Toll-like Receptor 4 mRNA Expression Research Articles

Suppression of TLR4 by miR-448 is involved in Diabetic development via regulating Macrophage polarization.

Lipopolysaccharide (LPS) contributed to the development and progression of type 2 diabetes mellitus (T2D), while TLR4 is reported to mediate the LPS-induced inflammation in macrophages. However, the potential molecular mechanisms for TLR4-mediated macrophages activation in T2D have not yet to be fully clarified. Type 2 diabetes models in C57BL/6J mice were generated by a combination administration of streptozotocin (STZ) and a high-fat diet (HFD). Cell proportions of M1 and M2 macrophages were analyzed using flow cytometry. Expression profiles of miR-448 and TLR 4 were determined by qRT-PCR and Western blot. LPS/IFN-γ significantly induced M1 polarization in macrophages characterized by the increased levels of TNF-α, IL-6, IL-12, iNOS and decreased levels of TNF-β, CCL-22, IL-10 and Arg-1, with a higher expression of toll-like receptor 4 (TLR4) in vitro. Consistently, T2D mice-derived macrophages had a significantly elevated expression of TLR4 mRNA and decreased expression of miR-448. We further confirmed that miR-448 could inhibit TLR4 expression by targeting the 3'-UTR of TLR4, rescuing the LPS/IFN-γ-induced M1 macrophage polarization. Taken together, our results indicated that decreased miR-448 in diabetic macrophages may contribute to LPS-induced M1 polarization by targeting TLR4, thereby modulating T2D development.

Plasmodium-Salmonella Coinfection Induces Intense Inflammatory Response, Oxidative Stress, and Liver Damage: A Mice Model Study for Therapeutic Strategy.

Impairment of host immune response in malaria favors bacteremia caused by typhoidal or nontyphoidal serovars of Salmonella enterica. Ofloxacin and Artesunate are the drugs that are clinically proven for treating typhoid and malaria, respectively. The study evaluates the host responses upon treatment with antibiotic (Ofloxacin) and antimalarial (Artesunate) in a standardized mice model harboring coinfection. BALB/c mice (18-22 g) were simultaneously coinfected with Plasmodium yoelii nigeriensis (Pyn) and S. enterica serovar Typhimurium (STm) and then treated with Ofloxacin or/and Artesunate from day 4 to day 7. The bacterial burden, liver function enzymes, oxidative stress, m-RNA expression of Toll-like receptors (TLR-2 and TLR-4), Th1/Th2 cytokines, hemeoxygenase-1, and NFкB were assessed. Ofloxacin treatment failed to counter the bacterial proliferation in Pyn-STm coinfected mice. However, upon controlling parasitemia with antimalarial, the efficacy of Ofloxacin could be regained. Elevated bacterial burden with malaria induces the expression of TLR-2 and TLR-4 triggering intense inflammatory response (NFκB, Th1/Th2 cytokines) in coinfected mice. This results in critical liver damage (ALT, AST, and ALP), oxidative stress (lipid peroxidation, total GSH, catalase, and super oxide dismutase), and hemeoxygenase-1 (HO-1). The study concludes that malaria infection aggravates the secondary infection of Salmonella serovars and the control of septicemia is critical in recovery of the coinfected subject.

Intranasal curcumin protects against LPS-induced airway remodeling by modulating toll-like receptor-4 (TLR-4) and matrixmetalloproteinase-9 (MMP-9) expression via affecting MAP kinases in mouse model.

Bacterial infections can exacerbate asthmatic inflammation depending on lipopolysaccharide (LPS) composition, the outermost component of cell wall, its exposure timings as well as host's immune status. In present study, Balb/c mice were exposed to antigen (ovalbumin) and LPS simultaneously to establish an asthmatic model. Curcumin (diferuloylmethane), well known for its anti-inflammatory potential, was administered through intranasal route 1h before LPS and OVA (ovalbumin) exposure to evaluate its efficacy against airway structural changes. Inflammatory cell infiltration in lungs was measured by flow cytometry and further eosinophils were especially measured by immunofluorescence detection of major basic protein (MBP) as marker of eosinophilc granule protein. We also measured reactive oxygen species (ROS) in BALF by spectrofluorometry. MMP-9 activity was evaluated by gelatin zymography and mRNA expressions of MMP-9, TIMP-1, TGF-β1, IL-13, Collagen-1 and TLR-4 were measured in lungs. Protein expression of MAP kinases (P-ERK, P-JNK, P-p38), TLR-4, Cox-2, Lox-5 and Eotaxin was measured by western blotting. Hydroxyproline level and masson's trichrome staining were used to evaluate collagen deposition in lung. Exposure to LPS (0.1µg) exacerbates airway inflammation and induces structural changes in lungs by enhanced ROS production, collagen deposition, expression of genes involved in airway remodeling and activation of MAP kinases pathway enzymes. Intranasal curcumin pretreatment had significantly suppressed inflammatory mediators and airway remodeling proteins. Our results strongly suggest that intranasal curcumin effectively protects LPS-induced airway inflammation and structural changes by modulating genes involved in airway remodeling in safer way; hence, it can be considered as supplementary alternative towards asthma treatments.

Medium-Chain Triglycerides Attenuate Liver Injury in Lipopolysaccharide-Challenged Pigs by Inhibiting Necroptotic and Inflammatory Signaling Pathways.

This study was conducted to investigate whether medium-chain triglycerides (MCTs) attenuated lipopolysaccharide (LPS)-induced liver injury by down-regulating necroptotic and inflammatory signaling pathways. A total of 24 pigs were randomly allotted to four treatments in a 2 × 2 factorial design including diet (0 and 4% MCTs) and immunological challenge (saline and LPS). After three weeks of feeding with or without 4% MCTs, pigs were challenged with saline or LPS. MCTs led to a significant increase in eicosapentaenoic acid, docosahexaenoic acid and total (n-3) polyunsaturated fatty acid concentrations. MCTs attenuated LPS-induced liver injury as indicated by an improvement in liver histomorphology and ultrastructural morphology of hepatocytes, a reduction in serum alanine aminotransferase and alkaline phosphatase activities as well as an increase in claudin-1 protein expression. In addition, MCTs also reduced serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 concentrations, liver TNF-α and IL-1β mRNA expression and protein concentrations and enhanced liver heat shock protein 70 protein expression in LPS-challenged pigs. Moreover, MCTs decreased mRNA expression of receptor-interacting serine/threonine-protein kinase (RIP) 3, mixed-lineage kinase domain-like protein (MLKL) and phosphoglycerate mutase 5 and inhibited MLKL phosphorylation in the liver. Finally, MCTs decreased liver mRNA expression of toll-like receptor (TLR) 4, nucleotide-binding oligomerization domain protein (NOD) 1 and multiple downstream signaling molecules. MCTs also suppressed LPS-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation and increased extracellular signal-related kinase 1/2 phosphorylation in the liver. These results indicated that MCTs are capable of attenuating LPS-induced liver damage by suppressing hepatic necroptotic (RIP1/RIP3/MLKL) and inflammatory (TLR4/NOD1/p38 MAPK) signaling pathways.

Astaxanthin protects lipopolysaccharide-induced inflammatory response in Channa argus through inhibiting NF-κB and MAPKs signaling pathways

The present study was conducted to evaluate the protective effects of astaxanthin against lipopolysaccharide (LPS)-induced inflammatory responses in Channa argus in vivo and ex vivo. Primary hepatocytes were exposed to different concentrations of LPS for 24 h to induce an inflammatory response, and the protective effects of astaxanthin against LPS-induced inflammation were studied ex vivo and in vivo. Hepatocytes exposed to LPS (5–20 μg mL−1) alone for 24 h resulted in a significant increase in lactate dehydrogenase release (LDH), Nitric oxide (NO) production and Malondialdehyde (MDA) content, 10 μg mL−1 LPS could induced inflammatory response in hepatocytes. Gene expression of TLR4, NFkBp65, MAPKp38, TNF-α, IL-6 and IL-1β mRNA expression were also enhanced ex vivo (p < 0.05). In vivo test demonstrated that pretreatment with astaxanthin prevented the LPS-induced upregulation of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β. Besides, astaxanthin blocked the expression of Toll-like receptor 4 (TLR4) and then suppressed the phosphorylation of nuclear transcription factor-kappa B (NF-κB) p65 and degradation inhibitor of NF-κBα (IκBα). Further study showed that astaxanthin could suppress the phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) in mitogen-activated protein kinase (MAPK) signal pathway. In conclusion, our results suggest that astaxanthin played an anti-inflammatory role by regulating TLR4 and the NF-κB and MAPK signaling pathways in C. argus.

Withaferin A Protects Against High-Fat Diet-Induced Obesity Via Attenuation of Oxidative Stress, Inflammation, and Insulin Resistance.

Withaferin A (WA), a bioactive constituent derived from Withania somnifera plant, has been shown to exhibit many qualifying properties in attenuating several metabolic diseases. The current investigation sought to elucidate the protective mechanisms of WA (1.25mg/kg/day) on pre-existing obese mice mediated by high-fat diet (HFD) for 12weeks. Following dietary administration of WA, significant metabolic improvements in hepatic insulin sensitivity, adipocytokines with enhanced glucose tolerance were observed. The hepatic oxidative functions of obese mice treated with WA were improved via augmented antioxidant enzyme activities. The levels of serum pro-inflammatory cytokines and hepatic mRNA expressions of toll-like receptor (TLR4), nuclear factor κB (NF-κB), tumor necrosis factor-α (TNF-α), chemokine (C-C motif) ligand-receptor, and cyclooxygenase 2 (COX2) in HFD-induced obese mice were reduced. Mechanistically, WA increased hepatic mRNA expression of peroxisome proliferator-activated receptors (PPARs), cluster of differentiation 36 (CD36), fatty acid synthase (FAS), carnitine palmitoyltransferase 1 (CPT1), glucokinase (GCK), phosphofructokinase (PFK), and phosphoenolpyruvate carboxykinase (PCK1) that were associated with enhanced lipid and glucose metabolism. Taken together, these results indicate that WA exhibits protective effects against HFD-induced obesity through attenuation of hepatic inflammation, oxidative stress, and insulin resistance in mice.

Aging leads to dysfunctional innate immune responses to TLR2 and TLR4 agonists.

Sepsis is more common in the elderly. TNF⍺ is recognized as an important mediator in sepsis and Toll-like receptors (TLRs) play an important role in initiating signaling cascades to produce TNF⍺. Little is known about how innate immunity is altered in healthy human aging that predisposes to sepsis. We tested the hypothesis that aging dysregulates the innate immune response to TLR 2 and 4 ligands. We performed whole blood assays on 554 healthy subjects aged 40-80years. TNFα production was measured at baseline and after stimulation with the TLR2 agonists: peptidoglycan, lipoteichoic acid, Pam3CysK, Zymosan A and the TLR4 agonist lipopolysaccharide (LPS). In a subset of subjects (n = 250), we measured Toll-like receptor (TLR) 2, 4 and MyD88 expression using real-time PCR. We measured a 2.5% increase per year in basal secretion of TNFα with aging (n = 554 p = 0.02). Likewise, TNFα secretion was increased with aging after stimulation with peptidoglycan (1.3% increase/year; p = 0.0005) and zymosan A (1.1% increase/year p = 0.03). We also examined the difference between baseline and stimulated TNFα for each individual. We found that the increase was driven by the elevated baseline levels. In fact, there was a diminished stimulated response to LPS (1.9% decrease/year; p = 0.05), lipoteichoic acid (2.1% decrease/year p = 0.03), and Pam3CysK (2.6% decrease/year p = 0.0007). There were no differences in TLR or MyD88 mRNA expression with aging, however, there was an inverse relationship between TLR expression and stimulated TNFα production. With aging, circulating leukocytes produce high levels of TNFα at baseline and have inadequate responses to TLR2 and TLR4 agonists. These defects likely contribute to the increased susceptibility to sepsis in older adults.

Expression of toll-like receptor 4 and its connection with type 2 diabetes mellitus

Toll-like receptor 4 (TLR4) plays an important role in modulating innate immunity. Type 2 diabetes mellitus (T2DM) is a chronic disease that is characterized by impaired insulin resistance and abnormal immune response. Genetic background and consequently genetic factors might have a key role in both onset and progression of T2DM-related complications. The aim of this work was to study the role of toll-like receptor 4 (TLR4) in the development of type 2 diabetes mellitus (T2DM). This study was carried out on 90 subjects, 30 type 2 diabetic patients, 30 patients with impaired glucose tolerance and 30 age and gender matched healthy controls. mRNA expression of (TLR4) was assessed by reverse transcriptase PCR (RT-PCR) using real time PCR.. Results showed significant statistical difference between the three studied groups regarding BMI, serum FBG, HDL, TGs, TC, LDL, HOMA -IR and mRNA expression of TLR4 with highest level of TLR4 mRNA expression in T2DM patients. From this study, it might be concluded that high expression of (TLR4) is associated with T2DM.

Detrimental effects of lipopolysaccharides on maturation of bovine oocytes.

ObjectiveGram-negative bacteria lipopolysaccharide (LPS) has been reported to be associated with uterine impairment, embryonic resorption, ovarian dysfunction, and follicle retardation. Here, we aimed to investigate the toxic effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes.MethodsFirst, we developed an in vitro model to study the response of bovine cumulus-oocyte complexes (COCs) to LPS stress. After incubating germinal vesicle COCs in 10 μg/mL of LPS, we analyzed the following three aspects: the expression levels of the LPS receptor toll-like receptor 4 (TLR4) in COCs, activities of intracellular signaling protein p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappa B (NF-κB); and the concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6. Furthermore, we determined the effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes.ResultsThe results revealed that LPS treatment significantly elevated TLR4 mRNA and protein expression levels in COCs. Exposure of COCs to LPS also resulted in a marked increase in activity of the intracellular signaling protein p-p38 MAPK and NF-κB. Furthermore, oocytes cultured in maturation medium containing LPS had significantly higher concentrations of the proinflammatory cytokines IL-1β, TNF-α, and IL-6. LPS exposure significantly decreased the first polar body extrusion rate. The cytoplasmic maturation, characterized by polar body extrusion and distribution of peripheral cortical granules, was significantly impaired in LPS-treated oocytes. Moreover, LPS exposure significantly increased intracellular reactive oxygen species levels and the relative mRNA abundance of the antioxidants thioredoxin (Trx), Trx2, and peroxiredoxin 1 in oocytes. Moreover, the early apoptotic rate and the release of cytochrome C were significantly increased in response to LPS. The cleavage, morula, and blastocyst formation rates were significantly lower in parthenogenetically activated oocytes exposed to LPS, while the incidence of apoptotic nuclei in blastocysts was significantly increased.ConclusionTogether, these results provide an underlying mechanism by which LPS impairs maturation potential in bovine oocytes.

18β-glycyrrhetinic acid inhibited mitochondrial energy metabolism and gastric carcinogenesis through methylation-regulated TLR2 signaling pathway.

The natural phenolic substance, 18β-glycyrrhetinic acid (GRA), has shown enormous potential in the chemoprevention of cancers with rich resources and biological safety, but the GRA-regulated genetic and epigenetic profiles are unclear. Deregulated mitochondrial cellular energetics supporting higher adenosine triphosphate provisions relative to the uncontrolled proliferation of cancer cells is a cancer hallmark. The Toll-like receptor 2 (TLR2) signaling pathway has emerged as a key molecular component in gastric cancer (GC) cell proliferation and epithelial homeostasis. However, whether TLR2 influenced GC cell energy metabolism and whether the inhibition effects of GRA on GC relied on TLR2 signaling were not illustrated. In the present study, TLR2 mRNA and protein expression levels were elevated in gastric tumors in the K19-Wnt1/C2mE (Gan) mice model, GC cell lines and human GCs, and the overexpression of TLR2 was correlated with the high histological grade and was a poor prognostic factor in GC patients. Further gain and loss of function showed that TLR2 activation induced GC cell proliferation and promoted reactive oxygen species (ROS) generation, Ca2+ accumulation, oxidative phosphorylation and the electron transport chain, while blocking TLR2 inhibited mitochondrial function and energy metabolism. Furthermore, GRA pretreatment inhibited TLR2-activated GC cell proliferation, energy metabolism and carcinogenesis. In addition, expression of TLR2 was found to be downregulated by GRA through methylation regulation. Collectively, the results demonstrated that GRA inhibited gastric tumorigenesis through TLR2-accelerated energy metabolism, suggesting GRA as a promising therapeutic agency targeting TLR2 signaling in GC.

The excretory-secretory products of Echinococcus granulosus protoscoleces stimulated IL-10 production in B cells via TLR-2 signaling

BackgroundExcretory-secretory products released by Echinococcus granulosus protoscoleces (EgPSC-ESPs) are well-known to regulate T cell responses. However, their direct influence on the differentiation of B cell subsets remains largely elusive. This study investigated the effects of EgPSC-ESPs on the differentiation of IL-10-producing B cells (B10), and explored the possible role of Toll-like receptor 2 (TLR-2) signaling in this process.ResultsIn comparison to phosphate buffered saline (PBS), B cells exposed to the excretory–secretory products (ESPs) generated higher percentages of B10 cells, with higher expression of IL-10 mRNA, and larger amount of IL-10 production, which were in a dose dependent way. The mRNA and protein expression of TLR-2 in the ESPs-stimulated B cells were significantly higher than those in PBS, which was consistent to the results in B cells isolated from EgPSC infected mice. Moreover, TLR-2−/− B cells in response to ESPs stimulation expressed lower levels of IL-10 mRNA and produced undetectable IL-10 in comparison to those in normal B cells. In addition, Phosphatase and tensin homolog deleted on chromosome ten/AKT/Phosphatidylinositol-3 kinase (PTEN/AKT/PI3K) pathway was activated in ESPs-treated B cells, which was also dependent on TLR-2 signaling. Pam3CSK4, the agonist of TLR-2, could mock the effects of ESPs on the expression of PTEN, AKT and PI3K.ConclusionOverall, this study revealed that TLR-2 signaling was required for B10 induction mediated by EgPSC-ESPs, which might be an immunomodulatory target against the parasite infection.

MMP-9 Upregulation is Attenuated by the Monoclonal TLR2 Antagonist T2.5 After Oxygen–Glucose Deprivation and Reoxygenation in Rat Brain Microvascular Endothelial Cells

BackgroundBlood-brain barrier (BBB) disruption plays a key role in the pathophysiology of acute ischemic stroke. Matrix metalloproteinases-2/9 (MMP-2/9) have been shown to participate in the disruption of the BBB and hemorrhagic transformation after cerebral ischemia. Toll-like receptor 2 (TLR2) may also be correlated with endothelial cell injury during ischemia-reperfusion events. However, the correlation between MMP-2/9 and TLR2 on endothelial cells after ischemia has not yet been evaluated. The aim of the study was to evaluate the impact of TLR2 and MMP-2/9 on tight junction proteins (TJs) after oxygen–glucose deprivation and reoxygenation (OGDR). Materials and methodsRat primary brain microvascular endothelial cells (BMECs) were cultured. Quantitative real-time PCR and western blotting were used to measure the mRNA and proteins expression of TLR2 and MMP-2/-9. The protein expression of TJs was detected by western blotting and immunofluorescence. ResultsMMP-9 significantly increased after OGDR. Protein and mRNA expression of TLR2 was also upregulated. However, claudin-5, occludin, collagen-Ⅳ, and ZO-1 were decreased after OGDR. When monoclonal anti-TLR2 antibody (T2.5) was added to BMECs after OGDR, MMP-9 was significantly downregulated, whereas occludin and collagen-Ⅳ had a tendency to increase. ConclusionTLR2 antagonist T2.5 is able to downregulate the expression of MMP-9, and may constitute a therapeutic option for restoration of the BBB after OGDR.

Innate immunity in the postmortem brain of depressed and suicide subjects: Role of Toll-like receptors

Abnormalities of Toll-like receptors (TLRs) have been implicated in the pathophysiology of depression and suicide. Interactions of TLRs with pathogen-associated molecular patterns (PAMP) and damage-associated molecular patterns (DAMP) initiate signaling through myeloid differentiation primary response-88 (MyD88) and produce cytokines through the activation of the transcription factor nuclear factor kappa beta (NF-kB). We have earlier shown an increase in the protein and mRNA expression of TLR3 and TLR4 in the prefrontal cortex (PFC) of depressed suicide (DS) subjects compared with normal control (NC) subjects. To examine if other TLRs are altered in postmortem brain, we have now determined the protein and mRNA expression of other TLRs (TLR1, TLR2, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10) in the PFC of DS, depressed non-suicide (DNS), non-depressed suicide (NDS) and NC subjects. We determined the protein expression by Western blot and mRNA expression levels by real-time PCR (qPCR) in the PFC of 24 NC, 24 DS, 12 DNS and 11 NDS subjects. Combined with our previous study of TLR3 and TLR4, we found that the protein expression of TLR2, TLR3, TLR4, TLR6 and TLR10, and mRNA expression of TLR2 and TLR3 was significantly increased in the DS group compared with NC group. This study demonstrated that certain specific TLRs are altered in DS subjects, and hence those TLRs may be appropriate targets for the development of therapeutic agents for the treatment of suicidal behavior.

Effect and mechanism of MicroRNA-146a on TLR4 inflammatory signal pathway in the lung tissues of rats with mechanical ventilator-induced lung injury

Objective: To investigate the effect and mechanism of microRNA-146a (miR-146a) on Toll-Like Receptor 4 (TLR4) inflammatory signal pathway in the lung tissues of rats with mechanical ventilator-induced lung injury. Methods: Thirty-two healthy male Sprague-Dawley rats were randomly divided into 4 groups (n=8 each): group A, normal control group, no mechanical ventilation, spontaneous breathing; group B, mechanical ventilation injury; group C, mechanical ventilation injury plus no-load virus transfection; group D, mechanical ventilation injury plus virus transfection; in group B, C, and D, mechanical ventilation were performed, respiratory rate was controlled at 80 beats/min, tidal volume was 40 ml/kg, inhaled oxygen concentration (FiO2) was 21%, inhalation/expiration ratio was 1∶2, positive end expiratory pressure ventilation (PEEP) was 0, each group were ventilated 4 hours daily, 7 days continuously to establish ventilator induced lung injury (VILI) rat model. Paraffin-embedded sections of lung tissue were stained with HE, the morphology and damage of lung tissue were observed under microscope. The lungs wet and dry ratio (W/D), the levels of inflammatory cytokines interleukin (IL)-1β, IL-2 and tumor necrosis factor (TNF)-α were determined. Real-time PCR was used to detect the expression of TLR4 mRNA. The level of TLR4 protein was determined by Western blot. Results: The levels of lung tissue W/D and lung injury scores in group B (6.41±0.10, 11.38±0.92), group C (6.45±0.19, 11.75±1.04), group D (5.95±0.14, 7.53±4.78) were significantly increased than those in group A (4.33±0.08, 0.25±0.46), and in group D they were significantly decreased than group C (all P<0.01). The levels of IL-1β, IL-2, TNF-α in group B[(36.07±4.28) pg/ml, (5.02±0.63) ng/ml, (382.57±35.41) ng/ml], group C[(35.82±5.47) pg/ml, (4.98±0.71) ng/ml, (375.13±36.95) ng/ml], group D[(27.01±3.18) pg/ml, (3.96±0.82) ng/ml, (297.56±39.08) ng/ml]were significantly increased than those in group A[(21.46±3.15) pg/ml, (2.45±0.17) ng/ml, (195.92±18.07) ng/ml], and in group D they were significantly decreased than group C (all P<0.01). The relative expression levels of TLR4 mRNA and TLR4 proteins in group B (29.57±5.10, 0.75±0.110), group C (27.27±4.72, 0.77±0.130), group D (12.89±2.58, 0.48±0.057) were significantly increased than those in group A (1.02±0.13, 0.18±0.025), and in group D they were significantly decreased than group C (all P<0.01). There was no significant difference of all the above indicators between group B and C (all P>0.05). Conclusions: MiR-146a can reduce acute lung inflammation and TLR4 expression in lungs of rats with mechanical ventilator-induced lung injury. MiR-146a may inhibit the inflammatory response through TLR4 signaling pathway.

Effects of TLR4-PI3K-Rac1 pathway on cytoskeleton rearrangement and phagocytosis of macrophage

Objective: To investigate the effects of Toll-like receptor 4 (TLR4)-phosphatidylinositol 3-kinase (PI3K) -Ras-related C3 botulinum toxin 1 (Rac1) signaling pathway on macrophage cytoskeleton rearrangement and phagocytosis. Methods: Mouse macrophage cell line RAW264.7 was divided into blank group, negative control group and TLR4-RNA interference (RNAi) group. The lentivirus carrying TLR4 short hairpin RNA (shRNA) and nonsense control sequence were respectively transfected into TLR4-RNAi group and negative control group. The cells in blank group were not transfected. The silencing efficiency of TLR4 was detected by Western blot. Real-time fluorescence quantitative PCR was used to detect the expression of PI3K and Rac1 mRNA in each group. The expressions of PI3K, p-Rac1 and Rac1 protein were detected by Western blot. Cytoskeleton was observed by laser scanning confocal microscopy. Mean fluorescence intensity (MFI) and the percentage of cells phagocytosing flurescein inothiocyanate-labeled Eseherichina coli (FITC-E.coli) (Phagocytosed cell %) were detected by flow cytometry. Results: The RAW264.7 cells can be successfully transfected by TLR4-shRNA lentivirus, and the transfection efficiency ranged from 80% to 90%. The silencing efficiency of TLR4 gene was (63±4)%. After silencing the TLR4 gene, the relative expression of TLR4 mRNA and protein (0.20±0.03, 0.37±0.04), PI3K mRNA and protein (0.64±0.06, 0.75±0.06), Rac1 mRNA, protein and p-Rac1 protein (0.75±0.04, 0.76±0.01, 0.74±0.05) in TLR4-RNAi group were significantly lower than those in negative control group and blank group (all P<0.01). The change of cytoskeleton: after silencing the TLR4 gene, the celluar pseudopods were short and stiff, with the impaired capacity of phagocytosing FITC-E.coli. Cells in blank group and negative control group extended good pseudopodia, and the capacity of phagocytosing FITC-E.coli was normal. The MFI and Phagocytosed cells % of TLR4-RNAi group[(7 453±564), (70.20±2.27)%]were significantly lower than those in the blank group and the negative control group (all P<0.01). Positive correlations were existed between mRNA, protein expression of TLR4, PI3K, Rac1 and MFI, Phagocytosed cells% (all P<0.05) in all groups. Conclusion: TLR4-PI3K-Rac1 pathway involves in the cytoskeleton rearrangement and impairs the phagocytosis of macrophages.

Low expression of miR-203 promoted diabetic nephropathy via increasing TLR4.

To investigate the relationship between microRNA-203 (miR-203) and diabetic nephropathy and its potential mechanism. The expression of microRNA-203 in mice with diabetic nephropathy and M4200 cells cultured with high glucose was detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Toll-like receptor 4 (TLR4), the target gene of microRNA-203, was predicted and screened by bioinformatics method. Real-time quantitative PCR and Western blot were used to detect the endogenous TLR4 level in renal cortex of db/db mice with diabetic nephropathy and glomerular mesangial cells cultured in high glucose or low glucose. The expression of microRNA-203 and TLR4 mRNA were evaluated by RT-PCR after treatment of miR-203 mimics and inhibitor. The protein of TLR4 level was detected by Western blot. Additionally, the proliferation ability of cells was evaluated by Cell Counting Kit-8 (CCK8). The target relationship between microRNA-203 and TLR4 3' UTR was confirmed by luciferase reporter assay RESULTS: The expression of miR-203 was significantly decreased in the kidney of mice with diabetic nephropathy and M4200 cells cultured in high glucose. On the contrary, TLR4 expression was significantly increased. Results of in vitro experiments showed that miR-203 could bind to 3'UTR region of TLR4. Overexpression of microRNA-203 significantly decreased the levels of TLR4 mRNA and protein. Meanwhile, low expression of miR-203 leaded to increased TLR4 expression, resulting in an enhanced proliferation of M4200 cells. The downregulation of microRNA-203 leaded to an increased level of TLR4, thus promoting proliferation of M4200 cells in the pathogenesis of diabetic nephropathy.

Toll-like receptors TLR-2, TLR-4, TLR-7, and TLR-9 in tumor tissue and serum of the patients with esophageal squamous cell carcinoma and gastro-esophageal junction cancer.

Stimulation of toll-like receptors (TLRs) has been linked to the development of esophageal and gastric cancers. The aim of the study was to evaluate the clinical significance of tissue expression and serum concentration of TLR-2, TLR-4, TLR-7 and TLR-9 in patients with esophageal squamous cell carcinoma and gastro-esophageal junction adenocarcinoma. The study group consisted of 97 individuals: 32 with esophageal squamous cell carcinoma, 27 with gastro-esophageal junction cancer, and 38 ageand gender-matched controls. The mRNA expression and protein concentration of TLRs in tissues and sera were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) tests. In esophageal cancer patients, mRNA expressions of TLR-2, TLR-4 and TLR-7, and protein concentrations of all TLRs were significantly higher in tumor than in control tissue (p < 0.05). In esophageal cancer patients with lymph node metastasis, a tendency toward higher protein concentrations of tumor TLR-4 was observed. In gastro-esophageal junction adenocarcinoma subgroup, only the mRNA expression of TLR-7 and protein concentrations of TLR-4, TLR-7 and TLR-9 were significantly higher in tumors than in normal mucosa (p < 0.05). Protein concentration of TLR-9 was significantly higher in tumors of gastro-esophageal junction cancer with lymph node metastasis and depth of tumor invasion. Diagnostic potential of serum TLR-4 as a marker of gastro-esophageal junction cancer presence was reported. We demonstrated differences in the expression patterns of TLRs between esophageal squamous cell carcinoma and adenocarcinoma of gastro-esophageal junction, and showed circulating TLR-4 to be a potential marker of gastro-esophageal junction cancer.

The novel estrogenic receptor GPR30 alleviates ischemic injury by inhibiting TLR4-mediated microglial inflammation

BackgroundThe steroid hormone estrogen (17-β-estradiol, E2) provides neuroprotection against cerebral ischemic injury by activating estrogen receptors. The novel estrogen receptor G protein-coupled receptor 30 (GPR30) is highly expressed in the brain and provides acute neuroprotection against stroke. However, the underlying mechanisms remain unclear.MethodsIn this study, ovariectomized female mice were subjected to middle cerebral artery occlusion (MCAO), and E2, G1, and ICI182780 were administered immediately upon reperfusion. The infarction volume, neurological scores, and neuronal injuries were examined. Primary microglial cells were subjected to oxygen-glucose deprivation (OGD), and the drugs were administered immediately upon reintroduction. The pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in penumbra and microglia were assessed by ELISA. The cell viability and lactose dehydrogenase (LDH) release of neurons co-cultured with microglia were analyzed using cell counting kit-8 (CCK8) and LDH release assays. Microglial activation as well as GPR30, Iba1, and Toll-like receptor 4 (TLR4) protein expression and TLR4 mRNA expression were detected. Additionally, NF-κB activity was detected in lipopolysaccharide (LPS)-activated microglia after the activation of GPR30.ResultsGPR30 was highly expressed in microglia and significantly increased after ischemic injury. The activation of GPR30 significantly reduced the infarction volume, improved the neurological deficit, and alleviated neuronal injuries. Moreover, GPR30 activation significantly reduced the release of TNF-α, IL-1β, and IL-6 from ischemic penumbra and microglia subjected to OGD and alleviated neuronal injury as assessed using the CCK8 and LDH assays. Finally, the activation of GPR30 relieved microglial activation, reduced Iba1 and TLR4 protein expression and TLR4 mRNA levels, and inhibited NF-κB activity.ConclusionsMicroglial GPR30 exerts acute neuroprotective effects by inhibiting TLR4-mediated microglial inflammation, which indicates that GPR30 may be a potential target for the treatment of ischemic stroke.

Diverse Profiles of Toll-Like Receptors 2, 4, 7, and 9 mRNA in Peripheral Blood and Biopsy Specimens of Patients with Celiac Disease.

Background and Aims Both adaptive and innate immunity are involved in the development of celiac disease (CD). Altered Toll-like receptors (TLR) expression and activation may be partially responsible for the inflammation and subsequently crypt hyperplasia, but the main driver for inflammation is gliadin-reactive T-cells. Therefore, the aim of this study was to investigate the TLRs 2, 4, 7, and 9 gene expressions in both peripheral blood and intestinal mucosa of patients with celiac disease compared to healthy control (HC). Material and Methods Blood samples from 120 confirmed active CD patients and 120 age- and sex-matched healthy volunteers served as control group were collected during 2015-2016. Also, 20 biopsy specimens from the study group were randomly collected. Total RNA was isolated using a standard commercial kit. The mRNA expression of TLRs was quantified by relative qPCR with β2 microglobulin (β2m) as a reference gene. Results TLR4 (P = 0.01) and TLR9 (P = 0.02) mRNA were significantly elevated in blood samples from CD patients compared to the healthy controls. Moreover, TLR2 (P = 0.03) and TLR4 (P = 0.0003) expression level was increased in CD biopsy specimens compared to controls, whereas expression of TLR9 mRNA was significantly decreased in CD patients. There was no significant difference in the expression of TLR7 in biopsy and blood specimens. Conclusions The alteration of TLR4 and TLR9 expression in the blood and biopsy samples of patients with CD supports the critical role of the innate immune system in the pathogenesis of this disease. Upregulation of TLR4 and TLR9 suggests the contribution of gut microbiota or dysregulation of the immune response to commensal flora in small bowel mucosa in celiac patients.

Varying magnesium concentration elicits changes in inflammatory response in human umbilical vein endothelial cells (HUVECs).

The aims of this study were to determine whether low concentrations of magnesium in vitro exacerbated the human umbilical vein endothelial cell (HUVEC) response to inflammatory challenge, and whether expression of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) through the toll-like receptor 4 (TLR4) played a role in this process. HUVECs were incubated with different concentrations of Mg (low- 0.1mM, control- 1mM, high- 5mM) for 72 h before being stimulated with bacterial lipopolysaccharide (LPS) for 4 h. The response of cells to LPS was greater in cells cultured in low Mg, relative to control cells and suppressed in high Mg. Expression of NF-κB was increased in low-Mg and decreased with high Mg. Low Mg increased the expression of TLR4 mRNA, but only in the presence of LPS. Antibody blockade of TLR4 but not TLR2 blunted the response of cells to LPS in low Mg, such that they were similar to unblocked 1mM Mg cells. Associations of Mg with cardiovascular disease may therefore relate to inflammatory responses mediated through the TLR4/NF-κB pathway.