Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In the present study, the first TLR5 gene in duck was cloned. The open reading frame (ORF) of duck TLR5 (dTLR5) cDNA is 2580 bp and encodes a polypeptide of 859 amino acids. We also cloned partial sequences of myeloid differentiation factor 88, 2'-5'-oligoadenylate synthetase (OAS), and myxovirus resistance (Mx) genes from duck. dTLR5 mRNA was highly expressed in the bursa of Fabricius, spleen, trachea, lung, jejunum, rectum, and skin; moderately expressed in the muscular and glandular tissues, duodenum, ileum, caecum, and pancreas; and minimally expressed in the heart, liver, kidney, and muscle. DF-1 or HeLa cells transfected with DNA constructs encoding dTLR5 can activate NF-kappaB leading to the activation of interleukin-6 (IL-6) promoter. When we challenged ducks with a Herts33 Newcastle disease virus (NDV), mRNA transcription of the antiviral molecules Mx, Double stranded RNA activated protein kinase (PKR), and OAS was up-regulated in the liver, lung, and spleen 1 and 2 days post-inoculation.
2',5'-Oligoadenylate Synthetase/genetics/metabolism ; Animals ; Cell Line ; *Cloning, Molecular ; Ducks ; Gene Expression Regulation/*physiology ; Humans ; Immunity, Innate ; Myeloid Differentiation Factor 88/genetics/metabolism ; Myxovirus Resistance Proteins/genetics/metabolism ; Newcastle Disease/metabolism ; Newcastle disease virus/classification ; RNA, Messenger/genetics/metabolism ; Species Specificity ; Toll-Like Receptor 5/genetics/*metabolism

miR-146a is an immunoregulatory microRNA closely associated with viral infection. This study investigated the expression changes of miR-146a in peripheral blood monocytes of HCV-infected patients and the mechanism by which the THP-1 cells were stimulated with HCV core protein in vitro. It was found that in the peripheral blood monocytes of HCV-infected patients, miR-146a expression was upregulated. After treated by interferon/ribavirin, miR-146a expression was decreased when HCV RNA became undetectable. HCV core could directly stimulate THP-1 cells to produce miR-146a. Silencing TLR2 and MyD88 could significantly inhibit the expression of miR-146a. It was concluded that the expression of miR-146a in peripheral blood monocytes of HCV-infected patients was abnormally increased. The TLR2-MyD88 signaling pathway may take part in the overexpression of miR-146a in monocytes stimulated with HCV core protein.
Adult ; Base Sequence ; Cell Line ; DNA Primers ; Female ; Hepatitis C, Chronic ; blood ; Humans ; Male ; MicroRNAs ; blood ; Middle Aged ; Monocytes ; metabolism ; Myeloid Differentiation Factor 88 ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Toll-Like Receptor 2 ; physiology ; Young Adult

To study the preventive effect of sophocarpine (Soc) on dextran sulfate sodium (DSS)-induced colitis in mice, in order to analyze the influence of Soc on toll like receptor 4 (TLR4)/mitogen-activated protein kinases (MAPKs) and janus tyrosine kinase 2 signal transducer and activator of transcription 3 (JAK2/STAT3) signal pathways in mice intestinal tissues. The mice was given 2.5% DSS for 6 days to induce the acute colitis model. The Soc-treated group was intraperitoneally injected with sophocarpine 30 mg · kg(-1) · d(-1) since the day before the experiment to the end. The disease activity index (DAI) was assessed everyday, and the colonic morphology and histological damage were observed with HE staining. The mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were detected by real-time RT-PCR. The changes in key protein kinase p38 mitogen-activated protein kinase (p38MAPK), c-Jun NH2-terminal protein kinase1/2 (JNK1/2), extracellular signal-regulated kinase1/2 (ERK1/2), JAK2, STAT3 in TLR4/MAPKs and JAK2/STAT3 signaling pathways were detected by western blot. The result showed that the model group showed statistical significance in body weight, DAI, colon length and histopathological changes compared with the normal group (P <0.05); however, the Soc-treated group showed significant improvements in the above indexes compared with the model group (P <0.05). TNF-α, IL-1β and IL-6 in the model group was significantly higher than that in the normal group (P <0.05), but lowered in the Soc-treated group to varying degrees (P <0.05). In the normal group, the expressions of TLR4 and the phosphorylation of P38, JNK1/2, JAK2, STAT3 were at low levels; in the model group, the phosphorylation of P38, JNK1/2, JAK2, STAT3 increased; the Soc-treated group showed a decrease in TLR4 expression compared with the model group, with notable declines in the phosphorylation of TLR4, P38, JNK1/2, JAK2, STAT3. These findings indicate that Soc can inhibit TLR4/MAPKs, K2/STAT3 signaling pathway activation, reduce the expression of proinflammatory cytokines TNF-α, IL-1β and IL-6 and relieve inflammatory reactions, so as to effectively prevent experimental colitis.
Alkaloids ; pharmacology ; therapeutic use ; Animals ; Colitis ; drug therapy ; immunology ; pathology ; Cytokines ; genetics ; Janus Kinase 2 ; antagonists & inhibitors ; physiology ; Male ; Mice ; Mice, Inbred BALB C ; Phosphorylation ; STAT3 Transcription Factor ; antagonists & inhibitors ; physiology ; Toll-Like Receptor 4 ; antagonists & inhibitors ; physiology

Chronic neuroinflammation is an integral pathological feature of major neurodegenerative diseases. The recruitment of microglia to affected brain regions and the activation of these cells are the major events leading to disease-associated neuroinflammation. In a previous study, we showed that neuron-released alpha-synuclein can activate microglia through activating the Toll-like receptor 2 (TLR2) pathway, resulting in proinflammatory responses. However, it is not clear whether other signaling pathways are involved in the migration and activation of microglia in response to neuron-released alpha-synuclein. In the current study, we demonstrated that TLR2 activation is not sufficient for all of the changes manifested by microglia in response to neuron-released alpha-synuclein. Specifically, the migration of and morphological changes in microglia, triggered by neuron-released alpha-synuclein, did not require the activation of TLR2, whereas increased proliferation and production of cytokines were strictly under the control of TLR2. Construction of a hypothetical signaling network using computational tools and experimental validation with various peptide inhibitors showed that beta1-integrin was necessary for both the morphological changes and the migration. However, neither proliferation nor cytokine production by microglia was dependent on the activation of beta1-integrin. These results suggest that beta1-integrin signaling is specifically responsible for the recruitment of microglia to the disease-affected brain regions, where neurons most likely release relatively high levels of alpha-synuclein.
Animals ; Antigens, CD29/genetics/*metabolism ; Cell Line, Tumor ; *Cell Movement ; Cells, Cultured ; Culture Media, Conditioned/*pharmacology ; Gene Regulatory Networks ; Humans ; Mice ; Mice, Inbred C57BL ; Microglia/drug effects/metabolism/*physiology ; Neurons/*metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Toll-Like Receptor 2/metabolism ; alpha-Synuclein/*pharmacology

BACKGROUNDTrichophyton rubrum (T. rubrum) represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that it may result from the failure of epithelial cells to recognize T. rubrum effectively and initiate effective immune responses. The C-type lectin receptors (CLR) and toll-like receptors (TLR) are the two major pattern recognition receptors (PRRs) that recognize fungal components. Therefore, the purpose of the study was to analyze the expression of those PRRs and the cytokines in HaCaT cells stimulated with heat-inactivated T. rubrum conidia and hyphae, respectively.

METHODSHaCaT cells were unstimulated or stimulated with heat-inactivated T. rubrum conidia and hyphae (1×10(6) and 1.5×10(5) colony-forming unit (CFU) in 2 ml medium, respectively) for 6, 12 and 24 hours. The mRNA expression of PRRs involved in recognizing fungal pathogen-associated molecular patterns (PAMPs) and signaling molecules were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). Meanwhile, surface toll-like receptor (TLR) 2, TLR4 and Dectin-1 were analyzed by fluorescence-activated cell sorter (FACS) 24 hours after treatment. The cytokines were detected in cell culture supernatants of HaCaT cells in 12 and 24 hours after treatment.

RESULTSHaCaT cells constitutively expressed mRNA of membrane-bound TLR1, 2, 4 and 6, Dectin1 and DC-SIGN, but not Dectin-2 or Mincle. Heat-killed T. rubrum did not significantly upregulate gene transcriptions of the PRRs of HaCaT cells. Heat-inactivated T. rubrum conidia significantly reduced the surface expression of TLR2 and Dectin-1, and suppressed the secretions of interferon-inducible protein-10 (IP-10) and monocyte chemotactic protein-1 (MCP-1) of HaCaT cells, while heat-killed T. rubrum hyphae significantly induced the secretions of IP-10 and MCP-1.

CONCLUSIONThe cell-wall antigens of T. rubrum fail to activate transcriptional expression of PRRs and induce a lower immune response of HaCaT cells by limited cytokines secretion.

Cells, Cultured ; Cytokines ; biosynthesis ; Humans ; Keratinocytes ; immunology ; Lectins, C-Type ; genetics ; physiology ; RNA, Messenger ; analysis ; Receptors, Pattern Recognition ; genetics ; physiology ; Toll-Like Receptor 2 ; physiology ; Trichophyton ; immunology

In the last decade, substantial progress has been made in understanding the molecular mechanisms involved in the initial host responses to viral infections. Herpesviral infections can provoke an inflammatory cytokine response, however, the innate pathogen-sensing mechanisms that transduce the signal for this response are poorly understood. In recent years, it has become increasingly evident that the Toll-like receptors (TLRs), which are germline-encoded pattern recognition receptors (PRRs), function as potent sensors for infection. TLRs can induce the activation of the innate immunity by recruiting specific intracellular adaptor proteins to initiate signaling pathways, which then culminating in activation of the nuclear factor kappa B (NF-κB) and interferon-regulatory factors (IRFs) that control the transcription of genes encoding type I interferon (IFN I) and other inflammatory cytokines. Furthermore, activation of innate immunity is critical for mounting adaptive immune responses. In parallel, common mechanisms used by viruses to counteract TLR-mediated responses or to actively subvert these pathways that block recognition and signaling through TLRs for their own benefit are emerging. Recent findings have demonstrated that TLR2 plays a crucial role in initiating the inflammatory process, and surprisingly that the response TLR2 triggers might be overzealous in its attempt to counter the attack by the virus. In this review, we summarize and discuss the recent advances about the specific role of TLR2 in triggering inflammatory responses in herpesvirus infection and the consequences of the alarms raised in the host that they are assigned to protect.
Adaptive Immunity ; Gene Expression Regulation ; immunology ; Herpesviridae ; physiology ; Herpesviridae Infections ; genetics ; immunology ; virology ; Host-Pathogen Interactions ; Humans ; Immune Evasion ; Immunity, Innate ; Interferon Regulatory Factors ; genetics ; metabolism ; Interferon Type I ; biosynthesis ; immunology ; NF-kappa B ; genetics ; metabolism ; Signal Transduction ; genetics ; immunology ; Toll-Like Receptor 2 ; genetics ; immunology

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

OBJECTIVETo study the mechanism of protective effect of Jinqiaomai (JQM) on lung injury induced by Klebsiella pneumonia in rats.

METHODThe model of rats with Klebsiella pneumonia was established. The male SD rats were randomly divided into control group, model group, JQM (6, 3, 1.5 g x kg(-1)) three groups, levofloxacin (25 mg x kg(-1)) group, JQM (3 g x kg(-1)) + levofloxacin (25 mg x kg(-1)) group. The contents of IL-1beta, ICAM-1 and INF-gamma in the lung tissue homogenate were measured by radio-immunoassay and Elisa. TLR2/4 mRNA and MyD88 mRNA expression were detected by RT-PCR. IkappaB-alpha expression was detected by Western Blot.

RESULTThe rats of model group had obvious lung injury, but those of JQM, JQM + levofloxacin and levofloxacin groups had less injury. The contents of IL-1beta, ICAM-1 and INF-gamma in lung tissue homogenate and the expressions of TLR2/4 mRNA, MyD88 mRNA and IkappaB-alpha in lung of model group were significantly higher than those in the control group (P < 0.05 or P < 0.01), while IL-1beta, ICAM-1 and INF-gamma of JQM groups were significantly lower than those of model group (P < 0.05 or P < 0.01). The expressions of TLR2/4 mRNA, MyD88 mRNA and IkappaB-alpha of JQM (6.3 g x kg(-1)) groups were significantly lower than those of model group(P <0. 05 or P <0. 01).

CONCLUSIONThe lung injury induced by Klebsiella pneumonia is related to TLR2/4, MyD88 mRNA and IkappaB-alpha. To decrease the excessive expression of TLR2/4, MyD88 mRNA and IkappaB-alpha might be the main mechanism of protective effect of Jinqiaomai on lung injury.

Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Gene Expression ; drug effects ; Humans ; I-kappa B Proteins ; genetics ; metabolism ; Klebsiella Infections ; drug therapy ; genetics ; metabolism ; microbiology ; Klebsiella pneumoniae ; drug effects ; physiology ; Lung ; drug effects ; metabolism ; microbiology ; Male ; Myeloid Differentiation Factor 88 ; genetics ; metabolism ; NF-KappaB Inhibitor alpha ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Toll-Like Receptor 2 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism

Anti-cancer drug bleomycin (BLM) can cause acute lung injury (ALI) which often results in pulmonary fibrosis due to a failure of resolving acute inflammatory response. The aim of this study is to investigate whether toll-like receptor (TLR) 2 mediates BLM-induced ALI, inflammation and fibrosis. BLM-induced dendritic cells (DCs) maturation was analyzed by flow cytometry and cytokine secretion was detected by the ELISA method. The expression and activity of p38 and ERK MAPK were determined with Western blotting. The roles of TLR2 in ALI, inflammation and fibrosis were investigated in C57BL/6 mice administered intratracheally with BLM. The results demonstrated that BLM-administered mice had higher expression of TLR2 (P<0.001) and its signaling molecules. Blocking TLR2 significantly inhibited the maturation of DCs and reversed BLM-stimulated secretion of cytokines in DCs, such as IL-6 (P<0.001), IL-17 (P<0.05) and IL-23 (P<0.05). TLR2 inhibition attenuated BLM-induced increase of inflammatory cells in bronchoalveolar lavage fluid (BALF), and reversed the immunosuppressive microenvironment by enhancing TH1 response (P<0.05) and inhibiting TH2 (P<0.001), Treg (P<0.01) and TH17 (P<0.01) responses. Importantly, blocking TLR2 in vivo significantly protected BLM-administered mice from pulmonary injury, inflammation and fibrosis and subsequently increased BLM-induced animal survival (from 50% to 92%). Therefore, TLR2 is a novel potential target for ALI and pulmonary fibrosis.
Acute Lung Injury ; chemically induced ; metabolism ; pathology ; Animals ; Bleomycin ; toxicity ; Bronchoalveolar Lavage Fluid ; Cells, Cultured ; Cytokines ; secretion ; Dendritic Cells ; cytology ; metabolism ; Inflammation ; chemically induced ; metabolism ; pathology ; Interleukin-17 ; secretion ; Interleukin-23 ; secretion ; Interleukin-6 ; secretion ; Lung ; metabolism ; MAP Kinase Signaling System ; Male ; Mice ; Mice, Inbred C57BL ; Pulmonary Fibrosis ; chemically induced ; metabolism ; pathology ; T-Lymphocytes, Regulatory ; drug effects ; Th1 Cells ; drug effects ; Th2 Cells ; drug effects ; Toll-Like Receptor 2 ; metabolism ; physiology

BACKGROUNDInvasive pulmonary aspergillosis (IPA) is a severe and frequently fatal disease in patients receiving treatment with immunosuppressive agents such as cyclophosphamide. Aspergillus fumigatus (A. fumigatus) now is a leading cause of IPA. Dectin-1 and Toll-like receptor 2 (TLR2) are important pattern recognition receptors involved in immune responses to A. fumigatus in vitro. However, the expression of the two receptors during the infection of A. fumigatus in vivo is not completely understood. The effects of cyclophosphamide treatment on the expression of the receptors need to be further studied.

METHODSWe established different immune status in mice models with or without A. fumigatus infection. On days 1, 3 and 5 post inoculation, pulmonary tissues from mice of the different groups were harvested. Dectin-1 and TLR2 mRNA expression in the lungs of the mice were investigated by real-time PCR. The pulmonary fungal burden in the mice with A. fumigatus infection was also evaluated.

RESULTSIn the immunocompetent mice, three days after A. fumigatus inoculation, dectin-1 and TLR2 expression increased markedly compared with the normal control group. Cyclophosphamide inhibited the clearance of pathogens and the expression of dectin-1 with or without A. fumigatus infection in the lungs as well. There was no statistical difference in TLR2 expression between the different immune status groups.

CONCLUSIONSOur results suggest that in vivo, dectin-1 and TLR2 are activated during the experimental period which would provide a broad range of possibilities for a specific and effective inflammatory response to kill A. fumigatus. Inhibition of dectin-1 expression may be one of the mechanisms of cyclophosphamide in the development of IPA.

Animals ; Aspergillosis ; immunology ; microbiology ; Aspergillus fumigatus ; immunology ; physiology ; Cyclophosphamide ; pharmacology ; Immunosuppressive Agents ; pharmacology ; Lectins, C-Type ; Lung ; drug effects ; metabolism ; microbiology ; Male ; Membrane Proteins ; genetics ; Mice ; Mice, Inbred BALB C ; Nerve Tissue Proteins ; genetics ; Polymerase Chain Reaction ; Toll-Like Receptor 2 ; genetics