RESUMEN
NLRP3 is a pattern recognition receptor with a well-documented role in inducing inflammasome assembly in response to cellular stress. Deregulation of its activity leads to many inflammatory disorders including gouty arthritis, Alzheimer disease, and cancer. Whereas its role in the context of cancer has been mostly explored in the immune compartment, whether NLRP3 exerts functions unrelated to immunity in cancer development remains unexplored. Here, we demonstrate that NLRP3 interacts with the ATM kinase to control the activation of the DNA damage response, independently of its inflammasome activity. NLRP3 down-regulation in both broncho- and mammary human epithelial cells significantly impairs ATM pathway activation, leading to lower p53 activation, and provides cells with the ability to resist apoptosis induced by acute genotoxic stress. Interestingly, NLRP3 expression is down-regulated in non-small cell lung cancers and breast cancers, and its expression positively correlates with patient overall survival. Our findings identify a novel non-immune function for NLRP3 in maintaining genome integrity and strengthen the concept of a functional link between innate immunity and DNA damage sensing pathways to maintain cell integrity.
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Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Humanos , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inmunidad Innata , Daño del ADN , Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismoRESUMEN
Inflammasomes are molecular complexes that trigger an inflammatory response upon detection of pathogens or danger signals. Recent studies suggest that they are also involved in cancer progression. However, their roles during tumorigenesis remain poorly understood and controversial. Here, we investigated whether inflammasome activation supports mammary tumor growth. Using mouse models of invasive breast cancer, our results demonstrate that the absence of a functional inflammasome impairs tumor growth. Importantly, tumors implanted into inflammasome-deficient mice recruited significantly less neutrophils and more natural killer (NK) cells, and these latter cells displayed a more active phenotype. Interestingly, NK cell depletion abolished the anti-tumoral effect observed in inflammasome-deficient mice, although inflammasome-regulated cytokine neutralization had no effect. Thus, our work identifies a novel role for the inflammasome in supporting mammary tumor growth by attenuating NK cell recruitment and activity. These results suggest that inflammasome inhibition could be a putative target for treating invasive breast cancers.
RESUMEN
Although immune checkpoint-targeted therapies are currently revolutionizing cancer care, only a minority of patients develop durable objective responses to anti-PD-1, PD-L1, and CTLA-4 therapy. Therefore, new therapeutic interventions are needed to increase the immunogenicity of tumors and overcome the resistance to these immunotherapies. Oncolytic properties of common viruses can be exploited for the priming of antitumor immunity, and such oncolytic viruses are currently in active clinical development in combination with immune checkpoint-targeted therapies. However, the routine implementation of these therapies is limited by their manufacturing constraints, the risk of exposure of clinical staff, and the ongoing regulations on genetically modified organisms. We sought to determine whether anti-infectious disease vaccines could be used as a commercially available source of immunostimulatory agents for cancer immunotherapy. We found that rotavirus vaccines have both immunostimulatory and oncolytic properties. In vitro, they can directly kill cancer cells with features of immunogenic cell death. In vivo, intratumoral rotavirus therapy has antitumor effects that are dependent on the immune system. In several immunocompetent murine tumor models, intratumoral rotavirus overcomes resistance to and synergizes with immune checkpoint-targeted therapy. Heat- and UV-inactivated rotavirus lost their oncolytic activity but kept their synergy with immune checkpoint-targeted antibodies through the up-regulation of the double-stranded RNA receptor retinoic acid-induced gene 1 (RIG-I). Rotavirus vaccines are clinical-grade products used in pediatric and adult populations. Therefore, in situ immunization strategies with intratumoral-attenuated rotavirus could be implemented quickly in the clinic.
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Vacunas contra el Cáncer/uso terapéutico , Inmunoterapia/métodos , Vacunas contra Rotavirus/uso terapéutico , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Proteína 58 DEAD Box/metabolismo , Femenino , Citometría de Flujo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Receptores InmunológicosRESUMEN
BACKGROUND: Several unautomated anti-HEV diagnostic tests are presently available. OBJECTIVE: We have evaluated the performance of the new automated VIDAS® ANTI-HEV IgM and IgG assays. STUDY DESIGN: We assessed the reproducibility and cross-reactivity of both VIDAS assays and the analytical sensitivity and linearity of the VIDAS IgG assay. We also tested the VIDAS and comparator assays Wantai IgG and IgM on immunocompetent and immunocompromised patients. Data were analysed according to the infectious profile, with samples from viremic phase (HEV RNA/IgM positive) and post-viremic phase (HEV RNA negative, IgM positive) infections, and uninfected patients (HEV RNA/IgM negative). RESULTS: Within-run reproducibility was <10% and between-run reproducibility was <12% for both assays. We found no cross-reactivity, except for the VIDAS IgG assay in some patients with HBV (1/10) or malaria (3/23) infections and for the VIDAS IgM assay in some HIV-infected patients (1/10). The VIDAS IgG assay was linear over 0.10-10.0 U/mL. Analytical sensitivity of the IgG assay was 0.71 IU/ml (probit analysis). The clinical sensitivity of the VIDAS IgM assay was 97.65% for viremic samples (83/85) and 59.15% (42/71) for post-viremic samples from immunocompetent patients. It was 78.95% (45/57) for acute phase samples and 77.78% (28/36) for post-viremic samples from immunocompromised patients. Specificity was excellent (>99%) in both populations. CONCLUSION: The analytical and clinical performance of the new VIDAS® ANTI-HEV assays was excellent. These rapid, automated assays for detecting HEV antibodies will strengthen the arsenal for diagnosing HEV infections.
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Automatización de Laboratorios/normas , Anticuerpos Antihepatitis/sangre , Hepatitis E/diagnóstico , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Juego de Reactivos para Diagnóstico/normas , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Reacciones Cruzadas , Femenino , Virus de la Hepatitis E/inmunología , Humanos , Lactante , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Pruebas Serológicas/normas , Adulto JovenRESUMEN
Human papillomavirus type 16 (HPV16) and other oncoviruses have been shown to block innate immune responses and to persist in the host. However, to avoid viral persistence, the immune response attempts to clear the infection. IL-1ß is a powerful cytokine produced when viral motifs are sensed by innate receptors that are members of the inflammasome family. Whether oncoviruses such as HPV16 can activate the inflammasome pathway remains unknown. Here, we show that infection of human keratinocytes with HPV16 induced the secretion of IL-1ß. Yet, upon expression of the viral early genes, IL-1ß transcription was blocked. We went on to show that expression of the viral oncoprotein E6 in human keratinocytes inhibited IRF6 transcription which we revealed regulated IL-1ß promoter activity. Preventing E6 expression using siRNA, or using E6 mutants that prevented degradation of p53, showed that p53 regulated IRF6 transcription. HPV16 abrogation of p53 binding to the IRF6 promoter was shown by ChIP in tissues from patients with cervical cancer. Thus E6 inhibition of IRF6 is an escape strategy used by HPV16 to block the production IL-1ß. Our findings reveal a struggle between oncoviral persistence and host immunity; which is centered on IL-1ß regulation.
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Regulación de la Expresión Génica/inmunología , Evasión Inmune/inmunología , Factores Reguladores del Interferón/metabolismo , Interleucina-1beta/biosíntesis , Infecciones por Papillomavirus/inmunología , Papillomavirus Humano 16/inmunología , Humanos , Factores Reguladores del Interferón/inmunología , Interleucina-1beta/inmunología , Queratinocitos/inmunología , Queratinocitos/metabolismo , Queratinocitos/virología , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/metabolismo , Proteínas Represoras/metabolismoRESUMEN
BACKGROUND: High performance anti-hepatitis E virus (HEV) IgG assays are crucial for epidemiology. OBJECTIVE: To evaluate the performance of 2 prototypes developed for the VIDAS® automatic system for detecting anti-HEV IgG, one based on the ORF2 antigen (ORF2 prototype) and the other on the ORF2 and ORF3 antigens (ORF2/3 prototype), with reference to the Wantai anti-HEV IgG assay. STUDY DESIGN: The sensitivity of each assay was determined by testing 113 blood samples, 63 from immunocompetent and 50 from immunocompromised patients, with a proven HEV infection defined by detecting HEV RNA. Their specificity was assessed with 103 blood samples that the Wantai assay indicated was negative for anti-HEV IgM and IgG, and negative for HEV-RNA. Cross reactivity was assessed using samples that were positive for hepatitis A virus IgG (n=16), hepatitis C virus antibodies (n=15), hepatitis B virus antigen and anti-HBc antibodies (n=16), rheumatoid factor (n=14), and negative for anti-HEV IgG with the Wantai assay. RESULTS: The sensitivities in immunocompetent patients were: 95.2% (ORF2), 96.8% (ORF2/3), and 93.6% (Wantai); in immunocompromised patients they were: 66% (ORF2), 72% (ORF2/3), and 68% (Wantai). Both VIDAS prototypes detected low concentrations of anti-HEV IgG. The overall specificity was 100% (ORF2 prototype) and 98.1% (ORF2/3 prototype). Both VIDAS prototypes cross-reacted in five samples (9.6%), mainly those containing HCV antibodies or rheumatoid factor. CONCLUSION: Both VIDAS® prototypes performed very well and appear to be suitable for routine detection of anti-HEV IgG.
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Anticuerpos Antihepatitis/sangre , Hepatitis E/diagnóstico , Inmunoensayo/métodos , Automatización de Laboratorios/métodos , Humanos , Inmunoglobulina G/sangre , Sensibilidad y EspecificidadRESUMEN
Dendritic cells (DCs) cross-present antigen (Ag) to initiate T-cell immunity against most infections and tumors. Natural killer (NK) cells are innate cytolytic lymphocytes that have emerged as key modulators of multiple DC functions. Here, we show that human NK cells promote cross-presentation of tumor cell-derived Ag by DC leading to Ag-specific CD8(+) T-cell activation. Surprisingly, cytotoxic function of NK cells was not required. Instead, we highlight a critical and nonredundant role for IFN-γ and TNF-α production by NK cells to enhance cross-presentation by DC using two different Ag models. Importantly, we observed that NK cells promote cell-associated Ag cross-presentation selectively by monocytes-derived DC (Mo-DC) and CD34-derived CD11b(neg) CD141(high) DC subsets but not by myeloid CD11b(+) DC. Moreover, we demonstrate that triggering NK cell activation by monoclonal antibodies (mAbs)-coated tumor cells leads to efficient DC cross-presentation, supporting the concept that NK cells can contribute to therapeutic mAbs efficiency by inducing downstream adaptive immunity. Taken together, our findings point toward a novel role of human NK cells bridging innate and adaptive immunity through selective induction of cell-associated Ag cross-presentation by CD141(high) DC, a process that could be exploited to better harness Ag-specific cellular immunity in immunotherapy.
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Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Inmunidad Celular/inmunología , Células Asesinas Naturales/inmunología , Neoplasias/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Células Dendríticas/patología , Humanos , Células Asesinas Naturales/patología , Neoplasias/patología , Células Tumorales CultivadasRESUMEN
Plasmacytoid dendritic cells (pDC) are key regulators of antiviral immunity. In previous studies, we reported that pDC-infiltrating human primary breast tumors represent an independent prognostic factor associated with poor outcome. To understand this negative impact of tumor-associated pDC (TApDC), we developed an orthotopic murine mammary tumor model that closely mimics the human pathology, including pDC and regulatory T cell (Treg) infiltration. We showed that TApDC are mostly immature and maintain their ability to internalize antigens in vivo and to activate CD4(+) T cells. Most importantly, TApDC were specifically altered for cytokine production in response to Toll-like receptor (TLR)-9 ligands in vitro while preserving unaltered response to TLR7 ligands (TLR7L). In vivo pDC depletion delayed tumor growth, showing that TApDC provide an immune-subversive environment, most likely through Treg activation, thus favoring tumor progression. However, in vivo intratumoral administration of TLR7L led to TApDC activation and displayed a potent curative effect. Depletion of pDC and type I IFN neutralization prevented TLR7L antitumoral effect. Our results establish a direct contribution of TApDC to primary breast tumor progression and rationalize the application of TLR7 ligands to restore TApDC activation in breast cancer. Cancer Res; 73(15); 4629-40. ©2013 AACR.
Asunto(s)
Células Dendríticas/inmunología , Activación de Linfocitos/inmunología , Neoplasias Mamarias Experimentales/inmunología , Glicoproteínas de Membrana/inmunología , Receptor Toll-Like 7/inmunología , Animales , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Femenino , Ligandos , Prueba de Cultivo Mixto de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Receptor Toll-Like 7/metabolismoRESUMEN
Human papillomavirus type 16 (HPV16) and other oncogenic viruses have been reported to deregulate immunity by suppressing the function of the double-stranded DNA innate sensor TLR9. However, the mechanisms leading to these events remain to be elucidated. We show that infection of human epithelial cells with HPV16 promotes the formation of an inhibitory transcriptional complex containing NF-κBp50-p65 and ERα induced by the E7 oncoprotein. The E7-mediated transcriptional complex also recruited the histone demethylase JARID1B and histone deacetylase HDAC1. The entire complex bound to a specific region on the TLR9 promoter, which resulted in decreased methylation and acetylation of histones upstream of the TLR9 transcriptional start site. The involvement of NF-κB and ERα in the TLR9 down-regulation by HPV16 E7 was fully confirmed in cervical tissues from human patients. Importantly, we present evidence that the HPV16-induced TLR9 down-regulation affects the interferon response which negatively regulates viral infection. Our studies highlight a novel HPV16-mediated mechanism that combines epigenetic and transcriptional events to suppress a key innate immune sensor.
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Papillomavirus Humano 16/inmunología , Papillomavirus Humano 16/patogenicidad , Proteínas E7 de Papillomavirus/inmunología , Receptor Toll-Like 9/genética , Secuencia de Bases , Línea Celular Tumoral , Cuello del Útero/inmunología , Cuello del Útero/metabolismo , Cuello del Útero/virología , Regulación hacia Abajo/genética , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Femenino , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Datos de Secuencia Molecular , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/virología , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismoRESUMEN
We previously reported that plasmacytoid dendritic cells (pDCs) infiltrating breast tumors are impaired for their interferon-α (IFN-α) production, resulting in local regulatory T cells amplification. We designed our study to decipher molecular mechanisms of such functional defect of tumor-associated pDC (TApDC) in breast cancer. We demonstrate that besides IFN-α, the production by Toll-like receptor (TLR)-activated healthy pDC of IFN-ß and TNF-α but not IP-10/CXCL10 nor MIP1-α/CCL3 is impaired by the breast tumor environment. Importantly, we identified TGF-ß and TNF-α as major soluble factors involved in TApDC functional alteration. Indeed, recombinant TGF-ß1 and TNF-α synergistically blocked IFN-α production of TLR-activated pDC, and neutralization of TGF-ß and TNF-α in tumor-derived supernatants restored pDCs' IFN-α production. The involvment of tumor-derived TGF-ß was further confirmed in situ by the detection of phosphorylated Smad2 in the nuclei of TApDC in breast tumor tissues. Mechanisms of type I IFN inhibition did not involve TLR downregulation but the inhibition of IRF-7 expression and nuclear translocation in pDC after their exposure to tumor-derived supernatants or recombinant TGF-ß1 and TNF-α. Our findings indicate that targeting TApDC to restore their IFN-α production might be an achievable strategy to induce antitumor immunity in breast cancer by combining TLR7/9-based immunotherapy with TGF-ß and TNF-α antagonists.
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Neoplasias de la Mama/metabolismo , Células Dendríticas/metabolismo , Interferón-alfa/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Quimiocina CCL3/biosíntesis , Quimiocina CXCL10/biosíntesis , Femenino , Humanos , Factor 7 Regulador del Interferón/biosíntesis , Interferón beta/biosíntesis , Fosforilación , Transporte de Proteínas , Proteínas Recombinantes/farmacología , Proteína Smad2/metabolismo , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 9/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Infiltration and dysfunction of immune cells have been documented in many types of cancers. We previously reported that plasmacytoid dendritic cells (pDC) within primary breast tumors correlate with an unfavorable prognosis for patients. The role of pDC in cancer remains unclear but they have been shown to mediate immune tolerance in other pathophysiologic contexts. We postulated that pDC may interfere with antitumor immune response and favor tolerance in breast cancer. The present study was designed to decipher the mechanistic basis for the deleterious impact of pDC on the clinical outcome. Using fresh human breast tumor biopsies (N = 60 patients), we observed through multiparametric flow cytometry increased tumor-associated (TA) pDC (TApDC) rates in aggressive breast tumors, i.e., those with high mitotic index and the so-called triple-negative breast tumors (TNBT). Furthermore, TApDC expressed a partially activated phenotype and produced very low amounts of IFN-α following toll-like receptor activation in vitro compared with patients' blood pDC. Within breast tumors, TApDC colocalized and strongly correlated with TA regulatory T cells (TATreg), especially in TNBT. Of most importance, the selective suppression of IFN-α production endowed TApDC with the unique capacity to sustain FoxP3(+) Treg expansion, a capacity that was reverted by the addition of exogenous IFN-α. These findings indicate that IFN-α-deficient TApDC accumulating in aggressive tumors are involved in the expansion of TATreg in vivo, contributing to tumor immune tolerance and poor clinical outcome. Thus, targeting pDC to restore their IFN-α production may represent an attractive therapeutic strategy to overcome immune tolerance in breast cancer.
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Neoplasias de la Mama/inmunología , Células Dendríticas/metabolismo , Interferón-alfa/biosíntesis , Linfocitos T Reguladores/inmunología , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Femenino , Humanos , Inmunohistoquímica , Microambiente TumoralRESUMEN
The Gram-negative bacteria Yersinia pestis, causative agent of plague, is extremely virulent. One mechanism contributing to Y. pestis virulence is the presence of a type-three secretion system, which injects effector proteins, Yops, directly into immune cells of the infected host. One of these Yop proteins, YopJ, is proapoptotic and inhibits mammalian NF-κB and MAP-kinase signal transduction pathways. Although the molecular mechanism remained elusive for some time, recent work has shown that YopJ acts as a serine/threonine acetyl-transferase targeting MAP2 kinases. Using Drosophila as a model system, we find that YopJ inhibits one innate immune NF-κB signaling pathway (IMD) but not the other (Toll). In fact, we show YopJ mediated serine/threonine acetylation and inhibition of dTAK1, the critical MAP3 kinase in the IMD pathway. Acetylation of critical serine/threonine residues in the activation loop of Drosophila TAK1 blocks phosphorylation of the protein and subsequent kinase activation. In addition, studies in mammalian cells show similar modification and inhibition of hTAK1. These data present evidence that TAK1 is a target for YopJ-mediated inhibition.
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Proteínas Bacterianas/metabolismo , Inmunidad Innata , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas , Serina O-Acetiltransferasa/metabolismo , Yersinia pestis/enzimología , Acetilación , Animales , Proteínas Bacterianas/inmunología , Drosophila melanogaster , Células HEK293 , Humanos , Quinasas Quinasa Quinasa PAM/inmunología , FN-kappa B/inmunología , FN-kappa B/metabolismo , Peste/inmunología , Peste/metabolismo , Serina O-Acetiltransferasa/inmunología , Yersinia pestis/inmunología , Yersinia pestis/patogenicidadRESUMEN
Pattern recognition receptors (PRRs) are known for many years for their role in the recognition of microbial products and the subsequent activation of the immune system. The 2011 Nobel Prize for medicine indeed rewarded J. Hoffmann/B. Beutler and R. Steinman for their revolutionary findings concerning the activation of the immune system, thus stressing the significance of understanding the mechanisms of activation of the innate immunity. Such immunostimulatory activities are of major interest in the context of cancer to induce long-term antitumoral responses. Ligands for the toll-like receptors (TLRs), a well-known family of PRR, have been shown to have antitumoral activities in several cancers. Those ligands are now undergoing extensive clinical investigations both as immunostimulant molecules and as adjuvant along with vaccines. However, when considering the use of these ligands in tumor therapy, one shall consider the potential effect on the tumor cells themselves as well as on the entire organism. Recent data indeed demonstrate that TLR activation in tumor cells could trigger both pro- or antitumoral effect depending on the context. This review discusses this balance between the intrinsic activation of PRR in tumor cells and the extrinsic microenvironment activation in term of overall effect of PRR ligands on tumor development. We review recent advances in the field and underline appealing prospects for clinical development of PRR agonists in the light of our current knowledge on their expression and activation.
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Antineoplásicos/uso terapéutico , Inmunoterapia Activa/métodos , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Receptores Toll-Like/agonistas , Animales , Descubrimiento de Drogas , Humanos , Inmunidad Innata , Ligandos , Escape del Tumor , Microambiente TumoralRESUMEN
Staphylococcus aureus is a major pathogen responsible for both nosocomial and community-acquired infections. Central to its virulence is its ability to secrete haemolysins, pore-forming toxins and cytolytic peptides. The large number of membrane-damaging toxins and peptides produced during S. aureus infections has hindered a precise understanding of their specific roles in diseases. Here, we used comprehensive libraries of recombinant toxins and synthetic cytolytic peptides, of S. aureus mutants and clinical strains to investigate the role of these virulence factors in targeting human macrophages and triggering IL-1ß release. We found that the Panton Valentine leukocidin (PVL) is the major trigger of IL-1ß release and inflammasome activation in primary human macrophages. The cytolytic peptides, δ-haemolysin and PSMα3; the pore-forming toxins, γ-haemolysin and LukDE; and ß-haemolysin synergize with PVL to amplify IL-1ß release, indicating that these factors cooperate with PVL to trigger inflammation. PVL(+) S. aureus causes necrotizing pneumonia in children and young adults. The severity of this disease is due to the massive recruitment of neutrophils that cause lung damage. Importantly, we demonstrate that PVL triggers IL-1ß release in human alveolar macrophages. Furthermore, IL-1ß released by PVL-intoxicated macrophages stimulates the secretion of the neutrophil attracting chemokines, IL-8 and monocyte chemotactic protein-1, by lung epithelial cells. Finally, we show that PVL-induced IL-8/monocyte chemotactic protein-1 release is abolished by the inclusion of IL-1 receptor antagonist (IL-1Ra) in a mixed culture of lung epithelial cells and macrophages. Together, our results identify PVL as the predominant S. aureus secreted factor for triggering inflammasome activation in human macrophages and demonstrate how PVL-intoxicated macrophages orchestrate inflammation in the lung. Finally, our work suggests that anakinra, a synthetic IL-1Ra, may be an effective therapeutic agent to reduce the massive neutrophils infiltration observed during necrotizing pneumonia and decrease the resulting host-mediated lung injury.
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Toxinas Bacterianas/metabolismo , Quimiocinas/metabolismo , Células Epiteliales/inmunología , Exotoxinas/metabolismo , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Leucocidinas/metabolismo , Macrófagos/microbiología , Staphylococcus aureus/patogenicidad , Animales , Niño , Humanos , Pulmón/inmunología , Pulmón/patología , Macrófagos/inmunología , Neutrófilos/inmunología , Staphylococcus aureus/inmunología , Factores de Virulencia/metabolismo , Adulto JovenRESUMEN
In breast carcinomas, patient survival seems to be negatively affected by the recruitment of regulatory T cells (T(reg)) within lymphoid aggregates by CCL22. However, the mechanisms underpinning this process, which may be of broader significance in solid tumors, have yet to be described. In this study, we determined how CCL22 production is controlled in tumor cells. In human breast carcinoma cell lines, CCL22 was secreted at low basal levels that were strongly increased in response to inflammatory signals [TNF-α, IFN-γ, and interleukin (IL)-1ß], contrasting with CCL17. Primary breast tumors and CD45(+) infiltrating immune cells appeared to cooperate in driving CCL22 secretion, as shown clearly in cocultures of breast tumor cell lines and peripheral blood mononuclear cells (PBMC) or their supernatants. We determined that monocyte-derived IL-1ß and TNF-α are key players as monocyte depletion or neutralization of these cytokines attenuated secretion of CCL22. However, when purified monocytes were used, exogenous human IFN-γ was also required to generate this response suggesting a role for IFN-γ-producing cells within PBMCs. In this setting, we found that human IFN-γ could be replaced by the addition of (i) IL-2 or K562-activated natural killer (NK) cells or (ii) resting NK cells in the presence of anti-MHC class I antibody. Taken together, our results show a dialogue between NK and tumor cells leading to IFN-γ secretion, which in turn associates with monocyte-derived IL-1ß and TNF-α to drive production of CCL22 by tumor cells and subsequent recruitment of T(reg). As one validation of this conclusion in primary breast tumors, we showed that NK cells and macrophages tend to colocalize within tumors. In summary, our findings suggest that at early times during tumorigenesis, the detection of tumor cells by innate effectors (monocytes and NK cells) imposes a selection for CCL22 secretion that recruits T(reg) to evade this early antitumor immune response.
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Neoplasias de la Mama/inmunología , Quimiocina CCL22/biosíntesis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Comunicación Celular , Línea Celular Tumoral , Citotoxicidad Inmunológica/inmunología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Inmunidad Innata , Interferón gamma/farmacología , Interleucina-1beta/farmacología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Although Toll-like receptor 9 (TLR9) has been implicated in cytokine and type I interferon (IFN) production during malaria in humans and mice, the high AT content of the Plasmodium falciparum genome prompted us to examine the possibility that malarial DNA triggered TLR9-independent pathways. Over 6000 ATTTTTAC ("AT-rich") motifs are present in the genome of P. falciparum, which we show here potently induce type I IFNs. Parasite DNA, parasitized erythrocytes and oligonucleotides containing the AT-rich motif induce type I IFNs via a pathway that did not involve the previously described sensors TLR9, DAI, RNA polymerase-III or IFI16/p204. Rather, AT-rich DNA sensing involved an unknown receptor that coupled to the STING, TBK1 and IRF3-IRF7 signaling pathway. Mice lacking IRF3, IRF7, the kinase TBK1 or the type I IFN receptor were resistant to otherwise lethal cerebral malaria. Collectively, these observations implicate AT-rich DNA sensing via STING, TBK1 and IRF3-IRF7 in P. falciparum malaria.
Asunto(s)
Secuencia Rica en At/genética , ADN Protozoario/genética , Malaria Falciparum/inmunología , Oligonucleótidos/genética , Plasmodium falciparum/fisiología , Animales , ADN Protozoario/metabolismo , Perfilación de la Expresión Génica , Humanos , Inmunidad Innata/genética , Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Malaria Falciparum/parasitología , Malaria Falciparum/fisiopatología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Oligonucleótidos/metabolismo , Plasmodium falciparum/patogenicidad , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor de Interferón alfa y beta/genética , Transducción de Señal/genéticaRESUMEN
TBK1 is critical for immunity against microbial pathogens that activate TLR4- and TLR3-dependent signaling pathways. To address the role of TBK1 in inflammation, mice were generated that harbor two copies of a mutant Tbk1 allele. This Tbk1(Δ) allele encodes a truncated Tbk1(Δ) protein that is catalytically inactive and expressed at very low levels. Upon LPS stimulation, macrophages from Tbk1(Δ/Δ) mice produce normal levels of proinflammatory cytokines (e.g., TNF-α), but IFN-ß and RANTES expression and IRF3 DNA-binding activity are ablated. Three-month-old Tbk1(Δ/Δ) mice exhibit mononuclear and granulomatous cell infiltrates in multiple organs and inflammatory cell infiltrates in their skin, and they harbor a 2-fold greater amount of circulating monocytes than their Tbk1(+/+) and Tbk1(+/Δ) littermates. Skin from 2-week-old Tbk1(Δ/Δ) mice is characterized by reactive changes, including hyperkeratosis, hyperplasia, necrosis, inflammatory cell infiltrates, and edema. In response to LPS challenge, 3-month-old Tbk1(Δ/Δ) mice die more quickly and in greater numbers than their Tbk1(+/+) and Tbk1(+/Δ) counterparts. This lethality is accompanied by an overproduction of several proinflammatory cytokines in the serum of Tbk1(Δ/Δ) mice, including TNF-α, GM-CSF, IL-6, and KC. This overproduction of serum cytokines in Tbk1(Δ/Δ) mice following LPS challenge and their increased susceptibility to LPS-induced lethality may result from the reactions of their larger circulating monocyte compartment and their greater numbers of extravasated immune cells.
Asunto(s)
Lipopolisacáridos/toxicidad , Monocitos/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Quimiocina CCL2/biosíntesis , Femenino , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Human metapneumoviruses (HMPVs) are recently identified Paramyxoviridae that contribute to respiratory tract infections in children. No effective treatments or vaccines are available. Successful defense against virus infection relies on early detection by germ line-encoded pattern recognition receptors and activation of cytokine and type I IFN genes. Recently, the RNA helicase retinoic acid-inducible gene I (RIG-I) has been shown to sense HMPV. In this study, we investigated the abilities of two prototype strains of HMPV (A1 [NL\1\00] and B1 [NL\1\99]) to activate RIG-I and induce type I IFNs. Despite the abilities of both HMPV-A1 and HMPV-B1 to infect and replicate in cell lines and primary cells, only the HMPV-A1 strain triggered RIG-I to induce IFNA/B gene transcription. The failure of the HMPV-B1 strain to elicit type I IFN production was dependent on the B1 phosphoprotein, which specifically prevented RIG-I-mediated sensing of HMPV viral 5' triphosphate RNA. In contrast to most cell types, plasmacytoid dendritic cells displayed a unique ability to sense both HMPV-A1 and HMPV-B1 and in this case sensing was via TLR7 rather than RIG-I. Collectively, these data reveal differential mechanisms of sensing for two closely related viruses, which operate in cell type-specific manners.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Metapneumovirus/inmunología , Fosfoproteínas/metabolismo , Receptor Toll-Like 7/metabolismo , Interferencia Viral/inmunología , Animales , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/fisiología , Regulación Viral de la Expresión Génica/inmunología , Humanos , Inmunidad Innata , Interferón-alfa/biosíntesis , Interferón-alfa/genética , Interferón beta/biosíntesis , Interferón beta/genética , Ligandos , Metapneumovirus/genética , Metapneumovirus/patogenicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Paramyxoviridae/inmunología , Infecciones por Paramyxoviridae/metabolismo , Infecciones por Paramyxoviridae/virología , Fosfoproteínas/genética , ARN Viral/genética , Receptores Inmunológicos , Especificidad de la Especie , Receptor Toll-Like 7/deficiencia , Receptor Toll-Like 7/fisiología , Células VeroRESUMEN
Detection of non-self RNA by TLRs within endosomes and by retinoic acid-inducible gene I (RIG-I)-like helicases in the cytosol is central to mammalian antiviral immunity. In this study, we used pathway-specific agonists and targeted delivery to address RNA immunorecognition in primary human immune cells. Within PBMC, plasmacytoid dendritic cells (pDC) and monocytes were found to be responsible for IFN-alpha production upon immunorecognition of RNA. The mechanisms of RNA recognition in pDC and monocytes were distinct. In pDC, recognition of ssRNA and dsRNA oligonucleotides was TLR7-dependent, whereas a 5' triphosphate moiety (RIG-I ligand activity) had no major contribution to IFN-alpha production. In monocytes, the response to RNA oligonucleotides was mediated by either TLR8 or RIG-I. TLR8 was responsible for IL-12 induction upon endosomal delivery of ssRNA oligonucleotides and RIG-I was responsible for IFN-alpha production upon delivery of 5' triphosphate RNA into the cytosol. In conclusion, the dissection of these pathways by selecting the appropriate structure and delivery of RNA reveals pDC as major producer of IFN-alpha upon TLR-mediated stimulation and monocytes as major producer of IFN-alpha upon RIG-I-mediated stimulation. Furthermore, our results uncover the potential of monocytes to function as major producers of IL-12p70, a key Th1 cytokine classically ascribed to myeloid dendritic cells that cannot be induced by CpG oligonucleotides in the human system.
Asunto(s)
ARN Helicasas DEAD-box/fisiología , Interleucina-12/biosíntesis , Monocitos/inmunología , Oligorribonucleótidos/farmacología , ARN/inmunología , Receptor Toll-Like 7/efectos de los fármacos , Receptor Toll-Like 8/efectos de los fármacos , Células Cultivadas , Proteína 58 DEAD Box , Células Dendríticas/inmunología , Humanos , Interferón-alfa/biosíntesis , Oligorribonucleótidos/inmunología , ARN/farmacología , Receptores InmunológicosRESUMEN
In the case of an influenza pandemic, the current global influenza vaccine production capacity will be unable to meet the demand for billions of vaccine doses. The ongoing threat of an H5N1 pandemic therefore urges the development of highly immunogenic, dose-sparing vaccine formulations. In unprimed individuals, inactivated whole virus (WIV) vaccines are more immunogenic and induce protective antibody responses at a lower antigen dose than other formulations like split virus (SV) or subunit (SU) vaccines. The reason for this discrepancy in immunogenicity is a long-standing enigma. Here, we show that stimulation of Toll-like receptors (TLRs) of the innate immune system, in particular stimulation of TLR7, by H5N1 WIV vaccine is the prime determinant of the greater magnitude and Th1 polarization of the WIV-induced immune response, as compared to SV- or SU-induced responses. This TLR dependency largely explains the relative loss of immunogenicity in SV and SU vaccines. The natural pathogen-associated molecular pattern (PAMP) recognized by TLR7 is viral genomic ssRNA. Processing of whole virus particles into SV or SU vaccines destroys the integrity of the viral particle and leaves the viral RNA prone to degradation or involves its active removal. Our results show for a classic vaccine that the acquired immune response evoked by vaccination can be enhanced and steered by the innate immune system, which is triggered by interaction of an intrinsic vaccine component with a pattern recognition receptor (PRR). The insights presented here may be used to further improve the immune-stimulatory and dose-sparing properties of classic influenza vaccine formulations such as WIV, and will facilitate the development of new, even more powerful vaccines to face the next influenza pandemic.