Toll-like Receptor 4 mRNA Expression Research Articles

Toll-like receptor 1 as a possible target in non-alcoholic fatty liver disease

Toll-like receptors (TLRs) in the liver compartment have repeatedly been attributed to the development of non-alcoholic fatty liver disease (NAFLD). Knowledge on TLR expression in blood cells and their relation to intestinal microbiota and NAFLD development is limited. Here, we determined TLR expression patterns in peripheral blood mononuclear cells (PBMCs) of NAFLD patients and controls, their relation to intestinal microbiota and the impact of TLRs found altered in NAFLD development. Markers of intestinal permeability in blood and TLR mRNA expression in PBMCs were determined in 37 NAFLD patients and 15 age-matched healthy controls. Fecal microbiota composition was evaluated in 21 NAFLD patients and 9 controls using 16S rRNA gene amplicon sequencing. Furthermore, TLR1−/− and C57BL/6 mice (n = 5–6/group) were pair-fed a liquid control or a fat-, fructose- and cholesterol-rich diet. Intestinal microbiota composition and markers of intestinal permeability like zonulin and bacterial endotoxin differed significantly between groups with the latter markers being significantly higher in NAFLD patients. Expression of TLR1-8 and 10 mRNA was detectable in PBMCs; however, only TLR1 expression, being higher in NAFLD patients, were significantly positively correlated with the prevalence of Holdemanella genus while negative correlations were found with Gemmiger and Ruminococcus genera. TLR1−/− mice were significantly protected from the development of diet-induced NAFLD when compared to wild-type mice. While intestinal microbiota composition and permeability differed significantly between NAFLD patients and healthy subjects, in PBMCs, only TLR1 expression differed between groups. Still, targeting these alterations might be a beneficial approach in the treatment of NAFLD in some patients.

Valsartan Prevented Neointimal Hyperplasia and Inhibited SRSF1 Expression and the TLR4-iNOS-ERK-AT1 Receptor Pathway in the Balloon-injured Rat Aorta.

Valsartan has the potential to attenuate neointimal hyperplasia and to suppress the inflammatory response. This study aimed to evaluate the role of valsartan in neointimal hyperplasia and the toll-like receptor 4 (TLR4)-nitric oxide synthase (NOS) pathway in the balloon-injured rat aorta.Forty-eight Wistar rats were randomly allocated to three groups: sham control (control), balloon-injured group (surgery), and balloon-injured+valsartan-treated group (valsartan). Rats were killed at 14 and 28 days after balloon-injury, and then the aortic tissues were collected for morphometric analysis as well as for measurements of the mRNA or protein expression of angiotensin II, angiotensin II type 1 (AT1) receptor, angiotensin II type 2 (AT2) receptor, TLR4, endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), serine/arginine-rich splicing factor 1(SRSF1) and extracellular signal regulated kinase (ERK). Valsartan at a dose of 20 mg/kg/day markedly decreased neointimal hyperplasia in the aorta of balloon-injured rats, and significantly reduced the mRNA or protein expression of TLR4, AT1 receptor, SRSF1 and phosphorylated-ERK (p-ERK) as well as the aortic levels of iNOS (all p < 0.05). Moreover, valsartan increased the eNOS level and AT2 receptor mRNA and protein expression levels (all p < 0.05). Valsartan prevented neointimal hyperplasia and inhibited SRSF1 expression and the TLR4-iNOS-ERK-AT1 receptor pathway in the balloon-injured rat aorta.

Vitamin D3 Attenuates Viral-Induced Inflammation and Fibrotic Responses in Bronchial Smooth Muscle Cells.

Toll-like receptor 3 (TLR3) activation by viral infections plays a key role in promoting inflammatory immune responses that contribute to pulmonary fibrosis in chronic inflammatory respiratory diseases. Vitamin D3 has been shown to be beneficial to patients with asthma and chronic obstructive pulmonary disease (COPD) through its anti-inflammatory and anti-fibrotic properties. Smooth muscle cells are one of the major contributors to airway remodeling in asthma and COPD. We therefore aimed to investigate the effect of vitamin D3 treatment on viral-induced TLR3 responses in Bronchial Smooth Muscle Cells (BSMCs) as a mechanism contributing to pulmonary fibrosis in asthma and COPD. Primary BSMCs from patients with asthma (n=4), COPD (n=4), and healthy control subjects (n=6) were treated with polyinosinic: polycytidylic acid (polyI:C), TLR3 agonist in the presence or absence of vitamin D3 (1,25D3). Here we report the mRNA expression and protein levels of pro-inflammatory and pro-fibrotic markers (IL-6, IFN-β1, CCL2/MCP-1, fibronectin 1 and type I collagen) among BSMCs groups: asthma, COPD, and healthy controls. We show that at the baseline, prior to polyI:C stimulation, asthma and COPD BSMCs presented increased pro-inflammatory and pro-fibrotic state compared to healthy control subjects, as measured by quantitative PCR and immunoassays (ELISA/Flow Cytometry. Ligation of TLR3 by polyI:C in BSMCs was associated with increased TLR3 mRNA expression, and 1,25D3 treatment significantly reduced its expression. In addition, 1,25D3 decreased the expression of IL-6, IFN-β1, CCL2, FN1 and COL1A1 induced by polyI:C in BSMCs. The regulatory effect of 1,25D3 treatment on polyI:C-stimulated BSMCs was further confirmed at protein levels. Our findings suggest that vitamin D3 attenuates TLR3 agonist-induced inflammatory and fibrotic responses in BSMCs and support the clinical relevance of vitamin D3 supplementation in patients with viral infections having chronic respiratory diseases, such as asthma and COPD.

Galacto-oligosaccharides improve barrier function and relieve colonic inflammation via modulating mucosa-associated microbiota composition in lipopolysaccharides-challenged piglets

BackgroundGalacto-oligosaccharides (GOS) have been shown to modulate the intestinal microbiota of suckling piglets to exert beneficial effects on intestinal function. However, the modulation of intestinal microbiota and intestinal function by GOS in intestinal inflammation injury models has rarely been reported. In this study, we investigated the effects of GOS on the colonic mucosal microbiota composition, barrier function and inflammatory response of lipopolysaccharides (LPS)-challenged suckling piglets.MethodsA total of 18 newborn suckling piglets were divided into three groups, the CON group, the LPS-CON group and the LPS-GOS group. Piglets in the LPS-GOS group were orally fed with 1 g/kg body weight of GOS solution every day. On the d 14, piglets in the LPS-CON and LPS-GOS group were challenged intraperitoneally with LPS solution. All piglets were slaughtered 2 h after intraperitoneal injection and sampled.ResultsWe found that the colonic mucosa of LPS-challenged piglets was significantly injured and shedding, while the colonic mucosa of the LPS-GOS group piglets maintained its structure. Moreover, GOS significantly reduced the concentration of malondialdehyde (MDA) and the activity of reactive oxygen species (ROS) in the LPS-challenged suckling piglets, and significantly increased the activity of total antioxidant capacity (T-AOC). GOS significantly increased the relative abundance of norank_f__Muribaculaceae and Romboutsia, and significantly decreased the relative abundance of Alloprevotella, Campylobacter and Helicobacter in the colonic mucosa of LPS-challenged suckling piglets. In addition, GOS increased the concentrations of acetate, butyrate and total short chain fatty acids (SCFAs) in the colonic digesta of LPS-challenged suckling piglets. GOS significantly reduced the concentrations of interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α) and cluster of differentiation 14 (CD14), and the relative mRNA expression of Toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) in the LPS-challenged suckling piglets. In addition, GOS significantly reduced the relative mRNA expression of mucin2 (MUC2), and significantly increased the protein expression of Claudin-1 and zonula occluden-1 (ZO-1) in LPS-challenged suckling piglets.ConclusionsThese results suggested that GOS can modulate the colonic mucosa-associated microbiota composition and improve the intestinal function of LPS-challenged suckling piglets.

Activation of 5-HT 1b/d receptor restores the cognitive function by reducing glutamate release, deposition of β-amyloid and TLR-4 pathway in the brain of scopolamine-induced dementia in rat.

This study evaluates the effect of 5-HT 1b/d agonist on cognitive function in scopolamine (SPN)-induced dementia in the rat. Dementia was induced by administration of SPN 2 mg/kg/day, intraperitoneally, for a duration of 21 days. The effect of zolmitriptan (ZMT) 30 mg/kg, intraperitoneally, was observed on cognitive function, and the parameters of oxidative stress like malondialdehyde (MDA) level, nitric oxide (NO), superoxide dismutase (SOD) and glutathione peroxidase (GPX) were estimated at the end. Histopathology study of brain tissue was performed for the determination of β-amyloid peptide, and qRT-PCR was used to determine the mRNA expression of Toll-like receptor 4 (TLR-4), IL-17 and β-amyloid. Data of the study suggested that treatment with ZMT alone and in combination with DMP (dextromethorphan) significantly (P < 0.01) decreases the escape latency in conditioned avoidance response (CAR) and transfer latency in elevated plus maze (EPM) as compared with negative control group. Moreover, the result of Morris water maze (MWM) shows an increase in retention time and a decrease in escape latency in ZMT alone and in combination with DMP-treated group of SPN-induced dementia than in the negative control group. There was a significant decrease in MDA and NO and increase in SOD and GPX in the brain tissues of ZMT and ZMT + DMP-treated group than negative control group. Histopathology study also suggested that the concentration of Aβ peptide decreases in the brain tissues in ZMT and ZMT + DMP-treated group than the negative control group. Moreover, ZMT treatment ameliorates the altered mRNA expression of TLR-4 and IL-17 in the brain tissue of SPN-induced dementia rat. In conclusion, ZMT restores the cognitive functions and impaired memory in SPN-induced dementia in the rat by decreasing oxidative stress and Aβ peptide in the brain tissue of rat.

A Comparative Study on Changes in Total and Differential Milk Cell Counts, Activity, and Expression of Milk Phagocytes of Healthy and Mastitic Indigenous Sahiwal Cows.

Milk somatic cell counts (SCCs) have been used as a gold standard to monitor mammary health as well as an indicator of raw milk quality. The present work was undertaken to compare the changes in the milk SCC, milk differential leukocyte counts (DLCs), phagocytic activity (PA) of milk neutrophils and macrophages (by nitroblue tetrazolium assay), extracellular trap formation (PicoGreen assay) and mRNA expression of various genes in milk neutrophils and macrophages (reverse transcription–polymerase chain reaction), and milk plasma cortisol concentration (enzyme-linked immunosorbent assay) in healthy, subclinical mastitis (SCM), and clinical mastitis (CM) cows. Milk was collected from healthy, SCM, and CM cows grouped based on their SCCs and California mastitis test with eight cows in each group. Milk SCC was estimated by SCC counter, and DLC was done after staining the milk slide under a microscope at 100×. Total SCCs in healthy, SCM, and CM cows were on an average of 128.30, 300.3, and 694.40 × 103 cells/mL, respectively. Milk DLCs indicated a lower percentage of macrophage and lymphocytes and a higher (p < 0.05) percentage of neutrophils in SCM and CM compared to healthy milk. The percentage of mature segmented neutrophils was lower, whereas immature band neutrophils were higher (p < 0.05) in the SCM and CM groups as compared to healthy cows. The viability, in vitro PA, and extracellular trap formation of neutrophils were lower (p < 0.05) in SCM and CM milk samples as compared to healthy samples. However, the PA of macrophage remained unchanged in all the studied groups. The relative mRNA expression of Toll-like receptors (TLR2, TLR4), myeloperoxidase, and interleukin 2α (IL-2α) receptor (CD25) were minimum in healthy samples and increased (p < 0.05) with the progress of mammary inflammation. However, CD44 decreased (p < 0.05), and CD62L remained unchanged in mastitis as compared to healthy cows. Plasma cortisol concentrations were higher (p < 0.05) in mastitis as compared to healthy cows and were negatively correlated with the number of milk macrophages and the functions of milk phagocytes. Estimation of total SCC, milk DLC, and activity of milk phagocytes is essential for effective control and prevention of incidence of mastitis in dairy cows.

MyD88 determines the protective effects of fish oil and perilla oil against metabolic disorders and inflammation in adipose tissue from mice fed a high-fat diet

BackgroundThe beneficial effects of ω−3 polyunsaturated fatty acids (PUFA) vary between different sources. However, there is a paucity of comparative studies regarding the effects and mechanisms of marine and plant ω−3 PUFA on obesity.ObjectiveThe aim of this study was to evaluate the effects of fish oil (FO) and perilla oil (PO) on glucolipid metabolism, inflammation, and adipokine in mice fed a high-fat (HF) diet in association with the contribution of toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88) pathway.MethodsC57BL/6J mice and MyD88−/− mice were randomly divided into 4 groups: normal chow diet, HF diet, HF diet accompanied by daily gavage with either FO or PO. After 4 weeks, blood biochemistries, adipocyte histology, mRNA, and protein expression of MyD88-dependent and -independent pathways of TLR4 signaling in epididymal adipose tissue were measured.ResultsIn C57BL/6J mice, there were no statistical differences between FO and PO in decreasing body weight, glucose, insulin, triglyceride, total cholesterol, interleukin-6, and increasing adipocyte counts. FO and PO decreased mRNA and protein expression of TLR4, MyD88, tumor necrosis factor receptor-associated factor 6, inhibitor of nuclear factor kappa B kinase beta and nuclear factor-kappa B p65. In MyD88−/− mice, the beneficial effects of FO and PO on HF diet-induced metabolism abnormalities and inflammation were abolished. FO and PO had no impacts on mRNA and protein expression of receptor-interacting protein-1, interferon regulate factor 3, and nuclear factor-kappa B p65.ConclusionFO and PO exhibit similar protective effects on metabolic disorders and inflammation through inhibiting TLR4 signaling in a manner dependent on MyD88. These findings highlight plant ω−3 PUFA as an attractive alternative source of marine ω−3 PUFA and reveal a mechanistic insight for preventive benefits of ω−3 PUFA in obesity and related metabolic diseases.

Cultured Human Uveal Melanocytes Express/secrete CXCL1 and CXCL2 Constitutively and Increased by Lipopolysaccharide via Activation of Toll-like Receptor 4

ABSTRACT Purpose: Lipopolysaccharide (LPS) can activate Toll-like receptor 4 (TLR4) and increase the expression of CXCL1 and CXCL2, the potent neutrophils chemoattractants, in various cell types. These effects have not been previously reported in the uveal melanocytes. This study was designed to investigate the effects of LPS on the activation of TLR4 and expression of CXCL1/CXCL2 in cultured human uveal melanocytes and the relevant signal pathways. Methods: Effects of LPS on the expression of TLR4 were tested using real-time PCR, flow cytometry and fluorescence immunostaining. Effects of LPS-induced expression/secretion of CXCL1/CXCL2 were studied using real-time PCR in cell lysates and ELISA in conditioned media of cultured uveal melanocytes. Activated NF-κB and phosphorylated MAPK signals were tested in cells with and without LPS treatment using flow cytometry. Effects of various signal inhibitors on p38, ERK1/2, JNK1/2 and NF-κB on the secretion of CXCL1/CXCL2 were tested by ELISA. The effects of neutralized antibodies of CXCL1/CXCL2 on the severity of LPS-induced uveitis were tested in a mouse model. Results: LPS stimulation increased the expression of TLR4 mRNA and protein in culture uveal melanocytes. Constitutive secretion of CXCL1/CXCL2 was detected in uveal melanocytes and was significantly increased dose- and time-dependently by LPS stimulation. LPS mainly increased the activated NF-κB and phosphorylated JNK1/2. LPS-induced expression of CXCL1/CXCL2 was blocked by NF-κB and JNK1/2 inhibitors. The severity of LPS-induced uveitis was significantly inhibited by neutralizing antibody to CXCL1/CXCL2 Conclusions: This is the first report on the LPS-induced expression of CXCL1 and CXCL2 by uveal melanocytes via the activation of TLR4. These results suggest that uveal melanocytes may play a role in the immune reaction that eliminates the invading pathogens. Conversely, an excessive LPS-induced inflammatory reaction may also lead to the development of inflammatory ocular disorders, such as non-infectious uveitis.

Systematic Pharmacology Reveals the Antioxidative Stress and Anti-Inflammatory Mechanisms of Resveratrol Intervention in Myocardial Ischemia-Reperfusion Injury.

Objective To explore the oxidative stress and inflammatory mechanisms of resveratrol intervention in myocardial ischemia-reperfusion injury (MIRI). Methods The potential targets of resveratrol were predicted by PharmMapper. The MIRI genes were collected by Online Mendelian Inheritance in Man (OMIM), GeneCards is used to collect related disease genes, and String is used for enrichment analysis. Animal experiments were then performed to verify the systematic pharmacological results. Hematoxylin-eosin (HE) staining was used to observe myocardial damage. The levels of serum interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in each experimental group were detected. The protein and mRNA expressions of Toll-like receptor 4 (TLR4), nuclear factor-kappa (NF-κB) p65, IL-1β, IL-6, and TNF-α in rat myocardial tissue were measured. Results The results of systematic pharmacology showed that insulin resistance, FoxO signaling pathway, adipocytokine signaling pathway, insulin signaling pathway, PI3K-Akt signaling pathway, ErbB signaling pathway, T-cell receptor signaling pathway, peroxisome proliferator-activated receptors (PPAR) signaling pathway, Ras signaling pathway, TNF signaling pathway, and so on were regulated to improve MIRI. The results of animal experiments showed that the myocardial cells of the sham operation group were arranged in fibrous form, and the myocardial ischemia-reperfusion injury group had obvious cell morphology disorder. Compared with the MIRI group, the resveratrol group had a certain degree of relief. Compared with the MIRI group, serum IL-1β, TNF-α, and IL-6 in the resveratrol group was significantly reduced (P < 0.05), and myocardial tissue TLR4, NF-κB p65, IL-1β, IL-6, and TNF-α mRNA and protein expressions were significantly reduced (P < 0.05). Conclusion Resveratrol can effectively improve MIRI, and its mechanism may be related to antioxidative stress and anti-inflammatory.

The Expression of Toll-Like Receptor 4 mRNA in PBMCs Is Upregulated in Smokers and Decreases Upon Smoking Cessation.

BackgroundStudies have shown in vitro that cigarette smoke condensate stimulates monocytes to express toll-like receptor 4 (TLR4), tumor necrosis factor-α (TNF-α), and intercellular adhesion molecule 1 (ICAM-1), and enhances their adhesion to the endothelium. However, the same effects of cigarette smoking have not been explored in vivo. This study is to investigate the effect of cigarette smoking and smoking cessation on their mRNA expression in human peripheral blood mononuclear cells (PBMCs).MethodsA group of 97 smokers and 62 nonsmokers were enrolled. The RNA from PBMCs was assessed with real-time polymerase chain reaction (PCR) to determine the levels of ICAM-1, TNF-α, and TLR4. The same markers in PBMCs of 87 quitters were examined before and at one week, one month, and two months after smoking cessation.ResultsOf the 97 smokers, 85 (87.6%) were males, and 30 (48.4%) of the nonsmokers were males (p < 0.0001). The mean (SD) age of the smokers was 43.24 (10.89) years, which was younger than 43.45 (11.41) years of nonsmokers (p < 0.0001). The incidence of cardiovascular diseases was 13.4% in smokers, which was higher than 1.6% in nonsmokers (p < 0.05). Both ICAM-1 and TNF-α mRNA levels in PBMCs were higher among the smokers (p < 0.0001). In addition, TLR4 mRNA levels in PBMCs were statistically elevated in the smokers (p < 0.0001) comparing with those in the nonsmokers. The mRNA levels of TLR4 and TNF-α in PBMCs decreased in those who had quit smoking for 2 months (p < 0.0001).ConclusionsICAM-1, TNF-α, and TLR4 mRNA expression levels in PBMCs increased in smokers and decreased after being on a smoking cessation program for 2 months. This finding suggested that TLR4 expression may mediate the atherogenic inflammatory process induced by smoking.

Expression of toll-like receptor 4 gene polymorphism and inflammatory factors in peripheral blood on patients with sepsis

Objective: To observe the presence of toll-like receptor 4 (TLR4) gene polymorphism in the venous blood in patients with sepsis, meanwhile, to observe the expression and the diagnostic value of TLR4 mRNA, interferon-γ (IFN-γ), interleukin-23 (IL-23), procalcitonin (PCT) and C-reactive protein (CRP) in patients with different degrees of sepsis.Methods: The peripheral blood samples from the subjects were collected to extract genomic DNA, gene sequencing and enzyme digestion method were applied to the detection of TLR4 Asp299Gly loci polymorphism. The expression of TLR4 mRNA in the peripheral blood was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The expression levels of IFN-γ, IL-23, PCT and CRP were measured by enzyme linked immunosorbent assay (ELISA).Results: (1) The gene polymorphism was not present in TLR4 Asp299Gly loci; (2) The expression of TLR4 mRNA in the peripheral blood in patients with sepsis: on d1, there were statistically significant differences between the normal group and the group (APACHE II ≤ 20), between the normal group and the group (APACHE II &gt; 20), between the group (APACHE II ≤ 20) and the group (AAPACHE II &gt; 20) (t = 5.741, 14.780 and 10.500, all p &lt; .01). APACHE II stands for Acute Physiology and Chronic Health Evaluation II. On d7, there were statistically significant differences between the normal group and the group (APACHE II ≤ 20), between the normal group and the group (APACHE II &gt; 20), between the group (APACHE II ≤ 20) and the group (APACHE II &gt; 20) (t = 4.186, 13.830 and 9.645, all p &lt; .01); (3) The expression levels of IFN-γ, IL-23, PCT and CRP were obviously upregulated (all p &lt; .01), and TLR4 was positively correlated with IFN-γ and IL-23 (all p &lt; .01); (4) The best cutoff value of TLR4 mRNA at baseline was 891.6 μg/L, with a sensitivity of 100% and a diagnostic specificity of 57%. The best cutoff value of IFN-γ at baseline was 84.5 μg/L, with a sensitivity of 100% and a diagnostic specificity of 57%. The best cutoff value of IL-23 at baseline was 861 μg/L, with a sensitivity of 100% and a diagnostic specificity of 97%.Conclusions: (1) The Asp299Gly polymorphism is not present in TLR4 gene; (2) The expression levels of TLR4 mRNA, IFN-γ and IL-23 are relatively high in patients with sepsis, and will be higher with the increased severity of sepsis; (3) TLR4 mRNA, IFN-γ and IL-23 can be used as molecular biomarkers for the early diagnosis of sepsis.

Osteogenesis, Osteoclastogenesis and their Crosstalk in Lipopolysaccharide-induced Periodontitis in Mice.

To determine the crosstalk of osteogenesis and osteoclastogenesis of alveolar bone in lipopolysaccharide (LPS)-induced periodontitis in mice. A representative periodontitis model was established by treating mice with LPS, and osteoblasts and osteoclasts were cultured. Osteoblasts and osteoclasts were cocultured to determine the effects of LPS on the crosstalk of osteogenesis and osteoclastogenesis. Quantitative polymerase chain reaction (qPCR) was performed to determine the expression of osteoclastogenesis makers underlying the potential mechanisms. The morphological and pathological changes in alveolar bone were observed in LPSinduced mice and LPS dose-dependently suppressed osteogenesis. The mRNA expression of cathepsin K, as a marker of osteoclasts, was accordingly downregulated in the coculture. The mRNA expression of osteoprotegerin was increased, while that of receptor activator of nuclear factor-κB ligand (RANKL) was decreased with an increased concentration of LPS. Moreover, the mRNA expression of toll-like receptor 4 (TLR4) was upregulated by LPS, whereas TLR4 knockout partially recovered osteoclast differentiation in the upper layer of the coculture. LPS dose-dependently suppressed osteogenesis but had a bidirectional effect on osteoclastogenesis. The combined effects of LPS on osteogenesis, osteoclastogenesis and their crosstalk via TLR4 account for alveolar bone loss in periodontitis.

Rhamnetin ameliorates macrophage-mediated inflammation and pro-atherosclerosis pathways in apolipoprotein E-deficient mice.

The present study was performed to examine the protective effects of rhamnetin against the development of atherosclerosis and its effects on the regulation of several pathways. The anti-atherosclerosis effect of rhamnetin was examined in cells stimulated with oxidized low-density lipoprotein (ox-LDL) by estimating the percentages of foam cell formation and apoptotic cells and determining the expression of specific proteins. As an in vivo model of atherosclerosis, apolipoprotein E-deficient (ApoE-/-) mice were treated with intragastric rhamnetin at 100 or 200 mg/kg for 8 weeks. The levels of inflammatory cytokines and oxidative stress parameters were determined in the aortic tissue of rhamnetin-treated ApoE-/- mice. The effects of rhamnetin on the Toll-like receptor 4 (TLR-4) pathway were assessed by quantitative reverse transcription polymerase chain reaction. The results of this study suggest that rhamnetin reduces the percentages of foam cells and apoptotic cells and the expression of CD36 and TLR-4 protein in ox-LDL-stimulated macrophages. The rhamnetin treatment group showed reductions in the lipid profile and levels of parameters of liver function compared to ApoE-/- mice. The levels of inflammatory cytokines and oxidative stress were reduced in the aortic tissue of the rhamnetin treatment group compared to ApoE-/- mice. Treatment with rhamnetin ameliorated the expression of TLR-4 mRNA and components of the TLR-4 pathway in the aortic tissue of ApoE-/- mice. In conclusion, the results of this study suggest that rhamnetin treatment inhibits the inflammatory and pro-atherosclerosis pathways in ApoE-/- mice.

Toll-Like Receptor Expression Profiles in Koala (Phascolarctos cinereus) Peripheral Blood Mononuclear Cells Infected with Multiple KoRV Subtypes.

Simple SummaryKoala retrovirus (KoRV) is a major pathogen of koala. Toll-like receptors (TLRs) are important innate immune component that are evolutionary conserved and play a crucial role in the early defense against invading pathogens. The expression profile of TLRs in KoRV infection in koalas is not characterized yet. Therefore, in this study, we characterized TLR expression patterns in koalas infected with KoRV-A only vs. KoRV-A with KoRV-B and/or -C. Using qRT-PCR, we measured TLR2–10 and TLR13 mRNA expression in peripheral blood mononuclear cells (PBMCs) and/or tissues from captive koalas in Japanese zoos. We observed variations in TLR expression in koalas with a range of subtype infection profiles (KoRV-A only vs. KoRV-A with KoRV-B and/or -C). The findings of this study might improve our current understanding of koala’s immune response to KoRV infection.Toll-like receptors (TLRs), evolutionarily conserved pattern recognition receptors, play an important role in innate immunity by recognizing microbial pathogen-associated molecular patterns. Koala retrovirus (KoRV), a major koala pathogen, exists in both endogenous (KoRV-A) and exogenous forms (KoRV-B to J). However, the expression profile of TLRs in koalas infected with KoRV-A and other subtypes is yet to characterize. Here, we investigated TLR expression profiles in koalas with a range of subtype infection profiles (KoRV-A only vs. KoRV-A with KoRV-B and/or -C). To this end, we cloned partial sequences for TLRs (TLR2–10 and TLR13), developed real-time PCR assays, and determined TLRs mRNA expression patterns in koala PBMCs and/or tissues. All the reported TLRs for koala were expressed in PBMCs, and variations in TLR expression were observed in koalas infected with exogenous subtypes (KoRV-B and KoRV-C) compared to the endogenous subtype (KoRV-A) only, which indicates the implications of TLRs in KoRV infection. TLRs were also found to be differentially expressed in koala tissues. This is the first report of TLR expression profiles in koala, which provides insights into koala’s immune response to KoRV infection that could be utilized for the future exploitation of TLR modulators in the maintenance of koala health.

Centella asiatica Extract Attenuates Kidney Fibrosis Through Reducing Mesenchymal Transition and Inflammation in Ureteral Ligation Model in Mice

Background: Kidney fibrosis is the common final pathway of chronic kidney disease (CKD), and is characterized by inflammation, mesenchymal transition with myofibroblast formation and epithelial to mesenchymal transition (EMT). Centella asiatia (CeA) is an herb that has a reno-protective effect. However, its mechanism of action in kidney fibrosis has not been elucidated. Aim: To elucidate the effect of CeA in amelioration of kidney fibrosis in a unilateral ureteral obstruction (UUO) model and focus on mesenchymal transition and inflammation. Methods: Unilateral ureteral obstruction was performed in male Swiss-background mice (age: 2–3 months, weight: 30–40 g, UUO group n = 6) to induce kidney fibrosis. Two doses of CeA extract with oral administration, 210 and 840 mg/kg body weight were added in UUO (U+C210 and U+C840 groups, each n = 6). The sham operation procedure was performed for the control group (SO, n = 6). The mice were euthanized at day-14 after operation. Tubular injury and interstitial fibrosis area fractions in kidney tissues of the mice were quantified based on periodic acid-Schiff (PAS) and Sirius Red (SR) staining. Immunostaining was performed for examination of fibroblast (PDGFR-β), myofibroblast (α-SMA), Monocyte Chemoattractant Protein-1 (MCP-1) and macrophage (CD68), meanwhile double immunofluorescence was performed with PDGFR-β and α-SMA. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to examine mRNA expression of TGF-β, Collagen-1, Snail, E-cadherin, vimentin, fibroblast-specific protein 1 (FSP-1), CD68, toll-like receptor 4 (TLR4), and MCP-1. Results: We observed a significantly higher interstitial fibrosis area fraction and tubular injury (p < 0.001) with fibroblast expansion and myofibroblast formation in the UUO group than in the SO group. These findings were associated with higher mRNA expression of TGF-β, Collagen-1, Snail, vimentin, FSP-1, CD68, TLR4, and MCP-1 and lower mRNA expression of E-cadherin. The U+C840 group had a significantly lower tubular injury score and interstitial fibrosis area fraction, which associated with downregulation of mRNA expression of TGF-β, Collagen-1, Snail, vimentin, FSP-1, CD68, TLR4, and MCP-1, with upregulation of mRNA expression of E-cadherin. Immunostaining observation revealed the U+C840 group demonstrated reduction of macrophage infiltration and myofibroblast expansion. Conclusion: CeA treatment with dose-dependently ameliorates mesenchymal transition and inflammation in kidney fibrosis in mice.

Age-dependent messenger RNA expression of toll-like receptor 4 and intercellular adhesion molecule-1 in peripheral blood mononuclear cells.

Inflammation plays an important role in the ageing process in which monocytes/macrophages are important players. Intercellular adhesion molecule-1 (ICAM-1), tumour necrosis factor-α (TNF-α) and Toll-like receptor 4 (TLR4) are well-known inflammatory markers. This study aimed to investigate the relationship between age and the expression and correlation of ICAM-1, TNF-α and TLR4 mRNA in peripheral blood mononuclear cells (PBMCs). A total of 239 participants were recruited in a medical centre in Taiwan. The mRNA isolated from the PBMCs was used to determine the levels of ICAM-1, TNF-α and TLR4 mRNAs with real-time polymerase chain reaction (PCR). The propensity-matched analysis was also applied for subgroup analysis. When compared 189 older adults (≧65years) to 50 younger adults (<65years), the ICAM-1, TNF-α and TLR4 mRNA levels in PBMCs were significantly higher in older adults (2.00±0.72 vs 0.87±0.34 for ICAM-1, 2.32±0.69 vs 1.15±0.44 for TNF-α and 1.56±0.47 vs 1.05±0.51 for TLR4, and all P<.0001). Also, both age and TLR4 were independent factors affecting mononuclear cell ICAM-1 in the multiple linear regression analysis (P<.0001). The mRNA levels of ICAM-1 and TLR4 in PBMCs are higher in older adults than those in younger adults. TLR4 is an independent factor affecting ICAM-1 expression in PBMCs, especially in older adults. This may suggest that ICAM-1 and TLR4 in PBMCs are potential biomarkers and their relationship may shed some light on the ageing process.

Analysis of PI3K Pathway Associated Molecules Reveals Dysregulated Innate and Adaptive Functions of B Cells in Early Diffuse Cutaneous Systemic Sclerosis.

B cell activation is an early event in the development of systemic sclerosis (SSc). The classical activation of B cells downstream of the B-cell receptor (BCR) involves the phosphatidylinositol-3 kinase (PI3K) pathway that integrates the effects of multiple co-stimulatory receptors. Our analysis of PI3K pathway associated molecules in peripheral blood B cells of early diffuse cutaneous SSc (dcSSc) patients showed altered mRNA expression of Toll-like receptor (TLR) homolog CD180, TLR4, complement component 3, IL-4 receptor and secreted phosphoprotein 1 (SPP1). Parallel to this, we found elevated basal SPP1 secretion in dcSSc B cells, but, with BCR + IL-4 receptor co-stimulation, we could not induce further secretion. CD180 stimulation alone resulted in NF-κB activation in more B cells than CD180 + BCR co-stimulation both in dcSSc and healthy control (HC), but the co-engagement increased the phosphorylation of NF-κB only in dcSSc B cells. Additionally, in contrast with HC B cells, the lower basal production of IL-10 by dcSSc B cells could not be elevated with CD180 stimulation. Furthermore, activation via CD180 increased the percentage of CD86+ switched memory (CD27+IgD−) B cells in dcSSc compared to HC. Our results suggest that alternative B cell activation and CD180 dysfunction cause imbalance of regulatory mechanisms in dcSSc B cells.

A novel stepwise integrative analysis pipeline reveals distinct microbiota-host interactions and link to symptoms in irritable bowel syndrome

Although incompletely understood, microbiota-host interactions are assumed to be altered in irritable bowel syndrome (IBS). We, therefore, aimed to develop a novel analysis pipeline tailored for the integrative analysis of microbiota-host interactions and association to symptoms and prove its utility in a pilot cohort. A multilayer stepwise integrative analysis pipeline was developed to visualize complex variable associations. Application of the pipeline was demonstrated on a dataset of IBS patients and healthy controls (HC), using the R software package to analyze colonic host mRNA and mucosal microbiota (16S rRNA gene sequencing), as well as gastrointestinal (GI) and psychological symptoms. In total, 42 IBS patients (57% female, mean age 33.6 (range 18–58)) and 20 HC (60% female, mean age 26.8 (range 23–41)) were included. Only in IBS patients, mRNA expression of Toll-like receptor 4 and genes associated with barrier function (PAR2, OCLN, TJP1) intercorrelated closely, suggesting potential functional relationships. This host genes-based “permeability cluster” was associated to mucosa-adjacent Chlamydiae and Lentisphaerae, and furthermore associated to satiety as well as to anxiety, depression and fatigue. In both IBS patients and HC, chromogranins, secretogranins and TLRs clustered together. In IBS patients, this host genes-based “immune-enteroendocrine cluster” was associated to specific members of Firmicutes, and to depression and fatigue, whereas in HC no significant association to microbiota was identified. We have developed a stepwise integrative analysis pipeline that allowed identification of unique host-microbiota intercorrelation patterns and association to symptoms in IBS patients. This analysis pipeline may aid in advancing the understanding of complex variable associations in health and disease.

Fatty acid-binding protein 4 silencing protects against lipopolysaccharide-induced cardiomyocyte hypertrophy and apoptosis by inhibiting the Toll-like receptor 4-nuclear factor-κB pathway.

ObjectiveTo explore the effects and potential mechanisms of fatty acid-binding protein 4 (FABP4) in a lipopolysaccharide (LPS)-induced in vitro septic cardiomyopathy model.MethodsRat cardiomyocyte H9c2 cells were transfected with small interfering RNA (siRNA) against FABP4 (siFABP4), then induced with LPS. The following parameters were measured: cell viability, lactate dehydrogenase release, cardiac hypertrophy and related marker expression, apoptosis, inflammatory cytokine release and expression, and the activation of Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) pathways.ResultsLPS increased the mRNA and protein expression of FABP4 in H9c2 cells. FABP4 silencing by siFABP4 significantly inhibited LPS-induced cardiac hypertrophy and reduced the mRNA expression of the myocardial hypertrophy markers atrial natriuretic peptide and brain natriuretic peptide. siFABP4 also attenuated LPS-induced increase in TUNEL-positive apoptotic cells, caspase-3 and caspase-9 activities, and the release and expression of proinflammatory cytokines. Mechanistically, we found that FABP4 silencing inhibited the mRNA and protein expression of TLR4 and suppressed the NF-kappa B signaling pathway, as evidenced by reduced nuclear NF-κB p65 and increased cytoplasmic I-κBα expression in LPS-stimulated H9c2 cells.ConclusionFABP4 silencing reduces LPS-induced cardiomyocyte hypertrophy and apoptosis by down-regulating the TLR4/NF-κB axis.

Specific TLR4 Blocking Effect of a Novel 3,4-Dihydropyrimidinone Derivative.

Background: Toll-like receptor 4 (TLR4) initiates both innate and adaptive immune responses, which plays an important protective role in self-defense mechanisms. Excessive or inappropriate TLR4 activation causes the development of many autoimmune diseases. Dihydropyrimidinone derivatives are medicinally important molecules with diverse pharmacological activities, including anti-inflammatory activity. The present study focused on novel synthesized 3,4-dihydropyrimidinone derivatives and evaluated their inhibitory effects on TLR4. Methods: A series of 3,4-dihydropyrimidinone derivatives were recently synthesized and evaluated for their TLR4 inhibition activities and cytotoxic on HEK-BlueTM hTLR4 cells with the help of QUANTI-Blue assay and MTS assay. Selected compound 3 was analyzed for its molecular docking with TLR4 by using Autodock vina 1.1.2. Its effect on the TLR4 pathway related cytokines was also evaluated in THP-1 cells and human peripheral blood mononuclear cells by using real-time PCR, ELISA and western blot. Results: Five compounds were synthesized and characterized for effectiveness based on 3,4-dihydropyrimidinone. Compound 3 was found to be the potent hybrid among the synthesized compounds, with high TLR4 inhibition activities and low cytotoxic activities against HEK-BlueTM hTLR4 cells. Molecular docking analysis showed that two hydrogen bonds between compound 3 and residues Asp209(TLR4) and Asp99(MD-2) mainly contribute to the TLR4 inhibition. In addition, compound 3 suppressed LPS-induced of the mRNA expression of TLR4, IP-10, TNF-α, IL-6, IL-12A, and IL-12B, the protein expression of pIRF3 and pNFκB and the secretion of IP-10, TNF-α in THP-1 cell line. Compound 3 also inhibited LPS-induced expression of TNF-α, IL-6, and IL-1β but increased IP-10 at mRNA levels in human peripheral blood mononuclear cells. Conclusion: Our study reveals compound 3, a novel 3,4-dihydropyrimidinone derivative, is a potential TLR4 antagonist, which opens up new research avenues for the development of promising therapeutic agents for inflammatory and autoimmune diseases.