RESUMEN

TLRs are the most thoroughly studied group of pattern-recognition receptors that play a central role in innate immunity. Among them, TLR10 (CD290) remains the only TLR family member without a known ligand and clearly defined functions. One major impediment to studying TLR10 is its absence in mice. A recent study on TLR10 knock-in mice demonstrated its intrinsic inhibitory role in B cells, indicating that TLR10 is a potential drug target in autoimmune diseases. In this study, we interrogated the expression and function of TLR10 in human plasmacytoid dendritic cells (pDCs). We have seen that primary human pDCs, B cells, and monocytes constitutively express TLR10. Upon preincubation with an anti-TLR10 Ab, production of cytokines in pDCs was downregulated in response to stimulation with DNA and RNA viruses. Upon further investigation into the possible mechanism, we documented phosphorylation of STAT3 upon Ab-mediated engagement of TLR10. This leads to the induction of inhibitory molecule suppressor of cytokine signaling 3 (SOCS3) expression. We have also documented the inhibition of nuclear translocation of transcription factor IFN regulatory factor 7 (IRF7) in pDCs following TLR10 engagement. Our data provide the (to our knowledge) first evidence that TLR10 is constitutively expressed on the surface of human pDCs and works as a regulator of their innate response. Our findings indicate the potential of harnessing the function of pDCs by Ab-mediated targeting of TLR10 that may open a new therapeutic avenue for autoimmune disorders.

Asunto(s)

Células Dendríticas , Factor 7 Regulador del Interferón , Factor de Transcripción STAT3 , Proteína 3 Supresora de la Señalización de Citocinas , Receptor Toll-Like 10 , Humanos , Células Dendríticas/inmunología , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/genética , Proteína 3 Supresora de la Señalización de Citocinas/inmunología , Factor 7 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/inmunología , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/inmunología , Receptor Toll-Like 10/inmunología , Receptor Toll-Like 10/genética , Inmunidad Innata/inmunología , Citocinas/metabolismo , Citocinas/inmunología , Células Cultivadas , Fosforilación , Animales , Linfocitos B/inmunología , Transducción de Señal/inmunología , Ratones , Monocitos/inmunología

RESUMEN

TLRs are important pattern-recognition receptors involved in the activation of innate immune responses against foreign pathogens. TLR10 is the only TLR family member without a known ligand, signaling pathway, or clear cellular function. Previous work has shown that TLR10 suppresses proinflammatory cytokine production in response to TLR agonists in a mixed human mononuclear cell population. We report that TLR10 is preferentially expressed on monocytes and suppresses proinflammatory cytokine production resulting from either TLR or CD40 stimulation. TLR10 engagement affects both the MAPK and Akt signaling pathways, leading to changes in the transcriptome of isolated human monocytes. Differentiation of monocytes into dendritic cells in the presence of an αTLR10 mAb reduced the expression of maturation markers and the induction of proinflammatory cytokines, again in response to either TLR or CD40 stimulation. Finally, in coculture experiments, TLR10 differentiated dendritic cells exhibited a decreased capacity to activate T cells as measured by IL-2 and IFN-γ production. These data demonstrate that TLR10 is a novel regulator of innate immune responses and of the differentiation of primary human monocytes into effective dendritic cells.

Asunto(s)

Células Dendríticas/inmunología , Monocitos/inmunología , Linfocitos T/inmunología , Receptor Toll-Like 10/inmunología , Transcriptoma/inmunología , Inmunidad Adaptativa , Anticuerpos Monoclonales/farmacología , Antígenos CD40/genética , Antígenos CD40/inmunología , Diferenciación Celular , Técnicas de Cocultivo , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Inmunidad Innata , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/inmunología , Monocitos/citología , Monocitos/efectos de los fármacos , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Transducción de Señal , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Receptor Toll-Like 10/agonistas , Receptor Toll-Like 10/genética , Transcriptoma/genética

RESUMEN

Toll-like receptors play a central role in the initiation of adaptive immune responses with several TLR agonists acting as known B cell mitogens. Despite thousands of publications on TLRs, the function of TLR10 remains unknown. We have found that Ab-mediated engagement of TLR10 on primary human B cells suppresses B cell proliferation, cytokine production, and signal transduction. When challenged with either a T independent or T dependent Ag, TLR10 transgenic mice exhibit diminished Ab responses. Adoptive transfer of splenic B cells into B cell-deficient mice revealed that the suppressive effects on Ag-specific humoral immune responses are entirely B cell intrinsic. Our results demonstrate that TLR10 has a functional role within the B cell lineage that is distinct from that of other TLR family members and may provide a potential therapeutic target for diseases characterized by dysregulated B cell activity.

Asunto(s)

Inmunidad Adaptativa/inmunología , Linfocitos B/inmunología , Activación de Linfocitos/inmunología , Receptor Toll-Like 10/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnicas de Sustitución del Gen , Humanos , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Yersinia pestis is a Gram-negative bacterium that is the causative agent of bubonic and pneumonic plague. It is commonly acquired by mammals such as rodents and humans via the bite of an infected flea. We previously reported that multiple substrains of the 129 mouse background are resistant to pigmentation locus-negative (pgm(-)) Yersinia pestis and that this phenotype maps to a 30-centimorgan (cM) region located on chromosome 1. In this study, we have further delineated this plague resistance locus to a region of less than 20 cM through the creation and phenotyping of recombinant offspring arising from novel crossovers in this region. Furthermore, our experiments have revealed that there are at least two alleles in this initial locus, both of which are required for resistance on a susceptible C57BL/6 background. These two alleles work in trans since resistance is restored in offspring possessing one allele contributed by each parent. Our studies also indicated that the Slc11a1 gene (formerly known as Nramp1) located within the chromosome1 locus is not responsible for conferring resistance to 129 mice.

Asunto(s)

Cromosomas de los Mamíferos , Resistencia a la Enfermedad/genética , Inmunidad Innata/genética , Yersinia pestis , Alelos , Animales , Mapeo Cromosómico , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Virulencia

RESUMEN

TLRs are central components of the innate immune system which, upon recognition of bacterial, fungal or viral components, activate intracellular signals that lead to protective inflammatory responses. Among the 10-member human TLR family, TLR10 is the only remaining orphan receptor without a known ligand or signaling function. Murine TLR10 is a disrupted pseudogene, which precludes investigation using classic gene knockout approaches. We report here that TLR10 suppressed the production of an array of cytokines in stably transfected human myelomonocytic U937 cells in response to other TLR agonists. This broad TLR suppressive activity affects both MyD88- and TRIF-inducing IFN-ß-mediated signaling pathways upstream of IκB and MAPK activation. Compared with nontransgenic littermate controls, monocytes of TLR10 transgenic mice exhibited blunted IL-6 production following ex vivo blood stimulation with other TLR agonists. After i.p. injection of LPS, lower levels of TNFα, IL-6, and type 1 IFN were measured in the serum of TLR10 transgenic mice compared to nontransgenic mice, but did not affect mouse survival in an LPS-induced septic shock model. Finally, treatment of human mononuclear cells with a monoclonal anti-TLR10 Ab suppressed proinflammatory cytokines released by LPS stimulation. These results demonstrate that TLR10 functions as a broad negative regulator of TLR signaling and suggests that TLR10 has a role in controlling immune responses in vivo.

Asunto(s)

Células Mieloides/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 10/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Humanos , Inmunidad Innata , Mediadores de Inflamación/metabolismo , Interferón beta/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal , Receptor Toll-Like 10/genética , Transgenes/genética , Factor de Necrosis Tumoral alfa/metabolismo , Células U937

RESUMEN

Bacterial lipopolysaccharide (LPS) activates the innate immune system by forming a complex with myeloid differentiation factor 2 (MD-2) and Toll-like receptor 4 (TLR4), which is present on antigen presenting cells. MD-2 plays an essential role in this activation of the innate immune system as a member of the ternary complex, TLR4:MD-2:LPS. With the goal of further understanding the molecular details of the interaction of MD-2 with LPS and TLR4, and possibly toward engineering dominant negative regulators of the MD-2 protein, here we subjected MD-2 to a mutational analysis using yeast display. The approach included generation of site-directed alanine mutants, and ligand-driven selections of MD-2 mutant libraries. Our findings showed that: (1) proline mutations in the F119-K132 loop that binds LPS were strongly selected for enhanced yeast surface stability, (2) there was a preference for positive-charged side chains (R/K) at residue 120 for LPS binding, and negative-charged side chains (D/E) for TLR4 binding, (3) aromatic residues were strongly preferred at F119 and F121 for LPS binding, and (4) an MD-2 mutant (T84N/D101A/S118A/S120D/K122P) exhibited increased binding to TLR4 but decreased binding to LPS. These studies revealed the impact of specific residues and regions of MD-2 on the binding of LPS and TLR4, and they provide a framework for further directed evolution of the MD-2 protein.

Asunto(s)

Lipopolisacáridos/metabolismo , Saccharomyces cerevisiae/genética , Receptor Toll-Like 4/genética , Secuencia de Aminoácidos , Sitios de Unión , Expresión Génica , Humanos , Inmunidad Innata , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Antígeno 96 de los Linfocitos/química , Antígeno 96 de los Linfocitos/genética , Antígeno 96 de los Linfocitos/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Biblioteca de Péptidos , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Saccharomyces cerevisiae/metabolismo , Electricidad Estática , Relación Estructura-Actividad , Receptor Toll-Like 4/química , Receptor Toll-Like 4/inmunología

RESUMEN

Bacterial lipoproteins are the most potent microbial agonists for the Toll-like receptor 2 (TLR2) subfamily, and this pattern recognition event induces cellular activation, leading to host immune responses. Triacylated bacterial lipoproteins coordinately bind TLR1 and TLR2, resulting in a stable ternary complex that drives intracellular signaling. The sensitivity of TLR-expressing cells to lipoproteins is greatly enhanced by two lipid-binding serum proteins known as lipopolysaccharide-binding protein (LBP) and soluble CD14 (sCD14); however, the physical mechanism that underlies this increased sensitivity is not known. To address this, we measured the ability of LBP and sCD14 to drive ternary complex formation between soluble extracellular domains of TLR1 and TLR2 and a synthetic triacylated lipopeptide agonist. Importantly, addition of substoichiometric amounts of either LBP or sCD14 significantly enhanced formation of a TLR1·TLR2 lipopeptide ternary complex as measured by size exclusion chromatography. However, neither LBP nor sCD14 was physically associated with the final ternary complex. Similar results were obtained using outer surface protein A (OspA), a naturally occurring triacylated lipoprotein agonist from Borrelia burgdorferi. Activation studies revealed that either LBP or sCD14 sensitized TLR-expressing cells to nanogram levels of either the synthetic lipopeptide or OspA lipoprotein agonist. Together, our results show that either LBP or sCD14 can drive ternary complex formation and TLR activation by acting as mobile carriers of triacylated lipopeptides or lipoproteins.

Asunto(s)

Receptores de Lipopolisacáridos/biosíntesis , Receptor Toll-Like 1/metabolismo , Receptor Toll-Like 2/metabolismo , Proteínas de Fase Aguda , Bacteriemia/microbiología , Proteínas Bacterianas/metabolismo , Unión Competitiva , Proteínas Portadoras , Ensayo de Inmunoadsorción Enzimática/métodos , Células HEK293 , Humanos , Inmunidad Innata , Inflamación , Lipopéptidos/química , Glicoproteínas de Membrana , Unión Proteica , Proteínas Recombinantes/química , Sepsis/microbiología , Transducción de Señal

RESUMEN

Human monocyte differentiation Ag CD14 is a pattern recognition receptor that enhances innate immune responses to infection by sensitizing host cells to bacterial LPS (endotoxin), lipoproteins, lipoteichoic acid, and other acylated microbial products. CD14 physically delivers these lipidated microbial products to various TLR signaling complexes that subsequently induce intracellular proinflammatory signaling cascades upon ligand binding. The ensuing cellular responses are usually protective to the host but can also result in host fatality through sepsis. In this work, we have determined the x-ray crystal structure of human CD14. The structure reveals a bent solenoid typical of leucine-rich repeat proteins with an amino-terminal pocket that presumably binds acylated ligands including LPS. Comparison of human and mouse CD14 structures shows great similarity in overall protein fold. However, compared with mouse CD14, human CD14 contains an expanded pocket and alternative rim residues that are likely to be important for LPS binding and cell activation. The x-ray crystal structure of human CD14 presented in this article may foster additional ligand-bound structural studies, virtual docking studies, and drug design efforts to mitigate LPS-induced sepsis and other inflammatory diseases.

Asunto(s)

Receptores de Lipopolisacáridos/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Lipopolisacáridos/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Relación Estructura-Actividad

RESUMEN

We recently identified I602S as a frequent single-nucleotide polymorphism of human TLR1 that greatly inhibits cell surface trafficking, confers hyporesponsiveness to TLR1 agonists, and protects against the mycobacterial diseases leprosy and tuberculosis. Because mycobacteria are known to manipulate the TLR system to their advantage, we hypothesize that the hyporesponsive 602S variant may confer protection by enabling the host to overcome this immune subversion. We report that primary human monocytes and macrophages from homozygous TLR1 602S individuals are resistant to mycobacterial-induced downregulation of macrophage MHC class II, CD64, and IFN-γ responses compared with individuals who harbor the TLR1 602I variant. Additionally, when challenged with mycobacterial agonists, macrophages from TLR1 602S/S individuals resist induction of host arginase-1, an enzyme that depletes cellular arginine stores required for the production of antimicrobial reactive nitrogen intermediates. The differences in cell activation mediated by TLR1 602S and TLR1 602I are observed upon stimulation with soluble mycobacterial-derived agonists but not with whole mycobacterial cells. Taken together, these results suggest that the TLR1 602S variant protects against mycobacterial disease by preventing soluble mycobacterial products, perhaps released from granulomas, from disarming myeloid cells prior to their encounter with whole mycobacteria.

Asunto(s)

Macrófagos/inmunología , Monocitos/inmunología , Mycobacterium tuberculosis/inmunología , Polimorfismo de Nucleótido Simple/genética , Receptor Toll-Like 1/metabolismo , Arginasa/genética , Arginasa/inmunología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Membrana Celular/microbiología , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Isoleucina/genética , Isoleucina/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Monocitos/efectos de los fármacos , Monocitos/microbiología , Mycobacterium tuberculosis/patogenicidad , Polimorfismo de Nucleótido Simple/inmunología , Transporte de Proteínas/efectos de los fármacos , Receptores de IgG/genética , Receptores de IgG/inmunología , Serina/genética , Serina/inmunología , Receptor Toll-Like 1/genética , Receptor Toll-Like 1/inmunología

RESUMEN

Genetic association studies of leprosy cohorts across the world have identified numerous polymorphisms which alter susceptibility and outcome to infection with Mycobacterium leprae. As expected, many of the polymorphisms reside within genes that encode components of the innate and adaptive immune system. Despite the preponderance of these studies, our understanding of the mechanisms that underlie these genetic associations remains sparse. Toll-like receptors (TLRs) have emerged as an essential family of innate immune pattern recognition receptors which play a pivotal role in host defense against microbes, including pathogenic strains of mycobacteria. This paper will highlight studies which have uncovered the association of specific TLR gene polymorphisms with leprosy or tuberculosis: two important diseases resulting from mycobacterial infection. This analysis will focus on the potential influence these polymorphic variants have on TLR expression and function and how altered TLR recognition or signaling may contribute to successful antimycobacterial immunity.

RESUMEN

The subcellular localization of Toll-like receptors (TLRs) is critical to their ability to function as innate immune sensors of microbial infection. We previously reported that an I602S polymorphism of human TLR1 is associated with aberrant trafficking of the receptor to the cell surface, loss of responses to TLR1 agonists, and differential susceptibility to diseases caused by pathogenic mycobacteria. Through an extensive analysis of receptor deletion and point mutants we have discovered that position 602 resides within a short 6 amino acid cytoplasmic region that is required for TLR1 surface expression. This short trafficking motif, in conjunction with the adjacent transmembrane domain, is sufficient to direct TLR1 to the cell surface. A serine at position 602 interrupts this trafficking motif and prevents cell surface expression of TLR1. Additionally, we have found that ER-resident TLR chaperones, PRAT4A and PRAT4B, act as positive and negative regulators of TLR1 surface trafficking, respectively. Importantly, either over-expression of PRAT4A or knock-down of PRAT4B rescues cell surface expression of the TLR1 602S variant. We also report that IFN-γ treatment of primary human monocytes derived from homozygous 602S individuals rescues TLR1 cell surface trafficking and cellular responses to soluble agonists. This event appears to be mediated by PRAT4A whose expression is strongly induced in human monocytes by IFN-γ. Collectively, these results provide a mechanism for the differential trafficking of TLR1 I602S variants, and highlight the distinct roles for PRAT4A and PRAT4B in the regulation of TLR1 surface expression.

Asunto(s)

Regulación de la Expresión Génica/fisiología , Chaperonas Moleculares/metabolismo , Monocitos/metabolismo , Receptor Toll-Like 1/biosíntesis , Sustitución de Aminoácidos , Animales , Antivirales/farmacología , Células COS , Chlorocebus aethiops , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Interferón gamma/farmacología , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/genética , Mutación Missense , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Receptor Toll-Like 1/genética

RESUMEN

Toll-like receptors (TLRs) play a central role in host defense by inducing inflammatory and adaptive immune responses following infection. Drugs that target TLRs are of considerable interest as potential inflammatory regulators, vaccine adjuvants, and novel immunotherapeutics. TLR2, in cooperation with either TLR1 or TLR6, mediates responses to a wide variety of microbial products as well as products of host tissue damage. In an effort to understand the structural basis of TLR2 recognition and uncover novel TLR2 agonists, a synthetic chemical library of 24,000 compounds was screened using an IL-8-driven luciferase reporter in cells expressing these human receptors. The screening yielded several novel TLR2-dependent activators that utilize TLR1, TLR6, or both as co-receptors. These novel small molecule compounds are aromatic in nature and structurally unrelated to any known TLR2 agonists. The three most potent compounds do not exhibit synergistic activity, nor do they act as pseudoantagonists toward natural TLR2 activators. Interestingly, two of the compounds exhibit species specificity and are inactive toward murine peritoneal macrophages. Mutational analysis reveals that although the central extracellular region of TLR1 is required for stimulation, there are subtle differences in the mechanism of stimulation mediated by the synthetic compounds in comparison with natural lipoprotein agonists. The three most potent compounds activate cells in the nanomolar range and stimulate cytokine production from human peripheral blood monocytes. Our results confirm the utility of high throughput screens to uncover novel synthetic TLR2 agonists that may be of therapeutic benefit.

Asunto(s)

Receptor Toll-Like 2/agonistas , Receptor Toll-Like 2/química , Animales , Técnicas Químicas Combinatorias/métodos , Simulación por Computador , Citocinas/metabolismo , Análisis Mutacional de ADN , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Inmunoterapia/métodos , Interleucina-8/química , Macrófagos/metabolismo , Ratones , Monocitos/metabolismo , Relación Estructura-Actividad

RESUMEN

TLRs are central receptors of the innate immune system that drive host inflammation and adaptive immune responses in response to invading microbes. Among human TLRs, TLR10 is the only family member without a defined agonist or function. Phylogenetic analysis reveals that TLR10 is most related to TLR1 and TLR6, both of which mediate immune responses to a variety of microbial and fungal components in cooperation with TLR2. The generation and analysis of chimeric receptors containing the extracellular recognition domain of TLR10 and the intracellular signaling domain of TLR1, revealed that TLR10 senses triacylated lipopeptides and a wide variety of other microbial-derived agonists shared by TLR1, but not TLR6. TLR10 requires TLR2 for innate immune recognition, and these receptors colocalize in the phagosome and physically interact in an agonist-dependent fashion. Computational modeling and mutational analysis of TLR10 showed preservation of the essential TLR2 dimer interface and lipopeptide-binding channel found in TLR1. Coimmunoprecipitation experiments indicate that, similar to TLR2/1, TLR2/10 complexes recruit the proximal adaptor MyD88 to the activated receptor complex. However, TLR10, alone or in cooperation with TLR2, fails to activate typical TLR-induced signaling, including NF-kappaB-, IL-8-, or IFN-beta-driven reporters. We conclude that human TLR10 cooperates with TLR2 in the sensing of microbes and fungi but possesses a signaling function distinct from that of other TLR2 subfamily members.

Asunto(s)

Inmunidad Innata , Modelos Inmunológicos , Transducción de Señal/inmunología , Receptor Toll-Like 10/fisiología , Receptor Toll-Like 1/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Línea Celular Tumoral , Espacio Extracelular/química , Espacio Extracelular/genética , Espacio Extracelular/inmunología , Humanos , Inmunidad Innata/genética , Lipopéptidos/síntesis química , Lipopéptidos/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Multimerización de Proteína/genética , Multimerización de Proteína/inmunología , Estructura Terciaria de Proteína/genética , Seudogenes/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/genética , Receptor Toll-Like 1/agonistas , Receptor Toll-Like 1/química , Receptor Toll-Like 1/deficiencia , Receptor Toll-Like 10/agonistas , Receptor Toll-Like 10/química , Receptor Toll-Like 10/deficiencia , Receptor Toll-Like 2/química , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 2/fisiología

RESUMEN

Peripheral activation of the immune system by infectious agents triggers the brain-cytokine system causing sickness behaviors which profoundly impact well-being. Dietary fiber is a beneficial foodstuff that, from a gastrointestinal tract perspective, exists in both insoluble and soluble forms. We show that a diet rich in soluble fiber protects mice from endotoxin-induced sickness behavior by polarizing mice Th2 when compared to a diet containing only insoluble fiber. Mice fed soluble fiber became less sick and recovered faster from endotoxin-induced sickness behaviors than mice fed insoluble fiber. In response to intraperitoneal endotoxin, mice fed soluble fiber had up-regulated IL-1RA and reduced IL-1beta and TNF-alpha in the brain as compared to mice fed insoluble fiber. Importantly, mice fed soluble fiber had a basal increase in IL-4 in the ileum and spleen which was absent in MyD88 knockout mice. Con-A stimulated splenocytes from mice fed soluble fiber showed increased IL-4 and IL-5 and decreased IL-2, IL-12 and IFN-gamma when compared to mice fed insoluble fiber. Likewise, endotoxin-stimulated macrophages from mice fed soluble fiber demonstrated decreased IL-1beta, TNF-alpha, IFN-gamma, IL-12 and nitrate and increased IL-1RA, arginase 1 and Ym1 when compared to mice fed insoluble fiber. Finally, the behavioral protection afforded by feeding mice soluble fiber was reduced in IL-4 knockout mice, as was the impact of soluble fiber on Con-A stimulated splenocytes and endotoxin activated macrophages. These data show that a diet rich in soluble fiber protects against endotoxin-induced sickness behavior by polarizing mice Th2 and promoting alternative activation of macrophages.

Asunto(s)

Citocinas/metabolismo , Dietoterapia/métodos , Fibras de la Dieta/farmacología , Endotoxinas/farmacología , Conducta de Enfermedad , Interleucina-4/metabolismo , Células Th2/metabolismo , Animales , Antidiarreicos/farmacología , Citocinas/genética , Citocinas/inmunología , Fibras de la Dieta/clasificación , Endotoxinas/administración & dosificación , Ensayo de Inmunoadsorción Enzimática , Íleon/citología , Íleon/efectos de los fármacos , Íleon/inmunología , Inyecciones Intraperitoneales , Interferón gamma/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Interleucina-12/genética , Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Interleucina-4/deficiencia , Interleucina-4/genética , Interleucina-4/inmunología , Interleucina-6/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/genética , Pectinas/farmacología , Reacción en Cadena de la Polimerasa , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inervación , Células Th2/efectos de los fármacos , Células Th2/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba

RESUMEN

Toll-like receptors (TLRs) are important sensors of foreign microbial components as well as products of damaged or inflamed self tissues. Upon sensing these molecules, TLRs initiate a series of downstream signaling events that drive cellular responses including the production of cytokines, chemokines, and other inflammatory mediators. This outcome results from the intracellular assembly of protein complexes that drive phosphorylation and other signaling cascades ultimately leading to chromatin remodeling and transcription factor activation. In addition to driving inflammatory responses, TLRs also regulate cell proliferation and survival which serves to expand useful immune cells and integrate inflammatory responses and tissue repair processes. In this context, central TLR signaling molecules, such as the mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinase (PI3K), play key roles. In addition, four major groups of transcription factors which are targets of TLR activation also control cell fate. This review focuses on the role of TLR signaling as it relates to cell proliferation and survival. This topic not only has important implications for understanding host defense and tissue repair, but also cancer which is often associated with conditions of chronic inflammation.

Asunto(s)

Proliferación Celular , Supervivencia Celular , Transducción de Señal/fisiología , Receptores Toll-Like/metabolismo , Activación Enzimática , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/metabolismo , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Regeneración/fisiología

RESUMEN

The expansion of sensing function by cell surface Toll-like receptors (TLRs) has grown to include not only more diverse viral, bacterial, fungal and protozoan surface components, but also a plethora of endogenous molecules arising from host cell and tissue damage as well as the inflammatory response itself. This flexibility in recognition is accommodated not only by physical and structural features of the TLRs themselves, but also by additional innate immune receptors, soluble molecules and subcellular trafficking mechanisms. These events have begun to reveal a remarkable plasticity and complexity within this critical arm of the host innate immune system.

Asunto(s)

Inmunidad Innata , Receptores Toll-Like/inmunología , Animales , Antígenos CD/inmunología , Humanos , Lipopolisacáridos/inmunología , Unión Proteica , Transducción de Señal , Receptores Toll-Like/metabolismo

RESUMEN

The pentameric B subunit of type IIb Escherichia coli enterotoxin (LT-IIb-B(5)), a doughnut-shaped oligomeric protein from enterotoxigenic E. coli, activates the TLR2/TLR1 heterodimer (TLR2/1). We investigated the molecular basis of the LT-IIb-B(5) interaction with TLR2/1 to define the structure-function relationship of LT-IIb-B(5) and, moreover, to gain an insight into how TLR2/1 recognizes large, nonacylated protein ligands that cannot fit within its lipid-binding pockets, as previously shown for the Pam(3)CysSerLys(4) (Pam(3)CSK(4)) lipopeptide. We first identified four critical residues in the upper region of the LT-IIb-B(5) pore. Corresponding point mutants (M69E, A70D, L73E, S74D) were defective in binding TLR2 or TLR1 and could not activate APCs, despite retaining full ganglioside-binding capacity. Point mutations in the TLR2/1 dimer interface, as determined in the crystallographic structure of the TLR2/1-Pam(3)CSK(4) complex, resulted in diminished activation by both Pam(3)CSK(4) and LT-IIb-B(5). Docking analysis of the LT-IIb-B(5) interaction with this apparently predominant activation conformation of TLR2/1 revealed that LT-IIb-B(5) might primarily contact the convex surface of the TLR2 central domain. Although the TLR1/LT-IIb-B(5) interface is relatively smaller, the leucine-rich repeat motifs 9-12 in the central domain of TLR1 were found to be critical for cooperative TLR2-induced cell activation by LT-IIb-B(5). Moreover, the putative LT-IIb-B(5) binding site overlaps partially with that of Pam(3)CSK(4); consistent with this, Pam(3)CSK(4) suppressed TLR2 binding of LT-IIb-B(5), albeit not as potently as self-competitive inhibition. We identified the upper pore region of LT-IIb-B(5) as a TLR2/1 interactive domain, which contacts the heterodimeric receptor at a site that is distinct from, although it overlaps with, that of Pam(3)CSK(4).

Asunto(s)

Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Escherichia coli Enterotoxigénica/química , Enterotoxinas/química , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Mapeo Peptídico , Receptor Toll-Like 1/metabolismo , Receptor Toll-Like 2/metabolismo , Animales , Toxinas Bacterianas/genética , Línea Celular Tumoral , Células Cultivadas , Cristalografía por Rayos X , Dimerización , Escherichia coli Enterotoxigénica/genética , Escherichia coli Enterotoxigénica/metabolismo , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Humanos , Lipopéptidos/química , Lipopéptidos/genética , Lipopéptidos/metabolismo , Ratones , Ratones Endogámicos BALB C , Mapeo Peptídico/métodos , Mutación Puntual , Unión Proteica/genética , Unión Proteica/inmunología , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptor Toll-Like 1/antagonistas & inhibidores , Receptor Toll-Like 1/química , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 2/química

RESUMEN

Spirochaeta aurantia is a free-living saprophytic spirochete that grows easily in simple laboratory media, and thus can be used as a model for the investigation of surface carbohydrate structures in spirochetae, which are normally not available in sufficient amounts. Freeze-substitution electron microscopy indicated the presence of a capsule-like material projecting from the surface of S. aurantia. Extraction of cells gave two major glycolipids, the one with a higher molecular mass glycolipid was designated large glycolipid A (LGLA). LGLA contained small amount of branched and unsaturated O-linked fatty acids, L: -rhamnose, L: -fucose, D: -xylose, D: -mannose, D: -glucosamine, D: -glycero-D: -gluco-heptose (DDglcHep), D: -glycero-D: -manno-heptose (DDHep), and a novel branched tetradeoxydecose monosaccharide, which we proposed to call aurantose (Aur). The carbohydrate structure of LGLA was extremely complex and consisted of the repeating units built of 11 monosaccharides, arrangement of nine of them was determined as: - [- 3 - beta - DDglcHep - 3 - beta - D - GlcNAc - 2 - beta - D - Man - ] - which wasdeduced from the NMR and chemical data on the LGLA and its fragments, obtained by various degradations. Tentative position of two remaining sugars is proposed. LGLA was negative for gelation of Limulus amebocyte lysate, did not contain lipid A, and was unable to activate any known Toll-like receptors.

Asunto(s)

Membrana Celular/metabolismo , Glucolípidos/análisis , Spirochaeta/metabolismo , Secuencia de Carbohidratos , Membrana Celular/ultraestructura , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Cromatografía de Gases y Espectrometría de Masas , Glucolípidos/química , Glucolípidos/aislamiento & purificación , Células HeLa , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Spirochaeta/ultraestructura , Receptores Toll-Like/metabolismo

RESUMEN

Recent culture-independent studies have revealed that a healthy vaginal ecosystem harbors a surprisingly complex assemblage of microorganisms. However, the spatial distribution and composition of vaginal microbial populations have not been investigated using molecular methods. Here, we evaluated site-specific microbial composition within the vaginal ecosystem and examined the influence of sampling technique in detection of the vaginal microbiota. 16S rRNA gene clone libraries were prepared from samples obtained from different locations (cervix, fornix, outer vaginal canal) and by different methods (swabbing, scraping, lavaging) from the vaginal tracts of eight clinically healthy, asymptomatic women. The data reveal that the vaginal microbiota is not homogenous throughout the vaginal tract but differs significantly within an individual with regard to anatomical site and sampling method used. Thus, this study illuminates the complex structure of the vaginal ecosystem and calls for the consideration of microenvironments when sampling vaginal microbiota as a clinical predictor of vaginal health.

Asunto(s)

Bacterias/clasificación , Bacterias/aislamiento & purificación , Biodiversidad , Vagina/microbiología , Adulto , Bacterias/genética , Femenino , Biblioteca de Genes , Humanos , Persona de Mediana Edad , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Adulto Joven

RESUMEN

The immune response to infection includes activation of the blood clotting system, leading to extravascular fibrin deposition to limit the spread of invasive microorganisms. Some bacteria have evolved mechanisms to counteract this host response. Pla, a member of the omptin family of Gram-negative bacterial proteases, promotes the invasiveness of the plague bacterium, Yersinia pestis, by activating plasminogen to plasmin to digest fibrin. We now show that the endogenous anticoagulant tissue factor pathway inhibitor (TFPI) is also highly sensitive to proteolysis by Pla and its orthologs OmpT in Escherichia coli and PgtE in Salmonella enterica serovar Typhimurium. Using gene deletions, we demonstrate that bacterial inactivation of TFPI requires omptin expression. TFPI inactivation is mediated by proteolysis since Western blot analysis showed that TFPI cleavage correlated with loss of anticoagulant function in clotting assays. Rates of TFPI inactivation were much higher than rates of plasminogen activation, indicating that TFPI is a better substrate for omptins. We hypothesize that TFPI has evolved sensitivity to proteolytic inactivation by bacterial omptins to potentiate procoagulant responses to bacterial infection. This may contribute to the hemostatic imbalance in disseminated intravascular coagulation and other coagulopathies accompanying severe sepsis.

Asunto(s)

Proteínas Bacterianas/metabolismo , Bacterias Gramnegativas/metabolismo , Infecciones por Bacterias Gramnegativas/metabolismo , Lipoproteínas/metabolismo , Serina Endopeptidasas/metabolismo , Proteínas Bacterianas/genética , Coagulación Intravascular Diseminada/metabolismo , Coagulación Intravascular Diseminada/microbiología , Eliminación de Gen , Bacterias Gramnegativas/genética , Infecciones por Bacterias Gramnegativas/genética , Humanos , Lipoproteínas/genética , Activadores Plasminogénicos/genética , Activadores Plasminogénicos/metabolismo , Sepsis/genética , Sepsis/metabolismo , Sepsis/microbiología , Serina Endopeptidasas/genética