RESUMEN
While Tn-Seq is a powerful tool to determine genome-wide bacterial fitness in high-throughput, culturing transposon-mutant libraries in pools can mask community or other complex single-cell phenotypes. Droplet Tn-Seq (dTn-Seq) solves this problem by microfluidics facilitated encapsulation of individual transposon mutants into growth medium-in-oil droplets, thereby enabling isolated growth, free from the influence of the population. Here we describe and validate microfluidic chip design, production, encapsulation, and dTn-Seq sample preparation. We determine that 1-3% of mutants in Streptococcus pneumoniae have a different fitness when grown in isolation and show how dTn-Seq can help identify leads for gene function, including those involved in hyper-competence, processing of alpha-1-acid glycoprotein, sensitivity against the human leukocyte elastase and microcolony formation. Additionally, we show dTn-Seq compatibility with microscopy, FACS and investigations of bacterial cell-to-cell and bacteria-host cell interactions. dTn-Seq reduces costs and retains the advantages of Tn-Seq, while expanding the method's original applicability.
Asunto(s)
Elementos Transponibles de ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Técnicas Analíticas Microfluídicas/métodos , Análisis de Secuencia de ADN/métodos , Análisis de la Célula Individual/métodos , ADN Bacteriano/genética , Biblioteca de Genes , Genes Bacterianos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Mutación , Análisis de la Célula Individual/instrumentación , Streptococcus pneumoniae/genéticaRESUMEN
Streptococcus pneumoniae (Spn) is an asymptomatic colonizer of the human nasopharynx but can also cause disease in the inner ear, meninges, lung and blood. Although various mechanisms contribute to the effective clearance of Spn, opsonophagocytosis by neutrophils is perhaps most critical. Upon phagocytosis, Spn is exposed to various degradative molecules, including a family of neutrophil serine proteases (NSPs) that are stored within intracellular granules. Despite the critical importance of NSPs in killing Spn, the bacterial proteins that are degraded by NSPs leading to Spn death are still unknown. In this report, we identify a 90kDa protein in a purified cell wall (CW) preparation, aminopeptidase N (PepN) that is degraded by the NSP neutrophil elastase (NE). Since PepN lacked a canonical signal sequence or LPxTG motif, we created a mutant expressing a FLAG tagged version of the protein and confirmed its localization to the CW compartment. We determined that not only is PepN a CW-localized protein, but also is a substrate of NE in the context of intact Spn cells. Furthermore, in comparison to wild-type TIGR4 Spn, a mutant strain lacking PepN demonstrated a significant hyper-resistance phenotype in vitro in the presence of purified NE as well as in opsonophagocytic assays with purified human neutrophils ex vivo. Taken together, this is the first study to demonstrate that PepN is a CW-localized protein and a substrate of NE that contributes to the effective killing of Spn by NSPs and human neutrophils.
Asunto(s)
Aminopeptidasas/metabolismo , Proteínas Bacterianas/metabolismo , Neutrófilos/metabolismo , Streptococcus pneumoniae/metabolismo , Aminopeptidasas/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Antígenos CD13/metabolismo , Pared Celular/metabolismo , Voluntarios Sanos , Humanos , Elastasa de Leucocito/metabolismo , Pulmón/metabolismo , Fagocitosis , Serina Proteasas/metabolismo , Streptococcus pneumoniae/patogenicidad , Adulto JovenRESUMEN
UNLABELLED: The polysaccharide capsule of Streptococcus pneumoniae is required for nasopharyngeal colonization and for invasive disease in the lungs, blood, and meninges. In contrast, the vast majority of conjunctival isolates are acapsular. The first serotype-specific gene in the capsule operon, cpsE, encodes the initiating glycosyltransferase and is one of the few serotype-specific genes that can tolerate null mutations. This report characterizes a spontaneously arising TIGR4 mutant exhibiting a reduced capsule, caused by a 6-nucleotide duplication in cpsE which results in duplication of Ala and Ile at positions 45 and 46. This strain (AI45dup) possessed more exposed phosphorylcholine and was hypersusceptible to C3 complement deposition compared to the wild type. Accordingly, the mutant was significantly better at forming abiotic biofilms and binding epithelial cells in vitro but was avirulent in a sepsis model. In vitro serial passaging of the wild-type strain failed to reproduce the AI45dup mutation but instead led to a variety of mutants with reduced capsule harboring single nucleotide polymorphisms (SNPs) in cpsE. A single passage in the sepsis model after high-dose inoculation readily yielded revertants of AI45dup with restored wild-type capsule level, but the majority of SNP alleles of cpsE could not revert, suppress, or bypass. Analysis of cpsE in conjunctival isolates revealed a strain with a single missense mutation at amino acid position 377, which was responsible for reduced encapsulation. This study supports the hypothesis that spontaneous, nonreverting mutations in cpsE serve as a form of adaptive mutation by providing a selective advantage to S. pneumoniae in niches where expression of capsule is detrimental. IMPORTANCE: While the capsule of Streptococcus pneumoniae is required for colonization and invasive disease, most conjunctival isolates are acapsular by virtue of deletion of the entire capsular operon. We show that spontaneous acapsular mutants isolated in vitro harbor mostly nonrevertible single nucleotide polymorphism (SNP) null mutations in cpsE, encoding the initiating glycosyltransferase. From a small collection of acapsular conjunctival isolates, we identified one strain with a complete capsular operon but containing a SNP in cpsE that we show is responsible for the acapsular phenotype. We propose that acapsular conjunctival isolates may arise initially from such nonreverting SNP null mutations in cpsE, which can be followed later by deletion of portions or all of the cps operon.
Asunto(s)
Cápsulas Bacterianas/metabolismo , Glicosiltransferasas/metabolismo , Mutación , Pase Seriado , Streptococcus pneumoniae/enzimología , Streptococcus pneumoniae/metabolismo , Conjuntiva/microbiología , Glicosiltransferasas/genética , Mutagénesis Insercional , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificación , VirulenciaRESUMEN
In murine schistosomiasis, immunopathology and cytokine production in response to parasite eggs are uneven and strain dependent. CBA/J (CBA) mice develop severe hepatic granulomatous inflammation associated with prominent Th17 cell responses driven by dendritic cell (DC)-derived IL-1ß and IL-23. Such Th17 cells fail to develop in low-pathology C57BL/6 (BL/6) mice, and the reasons for these strain-specific differences in APC reactivity to eggs remain unclear. We show by gene profiling that CBA DCs display an 18-fold higher expression of the C-type lectin receptor CD209a, a murine homolog of human DC-specific ICAM-3-grabbing nonintegrin, compared with BL/6 DCs. Higher CD209a expression was observed in CBA splenic and granuloma APC subpopulations, but only DCs induced Th17 cell differentiation in response to schistosome eggs. Gene silencing in CBA DCs and overexpression in BL/6 DCs demonstrated that CD209a is essential for egg-elicited IL-1ß and IL-23 production and subsequent Th17 cell development, which is associated with SRC, RAF-1, and ERK1/2 activation. These findings reveal a novel mechanism controlling the development of Th17 cell-mediated severe immunopathology in helminthic disease.
Asunto(s)
Moléculas de Adhesión Celular/inmunología , Células Dendríticas/inmunología , Regulación de la Expresión Génica/inmunología , Lectinas Tipo C/inmunología , Receptores de Superficie Celular/inmunología , Schistosoma/inmunología , Esquistosomiasis/inmunología , Células Th17/inmunología , Animales , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Línea Celular , Células Dendríticas/metabolismo , Células Dendríticas/patología , Activación Enzimática/genética , Activación Enzimática/inmunología , Femenino , Regulación de la Expresión Génica/genética , Silenciador del Gen/inmunología , Granuloma/genética , Granuloma/inmunología , Granuloma/patología , Humanos , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Interleucina-23/genética , Interleucina-23/inmunología , Interleucina-23/metabolismo , Lectinas Tipo C/biosíntesis , Lectinas Tipo C/genética , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/inmunología , Ratones , Ratones Endogámicos CBA , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/inmunología , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Proteínas Proto-Oncogénicas pp60(c-src)/inmunología , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Schistosoma/genética , Schistosoma/metabolismo , Esquistosomiasis/genética , Esquistosomiasis/metabolismo , Esquistosomiasis/patología , Bazo/inmunología , Bazo/metabolismo , Bazo/patología , Células Th17/metabolismo , Células Th17/patologíaRESUMEN
Streptococcus pneumoniae is a commensal of the human nasopharynx but can cause invasive diseases, including otitis media, pneumonia, sepsis, and meningitis. The capsular polysaccharide (capsule) is a critical virulence factor required for both asymptomatic colonization and invasive disease, yet the expression level is different in each anatomical site. During colonization, reduced levels of capsule promote binding to the host epithelium and biofilm formation, while during systemic infection, increased capsule is required to evade opsonophagocytosis. How this regulation of capsule expression occurs is incompletely understood. To investigate the contribution of transcriptional regulation on capsule level in the serotype 4 strain TIGR4, we constructed two mutants harboring a constitutive promoter that was either comparably weaker (Pcat) or stronger (PtRNAGlu) than the wild-type (WT) capsule promoter, Pcps. Mild reductions in cpsA and cpsE transcript levels in the Pcat promoter mutant resulted in a 2-fold reduction in total amounts of capsule and in avirulence in murine models of lung and blood infection. Additionally, the PtRNAGlu mutant revealed that, despite expressing enhanced levels of cpsA and cpsE and possessing levels of capsule comparable to those of WT TIGR4, it was still significantly attenuated in all tested in vivo niches. Further analysis using chimeric promoter mutants revealed that the WT -10 and -35 boxes are required for optimal nasopharyngeal colonization and virulence. These data support the hypothesis that dynamic transcriptional regulation of the capsule operon is required and that the core promoter region plays a central role in fine-tuning levels of capsule to promote colonization and invasive disease.
Asunto(s)
Cápsulas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Operón , Regiones Promotoras Genéticas , Streptococcus pneumoniae/fisiología , Streptococcus pneumoniae/patogenicidad , Factores de Virulencia/metabolismo , Animales , Bacteriemia/microbiología , Bacteriemia/patología , Cápsulas Bacterianas/genética , Portador Sano/microbiología , Modelos Animales de Enfermedad , Femenino , Ratones , Mutación , Infecciones Neumocócicas/microbiología , Neumonía Neumocócica/microbiología , Neumonía Neumocócica/patología , Streptococcus pneumoniae/genética , Transcripción Genética , Virulencia , Factores de Virulencia/genéticaRESUMEN
The vacuolar (H(+))-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation.
Asunto(s)
Células de la Médula Ósea/enzimología , Células Dendríticas/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Androstadienos/farmacología , Animales , Células de la Médula Ósea/citología , Células Dendríticas/citología , Femenino , Inmunosupresores/farmacología , Lisosomas/enzimología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos BALB C , Complejos Multiproteicos/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , WortmaninaRESUMEN
Schistosomiasis is a major tropical disease caused by trematode helminths in which the host mounts a pathogenic immune response against tissue-trapped parasite eggs. The immunopathology consists of egg antigen-specific CD4 T cell-mediated granulomatous inflammation that varies greatly in magnitude in humans and among mouse strains in an experimental model. New evidence, covered in this review, intimately ties the development of severe pathology to IL-17-producing CD4 T helper (Th17) cells, a finding that adds a new dimension to the traditional CD4 Th1 vs. Th2 cell paradigm. Most examined mouse strains, in fact, develop severe immunopathology with substantial Th17 as well as Th1 and Th2 cell responses; a solely Th2-polarized response is an exception that is only observed in low-pathology strains such as the C57BL/6. The ability to mount pathogenic Th17 cell responses is genetically determined and depends on the production of IL-23 and IL-1ß by antigen presenting cells following recognition of egg antigens; analyses of several F2 progenies of (high × low)-pathology strain crosses demonstrated that quantitative trait loci governing IL-17 levels and disease severity vary substantially from cross to cross. Low pathology is dominant, which may explain the low incidence of severe disease in humans; however, coinfection with intestinal nematodes can also dampen pathogenic Th17 cell responses by promoting regulatory mechanisms such as those afforded by alternatively activated macrophages and T regulatory cells. A better understanding of the pathways conducive to severe forms of schistosomiasis and their regulation should lead to interventions similar to those presently used to manage other immune-mediated diseases.
Asunto(s)
Interacciones Huésped-Parásitos/inmunología , Esquistosomiasis/inmunología , Células Th17/inmunología , Animales , Antígenos Helmínticos/inmunología , Modelos Animales de Enfermedad , Interacciones Huésped-Parásitos/genética , Humanos , Ratones , Esquistosomiasis/genéticaRESUMEN
CBA/J mice infected with the helminth Schistosoma mansoni develop severe CD4 T cell-mediated hepatic granulomatous inflammation against parasite eggs associated with a robust Th17 cell response. We investigated the requisites for Th17 cell development using novel CD4 T cells expressing a transgenic TCR specific for the major Sm-p40 egg Ag, which produce IL-17 when stimulated with live schistosome eggs. Neutralization of IL-23 or blockade of the IL-1 receptor, but not IL-6 neutralization, abrogated egg-induced IL-17 secretion by transgenic T cells, whereas exogenous IL-23 or IL-1ß reconstituted their ability to produce IL-17 when stimulated by syngeneic IL-12p40-deficient dendritic cells. Kinetic analysis demonstrated that IL-17 production was initiated by IL-23 and amplified by IL-1ß. Significantly, schistosome-infected IL-12p40-deficient or IL-1R antagonist-treated CBA/J mice developed markedly reduced hepatic immunopathology with a dampened egg Ag-specific IL-17 response. These results demonstrate that the IL-23-IL-1-IL-17 axis has a central role in the development of severe schistosome egg-induced immunopathology.
Asunto(s)
Antígenos Helmínticos/fisiología , Proteínas del Helminto/fisiología , Interleucina-1beta/fisiología , Interleucina-23/fisiología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/parasitología , Células Th17/inmunología , Células Th17/parasitología , Animales , Epítopos de Linfocito T/inmunología , Interferón gamma/biosíntesis , Interleucina-17/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Esquistosomiasis mansoni/metabolismo , Células Th17/patologíaRESUMEN
Infection with the trematode parasite Schistosoma mansoni results in a distinct heterogeneity of disease severity, both in humans and in an experimental mouse model. Severe disease is characterized by pronounced hepatic egg-induced granulomatous inflammation in a proinflammatory cytokine environment, whereas mild disease corresponds with reduced hepatic inflammation in a Th2 skewed cytokine environment. This marked heterogeneity indicates that genetic differences play a significant role in disease development, yet little is known about the genetic basis of dissimilar immunopathology. To investigate the role of genetic susceptibility in murine schistosomiasis, quantitative trait loci analysis was performed on F(2) progeny derived from SJL/J and C57BL/6 mice, which develop severe and mild pathology, respectively. In this study, we show that severe liver pathology in F(2) mice 7 wk after infection significantly correlated with an increase in the production of the proinflammatory cytokines IL-17, IFN-gamma, and TNF-alpha by schistosome egg Ag-stimulated mesenteric lymph node cells. Quantitative trait loci analysis identified several genetic intervals controlling immunopathology as well as IL-17 and IFN-gamma production. Egg granuloma size exhibited significant linkage to two loci, D4Mit203 and D17Mit82, both of which were inherited in a BL/6 dominant manner. Furthermore, a significant reduction of hepatic granulomatous inflammation and IL-17 production in interval-specific congenic mice demonstrated that the two identified genetic loci have a decisive effect on the development of immunopathology in murine schistosomiasis.
Asunto(s)
Interleucina-17/biosíntesis , Parasitosis Hepáticas/inmunología , Parasitosis Hepáticas/patología , Óvulo/inmunología , Sitios de Carácter Cuantitativo/inmunología , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/patología , Animales , Cruzamientos Genéticos , Femenino , Ligamiento Genético/inmunología , Granuloma/genética , Granuloma/inmunología , Granuloma/patología , Granuloma/prevención & control , Interleucina-17/antagonistas & inhibidores , Parasitosis Hepáticas/genética , Parasitosis Hepáticas/prevención & control , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Mapeo Físico de Cromosoma , Esquistosomiasis mansoni/genética , Esquistosomiasis mansoni/prevención & control , Especificidad de la EspecieRESUMEN
Infection with schistosomes results in a CD4 T cell-mediated inflammatory reaction against parasite eggs that varies greatly in magnitude both in humans as well as in mice. In the murine disease, the severe form of immunopathology correlates with high levels of IL-17. We now report that live schistosome eggs stimulate dendritic cells from high pathology-prone CBA mice to produce IL-12p40, IL-6, and TGF-beta, whereas those from low pathology-prone BL/6 mice only make TGF-beta. Moreover, egg-stimulated dendritic cells plus naive CD4 T cells from CBA mice resulted in increased levels of IL-6, IL-23, IL-1beta, as well as IL-17 and the chemokines CXCL1, CXCL2, and CCL2, whereas similarly treated BL/6 cell cocultures instead expressed higher IL-4, IL-5, IL-10, and the transcription factor Foxp3. Neutralization of IL-23 and IL-1, but not of IL-6 or IL-21, profoundly inhibited egg-induced IL-17 production in the CBA cocultures. Conversely, stimulation with schistosome eggs in the presence of exogenous IL-23 and IL-1beta induced BL/6 cells to make IL-17. These findings identify IL-23 and IL-1 as critical host factors that drive IL-17 production, and suggest that parasite recognition followed by a genetically determined innate proinflammatory response induces the development of Th17 cells and thus controls the outcome of immunopathology in schistosomiasis.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Interleucina-17/biosíntesis , Interleucina-1/biosíntesis , Interleucina-23/biosíntesis , Óvulo/inmunología , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/patología , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/parasitología , Células Cultivadas , Técnicas de Cocultivo , Citocinas/biosíntesis , Células Dendríticas/metabolismo , Células Dendríticas/parasitología , Femenino , Predisposición Genética a la Enfermedad , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Óvulo/metabolismo , Esquistosomiasis mansoni/genética , Índice de Severidad de la EnfermedadRESUMEN
Infection with the trematode helminth Schistosoma mansoni results in a parasite egg-induced, CD4 T-cell-mediated, hepatointestinal granulomatous and fibrosing inflammation that varies greatly in severity, with a higher frequency of milder forms typically occurring in regions where the disease is endemic. One possible explanation for this is that in these regions the degree of inflammation is lessened by widespread concurrent infection with gastrointestinal nematodes. We tested this hypothesis by establishing a murine coinfection model in which mice were infected with the intestinal nematode parasite Heligmosomoides polygyrus prior to infection with S. mansoni. In CBA mice that naturally display a severe form of schistosomiasis, preinfection with H. polygyrus resulted in a marked reduction in schistosome egg-induced hepatic immunopathology, which was associated with significant decreases in the levels of interleukin-17 (IL-17), gamma interferon, tumor necrosis factor alpha, IL-23, IL-6, and IL-1beta and with increases in the levels of IL-4, IL-5, IL-10, and transforming growth factor beta in mesenteric lymph node cells, purified CD4 T cells, and isolated liver granuloma cells. There also were increases in liver Ym1 and forkhead box P3 transcription factor expression. In another model of high-pathology schistosomiasis induced in C57BL/6 mice by immunization with schistosome egg antigens in complete Freund's adjuvant, coinfection with the nematodes also resulted in a marked inhibition of hepatic immunopathology accompanied by similar shifts in cytokine production. These findings demonstrate that intestinal nematodes prevent Th1- and Th17-cell-mediated inflammation by promoting a strong Th2-polarized environment associated with increases in the levels of alternatively activated macrophages and T regulatory cells, which result in significant amelioration of schistosome-induced immunopathology.
Asunto(s)
Citocinas/biosíntesis , Parasitosis Hepáticas/inmunología , Hígado/parasitología , Esquistosomiasis mansoni/complicaciones , Infecciones por Strongylida/complicaciones , Animales , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Hígado/inmunología , Hígado/patología , Parasitosis Hepáticas/parasitología , Parasitosis Hepáticas/patología , Macrófagos/inmunología , Ratones , Nematospiroides dubius/inmunología , Óvulo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/patología , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/patología , Linfocitos T Reguladores/inmunologíaRESUMEN
In infection with the trematode helminth Schistosoma mansoni, the severity of CD4 T cell-mediated hepatic granulomatous and fibrosing inflammation against parasite eggs varies considerably in humans and among mouse strains. In mice, either the natural high pathology, or high pathology induced by concomitant immunization with schistosome egg Ags (SEA) in CFA (SEA/CFA), results from a failure to contain a net proinflammatory cytokine environment. We previously demonstrated that the induction of severe immunopathology was dependent on the IL-12/IL-23 common p40 subunit, and correlated with an increase in IL-17, thus implying IL-23 in the pathogenesis. We now show that mice lacking the IL-23-specific subunit p19 are impaired in developing severe immunopathology following immunization with SEA/CFA, which is associated with a marked drop of IL-17 in the granulomas, but not in the draining mesenteric lymph nodes, and with a markedly suppressed SEA-specific IFN-gamma response regulated by a striking increase in IL-10. The granulomas are characterized by a significant reduction in Gr-1(+) cell recruitment and by alternative macrophage activation. Taken together, these results demonstrate that IL-23 per se is not necessary for the generation of IL-17-producing T cells, but is essential for the development of severe schistosome egg-induced immunopathology, and its absence cannot be overcome with other possible compensatory mechanisms.
Asunto(s)
Interleucina-17/biosíntesis , Interleucina-17/genética , Subunidad p19 de la Interleucina-23/fisiología , Óvulo/inmunología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/patología , Animales , Antígenos Helmínticos/administración & dosificación , Antígenos Helmínticos/inmunología , Células Cultivadas , Regulación de la Expresión Génica/inmunología , Interleucina-17/antagonistas & inhibidores , Subunidad p19 de la Interleucina-23/deficiencia , Subunidad p19 de la Interleucina-23/genética , Parasitosis Hepáticas/inmunología , Parasitosis Hepáticas/metabolismo , Parasitosis Hepáticas/patología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/parasitología , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esquistosomiasis mansoni/genética , Esquistosomiasis mansoni/mortalidad , Índice de Severidad de la Enfermedad , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/parasitología , Subgrupos de Linfocitos T/patologíaRESUMEN
In schistosomiasis mansoni, parasite eggs cause hepatointestinal granulomatous inflammation and fibrosis mediated by CD4 T cells specific for egg antigens. The severity of disease varies extensively in humans and among mouse strains. Marked disease exacerbation induced in typically low-pathology C57BL/6 mice by immunization with schistosome egg antigens (SEA) in complete Freund's adjuvant (SEA/CFA) correlates with elevated production of the proinflammatory cytokines gamma interferon (IFN-gamma) and interleukin-17 (IL-17), which are regulated by IL-12 and IL-23, respectively. Here we examined the effect on the schistosome infection of a third member of the IL-12 family of heterodimeric cytokines, IL-27, using SEA/CFA-immunized and unimmunized mice deficient in the IL-27 receptor chain WSX-1 (WSX-1(-/-)). SEA-stimulated bulk mesenteric lymph node cells or CD4 T cells from 7-week-infected WSX-1(-/-) mice produced significantly less IFN-gamma than did those from C57BL/6 mice, even though there was no difference between these mice in exacerbated hepatic egg-induced granulomatous inflammation or in the levels of IL-17 induced by immunization with SEA/CFA. A fraction of the cells in the granulomas stained positive for IL-27, but there were no significant differences between WSX-1(-/-) and BL/6 mice, nor were there differences in the number of CD4 T cells and eosinophils. A 24-week chronic infection resulted in markedly reduced levels of proinflammatory cytokines, including IFN-gamma, in WSX-1(-/-) mice, but again the magnitude of immunopathology was not significantly different between the two groups. These findings indicate that despite the impaired IFN-gamma production, IL-27 signaling has no significant effect on either the magnitude of egg-induced immunopathology or on its closest in vitro correlate, IL-17.
Asunto(s)
Interferón gamma/biosíntesis , Interleucina-17/fisiología , Schistosoma mansoni , Esquistosomiasis/inmunología , Esquistosomiasis/patología , Transducción de Señal/fisiología , Animales , Inflamación/prevención & control , Interferón gamma/inmunología , Interleucina-17/análisis , Interleucina-17/deficiencia , Interleucinas/análisis , Ratones , Esquistosomiasis/metabolismoRESUMEN
To study the effects of tumor-derived monocyte chemoattractant protein-1 (MCP-1, CCL2) on the anti-tumor immune response we used the 4T1 murine mammary carcinoma which constitutively expresses CCL2. We generated 4T1 that do not express detectable levels of CCL2 and found that the T cell response to the tumors were altered. Lymph nodes draining the CCL2- tumor contained CD62Llo cells that produced greater levels of INF-gamma in response to the tumor than CD62Llo cells from lymph nodes draining a tumor that produced CCL2. Moreover, exposure of splenic T cells to recombinant CCL2 in vitro decreased the ability of the T cells to produce IFN-gamma. However, despite the enhanced effector function evident in the absence of CCL2, vaccination/challenge experiments failed to reveal an increase in immunogenicity of the CCL2 null cells relative to the CCL2+ cells. Collectively, these data indicate that tumor-derived CCL2 could decrease T cell effector function, yet not the overall immunogenicity of the tumor.