RESUMEN
Inflammasome activation is associated with numerous diseases. However, in vivo detection of the activated inflammasome complex has been limited by a dearth of tools. We have developed transgenic mice that ectopically express the fluorescent adaptor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and characterized the formation of assembled inflammasome complexes ("specks") in primary cells and tissues. In addition to hematopoietic cells, we have found that a stromal population in the lung tissues formed specks during the early phase of influenza infection, whereas myeloid cells showed speck formation after 2 days. In a peritonitis and group B streptococcus infection model, a higher percentage of neutrophils formed specks at early phases of infection, while dendritic cells formed specks at later time points. Furthermore, speck-forming cells underwent pyroptosis and extensive release of specks to the extracellular milieu in vivo. These data underscore the importance of free specks during inflammatory processes in vivo.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/genética , Inflamasomas/metabolismo , Animales , Proteínas Adaptadoras de Señalización CARD/biosíntesis , Femenino , Expresión Génica , Genes Reporteros , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/metabolismo , Peritonitis/inmunología , Peritonitis/metabolismo , Multimerización de Proteína , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/metabolismoRESUMEN
The CD200R1:CD200 axis is traditionally considered to limit tissue inflammation by down-regulating pro-inflammatory signaling in myeloid cells bearing the receptor. We generated CD200R1(-/-) mice and employed them to explore both the role of CD200R1 in regulating macrophage signaling via TLR2 as well as the host response to an in vivo, TLR2-dependent model, herpes simplex virus 1 (HSV-1) infection. CD200R1(-/-) peritoneal macrophages demonstrated a 70-75% decrease in the generation of IL-6 and CCL5 (Rantes) in response to the TLR2 agonist Pam(2)CSK(4) and to HSV-1. CD200R1(-/-) macrophages could neither up-regulate the expression of TLR2, nor assemble a functional inflammasome in response to HSV-1. CD200R1(-/-) mice were protected from HSV-1 infection and exhibited dysfunctional TLR2 signaling. Finally, both CD200R1(-/-) mice and CD200R1(-/-) fibroblasts and macrophages showed a markedly reduced ability to support HSV-1 replication. In summary, our data demonstrate an unanticipated and novel requirement for CD200R1 in "licensing" pro-inflammatory functions of TLR2 and in limiting viral replication that are supported by ex vivo and in vivo evidence.
Asunto(s)
Antígenos de Superficie/metabolismo , Herpesvirus Humano 1/fisiología , Inflamación/inmunología , Receptores de Superficie Celular/metabolismo , Transducción de Señal/inmunología , Receptor Toll-Like 2/metabolismo , Replicación Viral/fisiología , Animales , Encéfalo/inmunología , Encéfalo/patología , Encéfalo/virología , Embrión de Mamíferos/citología , Encefalitis/inmunología , Encefalitis/patología , Encefalitis/virología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/virología , Marcación de Gen , Inflamación/patología , Interferón Tipo I/biosíntesis , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Macrófagos Peritoneales/virología , Ratones , Receptores de Orexina , Receptores de Superficie Celular/deficiencia , Carga Viral/inmunologíaRESUMEN
It is estimated that half of the world's population is infected by Helicobacter pylori (H. pylori) (Polk and Peek, Nat Rev Cancer 10:403-414, 2010; Peek et al., Physiol Rev 90:831-858, 2010). Following infection, H. pylori induces a chronic innate immune response that is thought to contribute to gastric complications. Due to the widespread prevalence of H. pylori, it is important to study the innate immune responses that result from the infection. A variety of in vitro and in vivo techniques have been developed by our laboratory to study this immune response (Fox et al., Am J Pathol 171:1520-1528, 2007; Kurt-Jones et al., Infect Immun 75:471-480, 2007; Kurt-Jones et al., J Endotoxin Res 10:419-424, 2004). These methods are described here.
Asunto(s)
Infecciones por Helicobacter/inmunología , Helicobacter pylori/inmunología , Inmunidad Innata , Animales , Técnicas de Cultivo de Célula , Separación Celular , Citocinas/metabolismo , Disección , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Infecciones por Helicobacter/microbiología , Helicobacter pylori/patogenicidad , Humanos , Luciferasas/genética , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Especificidad de la Especie , Estómago/microbiología , Estómago/patología , TransfecciónRESUMEN
Type I interferons (IFNs), predominantly IFN-α and -ß, play critical roles in both innate and adaptive immune responses against viral infections. Interferon regulatory factor 7 (IRF7), a key innate immune molecule in the type I IFN signaling pathway, is essential for the type I IFN response to many viruses, including lymphocytic choriomeningitis virus (LCMV). Here, we show that although IRF7 knockout (KO) mice failed to control the replication of LCMV in the early stages of infection, they were capable of clearing LCMV infection. Despite the lack of type I IFN production, IRF7 KO mice generated normal CD4(+) T cell responses, and the expansion of naïve CD8(+) T cells into primary CD8(+) T cells specific for LCMV GP(33-41) was relatively normal. In contrast, the expansion of the LCMV NP(396)-specific CD8(+) T cells was severely impaired in IRF7 KO mice. We demonstrated that this defective CD8(+) T cell response is due neither to an impaired antigen-presenting system nor to any intrinsic role of IRF7 in CD8(+) T cells. The lack of a type I IFN response in IRF7 KO mice did not affect the formation of memory CD8(+) T cells. Thus, the present study provides new insight into the impact of the innate immune system on viral pathogenesis and demonstrates the critical contribution of innate immunity in controlling virus replication in the early stages of infection, which may shape the quality of CD8(+) T cell responses.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Factor 7 Regulador del Interferón/inmunología , Interferón Tipo I/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Inmunidad Adaptativa , Animales , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , Inmunidad Innata , Factor 7 Regulador del Interferón/genética , Activación de Linfocitos , Coriomeningitis Linfocítica/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Replicación ViralRESUMEN
The toll-like receptors comprise one of the most conserved components of the innate immune system, signaling the presence of molecules of microbial origin. It has been proposed that signaling through TLR4, which requires CD14 to recognize bacterial lipopolysaccharide (LPS), may generate low-grade inflammation and thereby affect insulin sensitivity and glucose metabolism. To examine the long-term influence of partial innate immune signaling disruption on glucose homeostasis, we analyzed knockout mice deficient in CD14 backcrossed into the diabetes-prone C57BL6 background at 6 or 12 months of age. CD14-ko mice, fed either normal or high-fat diets, displayed significant glucose intolerance compared to wild type controls. They also displayed elevated norepinephrine urinary excretion and increased adrenal medullary volume, as well as an enhanced norepinephrine secretory response to insulin-induced hypoglycemia. These results point out a previously unappreciated crosstalk between innate immune- and sympathoadrenal- systems, which exerts a major long-term effect on glucose homeostasis.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Glucosa/metabolismo , Homeostasis , Receptores de Lipopolisacáridos/metabolismo , Adiposidad/efectos de los fármacos , Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/patología , Animales , Peso Corporal/efectos de los fármacos , Grasas de la Dieta/farmacología , Ayuno/sangre , Ácidos Grasos/farmacología , Prueba de Tolerancia a la Glucosa , Homeostasis/efectos de los fármacos , Humanos , Hipoglucemia/sangre , Hipoglucemia/patología , Hipoglucemia/fisiopatología , Insulina/sangre , Lípidos/sangre , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/efectos de los fármacosRESUMEN
Herpes simplex virus 1 (HSV-1) causes a spectrum of disease, including herpes labialis, herpes keratitis, and herpes encephalitis, which can be lethal. Viral recognition by pattern recognition receptors plays a central role in cytokine production and in the generation of antiviral immunity. The relative contributions of different Toll-like receptors (TLRs) in the innate immune response during central nervous system infection with HSV-1 have not been fully characterized. In this study, we investigate the roles of TLR2, TLR9, UNC93B1, and the type I interferon (IFN) receptor in a murine model of HSV-1 encephalitis. TLR2 is responsible for detrimental inflammatory cytokine production following intracranial infection with HSV-1, and the absence of TLR2 expression leads to increased survival in mice. We prove that inflammatory cytokine production by microglial cells, astrocytes, neutrophils, and monocytes is mediated predominantly by TLR2. We also demonstrate that type I IFNs are absolutely required for survival following intracranial HSV-1 infection, as mice lacking the type I IFN receptor succumb rapidly following infection and have high levels of HSV in the brain. However, the absence of TLR9 does not impact survival, type I IFN levels, or viral replication in the brain following infection. The absence of UNC93B1 leads to a survival disadvantage but does not impact viral replication or type I IFN levels in the brain in HSV-1-infected mice. These results illustrate the complex but important roles that innate immune receptors play in host responses to HSV-1 during infection of the central nervous system.
Asunto(s)
Sistema Nervioso Central/inmunología , Herpes Simple/inmunología , Herpesvirus Humano 1/fisiología , Inmunidad Innata , Animales , Sistema Nervioso Central/virología , Herpes Simple/virología , Herpesvirus Humano 1/inmunología , Humanos , Interferón Tipo I/inmunología , Ratones , Ratones Endogámicos C57BL , Receptor de Interferón alfa y beta/inmunología , Receptores Toll-Like/inmunologíaRESUMEN
UNC93B1 associates with TLR3, 7, and 9, mediating their translocation from the endoplasmic reticulum to the endolysosome, thus allowing proper activation by microbial nucleic acids. We found that the triple-deficient 3d mice, which lack functional UNC93B1 as well as functional endosomal TLRs, are highly susceptible to infection with Trypanosoma cruzi. The enhanced parasitemia and mortality in 3d animals were associated with impaired proinflammatory response, including reduced levels of IL-12p40 and IFN-γ. Importantly, the phenotype of 3d mice was intermediary between MyD88(-/-) (highly susceptible) and TLR9(-/-) (moderately susceptible), indicating the involvement of an additional UN93B1-dependent TLR(s) on host resistance to T. cruzi. Hence, our experiments also revealed that TLR7 is a critical innate immune receptor involved in recognition of parasite RNA, induction of IL-12p40 by dendritic cells, and consequent IFN-γ by T lymphocytes. Furthermore, we show that upon T. cruzi infection, triple TLR3/7/9(-/-) mice had similar phenotype than 3d mice. These data imply that the nucleic acid-sensing TLRs are critical determinants of host resistance to primary infection with T. cruzi.
Asunto(s)
Enfermedad de Chagas/inmunología , Inmunidad Innata , Glicoproteínas de Membrana/inmunología , Proteínas de Transporte de Membrana/inmunología , Receptor Toll-Like 7/inmunología , Trypanosoma cruzi/inmunología , Animales , Enfermedad de Chagas/genética , Interferón gamma/genética , Interferón gamma/inmunología , Subunidad p40 de la Interleucina-12/genética , Subunidad p40 de la Interleucina-12/inmunología , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , Parasitemia/genética , Parasitemia/inmunología , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 7/genética , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/inmunologíaRESUMEN
Exciting discoveries related to IL-1R/TLR signaling in the development of atherosclerosis plaque have triggered intense interest in the molecular mechanisms by which innate immune signaling modulates the onset and development of atherosclerosis. Previous studies have clearly shown the definitive role of proinflammatory cytokine IL-1 in the development of atherosclerosis. Recent studies have provided direct evidence supporting a link between innate immunity and atherogenesis. Although it is still controversial about whether infectious pathogens contribute to cardiovascular diseases, direct genetic evidence indicates the importance of IL-1R/TLR signaling in atherogenesis. In this study, we examined the role of IL-1R-associated kinase 4 (IRAK4) kinase activity in modified low-density lipoprotein (LDL)-mediated signaling using bone marrow-derived macrophage as well as an in vivo model of atherosclerosis. First, we found that the IRAK4 kinase activity was required for modified LDL-induced NF-κB activation and expression of a subset of proinflammatory genes but not for the activation of MAPKs in bone marrow-derived macrophage. IRAK4 kinase-inactive knockin (IRAK4KI) mice were bred onto ApoE(-/-) mice to generate IRAK4KI/ApoE(-/-) mice. Importantly, the aortic sinus lesion formation was impaired in IRAK4KI/ApoE(-/-) mice compared with that in ApoE(-/-) mice. Furthermore, proinflammatory cytokine production was reduced in the aortic sinus region of IRAK4KI/ApoE(-/-) mice compared with that in ApoE(-/-) mice. Taken together, our results indicate that the IRAK4 kinase plays an important role in modified LDL-mediated signaling and the development of atherosclerosis, suggesting that pharmacological inhibition of IRAK4 kinase activity might be a feasible approach in the development of antiatherosclerosis drugs.
Asunto(s)
Aterosclerosis/enzimología , Aterosclerosis/inmunología , Regulación de la Expresión Génica/inmunología , Mediadores de Inflamación/administración & dosificación , Quinasas Asociadas a Receptores de Interleucina-1/fisiología , Lipoproteínas LDL/administración & dosificación , FN-kappa B/metabolismo , Acetilación , Animales , Aorta Torácica/enzimología , Aorta Torácica/metabolismo , Aorta Torácica/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/patología , LDL-Colesterol/metabolismo , LDL-Colesterol/fisiología , Regulación de la Expresión Génica/genética , Inflamación/enzimología , Inflamación/genética , Inflamación/patología , Mediadores de Inflamación/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Quinasas Asociadas a Receptores de Interleucina-1/deficiencia , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/fisiologíaRESUMEN
UNC93B1 associates with Toll-Like Receptor (TLR) 3, TLR7 and TLR9, mediating their translocation from the endoplasmic reticulum to the endolysosome, hence allowing proper activation by nucleic acid ligands. We found that the triple deficient '3d' mice, which lack functional UNC93B1, are hyper-susceptible to infection with Toxoplasma gondii. We established that while mounting a normal systemic pro-inflammatory response, i.e. producing abundant MCP-1, IL-6, TNFα and IFNγ, the 3d mice were unable to control parasite replication. Nevertheless, infection of reciprocal bone marrow chimeras between wild-type and 3d mice with T. gondii demonstrated a primary role of hemopoietic cell lineages in the enhanced susceptibility of UNC93B1 mutant mice. The protective role mediated by UNC93B1 to T. gondii infection was associated with impaired IL-12 responses and delayed IFNγ by spleen cells. Notably, in macrophages infected with T. gondii, UNC93B1 accumulates on the parasitophorous vacuole. Furthermore, upon in vitro infection the rate of tachyzoite replication was enhanced in non-activated macrophages carrying mutant UNC93B1 as compared to wild type gene. Strikingly, the role of UNC93B1 on intracellular parasite growth appears to be independent of TLR function. Altogether, our results reveal a critical role for UNC93B1 on induction of IL-12/IFNγ production as well as autonomous control of Toxoplasma replication by macrophages.
Asunto(s)
Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/inmunología , Toxoplasmosis Animal/genética , Toxoplasmosis Animal/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Separación Celular , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Immunoblotting , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-12/biosíntesis , Interleucina-12/inmunología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Toxoplasma/genética , Toxoplasma/inmunologíaRESUMEN
Type I interferons (IFNs) play a critical role in the host defense against viruses. Lymphocytic choriomeningitis virus (LCMV) infection induces robust type I IFN production in its natural host, the mouse. However, the mechanisms underlying the induction of type I IFNs in response to LCMV infection have not yet been clearly defined. In the present study, we demonstrate that IRF7 is required for both the early phase (day 1 postinfection) and the late phase (day 2 postinfection) of the type I IFN response to LCMV, and melanoma differentiation-associated gene 5 (MDA5)/mitochondrial antiviral signaling protein (MAVS) signaling is crucial for the late phase of the type I IFN response to LCMV. We further demonstrate that LCMV genomic RNA itself (without other LCMV components) is able to induce type I IFN responses in various cell types by activation of the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5. We also show that expression of the LCMV nucleoprotein (NP) inhibits the type I IFN response induced by LCMV RNA and other RIG-I/MDA5 ligands. These virus-host interactions may play important roles in the pathogeneses of LCMV and other human arenavirus diseases.
Asunto(s)
Interacciones Huésped-Patógeno , Interferón Tipo I/antagonistas & inhibidores , Interferón Tipo I/biosíntesis , Virus de la Coriomeningitis Linfocítica/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , Humanos , Factor 7 Regulador del Interferón/metabolismo , Helicasa Inducida por Interferón IFIH1 , Ratones , Ratones Noqueados , Nucleoproteínas/inmunología , ARN Viral/inmunología , ARN Viral/metabolismo , Proteínas Virales/inmunologíaRESUMEN
Blockade of Toll-like receptor (TLR)-mediated inflammatory responses represents a new approach in the development of anti-inflammation therapeutics. In the present study, we have screened for TLR2-mediated inflammation inhibitors from small molecule compound libraries using a sensitive cell line stably expressing TLR2, CD14, and an NF-kappaB-driven-luciferase reporter gene. Lymphocytic choriomeningitis virus (LCMV) was used as a virus model. This arenavirus activates a TLR2/CD14-dependent NF-kappaB signaling pathway. We have identified 10 potential anti-inflammatory compounds out of 101,306 compounds. We further evaluated 1 of these positive compounds, E567. We demonstrated that compound E567 efficiently inhibits both LCMV and Herpes simplex virus 1 (HSV-1) induced cytokine responses in both human and mouse cell cultures. We also demonstrated that E567 inhibits cytokine responses in the mouse. Remarkably, E567 is also capable of inhibiting LCMV replication in mice. This is a new model for developing drugs for use in treating viral illnesses.
Asunto(s)
Herpesvirus Humano 1/inmunología , Factores Inmunológicos/farmacología , Receptores de Lipopolisacáridos/metabolismo , Virus de la Coriomeningitis Linfocítica/inmunología , FN-kappa B/antagonistas & inhibidores , Receptor Toll-Like 2/antagonistas & inhibidores , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Línea Celular , Evaluación Preclínica de Medicamentos/métodos , Femenino , Genes Reporteros , Herpesvirus Humano 1/patogenicidad , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Coriomeningitis Linfocítica/tratamiento farmacológico , Virus de la Coriomeningitis Linfocítica/patogenicidad , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacosRESUMEN
Ing4 is a member of the inhibitor of growth (ING) family of chromatin-modifying proteins. Biochemical experiments indicate that Ing4 is a subunit of the HB01-JADE-hEAF6 histone acetyltransferase complex responsible for most nucleosomal histone H4 acetylation in eukaryotes, and transfection studies suggest that Ing4 may regulate a wide variety of cellular processes, including DNA repair, apoptosis, cell-cycle regulation, metastasis, angiogenesis, and tumor suppression. However, in vivo evidence for a physiological role for Ing4 in cell-growth regulation is lacking. We have generated Ing4-deficient mice to explore the role of Ing4 in development, tumorigenesis, and in NF-kappaB signaling. Ing4-null mice develop normally and are viable. Although mice deficient for Ing4 fail to form spontaneous tumors, they are hypersensitive to LPS treatment and display elevated cytokine responses. Macrophages isolated from Ing4-null mice have increased levels of nuclear p65/RelA protein, resulting in increased RelA binding to NF-kappaB target promoters and up-regulation of cytokine gene expression. However, increased promoter occupancy by RelA in LPS-stimulated, Ing4-null cells does not always correlate with increased NF-kappaB target-gene expression, as RelA activation of a subset of cytokine promoters also requires Ing4 for proper histone H4 acetylation. Furthermore, activation of the IkappaB alpha promoter by RelA is also Ing4-dependent, and LPS-stimulated, Ing4-null cells have reduced levels of IkappaB alpha promoter H4 acetylation and IkappaB gene expression. Thus, Ing4 negatively regulates the cytokine-mediated inflammatory response in mice by facilitating NF-kappaB activation of IkappaB promoters, thereby suppressing nuclear RelA levels and the activation of select NF-kappaB target cytokines.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Homeodominio/metabolismo , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Proteínas Supresoras de Tumor/metabolismo , Animales , Núcleo Celular/metabolismo , Cruzamientos Genéticos , Citocinas/metabolismo , Inflamación , Lipopolisacáridos/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Transducción de Señal , Factor de Transcripción ReIA/metabolismoRESUMEN
Complement-containing immune complexes can be presented to phagocytes by human erythrocytes bearing complement receptor 1 (CR1). Although this has long been assumed to be a mechanism by which humans are able to protect themselves from "extracellular" bacteria such as pneumococci, there is little direct evidence. In these studies we have investigated this question by comparing results for erythrocytes from transgenic mice expressing human CR1 on their erythrocytes to the results for wild-type mouse erythrocytes that do not express CR1. We demonstrate that human CR1 expression on murine erythrocytes allows immune adherence to beads opsonized with either mouse or human serum as a source of complement. The role of CR1 in immune adherence was supported by studies showing that it was blocked by the addition of antibody to human CR1. Furthermore, human CR1 expression enhances the immune adherence of opsonized pneumococci to erythrocytes in vitro, and the pneumococci attached to erythrocytes via CR1 can be transferred in vitro to live macrophages. Even more importantly, we observed that if complement-opsonized pneumococci are injected intravenously with CR1(+) mouse erythrocytes into wild-type mice (after a short in vitro incubation), they are cleared faster than opsonized pneumococci similarly injected with wild-type mouse erythrocytes. Finally, we have shown that the intravenous (i.v.) injection of pneumococci into CR1(+) mice also results in more rapid blood clearance than in wild-type mice. These data support that immune adherence via CR1 on erythrocytes likely plays an important role in the clearance of opsonized bacteria from human blood.
Asunto(s)
Membrana Eritrocítica/fisiología , Infecciones Neumocócicas/inmunología , Receptores de Complemento/biosíntesis , Animales , Complejo Antígeno-Anticuerpo/inmunología , Complejo Antígeno-Anticuerpo/fisiología , Línea Celular , Membrana Eritrocítica/inmunología , Expresión Génica/inmunología , Expresión Génica/fisiología , Humanos , Macrófagos/inmunología , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fagocitosis/inmunología , Fagocitosis/fisiología , Receptores de Complemento/fisiología , Streptococcus pneumoniae/inmunologíaRESUMEN
Neoplastic epithelia may remain dormant and clinically unapparent in human patients for decades. Multiple risk factors including mutations in tumor cells or the stromal cells may affect the switch from dormancy to malignancy. Gene mutations, including p53 mutations, within the stroma of tumors are associated with a worse clinical prognosis; however, it is not known if these stromal mutations can promote tumors in genetically at-risk tissue. To address this question, Apc(Min/+) and Apc(Min/+) Rag2(-/-) mice, which have a predilection to mammary carcinoma (as well as wild-type (wt) mice), received mesenchymal stem cells (MSC) with mutant p53 (p53MSC) transferred via tail vein injection. In the wt mouse, p53MSC circulated in the periphery and homed to the marrow cavity where they could be recovered up to a year later without apparent effect on the health of the mouse. No mammary tumors were found. However, in mice carrying the Apc(Min/+) mutation, p53MSC homed to mammary tissue and significantly increased the incidence of mammary carcinoma. Tumor necrosis factor (TNF)-alpha-dependent factors elaborated from mesenchymal cells converted quiescent epithelia into clinically apparent disease. The increased cancer phenotype was completely preventable with neutralization of TNF-alpha or by transfer of CD4(+) regulatory T cells from immune competent donors, demonstrating that immune competency to regulate inflammation was sufficient to maintain neoplastic dormancy even in the presence of oncogenic epithelial and stromal mutations. The significant synergy between host immunity and mesenchymal cells identified here may restructure treatments to restore an anticancer microenvironment.
Asunto(s)
Células de la Médula Ósea/patología , Neoplasias Mamarias Animales/etiología , Células Madre Mesenquimatosas/patología , Mutación/genética , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos CD/metabolismo , Médula Ósea , Células de la Médula Ósea/metabolismo , Neoplasias de la Mama/etiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/etiología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/metabolismo , Medios de Cultivo Condicionados/farmacología , Proteínas de Unión al ADN/genética , Epitelio/patología , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Genes APC , Humanos , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/inmunología , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Mutantes , Ratones SCID , Persona de Mediana Edad , Mutación Missense/genética , Trasplante de Neoplasias/patología , Células del Estroma/metabolismo , Células del Estroma/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/trasplante , Trasplante Heterólogo/patología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Coxsackie B viruses (CVB) are enteroviruses that have been associated with a variety of human diseases, including myocarditis. In the present study, we found that MDA5 and its adaptor molecule MAVS are critical for type I interferon responses to CVB, since the absence of either MAVS or MDA5 leads to deficient type I interferon production and early mortality in mice infected with CVB. Pancreatic and hepatic necrosis were observed on histopathological examination of MAVS and MDA5 knockout mice infected with CVB. Inflammatory cytokine production in response to systemic CVB infection was independent of MAVS. Surprisingly, virus titers were not elevated in MAVS-deficient mice, despite significant reductions in type I interferon levels. These data highlight the importance of type I interferon in host defense and provide insight on the mechanisms of innate immune responses following coxsackievirus infection.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , ARN Helicasas DEAD-box/inmunología , Enterovirus Humano B/inmunología , Interferón Tipo I/inmunología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Infecciones por Coxsackievirus/inmunología , Infecciones por Coxsackievirus/virología , Citocinas/biosíntesis , Inmunidad Innata , Helicasa Inducida por Interferón IFIH1 , Ratones , Ratones Noqueados , Carga ViralRESUMEN
Type 5 adenovirus (Ad5) is a human pathogen that has been widely developed for therapeutic uses, with only limited success to date. We report here the novel finding that human erythrocytes present Coxsackie virus-adenovirus receptor (CAR) providing an Ad5 sequestration mechanism that protects against systemic infection. Interestingly, erythrocytes from neither mice nor rhesus macaques present CAR. Excess Ad5 fiber protein or anti-CAR antibody inhibits the binding of Ad5 to human erythrocytes and cryo-electron microscopy shows attachment via the fiber protein of Ad5, leading to close juxtaposition with the erythrocyte membrane. Human, but not murine, erythrocytes also present complement receptor (CR1), which binds Ad5 in the presence of antibodies and complement. Transplantation of human erythrocytes into nonobese diabetic/severe combined immunodeficiency mice extends blood circulation of intravenous Ad5 but decreases its extravasation into human xenograft tumors. Ad5 also shows extended circulation in transgenic mice presenting CAR on their erythrocytes, although it clears rapidly in transgenic mice presenting erythrocyte CR1. Hepatic infection is inhibited in both transgenic models. Erythrocytes may therefore restrict Ad5 infection (natural and therapeutic) in humans, independent of antibody status, presenting a formidable challenge to Ad5 therapeutics. "Stealthing" of Ad5 using hydrophilic polymers may enable circumvention of these natural virus traps.
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Adenovirus Humanos/inmunología , Eritrocitos/inmunología , Eritrocitos/metabolismo , Receptores de Complemento/inmunología , Receptores Virales/inmunología , Inactivación de Virus , Infecciones por Adenovirus Humanos/sangre , Infecciones por Adenovirus Humanos/inmunología , Adenovirus Humanos/metabolismo , Adenovirus Humanos/fisiología , Animales , Presentación de Antígeno/inmunología , Presentación de Antígeno/fisiología , Sitios de Unión , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Eritrocitos/virología , Femenino , Células HT29 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Modelos Biológicos , Células Tumorales CultivadasRESUMEN
Respiratory syncytial virus (RSV) is a common cause of infection that is associated with a range of respiratory illnesses, from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children <1 year of age. Host innate and acquired immune responses activated following RSV infection have been suspected to contribute to RSV disease. Toll-like receptors (TLRs) activate innate and acquired immunity and are candidates for playing key roles in the host immune response to RSV. Leukocytes express TLRs, including TLR2, TLR6, TLR3, TLR4, and TLR7, that can interact with RSV and promote immune responses following infection. Using knockout mice, we have demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting tumor necrosis factor alpha, interleukin-6, CCL2 (monocyte chemoattractant protein 1), and CCL5 (RANTES). As previously noted, TLR4 also contributes to cytokine activation (L. M. Haynes, D. D. Moore, E. A. Kurt-Jones, R. W. Finberg, L. J. Anderson, and R. A. Tripp, J. Virol. 75:10730-10737, 2001, and E. A. Kurt-Jones, L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, and R. W. Finberg, Nat. Immunol. 1:398-401, 2000). Furthermore, we demonstrated that signals generated following TLR2 and TLR6 activation were important for controlling viral replication in vivo. Additionally, TLR2 interactions with RSV promoted neutrophil migration and dendritic cell activation within the lung. Collectively, these studies indicate that TLR2 is involved in RSV recognition and subsequent innate immune activation.
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Inmunidad Innata , Virus Sincitiales Respiratorios/fisiología , Receptor Toll-Like 2/fisiología , Animales , Líquido del Lavado Bronquioalveolar , Células Dendríticas/inmunología , Humanos , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunologíaRESUMEN
Myeloid differentiation factor 88 (MyD88) is an essential adaptor protein in the Toll-like receptor-mediated innate signaling pathway, as well as in interleukin-1 receptor (IL-1R) and IL-18R signaling. The importance of MyD88 in the regulation of innate immunity to microbial pathogens has been well demonstrated. However, its role in regulating acquired immunity to viral pathogens and neuropathogenesis is not entirely clear. In the present study, we examine the role of MyD88 in the CD4(+) T-cell response following lymphocytic choriomeningitis virus (LCMV) infection. We demonstrate that wild-type (WT) mice developed a CD4(+) T-cell-mediated wasting disease after intracranial infection with LCMV. In contrast, MyD88 knockout (KO) mice did not develop wasting disease in response to the same infection. This effect was not the result of MyD88 regulation of IL-1 or IL-18 responses since IL-1R1 KO and IL-18R KO mice were not protected from weight loss. In the absence of MyD88, naïve CD4(+) T cells failed to differentiate to LCMV-specific CD4 T cells. We demonstrated that MyD88 KO antigen-presenting cells are capable of activating WT CD4(+) T cells. Importantly, when MyD88 KO CD4(+) T cells were reconstituted with an MyD88-expressing lentivirus, the rescued CD4(+) T cells were able to respond to LCMV infection and support IgG2a antibody production. Overall, these studies reveal a previously unknown role of MyD88-dependent signaling in CD4(+) T cells in the regulation of the virus-specific CD4(+) T-cell response and in viral infection-induced immunopathology in the central nervous system.
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Infecciones por Arenaviridae/inmunología , Linfocitos T CD4-Positivos/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Factor 88 de Diferenciación Mieloide/inmunología , Animales , Anticuerpos Antivirales/sangre , Inmunoglobulina G/sangre , Interleucina-1/inmunología , Interleucina-18/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/deficienciaRESUMEN
Influenza virus infection of the respiratory tract is characterized by a neutrophil infiltrate accompanied by inflammatory cytokine and chemokine production. We and others have reported that Toll-like receptor (TLR) proteins are present on human neutrophils and that granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment enhances IL-8 (CXCL8) secretion in response to stimulation with TLR ligands. We demonstrate that influenza virus can induce IL-8 and other inflammatory cytokines from GM-CSF-primed human neutrophils. Using heat inactivation of influenza virus, we show that viral entry but not replication is required for cytokine induction. Furthermore, endosomal acidification and viral uncoating are necessary. Finally, using single-cell analysis of intracellular cytokine accumulation in neutrophils from knockout mice, we prove that TLR7 is essential for influenza viral recognition and inflammatory cytokine production by murine neutrophils. These studies demonstrate neutrophil activation by influenza virus and highlight the importance of TLR7 and TLR8 in that response.
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Subtipo H3N2 del Virus de la Influenza A/inmunología , Neutrófilos/inmunología , Neutrófilos/virología , Receptores Toll-Like/metabolismo , Animales , Línea Celular , Citocinas/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Inmunidad Innata , Técnicas In Vitro , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Subtipo H3N2 del Virus de la Influenza A/fisiología , Ligandos , Macrólidos/farmacología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Activación Neutrófila/efectos de los fármacos , Activación Neutrófila/inmunología , Neutrófilos/efectos de los fármacos , ARN Viral/inmunología , ARN Viral/metabolismo , Proteínas Recombinantes , Receptor Toll-Like 7/deficiencia , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/metabolismo , Receptores Toll-Like/agonistas , Internalización del Virus , Replicación ViralRESUMEN
In response to invading pathogens, Toll-like receptors (TLR) play a critical role in the initiation of the innate immune response, which can be either beneficial or detrimental to the host. In the present study, we demonstrated that central nervous system (CNS) glial cells are activated by Lymphocytic Choriomeningitis Virus (LCMV) in a TLR2-MyD88/Mal-dependent manner. Specifically, in response to LCMV, both astrocytes and microglial cells isolated from wild-type (WT) mice produced chemokines, such as MCP-1, RANTES and TNF-alpha. Similar responses occurred in TLR3 KO and TLR4 KO glial cells. In striking contrast, both astrocytes and microglial cells isolated from mice deficient in TLR2, MyD88, and Mal did not produce any of these chemokines. In addition, LCMV infection of glial cells induced up-regulation of TLR2, MHC class-I and II, CD40, CD86 in a MyD88-dependent manner. These results define a functional role for TLR signaling in viral infection-induced activation of CNS glial cells as well as for the immunopathology in the CNS.