Toll-like Receptor 4 mRNA Expression Research Articles

Beneficial effects of dietary β-glucan on growth and health status of Pacific white shrimp Litopenaeus vannamei at low salinity

An 8-week trial was conducted to evaluate the effect of dietary β-glucan supplement (0, 0.01%, 0.02%, or 0.04%) on growth and health of Pacific white shrimp Litopenaeus vannamei at low salinity of 3 practical salinity unit (psu). The L. vannamei fed 0.02% and 0.04% β-glucan gained more weight and showed higher activities of protease, amylase, superoxide dismutase, and glutathione peroxidase in the intestine than in the control (0% β-glucan). The L. vannamei fed 0.04% β-glucan had a higher condition factor than those fed the control diet. Amylase activity in the hepatopancreas of L. vannamei fed 0.02% β-glucan was higher than those fed the control diet. Dietary β-glucan supplement increased the mRNA expressions of Toll-like receptor, myostatin, immune deficiency or heat shock protein 70, but decreased the mRNA expressions of tumor necrosis factor-α and C-type lectin 3 in both hepatopancreas and intestine. The response of intestine microbiota in L. vannamei fed 0.04% β-glucan was further compared to the control. The 0.04% β-glucan supplement reduced richness and diversity of the intestinal microbial community as indicated by the low values of Chao1 estimator, ACE estimator, Simpson index and Shannon diversity index. Abundances of Bacillus, Chitinibacter, Geobacillus and Vibrio in the intestine increased, while Flavobacterium, Microbacterium and Mycobacterium decreased significantly in L. vannamei fed 0.04% β-glucan compared to the control. This study indicates that dietary β-glucan supplement at 0.02%–0.04% can significantly improve digestibility, antioxidant capacity and immunity in L. vannamei, and thus improve growth performance and survival at low salinity. These beneficial effects of β-glucan probably are related to the dominance of probiotics over potential pathogens in the intestine.

TLR4 and nucleolin influence cell injury, apoptosis and inflammatory factor expression in respiratory syncytial virus-infected N2a neuronal cells.

Respiratory syncytial virus (RSV) infection was recently reported to be associated with central nervous system (CNS) symptoms and neurological complications; however, related studies are very limited. Moreover, the molecular mechanism underlying RSV neuropathogenesis is still unclear. Our previous study revealed that toll-like receptor 4 (TLR4) and nucleolin (C23) could be modulated and that they played a role during RSV infection in mouse neuronal-2a (N2a) cells. In the present study, the effects of silencing of TLR4 and C23 on RSV propagation and N2a cellular responses were examined by using RNA interference technology. Four N2a cell treatment groups were established, namely, a normal control group, RSV control group, TLR4 siRNA + RSV group, and C23 siRNA + RSV group. Expression changes in NeuN protein and colocalization of C23 and TLR4 with RSV F protein were assessed using confocal microscopy. Changes in TLR4 and C23 mRNA expression, TLR4, C23, TLR3, TLR7, and p-NF-κB protein expression, and interleukin (IL)-8, IL-6, and tumor necrosis factor (TNF-α) cytokine secretion was measured using quantitative real-time reverse-transcription polymerase chain reaction, Western blot analysis, and enzyme-linked immunosorbent assay, respectively. RSV titers and the apoptotic status of N2a cells were monitored using plaque formation assays and flow cytometry, respectively. The results indicated that TLR4 and C23 gene knockdown decreased the amount of F protein in RSV-infected N2a cells, inhibited RSV propagation, attenuated N2a neuronal injury, diminished cell apoptosis levels, downregulated TLR3 and TLR7 protein expression, and reduced inflammatory protein expression. Therefore, TLR4 and C23 knockdown influences cell injury, apoptosis and inflammatory protein expression in RSV-infected N2a cells.

Effect of isotretinoin on peptidoglycan-induced expression of inflammatory genes in human SZ95 sebocytes

Objective To evaluate the effect of isotretinoin on expression of acne-associated inflammatory genes induced by peptidoglycan in human SZ95 sebocytes, and to explore the molecular mechanism underlying the treatment of acne with isotretinoin. Methods Cultured SZ95 sebocytes were divided into 3 groups: control group receiving no treatment, peptidoglycan group treated with 20 mg/L peptidoglycan alone, and costimulation group treated with 20 mg/L peptidoglycan combined with 10-5 mol/L isotretinoin. After 3-hour treatment, real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of pro-inflammatory cytokines including interleukin (IL) -1α, IL-1β, IL-6, IL-8, tumor necrosis factor (TNF) -α, Toll-like receptor 2 (TLR2) and MyD88 (a downstream gene of TLR2) in SZ95 sebocytes in the above groups. After 24-hour treatment, enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of IL-1α, IL-1β, IL-6, IL-8 and TNF-α in the cell culture supernatant in the above groups. After 48-hour treatment, Western blot analysis was conducted to determine the protein expression of TLR2 and MyD88. Statistical analysis was carried out with SPSS 23 software by one-way analysis of variance (ANOVA) for the comparison among the 3 groups, and by Bonferroni method for multiple comparisons. Results The mRNA and protein expression of pro-inflammatory cytokines including IL-1α, IL-1β, IL-6, IL-8 and TNF-α all significantly differed among the 3 groups (all P < 0.01) , and was significantly higher in the peptidoglycan group than in the control group and costimulation group (both P < 0.016 7) . The mRNA expression of MyD88 also significantly differed among the control group, peptidoglycan group and costimulation group (6.707 ± 0.950, 10.270 ± 0.477, 7.892 ± 0.900 respectively, F = 10.17, P < 0.01) , and was significantly higher in the peptidoglycan group than in the control group and costimulation group (t = 4.740, 3.298 respectively, both P < 0.016 7) . The mRNA and protein expression of TLR2 were markedly higher in the peptidoglycan group than in the control group, but did not differ between the peptidoglycan group and the costimulation group. Conclusion Isotretinoin can inhibit peptidoglycan-induced expression of inflammatory factors possibly associated with the occurrence of acne in human SZ95 sebocytes, likely by inhibiting the expression of MyD88, but not TLR2, in the innate immune response, which may be one of the mechanisms underlying the treatment of acne with isotretinoin. Key words: Acne vulgaris; Isotretinoin; Molecular mechanisms of pharmacological action; Peptidoglycan; Cytokines; Toll-like receptor 2; Immunity, innate; Human sebocytes

Heterogeneous expression of Toll-like receptors genes in lymphoid tissues of different ages pigs

Toll-like receptors (TLRs), known as the pathogen recognition receptors, recognize the microbial components. The details expression kinetics of TLRs may help to understand the immune responsiveness of pigs, but the expression patterns of all TLRs (TLR1–10) have not yet been studied in pigs. Therefore, this study unravels the expression patterns of the TLR family set of genes (TLR1–10) in different lymphoid tissues collected from pigs at different ages. A total of nine clinically healthy Pietrain pigs at three age groups were selected for this experiment. Each age group consisted of three animals. Cervical lymph node (CLN), thymus, liver, spleen, lung, heart, skin tissue and peripheral blood mononuclear cells (PBMC) were collected for both mRNA and protein isolation. The mRNA expression of TLRs (1−10) was quantified in all tissues. TLR3 mRNA was the most abundant in all tissues. It was expressed highly in thymus, kidney, lungs and liver tissues. Western blot and immunofluorescence was performed for protein expression and localization of selected TLRs (TLR2, 3 and 9) in selected tissues (CLN, spleen and lungs). The Western blot results of TLR2, 3 and 9 in selected tissues appeared to be consistent with the mRNA expression results. Cells in lungs, spleen and CLN were positively immunostained for TLR2, 3 and 9. This study sheds light on the expression patterns of TLR (1–10) genes in important lymphoid tissues in pigs of different ages. The heterogeneous expression of TLRs with ages may contribute to the altered immune responsiveness of pigs with ages.

The role of toll-like receptor 4 and mast cell in the ameliorating effect of electroacupuncture on visceral hypersensitivity in rats.

Visceral hypersensitivity is one of the main pathogenesis of irritable bowel syndrome (IBS) and mast cell activation is closely related to visceral hypersensitivity. As a critical molecule of the innate immune system, toll-like receptor 4 (TLR4) may modulate the activation of mast cell. Alleviating effect of electroacupuncture (EA) on visceral hypersensitivity has been proved, whereas, whether the TLR4 and mast cell is involved in this process remains unclear. Forty Sprague-Dawley rats were randomly divided into five groups: control group, model group, EA group, sham EA group, and mast cell stabilizer (MCS) group. Visceral sensitivity during colorectal distension was assessed by the measurement of visceral motor reflex (VMR). TLR4 mRNA and protein expression were assessed by real-time PCR and immunohistochemistry, respectively. Mast cell number and mast cell tryptase (MCT) expression were detected. The level of inflammatory cytokine in serum was detected with ELISA. Visceral sensitivity was significantly higher in the model group than in the control group. EA and MCS significantly reduced VMR score at 0.8mL and 1.2mL distention pressures. Compared with model group, TLR4 mRNA expression, the protein expression of TLR4 and MCT, and the number of mast cells with degranulation in the colonic tissue, serum concentration of IL-1β and IL-8 were all significantly decreased in EA and MCS group. Electroacupuncture ameliorated visceral hypersensitivity in colon-sensitized model probably via decreasing the level of pro-inflammatory cytokines released by mast cell which were decreased when the TLR4 expression in the colonic tissue was downregulated by EA.

Lipopolysaccharide and palmitic acid synergistically induced MCP-1 production via MAPK-meditated TLR4 signaling pathway in RAW264.7 cells

BackgroundObesity increases the risk of developing diabetes mellitus. Clinical studies suggest that risk factors like palmitic acid (PA) and lipopolysaccharide (LPS) exist simultaneously in diabetes with obesity. Combination of PA and LPS even at low concentration can induce strong inflammatory reaction. Monocyte chemoattractant protein-1 (MCP-1) is an important inflammatory chemokine related to insulin resistance and type II diabetes. Our previous study using PCR array revealed that LPS and PA synergistically induce MCP-1 mRNA expression in macrophage cells RAW264.7, while the protein expression of MCP-1 in this case was not investigated. Moreover, the underling mechanism in the synergistic effect of MCP-1 expression or production induced by treatment of LPS and PA combination remains unclear.MethodsProtein secretion of MCP-1 was measured by the enzyme-linked immunosorbent assay (ELISA) and mRNA levels of MCP-1 and Toll-like receptor 4 (TLR4) were measured by real-time PCR. Statistical analysis was conducted using SPSS software.ResultsLPS could increase MCP-1 transcription as well as secretion in RAW264.7, and PA amplified this effect obviously. Meanwhile, combination of LPS with PA increased TLR4 mRNA expression while LPS alone or PA alone could not, TLR4 knockdown inhibited MCP-1 transcription/secretion induced by LPS plus PA. Moreover, not NF-κB inhibitor but inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPK were found to block MCP-1 generation stimulated by LPS plus PA.ConclusionLPS and PA synergistically induced MCP-1 secretion in RAW264.7 macrophage cells, in which MCP-1 transcription mediated by MAPK/TLR4 signaling pathways was involved. Combined treatment of PA and LPS in RAW264.7 cells mimics the situation of diabetes with obesity that has higher level of PA and LPS, MAPK/TLR4/ MCP-1 might be potential therapeutic targets for diabetes with obesity.

Visualizing neuroinflammation with fluorescence and luminescent lanthanide-based in situ hybridization

BackgroundNeurokine signaling via the release of neurally active cytokines arises from glial reactivity and is mechanistically implicated in central nervous system (CNS) pathologies such as chronic pain, trauma, neurodegenerative diseases, and complex psychiatric illnesses. Despite significant advancements in the methodologies used to conjugate, incorporate, and visualize fluorescent molecules, imaging of rare yet high potency events within the CNS is restricted by the low signal to noise ratio experienced within the CNS. The brain and spinal cord have high cellular autofluorescence, making the imaging of critical neurokine signaling and permissive transcriptional cellular events unreliable and difficult in many cases.MethodsIn this manuscript, we developed a method for background-free imaging of the transcriptional events that precede neurokine signaling using targeted mRNA transcripts labeled with luminescent lanthanide chelates and imaged via time-gated microscopy. To provide examples of the usefulness this method can offer to the field, the mRNA expression of toll-like receptor 4 (TLR4) was visualized with traditional fluorescent in situ hybridization (FISH) or luminescent lanthanide chelate-based in situ hybridization (LISH) in mouse BV2 microglia or J774 macrophage phenotype cells following lipopolysaccharide stimulation. TLR4 mRNA staining using LISH- and FISH-based methods was also visualized in fixed spinal cord tissues from BALB/c mice with a chronic constriction model of neuropathic pain or a surgical sham model in order to demonstrate the application of this new methodology in CNS tissue samples.ResultsSignificant increases in TLR4 mRNA expression and autofluorescence were visualized over time in mouse BV2 microglia or mouse J774 macrophage phenotype cells following lipopolysaccharide (LPS) stimulation. When imaged in a background-free environment with LISH-based detection and time-gated microscopy, increased TLR4 mRNA was observed in BV2 microglia cells 4 h following LPS stimulation, which returned to near baseline levels by 24 h. Background-free imaging of mouse spinal cord tissues with LISH-based detection and time-gated microscopy demonstrated a high degree of regional TLR4 mRNA expression in BALB/c mice with a chronic constriction model of neuropathic pain compared to the surgical sham model.ConclusionsAdvantages offered by adopting this novel methodology for visualizing neurokine signaling with time-gated microscopy compared to traditional fluorescent microscopy are provided.

Differential expression of inflammatory cytokines and chemokines in lipopolysaccharide-stimulated melanocytes from lightly and darkly pigmented skin.

Increasing evidence suggests that human epidermal melanocytes play an important role in the skin immune system; however, a role of their pigmentation in immune and inflammatory responses is poorly examined. In the study, the expression of Toll-like receptor 4 (TLR4) and inflammatory cytokines and chemokines by cultured normal melanocytes derived from lightly and darkly pigmented skin was investigated after cell stimulation with lipopolysaccharide (LPS). The basal TLR4 mRNA level in heavily pigmented cells was higher as compared to their lightly pigmented counterparts. Melanocyte exposure to LPS upregulated the expression of TLR4 mRNA and enhanced the DNA-binding activity of NF-κB p50 and p65. We found substantial differences in the LPS-stimulated expression of numerous genes encoding inflammatory cytokines and chemokines between the cells with various melanin contents. In lightly pigmented melanocytes, the most significantly upregulated genes were nicotinamide phosphoribosyltransferase (NAMPT/visfatin), the chemokines CCL2 and CCL20, and IL6, while the genes for CXCL12, IL-16 and the chemokine receptor CCR4 were the most significantly upregulated in heavily pigmented cells. Moreover, the lightly pigmented melanocytes secreted much more NAMPT, CCL2 and IL-6. The results of our study suggest modulatory effect of melanogenesis on the immune properties of normal epidermal melanocytes.

The anti-diabetic activities, gut microbiota composition, the anti-inflammatory effects of Scutellaria–coptis herb couple against insulin resistance-model of diabetes involving the toll-like receptor 4 signaling pathway

Ethnopharmacological relevanceScutellaria–coptis herb couple (SC) is one of the well-known herb couples in many traditional Chinese compound formulas used for the treatment of diabetes mellitus (DM), which has been used to treat DM for thousands of years in China. Aim of the studyFew studies have confirmed in detail the anti-diabetic activities of SC in vivo and in vitro. The present investigations aimed to evaluate the anti-diabetic activity of SC in type 2 diabetic KK-Ay mice and in RAW264.7 macrophages to understand its possible mechanism. Materials and methodsHigh-performance liquid chromatography with ultraviolet detection (HPLC-UV) and LC-LTQ-Orbitrap Pro mass spectrometry were used to analyze the active ingredients of SC extracts and control the quality. A type 2 diabetic KK-Ay mice model was established by high-fat diet. Body weight, fasting blood glucose levels, fasting blood insulin levels, glycosylated hemoglobin and glycosylated serum protein were measured. The effects of SC on total cholesterol (TC), high-density lipoprotein (HDL) and triglyceride (TG) levels were examined. The lipopolysaccharide (LPS), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-α) levels were measured. Gut microbial communities were assayed by polymerase chain reaction (PCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) methods. The expressions of Toll-like receptor 4 (TLR4) and MyD88 protein in the colons were measured by western blot. In RAW264.7 macrophages, IL-6, TNF-α, TLR4 and MyD88 protein levels were measured by enzyme-linked immunosorbent assay (ELISA) kits or western blot, and the mRNA expression of IL-6, TNF-α and TLR4 was examined by the real time PCR. ResultsThe present results showed that the SC significantly increased blood HDL and significantly reduced fasting blood glucose, fasting blood insulin, glycosylated hemoglobin, glycosylated serum protein, TC, TG, LPS, IL-6 and TNF-α levels (P < 0.05 or P < 0.01) in type-2 diabetic KK-Ay mice. Furthermore, SC could regulate the structure of intestinal flora. Additionally, the expressions of TLR4 and MyD88 protein in the colons were significantly decreased in the model group (P < 0.05 or P < 0.01). However, SC had no significant effect on weight gain. In RAW264.7 macrophages, SC containing serum (SC-CS) (5%, 10% and 20%) significantly decreased IL-6, TNF-α, TLR4 and MyD88 protein levels and the mRNA expression of IL-6, TNF-α and TLR4 (P < 0.05 or P < 0.01). ConclusionsThe anti-diabetic effects of SC were attributed to its regulation of intestinal flora and anti-inflammation involving the TLR4 signaling pathway. These findings provide a new insight into the anti-diabetic application for SC in clinical settings and display the potential of SC in the treatment of DM.

Association of toll-like receptor 9 expression with prognosis of systemic lupus erythematosus.

The current study assessed the association between toll-like receptor 9 (TLR9) and systemic lupus erythematosus (SLE) and subsequently determined the predictive value of TLR9 in assessing the prognosis of SLE. A total of 90 newly diagnosed patients with SLE and 49 healthy control subjects were enrolled in the current study. The expression of TLR9 mRNA was measured in whole blood samples from patients and controls. All patients were followed up for ≥2 years and their clinical parameters were recorded. After 2 years, 30 patients were randomly chosen from patient subgroups with high (n=20) or low (n=10) TLR9 levels and the expression of TLR9 mRNA were measured again. Cox proportional hazards regression was used to identify the risk factors of SLE prognosis. Patients with SLE and high SLE disease activity exhibited significantly increased TLR9 expression (P<0.05). Persistent proteinuria of >0.5 g/day [hazard ratio (HR), 6.314; 95% confidence interval (CI), 2.858-13.947], C-reactive protein levels (HR, 1.013; 95% CI, 1.007-1.019) and high-TLR9 mRNA expression (HR, 3.852; 95% CI, 1.931-7.684) were independent risk factors of poor prognosis during a 2-year follow-up period, whereas patient treatment with >1 immunosuppressant (HR, 0.374; 95% CI, 0.173-0.808) was a factor indicating favorable prognosis. Furthermore, the expression of TLR9 mRNA remained high in patients with poor prognosis at the end of a 2-year follow-up period but in patients with a favorable prognosis, TLR9 mRNA expression was significantly reduced compared with the levels measured at SLE onset (P<0.0001). Therefore, the expression of TLR9 mRNA in whole blood samples at SLE onset is associated with SLE disease activity and its expression may be used as an indicator of poor prognosis in patients with SLE.

Increased TLR4 Expression Aggravates Sepsis by Promoting IFN-γ Expression in CD38-/- Mice.

Gram-negative bacterial sepsis accounts for up to 50% worldwide sepsis that causes hospital mortality. Acute kidney injury (AKI), a common complication of Gram-negative bacterial sepsis, is caused by Toll-like receptor 4 (TLR4) activation. Lipopolysaccharide (LPS) is an endotoxin in Gram-negative bacteria and is recognized specifically by TLR4, which initiates innate immune response. Also, TLR4 signaling pathway activation is essential in response to LPS infection. CD38 is one of the well-known regulators of innate immunity, whose dysregulation contributes to sepsis. Many studies have proven that an attenuated Gram-positive bacterium induces sepsis in a CD38-blocking model. However, the pathogenesis of Gram-negative bacteria-induced sepsis in a CD38−/− mouse model remains unclear. The aim of this study is to investigate whether kidney injury is still attenuated in a LPS-induced CD38−/− sepsis model and identify the potential mechanism. We assess the severity of kidney injury related to proinflammatory cytokine expressions (IFN-γ, TNF-α, IL-1β, and IL-6) in WT and CD38−/− mice. Our results showed more aggravated kidney damage in CD38−/− mice than in WT mice, accompanied with an increase of proinflammatory cytokine expression. In addition, compared with CD38−/−TLR4mut mice, we found an increase of TLR4 expression and mRNA expression of these cytokines in the kidney of CD38−/− mice, although only increased IFN-γ level was detected in the serum. Taken together, these results demonstrated that an increased TLR4 expression in CD38−/− mice could contribute to the aggravation of AKI through boosting of the production of IFN-γ.

Gut derived-endotoxin contributes to inflammation in severe ischemic acute kidney injury

BackgroundRecent researches indicate that the intestinal consequences of renal ischemia reperfusion (IR) would predispose to the translocation of gut-derived endotoxin. Here, we designed experiments to test the hypothesis that the gut-derived endotoxin has a potential role in mediating local inflammatory processes in the acutely injured kidney.MethodsRats were performed sham or renal IR surgery (60 min of bilateral renal ischemia, then 24 h of reperfusion) (n = 5). The intestinal structural and mucosa permeability were evaluated. Serum endotoxin and bacterial load in liver and mesenteric lymph nodes (MLN) were measured. Separate groups were pretreated with oral norfloxacin 20 mg/kg/day or saline for 4 weeks and divided into sham plus saline, sham plus norfloxacin, renal IR plus saline and renal IR plus norfloxacin group. Serum biochemistry and endotoxin were determined. Kidney pathological changes were scored. Protein or mRNA expression of toll-like receptor 4 (TLR4) and proinflammatory mediators were measured in kidney homogenate.ResultsRenal IR led to marked intestinal integrity disruption and increase in intestinal permeability. These are accompanied by low grade of endotoxemia as well as increased bacterial load in liver and MLN. The group pretreated with norfloxacin showed significant attenuation of the increase in serum urea, ALAT, ASAT and endotoxin. The increased renal protein or mRNA of TLR4 and proinflammatory mediators (IL-6 and MCP-1) in the unpretreated animals was significantly attenuated in the norfloxacin-pretreated animals. However, norfloxacin pretreatment did not produce any protective effects on renal tubular integrity.ConclusionsOur results show for the first time that gut-derived endotoxin, resulting from an increased intestinal permeability after severe renal IR, subsequently amplifies intrarenal inflammatory response by activation renal TLR4 signaling. Our study results do not establish that antibiotic administration was effective in improving the overall renal outcome. However, our findings may be the first step to understanding how to tailor therapies to mitigate intrarenal inflammation in select groups of patients.

Escherichiacoli promotes DSS‑induced murine colitis recovery through activation of the TLR4/NF‑κB signaling pathway.

Increasing evidence suggests that intestinal microbiota have critical function in the pathogenesis of inflammatory bowel disease. This present study investigated the effects of Escherichia coli (E. coli) in mice with dextran sulfate sodium (DSS)-induced colitis. Furthermore, Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) gene expression was measured by reverse transcription-quantitative polymerase chain reaction. In total, two experiments were performed. In the first experiment, four groups were established in BALB/c mice: i) Group A, control (no treatments); ii) group B, DSS-induced colitis; iii) group C, DSS-induced colitis bacteria depleted (BD) mice; and iv) group D, E. coli-treated DSS-induced colitis BD mice. In the second experiment, there were three groups: i) Group A1, control C57BL/6 mice; ii) group B1, E. coli-treated DSS-induced colitis BD C57BL/6 mice; and iii) E. coli-treated DSS-induced colitis BD TLR4−/− mice. Clinical outcomes, colon and immune histopathology and tissue myeloperoxidase activity were assessed. Mice with DSS-induced colitis that were treated with E. coli exhibited enhanced recovery, with significantly improved clinical and histological scores compared with the DSS only group. The mRNA expression of TLR4 and NF-κB in the E. coli-treated group was also significantly higher. These effects were abolished in TLR4−/− mice, suggesting that E. coli may have promoted recovery through the TLR4 pathway. The present study indicated that E. coli promoted recovery from DSS-induced colitis in mice, potentially through activation of the TLR4/NF-κB signaling pathway.

Inhibition of HMGB1 improves necrotizing enterocolitis by inhibiting NLRP3 via TLR4 and NF-κB signaling pathways.

To explore the relationship between high-mobility group box 1 (HMGB1) and NLR pyrin domain containing 3 (NLRP3) in the development of necrotizing enterocolitis (NEC). NEC rat models were constructed and treated with HMGB1 inhibitor glycyrrhizin (GL) with different concentration. An inflammatory condition of intestinal tissue in newborn NEC rats was observed by hematoxylin and eosin staining. The messenger RNA (mRNA) and protein expression of HMGB1, NLRP3, toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), and caspase 1 were determined by real-time polymerase chain reaction and western blot analysis, respectively. The content of interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) was determined by enzyme-linked immunosorbent assay. Human intestinal epithelial cell lines were induced to NEC by lipopolysaccharides (LPSs). LPS-induced cells were transfected with small interfering RNA-HMGB1 and NLRP3 plasmid vector. The mRNA and protein expression of HMGB1, NLRP3, TLR4, NF-κB, caspase 1, IL-1β, and TNF-α were determined by real-time PCR and western blot analysis, respectively. The mRNA and protein expression of HMGB1 and NLRP3 in the NEC group was significantly higher than the control group. Inhibition of HMGB1 expression improved intestinal inflammation in newborn NEC rats. The expression of HMGB1, NLRP3, TLR4, NF-κB, and caspase 1 was upregulated in NEC and was weakened after treating with GL. LPS induction to intestinal epithelial cells markedly increased the expression of HMGB1, NLRP3, TLR4, NF-κB, caspase 1, IL-1β, and TNF-α. The knockdown of HMGB1 abolished the increase of expression, whereas further transfection with NLRP3 plasmid vector recovered the increase. HMGB1 and NLRP3 were all upregulated in the development of NEC. Inhibition on HMGB1 could improve the intestinal inflammation in NEC by inhibiting NLRP3 via TLR4 and NF-κB signaling pathways.

Up-regulated mRNA expression of some anti-inflammatory mediators in bovine oviduct epithelial cells by urea in vitro: Cellular pathways by Reactome analysis

Increased urea concentration is a major cause of low fertility in dairy cows fed high-protein diets. A strong correlation exists between the urea concentration in the blood and oviduct fluid of dairy cows. In this study, bovine oviduct epithelial cells (BOECs) were incubated with varying concentrations of urea (0, 20, 40, and 80 mg/dL) in the absence of ovarian sex steroids (estradiol and progesterone) and luteinizing hormone. The 80 mg/dL urea reduced the cell viability, and thus was excluded in further analysis. Compared to the control (U0), the 20 mg/dL urea (U20) increased the mRNA expression of Toll-like receptor (TLR) 4, interleukin (IL) 10, IL4, and prostaglandin (PG) E synthase (mPGES) but decreased the mRNA expression of tumor necrosis factor α (TNFA). Compared to U0, the 40 mg/dL urea (U40) decreased the mRNA expression of TNFA and increased alpha-1-acid glycoprotein (AGP). U40 also increased TLR2, IL10, and IL4 mRNA expression compared to U0. In addition, compared to U20, the U40 decreased the mRNA expression of TLR4 and IL1B but increased that of AGP and TLR2. Subsequently, the mRNA expression data were then projected into the Reactome database. The Reactome analysis showed that pathways, including cytokine signaling in the immune system (i.e., TNFs bind their physiological receptors) and death receptor signaling (i.e., TNF signaling), were down-regulated in the presence of urea compared to the U0 group. These in vitro data implied that high urea level can alter the balance between pro- and anti-inflammatory responses in BOECs, thus providing a suboptimal environment for the early reproductive events or a weakened innate immune system, predisposing the oviduct to infections.

Glycyrrhizin and Omega-3 fatty acids have hepatoprotective roles through toll-like receptor-4

ABSTRACTThis study investigated the hepatoprotective role of natural compounds [glycyrrhizin (GL) and omega-3 fatty acids (ω-3)] against thioacetamide (TAA)-induced liver fibrosis and clarified their underlying molecular mechanism through their effect on oxidative stress and toll-like receptor-4 (TLR-4). Forty male Wistar rats were randomized to five groups: Control group and four groups received TAA 200 mg/kg i.p. twice weekly for 8 weeks: TAA group, TAA+GL group (received GL 25 mg/kg daily by oral tube), TAA+ω-3 group (received ω-3150 mg/kg daily by oral tube), TAA+GL+ω-3 group (received similar combined doses of both natural compounds). GL, ω-3 and their combination protected the liver from TAA-hepatotoxic effects. Their hepatoprotective effects were confirmed by histopathological analysis as they significantly reduced the necroinflammatory scores and the extent of fibrosis. They act as anti-oxidant as they significantly decreased the liver malondialdehyde level (P < 0.005). They also significantly increased liver inactive TLR-4 concentration (P < 0.005) which indicates inhibition of TLR-4 pathway. This is confirmed by a significant decrease in liver TLR-4 mRNA expression (P < 0.005). In conclusion, glycyrrhizin, omega-3 fatty acids and their combination have potent anti-inflammatory, anti-oxidant and anti-fibrotic effects through inhibition of TLR-4.

Immunomodulation Properties of Solid-State Fermented Laetiporussulphureus Ethanol Extracts in Chicken Peripheral Blood Monocytes In Vitro

ABSTRACT Laetiporus sp. is recognized as a fungal species traditionally used for medicinal purposes. This study investigated the in-vitro effects of solid-state fermented Laetiporussulphureus ethanol extracts (LSE) for their immunomodulatory potential. Bioactive levels detected in the LSE on different days throughout the fermentation period revealed that the 12th day was the most efficient, with 7.19 ± 0.66 GAE/g DM crude phenolic content, 2.71 ± 0.03 UAE/g DM crude triterpenoid content, 12.93 ± 0.88 GCE/g DM crude polysaccharides, and 96.44 ± 0.2 mg/g DM ergosterol content. In-vitroLSE tests on chPBMC showed no cytotoxicity within a range of 0.05-1 mg/mL, but LPS-inhibited cell viability was improved, as well as LPS-induced nitric oxide (NO) production and mRNA levels of nuclear factor kappa B (NFκB), Toll-like receptor 4 (TLR4), inducible nitric oxide synthase (iNOS), and interleukin (IL)-1βwere attenuated Furthermore, the direct application of LSE on chPBMC showed a small but not significant increase in NFκB, TLR4, and iNOS mRNA expression compared with the control group. These results indicate the potential of LSE to modulate LPS-triggered inflammation processes involving TLR4 and NFκB mediation. However, further experiments are required to determine the specific pathway.

Heat stress combined with lipopolysaccharide alter the activity and superficial molecules of peripheral monocytes.

The purpose of this study was to focus on the underlying relationship between the hyperactivity for the peripheral monocytes and heat stroke by investigating the inflammatory oxidative activity of and the expression of superficial molecules. Peripheral blood samples were collected from 10 healthy adult volunteers. Human blood monocytes were isolated by density gradient centrifugation and sequent adherent culture. The objectives were divided into four groups: 43°C heat stress combined with lipopolysaccharide (LPS) group, 43°C heat stress group, LPS group, and control group. There were 10 cases in each group. An enzyme-linked immunosorbent assay (ELISA) test was used to measure the concentrations of supernatant inflammatory mediators (tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-10 (IL-10)). After loaded by 2,7-Dichlorodi-hydrofluorescein-diacetate (DCFHDA) fluorescent probe, intracellular reactive oxygen species (ROS) levels were determined by a flow cytometry. After fluorescent microspheres incubation, the phagocytosis of monocytes was observed under a fluorescent microscope. Respectively, the flow cytometry and Western blot were used to evaluate the level of triggering receptor expressed on myeloid cells-1 (TREM-1) and Toll-like receptor-4 (TLR-4) on the monocytes. Furthermore, the mRNA expression of TREM-1 and TLR-4 was detected by real-time polymerase chain reaction (RT-PCR). The heat stress combined with LPS stimulation promoted the peripheral monocytes to produce inflammatory mediators (TNF-α, IL-1β, and IL-10) and release ROS. Otherwise, such complex strike significantly suppressed the phagocytic activity of monocytes in peripheral blood. Moreover, the expression of TREM-1, TLR-4 and CD86 was measured by the flow cytometry on peripheral monocytes which were respectively promoted by the union of heat stress and LPS. The results of Western blot and RT-PCR demonstrated the similar kinetics on these superficial molecules (TREM-1, TLR-4, and CD86) stimulated by the combination of heat stress and LPS. The underlying mechanism of the dysfunction for the peripheral monocytes may be related to the abnormal expression of superficial molecules TREM-1, TLR-4, and CD86 on the monocytes induced by heat stress and LPS.

The expression of toll-like receptors in peripheral blood mononuclear cells is altered in schizophrenia

Increasing evidence suggests that in addition to neurochemical abnormalities, various immunological alterations are related to the pathogenesis of schizophrenia. Toll-like receptors (TLRs) actively mediate immune/inflammatory processes and play a pivotal role in damage/danger recognizing. Therefore, the aim of this study was to compare the expression of TLRs in peripheral blood mononuclear cells (PBMCs) in schizophrenic patients with those of healthy subjects. It also measures the metabolic status of the study subjects. Twenty-seven adult European Caucasian patients with paranoid schizophrenia and twenty-nine healthy volunteers were included in this prospective study. qRT-PCR assessed TLR mRNA expression levels. Body composition was measured using two methods: bioimpedance analysis (BIA) and dual-energy X-ray absorptiometry (DXA). The TLR1, TLR2, TLR4, TLR6, and TLR9 expression were down-regulated, in opposite to TLR3 and TLR7 which manifested higher expression in patients with schizophrenia. TLR5 and TLR8 mRNAs did not differ between groups. TLR mRNA expression was highly correlated. Decreased TLR expression may protect against excessive cell stimulation via exogenous and/or endogenous ligands, and may be recognized as a counterbalancing mechanism limiting the excessive development of inflammation.

Can the Toll-like receptors 4 expression in peripheral blood mononuclear cells help assess the effectiveness of immunosuppression and the chance of a future good renal transplant function?

BackgroundA small percentage of peripheral blood mononuclear cells (PBMCs) circulating during the kidney transplantation (KT) period remain in the blood long after transplantation. A part of the PBMCs penetrates the graft. AimTo examine if the choice of immunosuppression may change TLR4ex and how TLR4ex affects the transplant function in the future. MaterialThe study population-143 transplanted patients (pts) (55 females, 88 males), mean age on recruitment day 50.33 ± 12.8 years old, mean BMI 25.04 ± 4.18. 41 pts. experienced delayed graft function (DGF+). 55 pts. were treated with cyclosporine A (CsA) and 88 with tacrolimus (Tac). All were treated with mofetil mycophenolate (MMF). The PBMCs acquisition and starting point of the follow-up (TLR-day) was at least one month after KT. MethodWe investigated averaged mRNA expression of Toll-like receptors 4 (TLR4ex) in non-stimulated peripheral blood mononuclear cells with the use of real-time polymerase chain reaction. The KT pts. (All, Tac, CsA, DGF+) were divided by the respective median of their TLR4ex (lower: L-TLR4ex, higher: H-TLR4ex). Main clinical parameters and transplant biopsy files (if available) were assessed on TLR-day and post follow-up. ResultsWe found that TLR4ex was reduced for a long time in patients who experienced delayed graft function. L-TLR4ex had a higher proportion of DGF+ patients, and patients treated with CsA but lower of those treated with Tac than in H-TLR4ex. The amplitude of changes in renal function parameters (ΔEGFR%/ΔsCr/ΔsCr%) was clearly less favorable for L-TLR4ex. Tacrolimus expressed a stabilizing effect. Both the positive vasculitis score and chronic graft nephropathy were more frequent in the L-TLR4ex group. On TLR-day an association of renal function and Tac concentration with TLR4ex was clear only in the tacrolimus population. The TLR4ex was lower in patients with a future deterioration of the graft function. ConclusionIn kidney transplant recipients the occurrence of DGF results in a long-term reduction of the averaged TLR4ex in PBMC. Tacrolimus exerts a clear, stabilizing, positive and dose-dependent effect on TLR4ex. An improvement in renal transplant function may be expected in KT patients with high TLR4ex. Evaluation of the averaged TLR4ex can be used to assess the efficacy of immunosuppression in the treatment with tacrolimus and to estimate the likelihood of deterioration in renal function.