BACKGROUNDThe Toll-like receptors (TLRs) represent a group of single-pass transmembrane receptors expressed on sentinel cells that are central to innate immune responses.The aim of this study was to investigate the presence of soluble TLRs in pleural effusions, and the diagnostic values of TLRs for pleural effusion with various etiologies.

METHODSPleural effusion and serum samples were collected from 102 patients (36 with malignant pleural effusion, 36 with tuberculous pleural effusion, 18 with bacterial pleural effusion, and 12 with transudative pleural effusion). The concentrations of TLR1 to TLR10 were determined in effusion and serum samples by enzyme linked immunosorbent assay. Four classical parameters (protein, lactate dehydrogenase, glucose and C-reactive protein (CRP)) in the pleural fluid were also assessed. Receiver-operating characteristic curves were used to assess the sensitivity and specificity of pleural fluid TLRs and biochemical parameters for differentiating bacterial pleural effusion.

RESULTSThe concentrations of TLR1, TLR3, TLR4, TLR7 and TLR9 in bacterial pleural effusion were significantly higher than those in malignant, tuberculous, and transudative groups, respectively. Analysis of receiver operating characteristic curves revealed that the area under the curves of TLR1, TLR3, TLR4, TLR7 and TLR9 were 0.831, 0.843, 0.842, 0.883 and 0.786, respectively, suggesting that these TLRs play a role in the diagnosis of bacterial pleural effusion. Also, the diagnostic value of TLRs for bacterial pleural effusions was much better than that of biochemical parameters (protein, lactate dehydrogenase, glucose and CRP).

CONCLUSIONSThe concentrations of TLR1, TLR3, TLR4, TLR7 and TLR9 appeared to be increased in bacterial pleural effusion compared to non-bacterial pleural effusions. Determination of these pleural TLRs may improve the ability of clinicians to differentiate pleural effusion patients of bacterial origin from those with other etiologies.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bacterial Infections ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Male ; Middle Aged ; Pleural Effusion ; metabolism ; microbiology ; Prospective Studies ; Toll-Like Receptor 1 ; metabolism ; Toll-Like Receptor 3 ; metabolism ; Toll-Like Receptor 4 ; metabolism ; Toll-Like Receptor 7 ; metabolism ; Toll-Like Receptor 9 ; metabolism ; Toll-Like Receptors ; metabolism ; Young Adult

Toll-like receptors (TLRs) play a central role of innate immunity as well as of enhancing the adaptive immunity. TLR1 interacts with TLR2 to recognize the mycobacterial 19-kD lipoprotein. Danio rerio genomic sequences predicted TLR family genes. Danio rerio TLR1 gene was cloned with RACE-PCR and RT-PCR methods to get more sights of immunity against mycobacterial infection such as leprosy. Cloned TLR1 showed insertion of 22 amino acid residues comparing to NCBI- predicted TLR1, and an open reading frame composing of deduced 795 amino acids. Toll-interlukin1 receptor domain and leucine-rich repeats were in the cloned TLR1 as were noted in NCBI-predicted TLR1. Cloned zebrafish TLR1 had high similarity to human TLR1 with 35% amino acid identities and 57% amino acid positivities across the entire sequence (E=1e-130). The cloned sequence might help to study the innate and adaptive immunity of mycobacterial infection.
Adaptive Immunity ; Amino Acids ; Clone Cells* ; Cloning, Organism* ; Humans ; Immunity, Innate ; Leprosy ; Lipoproteins ; Open Reading Frames ; Toll-Like Receptor 1* ; Toll-Like Receptors* ; Zebrafish*

The study investigated the inhibitory effect and mechanism of tectorigenin derivative(SGY) against herpes simplex virus type Ⅰ(HSV-1) by in vitro experiments. The cytotoxicity of SGY and positive drug acyclovir(ACV) on African green monkey kidney(Vero) cells and mouse microglia(BV-2) cells was detected by cell counting kit-8(CCK-8) method, and the maximum non-toxic concentration and median toxic concentration(TC_(50)) of the drugs were calculated. After Vero cells were infected with HSV-1, the virulence was determined by cytopathologic effects(CPE) to calculate viral titers. The inhibitory effect of the tested drugs on HSV-1-induced cytopathy in Vero cells was measured, and their modes of action were initially explored by virus adsorption, replication and inactivation. The effects of the drugs on viral load of BV-2 cells 24 h after HSV-1 infection and the Toll-like receptor(TLR) mRNA expression were detected by real-time fluorescence quantitative PCR(RT-qPCR). The maximum non-toxic concentrations of SGY against Vero and BV-2 cells were 382.804 μg·mL~(-1) and 251.78 μg·mL~(-1), respectively, and TC_(50) was 1 749.98 μg·mL~(-1) and 2 977.50 μg·mL~(-1), respectively. In Vero cell model, the half maximal inhibitory concentration(IC_(50)) of SGY against HSV-1 was 54.49 μg·mL~(-1), and the selection index(SI) was 32.12, with the mode of action of significantly inhibiting replication and directly inactivating HSV-1. RT-qPCR results showed that SGY markedly reduced the viral load in cells. The virus model group had significantly increased relative expression of TLR2, TLR3 and tumor necrosis factor receptor-associated factor 3(TRAF3) and reduced relative expression of TLR9 as compared with normal group, and after SGY intervention, the expression of TLR2, TLR3 and TRAF3 was decreased to different degrees and that of TLR9 was enhanced. The expression of inflammatory factors inducible nitric oxide synthase(iNOS), tumor necrosis factor-α(TNF-α), and interleukin-1β(IL-1β) was remarkably increased in virus model group as compared with that in normal group, and the levels of these inflammatory factors dropped after SGY intervention. In conclusion, SGY significantly inhibited and directly inactivated HSV-1 in vitro. In addition, it modulated the expression of TLR2, TLR3 and TLR9 related pathways, and suppressed the increase of inflammatory factor levels.
Animals ; Antiviral Agents/therapeutic use* ; Chlorocebus aethiops ; Herpes Simplex/pathology* ; Herpesvirus 1, Human/metabolism* ; Isoflavones ; Mice ; TNF Receptor-Associated Factor 3/pharmacology* ; Toll-Like Receptor 2/metabolism* ; Toll-Like Receptor 3/metabolism* ; Toll-Like Receptor 9/metabolism* ; Toll-Like Receptors/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Vero Cells ; Virus Replication

The innate immune response in patients who develop inflammatory bowel disease (IBD) may be abnormal. However, the exact role of Toll-like receptors (TLRs) / CD14 gene in the pathogenesis of IBD has not been fully elucidated. We aimed to investigate the association between polymorphisms of TLR1, 2, 4, 6, and CD14 gene and susceptibility to IBD in Korean population. A total 144 patients of IBD (99 patients with ulcerative colitis, 45 patients with Crohn's disease) and 178 healthy controls were enrolled. Using a PCR-RFLP, we evaluated mutations of TLR1 (Arg80Thr), TLR2 (Arg753Gln and Arg677Trp), TLR4 (Asp299Gly and Thr399Ile), TLR6 (Ser249Pro) genes and the -159 C/T promoter polymorphism of CD14 gene. No TLR polymorphisms were detected in Korean subjects. T allele and TT genotype frequencies of CD14 gene were significantly higher in IBD patients than in healthy controls. In subgroup analysis, T allelic frequency was higher in pancolitis phenotype of ulcerative colitis. In Korean population, the promoter polymorphism at -159 C/T of the CD14 gene is positively associated with IBD, both ulcerative colitis and Crohn's disease.
Adult ; Aged ; Alleles ; Antigens, CD14/*genetics ; Asian Continental Ancestry Group/*genetics ; Colitis, Ulcerative/genetics ; Crohn Disease/genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Inflammatory Bowel Diseases/*genetics ; Male ; Middle Aged ; Phenotype ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Republic of Korea ; Toll-Like Receptor 1/genetics ; Toll-Like Receptor 2/genetics ; Toll-Like Receptor 4/genetics ; Toll-Like Receptor 6/genetics ; Toll-Like Receptors/*genetics

OBJECTIVETo investigate the mechanism of Mycobacterium tuberculosis invasion to mouse dendritic cells (DC).

METHODSMycobacterium tuberculosis strain H37Rv was co-cultured with mouse DC2.4 cells.The mRNA expression of Toll-like receptor 2/4(TLR2/4) in DC2.4 cells was detected by fluorescent quantitative real-time PCR and the protein expression of nuclear factor κB(NF-κB) was assessed by Western blotting.The extracellular concentration of tumor necrosis factor α(TNF-α) was measured by ELISA methods during Mycobacterium Tuberculosis invasion.Indirect immunofluorescent staining and flow cytometry assay were used to detect the expression of CD80 and CD86 on DC2.4 cells before and after invasion.

RESULTSThe invasion of Mycobacterium tuberculosis in DC2.4 cells was observed after 2 h of co-incubation.The rates of invasion were (37.9±5.6)%,(51.2±7.6)%,(57.2±8.9)% and(63.9±6.8)% at 6,8,10 and 12 h after co-incubation,respectively.The mRNA expression level of TLR2 /4 was significantly increased at 6 h but decreased at 10 h after co-incubation.The expressions of NF-κB p65 and TNF-α were higher in DC2.4 cells after being invaded by 6,8,and 10 h and then gradually decreased.CD80 and CD86 expression were increased on DC2.4 at 6 h after co-incubation.

CONCLUSIONInvasion of Mycobacterium tuberculosis strain H37Rv to DC might enhance its antigen-presenting function through activation of TLR2/4-NF-kB signaling pathway.

Animals ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; Cells, Cultured ; Dendritic Cells ; immunology ; metabolism ; Mice ; Mycobacterium tuberculosis ; NF-kappa B ; metabolism ; Signal Transduction ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 4 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVECelastrol has been established as a nuclear factor-κB (NF-κB) activation inhibitor; however, the exact mechanism behind this action is still unknown. Using text-mining technology, the authors predicted that interleukin-1 receptor-associated kinases (IRAKs) are potential celastrol targets, and hypothesized that targeting IRAKs might be one way that celastrol inhibits NF-κB. This is because IRAKs are key molecules for some crucial pathways to activate NF-κB (e.g., the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily).

METHODSThe human hepatocellular cell line (HepG2) treated with palmitic acid (PA) was used as a model for stimulating TLR4/NF-κB activation, in order to observe the potential effects of celastrol in IRAK regulation and NF-κB inhibition. The transfection of small interfering RNA was used for down-regulating TLR4, IRAK1 and IRAK4, and the Western blot method was used to detect changes in the protein expressions.

RESULTSThe results showed that celastrol could effectively inhibit PA-caused TLR4-dependent NF-κB activation in the HepG2 cells; PA also activated IRAKs, which were inhibited by celastrol. Knocking down IRAKs abolished PA-caused NF-κB activation.

CONCLUSIONThe results for the first time show that targeting IRAKs is one way in which celastrol inhibits NF-κB activation.

Hep G2 Cells ; Humans ; Interleukin-1 Receptor-Associated Kinases ; antagonists & inhibitors ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Phosphorylation ; Toll-Like Receptor 4 ; antagonists & inhibitors ; physiology ; Triterpenes ; pharmacology

TLR2 activity plays an important role in the pathogenesis of autoimmune diseases, tumor carcinogenesis and cardio-cerebrovascular diseases. To establish a TLR2 receptor-based cell screening model, NF-kappaB promoter-driven luciferase reporter plasmids were transfected into human embryonic kidney cells (HEK293) stably expressing human TLR2 and co-receptors CD14, TLR1 and TLR6. Single clones were then isolated and characterized. Using this screening system, a human TLR2-binding peptide C8 was obtained from the Ph.D.-7 Phage Display Peptide Library through biopanning and rapid analysis of selective interactive ligands (BRASIL). The binding characteristic of C8 with human TLR2 was evaluated by ELISA, flow cytometry and immunofluorescence. The NF-kappaB luciferase activity assay showed that C8 could activate the TLR2/TLR1 signaling pathway and induce the production of cytokines TNF-alpha and IL-6. In conclusion, the TLR2 receptor-based cell screening system is successfully established and a new TLR2-binding peptide is identified by using this system.
Bacteriophages ; Drug Evaluation, Preclinical ; Genes, Reporter ; HEK293 Cells ; Humans ; Interleukin-6 ; metabolism ; Lipopolysaccharide Receptors ; metabolism ; Luciferases ; genetics ; metabolism ; Peptide Library ; Peptides ; metabolism ; pharmacology ; Promoter Regions, Genetic ; Protein Binding ; Signal Transduction ; drug effects ; Toll-Like Receptor 1 ; metabolism ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 6 ; metabolism ; Transfection ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVES: To study the role and mechanism of autophagy in lipopolysaccharide (LPS)-induced inflammatory response of human alveolar epithelial A549 cells. METHODS: A549 cells were stimulated with LPS to establish a cell model of inflammatory response, and were then grouped (n=3 each) by concentration (0, 1, 5, and 10 μg/mL) and time (0, 4, 8, 12, and 24 hours). The A549 cells were treated with autophagy inhibitor 3-methyladenine (3-MA) to be divided into four groups (n=3 each): control, LPS, 3-MA, and 3-MA+LPS. The A549 cells were treated with autophagy agonist rapamycin (RAPA) to be divided into four groups (n=3 each): control, LPS, RAPA, and RAPA+LPS. The A549 cells were transfected with the Toll-like receptor 4 (TLR4) overexpression plasmid to be divided into four groups (n=3 each): TLR4 overexpression control, TLR4 overexpression, TLR4 overexpression control+LPS, and TLR4 overexpression+LPS. The A549 cells were transfected with TLR4 siRNA to be divided into four groups (n=3 each): TLR4 silencing control,TLR4 silencing, TLR4 silencing control+LPS, and TLR4 silencing+LPS. CCK-8 assay was used to measure cell viability. Western blot was used to measure the protein expression levels of inflammatory indicators (NLRP3, Caspase-1, and ASC), autophagic indicators (LC3B, Beclin-1, and P62), and TLR4. RESULTS: After stimulation with 1 μg/mL LPS for 12 hours, the levels of inflammatory indicators (NLRP3, Caspase-1, and ASC), autophagic indicators (LC3B, Beclin-1, and P62), and TLR4 increased and reached the peak (P<0.05). Compared with the LPS group, the 3-MA+LPS group had reduced expression of autophagy-related proteins and increased expression of inflammation-related proteins and TLR4, while the RAPA+LPS group had increased expression of autophagy-related proteins and reduced inflammation-related proteins and TLR4 (P<0.05). The TLR4 overexpression+LPS group had reduced autophagy-related proteins and increased inflammation-related proteins compared with the TLR4 overexpression control+LPS group, and the TLR4 silencing+LPS group had increased autophagy-related proteins and reduced inflammation-related proteins compared with the TLR4 silencing control+LPS group (P<0.05). CONCLUSIONS In the LPS-induced inflammatory response of human alveolar epithelial A549 cells, autophagic flux has a certain protective effect on A549 cells. TLR4-mediated autophagic flux negatively regulates the LPS-induced inflammatory response of A549 cells.
Humans ; A549 Cells ; Autophagy ; Beclin-1/metabolism* ; Caspase 1/metabolism* ; Inflammation ; Lipopolysaccharides/pharmacology* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Toll-Like Receptor 4/metabolism*

Microglial cells were purified from a mixed neuroglia culture prepared from the neonatal chicken brain in vitro, and were infected with the vvMDV YL040920 isolate and an attenuated MDV vaccine strain CVI988/Rispens, respectively. The presence of cytopathic effect (CPE) was examined daily, and the MEQ expression in MDV-infected microglia was detected by immunohistochemistry assay. DNA replication of the MDV meq gene and transcription of the gB gene were determined by real-time quantitative PCR (qPCR) and qRT-PCR, respectively. The transcripts of Toll-like receptor (TLR) mRNA in microglia post MDV infection were quantified by qRT-PCR. The results of this study showed that both vvMDV YL040920 and attenuated vaccine strain CVI988/Rispens could infect microglia and produce characteristic CPE with plaque formation. The plaques were formed due to cells shedding at multi-sites, then quickly expanded and integrated. Furthermore, the MEQ protein was detected in nuclei of YL040920 and CVI988/ Rispens-infected microglia, and MDV meq DNA replication and gB gene transcription in MDV-infected microglia were also confirmed. Although both MDV DNA copies and gB transcripts were increased in the virus-infected microglia, the higher viral DNA load and gB transcript were observed for CVI988/Rispens than for YL040920 in vitro (P < or = 0.05/0.001). The transcriptions of TLR15 and TLR1LB gene were found to be up-regulated in microglia following MDV infection in vitro. Purified microglia infected with YL040920 was observed increased TLR15 and TLR1LB transcripts as early as 1 day post infection (dpi), and reached its peak level at 3 dpi, then decreased mildly at 5 dpi. For CVI988/Rispens, it induced an increase of TLR15 transcript as early as 1 dpi, and rose rapidly at 3 dpi, and then decreased slightly at 5 dpi. At the same time, CVI988/Rispens induced the increase of chTLR1LB transcript at 3 dpi and decreased at 5 dpi. By comparing the TLRs transcription between YL040920 and CVI988/Rispens-infected microglia, it was suggested that vvMDV YL040920 might induce more TLR15 transcript than the attenuated vaccine strain CVI988/Rispens (P < or = 0.01/0.001), while CVI988/Rispens induced more TLR1LB transcript than YL040920 (P < or = 0.001).
Animals ; Brain ; metabolism ; virology ; Chickens ; Gene Expression ; Herpesvirus 2, Gallid ; genetics ; physiology ; Marek Disease ; genetics ; metabolism ; virology ; Microglia ; metabolism ; virology ; Poultry Diseases ; genetics ; metabolism ; virology ; Toll-Like Receptor 1 ; genetics ; metabolism ; Toll-Like Receptors ; genetics ; metabolism ; Transcription, Genetic

INTRODUCTION: TLR-5, a member of the toll-like receptor (TLR) family, is a element of the type I transmembrane receptors, which are characterized by an intracellular signaling domain homolog to the interleukin-1 receptor. These receptors recognize microbial components, particularly bacterial flagellin. All-trans retinoic acid (atRA, tretinoin), a natural metabolite of vitamin A, acts as a growth and differentiation factor in many tissues, and is also needed for immune functions. In this study, THP-1 human macrophage-monocytes were used to examine the mechanisms by which atRA regulated the expression of TLR-5. Because the molecular mechanism underlying this regulation at the transcriptional level is also unclear, this study examined which putative transcription factors are responsible for TLR-5 expression by atRA in immune cells. MATERIALS AND METHODS: This study examined whether atRA induces the expression of TLR-5 in THP-1 cells using reverse transcription-polymerase chain reaction (RT-PCR), and which transcription factors are involved in regulating the TLR-5 promoter in RAW264.7 cells using a reporter assay system. Western blot analysis was used to determine which signal pathway is involved in the expression of TLR-5 in atRA-treated THP-1 cells. RESULTS: atRA at a concentration of 10 nM greatly induced the expression of TLR-5 in THP-1 cells. Human TLR-5 promoter contains three Sp-1/GC binding sites around -50 bp and two NF-kB binding sites at -380 bp and -160 bp from the transcriptional start site of the TLR-5 gene. Sp-1/GC is primarily responsible for the constitutive TLR-5 expression, and may also contribute to NF-kB at -160 bp to induce TLR-5 after atRA stimulation in THP-1 cells. The role of NF-kB in TLR-5 expression was further confirmed by inhibitor pyrrolidine dithiocarbamate (PDTC) experiments, which greatly reduced the TLR-5 transcription by 70-80%. CONCLUSION: atRA induces the expression of the human TLR-5 gene and NF-kB is a critical transcription factor for the atRA-induced expression of TLR-5. Accordingly, it is conceivable that retinoids are required for adequate innate and adaptive immune responses to agents of infectious diseases. atRA and various synthetic retinoids have been used therapeutically in human diseases, such as leukemia and other cancers due to the antiproliferative and apoptosis inducing effects of retinoids. Therefore, understanding the molecular regulatory mechanism of TLR-5 may assist in the design of alternative strategies for the treatment of infectious diseases, leukemia and cancers.
Apoptosis ; Binding Sites ; Blotting, Western ; Communicable Diseases ; Flagellin ; Humans ; Immunity, Cellular ; Interleukin-1 ; Leukemia ; NF-kappa B ; Pyrrolidines ; Retinoids ; Signal Transduction ; Thiocarbamates ; Toll-Like Receptor 5 ; Toll-Like Receptors ; Transcription Factors ; Tretinoin ; Vitamin A