To investigate the expression of Toll-like receptor (TLR) 2 and 4 mRNA in local tissues of model of oropharyngeal candidiasis in mice and to explore the potential role of TLR2 and TLR4 in earlier period of immune response, a murine model of oropharyngeal candidiasis inoculated by cotton wool balls saturated with Candida albicans was established. Mice were sacrificed at the indicated time points and the oropharyngeal tissues were excised. The expression of TLR2 and TLR4 mRNA was detected by RT-PCR. The results showed that low level of TLR2/4 mRNA could be detected in oropharyngeal tissues, but they were markedly up-regulated 6 h after inoculation, peaking after 12-24 h. Tissue TLR4 mRNA was gradually down-regulated 24-48 h, while TLR2 mRNA levels remained high up to the 72nd h. These data suggested that oropharyngeal infection of Candida albicans could result in up-regulation of TLR2/4 mRNA expression in local tissues, which might play important roles in earlier period of immune response.
Candidiasis/metabolism ; Candidiasis, Oral/*metabolism ; Mouth Mucosa/*metabolism ; Pharyngitis/metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Random Allocation ; Toll-Like Receptor 2/*biosynthesis ; Toll-Like Receptor 2/genetics ; Toll-Like Receptor 4/*biosynthesis ; Toll-Like Receptor 4/genetics

Activation pattern recognition receptors can cause the startup of downstream signaling pathways, the expression of inflammatory factors, and finally immunological inflammatory reaction. Either exogenous pathogenic microorganisms or endogenous tissue components can activate these pattern recognition receptors as ligands at varying degrees, and then cause the immunological inflammatory reaction. Therefore, it is of great significance to inhibit relevant receptors, as well as the immunological inflammatory reaction, in order to avoid tissue injury during the course of disease. Baicalin is able to specifically inhibit the expression of TLR2/4-NOD2, inhibit the expression of inflammatory factors IL-1beta, IL-6 and TNF-alpha, and thereby reducing the injury of the tissue cells during the course of disease. This effect is non-specific with tissues, which is of great theoretical and practical significance in druggability. In addition, the drug metabolism and toxicity of baicalin are also discussed for its druggability in this article.
Animals ; Flavonoids ; pharmacology ; Humans ; Nod2 Signaling Adaptor Protein ; metabolism ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 4 ; metabolism

OBJECTIVEThe aim of this study was to survey the influence of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) repression to receptor activator of nuclear factor-kappaB ligand (RANKL) expression of human periodontal ligament fibroblasts (HPDLFs) under the stimulation of lipopolysaccharide (LPS).

METHODSThe level of RANKL in HPDLFs stimulated by 100 ng x mL(-1), 1 microg x mL(-1) and 10 microg x mL(-1) Escherichia coli (E. coli) LPS after 6, 12, 24 and 48 h was detected by enzyme linked immunosorbent assay (ELISA). The level of RANKL in HPDLFs stimulated by 1 microg x mL(-1) E. coli LPS after pretreatment with different titre anti-TLR2+anti-TLR4, anti-TLR2 and anti-TLR4 antibody were observed respectively.

RESULTSRANKL was detected at 6 h after stimulation with LPS, and the levels of these cytokine were highest at 24 h, and then gradually decreased. The regularity of each LPS concentration was approximately similar. After pretreatment with anti-TLR2+anti-TLR4, anti-TLR2 and anti-TLR4 antibody, the level of RANKL was significantly decreased under the stimulation of 1 microg x mL(-1) LPS (P<0.05). In the three groups, the expression of RANKL was significantly different (P<0.05). The level of RANKL in anti-TLR2+anti-TLR4 antibody pretreatment group was the lowest, the level in anti-TLR4 antibody pretreatment group was higher, and the level in anti-TLR2 antibody pretreatment group was the highest.

CONCLUSIONTLR2 and TLR4 participate in the process of RANKL expres-in HPDLFs induced by LPS. Anti-TLR4 antibody has better inhibition effect to RANKL expression of HPDLFs stimulated by LPS than anti-TLR2.

Escherichia coli ; Fibroblasts ; Humans ; Lipopolysaccharides ; Periodontal Ligament ; RANK Ligand ; Toll-Like Receptor 2 ; Toll-Like Receptor 4

OBJECTIVE: The aim of this study was to clarify whether stimulation of recombinant IL-17, TLR2 and TLR4 by their specific ligands induces the production of RANKL and IL-6 in the fibroblast-like synoviocytes (FLSs) from RA patients. METHODS: FLSs were isolated from RA synovial tissues and they were stimulated with the IL-17, TLR2 ligand bacterial peptidoglycan (PGN) and TLR4 ligand lipopolysaccharide (LPS). The RANKL levels were assessed by RT-PCR and western blotting. The expressions of IL-17, TLR2, TLR4, RANKL and IL-6 in the RA synovium were quantified by immunohistochemistry and these values were compared with the values obtained in the osteoarthritis synovium. The increased IL-6 production in the culture supernatants of the RA FLSs was quantified by sandwich ELISA. RESULTS: The mRNA and protein levels of RANKL and IL-6 increased in the RA FLSs stimulated with PGN, LPS and IL-17, or PGN plus IL-17 or LPS plus IL-17. The expressions of IL-17, TLR2, TLR4, RANKL and IL-6 were much higher in the RA synovium than those in the osteoarthritis (OA) synovium. CONCLUSION: We observed synergistic effects of TLR-2, TLR-4 and IL-17 upon the induction of RANKL. In conclusion, our data supports the previous evidence of an important role of TLR-2, TLR-4 and IL-17 in the pathogenesis of RA.
Blotting, Western ; Fibroblasts ; Humans ; Immunohistochemistry ; Interleukin-17 ; Interleukin-6 ; Ligands ; Osteoarthritis ; Peptidoglycan ; RNA, Messenger ; Synovial Membrane ; Toll-Like Receptor 2 ; Toll-Like Receptor 4 ; Toll-Like Receptors

Synucleinopathies are a collection of neurological diseases that are characterized by deposition of α-synuclein aggregates in neurons and glia. These diseases include Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. Although it has been increasingly clear that α-synuclein is implicated in the pathogenesis of PD and other synucleinopathies, the precise mechanism underlying the disease process remains to be unraveled. The past studies on how α-synuclein exerts pathogenic actions have focused on its direct, cell-autonomous neurotoxic effects. However, recent findings suggested that there might be indirect, non-cell-autonomous pathways, perhaps through the changes in glial cells, for the pathogenic actions of this protein. Here, we present evidence that α-synuclein can cause neurodegeneration through a non-cell-autonomous manner. We show that α-synuclein can be secreted from neurons and induces inflammatory responses in microglia, which in turn secreted neurotoxic agents into the media causing neurodegeneration. The neurotoxic response of microglia was mediated by activation of toll-like receptor 2 (TLR2), a receptor for neuron-derived α-synuclein. This work suggests that TLR2 is the key molecule that mediates non-cell-autonomous neurotoxic effects of α-synuclein, hence a candidate for the therapeutic target.
Dementia ; Lewy Bodies ; Microglia ; Multiple System Atrophy ; Neuroglia ; Neurons ; Parkinson Disease ; Toll-Like Receptor 2* ; Toll-Like Receptors*

To investigate TLR2 (Toll-like receptor 2) mRNA expression in ischemic hepatic lobes under the condition of partial hepatic ischemia/reperfusion injury in BALB/c mice and its relationship with liver function impairment. A partial ischemia/reperfusion injury model was established. The portal vein and hepatic artery supply to the median and left lobes of the liver were obstructed by an atraumatic artery micro-clip, with the obstruction lasting for about 60 min. Then reperfusion was fulfilled by removal of the clip. The liver samples were collected at the 4th h after the restoration of blood inflow. Total RNA was extracted from the liver samples and analyzed quantitatively by method of real-time PCR. At the same time, portal vein serum and plasma were taken respectively for further detection of the level of endotoxin, tumor necrosis factor alpha (TNF-alpha) and plasmic alanine aminotransferase (pALT). The results indicated that TLR2 mRNA in ischemic lobe was up-regulated markedly in mice partial liver ischemia/reperfusion injury model compared to that in sham operation group (deltaCt: 1.05 +/- 1.02 vs 5.08 +/- 1.36, P<0.001). The level of portal vein pALT and TNF-alpha increased significantly (112.32 +/- 17.56 pg/ml vs 6.07 +/- 5.33 pg/ml, P<0.01; 890 +/- 127 microm/L vs 30 +/- 5 microm/L, P<0.001) . However, the level of portal vein endotoxin remained below the normal line, suggesting a state of non-endotoxemia. TLR2 mRNA expression in ischemic lobe, as well as portal vein pALT and TNF-alpha, was up-regulated in the model of mice partial ischemia/reperfusion injury, suggesting the involvement of TLR2 in ischemia/reperfusion pathological process.
Liver/*blood supply ; Liver/metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; RNA, Messenger/physiology ; Reperfusion Injury/etiology ; Reperfusion Injury/*metabolism ; Toll-Like Receptor 2/*biosynthesis ; Toll-Like Receptor 2/genetics ; Toll-Like Receptor 2/physiology ; Up-Regulation

OBJECTIVEThis study was aimed to investigate the possible effect of Toll-like receptors 2 (TLR2) and Toll-like receptors 4 (TLR4) on the migration function of umbilical cord blood (UCB) CD34+ hematopoietic stem/progenitor cells induced by bone marrow-derived mesenchymal stem cells (MSCs) and to explore the underlying mechanism.

METHODSThe expression of TLR2 and TLR4 on MSC was detected with flow cytometry. After the MSC were pretreated with TLR2 agonist (PAM3CSK4) and/or TLR4 agonist (LPS), the supernatants were collected. The effect of the supernatants on the migration of CD34+ cells was evaluated with chemotaxis assays. Alterations of chemokine (SDF-1) secreted by MSC in the supernatants were assayed by ELISA.

RESULTSThe expression levels of TLR2 and TLR4 were (31.5±4.6)% and (85.6±6.7)% respectively. Compared with the blank group, the migration ability of CD34+ cells increased significantly in control, LPS and/or PAM3CSK4 groups (P<0.01). Further study found that LPS and/or PAM3CSK4 enhanced the chemotactic ability of CD34+ cells (P<0.05), but the concentration of SDF-1 was not changed significantly in all of LPS and/or PAM3CSK4 groups (P>0.05) in comparison with the control group.

CONCLUSIONTLR2 and TLR4 signalings may indirectly increase the migration of CD34+ hematopoietic stem/progenitor cells by modulating BM-MSC functions, which may not significantly correlate with the production of chemokine SDF-1 by MSCs.

Antigens, CD34 ; Cell Movement ; Cells, Cultured ; Chemokine CXCL12 ; Fetal Blood ; Hematopoietic Stem Cells ; Humans ; Mesenchymal Stromal Cells ; Signal Transduction ; Toll-Like Receptor 2 ; Toll-Like Receptor 4

OBJECTIVETo explore the role of toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) in myocardial ischemia/reperfusion injury (MI/RI) by observing the dynamic expression changes at mRNA and protein levels early after myocardial ischemia/reperfusion (I/ R).

METHODSThe Wistar rats were randomly divided into Sham and I/R group (n = 42), and killed according to different reperfusion time (1, 2, 4, 6, 12, 24 h and 7 d). Structural and morphous changes of myocytes were observed under optical microscope. The mRNA and protein levels of TLR2 and TLR4 were detected using real-time PCR (RT-PCR). Monocyte chemokine protein-1 (MCP-1) and interleukine-6 (IL-6) mRNA levels were measured by reverse transcriptase-polymerase chain reaction (rt-PCR).

RESULTS(1) With the extension of reperfusion time, the myocardial infarct size increased smoothly, and reached the plateau at 4 h, then stayed in the platform. After reperfusion for 7 d, the ventricular had been remodeled. (2) At the beginning of reperfusion, myocardial structure showed no significant change in Sham group, but had different degrees of injury in I/R group. In rats of the group reperfused for 7 d the left ventricular remodeling could be visible. (3) Compared to sham group,TIR2, TLR4, MCP-1, IL-6 mRNA level were increased in myocardium in I/R group. TLR2 and TLR4 both peaked at 4 h of reperfusion, IL6 peaked at 6 h, followed by a gradually decrease. TLR4 and IL-6 mRNA levels rose again at 7 d. MCP-1 level in I/R group remained fairly with sham group at the beginning of reperfusion, and markedly elevated at 7 d.

CONCLUSIONExpression of TLRs mRNA in myocardium during early after myocardial ischemia/reperfusion increased rapidly and activated TLRs might play an important role in MI/RI through promoting the generation of inflammatory factors. At the late reperfusion, TLRs levels raise again and the expression of inflammatory factors increase once again, Those may probably affect the remodeling of ventricular, and injure myocardial structure and function.

Animals ; Chemokine CCL2 ; metabolism ; Disease Models, Animal ; Interleukin-6 ; metabolism ; Male ; Myocardial Reperfusion Injury ; metabolism ; Rats ; Rats, Wistar ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 4 ; metabolism

Acetylcysteine ; pharmacology ; Animals ; Cell Line ; Gene Expression ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; Mice ; Toll-Like Receptor 2 ; genetics ; Toll-Like Receptor 4 ; genetics

OBJECTIVETo study the association between single nucleotide polymorphisms(SNP) in toll-like receptors (TLR) 2 and 5 genes and the susceptibility to neonatal sepsis.

METHODSOne hundred and fourteen newborn infants who were diagnosed with clinical sepsis (case group) between May 2011 and January 2014 and 172 newborn infants without infection(control group) were enrolled in this study. The polymorphisms of TLR2 (rs5743708 and rs3804099) and TLR5 (rs5744105) were analyzed using a SNaPshot multiplex reaction to compare the genotypic and allelic frequencies between two groups. The relationship between TLR genotypes and susceptibility to sepsis was analyzed by logistic regression models.

RESULTSSignificant differences in genotypic frequencies of TLR2 rs3804099 (C/T) and TLR5 rs5744105 (C/G) were found between the two groups (P<0.05), but there was no significant difference in allelic frequencies of all the SNPs above between the two groups (P>0.05). The genotype on TLR2 rs5743708 was GG and no mutation was found in both groups. In regression models, birth weight (OR=3.065; P<0.05) and gestational age (OR=3.301; P<0.05) were closely associated with neonatal sepsis. Sex (OR=1.107, P>0.05), polymorphisms in rs3804099 (OR=0.876; P>0.05) and polymorphisms in rs5744105 (OR=0.820; P>0.05) genes were not risk factors for neonatal sepsis.

CONCLUSIONSTLR2 and 5 polymorphisms (rs5743708, rs3804099 and rs5744105) may not serve as the susceptible gene for sepsis in newborn infants.

Female ; Genetic Predisposition to Disease ; Humans ; Infant, Newborn ; Logistic Models ; Male ; Polymorphism, Single Nucleotide ; Sepsis ; genetics ; Toll-Like Receptor 2 ; genetics ; Toll-Like Receptor 5 ; genetics