genomic analysis were used to investigate the cause(s) for the variable response to LPS. Fibroblast cultures from 2 cows, one with a low response phenotype (LRarray) and another with a high response phenotype (HRarray), were selected from our collection of fibroblast cultures established from 88 cows. The LRarray fibroblast culture produced approximately 5-fold less IL-8 and IL-6 protein in response to 24-h LPS treatment than the HRarray fibroblast culture. Genomic analysis of RNA obtained from 3 replicates of the 2 cultures before and after 8-h LPS treatment revealed a combined LPS-induced differential expression of 321 transcripts, indicating the robust response capability of the fibroblast cell. Under basal conditions, the microarray analysis revealed 2-fold less expression of toll-like receptor 4 (TLR4) in the LRarray fibroblasts compared with the HRarray fibroblasts, and this was associated with a marked reduction in expression of genes regulated by the TLR4-MyD88-dependent and TLR4-TRIF-dependent pathways (IL-8, IL-6, SAA3, CCL20, MX1, IRF1, and ISG20). The between-culture differential expression of TLR4 was confirmed and extended by quantitative PCR analysis (QPCR) that revealed a 33-fold lower expression of TLR4 in the LRarray fibroblast culture. After LPS treatment, the difference in TLR4 expression increased to almost 50-fold and was associated with more than 8-fold lower expression of IL-8 and IL-6. No DNA sequence variations were identified in the proximal 1,300-bp promoter region of the TLR4 gene, and microarray analysis did not reveal a molecular explanation for the reduced TLR4 expression under either basal conditions or following exposure to LPS. The attenuated innate immune response of the LRarray fibroblast culture to LPS may be caused by reduced TLR4 receptor expression. Also, the primary dermal fibroblast cells can be used to examine underlying causes for between-cow variations in key immune response pathways.

Comparison of Nasal and Bronchial Epithelial Cells Obtained from Patients with COPD

For in vitro studies of airway pathophysiology, primary epithelial cells have many advantages over immortalised cell lines. Nasal epithelial cells are easier to obtain than bronchial epithelial cells and can be used as an alternative for in vitro studies. Our objective was to compare nasal and bronchial epithelial cells from subjects with COPD to establish if these cells respond similarly to pro-inflammatory stimuli. Cell cultures from paired nasal and bronchial brushings (21 subjects) were incubated with cigarette smoke extract (CSE) prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide. IL-6 and IL-8 were measured by ELISA and Toll-like receptor 4 (TLR-4) message and expression by RT-PCR and FACS respectively. IL-8 release correlated significantly between the two cell types. IL-6 secretion was significantly less from bronchial compared to nasal epithelial cells and secreted concentrations did not correlate. A 4 h CSE incubation was immunosuppressive for both nasal and bronchial cells, however prolonged incubation for 24 h was pro-inflammatory solely for the nasal cells. CSE reduced TLR-4 expression in bronchial cells only after 24 h, and was without effect on mRNA expression. In subjects with COPD, nasal epithelial cells cannot substitute for in vitro bronchial epithelial cells in airway inflammation studies.

Toll-like receptor 4 is up-regulated by mTOR activation during THP-1 macrophage foam cells formation

Macrophage foam cells formation is the most important process in atherosclerotic plaque formation and development. Toll-like receptor 4 (TLR4) is one of the important innate immune sensors of endogenous damage signals and crucial for regulating inflammation. Growing evidence indicates that TLR4 plays a very important role in macrophage foam cells formation. However, the underlying mechanisms regulating TLR4 expression in macrophage are not fully understood. In this study, we induced THP-1 macrophage foam cells formation with oxidative modified low-density lipoprotein (ox-LDL). We observed that TLR4 mRNA and protein expression were markedly up-regulated, and the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream target p70S6K were promoted during foam cells formation. The mTOR inhibitor rapamycin blocked mTOR phosphorylation and inhibited TLR4 expression induced by ox-LDL. Silencing mTOR, rictor or raptor protein expression by small interfering RNA, also inhibited the up-regulation of TLR4 expression, respectively. Inhibition of mTOR with rapamycin reversed the down-regulation of cellular lipid efflux mediator ABCA1, which resulted from the activation of TLR4 by ligands. These data suggested that TRL4 expression was up-regulated by a mechanism dependent on mTOR signal pathway activation during THP-1 macrophage foam cells formation. Inhibition of ox-LDL induced mTOR activation reduced TLR4 expression, and improved the impaired lipid efflux.

Down-regulation of endothelial TLR4 signalling after apo A-I gene transfer contributes to improved survival in an experimental model of lipopolysaccharide-induced inflammation

The protective effects of high-density lipoprotein (HDL) under lipopolysaccharide (LPS) conditions have been well documented. Here, we investigated whether an effect of HDL on Toll-like receptor 4 (TLR4) expression and signalling may contribute to its endothelial-protective effects and to improved survival in a mouse model of LPS-induced inflammation and lethality. HDL cholesterol increased 1.7-fold (p < 0.005) and lung endothelial TLR4 expression decreased 8.4-fold (p < 0.005) 2 weeks after apolipoprotein (apo) A-I gene transfer. Following LPS administration in apo A-I gene transfer mice, lung TLR4 and lung MyD88 mRNA expression, reflecting TLR4 signalling, were 3.0-fold (p < 0.05) and 2.1-fold (p < 0.05) lower, respectively, than in LPS control mice. Concomitantly, LPS-induced lung neutrophil infiltration, lung oedema and mortality were significantly attenuated following apo A–I transfer. In vitro, supplementation of HDL or apo A–I to human microvascular endothelial cells-1 24 h before LPS administration reduced TLR4 expression, as assessed by fluorescent-activated cell sorting, and decreased the LPS-induced MyD88 mRNA expression and NF-κB activity, independently of LPS binding. In conclusion, HDL reduces TLR4 expression and signalling in endothelial cells, which may contribute significantly to the protective effects of HDL in LPS-induced inflammation and lethality.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-010-0690-6) contains supplementary material, which is available to authorized users.

Longitudinal, Diet-induced Weight Gain is Associated with Increased Blood Monocytes and Reduced TLR4 Expression

Excessive weight gain increases systemic inflammation resulting in increased disease risk. Toll-like receptor 4 (TLR4) reportedly mediates increases in inflammation; however, its role has not been fully evaluated. Objective. The purpose of this study was to determine the longitudinal effect of diet-induced weight gain on blood monocyte concentration and cell-surface TLR4 expression. Research Methods &amp; Procedures. Male CD-1 mice were randomly assigned to high-fat (HF, n = 12) or low-fat (LF, n = 13) groups. Non-lethal, saphenous vein blood samples were collected at 0, 4, 8 and 12 weeks of treatment. Three-color flow cytometry was used to measure monocyte (CD11b+/CD14+) concentration and TLR4 cells-surface expression. Data were analyzed with a repeated measures ANOVA; significance was set at P&lt;0.05. Results. Body weight at week 12 was 21% greater in HF than LF (P&lt;0.05). At week 12 HF had 155% more monocytes (P&lt;0.05) with 24% less TLR4; Monocyte concentration and body weight at week 12 was negatively correlated with TLR4 gMFI (P&lt;0.05). Conclusions. The observed effects of high-fat feeding on blood monocytes are consistent with a phenotype, which may be associated with premature morbidity. The observed monocyte responses may be associated with immune dysfunction and diminished response to infection.

P74: Toll-like receptor-4 signaling does not increase rate of metastatic tumor engraftment in a murine model of colorectal carcinoma

Introduction: Obesity and newer chemotherapeutic agents used to treat metastatic colorectal carcinoma are associated with hepatic steatosis and Toll-like receptor-4 (TLR4) is increased in fatty liver. TLR4 is known to have pro-neoplastic effects in primary malignancies; however, the effect of hepatic TLR4 on metastatic liver tumor engraftment is unknown. The aim of this study was to determine if reduced TLR4 expression would alter the rate of metastatic tumor engraftment in steatotic liver. Methods: TLR4 deficient and corresponding wild-type C57Bl/10 mice were fed a diet of 60% fat or control diet to generate 4 groups of animals: Group 1 TLR4 intact/lean; Group 2 TLR4 deleted/lean; Group 3 TLR4 intact/obese; Group 4, TLR4 deleted/obese. Following 5 weeks of diet manipulation the mice underwent laparotomy and intrasplenic injection of 100,000 MC38 mouse colon adenocarcinoma cells. The diets were maintained for 28 days following operation at which time the mice were sacrificed and total liver tumor number and volume were measured. Liver steatosis was determined on H&E and Oil Red-O sections. Serum and tissue levels of IL-6 and TNF-a were measured by enzyme-linked immunosorbent assay. Differences between groups were measured using one-way ANOVA with the LSD procedure, all numbers reported as mean +/− standard deviation. Results: 1) Mice fed a high fat diet were consistently larger and had marked steatosis histologically compared to mice fed control diet. 2) Mean tumor number and volume was similar for all groups (number: Group 1 7.0+/−3.4; Group 2 10.8+/−12.0; Group 3 6.8+/−8.3; Group 4 4.3+/−5.3: p > 0.05 for all comparisons and volume: Group 1 272.1+/−465.6mm3; Group 2 595.5+/−917.3 mm3; Group 3 738.5+/−980.4 mm3; Group 4 1357.1+/−3280.6 mm3: p > 0.05 for all comparisons). 3) Serum levels of IL-6 and TNF-a were not markedly elevated and did not significantly differ. 4) Liver tissue levels of IL-6 and TNF-a were generally higher than serum, but were not significantly different among the groups. Conclusions: These findings indicate that in the setting of hepatic steatosis,TLR4 does not play a significant role in metastatic tumor engraftment.