RESUMEN
BACKGROUND: Patients with severe, uncontrolled asthma, particularly those with a non-eosinophilic phenotype, have a great unmet need for new treatments that act on a broad range of inflammatory pathways in the airway. Tezepelumab is a human monoclonal antibody that blocks the activity of thymic stromal lymphopoietin, an epithelial cytokine. In the PATHWAY phase 2b study (NCT02054130), tezepelumab reduced exacerbations by up to 71% in adults with severe, uncontrolled asthma, irrespective of baseline eosinophilic inflammatory status. This article reports the design and objectives of the phase 2 CASCADE study. METHODS: CASCADE is an ongoing exploratory, phase 2, randomized, double-blind, placebo-controlled, parallel-group study aiming to assess the anti-inflammatory effects of tezepelumab 210 mg administered subcutaneously every 4 weeks for 28 weeks in adults aged 18-75 years with uncontrolled, moderate-to-severe asthma. The primary endpoint is the change from baseline to week 28 in airway submucosal inflammatory cells (eosinophils, neutrophils, T cells and mast cells) from bronchoscopic biopsies. Epithelial molecular phenotyping, comprising the three-gene-mean technique, will be used to assess participants' type 2 (T2) status to enable evaluation of the anti-inflammatory effect of tezepelumab across the continuum of T2 activation. Other exploratory analyses include assessments of the impact of tezepelumab on airway remodelling, including reticular basement membrane thickening and airway epithelial integrity. At the onset of the COVID-19 pandemic, the protocol was amended to address the possibility that site visits would be limited. The amendment allowed for: at-home dosing of study drug by a healthcare professional, extension of the treatment period by up to 6 months so patients are able to attend an onsite visit to undergo the end-of-treatment bronchoscopy, and replacement of final follow-up visits with a virtual or telephone visit. DISCUSSION: CASCADE aims to determine the mechanisms by which tezepelumab improves clinical asthma outcomes by evaluating the effect of tezepelumab on airway inflammatory cells and remodelling in patients with moderate-to-severe, uncontrolled asthma. An important aspect of this study is the evaluation of the anti-inflammatory effect of tezepelumab across patients with differing levels of eosinophilic and T2 inflammation. TRIAL REGISTRATION: NCT03688074 (ClinicalTrials.gov). Registered 28 September 2018.
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Antiasmáticos/uso terapéutico , Antiinflamatorios/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Asma/tratamiento farmacológico , Adolescente , Adulto , Anciano , Antiasmáticos/efectos adversos , Antiinflamatorios/efectos adversos , Anticuerpos Monoclonales Humanizados/efectos adversos , Asma/diagnóstico , Asma/inmunología , Ensayos Clínicos Fase II como Asunto , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Multicéntricos como Asunto , Ensayos Clínicos Controlados Aleatorios como Asunto , Resultado del Tratamiento , Adulto JovenRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1005907.].
RESUMEN
Crohn's disease (CD) is a chronic inflammatory condition of diverse etiology. Exposure to foodborne pathogens causing acute gastroenteritis produces a long-term risk of CD well into the post-infectious period but the mechanistic basis for this ongoing relationship to disease onset is unknown. We developed two novel models to study the comorbidity of acute gastroenteritis caused by Salmonella Typhimurium or Citrobacter rodentium in mice colonized with adherent-invasive Escherichia coli (AIEC), a bacterial pathobiont linked to CD. Here, we show that disease activity in the post-infectious period after gastroenteritis is driven by the tissue-associated expansion of the resident AIEC pathobiont, with an attendant increase in immunopathology, barrier defects, and delays in mucosal restitution following pathogen clearance. These features required AIEC resistance to host defense peptides and a fulminant inflammatory response to the enteric pathogen. Our results suggest that individuals colonized by AIEC at the time of acute infectious gastroenteritis may be at greater risk for CD onset. Importantly, our data identify AIEC as a tractable disease modifier, a finding that could be exploited in the development of therapeutic interventions following infectious gastroenteritis in at-risk individuals.
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Coinfección/complicaciones , Enfermedad de Crohn/microbiología , Gastroenteritis/complicaciones , Animales , Citrobacter rodentium , Modelos Animales de Enfermedad , Infecciones por Enterobacteriaceae/complicaciones , Escherichia coli , Infecciones por Escherichia coli/complicaciones , Femenino , Inmunohistoquímica , Inflamación/complicaciones , Inflamación/microbiología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Salmonelosis Animal/complicaciones , Salmonella typhimuriumRESUMEN
Chemokines have been shown to be effective bactericidal molecules against a variety of bacteria and fungi in vitro. These direct antimicrobial effects are independent of their chemotactic activities involving immunological receptors. However, the direct biological role that these proteins may play in host defense, particularly against intestinal pathogens, is poorly understood. Here, we show that CXCL9, an ELR- chemokine, exhibits direct antimicrobial activity against Citrobacter rodentium, an attaching/effacing pathogen that infects the gut mucosa. Inhibition of this antimicrobial activity in vivo using anti-CXCL9 antibodies increases host susceptibility to C. rodentium infection with pronounced bacterial penetration into crypts, increased bacterial load, and worsened tissue pathology. Using Rag1(-/-) mice and CXCR3(-/-) mice, we demonstrate that the role for CXCL9 in protecting the gut mucosa is independent of an adaptive response or its immunological receptor, CXCR3. Finally, we provide evidence that phagocytes function in tandem with NK cells for robust CXCL9 responses to C. rodentium. These findings identify a novel role for the immune cell-derived CXCL9 chemokine in directing a protective antimicrobial response in the intestinal mucosa.
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Quimiocina CXCL9/inmunología , Infecciones por Enterobacteriaceae/inmunología , Mucosa Intestinal/inmunología , Transducción de Señal/inmunología , Animales , Quimiocinas/inmunología , Citrobacter rodentium/inmunología , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Host defense peptides secreted by colonocytes and Paneth cells play a key role in innate host defenses in the gut. In Crohn's disease, the burden of tissue-associated Escherichia coli commonly increases at epithelial surfaces where host defense peptides concentrate, suggesting that this bacterial population might actively resist this mechanism of bacterial killing. Adherent-invasive E. coli (AIEC) is associated with Crohn's disease; however, the colonization determinants of AIEC in the inflamed gut are undefined. Here, we establish that host defense peptide resistance contributes to host colonization by Crohn's-associated AIEC. We identified a plasmid-encoded genomic island (called PI-6) in AIEC strain NRG857c that confers high-level resistance to α-helical cationic peptides and α- and ß-defensins. Deletion of PI-6 sensitized strain NRG857c to these host defense molecules, reduced its competitive fitness in a mouse model of infection, and attenuated its ability to induce cecal pathology. This phenotype is due to two genes in PI-6, arlA, which encodes a Mig-14 family protein implicated in defensin resistance, and arlC, an OmpT family outer membrane protease. Implicit in these findings are new bacterial targets whose inhibition might limit AIEC burden and disease in the gut.
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Péptidos Catiónicos Antimicrobianos/inmunología , Enfermedad de Crohn/microbiología , Farmacorresistencia Bacteriana , Proteínas de Escherichia coli/metabolismo , Escherichia coli/inmunología , Péptido Hidrolasas/metabolismo , Factores de Virulencia/metabolismo , Animales , Modelos Animales de Enfermedad , Proteínas de Escherichia coli/genética , Femenino , Eliminación de Gen , Islas Genómicas , Humanos , Ratones , Péptido Hidrolasas/genética , Plásmidos , Factores de Virulencia/genéticaRESUMEN
Extracellular attaching and effacing (A/E) pathogens including pathogenic Escherichia coli colonize the host gut causing diarrhea and inflammation. Although much is known regarding the pathogenesis of A/E bacteria, there remains an incomplete understanding of host immune responses to these microbes. NK cells are an important source of IFN-γ and are essential for early innate responses to viral pathogens; however, their role during extracellular bacterial infections is still largely unexplored. We studied the host response to the murine A/E pathogen Citrobacter rodentium to investigate NK-cell function during infection. NK1.1⺠cell depletions and analysis of colonic intestinal inflammation following Citrobacter infection demonstrated that CD3â»NK1.1⺠cells play an important role in the initial clearance of C. rodentium, as evidenced by higher bacterial load, intestinal pathology, and crypt hyperplasia at the peak of inflammation in depleted mice. Loss of CD3â»NK1.1⺠cells resulted in lower colonic IFN-γ, TNF-α, and IL-12, and a delay in homing of IFN-γâºCD4⺠T cells to the gut. Loss of this response resulted in lower anti-C. rodentium IgG in NK1.1-depleted mice. These data establish that CD3â»NK1.1⺠cells are critical for inducing an early Th1 response involved in clearance of a pathogen that is restricted to the gastrointestinal tract.
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Citrobacter rodentium/inmunología , Colon/inmunología , Infecciones por Enterobacteriaceae/inmunología , Escherichia coli/inmunología , Hiperplasia/inmunología , Células Asesinas Naturales/inmunología , Células TH1/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Ly/metabolismo , Carga Bacteriana/inmunología , Complejo CD3/metabolismo , Movimiento Celular/inmunología , Colon/microbiología , Colon/patología , Femenino , Hiperplasia/microbiología , Interferón gamma/genética , Interferón gamma/metabolismo , Activación de Linfocitos , Depleción Linfocítica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismoRESUMEN
Crohn's disease is a chronic inflammatory condition of the gastrointestinal tract in which alterations to the bacterial community contribute to disease. Adherent-invasive Escherichia coli are associated with human Crohn's disease; however, their role in intestinal immunopathology is unclear because of the lack of an animal model compatible with chronic timescales. Here we establish chronic adherent-invasive Escherichia coli infection in streptomycin-treated conventional mice (CD1, DBA/2, C3H, 129e and C57BL/6), enabling the study of host response and immunopathology. Adherent-invasive Escherichia coli induces an active T-helper 17 response, heightened levels of proinflammatory cytokines and fibrotic growth factors, with transmural inflammation and fibrosis. Depletion of CD8+ T cells increases caecal bacterial load, pathology and intestinal fibrosis in C57BL/6 mice, suggesting a protective role. Our findings provide evidence that chronic adherent-invasive Escherichia coli infections result in immunopathology similar to that seen in Crohn's disease. With this model, research into the host and bacterial genetics associated with adherent-invasive Escherichia coli-induced disease becomes more widely accessible.
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Adhesión Bacteriana , Enfermedad de Crohn/microbiología , Infecciones por Escherichia coli/patología , Escherichia coli/fisiología , Inflamación/patología , Intestinos/microbiología , Intestinos/patología , Animales , Carga Bacteriana , Linfocitos T CD8-positivos/metabolismo , Ciego/microbiología , Ciego/patología , Recuento de Células , Enfermedad Crónica , Colon/microbiología , Colon/patología , Enfermedad de Crohn/complicaciones , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/patología , Infecciones por Escherichia coli/complicaciones , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Fibrosis , Regulación de la Expresión Génica , Humanos , Inflamación/complicaciones , Inflamación/inmunología , Inflamación/microbiología , Intestinos/inmunología , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Coloración y Etiquetado , Células TH1/inmunología , Células Th17/inmunologíaRESUMEN
Influenza epidemics and pandemics cause significant morbidity and mortality worldwide associated with severe immunopathology in the lung, and the mechanisms of such immunopathogenesis still remain poorly understood. While human studies help to understand influenza immunopathology, they provide only limited mechanistic information. On the other hand, recent studies using experimental animal models have significantly enhanced our understanding of the complex mechanisms involved in the immunopathogenesis during primary influenza or influenza-associated bacterial superinfection. This includes the involvement of acute inflammatory responses (macrophages, neutrophils, dendritic cells, toll-like receptors, cytokines, chemokines), CD4 and CD8 T cells, tissue remodeling processes, and contribution of bacterial superinfection. In particular, progress has been made in uncoupling the mechanisms that are involved in both anti-viral host defense and in immunopathogenesis from those that solely contribute to lung immunopathology. Uncoupling such events will facilitate the discovery of new intervention strategies to treat pulmonary immunopathology associated with influenza infection.
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Gripe Humana/inmunología , Animales , Infecciones Bacterianas/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Humanos , Inflamación/inmunología , Sobreinfección/inmunologíaRESUMEN
Lung immunopathology is the main cause of influenza-mediated morbidity and death, and much of its molecular mechanisms remain unclear. Whereas tumor necrosis factor-α (TNF-α) is traditionally considered a proinflammatory cytokine, its role in influenza immunopathology is unresolved. We have investigated this issue by using a model of acute H1N1 influenza infection established in wild-type and TNF-α-deficient mice and evaluated lung viral clearance, inflammatory responses, and immunopathology. Whereas TNF-α was up-regulated in the lung after influenza infection, it was not required for normal influenza viral clearance. However, TNF-α deficiency led not only to a greater extent of illness but also to heightened lung immunopathology and tissue remodeling. The severe lung immunopathology was associated with increased inflammatory cell infiltration, anti-influenza adaptive immune responses, and expression of cytokines such as monocyte chemoattractant protein-1 (MCP-1) and fibrotic growth factor, TGF-ß1. Thus, in vivo neutralization of MCP-1 markedly attenuated lung immunopathology and blunted TGF-ß1 production following influenza infection in these hosts. On the other hand, in vivo transgenic expression of MCP-1 worsened lung immunopathology following influenza infection in wild-type hosts. Thus, TNF-α is dispensable for influenza clearance; however, different from the traditional belief, this cytokine is critically required for negatively regulating the extent of lung immunopathology during acute influenza infection.
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Subtipo H1N1 del Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/inmunología , Neumonía Viral/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Inmunidad Adaptativa , Animales , Peso Corporal , Líquido del Lavado Bronquioalveolar , Quimiocina CCL2/deficiencia , Quimiocina CCL2/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Inmunidad Celular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Immunopathology is a major cause of influenza-associated morbidity and mortality worldwide. However, the role and regulatory mechanisms of CD4 T cells in severe lung immunopathology following acute influenza infection are poorly understood. In this paper, we report that the emergence of immunopathogenic CD4 T cells is under the control of a transmembrane immunoadaptor DAP12 pathway during influenza infection. We find that the mice lacking DAP12 have unaltered viral clearance but easily succumb to influenza infection as a result of uncontrolled immunopathology. Such immunopathology is associated with markedly increased CD4 T cells displaying markedly increased cytotoxicity and Fas ligand expression. Furthermore, the immunopathogenic property of these CD4 T cells is transferrable. Thus, depletion of CD4 T cells or abrogation of Fas/Fas ligand signaling pathway improves survival and immunopathology. We further find that DAP12 expressed by dendritic cells plays an important role in controlling the immunopathogenic CD4 T cells during influenza infection. Our findings identify a novel pathway that controls the level of immune-pathogenic CD4 T cells during acute influenza infection.
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Proteínas Adaptadoras Transductoras de Señales/inmunología , Linfocitos T CD4-Positivos/inmunología , Subtipo H1N1 del Virus de la Influenza A , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/patología , Traslado Adoptivo , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía/inmunología , Neumonía/patología , Neumonía/virología , Infecciones del Sistema Respiratorio/virología , Transducción de Señal/inmunologíaRESUMEN
The granuloma, a hallmark of host defense against pulmonary mycobacterial infection, has long been believed to be an active type 1 immune environment. However, the mechanisms regarding why granuloma fails to eliminate mycobacteria even in immune-competent hosts, have remained largely unclear. By using a model of pulmonary Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection, we have addressed this issue by comparing the immune responses within the airway luminal and granuloma compartments. We found that despite having a similar immune cellular profile to that in the airway lumen, the granuloma displayed severely suppressed type 1 immune cytokine but enhanced chemokine responses. Both antigen-presenting cells (APCs) and T cells in granuloma produced fewer type 1 immune molecules including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and nitric oxide. As a result, the granuloma APCs developed a reduced capacity to phagocytose mycobacteria and to induce T-cell proliferation. To examine the molecular mechanisms, we compared the levels of immune suppressive cytokine IL-10 in the airway lumen and granuloma and found that both granuloma APCs and T cells produced much more IL-10. Thus, IL-10 deficiency restored type 1 immune activation within the granuloma while having a minimal effect within the airway lumen. Hence, our study provides the first experimental evidence that, contrary to the conventional belief, the BCG-induced lung granuloma represents a symbiotic host-microbe microenvironment characterized by suppressed type 1 immune activation.
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Granuloma/microbiología , Interleucina-10/metabolismo , Mycobacterium bovis/metabolismo , Animales , Células Presentadoras de Antígenos/metabolismo , Vacuna BCG/metabolismo , Antígeno CD11b/biosíntesis , Antígeno CD11c/biosíntesis , Proliferación Celular , Femenino , Sistema Inmunológico , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Simbiosis , Linfocitos T/citología , Linfocitos T/microbiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Influenza viral infection is well-known to predispose to subsequent bacterial superinfection in the lung but the mechanisms have remained poorly defined. We have established a murine model of heterologous infections by an H1N1 influenza virus and Staphylococcus aureus. We found that indeed prior influenza infection markedly increased the susceptibility of mice to secondary S. aureus superinfection. Severe sickness and heightened bacterial infection in flu and S. aureus dual-infected animals were associated with severe immunopathology in the lung. We further found that flu-experienced lungs had an impaired NK cell response in the airway to subsequent S. aureus bacterial infection. Thus, adoptive transfer of naive NK cells to the airway of prior flu-infected mice restored flu-impaired antibacterial host defense. We identified that TNF-alpha production of NK cells played an important role in NK cell-mediated antibacterial host defense as NK cells in flu-experienced lungs had reduced TNF-alpha expression and adoptive transfer of TNF-alpha-deficient NK cells to the airway of flu-infected mice failed to restore flu-impaired antibacterial host defense. Defected NK cell function was found to be an upstream mechanism of depressed antibacterial activities by alveolar macrophages as contrast to naive wild-type NK cells, the NK cells from flu-infected or TNF-alpha-deficient mice failed to enhance S. aureus phagocytosis by alveolar macrophages. Together, our study identifies the weakened NK cell response in the lung to be a novel critical mechanism for flu-mediated susceptibility to bacterial superinfection.
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Subtipo H1N1 del Virus de la Influenza A/inmunología , Células Asesinas Naturales/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/microbiología , Neumonía Bacteriana/inmunología , Neumonía Viral/inmunología , Infecciones Estafilocócicas/inmunología , Sobreinfección/inmunología , Animales , Susceptibilidad a Enfermedades/inmunología , Susceptibilidad a Enfermedades/patología , Femenino , Células Asesinas Naturales/microbiología , Células Asesinas Naturales/virología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/microbiología , Macrófagos Alveolares/patología , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/patología , Neumonía Bacteriana/patología , Neumonía Bacteriana/virología , Neumonía Viral/microbiología , Neumonía Viral/patología , Infecciones Estafilocócicas/patología , Infecciones Estafilocócicas/virología , Sobreinfección/microbiología , Sobreinfección/patología , Sobreinfección/virologíaRESUMEN
RATIONALE: The airway luminal memory CD8 T cells induced by respiratory mucosal immunization in a murine model have been found to be critical to antituberculosis immunity. However, the mechanisms of their maintenance on airway mucosal surface still remain poorly understood. OBJECTIVES: Using a model of adenovirus-based intranasal immunization we investigated the immune property and the mechanisms of maintenance of airway luminal CD8 T cells. METHODS: Immune properties of airway luminal Mycobacterium tuberculosis antigen-specific CD8 T cells were examined. Proliferation of airway luminal CD8 T cells was determined by in vivo T cell-labeling techniques. The role of peripheral T cell recruitment in maintaining airway luminal CD8 T cells was investigated by blocking lymphocyte trafficking from lymphoid and peripheral tissues. The requirement of M. tuberculosis antigens for in situ T cell proliferation was evaluated using a T cell transfer approach. An airway M. tuberculosis challenge model was used to study the relationship between CD8 T cell-mediated protection and peripheral T cell recruitment. MEASUREMENTS AND MAIN RESULTS: Intranasal immunization leads to elicitation of persisting M. tuberculosis antigen-specific CD8 T cells in the airway lumen, which display an activated effector memory phenotype different from those in peripheral tissues. Airway luminal T cells continuously proliferate in an antigen-dependent manner, and can be maintained even in the absence of peripheral T cell recruitment. The lungs equipped with such CD8 T cells are protected from airway M. tuberculosis challenge independent of both peripheral T cell supply and CD4 T cells. CONCLUSIONS: Vaccine-inducible airway luminal antituberculosis memory CD8 T cells are self-renewable in an antigen-dependent manner, and can be maintained independent of peripheral T cell supply.
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Bronquios/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Mucosa Respiratoria/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Pulmonar/prevención & control , Administración Intranasal , Traslado Adoptivo/métodos , Animales , Antígenos Bacterianos/efectos de los fármacos , Antígenos Bacterianos/inmunología , Bronquios/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Inmunización Secundaria , Memoria Inmunológica/efectos de los fármacos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Ratones , Ratones Endogámicos BALB C , Mucosa Respiratoria/efectos de los fármacos , Tuberculosis Pulmonar/inmunologíaRESUMEN
Recombinant virus-vectored vaccines hold great promise for tuberculosis (TB) vaccination strategies. However, there is a lack of side-by-side comparative investigations to dissect the functional differences and support the advantage of multivalent virus-vectored vaccine over its monovalent counterpart. We previously successfully developed a monovalent adenovirus (Ad)-vectored vaccine expressing Ag85a (AdAg85a) and demonstrated its superior protective efficacy in models of pulmonary TB. In this study, we have developed a bivalent Ad TB vaccine expressing Ag85a and TB10.4 antigens as a fusion protein (AdAg85a:TB10.4) and compared its T-cell-activating and immune protective efficacy with that by monovalent AdAg85a. A single intranasal (i.n.) administration of AdAg85a:TB10.4 induced robust T-cell responses toward the respective antigens within the airway lumen and spleen, although the level of Ag85a-specific T-cell responses in the airway lumen triggered by bivalent AdAg85a:TB10.4 was lower than that by its monovalent counterpart at earlier time points. Thus, a single i.n. delivery of AdAg85a:TB10.4 conferred a markedly improved and sustained level of protection in the lung against Mycobacterium tuberculosis (M.tb) challenge over that by AdAg85a or by conventional BCG immunization with similarly induced levels of protection in the spleen. Our results indicate a unique advantage of multivalent viral-vectored TB vaccines for immunization against pulmonary TB.
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Adenoviridae/genética , Vectores Genéticos/genética , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Pulmonar/inmunología , Aciltransferasas/inmunología , Animales , Antígenos Bacterianos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Femenino , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Vacunas contra la Tuberculosis/genéticaRESUMEN
Protection by parenteral immunization with plasmid DNA vaccines against pulmonary tuberculosis (TB) is very modest. In this study, we have investigated the underlying mechanisms for the poor mucosal protective efficacy and the avenues and mechanisms to improve the efficacy of a single i.m. immunization with a monogenic plasmid DNA TB vaccine in a murine model. We show that i.m. DNA immunization fails to elicit accumulation of Ag-specific T cells in the airway lumen despite robust T cell responses in the spleen. Such systemically activated T cells cannot be rapidly mobilized into the airway lumen upon Mycobacterium tuberculosis exposure. However, airway deposition of low doses of soluble mycobacterial Ags in previously immunized mice effectively mobilizes the systemically activated T cells into the airway lumen. A fraction of such airway luminal T cells can persist in the airway lumen, undergo quick, robust expansion and activation and provide marked immune protection upon airway M. tuberculosis exposure. Airway mucosal deposition of soluble mycobacterial Ags was found to create a tissue microenvironment rich in proinflammatory molecules including chemokines and hence conducive to T cell recruitment. Thus, in vivo neutralization of MIP-1alpha or IFN-inducible protein-10 markedly inhibited the accumulation of Ag-specific T cells in the airway lumen. Our data suggest that immunoprotective efficacy on the mucosal surface by i.m. plasmid DNA immunization could be substantially improved by simple mucosal soluble Ag inoculation and restoration of mucosal luminal T cells. Our study holds implication for the future design of DNA vaccination strategies against intracellular infections.
Asunto(s)
Antígenos Bacterianos/farmacología , Inmunidad Mucosa/efectos de los fármacos , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/farmacología , Tuberculosis Pulmonar/prevención & control , Vacunas de ADN/farmacología , Administración Intranasal , Animales , Antígenos Bacterianos/inmunología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Quimiocina CCL3/inmunología , Quimiocina CXCL10/inmunología , Femenino , Humanos , Inmunidad Mucosa/inmunología , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Plásmidos/farmacología , Sistema Respiratorio/inmunología , Linfocitos T/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Pulmonar/inmunología , Vacunación/métodos , Vacunas de ADN/inmunologíaRESUMEN
BACKGROUND: The lung is divided into two major compartments: the alveolar space and the parenchyma. The alveolar macrophages are the first line of leukocytes in the lung taking up incoming microbes or microbial antigens whereas the parenchymal dendritic cells (DCs) are believed to be the sole potent antigen presenting cells (APCs) in the lung. Both resting alveolar macrophages and parenchymal DCs express CD11c. Several important questions remain to be elucidated: 1] to which extent the alveolar space and lung parenchymal CD11c+ APCs differ in their phenotype and ability to activate naïve T cells; 2] whether they differ in their ability to activate antigen-experienced or -primed T cells; and 3] whether these lung CD11c+ APC populations differ from the splenic CD11c+ APCs which have been commonly used for understanding APC biology. RESULTS: CD11c+ APCs from the alveolar space, lung parenchyma, and the spleen display differential co-stimulatory molecule expression and cytokine responsiveness upon stimulation. Alveolar space APCs are weak activators of naïve T cells compared to lung parenchymal and splenic CD11c+ APC populations. However, alveolar space APCs are able to potently activate the in vivo microbial antigen-primed T cells to a similar extent as lung parenchymal and splenic APCs. CONCLUSION: Together our findings indicate that alveolar CD11c+ APCs have a specialized T cell-activating function, capable of activating antigen-primed, but not naïve, T cells whereas lung CD11c+ APCs are capable of activating both the naïve and antigen-primed T cell populations.
Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Antígenos CD11/biosíntesis , Células Dendríticas/metabolismo , Pulmón/citología , Activación de Linfocitos/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos CD11/genética , Antígenos CD11/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Dendríticas/inmunología , Femenino , Memoria Inmunológica , Lipopolisacáridos/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , Ovalbúmina , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
In vitro manipulated dendritic cells (DC) have increasingly been used as a promising vaccine formulation against cancer and infectious disease. However, improved understanding of the immune mechanisms is needed for the development of safe and efficacious mucosal DC immunization. We have developed a murine model of respiratory mucosal immunization by using a genetically manipulated DC vaccine. Within 24 h of intranasal delivery, the majority of vaccine DCs migrated to the lung mucosa and draining lymph nodes and elicited a significant level of T cells capable of IFN-gamma secretion and CTL in the airway lumen as well as substantial T cell responses in the spleen. And such T cell responses were associated with enhanced protection against respiratory mucosal intracellular bacterial challenge. In comparison, parenteral i.m. DC immunization did not elicit marked airway luminal T cell responses and immune protection regardless of strong systemic T cell activation. Although repeated mucosal DC delivery boosted Ag-specific T cells in the airway lumen, added benefits to CD8 T cell activation and immune protection were not observed. By using MHC-deficient vaccine DCs, we further demonstrated that mucosal DC immunization-mediated CD8 and CD4 T cell activation does not require endogenous DCs. By using IL-12-deficient vaccine DCs, we also observed that IL-12(-/-) DCs failed to migrate to the lymph nodes but remained capable of T cell activation. Our observations indicate that mucosal delivery of vaccine DCs represents an effective approach to enhance mucosal T cell immunity, which may operate independent of vaccine IL-12 and endogenous DCs.
Asunto(s)
Traslado Adoptivo , Células Presentadoras de Antígenos/inmunología , Células Dendríticas/inmunología , Interleucina-12/deficiencia , Interleucina-12/fisiología , Activación de Linfocitos/inmunología , Mucosa Nasal/inmunología , Subgrupos de Linfocitos T/inmunología , Aciltransferasas/administración & dosificación , Aciltransferasas/genética , Aciltransferasas/inmunología , Administración Intranasal , Traslado Adoptivo/métodos , Secuencia de Aminoácidos , Animales , Células Presentadoras de Antígenos/metabolismo , Células Presentadoras de Antígenos/trasplante , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Células Dendríticas/microbiología , Células Dendríticas/trasplante , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos , Interleucina-12/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Mucosa Nasal/citología , Mucosa Nasal/metabolismo , Subgrupos de Linfocitos T/metabolismoRESUMEN
Staphylococcus aureus remains a common cause of nosocomial bacterial infections and are often antibiotic resistant. The role of NK cells and IL-15 and their relationship in host defense against extracellular bacterial pathogens including S. aureus remain unclear. We have undertaken several approaches to address this issue using wild type (WT), IL-15 gene knock-out (KO), and NK cell-depleted mouse models. Upon pulmonary staphylococcal infection WT mice had markedly increased activated NK cells, but not NKT or gammadelta T cells, in the airway lumen that correlated with IL-15 production in the airway and with alveolar macrophages. In vitro exposure to staphylococcal products and/or coculture with lung macrophages directly activated NK cells. In contrast, lung macrophages better phagocytosed S. aureus in the presence of NK cells. In sharp contrast to WT controls, IL-15 KO mice deficient in NK cells were found to be highly susceptible to pulmonary staphylococcal infection despite markedly increased neutrophils and macrophages in the lung. In further support of these findings, WT mice depleted of NK cells were similarly susceptible to staphylococcal infection while they remained fully capable of IL-15 production in the lung at levels similar to those of NK-competent WT hosts. Our study thus identifies a critical role for NK cells in host defense against pulmonary extracellular bacterial infection and suggests that IL-15 is involved in this process via its indispensable effect on NK cells, but not other innate cells. These findings hold implication for the development of therapeutics in treating antibiotic-resistant S. aureus infection.
Asunto(s)
Interleucina-15/metabolismo , Células Asesinas Naturales/inmunología , Macrófagos Alveolares/inmunología , Neumonía Estafilocócica/inmunología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Animales , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Interleucina-15/inmunología , Pulmón/inmunología , Pulmón/microbiología , Macrófagos Alveolares/microbiología , Ratones , Ratones Noqueados , Fagocitosis , Neumonía Estafilocócica/microbiología , Infecciones Estafilocócicas/microbiologíaRESUMEN
Gamma interferon (IFN-gamma) is a key cytokine in host defense against intracellular mycobacterial infection. It has been believed that both CD4 and CD8 T cells are the primary sources of IFN-gamma. However, the relative contributions of CD4 and CD8 T-cell subsets to IFN-gamma production and the relationship between CD4 and CD8 T-cell activation have not been examined. By using a model of pulmonary mycobacterial infection and various immunodetection assays, we found that CD4 T cells mounted a much stronger IFN-gamma response than CD8 T cells at various times after mycobacterial infection, and this pronounced IFN-gamma production by CD4 T cells was attributed to both greater numbers of antigen-specific CD4 T cells and a greater IFN-gamma secretion capacity of these cells. By using major histocompatibility complex class II-deficient or CD4-deficient mice, we found that the lack of CD4 T cells did not negatively affect primary or secondary CD8 T-cell IFN-gamma responses. The CD8 T cells activated in the absence of CD4 T cells were capable of immune protection against secondary mycobacterial challenge. Our results suggest that, whereas both CD4 and CD8 T cells are capable of IFN-gamma production, the former represent a much greater cellular source of IFN-gamma. Moreover, during mycobacterial infection, CD8 T-cell IFN-gamma responses and activation are independent of CD4 T-cell activation.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Interferón gamma/metabolismo , Activación de Linfocitos/inmunología , Mycobacterium bovis/patogenicidad , Tuberculosis Pulmonar/inmunología , Animales , Células Presentadoras de Antígenos , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Interferón gamma/inmunología , Ratones , Ratones Endogámicos C57BL , Tuberculosis Pulmonar/microbiologíaRESUMEN
Metarhizium anisopliae is an insect pathogenic fungus with a worldwide distribution. It is being developed and used as a biocontrol agent against a wide range of insect pests but relatively little is known of the life history of this fungus. We tested hypotheses concerning reproductive isolation and recombination in a sample of heat-active (ability to grow at 37 degrees C) and cold-active (ability to grow at 8 degrees C) sympatrically occurring isolates of M. anisopliae from Ontario, Canada by assaying nucleotide sequence variation at six polymorphic loci: the internally transcribed spacer (ITS) region of the nuclear ribosomal DNA repeat, and portions of calmodulin (CAL), chitin synthase (CHS), subtilisin-like protease (PR1), neutral trehalase (NTL) and actin (ACT)-encoding genes. The most parsimonious trees constructed showed a topology consistent with the heat-active and cold-active isolates as two monophyletic groups. We then applied Genealogical Concordance Phylogenetic Species Recognition (GCPSR) to the genealogical trees and concluded that the transition from concordance among branches to incongruity among branches delimited two species of M. anisopliae within Ontario. The GCPSR of two species was supported by intraspecific incongruity within each species when tested using the Partition Homogeneity test, indicating recombination. The GCPSR of two species also corresponded to the heat-active and cold-active groups. As the groups are morphologically indistinguishable we applied the term 'cryptic species'. Therefore, the sympatrically occurring heat-active and cold-active isolates represent different cryptic species with a history of recombination among isolates within each species.