RESUMEN

Toll-like receptors are pattern recognition receptors that bridge the innate and adaptive immune responses and are critical for host defense. Most studies of Toll-like receptors have focused upon their roles in myeloid cells. B lymphocytes express most Toll-like receptors and are responsive to Toll-like receptor ligands, yet Toll-like receptor-mediated signaling in B cells is relatively understudied. This is an important knowledge gap, as Toll-like receptor functions can be cell type specific. In striking contrast to myeloid cells, TRAF3 inhibits TLR-mediated functions in B cells. TRAF3-deficient B cells display enhanced IRF3 and NFκB activation, cytokine production, immunoglobulin isotype switching, and antibody production in response to Toll-like receptors 3, 4, 7, and 9. Here, we address the question of how TRAF3 impacts initial B-cell Toll-like receptor signals to regulate downstream activation. We found that TRAF3 in B cells associated with proximal Toll-like receptor 4 and 7 signaling proteins, including MyD88, TRAF6, and the tyrosine kinase Syk. In the absence of TRAF3, TRAF6 showed a greater association with several Toll-like receptor signaling proteins, suggesting that TRAF3 may inhibit TRAF6 access to Toll-like receptor signaling complexes and thus early Toll-like receptor signaling. In addition, our results highlight a key role for Syk in Toll-like receptor signaling in B cells. In the absence of TRAF3, Syk activation was enhanced in response to ligands for Toll-like receptors 4 and 7, and Syk inhibition reduced downstream Toll-like receptor-mediated NFκB activation and proinflammatory cytokine production. This study reveals multiple mechanisms by which TRAF3 serves as a key negative regulator of early Toll-like receptor signaling events in B cells.

Asunto(s)

Linfocitos B , Transducción de Señal , Factor 3 Asociado a Receptor de TNF , Factor 6 Asociado a Receptor de TNF , Receptores Toll-Like , Factor 3 Asociado a Receptor de TNF/metabolismo , Factor 3 Asociado a Receptor de TNF/genética , Linfocitos B/inmunología , Linfocitos B/metabolismo , Animales , Receptores Toll-Like/metabolismo , Ratones , Factor 6 Asociado a Receptor de TNF/metabolismo , Quinasa Syk/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Ratones Noqueados , Ratones Endogámicos C57BL , FN-kappa B/metabolismo

RESUMEN

ObjectiveMolecular docking and animal experiments were employed to explore the protective effect and mechanism of Da Chengqitang （DCQD） on intestinal barrier in septic mice. MethodText mining method was used to screen the active ingredients in DCQD. AutoDock Tools and Discovery Studio were used to study the interactions of active components with the core target proteins ［claudin-1， tumor necrosis factor （TNF）-α， interleukin （IL）-6， endogenous antimicrobial peptide mCRAMP， Toll-like receptor 4 （TLR4）， and myeloid differentiation primary response gene 88 （MyD88）］ in sepsis. Fifty C57BL/6 mice were randomized into sham， model， low- and high-dose （4 g∙kg-1 and 8 g∙kg-1） DCQD， and ulinastatin groups （n=10）. Before， during， and after the day of modeling surgery， each group was administrated with corresponding drugs. The mice in other groups except the model group were subjected to modeling by cecal ligation and puncture. Enzyme-linked immunosorbent assay （ELISA） was used measure the serum level of D-lactic acid to assess intestinal mucosa permeability. Hematoxylin-eosin staining was employed to observe the histopathological changes in the ileum and assess the intestinal mucosal damage and inflammatory infiltration. Western blotting was employed to determine the expression levels of tight junction proteins claudin-1 and occludin in the ileal tissue， which were indicative of the bowel barrier function. The TNF-α and IL-6 levels were measured by ELISA to assess the intestinal inflammation. The expression of mCRAMP in the ileal tissue was observed by immunohistochemistry. The mRNA levels of mCRAMP， TLR4， and MyD88 in mouse ileal tissue were determined by Real-time polymerase chain reaction， on the basis of which the mechanism of DCQD in protecting the intestinal barrier of septic mice was explored. ResultMolecular docking results showed that most of the 10 active ingredients of DCQD that were screened out by text mining could bind to sepsis targets by van der Waals force， hydrogen bonding， and other conjugated systems. The results of animal experiments showed that compared with the model group， low- or high-dose DCQD lowered the D-lactic acid level in the serum （P<0.01）， alleviated damage to the ileal tissue and mucosal edema， protected the small intestine villus integrity， reduced inflammatory cell infiltration， promoted the expression of claudin-1 （P<0.01）， lowered the IL-6 level （P<0.01）， up-regulated the mRNA and protein levels of mCRAMP （P<0.01）， and down-regulated the mRNA and protein levels of TLR4 and MyD88 （P<0.01） in the ileal tissue. In addition， high-dose DCQD lowered the TNF-α level and promoted the expression of occludin in the ileum tissue （P<0.01）， and low-dose DCQD up-regulated the protein level of occludin in the ileum tissue （P<0.05）. ConclusionDCQD has a protective effect on intestinal barrier in septic mice. It can reduce intestinal inflammation， repair intestinal mucosal damage， improve the tight junction protein level， and reduce intestinal mucosal permeability by up-regulating the mRNA and protein levels of mCRAMP and the down-regulating the expression of genes in the TLR4/MyD88 pathway.

RESUMEN

The placenta-specific 8 (PLAC8) gene, also known as ONZIN or C15, codes for a cysteine-rich peptide originally identified in mouse placental tissue and subsequently identified in a variety of epithelial tissues and immune cells. PLAC8 is also expressed in birds, such as ducks, where its functional roles remain unknown. Here, we aimed to determine the mRNA and protein expression profiles and the functional role of duck PLAC8 during the infection of duck hepatitis A virus type 1 (DHAV-1). We found that the duck PLAC8 is also a cysteine-rich polypeptide composed of 114 amino acid residues, with no signal peptide. Duck PLAC8 is highly expressed in the immune organs of young cherry valley ducks, including the thymus, bursa fabricius, and spleen. However, it has negligible expression level in liver, brain, kidney, and heart. Additionally, PLAC8 expression was considerably induced after DHAV-1 infection both in vitro and in vivo, especially in the immune organs of ducklings. This tissue expression distribution and induction upon infection suggest that PLAC8 might play a critical role in innate immunity. Our data showed that PLAC8 significantly suppressed the expression of Toll-like receptor 7 (TLR7), leading to decreased expression of downstream signaling molecules including myeloid differentiation primary response gene 88 (MyD88) and nuclear factor kappa-B (NF-κB). This ultimately resulted in low levels of type I interferon and interleukin 6 (IL-6). Additionally, PLAC8 positively regulated DHAV-1 replication levels. RNAi against PLAC8 in duck embryo fibroblasts considerably inhibited DHAV-1 propagation, while PLAC8 overexpression significantly facilitated DHAV-1 replication.

Asunto(s)

Patos , Virus de la Hepatitis del Pato , Hepatitis Viral Animal , Infecciones por Picornaviridae , Enfermedades de las Aves de Corral , Animales , Femenino , Ratones , Embarazo , Cisteína , Patos/genética , Patos/virología , Factor 88 de Diferenciación Mieloide/genética , Infecciones por Picornaviridae/veterinaria , Placenta , Transducción de Señal , Receptor Toll-Like 7/genética , Células HEK293 , Humanos

RESUMEN

OBJECTIVE: Long non-coding RNAs (lncRNAs) play critically in the pathogenesis of myocardial ischemia-reperfusion (I/R) injury. Thus, it was proposed to investigate the mechanism of LINC00461 in the disease through mediating microRNA-185-3p (miR-185-3p)/myeloid differentiation primary response gene 88 (Myd88) axis. METHODS: miR-185-3p, LINC00461 and Myd88 expression in mice with I/R injury was measured. Mice with I/R injury were injected with the gene expression-modified vectors, after which cardiac function, hemodynamics, myocardial enzyme, oxidative stress, and cardiomyocyte apoptosis were analyzed. RESULTS: I/R mice showed LINC00461 and Myd88 up-regulation and miR-185-3p down-regulation. Down-regulating LINC00461 or up-regulating miR-185-3p recovered cardiac function, reduced myocardial enzyme levels, and attenuated oxidative stress and cardiomyocyte apoptosis in mice with I/R. miR-185-3p overexpression rescued the promoting effect of LINC00461 upregulation on myocardial injury in I/R mice. CONCLUSION: LINC00461 knockdown attenuates myocardial I/R injury via elevating miR-185-3p expression to suppress Myd88 expression.

Asunto(s)

MicroARNs , Daño por Reperfusión Miocárdica , ARN Largo no Codificante , Daño por Reperfusión , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis/genética , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Daño por Reperfusión/metabolismo

RESUMEN

Toll-like receptors (TLRs) are essential for the protection of the host from pathogen infections by initiating the integration of contextual cues to regulate inflammation and immunity. However, without tightly controlled immune responses, the host will be subjected to detrimental outcomes. Therefore, it is important to balance the positive and negative regulations of TLRs to eliminate pathogen infection, yet avert harmful immunological consequences. This study revealed a distinct mechanism underlying the regulation of the TLR network. The expression of sex-determining region Y-box 4 (Sox4) is induced by virus infection in viral infected patients and cultured cells, which subsequently represses the TLR signaling network to facilitate viral replication at multiple levels by a distinct mechanism. Briefly, Sox4 inhibits the production of myeloid differentiation primary response gene 88 (MyD88) and most of the TLRs by binding to their promoters to attenuate gene transcription. In addition, Sox4 blocks the activities of the TLR/MyD88/IRAK4/TAK1 and TLR/TRIF/TRAF3/TBK1 pathways by repressing their key components. Moreover, Sox4 represses the activation of the nuclear factor kappa-B (NF-κB) through interacting with IKKα/α, and attenuates NF-kB and IFN regulatory factors 3/7 (IRF3/7) abundances by promoting protein degradation. All these contributed to the down-regulation of interferons (IFNs) and IFN-stimulated gene (ISG) expression, leading to facilitate the viral replications. Therefore, we reveal a distinct mechanism by which viral pathogens evade host innate immunity and discover a key regulator in host defense.

Asunto(s)

Inmunidad Innata/genética , Factores de Transcripción SOXC/genética , Factores de Transcripción SOXC/inmunología , Transducción de Señal/inmunología , Receptores Toll-Like/metabolismo , Virus/inmunología , Enterovirus Humano A/inmunología , Enterovirus Humano A/patogenicidad , Células Hep G2 , Humanos , Inmunidad Innata/inmunología , Virus de la Influenza A/inmunología , Virus de la Influenza A/patogenicidad , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Factor 88 de Diferenciación Mieloide/antagonistas & inhibidores , Factor 88 de Diferenciación Mieloide/inmunología , Transducción de Señal/genética , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Replicación Viral , Virus/patogenicidad

RESUMEN

OBJECTIVE: microRNAs (miRNAs) play critical roles in embryogenesis, cell differentiation and the pathogenesis of several human diseases, including systemic lupus erythematosus (SLE). Toll-like receptors (TLRs) are also known to exert crucial functions in the immune response activation occurring in the pathogenesis of autoimmune diseases like SLE. Herein, the current study aimed to explore the potential role of miR-152-3p in TLR-mediated inflammatory response in SLE. METHODS: We determined the miR-152-3p expression profiles in CD4+ T cells and peripheral blood mononuclear cells (PBMCs) harvested from patients with SLE and healthy controls, and analyzed the correlation between miR-152-3p expression and clinicopathological parameters. CD70 and CD40L expression patterns in CD4+ T cells were assessed by RT-qPCR and flow cytometry. ChIP was adopted to determine the enrichment of DNA methyltransferase 1 (DNMT1) in the promoter region of myeloid differentiation factor 88 (MyD88). RESULTS: The obtained findings revealed that miR-152-3p was highly-expressed in CD4+ T cells and PBMCs of patients with SLE, and this high expression was associated with facial erythema, joint pain, double-stranded DNA, and IgG antibody. DNMT1 could be enriched in the MyD88 promoter, and miR-152-3p inhibited the methylation of MyD88 by targeting DNMT1. We also found that silencing miR-152-3p inhibited MyD88 expression not only to repress the autoreactivity of CD4+ T cells and but also to restrain their cellular inflammation, which were also validated in vivo. CONCLUSION: Our study suggests that miR-152-3p promotes TLR-mediated inflammatory response in CD4+ T cells by regulating the DNMT1/MyD88 signaling pathway, which highlights novel anti-inflammatory target for SLE treatment.

Asunto(s)

Lupus Eritematoso Sistémico/genética , MicroARNs , Adolescente , Adulto , Anciano , Animales , Anticuerpos Antiidiotipos/sangre , Anticuerpos Antinucleares/sangre , Artralgia/genética , Artralgia/inmunología , Niño , Citocinas/inmunología , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/inmunología , Desmetilación , Eritema/genética , Eritema/inmunología , Cara , Femenino , Humanos , Inflamación/genética , Inflamación/inmunología , Leucocitos Mononucleares/inmunología , Lupus Eritematoso Sistémico/inmunología , Masculino , Ratones Endogámicos MRL lpr , Persona de Mediana Edad , Factor 88 de Diferenciación Mieloide/inmunología , Receptores Toll-Like/inmunología , Adulto Joven

RESUMEN

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. The activation of the toll-like receptor 4/myeloid differentiation primary response gene 88/nuclear factor-κB (TLR4/MyD88/NF-κB) pathway contributes to the development and progression of HCC. The ubiquitin-proteasome system regulates TLR4 expression. However, whether ubiquitin specific peptidase 13 (USP13) stabilizes TLR4 and facilitates HCC progression remains unclear. Here, quantitative real-time PCR (qRT-PCR) and immunohistochemistry analysis revealed that USP13 expression in HCC tissues was higher than in non-tumor liver tissues. Moreover, the elevated expression of USP13 was detected in HCC cells (SK-HEP-1, HepG2, Huh7, and Hep3B) compared to LO2 cells. Interestingly, the positive staining of USP13 was closely correlated with tumor size ≥ 5 cm and advanced tumor stage and conferred to significantly lower survival of HCC patients. Next, USP13 knockdown prominently reduced the proliferation, epithelial-mesenchymal transition (EMT), migration, and invasion of Hep3B and Huh7 cells, while USP13 overexpression enhanced these biological behaviors of HepG2 and LO2 cells. The silencing of USP13 significantly restrained the growth and lung metastasis of HCC cells in vivo. Mechanistically, the USP13 depletion markedly inhibited the TLR4/MyD88/NF-κB pathway in HCC cells. USP13 interacted with TLR4 and inhibited the ubiquitin-mediated degradation of TLR4. Significantly, TLR4 re-expression remarkably reversed the effects of USP13 knockdown on HCC cells. USP13 expression was markedly upregulated in HCC cells under hypoxia conditions. Notably, USP13 knockdown repressed hypoxia-induced activation of the TLR4/MyD88/NF-κB pathway in HCC cells. In conclusion, our study uncovered that hypoxia-induced USP13 facilitated HCC progression via enhancing TLR4 deubiquitination and subsequently activating the TLR4/MyD88/NF-κB pathway.

RESUMEN

Although a robust inflammatory response is needed to combat infection, this response must ultimately be terminated to prevent chronic inflammation. One mechanism that terminates inflammatory signaling is the production of alternative mRNA splice forms in the Toll-like receptor (TLR) signaling pathway. Whereas most genes in the TLR pathway encode positive mediators of inflammatory signaling, several, including that encoding the MyD88 signaling adaptor, also produce alternative spliced mRNA isoforms that encode dominant-negative inhibitors of the response. Production of these negatively acting alternatively spliced isoforms is induced by stimulation with the TLR4 agonist lipopolysaccharide (LPS); thus, this alternative pre-mRNA splicing represents a negative feedback loop that terminates TLR signaling and prevents chronic inflammation. In the current study, we investigated the mechanisms regulating the LPS-induced alternative pre-mRNA splicing of the MyD88 transcript in murine macrophages. We found that 1) the induction of the alternatively spliced MyD88 form is due to alternative pre-mRNA splicing and not caused by another RNA regulatory mechanism, 2) MyD88 splicing is regulated by both the MyD88- and TRIF-dependent arms of the TLR signaling pathway, 3) MyD88 splicing is regulated by the NF-κB transcription factor, and 4) NF-κB likely regulates MyD88 alternative pre-mRNA splicing per se rather than regulating splicing indirectly by altering MyD88 transcription. We conclude that alternative splicing of MyD88 may provide a sensitive mechanism that ensures robust termination of inflammation for tissue repair and restoration of normal tissue homeostasis once an infection is controlled.

Asunto(s)

Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Factor 88 de Diferenciación Mieloide/genética , FN-kappa B/metabolismo , Precursores del ARN/genética , Empalme del ARN/efectos de los fármacos , Empalme Alternativo/efectos de los fármacos , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Macrófagos/citología , Ratones , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Transcripción Genética/efectos de los fármacos

RESUMEN

Dental caries is a chronic, infectious, and destructive disease that allows bacteria to break into the dental pulp tissue. As caries-related bacteria invade the human dentinal tubules, odontoblasts are the first line of dental pulp that trigger the initial inflammatory and immune responses. DNA methylation is a key epigenetic modification that plays a fundamental role in gene transcription, and its role in inflammation-related diseases has recently attracted attention. However, whether DNA methylation regulates the inflammatory response of human odontoblasts is still unknown. In the present study, we investigated the expression of DNA methyltransferase (DNMT)-1 in lipoteichoic acid (LTA)-stimulated human odontoblast-like cells (hOBs) and found that DNMT1 expression showed a decline that is contrary to the transcription of inflammatory cytokines. Knockdown of the DNMT1 gene increased the expression of several cytokines, including IL-6 and IL-8, in the LTA-induced inflammatory response. DNMT1 knockdown increased the phosphorylation of IKKα/ß, IκBα, and p65 in the NF-κB pathway and the phosphorylation of p38 and ERK in the MAPK pathway; however, only the NF-κB pathway inhibitor PDTC suppressed both IL-6 and IL-8 expression, whereas inhibitors of the MAPK pathway (U0126, SB2035580, and SP600125) did not. Furthermore, DNMT1 knockdown upregulated the expression of MyD88 and TRAF6 but only attenuated the MyD88 gene promoter methylation in LTA-treated hOBs. Taken together, these results demonstrated that DNMT1 depletion caused hypomethylation and upregulation of MyD88, which resulted in activation of the NF-κB pathway and the subsequent release of LTA-induced inflammatory cytokines in hOBs. This study emphasizes the critical role of DNA methylation in the immune defense of odontoblasts when dental pulp reacted to caries.

Asunto(s)

ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN , Caries Dental/inmunología , Factor 88 de Diferenciación Mieloide/genética , Odontoblastos/inmunología , Adolescente , Adulto , Citocinas/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , Humanos , Inflamación/inducido químicamente , Inflamación/inmunología , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas , FN-kappa B/metabolismo , Odontoblastos/efectos de los fármacos , Fosforilación , Transducción de Señal , Ácidos Teicoicos/farmacología

RESUMEN

Toll-like receptors (TLRs) form part of the host innate immune system, in which they act as sensors of microbial and endogenous danger signals. Upon TLR activation, the intracellular Toll/interleukin-1 receptor domains of TLR dimers initiate oligomerization of a multiprotein signaling platform comprising myeloid differentiation primary response 88 (MyD88) and members of the interleukin-1 receptor-associated kinase (IRAK) family. Formation of this myddosome complex initiates signal transduction pathways, leading to the activation of transcription factors and the production of inflammatory cytokines. To date, little is known about the assembly and disassembly of the myddosome and about the mechanisms by which these complexes mediate multiple downstream signaling pathways. Here, we isolated myddosome complexes from whole-cell lysates of TLR-activated primary mouse macrophages and from IRAK reporter macrophages to examine the kinetics of myddosome assembly and disassembly. Using a selective inhibitor of IRAK4's kinase activity, we found that whereas TLR cytokine responses were ablated, myddosome formation was stabilized in the absence of IRAK4's kinase activity. Of note, IRAK4 inhibition had only a minimal effect on NF-κB and mitogen-activated protein kinase (MAPK) signaling. In summary, our results indicate that IRAK4 has a critical scaffold function in myddosome formation and that its kinase activity is dispensable for myddosome assembly and activation of the NF-κB and MAPK pathways but is essential for MyD88-dependent production of inflammatory cytokines. Our findings suggest that the scaffold function of IRAK4 may be an attractive target for treating inflammatory and autoimmune diseases.

Asunto(s)

Quinasas Asociadas a Receptores de Interleucina-1/genética , Factor 88 de Diferenciación Mieloide/genética , Receptores Toll-Like/genética , Animales , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/química , Macrófagos/química , Macrófagos/metabolismo , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Factor 88 de Diferenciación Mieloide/química , FN-kappa B/genética , Fosforilación , Transducción de Señal , Receptores Toll-Like/química

RESUMEN

Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a central regulator of innate immune signaling, controlling IL1R and Toll-like receptor (TLR)-mediated responses and containing both scaffolding and kinase activities. Humans deficient in IRAK4 activity have autosomal recessive primary immune deficiency (PID). Here, we characterized the molecular mechanism of dysfunction of two IRAK4 PID variants, G298D and the compound variant R12C (R12C/R391H/T458I). Using these variants and the kinase-inactive D329A variant to delineate the contributions of IRAK4's scaffolding and kinase activities to IL1R signaling, we found that the G298D variant is kinase-inactive and expressed at extremely low levels, acting functionally as a null mutation. The R12C compound variant possessed WT kinase activity, but could not interact with myeloid differentiation primary response 88 (MyD88) and IRAK1, causing impairment of IL-1-induced signaling and cytokine production. Quantitation of IL-1 signaling in IRAK4-deficient cells complemented with either WT or the R12C or D329A variant indicated that the loss of MyD88 interaction had a greater impact on IL-1-induced signaling and cytokine expression than the loss of IRAK4 kinase activity. Importantly, kinase-inactive IRAK4 exhibited a greater association with MyD88 and a weaker association with IRAK1 in IRAK4-deficient cells expressing kinase-inactive IRAK4 and in primary cells treated with a selective IRAK4 inhibitor. Loss of IRAK4 kinase activity only partially inhibited IL-1-induced cytokine and NF-κB signaling. Therefore, the IRAK4-MyD88 scaffolding function is essential for IL-1 signaling, but IRAK4 kinase activity can control IL-1 signal strength by modulating the association of IRAK4, MyD88, and IRAK1.

Asunto(s)

Síndromes de Inmunodeficiencia/genética , Quinasas Asociadas a Receptores de Interleucina-1/química , Quinasas Asociadas a Receptores de Interleucina-1/genética , Interleucina-1/genética , Factor 88 de Diferenciación Mieloide/genética , Línea Celular , Cristalografía por Rayos X , Humanos , Inmunidad Innata/genética , Síndromes de Inmunodeficiencia/inmunología , Síndromes de Inmunodeficiencia/patología , Interleucina-1/química , Quinasas Asociadas a Receptores de Interleucina-1/deficiencia , Mutación , Factor 88 de Diferenciación Mieloide/química , FN-kappa B/genética , Polimorfismo de Nucleótido Simple/genética , Receptores de Interleucina-1/química , Receptores de Interleucina-1/genética , Transducción de Señal

RESUMEN

Engagement of toll-like receptor (TLR)4 ligand, lipopolysaccharide (LPS) with TLR4 in mammals activates two downstream intracellular signaling routes; the myeloid differentiation primary response gene (MyD)88 dependent and independent pathways. However, existence of the later pathway leading to production of type I interferons (IFNs) in avian species has been debated due to conflicting observations. The objective of our study was to investigate whether LPS induces type I IFN production in chicken macrophages leading to antiviral response attributable to type I IFN. We found that LPS elicits type I IFN response dominated by IFN-ß production. We also found that reduction in infectious laryngotracheitis virus (ILTV) replication by LPS-mediated antiviral response is attributable to type I IFNs in addition to nitric oxide (NO). Our findings imply that LPS elicits both MyD88 dependent and independent pathways in chicken macrophages consequently eliciting anti-ILTV response attributable to production of both type I IFNs and NO.

Asunto(s)

Interferón Tipo I/metabolismo , Lipopolisacáridos/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Animales , Pollos , Herpesvirus Gallináceo 1/crecimiento & desarrollo , Herpesvirus Gallináceo 1/inmunología , Macrófagos/inmunología , Óxido Nítrico/metabolismo

RESUMEN

Toll-like receptor (TLR)-10 remains an orphan receptor without well-characterized ligands or functions. Here, we reveal that TLR10 is predominantly localized to endosomes and binds dsRNA in vitro at endosomal pH, suggesting that dsRNA is a ligand of TLR10. Recognition of dsRNA by TLR10 activates recruitment of myeloid differentiation primary response gene 88 for signal transduction and suppression of interferon regulatory factor-7 dependent type I IFN production. We also demonstrate crosstalk between TLR10 and TLR3, as they compete with each other for dsRNA binding. Our results suggest for the first time that dsRNA is a ligand for TLR10 and propose novel dual functions of TLR10 in regulating IFN signaling: first, recognition of dsRNA as a nucleotide-sensing receptor and second, sequestration of dsRNA from TLR3 to inhibit TLR3 signaling in response to dsRNA stimulation.

Asunto(s)

Interferones/metabolismo , ARN Bicatenario/metabolismo , Receptor Toll-Like 10/metabolismo , Receptor Toll-Like 3/metabolismo , Endosomas/metabolismo , Humanos , Transducción de Señal , Células THP-1

RESUMEN

Obesity-induced chronic inflammation is associated with metabolic disease. Results from mouse models utilizing a high-fat diet (HFD) have indicated that an increase in activated macrophages, including CD11c+ adipose tissue macrophages (ATMs), contributes to insulin resistance. Obesity primes myeloid cell production from hematopoietic stem cells (HSCs) and Toll-like receptor 4 (TLR4), and the downstream TIR domain-containing adapter protein-inducing interferon-ß (TRIF)- and MyD88-mediated pathways regulate production of similar myeloid cells after lipopolysaccharide stimulation. However, the role of these pathways in HFD-induced myelopoiesis is unknown. We hypothesized that saturated fatty acids and HFD alter myelopoiesis by activating TLR4 pathways in HSCs, differentially producing pro-inflammatory CD11c+ myeloid cells that contribute to obesity-induced metabolic disease. Results from reciprocal bone marrow transplants (BMTs) with Tlr4-/- and WT mice indicated that TLR4 is required for HFD-induced myelopoiesis and production of CD11c+ ATMs. Experiments with homozygous knockouts of Irakm (encoding a suppressor of MyD88 inactivation) and Trif in competitive BMTs revealed that MyD88 is required for HFD expansion of granulocyte macrophage progenitors and that Trif is required for pregranulocyte macrophage progenitor expansion. A comparison of WT, Tlr4-/-, Myd88-/-, and Trif-/- mice on HFD demonstrated that TLR4 plays a role in the production of CD11c+ ATMs, and both Myd88-/- and Trif-/- mice produced fewer ATMs than WT mice. Moreover, HFD-induced TLR4 activation inhibited macrophage proliferation, leading to greater accumulation of recruited CD11c+ ATMs. Our results indicate that HFD potentiates TLR4 and both its MyD88- and TRIF-mediated downstream pathways within progenitors and adipose tissue and leads to macrophage polarization.

Asunto(s)

Proteínas Adaptadoras del Transporte Vesicular/inmunología , Antígeno CD11c/inmunología , Macrófagos/patología , Factor 88 de Diferenciación Mieloide/inmunología , Mielopoyesis , Obesidad/patología , Receptor Toll-Like 4/inmunología , Tejido Adiposo/inmunología , Tejido Adiposo/patología , Animales , Dieta Alta en Grasa/efectos adversos , Inflamación/etiología , Inflamación/inmunología , Inflamación/patología , Macrófagos/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/complicaciones , Obesidad/etiología , Obesidad/inmunología

Asunto(s)

Antiinfecciosos , Aterosclerosis , Chlamydia , Humanos , Lípidos , Transducción de Señal , Receptor Toll-Like 4

RESUMEN

The role of resveratrol (trans-3,5,4'-trihydroxystilbene; RES) in lysophosphatidylcholine (LPC)­induced injury and inflammation in endothelial cells (regarded as an early event in arteriosclerosis) is unclear. The present study investigated whether RES reduces lactate dehydrogenase (LDH) activity and secretion of inflammatory cytokines such asinterleukin­6 and tumor necrosis factor­α, via the Toll­like receptor (TLR)­4/myeloid differentiation primary response gene 88 (MyD88)/nuclear factor (NF)­κB signal transduction pathway in LPC­induced damage and inflammation in human umbilical vein endothelial­12 (HUVE­12) cells. Using an ELISA and western blotting, the present study investigated the effects of RES on LDH activity and cytokine secretion. The effects of TLR­4 short hairpin (sh)RNA and TLR­4 cDNA transfection on NF­κB activation during LPC­induced damage and inflammation was also investigated in HUVE­12 cells. The results demonstrated that RES significantly inhibited the effect of LPC on enzyme activity, pro­inflammatory cytokine secretion, and expression of TLR­4, MyD88 and NF­κBp65 expression. In addition, RES and TLR­4 shRNA transfection suppressed LPC­induced injury and inflammation by blocking the TLR­4/MyD88/NF­κB signaling pathway Conversely, transfection with TLR­4 cDNA enhanced LPC­induced injury and inflammation, which abrogated the protective effects of RES. These data suggested that RES significantly suppressed LPC­induced damage and inflammation, via suppression of the TLR­4/MyD88/NF­κB signaling pathway, which may provide a new mechanistic evidence for the treatment of arteriosclerosis by RES.

Asunto(s)

Antiinflamatorios no Esteroideos/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Lisofosfatidilcolinas/antagonistas & inhibidores , Estilbenos/farmacología , Receptor Toll-Like 4/genética , Arteriosclerosis/genética , Arteriosclerosis/inmunología , Arteriosclerosis/patología , Línea Celular , ADN Complementario/genética , ADN Complementario/metabolismo , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Interleucina-6/antagonistas & inhibidores , Interleucina-6/genética , Interleucina-6/inmunología , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/inmunología , Lisofosfatidilcolinas/farmacología , Modelos Cardiovasculares , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , FN-kappa B/genética , FN-kappa B/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Resveratrol , Transducción de Señal , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología

RESUMEN

Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterised by a dysregulation of the immune system, which causes inflammation responses, excessive oxidative stress and a reduction in the number of cluster of differentiation (CD)4+CD25+forkhead box P3 (FoxP3)+ T cells. Supplementation with certain Lactobacillus strains has been suggested to be beneficial in the comprehensive treatment of SLE. However, little is known about the effect and mechanism of certain Lactobacillus strains on SLE. To investigate the effects of Lactobacillus on SLE, NZB/W F1 mice were orally gavaged with Lactobacillus paracasei GMNL-32 (GMNL-32), Lactobacillus reuteri GMNL-89 (GMNL-89) and L. reuteri GMNL-263 (GMNL-263). Supplementation with GMNL-32, GMNL-89 and GMNL-263 significantly increased antioxidant activity, reduced IL-6 and TNF-α levels and significantly decreased the toll-like receptors/myeloid differentiation primary response gene 88 signalling in NZB/W F1 mice. Notably, supplementation with GMNL-263, but not GMNL-32 and GMNL-89, in NZB/W F1 mice significantly increased the differentiation of CD4+CD25+FoxP3+ T cells. These findings reveal beneficial effects of GMNL-32, GMNL-89 and GMNL-263 on NZB/W F1 mice and suggest that these specific Lactobacillus strains can be used as part of a comprehensive treatment of SLE patients.

Asunto(s)

Antioxidantes/metabolismo , Linfocitos T CD4-Positivos/citología , Lactobacillus , Probióticos/administración & dosificación , Administración Oral , Animales , Linfocitos T CD4-Positivos/metabolismo , Modelos Animales de Enfermedad , Femenino , Factores de Transcripción Forkhead/metabolismo , Glutatión/sangre , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Interleucina-6/sangre , Lactobacillus/clasificación , Lupus Eritematoso Sistémico/terapia , Ratones , Ratones Endogámicos NZB , ARN Mensajero/sangre , Tiobarbitúricos/sangre , Receptores Toll-Like/sangre , Factor de Necrosis Tumoral alfa/sangre

RESUMEN

AIM: The present work aims to assess the effectiveness of lentiviral vector (LV) mediated Toll-like receptor 2 (TLR2) gene silencing in the survival of transplanted corneal allografts, against immune rejection, in rats. METHODS: LV mediated TLR2 small interference RNA (SiRNA) with enhanced green fluorescent protein (eGFP) [LV-TLR2-siRNA-eGFP] was realised and transfected to both rat corneal epithelial (EC) and stromal cells (SC). Multiplicity of infection (MOI) was optimized for transfection efficiency using flow cytometric (FCM) analysis. Viability of transfected cells and the success rate of TLR2 gene silencing were respectively determined by CCK-8 assay and western blot assay. The in-vivo experiments were subjected to a penetrating keratoplasty (PK) performed between host Sprague Dawley (SD) and donor Wistar/SD rats, randomly dividing them into 4 groups including the allograft, isograft, allograft treated with LV-eGFP (LV blank control) and allograft treated with LV-TLR2-siRNA-eGFP (LV treated group). The rejection index (RI) was then recorded under a slit lamp, every day following surgery. Expression of the TLR2 and Myeloid differentiation primary response gene 88 (MyD88) were detected by using immunohistochemistry on day 9 post-surgery, whereas grafts's TLR2 and MyD88 mRNA were determined on day 5, 9, and 14 post-surgery performing RT-PCR and, normal rat corneas were included as additional controls. RESULTS: Transfected cells showed the strongest eGFP expression when MOI was 200 with an efficiency of 77.5% for EC and 76.3% for SC. CCK-8 assay, as measured at 72 and 96h post transfection, showed no significant changes in the cell viability (both EC and SC) between the transfected and the control group (p>0.05, p>0.05). Western blot results demonstrated a successful down regulation of TLR2 expression by LV-TLR2-SiRNA-eGFP, in both EC and SC. In vivo, compared to allograft group, LV treated group demonstrated less edema, opacity and neovascularization. Immunohistochemical analysis revealed a lower expression of TLR2 and MyD88 in isograft and LV treated group as compared to allograft group. TLR2 and MyD88 mRNA were detected in all grafts, and increased over time. With its highest expression in allograft group (peak on day 9), the mRNA expression for TLR2 and MyD88 showed a significant difference amongst the groups, on both day 9 and 14 (p<0.001). CONCLUSIONS: LV mediated TLR2 siRNA could effectively down regulate the TLR2 expression via RNA interference and prolong the survival of corneal grafts, although not necessarily able to prevent the rejection.

Asunto(s)

Córnea/inmunología , Trasplante de Córnea , Silenciador del Gen , Marcación de Gen , Rechazo de Injerto/prevención & control , Lentivirus , Receptor Toll-Like 2 , Aloinjertos , Animales , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología

RESUMEN

Upon stimulation of toll-like receptors with various microbial ligands, induction of a variety of inflammatory genes is elicited by activation of a myeloid differentiation primary-response protein 88 (MyD88)-dependent signaling pathway. Interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK1) plays an essential role in this pathway by activating nuclear factor κB (NF-κB) and mitogen-activated kinases (MAPKs). Here, we identified optineurin (OPTN) as an IRAK1-binding protein by yeast two-hybrid screening using IRAK1 as bait. A C-terminal fragment of OPTN harboring a ubiquitin-binding domain was co-immunoprecipitated with IRAK1. In reporter analyses, overexpression of OPTN inhibited IL-1ß-, IRAK1-, and LPS-induced NF-κB activation. Consistently, OPTN deficiency resulted in increased NF-κB activation in response to IL-1ß/LPS stimulation. To address the mechanisms underlying the inhibitory effect of OPTN on NF-κB signaling, we focused on tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), which is an adaptor protein of IRAK1 and upon polyubiquitination plays a crucial role during NF-κB activation. Overexpression of OPTN prevented TRAF6 polyubiquitination. Furthermore, OPTN H486R mutant, which is unable to recruit the deubiquitinase CYLD, failed to inhibit IRAK1-induced NF-κB activation. These results suggest that the IRAK1-binding protein OPTN negatively regulates IL-1ß/LPS-induced NF-κB activation by preventing polyubiquitination of TRAF6.

Asunto(s)

Proteínas del Ojo/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal/fisiología , Factor de Transcripción TFIIIA/metabolismo , Sustitución de Aminoácidos , Animales , Proteínas de Ciclo Celular , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Enzima Desubiquitinante CYLD , Proteínas del Ojo/genética , Células HEK293 , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Péptidos y Proteínas de Señalización Intracelular , Lipopolisacáridos/farmacología , Proteínas de Transporte de Membrana , Ratones , Mutación Missense , Factor 88 de Diferenciación Mieloide/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Factor de Transcripción TFIIIA/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitinación/efectos de los fármacos , Ubiquitinación/fisiología

RESUMEN

Upon recognition of bacterial pathogens by pattern recognition receptors, cells are activated to produce pro-inflammatory cytokines and type I IFN by multiple signaling pathways. Every step of the process must be precisely regulated to prevent dysregulation. MicroRNAs (miRNAs) have been shown to be important regulators with profound effects on inflammatory response. Nevertheless, the miRNA-mediated regulatory mechanism remains unclear in fish species. Here, we addressed the role of miiuy croaker miR-214 in the bacteria triggered inflammatory response. miR-214 could significantly be up-regulated by Vibro harveyi and LPS stimulation. Up-regulating miR-214 subsequently inhibits the production of inflammatory cytokines by targeting myd88 to avoid excessive inflammation. Moreover, the negative regulatory mechanism of miR-214 has been demonstrated to be via the myd88-mediated NF-κB pathway. This is the first to focus on miR-214 acting as the negative regulator involved in the bacteria-triggered inflammatory response and thus may provide knowledge on the host-cell regulator responses to microbial infection.

Asunto(s)

Inflamación/microbiología , MicroARNs/inmunología , Factor 88 de Diferenciación Mieloide/genética , FN-kappa B/metabolismo , Animales , Infecciones Bacterianas/inmunología , Citocinas , Peces , Lipopolisacáridos , FN-kappa B/antagonistas & inhibidores , Transducción de Señal/inmunología