RESUMEN
Vaccines against infectious diseases have had great successes in the history of public health. Major breakthroughs have occurred in the development of vaccine-based interventions against viral and bacterial pathogens through the application of classical vaccine design strategies. In contrast the development of a malaria vaccine has been slow. Plasmodium falciparum malaria affects millions of people with nearly half of the world population at risk of infection. Decades of dedicated research has taught us that developing an effective vaccine will be time consuming, challenging, and expensive. Nevertheless, recent advancements such as the optimization of robust protein synthesis platforms, high-throughput immunoscreening approaches, reverse vaccinology, structural design of immunogens, lymphocyte repertoire sequencing, and the utilization of artificial intelligence, have renewed the prospects of an accelerated discovery of the key antigens in malaria. A deeper understanding of the major factors underlying the immunological and molecular mechanisms of malaria might provide a comprehensive approach to identifying novel and highly efficacious vaccines. In this review we discuss progress in novel antigen discoveries that leverage on the wheat germ cell-free protein synthesis system (WGCFS) to accelerate malaria vaccine development.
Asunto(s)
Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Triticum/química , Sistema Libre de Células/inmunología , Biosíntesis de ProteínasRESUMEN
Biochemical reconstitution has served as an important tool for understanding the mechanisms of many cellular processes including DNA replication, transcription, translation, vesicle trafficking, and ubiquitin-mediated proteolysis. Here, we demonstrate that biochemical reconstitution can be applied to studying a complex signaling pathway involving as many as 12 proteins or protein complexes acting at the surface of model membranes. We show that a temporal sequence of events in activated T cells beginning with phosphorylation of the T cell receptor and culminating in the activation of actin polymerization can be replicated in vitro. Our reconstitution demonstrates the sufficiency of these proteins in producing many of the complex behaviors observed during T cell activation. The ability to manipulate all of the components, measure reaction rates, and observe molecular behaviors, including at single molecule resolution, has enabled us to gain insight into some of the important biochemical features of this signaling pathway such as microcluster formation. The same system could be adapted to study other membrane-proximal signaling pathways, including growth factor receptors, death receptors, and Eph receptors.
Asunto(s)
Membrana Dobles de Lípidos/química , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/química , Transducción de Señal , Linfocitos T/química , Animales , Sistema Libre de Células/química , Sistema Libre de Células/inmunología , Humanos , Membrana Dobles de Lípidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunologíaRESUMEN
Vaccination contributes to improved herd health and production. Boosting immune development at a young age may have long-term effects by enhancing vaccine immune response and efficacy. In the bovine, colostrum is the sole source of maternal immunity, having a substantial effect on health status in the neonate. To date, colostral antibody concentration is used to evaluate colostrum quality. However, colostrum also contains proteins and cells, which may affect immune development and future responses to vaccines. To determine the effect of maternal colostral cells on immune development, 37 female Holstein and Jersey dairy calves were bottle-fed 4 quarts total of whole colostrum (WC) or cell-free colostrum (CFC) at birth. Calves were vaccinated with 2 series of multivalent vaccines. Series A consisted of vaccines given between 1 and 4mo of life. Series B consisted of vaccines given between 5 and 10mo of life. Calf peripheral blood samples were obtained before each vaccination series and monthly for 3mo after each vaccination series. Cellular blood parameters were determined by flow cytometry. Quantitative real-time PCR was used to determine cytokine gene expression in peripheral blood mononuclear cells before vaccination series B and once a month for 2mo after vaccination series B. Calves fed CFC had fewer numbers of B cells in mo 2 after vaccination series A when compared with WC-fed calves. Calves fed CFC had decreased gene expression levels of IL-2 in mo 1 and numbers of CD4(+)CD62L(+)CD45RO(-) and CD4(+)CD62L(+)CD45RO(+) T cells in mo 0 and 1 after vaccination series B as compared with WC-fed calves. Our findings indicate a greater response to vaccines up to 6 to 10mo post-WC feeding when compared with CFC. These data suggest that adoptive transfer of maternal colostral cells at birth has a long-term effect on development of the neonatal immune system.
Asunto(s)
Animales Recién Nacidos/inmunología , Bovinos/inmunología , Calostro/inmunología , Sistema Inmunológico/crecimiento & desarrollo , Inmunidad Innata , Vacunación/veterinaria , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Bovinos/crecimiento & desarrollo , Sistema Libre de Células/inmunología , FemeninoRESUMEN
The long term efficacy of tissue based heart valve grafts may be limited by progressive degeneration characterized by immune mediated inflammation and calcification. To avoid this degeneration, decellularized heart valves with functionalized surfaces capable of rapid in vivo endothelialization have been developed. The aim of this study is to examine the capacity of CD133 antibody-conjugated valve tissue to capture circulating endothelial progenitor cells (EPCs). Decellularized human pulmonary valve tissue was conjugated with CD133 antibody at varying concentrations and exposed to CD133 expressing NTERA-2 cl.D1 (NT2) cells in a microflow chamber. The amount of CD133 antibody conjugated on the valve tissue surface and the number of NT2 cells captured in the presence of shear stress was measured. Both the amount of CD133 antibody conjugated to the valve leaflet surface and the number of adherent NT2 cells increased as the concentration of CD133 antibody present in the surface immobilization procedure increased. The data presented in this study support the hypothesis that the rate of CD133(+) cell adhesion in the presence of shear stress to decellularized heart valve tissue functionalized by CD133 antibody conjugation increases as the quantity of CD133 antibody conjugated to the tissue surface increases.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Bioprótesis , Células Progenitoras Endoteliales/inmunología , Glicoproteínas/inmunología , Prótesis Valvulares Cardíacas , Péptidos/inmunología , Válvula Pulmonar/citología , Antígeno AC133 , Línea Celular , Separación Celular/métodos , Sistema Libre de Células/inmunología , Sistema Libre de Células/patología , Humanos , Válvula Pulmonar/inmunologíaRESUMEN
B cells are important in the pathogenesis of multiple sclerosis (MS) and some of the effects are not dependent on maturation of B cells into immunoglobulin (Ig) producing plasmablasts and plasma cells. B cells present antigen, activate T cells, and are involved in immunoregulation and cytokine secretion. To determine if B cells from MS patients secrete products that have deleterious effects on glial cells not mediated by Ig, and to compare effects with secretory products of normal controls (NC), we isolated B cells from 7 patients with relapsing remitting MS (RRMS) and 4 NC. B cells were cultured alone or after stimulation with CD40 ligand (CD40L), CD40L+cross-linking of the B cell antigen receptor (xBCR) and CD40L+xBCR+stimulation of toll like receptor 9 (TLR9). Supernatants were harvested and incubated with mixed central nervous system (CNS) neonatal rat glial cells. Supernatants from unstimulated NC B cells induced on average death of 7% (range 0-24%) of differentiated oligodendrocytes (OL); in contrast, supernatants from unstimulated B cells from RRMS patients induced death of 57% (range 35-74%) of OL. Supernatants of stimulated B cells from NC did not increase the minimal OL death whereas stimulation of B cells from RRMS had variable results compared to unstimulated B cells. Supernatants from both NC and RRMS induced microglial enlargement and loss of normal resting bipolar morphology. OL death did not correlate with levels of tumor necrosis alpha (TNF-α), lymphotoxin alpha (LT-α), interleukin 6 (IL-6), IL-10, transforming growth factor beta 1 (TGF-ß1) or any combination or ratio of these cytokines. Analysis of 26 supernatants from NC and RRMS patients failed to detect IgM. There were very low levels of IgG in 8 of the 26 supernatants, and no correlation between of OL death and presence or absence of IgG. Sera used in both the B cell and glial cell cultures were heated, which inactivates complement. The effects of B cell supernatants on OL could be direct and/or indirect involving either microglia and/or astrocytes. The identity of the toxic factor(s) is as yet unknown. Thus we have demonstrated that B cells from patients with RRMS but not NC secrete one or more factors toxic to OL. It is possible that such factors produced by peripheral blood B cells when within the CNS could contribute to demyelination in MS patients.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Muerte Celular/inmunología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Esclerosis Múltiple Recurrente-Remitente/patología , Oligodendroglía/inmunología , Oligodendroglía/patología , Animales , Animales Recién Nacidos , Subgrupos de Linfocitos B/patología , Sistema Libre de Células/inmunología , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Células Cultivadas , Humanos , Microglía/inmunología , Microglía/patología , Esclerosis Múltiple Recurrente-Remitente/metabolismo , Oligodendroglía/citología , RatasRESUMEN
The binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellular immune responses. We have developed a versatile high throughput cell-free method to measure MHC stability by capturing a variety of MHC products on the surface of streptavidin-coated particles followed by flow cytometry analysis. Arrays of peptide-MHC combinations, generated by exchanging conditional ligand-loaded MHC, could be probed in a single experiment, thus combining the molecular precision of biochemically purified MHCs with high content multiparametric flow cytometry-based assays. Semiquantitative determination of the peptide affinity for the restriction element could also be accomplished through competition experiments using this bead-based assay. Furthermore, the generated peptide-MHC reagents could directly be applied to antigen-specific CD8(+) T lymphocyte analysis. The combinatorial labeling of beads allowed straightforward identification by their unique fluorescent signatures and provided a convenient means for extended assay multiplexing.
Asunto(s)
Linfocitos T CD8-positivos/química , Epítopos de Linfocito T/química , Citometría de Flujo/métodos , Antígenos de Histocompatibilidad Clase I/química , Biblioteca de Péptidos , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Sistema Libre de Células/química , Sistema Libre de Células/inmunología , Epítopos de Linfocito T/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , HumanosRESUMEN
In response to cell injury, caused, for example, by trauma, several processes must be initiated simultaneously to achieve an acute inflammatory response designed to prevent sustained tissue damage and infection and to restore and maintain tissue homeostasis. Detecting cell injury is facilitated by the fact that damaged cells release intracellular molecules not normally present in the extracellular space. However, potential underlying mechanisms for the recognition of endogenous danger signals released upon cell injury have yet to be elucidated. In this study, we demonstrate that mast cells, potent promoters of acute inflammation, play a key role in responding to cell injury by recognizing IL-33 released from necrotic structural cells. In an in vitro model of cell injury, this recognition was shown to involve the T1/ST2 receptor and result in the secretion of proinflammatory leukotrienes and cytokines by mouse mast cells. Remarkably, of all of the components released upon necrosis, our results show that IL-33 alone is a key component responsible for initiating proinflammatory responses in mast cells reacting to cell injury. Our findings identify IL-33 as a key danger signal released by necrotic structural cells capable of activating mast cells, thus providing novel insights concerning the role of mast cells as sensors of cell injury.
Asunto(s)
Interleucinas/metabolismo , Mastocitos/inmunología , Mastocitos/patología , Animales , Sistema Libre de Células/inmunología , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Células Cultivadas , Citocinas/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Interleucina-33 , Interleucinas/deficiencia , Interleucinas/genética , Leucotrienos/metabolismo , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NecrosisRESUMEN
Posttranscriptional regulation is emerging as a key factor in glucocorticoid (GC)-mediated gene regulation. We investigated the role of the human GC receptor (GR) as an RNA-binding protein and its effect on mRNA turnover in human airway epithelial cells. Cell treatment with the potent GC budesonide accelerated the decay of CCL2 mRNA (t(1/2) = 8 ± 1 min versus 62 ± 17 min in DMSO-treated cells) and CCL7 mRNA (t(1/2) = 15 ± 4 min versus 114 ± 37 min), but not that of CCL5 mRNA (t(1/2)=231 ± 8 min versus 266 ± 5 min) in the BEAS-2B cell line. This effect was inhibited by preincubation with an anti-GR Ab, indicating that GR itself plays a role in the turnover of these transcripts. Coimmunoprecipitation and biotin pulldown experiments showed that GR associates with CCL2 and CCL7 mRNAs, but not CCL5 mRNA. These methods confirmed CCL2 mRNA targeting by GR in human primary airway epithelial cells. Association of the GR was localized to the 5' untranslated region of CCL2 mRNA and further mapped to nt 44-60. The collection of transcripts associated with GR, identified by immunoprecipitation of GR-mRNA complexes followed by microarray analysis, revealed 479 transcripts that associated with GR. Computational analysis of the primary sequence and secondary structures of these transcripts yielded a GC-rich motif, which was shown to bind to GR in vitro. This motif was used to predict binding of GR to an additional 7889 transcripts. These results indicate that cytoplasmic GR interacts with a subset of mRNA through specific sequences and can regulate turnover rates, suggesting a novel posttranscriptional role for GR as an RNA-binding protein.
Asunto(s)
Quimiocina CCL2/metabolismo , Quimiocina CCL7/metabolismo , Sistemas de Liberación de Medicamentos , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transcripción Genética/inmunología , Línea Celular , Sistema Libre de Células/inmunología , Sistema Libre de Células/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL7/genética , Biología Computacional , Citoplasma/genética , Citoplasma/inmunología , Citoplasma/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Estabilidad del ARN/genética , Estabilidad del ARN/inmunología , ARN Mensajero/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Human T cell leukemia virus type 1 (HTLV-1) is associated with two immunologically distinct diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. We observed previously that depletion of dendritic cells (DCs) in CD11c-diphtheria toxin receptor transgenic mice followed by infection with cell-free virus led to greater proviral and Tax mRNA loads and diminished cellular immune response compared with mice infected with cell-associated virus. To understand the significance of these in vivo results and explore the host-pathogen interaction between DCs and cell-free HTLV-1, we used FLT3 ligand-cultured mouse bone marrow-derived DCs (FL-DCs) and chimeric HTLV-1. Phenotypically, the FL-DCs upregulated expression of surface markers (CD80, CD86, and MHC class II) on infection; however, the level of MHC class I remained unchanged. We performed kinetic studies to understand viral entry, proviral integration, and expression of the viral protein Tax. Multiplex cytokine profiling revealed production of an array of proinflammatory cytokines and type 1 IFN (IFN-α) by FL-DCs treated with virus. Virus-matured FL-DCs stimulated proliferation of autologous CD3(+) T cells as shown by intracellular nuclear Ki67 staining and produced IFN-γ when cultured with infected FL-DCs. Gene expression studies using type 1 IFN-specific and DC-specific arrays revealed upregulation of IFN-stimulated genes, most cytokines, and transcription factors, but a distinct downregulation of many chemokines. Overall, these results highlight the critical early responses generated by FL-DCs on challenge with cell-free chimeric HTLV-1.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/virología , Infecciones por HTLV-I/inmunología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Inmunidad Celular/inmunología , Proteínas de la Membrana/fisiología , Animales , Línea Celular , Sistema Libre de Células/inmunología , Sistema Libre de Células/metabolismo , Sistema Libre de Células/virología , Células Cultivadas , Citocinas/biosíntesis , Células Dendríticas/metabolismo , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Regulación Viral de la Expresión Génica/inmunología , Infecciones por HTLV-I/metabolismo , Infecciones por HTLV-I/virología , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
In Guillain-Barré syndrome (GBS), ganglioside mimicry of Campylobacter jejuni lipo-oligosaccharide (LOS) drives the production of cross-reactive Abs to peripheral nerve gangliosides. We determined whether sialic acid residues in C. jejuni LOS modulate dendritic cell (DC) activation and subsequent B cell proliferation as a possible mechanism for the aberrant humoral immune response in GBS. Highly purified sialylated LOS of C. jejuni isolates from three GBS patients induced human DC maturation and secretion of inflammatory cytokines that were inhibited by anti-TLR4 neutralizing Abs. The extent of TLR4 signaling and DC activation was greater with LOS of the wild type isolates than with nonsialylated LOS of the corresponding sialyltransferase gene knockout (cst-II mutant) strains, indicating that sialylation boosts the DC response to C. jejuni LOS. Supernatants of LOS-activated DCs induced B cell proliferation after cross-linking of surface Igs in the absence of T cells. Lower B cell proliferation indices were found with DC supernatants after DC stimulation with cst-II mutant or neuraminidase desialylated LOS. This study showed that sialylation of C. jejuni LOS enhances human DC activation and subsequent B cell proliferation, which may contribute to the development of cross-reactive anti-ganglioside Abs found in GBS patients following C. jejuni infection.
Asunto(s)
Antígenos Bacterianos/metabolismo , Campylobacter jejuni/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Síndrome de Guillain-Barré/inmunología , Síndrome de Guillain-Barré/metabolismo , Lipopolisacáridos/metabolismo , Receptor Toll-Like 4/fisiología , Animales , Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/microbiología , Campylobacter jejuni/química , Secuencia de Carbohidratos , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Proliferación Celular , Sistema Libre de Células/inmunología , Células Cultivadas , Reacciones Cruzadas , Citocinas/metabolismo , Células Dendríticas/microbiología , Técnicas de Sustitución del Gen , Síndrome de Guillain-Barré/microbiología , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Ácidos Siálicos/química , Ácidos Siálicos/inmunología , Ácidos Siálicos/metabolismo , Sialiltransferasas/deficiencia , Sialiltransferasas/genética , Transducción de Señal/inmunologíaRESUMEN
Human T cell leukemia virus type 1 (HTLV-1) is associated with two immunologically distinct diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. The genesis of these diseases is believed to be associated with the route (mucosa versus blood) and mode (cell-free versus cell-associated) of primary infection as well as the modulation of dendritic cell (DC) functions. To explore the role of DCs during early HTLV-1 infection in vivo, we used a chimeric HTLV-1 with a replaced envelope gene from Moloney murine leukemia virus to allow HTLV-1 to fuse with murine cells, which are generally not susceptible to infection with human retroviruses. We also used a CD11c-diphtheria toxin receptor transgenic mouse model system that permits conditional transient depletion of CD11c(+) DCs. We infected these transgenic mice with HTLV-1 using both cell-free and cell-associated infection routes in the absence and presence of DCs. The ablation of DCs led to an enhanced susceptibility to infection with cell-free but not cell-associated HTLV-1 in both CD4 and non-CD4 fractions, as measured by the proviral load. Infection with cell-free virus in the absence of DCs was also found to have increased levels of Tax mRNA in the non-CD4 fraction. Moreover, depletion of DCs significantly dampened the cellular immune response (IFN-gamma(+)CD8(+) T cells) against both cell-free and cell-associated virus. These results uniquely differentiate the involvement of DCs in early cell-free versus late cell-associated infection of HTLV-1 and highlight a significant aspect of viral immunopathogenesis related to the progression of adult T cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis after the initial infection.
Asunto(s)
Antígeno CD11c/genética , Células Dendríticas/inmunología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Péptidos y Proteínas de Señalización Intercelular/genética , Leucaféresis , Paraparesia Espástica Tropical/inmunología , Animales , Muerte Celular/genética , Muerte Celular/inmunología , Línea Celular , Sistema Libre de Células/inmunología , Sistema Libre de Células/patología , Sistema Libre de Células/virología , Células Dendríticas/patología , Toxina Diftérica/genética , Toxina Diftérica/metabolismo , Susceptibilidad a Enfermedades/inmunología , Susceptibilidad a Enfermedades/virología , Factor de Crecimiento Similar a EGF de Unión a Heparina , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucaféresis/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Paraparesia Espástica Tropical/metabolismo , Paraparesia Espástica Tropical/virologíaRESUMEN
IFN-gamma has been shown to inhibit monocyte (Mo) differentiation into mature dendritic cells (DC). Because IFN-gamma also plays a role in tolerance induction, we asked whether this could be related to generation of tolerogenic DC (Tol-DC). Toward this aim, we cultured Mo with GM-CSF plus IL-4 in the presence or absence of IFN-gamma for 6 days and induced their maturation with TNF-alpha for 2 additional days. We showed that IFN-gamma deviated Mo differentiation from mature DC toward Tol-DC. Indeed, IFN-gamma-generated DC 1) expressed moderate levels of costimulatory molecules, but high levels of Langerin and CD123 molecules, 2) were maturation resistant, and 3) were unable to efficiently present alloantigen to T cells. More interestingly, naive CD4(+) T cells primed with IFN-gamma-generated DC expressed FoxP3 mRNA at high levels and exerted regulatory functions upon secondary stimulation with alloantigen. To address whether endogenously secreted IFN-gamma mediates a similar effect, we used the alloreaction as a model. We showed that cell-free supernatant harvested from an HLA-mismatched, but not HLA-identical, alloresponse induced differentiation of Mo into Tol-DC able to promote regulatory T cell generation. Moreover, when supplemented with GM-CSF plus IL-4, HLA-mismatched cell-free supernatant inhibited differentiation of Mo into mature DC. Finally, by adding Abs directed against inflammatory cytokines, we demonstrated that IFN-gamma plays a preponderant role in this inhibition. In conclusion, our results clearly demonstrate that exogenous or endogenous IFN-gamma, as well, induces differentiation of Mo toward Tol-DC, which results in FoxP3(+) regulatory T cell promotion.
Asunto(s)
Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Factores de Transcripción Forkhead/biosíntesis , Tolerancia Inmunológica , Interferón gamma/metabolismo , Monocitos/inmunología , Linfocitos T Reguladores/inmunología , Sistema Libre de Células/inmunología , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Humanos , Inmunofenotipificación , Interferón gamma/fisiología , Interleucina-4/fisiología , Prueba de Cultivo Mixto de Linfocitos , Monocitos/citología , Linfocitos T Reguladores/citologíaRESUMEN
Exacerbation of disease in systemic lupus erythematosus (SLE) is associated with bacterial infection. In conventional dendritic cells (cDCs), the TLR4 ligand bacterial LPS induces IFN-beta gene expression but does not induce IFN-alpha. We hypothesized that when cDCs are primed by cytokines, as may frequently be the case in SLE, LPS would then induce the production of IFN-alpha, a cytokine believed to be important in lupus pathogenesis. In this study we show that mouse cDCs and human monocytes produce abundant IFN-alpha following TLR4 engagement whether the cells have been pretreated either with IFN-beta or with a supernatant from DCs activated by RNA-containing immune complexes from lupus patients. This TLR4-induced IFN-alpha induction is mediated by both an initial TRIF-dependent pathway and a subsequent MyD88-dependent pathway, in contrast to TLR3-induced IFN-alpha production, which is entirely TRIF-dependent. There is also a distinct requirement for IFN regulatory factors (IRFs), with LPS-induced IFN-alpha induction being entirely IRF7- and partially IRF5-dependent, in contrast to LPS-induced IFN-beta gene induction which is known to be IRF3-dependent but largely IRF7-independent. This data demonstrates a novel pathway for IFN-alpha production by cDCs and provides one possible explanation for how bacterial infection might precipitate disease flares in SLE.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Interferón-alfa/biosíntesis , Interferón beta/fisiología , Lipopolisacáridos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Sistema Libre de Células/inmunología , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Células Cultivadas , Humanos , Inmunoglobulina G/farmacología , Interferón-alfa/fisiología , Ligandos , Lipopolisacáridos/farmacología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Transducción de Señal/inmunología , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genéticaRESUMEN
The skin is constantly exposed to commensal microflora and pathogenic microbes. The stratum corneum of the outermost skin layer employs distinct tools such as harsh growth conditions and numerous antimicrobial peptides (AMPs) to discriminate between beneficial cutaneous microflora and harmful bacteria. How the skin deals with microbes that have gained access to the live part of the skin as a result of microinjuries is ill defined. In this study, we report that the chemokine CXCL14 is a broad-spectrum AMP with killing activity for cutaneous gram-positive bacteria and Candida albicans as well as the gram-negative enterobacterium Escherichia coli. Based on two separate bacteria-killing assays, CXCL14 compares favorably with other tested AMPs, including human beta-defensin and the chemokine CCL20. Increased salt concentrations and skin-typical pH conditions did not abrogate its AMP function. This novel AMP is highly abundant in the epidermis and dermis of healthy human skin but is down-modulated under conditions of inflammation and disease. We propose that CXCL14 fights bacteria at the earliest stage of infection, well before the establishment of inflammation, and thus fulfills a unique role in antimicrobial immunity.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/fisiología , Quimiocinas CXC/fisiología , Enfermedades Cutáneas Infecciosas/inmunología , Enfermedades Cutáneas Infecciosas/terapia , Animales , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Candidiasis/inmunología , Candidiasis/microbiología , Candidiasis/terapia , Línea Celular Transformada , Línea Celular Tumoral , Sistema Libre de Células/inmunología , Sistema Libre de Células/microbiología , Quimiocinas CXC/antagonistas & inhibidores , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Relación Dosis-Respuesta Inmunológica , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/terapia , Infecciones por Bacterias Grampositivas/inmunología , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/terapia , Humanos , Mediadores de Inflamación/aislamiento & purificación , Mediadores de Inflamación/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Enfermedades Cutáneas Infecciosas/microbiología , Enfermedades Cutáneas Infecciosas/patologíaRESUMEN
The activation of neutrophil granulocytes has to be carefully controlled to balance desired activity against invading pathogens while avoiding overwhelming activation leading to host tissue damage. We now show that phospholipids are potential key players in this process by either enhancing or dampening the production of reactive oxygen species (ROS) during the oxidative burst. Unoxidized phospholipids induce the production of ROS, and they also work synergistically with FMLP in potentiating the oxidative burst in neutrophil granulocytes. Oxidation of these phospholipids, however, turns them into potent inhibitors of the oxidative burst. OxPls specifically inhibit ROS production by inhibiting the assembly of the phagocyte oxidase complex but do not alter neutrophil viability, nor do they interfere with MAPK activation. Furthermore, up-regulation of the activation marker Mac-1 and phagocytosis of bacteria is not affected. Therefore, phospholipids may act as sensors of oxidative stress in tissues and either positively or negatively regulate neutrophil ROS production according to their oxidation state.
Asunto(s)
Peroxidación de Lípido , Neutrófilos/metabolismo , Fosfolípidos/metabolismo , Estallido Respiratorio/inmunología , Sistema Libre de Células/efectos de los fármacos , Sistema Libre de Células/inmunología , Humanos , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/inmunología , Activación Neutrófila/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/fisiología , Fosfatidilgliceroles/metabolismo , Fosfatidilgliceroles/fisiología , Fosfatidilserinas/metabolismo , Fosfatidilserinas/fisiología , Fosfolípidos/clasificación , Fosfolípidos/fisiología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/farmacología , Estallido Respiratorio/efectos de los fármacosRESUMEN
The activation of Ag-specific T cells locally in the CNS could potentially contribute to the development of immune-mediated brain diseases. We addressed whether Ag-specific T cells could be stimulated in the CNS in the absence of peripheral lymphoid tissues by analyzing Ag-specific T cell responses in organotypic brain slice cultures. Organotypic brain slice cultures were established 1 h after intracerebral OVA Ag microinjection. We showed that when OVA-specific CD8(+) T cells were added to Ag-containing brain slices, these cells became activated and migrated into the brain to the sites of their specific Ags. This activation of OVA-specific T cells was abrogated by the deletion of CD11c(+) cells from the brain slices of the donor mice. These data suggest that brain-resident CD11c(+) cells stimulate Ag-specific naive CD8(+) T cells locally in the CNS and may contribute to immune responses in the brain.
Asunto(s)
Encéfalo/citología , Encéfalo/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Activación de Linfocitos/inmunología , Animales , Encéfalo/metabolismo , Antígeno CD11c/biosíntesis , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Movimiento Celular/genética , Movimiento Celular/inmunología , Proliferación Celular , Sistema Libre de Células/inmunología , Sistema Libre de Células/metabolismo , Técnicas de Cocultivo , Epítopos de Linfocito T/biosíntesis , Epítopos de Linfocito T/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Especificidad de Órganos/inmunología , Ovalbúmina/inmunologíaRESUMEN
Soluble HLA-G (sHLA-G) secretion by human preimplantation embryos in culture has been associated with successful embryo development, and therefore has potential to serve as a noninvasive marker of embryo viability. We have examined the spatial and temporal expression of HLA-G in embryos of varying developmental competence and the role of maternal factors in human embryonic HLA-G expression. Embryos that reached blastocyst stage on day 5 showed a higher frequency of sHLA-G secretion than those at morula or arrested stages (p < 0.05). There was no significant difference in sHLA-G secretion between normal embryos and those diagnosed as chromosomally abnormal by preimplantation genetic diagnosis. HLA-G detected in maternal plasma and follicular fluid did not appear to correlate with HLA-G expressed in the embryo or embryo supernatants. Confocal microscopy analysis indicated that HLA-G protein expression in embryos was not homogeneous; mostly, it was confined to blastocysts localized on trophectoderm and trophectoderm projections. Single-particle fluorescent imaging analysis of HLA-G on the cell surface of JEG-3 cells showed that HLA-G particles were mostly monomeric, but dimeric and higher order oligomers were also observed. These results suggest that HLA-G play an important role in preimplantation embryo development. However, the observed expression of HLA-G in arrested and chromosomally abnormal embryos indicates that HLA-G testing should be used with caution and in conjunction with conventional methods of embryo screening and selection.
Asunto(s)
Blastocisto/química , Blastocisto/inmunología , Líquido Folicular/química , Líquido Folicular/inmunología , Antígenos HLA/análisis , Antígenos HLA/genética , Antígenos de Histocompatibilidad Clase I/análisis , Antígenos de Histocompatibilidad Clase I/genética , Intercambio Materno-Fetal/inmunología , Diagnóstico Preimplantación , Blastocisto/metabolismo , Línea Celular Tumoral , Sistema Libre de Células/química , Sistema Libre de Células/inmunología , Sistema Libre de Células/metabolismo , Técnicas de Cultivo de Embriones , Femenino , Líquido Folicular/metabolismo , Antígenos HLA/biosíntesis , Antígenos HLA/sangre , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/sangre , Humanos , Microscopía Confocal , Microscopía Fluorescente , Óvulo/química , Óvulo/inmunología , Óvulo/metabolismo , Embarazo , Diagnóstico Preimplantación/instrumentación , Diagnóstico Preimplantación/métodosRESUMEN
Dendritic cells (DCs) are APCs that play an essential role by bridging innate and adaptive immunity. DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is one of the major C-type lectins expressed on DCs and exhibits high affinity for nonsialylated Lewis (Le) glycans. Recently, we reported the characterization of oligosaccharide ligands expressed on SW1116, a typical human colorectal carcinoma recognized by mannan-binding protein, which is a serum C-type lectin and has similar carbohydrate-recognition specificities as DC-SIGN. These tumor-specific oligosaccharide ligands were shown to comprise clusters of tandem repeats of Lea/Leb epitopes. In this study, we show that DC-SIGN is involved in the interaction of DCs with SW1116 cells through the recognition of aberrantly glycosylated forms of Lea/Leb glycans on carcinoembryonic Ag (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1). DC-SIGN ligands containing Lea/Leb glycans are also highly expressed on primary cancer colon epithelia but not on normal colon epithelia, and DC-SIGN is suggested to be involved in the association between DCs and colorectal cancer cells in situ by DC-SIGN recognizing these cancer-related Le glycan ligands. Furthermore, when monocyte-derived DCs (MoDCs) were cocultured with SW1116 cells, LPS-induced immunosuppressive cytokines such as IL-6 and IL-10 were increased. The effects were significantly suppressed by blocking Abs against DC-SIGN. Strikingly, LPS-induced MoDC maturation was inhibited by supernatants of cocultures with SW1116 cells. Our findings imply that colorectal carcinomas affecting DC function and differentiation through interactions between DC-SIGN and colorectal tumor-associated Le glycans may induce generalized failure of a host to mount an effective antitumor response.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular/inmunología , Neoplasias Colorrectales/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/patología , Glicoesfingolípidos/metabolismo , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/metabolismo , Antígenos CD/metabolismo , Antígeno CA-19-9 , Antígeno Carcinoembrionario/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Sistema Libre de Células/inmunología , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Técnicas de Cocultivo , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Glicosilación , Humanos , Antígenos del Grupo Sanguíneo de Lewis , Ligandos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Monocitos/metabolismo , Monocitos/patología , Células U937RESUMEN
Decellularization treatment of heart valves has been thought to eliminate tissue immunogenicity. Early failure of tissue-engineered xenogeneic heart valves was seen in children and has been a major drawback in this promising field of research. This study was designed to characterize the effects of acellular porcine heart valve tissue on immune activation in vitro. Incubation of decellularized porcine tissue with human plasma led to adsorption of IgG, activation of the classical complement pathway and adhesion of activated polymorphonuclear leukocytes (PMN). This inflammatory response was strongly inhibited by proteins extracted from native porcine tissue which might indicate that inhibitors of PMN activation present in the extracellular matrix (ECM) are lost during the decellularization process.
Asunto(s)
Activación de Complemento/inmunología , Vía Clásica del Complemento/inmunología , Granulocitos/inmunología , Válvulas Cardíacas/citología , Válvulas Cardíacas/inmunología , Inmunoglobulina G/inmunología , Anciano , Animales , Sistema Libre de Células/inmunología , Células Cultivadas , Femenino , Humanos , Masculino , PorcinosRESUMEN
Glycoprotein G (gG) of alphaherpesviruses has been described to function as a viral chemokine-binding protein (vCKBP). More recently, mutant viruses devoid of gG have been shown to result in increased virulence, but it remained unclear whether the potential of gG to serve as a vCKBP is responsible for this observation. In this study, we used equine herpesvirus type 1 (EHV-1) as a model to study the pathophysiological importance of vCKBP activity. First, in vitro chemotaxis assays studying migration of immune cells, an important function of chemokines, were established. In such assays, supernatants of EHV-1-infected cells significantly inhibited IL-8-induced chemotaxis of equine neutrophils. Identification of gG as the responsible vCKBP was achieved by repeating similar experiments with supernatants from cells infected with a gG-negative mutant, which were unable to alter IL-8-induced equine neutrophil migration. Furthermore, rEHV-1 gG was able to significantly reduce neutrophil migration, establishing gG as a bona fide vCKBP. Second, and importantly, in vivo analyses in a murine model of EHV-1 infection showed that neutrophil migration in the target organ lung was significantly reduced in the presence of gG. In summary, we demonstrate for the first time that EHV-1 gG not only binds to chemokines but is also capable of inhibiting their chemotactic function both in vitro and in vivo, thereby contributing to viral pathogenesis and virulence.