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1.
Arch Virol ; 164(5): 1433-1439, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30868265

RESUMO

Herpes simplex virus 1 (HSV-1), a double-stranded DNA virus, infects epithelial surfaces and establishes latency in the central nervous system, where astrocytes are a major immune cell type. Here, we report changes that occur in the expression of pathogen recognition receptors, such as Toll-like receptors, DNA and RNA sensors, interferons, and interferon-stimulated genes, when astrocytes are infected with HSV-1 strain F. We observed upregulation of Toll-like receptors 2, 6 and 9, MDA5, and DAI along with an increase in the expression of type I interferons and interferon-stimulated genes such as IFIT1, IFIT3 and RNase L. These genes encode proteins that mediate the antiviral immune response.


Assuntos
Astrócitos/imunologia , Astrócitos/virologia , Herpesvirus Humano 1/imunologia , Imunidade Inata/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Chlorocebus aethiops , Endorribonucleases/metabolismo , Helicase IFIH1 Induzida por Interferon/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas/metabolismo , Proteínas de Ligação a RNA , Receptor 2 Toll-Like/biossíntese , Receptor 6 Toll-Like/biossíntese , Receptor Toll-Like 9/biossíntese , Regulação para Cima/genética , Células Vero , Replicação Viral , eIF-2 Quinase/biossíntese
2.
Vet Immunol Immunopathol ; 206: 49-53, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30502912

RESUMO

In mares, placental diseases are a common cause of pregnancy failure and they can have an economic impact on the horse breeding industry. To our knowledge no published data on TLR expression in the equine placenta exist. This study examined the expression of TLR 2, 4 and 6 as transcript and protein in the placenta (chorioallantois) of 14 foals born alive. By PCR, all examined placental samples contained TLR 2, 4 and 6 transcripts. Using immunohistochemistry, trophoblasts and allantoic epithelium were immunopositive for TLR 2, 4 and 6 in all placental samples. The majority of placental samples contained TLR 4 and 6 positive stromal cells and vascular smooth muscle cells. Since these results confirm the expression of TLR 2, 4 and 6 in different cell populations of the equine placenta, they are the basis for studies into the pathogenesis of TLR-associated placental diseases in mares.


Assuntos
Cavalos/metabolismo , Placenta/metabolismo , Receptor 2 Toll-Like/biossíntese , Receptor 4 Toll-Like/biossíntese , Receptor 6 Toll-Like/biossíntese , Animais , Animais Recém-Nascidos , Feminino , Placenta/citologia , Gravidez , RNA Mensageiro/metabolismo , Estudos Retrospectivos , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Receptor 6 Toll-Like/genética
3.
Vet Immunol Immunopathol ; 185: 7-13, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28242004

RESUMO

Subfertility in mares is mainly caused by endometrial diseases. Alterations of Toll-like receptors (TLRs) are associated with endometrial disorders in women. This study investigated TLRs 2, 4 and 6 in the equine endometrium. Endometria of 21 mares were examined by histology, PCR and immunohistochemistry. Tissues from 2 mares were considered normal. The remaining showed endometritis, endometrosis and/or angiosclerosis. TLRs 2, 4 and 6 were expressed as transcripts and proteins in all endometria. Immunohistochemistry detected TLRs 2, 4 and 6 in mast cells, luminal and glandular epithelial cells, stromal cells, endothelia, vascular smooth muscle and/or inflammatory cells. Between examined endometria numbers of immunopositive epithelial cells varied considerably; TLRs were located in their cytoplasm and/or the nucleus. All other cell types displayed a cytoplasmic staining. Results indicate a complex and cell-type-specific modulation of TLRs 2, 4 and 6 in the equine endometrium. The lack of a detectable association between a particular disease and a distinct cellular expression may be explained by the often combined presence of several factors with a possible influence on TLRs. This study expands the basic knowledge on equine endometrial immunity and will assist to uncover if immunological alterations contribute to uterine diseases of mares.


Assuntos
Endométrio/metabolismo , Cavalos/metabolismo , Receptor 2 Toll-Like/biossíntese , Receptor 4 Toll-Like/biossíntese , Receptor 6 Toll-Like/biossíntese , Animais , Endométrio/patologia , Feminino , Doenças Uterinas/metabolismo , Doenças Uterinas/patologia , Doenças Uterinas/veterinária
4.
BMC Microbiol ; 17(1): 77, 2017 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-28356067

RESUMO

BACKGROUND: Salmonella enterica infections often exhibit a form of immune evasion. We previously observed that probiotic bacteria could prevent inhibition of lymphoproliferation and apoptosis responses of T cells associated with S. enterica infections in orally challenged mice. RESULTS: In this study, changes in expression of genes related to lymphocyte activation in mucosa-associated lymphoid tissues (MALT) of mice orally infected with S. enterica with and without treatment with probiotic bacteria were evaluated. Probiotic bacteria increased expression of mRNA for clusters of differentiation antigen 2 (Cd2), protein tyrosine phosphatase receptor type C (Ptprc), and Toll-like receptor 6 (Tlr6) genes related to T and B cell activation in mouse intestinal tissue. The probiotic bacteria were also associated with reduced mRNA expression of a group of genes (RelB, Myd88, Iκκa, Jun, Irak2) related to nuclear factor of kappa light chains enhancer in B cells (NF-κB) signal transduction pathway-regulated cytokine responses. Probiotic bacteria were also associated with reduced mRNA expression of apoptotic genes (Casp2, Casp12, Dad1, Akt1, Bad) that suggest high avidity lymphocyte sparing. Reduced CD2 immunostaining in mesenteric lymph nodes (MLN) was suggestive of reduced lymphocyte activation in probiotic-treated mice. Reduced immunostaining of TLR6 in MALT of probiotic-treated, S. enterica-infected mice suggests that diminished innate immune sensitivity to S. enterica antigens is associated with preventing lymphocyte deletion. CONCLUSIONS: The results of this study are consistent with prevention of S. enterica-induced deletion of lymphocytes by the influence of probiotic bacteria in mucosal lymphoid tissues of mice.


Assuntos
Proliferação de Células/efeitos dos fármacos , Imunomodulação , Probióticos/farmacologia , Salmonelose Animal/prevenção & controle , Salmonella enterica/patogenicidade , Linfócitos T/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linfócitos B/metabolismo , Antígenos CD2/biossíntese , Antígenos CD2/genética , Citocinas/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Terapia de Imunossupressão , Antígenos Comuns de Leucócito/biossíntese , Antígenos Comuns de Leucócito/genética , Linfonodos , Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Nódulos Linfáticos Agregados/efeitos dos fármacos , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/patologia , RNA Mensageiro/biossíntese , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Baço , Receptor 6 Toll-Like/biossíntese , Receptor 6 Toll-Like/genética
5.
Protein J ; 36(1): 28-35, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28161794

RESUMO

Toll-like receptors (TLRs) mediate immune responses upon recognition of a variety of ligands. To further elucidate the function of TLRs, it is important to identify novel ligands and their action mechanisms including polymer assembly. In this study, we propose an efficient method for preparation of the extracellular domain of human Toll-like receptor 6 (TLR6ED) in Escherichia coli using the bubbling cultivation method. Our preparation method improved the level of expression of TLR6ED into a soluble fraction as compared with typical cultivation using a rotary shaker. Circular dichroism (CD) experiments confirmed the structural formation of TLR6ED with secondary structure contents similar to leucine-rich repeat (LRR) modules. In addition, we also provided a procedure for preparing this recombinant protein using Sf9 insect cells, which ensures preservation of some key posttranslational modifications often lacking in bacteria-expressed proteins. These materials would be useful for analyzing novel molecules that bind directly to TLR6, complex formations with other regulators including TLR2 and TLR4, and the functional effects of N-linked glycosylation.


Assuntos
Proteínas Recombinantes/química , Receptor 6 Toll-Like/química , Animais , Dicroísmo Circular , Escherichia coli , Humanos , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera , Receptor 6 Toll-Like/biossíntese , Receptor 6 Toll-Like/genética
6.
Dev Comp Immunol ; 59: 39-47, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26797426

RESUMO

While Mycoplasma gallisepticum (MG) is a major pathogen that causes chronic respiratory diseases in chicken, the molecular mechanism of MG infection is not clear. In this study, we investigated the roles of Toll-like receptor 2 (TLR2) and 6 (TLR6) in MG infection. We found that TLR2 type 2 (TLR2-2) and TLR6 had differential expressions in chicken embryo fibroblasts (DF-1 cells), where TLR6 was highly expressed, but TLR2-2 was barely expressed. Upon MG infection, TLR6 expression was upregulated, followed by upregulation of downstream factors, MyD88, NF-κB, IL2, IL6, and TNF-α. Knockdown of TLR6 expression by shRNA abolished the MG-induced inflammatory responses. More interestingly, in the presence of TLR6, TLR2-2 didn't respond to MG infection in DF-1 cells. When TLR6 was knocked down by shRNA, however, TLR2 was upregulated upon MG infection, which was followed by upregulation of proinflammatory genes. Finally, we tested effects of the MG infection on expression of TLR2-2 and TLR6 in the lungs and trachea tissues of chicken embryos. We found both TLR2-2 and TLR6 were upregulated upon MG infection, followed by upregulation of the downstream NF-κB-mediated inflammatory responses. This study was the first to report the differential roles of TLR2-2 and TLR6 in MG-infected DF-1 cells and chicken embryos.


Assuntos
Inflamação/imunologia , Infecções por Mycoplasma/imunologia , Mycoplasma gallisepticum/imunologia , Doenças das Aves Domésticas/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 6 Toll-Like/imunologia , Animais , Linhagem Celular , Embrião de Galinha , Inflamação/genética , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/veterinária , NF-kappa B/imunologia , Doenças das Aves Domésticas/microbiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/biossíntese , Receptor 6 Toll-Like/biossíntese , Receptor 6 Toll-Like/genética
7.
Mol Immunol ; 68(2 Pt B): 476-83, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26463158

RESUMO

The present study describes and compares functional properties of Nuli-1 cells and primary human nasal epithelial cells (HNEC) including TLR expression and function. Differences in gene expression were identified for non-TLR genes that play a role in TLR response pathways. However, experiments comparing TLR gene expression for both Nuli-1 cells and HNECs indicated conserved expression in both cell types. Stimulation of the two cell types resulted in a conserved response to TLR3 agonists, but in differences in response to agonists for TLR5 and TLR6/2. HNECs were much more susceptible to infection with Staphylococcus aureus than NuLi-1 cells. Furthermore, when cultured at air-liquid interface (ALI), NuLi-1 cells possessed much lower trans-epithelial resistance than primary HNEC and did not exhibit maintenance of cell morphology or mucous production which was observed in HNECs. Nor did they produce the characteristic interconnecting pattern of tight junction complexes at the apicolateral margin of adjacent cells. Caution should therefore be exercised when selecting cell lines for immunological studies and a thorough screen of properties relevant to the study should always be carried out prior to commencement.


Assuntos
Células Epiteliais/imunologia , Mucosa Nasal/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Receptor 3 Toll-Like/biossíntese , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Expressão Gênica , Humanos , Imunidade Inata/imunologia , Mucosa Nasal/citologia , Mucosa Nasal/metabolismo , RNA Mensageiro/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/biossíntese , Receptor 3 Toll-Like/agonistas , Receptor 5 Toll-Like/agonistas , Receptor 5 Toll-Like/biossíntese , Receptor 6 Toll-Like/agonistas , Receptor 6 Toll-Like/biossíntese
8.
Cancer Immunol Immunother ; 64(3): 275-86, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25376541

RESUMO

Inflammation has been implicated in the initiation and progression of ovarian cancer (OC), the underlying mechanisms of which are still unclear. We hypothesized that the abnormal expression of Toll-like receptors (TLRs), which were potential activators of nuclear factor-kappa B p65 (NF-κB p65), could promote inflammation and tumorigenesis in OC. In this study, we characterized the expression of TLRs in peripheral blood mononuclear cells (PBMCs) and found TLR2 and TLR6 mRNAs levels to be higher in PBMCs from OC patients than in those from benign disease (BC) or healthy normal controls (NC). Flow cytometry analysis showed that TLR1, TLR2 and TLR6 were highly expressed in monocytes from OC patients, but not in those from control subjects. Consistently, inflammatory cytokines interleukin (IL)-1ß and IL-6 were up-regulated in PBMCs from OC patients upon stimulation with Pam3CSK4 (TLR1 ligand) and HKLM (TLR2 ligand), compared with unstimulated PBMCs. Stimulation of PBMCs with TLR ligands led to activation of downstream signaling molecules in TLRs (MyD88, TRAF6, TANK, NF-κB p65 and p-NF-κB p65). We also discovered that SK-OV-3-secreted factors were potent PBMCs activators, leading to the production of IL-1ß, IL-6 and IL-8 through activation of TLRs and downstream signaling molecules in PBMCs. Before coculturing with SK-OV-3, pretreatment of THP-1 cells or PBMCs with monoclonal antibodies against TLR1, TLR2 or TLR6 inhibited the production of IL-1ß and IL-6 and activation of MyD88, TRAF6, TANK, NF-κB p65 and p-NF-κB p65. Our results provided new evidence that TLR1, TLR2 and TLR6 signaling was linked with inflammation in OC microenvironment.


Assuntos
Leucócitos Mononucleares/metabolismo , Neoplasias Ovarianas/sangue , Receptores Toll-Like/sangue , Adulto , Estudos de Casos e Controles , Células Cultivadas , Feminino , Humanos , Inflamação/sangue , Inflamação/imunologia , Pessoa de Meia-Idade , Fator 88 de Diferenciação Mieloide/sangue , Neoplasias Ovarianas/imunologia , Receptor 2 Toll-Like/biossíntese , Receptor 2 Toll-Like/sangue , Receptor 6 Toll-Like/biossíntese , Receptor 6 Toll-Like/sangue , Receptores Toll-Like/biossíntese
9.
J Bone Joint Surg Am ; 96(20): 1692-8, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25320195

RESUMO

BACKGROUND: Toll-like receptors (TLRs) 1 and 6 are consistent molecular indicators of the host inflammatory response against bacterial infection. Our aims were to determine whether TLR elevation could be detected in infected periprosthetic tissues and to assess the utility of these biomarkers as tests for detecting a periprosthetic joint infection. METHODS: Fifty-nine patients undergoing revision total joint arthroplasty (twenty-seven hips and thirty-two knees) were prospectively evaluated for periprosthetic joint infection according to currently recommended diagnostic criteria. Nine patients were excluded because of insufficient work-up, leaving fifty available for study. Of these, twenty-one were categorized as infected and twenty-nine as noninfected. Periprosthetic tissues were collected intraoperatively, and total RNA was extracted by standard techniques. Expression of TLR messenger RNAs was assessed by first-strand complementary DNA synthesis from 1 µg of total RNA followed by real-time PCR (polymerase chain reaction). Results were normalized relative to the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Expression of TLRs 1, 6, and 10 in the infected and noninfected groups was compared with use of the Student t test. The receiver operating characteristic curve, area under the curve (AUC), sensitivity, specificity, positive likelihood ratio (LR+), and negative likelihood ratio (LR-) were calculated to determine the accuracy of each TLR for predicting periprosthetic joint infection at its optimal diagnostic threshold. RESULTS: Mean TLR1 mRNA expression was significantly elevated in infected compared with noninfected samples (0.600 compared with 0.005, p = 0.0003); the same was true of TLR6 (0.208 compared with 0.0165, p = 0.0059) but not of TLR10 (0.00019 compared with 0.00014, p = 0.6238). The AUC was 0.995 for TLR1, 0.883 for TLR6, and 0.546 for TLR10. The optimal threshold for diagnosing periprosthetic joint infection was 0.0924 for TLR1 (sensitivity = 95.2%, specificity = 100%, LR+ = 13.80, LR- = 0.91) and 0.0215 for TLR6 (sensitivity = 85.7%, specificity = 82.8%, LR+ = 4.98, LR- = 0.83). CONCLUSIONS: In our pilot study, TLR1 expression in periprosthetic tissues most accurately predicted periprosthetic joint infection. This measure of the host response may be particularly helpful in detecting culture-negative infections and avoiding false positives resulting from contamination. LEVEL OF EVIDENCE: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.


Assuntos
Artrite Infecciosa/metabolismo , Infecções Relacionadas à Prótese/metabolismo , Receptor 1 Toll-Like/biossíntese , Artroplastia de Substituição , Biomarcadores/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Pessoa de Meia-Idade , Falha de Prótese , RNA Mensageiro/biossíntese , Reoperação , Receptor 10 Toll-Like/biossíntese , Receptor 6 Toll-Like/biossíntese
10.
PLoS One ; 9(9): e106421, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25184331

RESUMO

Oxidative stress is key in the pathogenesis of several diseases including age-related macular degeneration (AMD), atherosclerosis, diabetes, and Alzheimer's disease. It has previously been established that a lipid peroxidation product, carboxyethylpyrrole (CEP), accumulates in the retinas of AMD patients. Retinal infiltrating macrophages also accumulate in the retinas of both AMD patients and in a murine model of AMD. We therefore investigated the ability of CEP-adducts to activate innate immune signaling in murine bone-marrow derived macrophages (BMDMs). We found that CEP specifically synergizes with low-dose TLR2-agonists (but not agonists for other TLRs) to induce production of inflammatory cytokines. Moreover, CEP selectively augments TLR2/TLR1-signaling instead of TLR2/TLR6-signaling. These studies uncover a novel synergistic inflammatory relationship between an endogenously produced oxidation molecule and a pathogen-derived product, which may have implications in the AMD disease process and other oxidative stress-driven pathologies.


Assuntos
Degeneração Macular/imunologia , Pirróis/administração & dosagem , Receptor 1 Toll-Like/biossíntese , Receptor 2 Toll-Like/biossíntese , Animais , Regulação da Expressão Gênica , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Degeneração Macular/genética , Degeneração Macular/patologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Pirróis/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 1 Toll-Like/genética , Receptor 2 Toll-Like/genética , Receptor 6 Toll-Like/biossíntese , Receptor 6 Toll-Like/genética
11.
Eur J Obstet Gynecol Reprod Biol ; 171(1): 12-7, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24125907

RESUMO

OBJECTIVES: To determine whether histologic chorioamnionitis is associated with changes in gene expression of TLR-1, -2, -4 and -6, and to describe the localization of these receptors in fetal membranes. STUDY DESIGN: A total of 135 amniochorion membranes with or without histologic chorioamnionitis from preterm or term deliveries were included. Fragments of membranes were submitted to total RNA extraction. RNA was reverse transcribed and the quantification of TLRs expression measured by real time PCR. RESULTS: All amniochorion membranes expressed TLR-1 and TLR-4, whereas 99.1% of membranes expressed TLR-2 and 77.4% expressed TLR-6. TLR-1 and TLR-2 expressions were significantly higher in membranes with histologic chorioamnionitis as compared to membranes without chorioamnionitis in preterm pregnancies (p=0.003 and p<0.001, respectively). Among the membranes of term pregnancies there were no differences in the expressions of such receptors regardless of inflammatory status. Regarding TLR-4 and TLR-6 expression, there was no difference among membranes with or without histologic chorioamnionitis, regardless gestational age at delivery. TLR-1, TLR-2, TLR-4 and TLR-6 expressions were observed in amniotic epithelial, chorionic and decidual cells. CONCLUSION: Amniochorion membranes express TLR-1, TLR-2, TLR-4 and TLR-6 and increased expression of TLR-1 and TLR-2 is related to the presence of histologic chorioamnionitis in preterm pregnancies. This study provides further evidence that amniochorion membranes act as a mechanical barrier to microorganisms and as components of the innate immune system.


Assuntos
Corioamnionite/genética , Receptores Toll-Like/biossíntese , Adulto , Âmnio/metabolismo , Corioamnionite/metabolismo , Córion/metabolismo , Estudos Transversais , Membranas Extraembrionárias/metabolismo , Feminino , Expressão Gênica , Humanos , Trabalho de Parto Prematuro/metabolismo , Gravidez , Nascimento Prematuro/fisiopatologia , Receptor 1 Toll-Like/biossíntese , Receptor 2 Toll-Like/biossíntese , Receptor 4 Toll-Like/biossíntese , Receptor 6 Toll-Like/biossíntese
12.
Cytotherapy ; 15(4): 423-33, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23352460

RESUMO

BACKGROUND AIMS: Toll-like receptors (TLRs) play an important role in innate and adaptive immunity by recognizing pathogen-associated molecular patterns (PAMPs). METHODS: In the present study, we investigated the expression and role of TLRs on human umbilical cord mesenchymal stromal cells (UC-MSCs). The proliferation, differentiation and immunoregulatory activity of UC-MSCs primed with or without TLR ligands were determined. RESULTS: At the RNA level, the expression of TLR2, 4, 6 and 9 was relatively higher than that of other TLRs. However, TLR3 and TLR4 expression were relatively higher at the protein level. UC-MSCs expressed functional TLRs by nuclear factor-κB activation and cytokine expression assay. Poly-inosinic acid:cytidylic acid [Poly(I:C)] stimulation inhibited the proliferation of UC-MSCs, but the ligand of other TLRs had no significant effect. Poly(I:C) stimulation enhanced the adipogenic differentiation capability of UC-MSCs, but lipopolysaccharide inhibited the adipogenic differentiation. Poly(I:C) and CpG-oligonucleotide promoted the immunosuppressive potentiality of UC-MSCs, accompanied with the phosphorylation of interferon regulatory factor 3 (IRF3) and increased expression of indoleamine 2,3-dioxygenase and interferon ß, whereas activation of other TLR ligands (synthetic analog fibroblast-stimulating lipopeptide-1 and lipopolysaccharide) failed to affect the immunoregulatory activity of UC-MSCs. CONCLUSIONS: Taken together, our data demonstrated that TLR activation influenced the function of UC-MSCs, which might have important implications in future efforts to explore the clinical potentials of UC-MSCs.


Assuntos
Células-Tronco Mesenquimais/citologia , Receptores Toll-Like/metabolismo , Cordão Umbilical/citologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/biossíntese , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/biossíntese , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Fosforilação , Poli I-C/farmacologia , Receptor 2 Toll-Like/biossíntese , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/biossíntese , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/biossíntese , Receptor 4 Toll-Like/metabolismo , Receptor 6 Toll-Like/biossíntese , Receptor 6 Toll-Like/metabolismo , Receptor Toll-Like 9/biossíntese , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/biossíntese
13.
Nutr Hosp ; 27(4): 1196-203, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23165562

RESUMO

INTRODUCTION: Pattern-recognition receptors (PRRs), which include Toll-like Receptor (TLRs) and Nacht leucine-rich repeat proteins (NLRP/NALPs), are molecules of innate immunity able to recognize a wide variety of ligands present in microorganisms and human tissues. Adipocytes (fat cells) may play an important role in the physiological regulation of their own immune responses via TLRs. During obesity, the inflammatory pathway is triggered and insulin responsiveness is altered in fat tissue as a result of TLR4 activation by dietary lipids. OBJECTIVE: Here, we investigate if other PRR family members could also participate in the inflammatory processes in the adipose tissue of obese mice. METHODS: The mRNA expression of TLRs, the NLRP3-inflammasome (NLRP3, ASC, caspase-1 and IL-lbeta), IL-6, and TNFα in the hepatic and adipose tissues of mice fed with a high fat diet (HFD) were studied by RT-PCR. RESULTS: Adipose tissue from mice fed with a HFD had decreased expression levels of TLR2, TLR6 and TLR7 and was similar to the pattern in hepatic tissue HFD mice. IL-6 and TNF-α expression also were decreased in adipose tissue of mice fed with a HFD. NLRP3-inflammasome expression was not modified. CONCLUSION: These results suggest that the low expression of TLR2, and TLR6 in the mice fed with a HFD could be regulating the inflammation induced by the diet employed in this study.


Assuntos
Tecido Adiposo/metabolismo , Dieta Hiperlipídica/efeitos adversos , Fígado/metabolismo , RNA Mensageiro/biossíntese , Receptor 2 Toll-Like/biossíntese , Receptor 6 Toll-Like/biossíntese , Animais , Peso Corporal/fisiologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Masculino , Camundongos , Camundongos Obesos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Reação em Cadeia da Polimerase em Tempo Real
14.
Arch Immunol Ther Exp (Warsz) ; 60(5): 373-82, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22915067

RESUMO

Zymosan-induced peritonitis represents a well-described model of acute inflammation. The binding of zymosan with its specific Toll-like receptors (TLR2 and TLR6) on leukocytes initiates activation and phosphorylation of nuclear factor (NF)-κB, which leads to accumulation of NF-κB p65 subunits in the nucleus and subsequently up-regulation of the proinflammatory cytokine genes expression. Intraperitoneal co-administration of zymosan and morphine significantly inhibits peritonitis in several strains of mice by decreasing the influx of exudatory cells; however, mechanisms of this action still remain unclear. We aimed to verify the effects of morphine on NF-κB and TLRs expression at messenger RNA and protein levels during the early stages of zymosan-induced peritonitis. Peritonitis was induced by a single injection of zymosan A or zymosan supplemented with morphine in Swiss mice. At selected time points, after stimulation, peritoneal leukocytes were harvested. The TLRs and NF-κB expression was assessed by real-time PCR and flow cytometry. In comparison with the mice injected with zymosan only, morphine co-injection significantly decreased the expression of phospho-NF-κB and TLR2 in all investigated immunocompetent cells as well as up-regulated the levels of nitric oxide (NO) in peritoneal fluid. Moreover, supplementation of zymosan with morphine altered the TLR, NF-κB and some proinflammatory cytokines (keratinocyte-derived chemokine, tumor necrosis factor-α) gene expression during ongoing inflammation. We may postulate that after morphine stimulation peritoneal leukocytes recognize less effectively zymosan antigens because of impaired TLRs expression. The lower TLR expression attenuates TLR-mediated signal transduction, which prevents NF-κB activation. Additionally, during zymosan-induced peritonitis, morphine may modulate the NF-κB expression, at least partially, by an up-regulated release of NO, as suggested by others.


Assuntos
Leucócitos/metabolismo , Morfina/farmacologia , NF-kappa B/biossíntese , Peritônio/citologia , Receptor 2 Toll-Like/biossíntese , Receptor 6 Toll-Like/biossíntese , Analgésicos Opioides/farmacologia , Animais , Linhagem Celular , Citometria de Fluxo/métodos , Queratinócitos/citologia , Masculino , Camundongos , Nitratos/química , Óxido Nítrico/metabolismo , Nitritos/química , Peritonite/imunologia , Fosforilação , RNA Mensageiro/metabolismo , Fatores de Tempo
16.
Am J Reprod Immunol ; 66(3): 209-22, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21385270

RESUMO

PROBLEM: Intrauterine bacterial infection during pregnancy may lead to adverse outcome. The objective of this study was to assess whether peptidoglycan (PGN) derived from Gram-positive bacteria induces trophoblast stem (TS) cell death or alters TS cell cytokine production. METHOD OF STUDY: Toll-like receptor (TLR) transcript expression was assessed by RT-PCR. Protein expression was determined by confocal microscopy or flow cytometry. 7-Aminoactinomycin D (7-AAD) staining was used to assess TS cell death. Morphological features of cell death were evaluated by transmission electron microscopy. The presence of cleaved caspase-3 and high mobility group box 1 (HMGB1) protein was examined by Western blot. Cytokine levels in cell supernatants were determined using a mouse cytokine 23-plex panel. RESULTS: Toll-like receptor 2 and TLR4 protein was expressed from the 1-cell stage through the blastocyst stage of murine embryo development. Murine TS cells expressed TLR2 and TLR6 but not TLR1 or TLR4 RNA. Only TLR2 protein was detected at the plasma membrane of TS cells. PGN induced TS cell death by a caspase-3-independent mechanism. The cell death pathway induced by PGN was morphologically consistent with necrosis. Finally, PGN induced HMGB1 release and increased MIP-1ß secretion while inhibiting the constitutive release of RANTES. CONCLUSION: Peptidoglycan-induced TS cell necrosis and the subsequent release of HMGB1 and MIP-1ß may regulate an infection-induced inflammatory response at the maternal-fetal interface and thus may play a role in the pathogenesis of infection-associated pregnancy complications.


Assuntos
Citocinas/imunologia , Peptidoglicano/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologia , Trofoblastos/efeitos dos fármacos , Trofoblastos/imunologia , Animais , Caspase 3/imunologia , Morte Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL5/biossíntese , Quimiocina CCL5/imunologia , Citocinas/biossíntese , Feminino , Masculino , Camundongos , Necrose/induzido quimicamente , Necrose/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Nucleares/imunologia , Gravidez , Receptor 1 Toll-Like/biossíntese , Receptor 1 Toll-Like/imunologia , Receptor 2 Toll-Like/biossíntese , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/biossíntese , Receptor 4 Toll-Like/imunologia , Receptor 6 Toll-Like/biossíntese , Receptor 6 Toll-Like/imunologia
17.
Crit Care ; 14(4): R160, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20731882

RESUMO

INTRODUCTION: Systemic inflammation in sepsis is initiated by interactions between pathogen molecular motifs and specific host receptors, especially toll-like receptors (TLRs). Flagellin is the main flagellar protein of motile microorganisms and is the ligand of TLR5. The distribution of TLR5 and the actions of flagellin at the systemic level have not been established. Therefore, we determined TLR5 expression and the ability of flagellin to trigger prototypical innate immune responses and apoptosis in major organs from mice. METHODS: Male Balb/C mice (n = 80) were injected intravenously with 1-5 µg recombinant Salmonella flagellin. Plasma and organ samples were obtained after 0.5 to 6 h, for molecular investigations. The expression of TLR5, the activation state of nuclear factor kappa B (NFκB) and mitogen-activated protein kinases (MAPKs) [extracellular related kinase (ERK) and c-jun-NH2 terminal kinase (JNK)], the production of cytokines [tumor necrosis alpha (TNFα), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), macrophage inhibitory protein-2 (MIP-2) and soluble triggering receptor expressed on myeloid cells (TREM-1)], and the apoptotic cleavage of caspase-3 and its substrate Poly(ADP-ribose) polymerase (PARP) were determined in lung, liver, gut and kidney at different time-points. The time-course of plasma cytokines was evaluated up to 6 h after flagellin. RESULTS: TLR5 mRNA and protein were constitutively expressed in all organs. In these organs, flagellin elicited a robust activation of NFκB and MAPKs, and induced significant production of the different cytokines evaluated, with slight interorgan variations. Plasma TNFα, IL-6 and MIP-2 disclosed a transient peak, whereas IL-1ß and soluble TREM-1 steadily increased over 6 h. Flagellin also triggered a marked cleavage of caspase-3 and PARP in the intestine, pointing to its ability to promote significant apoptosis in this organ. CONCLUSIONS: Bacterial flagellin elicits prototypical innate immune responses in mice, leading to the release of multiple pro-inflammatory cytokines in the lung, small intestine, liver and kidney, and also activates apoptotic signalling in the gut. Therefore, this bacterial protein may represent a critical mediator of systemic inflammation and intestinal barrier failure in sepsis due to flagellated micro-organisms.


Assuntos
Apoptose/efeitos dos fármacos , Flagelina/farmacologia , Imunidade Inata/efeitos dos fármacos , Síndrome de Resposta Inflamatória Sistêmica/induzido quimicamente , Animais , Caspase 3/metabolismo , Quimiocina CXCL2/biossíntese , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-1beta/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/biossíntese , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Receptores Imunológicos/biossíntese , Receptor 6 Toll-Like/biossíntese , Receptor Gatilho 1 Expresso em Células Mieloides , Fator de Necrose Tumoral alfa/biossíntese
18.
J Immunol ; 185(6): 3708-17, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20713893

RESUMO

Lipoteichoic acid (LTA), a ubiquitous cell wall component of Gram-positive bacteria, represents a potent immunostimulatory molecule. Because LTA of a mutant Staphylococcus aureus strain lacking lipoproteins (Deltalgt-LTA) has been described to be immunobiologically inactive despite a lack of ascertained structural differences to wild-type LTA (wt-LTA), we investigated the functional requirements for the recognition of Deltalgt-LTA by human peripheral blood cells. In this study, we demonstrate that Deltalgt-LTA-induced immune activation critically depends on the immobilization of LTA and the presence of human serum components, which, to a lesser degree, was also observed for wt-LTA. Under experimental conditions allowing LTA-mediated stimulation, we found no differences between the immunostimulatory capacity of Deltalgt-LTA and wt-LTA in human blood cells, arguing for a limited contribution of possible lipoprotein contaminants to wt-LTA-mediated immune activation. In contrast to human blood cells, TLR2-transfected human embryonic kidney 293 cells could be activated only by wt-LTA, whereas activation of these cells by Deltalgt-LTA required the additional expression of TLR6 and CD14, suggesting that activation of human embryonic kidney 293 cells expressing solely TLR2 is probably mediated by residual lipoproteins in wt-LTA. Notably, in human peripheral blood, LTA-specific IgG Abs are essential for Deltalgt-LTA-mediated immune activation and appear to induce the phagocytic uptake of Deltalgt-LTA via engagement of FcgammaRII. In this study, we have elucidated a novel mechanism of LTA-induced cytokine induction in human peripheral blood cells that involves uptake of LTA and subsequent intracellular recognition driven by TLR2, TLR6, and CD14.


Assuntos
Adjuvantes Imunológicos/sangue , Lipopolissacarídeos/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Ácidos Teicoicos/metabolismo , Receptor 2 Toll-Like/metabolismo , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/fisiologia , Reações Antígeno-Anticorpo , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Citocinas/biossíntese , Citocinas/sangue , Citocinas/metabolismo , Humanos , Imunoglobulina G/metabolismo , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Líquido Intracelular/microbiologia , Receptores de Lipopolissacarídeos/biossíntese , Receptores de Lipopolissacarídeos/fisiologia , Lipopolissacarídeos/sangue , Lipopolissacarídeos/imunologia , Lipoproteínas/deficiência , Lipoproteínas/genética , Proteínas Opsonizantes/metabolismo , Receptores de IgG/fisiologia , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/sangue , Ácidos Teicoicos/imunologia , Receptor 2 Toll-Like/sangue , Receptor 2 Toll-Like/fisiologia , Receptor 6 Toll-Like/biossíntese , Receptor 6 Toll-Like/fisiologia
19.
Immunology ; 128(4): 484-99, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19930041

RESUMO

We have characterized a Leishmania protein belonging to the silent information regulator 2 (SIR2) family [SIR2 related protein 1 (SIR2RP1)] that might play an immunoregulatory role during infection through its capacity to trigger B-cell effector functions. We report here that SIR2RP1 leads to the proliferation of activated B cells, causing increased expression of major histocompatibility complex (MHC) II and the costimulatory molecules CD40 and CD86, which are critical ligands for T-cell cross-talk during the development of adaptive immune responses. In contrast, B cells isolated from Toll-like receptor 2 (TLR2) knockout mice were unable to respond to the SIR2RP1 stimulus. Similarly, SIR2RP1 induced the maturation of dendritic cells (DCs) in a TLR2-dependent manner with the secretion of pro-inflammatory cytokines [interleukin (IL)-12 and tumour necrosis factor (TNF)-alpha] and enhanced the costimulatory properties of DCs. Nevertheless, immunization assays demonstrated that TLR2-deficient mice were able to mount a specific humoral response to SIR2RP1. Interestingly, further investigations showed that macrophages were activated by SIR2RP1 even in the absence of TLR2. Therefore, a different type of interplay between SIR2RP1 and the major antigen-presenting cells in vivo could explain the immune response observed in TLR2-deficient mice. Together, these results demonstrate that TLR2 signalling contributes to SIR2RP1 recognition by innate immune host cells.


Assuntos
Leishmania infantum/imunologia , Proteínas de Protozoários/imunologia , Sirtuína 1/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Linfócitos B/imunologia , Antígeno B7-2/biossíntese , Antígenos CD40/biossíntese , Proliferação de Células , Células Cultivadas , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II , Imunidade Inata , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/biossíntese , NF-kappa B/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Receptor 6 Toll-Like/biossíntese , Regulação para Cima/imunologia
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