RESUMEN
The genome of vertebrates contains endogenous retroviruses (ERVs) that are largely nonfunctional relicts of ancestral germline infection by exogenous retroviruses. However, in some mouse strains ERVs are actively involved in disease. Here we report that nucleic acid-recognizing Toll-like receptors 3, 7, and 9 (TLR 3, TLR7, and TLR9) are essential for the control of ERVs. Loss of TLR7 function caused spontaneous retroviral viremia that coincided with the absence of ERV-specific antibodies. Importantly, additional TLR3 and TLR9 deficiency led to acute T cell lymphoblastic leukemia, underscoring a prominent role for TLR3 and TLR9 in surveillance of ERV-induced tumors. Experimental ERV infection induced a TLR3-, TLR7-, and TLR9-dependent group of "acute-phase" genes previously described in HIV and SIV infections. Our study suggests that in addition to their role in innate immunity against exogenous pathogens, nucleic acid-recognizing TLRs contribute to the immune control of activated ERVs and ERV-induced tumors.
Asunto(s)
Retrovirus Endógenos/genética , Ácidos Nucleicos/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Viremia/genética , Animales , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Línea Celular , Retrovirus Endógenos/inmunología , Retrovirus Endógenos/metabolismo , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ácidos Nucleicos/inmunología , Ácidos Nucleicos/metabolismo , Oncogenes/genética , Oncogenes/inmunología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptores Toll-Like/inmunología , Viremia/inmunología , Viremia/metabolismoRESUMEN
Salmonella enterica is a food-borne pathogen able to cause a wide spectrum of diseases ranging from mild gastroenteritis to systemic infections. During almost all stages of the infection process Salmonella is likely to be exposed to a wide variety of host-derived antimicrobial peptides (AMPs). AMPs are important components of the innate immune response which integrate within the bacterial membrane, thus forming pores which lead ultimately to bacterial killing. In contrast to other AMPs Bactericidal/Permeability-increasing Protein (BPI) displayed only weak bacteriostatic or bactericidal effects towards Salmonella enterica sv. Typhimurium (STM) cultures. Surprisingly, we found that sub-antimicrobial concentrations of BPI fold-containing (BPIF) superfamily members mediated adhesion of STM depending on pre-formed type 1 fimbriae. BPIF proteins directly bind to type 1 fimbriae through mannose-containing oligosaccharide modifications. Fimbriae decorated with BPIF proteins exhibit extended binding specificity, allowing for bacterial adhesion on a greater variety of abiotic and biotic surfaces likely promoting host colonization. Further, fimbriae significantly contributed to the resistance against BPI, probably through sequestration of the AMP before membrane interaction. In conclusion, functional subversion of innate immune proteins of the BPIF family through binding to fimbriae promotes Salmonella virulence by survival of host defense and promotion of host colonization.
Asunto(s)
Salmonella enterica , Salmonella typhimurium , Fimbrias Bacterianas/metabolismo , Adhesión Bacteriana , Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
Endogenous retroviruses (ERVs) are an integral part of the mammalian genome. The role of immune control of ERVs in general is poorly defined as is their function as anti-cancer immune targets or drivers of autoimmune disease. Here, we generate mouse-strains where Moloney-Murine Leukemia Virus tagged with GFP (ERV-GFP) infected the mouse germline. This enables us to analyze the role of genetic, epigenetic and cell intrinsic restriction factors in ERV activation and control. We identify an autoreactive B cell response against the neo-self/ERV antigen GFP as a key mechanism of ERV control. Hallmarks of this response are spontaneous ERV-GFP+ germinal center formation, elevated serum IFN-γ levels and a dependency on Age-associated B cells (ABCs) a subclass of T-bet+ memory B cells. Impairment of IgM B cell receptor-signal in nucleic-acid sensing TLR-deficient mice contributes to defective ERV control. Although ERVs are a part of the genome they break immune tolerance, induce immune surveillance against ERV-derived self-antigens and shape the host immune response.
Asunto(s)
Linfocitos B , Retrovirus Endógenos , Animales , Ratones , Enfermedades Autoinmunes/genética , Linfocitos B/inmunología , Retrovirus Endógenos/genética , Mamíferos/genéticaRESUMEN
The reverse genetics system for influenza A viruses described by Hoffmann et al. (Virology 267(2):310-317, 2000, Proc Natl Acad Sci USA 97(11):6108-6113, 2000, ArchVirol 146(12):2275-2289, 2001) is one of the most commonly used. However, this cloning strategy is rather time-consuming and lacks a selection marker to identify positive clones carrying viral genes. We report here the optimization of the cloning protocol of viral genes into pHW2000 by (i) introducing a selection marker and (ii) simplifying the cloning strategy: now the cloning reaction takes only a few minutes and, in addition, is independent of internal restriction sites for BsmBI/Esp3I, BsaI or AarI. In order to accelerate the whole cloning protocol for the generation of recombinant viruses, we first introduced a lacP/Z-element (lac-promoter/lacZα-fragment) between the two BsmBI sites of pHW2000 to allow selection of positive clones by blue/white screening. Then we optimized the digestion/ligation-protocol: In our system, enzymatic digestion and ligation of PCR products into the vector is performed in a single "one-tube" reaction. Due to this strategy, time and material consumption is reduced by a great amount, as vector and cDNA do not have to be digested and purified prior to the ligation. Therefore, this one-tube reaction yields positive clones with high efficiency and fidelity, again saving time and material, which were formerly required for screening and analyzing clones. Finally, to add more versatility to the system, we also created an entry vector based on TA-cloning. This entry vector provides several advantages: inserted genes can easily be modified, e.g., by site-directed mutagenesis or tag attachment, and then subcloned into pHW2000 or other plasmids containing a similar cloning site (e.g., our modified pCAGGS-Esp-blue) by the same rapid and reliable one-tube reaction protocol described here. In fact, the presented protocol is suitable to be adapted to other reverse genetics systems (e.g., those for members of the order Mononegavirales or the family Bunyaviridae) or cloning of genes in general.
Asunto(s)
Virus de la Influenza A/genética , ARN Viral/genética , Clonación Molecular/métodos , ADN Complementario/genética , ADN Viral/genética , Plásmidos/genética , Reacción en Cadena de la PolimerasaRESUMEN
The human BPI (bactericidal/permeability-increasing protein), stored in primary azurophilic granula of neutrophil granulocytes and produced by mucosal epithelia, has been known for decades to bind LPS (lipopolysaccharide) with very high affinity and to efficiently kill Gram-negative bacteria. Thus BPI potentially represents a central component of the innate immune system to directly combat microbes and modulate subsequent adaptive immune responses. Especially in the lungs, which are frequently exposed to a variety of inhaled pathogens, antimicrobial innate defence molecules such as BPI, are of exceptional relevance. In the present review, we highlight possible functions of BPI during acute pneumonia and CF (cystic fibrosis)-associated chronic infections in the lung.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/metabolismo , Fibrosis Quística/inmunología , Inmunidad Innata , Neumonía Bacteriana/inmunología , Sistema Respiratorio/metabolismo , Animales , Fibrosis Quística/metabolismo , Humanos , Neumonía Bacteriana/metabolismoRESUMEN
The Lyme disease vaccine is based on the outer-surface lipoprotein (OspA) of the pathogen Borrelia burgdorferi, and 95% of vaccine recipients develop substantial titers of antibodies against OspA. Here, we identified seven individuals with very low antibody titers after vaccination (low responders). The macrophages of low responders produced less tumor necrosis factor-alpha and interleukin-6 after OspA stimulation and had lower cell-surface expression of Toll-like receptor (TLR) 1 as compared to normal cells, but normal expression of TLR2. TLRs activate innate responses to pathogens, and TLR2 recognizes lipoproteins and peptidoglycan (PGN). After OspA immunization, mice genetically deficient in either TLR2 (TLR2(-/-)) or TLR1 (TLR1(-/-)) produced low titers of antibodies against OspA. Notably, macrophages from TLR2(-/-) mice were unresponsive to OspA and PGN, whereas those from TLR1(-/-) mice responded normally to PGN but not to OspA. These data indicate that TLR1 and TLR2 are required for lipoprotein recognition and that defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Antígenos de Superficie/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Borrelia burgdorferi/inmunología , Proteínas de Drosophila , Vacunas contra Enfermedad de Lyme/inmunología , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Vacunas Bacterianas , Borrelia burgdorferi/química , Separación Celular , Células Cultivadas , Humanos , Interleucinas/metabolismo , Lipoproteínas/inmunología , Lipoproteínas/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Receptores de Superficie Celular/genética , Transducción de Señal , Receptor Toll-Like 1 , Receptor Toll-Like 2 , Receptores Toll-LikeRESUMEN
Infection and inflammation can augment local Na+ abundance. These increases in local Na+ levels boost proinflammatory and antimicrobial macrophage activity and can favor polarization of T cells towards a proinflammatory Th17 phenotype. Although neutrophils play an important role in fighting intruding invaders, the impact of increased Na+ on the antimicrobial activity of neutrophils remains elusive. Here we show that, in neutrophils, increases in Na+ (high salt, HS) impair the ability of human and murine neutrophils to eliminate Escherichia coli and Staphylococcus aureus. High salt caused reduced spontaneous movement, degranulation and impaired production of reactive oxygen species (ROS) while leaving neutrophil viability unchanged. High salt enhanced the activity of the p38 mitogen-activated protein kinase (p38/MAPK) and increased the interleukin (IL)-8 release in a p38/MAPK-dependent manner. Whereas inhibition of p38/MAPK did not result in improved neutrophil defense, pharmacological blockade of the phagocyte oxidase (PHOX) or its genetic ablation mimicked the impaired antimicrobial activity detected under high salt conditions. Stimulation of neutrophils with phorbol-12-myristate-13-acetate (PMA) overcame high salt-induced impairment in ROS production and restored antimicrobial activity of neutrophils. Hence, we conclude that high salt-impaired PHOX activity results in diminished antimicrobial activity. Our findings suggest that increases in local Na+ represent an ionic checkpoint that prevents excessive ROS production of neutrophils, which decreases their antimicrobial potential and could potentially curtail ROS-mediated tissue damage.
Asunto(s)
Infecciones Bacterianas/metabolismo , Infecciones Bacterianas/microbiología , Microambiente Celular , Neutrófilos/fisiología , Oxidorreductasas/metabolismo , Fagocitos/fisiología , Sodio/metabolismo , Animales , Infecciones Bacterianas/inmunología , Resistencia a la Enfermedad , Susceptibilidad a Enfermedades , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Interacciones Huésped-Patógeno , Humanos , Ratones , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Broad-spectrum antibiotics are widely used with patients in intensive care units (ICUs), many of whom develop hospital-acquired infections with Pseudomonas aeruginosa. Although preceding antimicrobial therapy is known as a major risk factor for P. aeruginosa-induced pneumonia, the underlying mechanisms remain incompletely understood. Here we demonstrate that depletion of the resident microbiota by broad-spectrum antibiotic treatment inhibited TLR-dependent production of a proliferation-inducing ligand (APRIL), resulting in a secondary IgA deficiency in the lung in mice and human ICU patients. Microbiota-dependent local IgA contributed to early antibacterial defense against P. aeruginosa. Consequently, P. aeruginosa-binding IgA purified from lamina propria culture or IgA hybridomas enhanced resistance of antibiotic-treated mice to P. aeruginosa infection after transnasal substitute. Our study provides a mechanistic explanation for the well-documented risk of P. aeruginosa infection following antimicrobial therapy, and we propose local administration of IgA as a novel prophylactic strategy.
Asunto(s)
Antibacterianos/farmacología , Deficiencia de IgA/tratamiento farmacológico , Inmunoglobulina A/farmacología , Neumonía Bacteriana/tratamiento farmacológico , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/inmunología , Animales , Humanos , Enfermedad Iatrogénica , Deficiencia de IgA/genética , Deficiencia de IgA/inmunología , Deficiencia de IgA/patología , Ratones , Ratones Noqueados , Neumonía Bacteriana/genética , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/patología , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/patologíaRESUMEN
Macrophages as immune cells prevent the spreading of pathogens by means of active phagocytosis and killing. We report here the presence of an antimicrobial protein, bactericidal/permeability-increasing protein (BPI) in human macrophages, which actively participates in engulfment and killing of Gram-negative pathogens. Our studies revealed increased expression of BPI in human macrophages during bacterial infection and upon stimulation with various pathogen-associated molecular patterns, viz., LPS and flagellin. Furthermore, during the course of an infection, BPI interacted with Gram-negative bacteria, resulting in enhanced phagocytosis and subsequent control of the bacterial replication. However, it was observed that bacteria which can maintain an active replicating niche (Salmonella Typhimurium) avoid the interaction with BPI during later stages of infection. On the other hand, Salmonella mutants, which cannot maintain a replicating niche, as well as Shigella flexneri, which quit the endosomal vesicle, showed interaction with BPI. These results propose an active role of BPI in Gram-negative bacterial clearance by human macrophages.
RESUMEN
In addition to their well-known antibacterial activity some antimicrobial peptides and proteins (AMPs) display also antiviral effects. A 27 aa peptide from the N-terminal part of human bactericidal/permeability-increasing protein (BPI) previously shown to harbour antibacterial activity inhibits the infectivity of multiple Influenza A virus strains (H1N1, H3N2 and H5N1) the causing agent of the Influenza pneumonia. In contrast, the homologous murine BPI-peptide did not show activity against Influenza A virus. In addition human BPI-peptide inhibits the activation of immune cells mediated by Influenza A virus. By changing the human BPI-peptide to the sequence of the mouse homologous peptide the antiviral activity was completely abolished. Furthermore, the human BPI-peptide also inhibited the pathogenicity of the Vesicular Stomatitis Virus but failed to interfere with HIV and measles virus. Electron microscopy indicate that the human BPI-peptide interferes with the virus envelope and at high concentrations was able to destroy the particles completely.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Antivirales/farmacología , Proteínas Sanguíneas/farmacología , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/patogenicidad , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Antivirales/metabolismo , Proteínas Sanguíneas/metabolismo , Células Cultivadas , Cricetinae , Perros , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Células de Riñón Canino Madin Darby , Neutrófilos/metabolismo , Fragmentos de Péptidos/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
The soluble fms-like tyrosine kinase 1 (sFlt-1), known to be increased in the serum of preeclamptic patients, is a relevant factor in causing maternal symptoms like hypertension and proteinuria. In this study, we aimed to reveal whether hypoxia is a cause of increased sFlt-1 levels and inflammation markers in vivo and whether these symptoms can be attenuated by interleukin 6 (IL-6) depletion. For this purpose, pregnant wild-type (wt) mice or IL-6(-/-) mice on embryonic day 16 were placed under either normoxic (20.9% oxygen) or hypoxic (6% oxygen) conditions for 6 hours. This led to a rise of sFlt-1 levels in maternal serum, independent of the IL-6 status of the dam. Increased maternal sFlt-1 serum levels were, however, not due to an increase in sFlt-1 messenger RNA levels in the placenta. Moreover, there was no increase in inflammatory markers in neither wt mice nor IL-6(-/-) mice. This suggests that hypoxia alone does not contribute to the induction of an inflammatory placenta. Also, the hypoxia-induced rise in sFlt-1 levels seems not to be mediated by IL-6 in vivo.
Asunto(s)
Hipoxia/enzimología , Inflamación/enzimología , Interleucina-6/deficiencia , Receptor 1 de Factores de Crecimiento Endotelial Vascular/sangre , Animales , Modelos Animales de Enfermedad , Femenino , Edad Gestacional , Hipoxia/sangre , Hipoxia/genética , Hipoxia/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inflamación/sangre , Inflamación/genética , Inflamación/inmunología , Interleucina-6/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Placenta/inmunología , Placenta/metabolismo , Embarazo , Regulación hacia Arriba , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Foreign RNA serves as pathogen-associated molecular pattern (PAMP) and is a potent immune stimulator for innate immune receptors. However, the role of single bacterial RNA species in immune activation has not been characterized in detail. We analyzed the immunostimulatory potential of transfer RNA (tRNA) from different bacteria. Interestingly, bacterial tRNA induced type I interferon (IFN) and inflammatory cytokines in mouse dendritic cells (DCs) and human peripheral blood mononuclear cells (PBMCs). Cytokine production was TLR7 dependent because TLR7-deficient mouse DCs did not respond and TLR7 inhibitory oligonucleotides inhibited tRNA-mediated activation. However, not all bacterial tRNA induced IFN-α because tRNA from Escherichia coli Nissle 1917 and Thermus thermophilus were non-immunostimulatory. Of note, tRNA from an E. coli knockout strain for tRNA (Gm18)-2'-O-methyltransferase (trmH) regained immunostimulatory potential. Additionally, in vitro methylation of this immunostimulatory Gm18-negative tRNA with recombinant trmH from T. thermophilus abolished its IFN-α inducing potential. More importantly, Gm18-modified tRNA acted as TLR7 antagonist and blocked IFN-α induction of influenza A virus-infected PBMCs.
Asunto(s)
Bacterias/genética , Guanosina/metabolismo , Inmunidad Innata/inmunología , Glicoproteínas de Membrana/inmunología , ARN de Transferencia/inmunología , Receptor Toll-Like 7/inmunología , ARNt Metiltransferasas/metabolismo , Animales , Bacterias/inmunología , Cromatografía Líquida de Alta Presión , Células Dendríticas/inmunología , Humanos , Inmunización , Interferón-alfa/metabolismo , Leucocitos Mononucleares/inmunología , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Oligonucleótidos , ARN de Transferencia/farmacología , Receptor Toll-Like 7/antagonistas & inhibidores , Receptor Toll-Like 7/genética , ARNt Metiltransferasas/genéticaRESUMEN
HIF1A is a transcription factor that plays a central role for the adaptation to tissue hypoxia and for the inflammatory response of myeloid cells, including DCs. HIF1A is stabilized by hypoxia but also by TLR ligands under normoxic conditions. The underlying signaling events leading to the accumulation of HIF1A in the presence of oxygen are still poorly understood. Here, we show that in contrast to hypoxic stabilization of HIF1A, normoxic, TLR-mediated HIF1A accumulation in DCs follows a different pathway that predominantly requires MYD88-dependent NF-κB activity. The TLR-induced HIF1A controls a subset of proinflammatory genes that are insufficiently induced following hypoxia-mediated HIF1A induction. Thus, TLR activation and hypoxia stabilize HIF1A via distinct signaling pathways, resulting in differential HIF1A-dependent gene expression.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/fisiología , Perfilación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia , Factor 88 de Diferenciación Mieloide/fisiología , Transducción de Señal , Receptor Toll-Like 4/fisiología , Animales , Biomarcadores/metabolismo , Western Blotting , Diferenciación Celular , Proliferación Celular , Inmunoprecipitación de Cromatina , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
By virtue of its direct contact with the environment, the lung is constantly challenged by infectious and non-infectious stimuli that necessitate a robust yet highly controlled host response coordinated by the innate and adaptive arms of the immune system. Mammalian Toll-like receptors (TLRs) function as crucial sentinels of microbial and non-infectious antigens throughout the respiratory tract and mediate host innate immunity. Selective induction of inflammatory responses to harmful environmental exposures and tolerance to innocuous antigens are required to maintain tissue homeostasis and integrity. Conversely, dysregulated innate immune responses manifest as sustained and self-perpetuating tissue damage rather than controlled tissue repair. In this article we review aspects of Toll-like receptor function that are relevant to the development of acute lung injury and chronic obstructive lung diseases as well as resistance to frequently associated microbial infections.
RESUMEN
Recognition of LPS by TLR4 initiates inflammatory responses inducing potent antimicrobial immunity. However, uncontrolled inflammatory responses can be detrimental. To prevent the development of septic shock during an infection with Gram-negative bacteria, the immune system has developed mechanisms to neutralize LPS by specialized proteins. In this study, we report the recombinant expression and functional characterization of the mouse homolog of human bactericidal/permeability-increasing protein (BPI). Purified recombinant mouse BPI was able to neutralize LPS-mediated activation of macrophages and to block LPS-dependent maturation of dendritic cells. Recombinant mouse BPI neutralized the capacity of Gram-negative bacteria to activate immune cells, but did not influence the stimulatory properties of Gram-positive bacteria. Unlike human BPI, mouse BPI failed to kill or inhibit the growth of Pseudomonas aeruginosa. Together, these data demonstrate that murine BPI is a potent LPS-neutralizing protein that may limit innate immune responses during Gram-negative infections.
Asunto(s)
Bacterias Gramnegativas/inmunología , Lipopolisacáridos/inmunología , Proteínas/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/inmunología , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/inmunología , Proteínas Sanguíneas/metabolismo , Línea Celular , Células Dendríticas/inmunología , Escherichia coli/crecimiento & desarrollo , Escherichia coli/inmunología , Escherichia coli/patogenicidad , Bacterias Gramnegativas/metabolismo , Humanos , Activación de Macrófagos , Ratones , Ratones Endogámicos C57BL , Proteínas/inmunología , Proteínas/aislamiento & purificación , Pseudomonas aeruginosa/crecimiento & desarrollo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Fusiogenic glycoprotein syncytin-1, expressed in human placenta, is a promising candidate for acquiring a basic knowledge of placental syncytialization. However, its cellular mode of action is unidentified. We investigated whether syncytin-1 may exert influence on apoptotic processes. Therefore, we incubated CHO cells after stable transfection with syncytin-1 (CHO-52) in the presence or absence of staurosporine (STS), a kinase inhibitor well characterized to induce apoptosis. When testing the phenotype of CHO-52 cells, we could demonstrate that the induction of apoptosis by STS was delayed over a period of up to 24 h. Furthermore, the cell death rate was decreased by approx 75% following transfection of syncytin-1 in CHO-52 compared to mock-treated cells. In detail, after 18h of incubation with 500 nM STS, 64 +/- 2% of CHO-52 cells were viable compared to 16 +/- 1% of CHO-mocks, after 24 h 43 +/- 3% vs 5 +/- 2%, respectively. CHO-52 cells exhibited a lower expression of active caspase 3 and anti-apoptotic Bcl-2 was found to be increased in CHO-52 cells at baseline and following STS treatment. Our study provides first evidence that syncytin-1 serves anti-apoptotic function under certain conditions. A lessened activation of caspase 3 and an increased expression of Bcl-2 are possible mechanisms.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Productos del Gen env/fisiología , Proteínas Gestacionales/fisiología , Estaurosporina/farmacología , Animales , Células CHO , Fusión Celular , Cricetinae , Cricetulus , Retrovirus Endógenos/genética , Retrovirus Endógenos/fisiología , Femenino , Productos del Gen env/genética , Humanos , Técnicas In Vitro , Microscopía Fluorescente , Embarazo , Proteínas Gestacionales/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Innate immunity provides a first line of host defence against infection through microbial recognition and killing while simultaneously activating a definitive adaptive immune response. Toll-like receptors (TLRs) are principal mediators of rapid microbial recognition and function mainly by detection of structural patterns that do not exist in the host. TLR2 and TLR4 were the first members of this innate immune receptor family to be strongly implicated in antibacterial host defence. Following the initial description of the mammalian TLR family, susceptibility to infection with numerous human microbial pathogens has been intensively studied using mice with engineered deletions of each of these molecules. While it has become quite clear that TLR activation is necessary for optimal host defence, a comprehensive understanding of the mechanisms by which this family of pattern recognition receptors engages protective immunity, particularly the adaptive response, is still evolving.
Asunto(s)
Infecciones Bacterianas/inmunología , Receptores Toll-Like/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Antígenos de Diferenciación/inmunología , Humanos , Inmunidad Activa , Inmunidad Innata , Factor 88 de Diferenciación Mieloide , Receptores Inmunológicos/inmunología , Receptor Toll-Like 4/inmunologíaRESUMEN
Antimicrobial effector proteins are a key mechanism for the innate immune system to combat pathogens once they infect the host. We report the identification and cloning of the mouse homologue of human bactericidal permeability/increasing protein (BPI). Mouse BPI is constitutively expressed in lymphatic organs and tissues as well as in mouse testis. Upon stimulation with different TLR ligands, mouse BPI is strongly expressed in granulocytes and, surprisingly, in bone marrow-derived dendritic cells. Mouse BPI is most strongly induced by bacterial LPS through a signaling pathway that is completely dependent on TLR4-Toll/IL-1R domain-containing adaptor inducing IFN-beta. Functional studies revealed that mouse BPI does have the potential to neutralize LPS and inhibits bacterial growth. Mouse BPI is expressed in granulocytes and bone marrow-derived dendritic cells, and the transcriptional activation is controlled by TLRs.
Asunto(s)
Proteínas Sanguíneas/genética , Proteínas Sanguíneas/inmunología , Endotoxinas/metabolismo , Interferón beta/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Receptores de Interleucina-1/metabolismo , Receptores Toll-Like/metabolismo , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos , Proteínas Sanguíneas/metabolismo , Northern Blotting , Células de la Médula Ósea/inmunología , Clonación Molecular , Células Dendríticas/inmunología , Granulocitos/inmunología , Granulocitos/metabolismo , Humanos , Interferón beta/inmunología , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Factor 88 de Diferenciación Mieloide , ARN Mensajero/análisis , Receptores de Interleucina-1/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Homología de Secuencia de Aminoácido , Receptores Toll-Like/inmunología , Transcripción Genética/inmunologíaRESUMEN
In cystic fibrosis (CF), the condition limiting the prognosis of affected children is the chronic obstructive lung disease accompanied by chronic and persistent infection with mostly mucoid strains of Pseudomonas aeruginosa. The majority of CF patients have antineutrophil cytoplasmic antibodies (ANCA) primarily directed against the bactericidal permeability-increasing protein (BPI) potentially interfering with antimicrobial effects of BPI. We analyzed the expression of BPI in the airways of patients with CF. In their sputum samples or bronchoalveolar lavage specimens, nearly all patients expressed BPI mRNA and protein, which were mainly products of neutrophil granulocytes as revealed by intracellular staining and subsequent flow cytometry. Repeated measurements revealed consistent individual BPI expression levels during several months quantitatively correlating with interleukin-8. In vitro, P. aeruginosa isolates from CF patients initiated the rapid release of BPI occurring independently of protein de novo syntheses. Furthermore, purified natural BPI as well as a 27-mer BPI-derived peptide displayed antimicrobial activity against even patient-derived mucoid P. aeruginosa strains and bacteria resistant against all antibiotics tested. Thus, BPI that is functionally active against mucoid P. aeruginosa strains is expressed in the airways of CF patients but may be hampered by autoantibodies, resulting in chronic infection.
Asunto(s)
Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacología , Fibrosis Quística/microbiología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Esputo/metabolismo , Secuencia de Aminoácidos , Antibacterianos/síntesis química , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos , Proteínas Sanguíneas/síntesis química , Proteínas Sanguíneas/química , Fibrosis Quística/metabolismo , Humanos , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/microbiología , Proteínas de la Membrana/síntesis química , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Neutrófilos/metabolismo , Infecciones por Pseudomonas/microbiología , Esputo/inmunologíaRESUMEN
PROBLEM: Toll-like receptors (TLRs) are expressed on placental cells. The aim of this study is to analyze signaling components activated in placenta cells after TLR ligand engagement. METHODS OF STUDY: In chorioncarcimoma cell lines the regulation of TLRs was determined by real time polymerase chain reaction as well as by fluorescence-activated cell sorter analysis. Activation of NF-kappaB was determined in a reporter assay system and the activation of the mitogen-activated protein kinase signaling pathways by immunoblot analysis. RESULTS: Both lipopolysaccharide (LPS) and DNA oligonucleotides containing unmethylated CpG motifs (CpG) induced the enhanced expression of TLR2 mRNA as well as a TLR2 surface protein expression. Functionally, incubation of JAR cells with microbial stimuli such as LPS activated NF-kappaB, as well as the phosphorylation of ERK1/2 and p38 MAP kinases and secretion of interleukin-8. CONCLUSION: The functional expression of TLRs on placental cells may play an important role in the initiation of an immune response in the developing fetus.