RESUMEN
During respiration, humans breathe in more than 10,000 liters of non-sterile air daily, allowing some pathogens access to alveoli. Interestingly, alveoli outnumber alveolar macrophages (AMs), which favors alveoli devoid of AMs. If AMs, like most tissue macrophages, are sessile, then this numerical advantage would be exploited by pathogens unless neutrophils from the blood stream intervened. However, this would translate to omnipresent persistent inflammation. Developing in vivo real-time intravital imaging of alveoli revealed AMs crawling in and between alveoli using the pores of Kohn. Importantly, these macrophages sensed, chemotaxed, and, with high efficiency, phagocytosed inhaled bacterial pathogens such as P. aeruginosa and S. aureus, cloaking the bacteria from neutrophils. Impairing AM chemotaxis toward bacteria induced superfluous neutrophil recruitment, leading to inappropriate inflammation and injury. In a disease context, influenza A virus infection impaired AM crawling via the type II interferon signaling pathway, and this greatly increased secondary bacterial co-infection.
Asunto(s)
Bacterias/inmunología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Animales , Femenino , Homeostasis , Humanos , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila , Neutrófilos/inmunología , Fagocitosis/inmunología , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/patogenicidad , Alveolos Pulmonares , Transducción de Señal , Staphylococcus aureus/inmunología , Staphylococcus aureus/patogenicidadRESUMEN
A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.
Asunto(s)
Dipeptidasas/metabolismo , Neutrófilos/fisiología , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Animales , Cilastatina/farmacología , Cilastatina/uso terapéutico , Dipeptidasas/antagonistas & inhibidores , Dipeptidasas/genética , Modelos Animales de Enfermedad , Endotoxemia/mortalidad , Endotoxemia/patología , Endotoxemia/prevención & control , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Lipopolisacáridos/farmacología , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/metabolismo , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Infiltración Neutrófila/efectos de los fármacos , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología , Tasa de SupervivenciaRESUMEN
Resident-tissue macrophages (RTMs) arise from embryonic precursors1,2, yet the developmental signals that shape their longevity remain largely unknown. Here we demonstrate in mice genetically deficient in 12-lipoxygenase and 15-lipoxygenase (Alox15-/- mice) that neonatal neutrophil-derived 12-HETE is required for self-renewal and maintenance of alveolar macrophages (AMs) during lung development. Although the seeding and differentiation of AM progenitors remained intact, the absence of 12-HETE led to a significant reduction in AMs in adult lungs and enhanced senescence owing to increased prostaglandin E2 production. A compromised AM compartment resulted in increased susceptibility to acute lung injury induced by lipopolysaccharide and to pulmonary infections with influenza A virus or SARS-CoV-2. Our results highlight the complexity of prenatal RTM programming and reveal their dependency on in trans eicosanoid production by neutrophils for lifelong self-renewal.
Asunto(s)
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Autorrenovación de las Células , Macrófagos Alveolares , Neutrófilos , Animales , Ratones , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Lesión Pulmonar Aguda , Animales Recién Nacidos , Araquidonato 12-Lipooxigenasa/deficiencia , Araquidonato 15-Lipooxigenasa/deficiencia , COVID-19 , Virus de la Influenza A , Lipopolisacáridos , Pulmón/citología , Pulmón/virología , Macrófagos Alveolares/citología , Macrófagos Alveolares/metabolismo , Neutrófilos/metabolismo , Infecciones por Orthomyxoviridae , Prostaglandinas E , SARS-CoV-2 , Susceptibilidad a EnfermedadesRESUMEN
Classic in vitro coculture assays of pathogens with host cells have contributed significantly to our understanding of the intracellular lifestyle of several pathogens. Coculture assays with pathogens and eukaryotic cells can be analyzed through various techniques including plating for colony-forming units (CFU), confocal microscopy, and flow cytometry. However, findings from in vitro assays require validation in an in vivo model. Several physiological conditions can influence host-pathogen interactions, which cannot easily be mimicked in vitro. Intravital microscopy (IVM) is emerging as a powerful tool for studying host-pathogen interactions by enabling in vivo imaging of living organisms. As a result, IVM has significantly enhanced the understanding of infection mediated by diverse pathogens. The versatility of IVM has also allowed for the imaging of various organs as sites of local infection. This chapter specifically focuses on IVM conducted on the lung for elucidating pulmonary immune response, primarily involving alveolar macrophages, to pathogens. Additionally, in this chapter we outline the protocol for lung IVM that utilizes a thoracic suction window to stabilize the lung for acquiring stable images.
Asunto(s)
Rastreo Celular , Microscopía Intravital , Macrófagos Alveolares , Macrófagos Alveolares/citología , Microscopía Intravital/métodos , Animales , Rastreo Celular/métodos , Ratones , Pulmón/citología , Interacciones Huésped-PatógenoRESUMEN
This study identified and characterized enteropathogenic Escherichia coli (EPEC) in the Canadian food supply. Eighteen of 450 E. coli isolates from food animal sources were identified as atypical EPEC (aEPEC). Several of the aEPEC isolates identified in this study possessed multiple virulence genes, exhibited adherence and attaching and effacing (A/E) lesion formation, disrupted tight junctions, and were coclassified with the extraintestinal pathogenic E. coli (ExPEC) and enterotoxigenic E. coli (ETEC) pathotypes.
Asunto(s)
Enfermedades de los Bovinos/epidemiología , Pollos , Escherichia coli Enteropatógena/genética , Infecciones por Escherichia coli/veterinaria , Carne/microbiología , Enfermedades de las Aves de Corral/epidemiología , Enfermedades de los Porcinos/epidemiología , Mataderos/normas , Animales , Western Blotting/veterinaria , Canadá/epidemiología , Bovinos , Enfermedades de los Bovinos/microbiología , Análisis por Conglomerados , Farmacorresistencia Microbiana/genética , Infecciones por Escherichia coli/epidemiología , Abastecimiento de Alimentos/normas , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa/veterinaria , Enfermedades de las Aves de Corral/microbiología , Porcinos , Enfermedades de los Porcinos/microbiologíaRESUMEN
Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) are food-borne pathogens that cause severe diarrhoeal disease in humans. Citrobacter rodentium is a related mouse pathogen that serves as a small animal model for EPEC and EHEC infections. EPEC, EHEC and C. rodentium translocate bacterial virulence proteins directly into host cells via a type III secretion system (T3SS). Non-LEE-encoded effector A (NleA) is a T3SS effector that is common to EPEC, EHEC and C. rodentium and is required for bacterial virulence. NleA localizes to the host cell secretory pathway and inhibits vesicle trafficking by interacting with the Sec24 subunit of mammalian coatamer protein II complex (COPII). Mammalian cells express four paralogues of Sec24 (Sec24A-D), which mediate selection of cargo proteins for transport and possess distinct, but overlapping cargo specificities. Here, we show that NleA binds Sec24A-D with two distinct mechanisms. An NleA protein variant with greatly diminished interaction with all Sec24 paralogues does not properly localize, does not inhibit COPII-mediated vesicle budding, and does not confer virulence in the mouse infection model. Together, this work provides strong evidence that the interaction and inhibition of COPII by NleA is an important aspect of EPEC- and EHEC-mediated disease.
Asunto(s)
Proteínas Bacterianas/metabolismo , Citrobacter rodentium/fisiología , Proteínas de Transporte Vesicular/metabolismo , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Vesículas Cubiertas por Proteínas de Revestimiento/microbiología , Citrobacter rodentium/metabolismo , Femenino , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Ratones , Ratones Endogámicos C3H , Dominios PDZ , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Transporte de Proteínas , Vías Secretoras , Eliminación de Secuencia , Proteínas de Transporte Vesicular/química , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
Resident macrophages play a unique role in the maintenance of tissue function. As phagocytes, they are an essential first line defenders against pathogens and much of the initial characterization of these cells was focused on their interaction with viral and bacterial pathogens. However, these cells are increasingly recognized as contributing to more than just host defense. Through cytokine production, receptor engagement and gap junction communication resident macrophages tune tissue inflammatory tone, influence adaptive immune cell phenotype and regulate tissue structure and function. This review highlights resident macrophages in the liver and lung as they hold unique roles in the maintenance of the interface between the circulatory system and the external environment. As such, we detail the developmental origin of these cells, their contribution to host defense and the array of tools these cells use to regulate tissue homeostasis.
Asunto(s)
Hígado , Macrófagos , Pulmón , Fagocitos , HomeostasisRESUMEN
In a world filled with microbes, some posing a threat to our body, our immune system is key to living a healthy life. The innate immune system is made of various cell types that act to guard our bodies. Unlike the adaptive immune system that has a specific response, our innate immune system encompasses cells that elicit unspecific immune responses, triggered whenever the right signals are detected. Our understanding of immunity started with the concept of our immune system only responding to "nonself" like the pathogens that invade our body. However, over the past few decades, we have learned that the immune system is more than an on/off switch that recognizes nonself. The innate immune system regularly patrols our bodies for pathogens and tissue damage. Our innate immune system not only seeks to resolve infection but also repair tissue injury, through phagocytosing debris and initiating the release of growth factors. Recently, we are starting to see that it is not just recognizing danger, our innate immune system plays a crucial role in repair. Innate immune cells phenotypically change during repair. In the context of severe injury or trauma, our innate immune system is modified quite drastically to help repair, resulting in reduced infection control. Moreover, these changes in immune cell function can be modified by sex as a biological variable. From past to present, in this overview, we provide a summary of the innate immune cells and pathways in infection and tissue repair. This article is categorized under: Immune System Diseases > Molecular and Cellular Physiology.
Asunto(s)
Sistema Inmunológico , Inmunidad Innata , Inmunidad Innata/fisiología , FagocitosisRESUMEN
Enteropathogenic Escherichia coli (EPEC) is a diarrhoeal pathogen that adheres to epithelial cells of the small intestine and uses a type III secretion system to inject effector proteins into host cells. EPEC infection leads to disruption of host intestinal tight junctions that are important for maintaining intestinal barrier function. This disruption is dependent on the bacterial type III secretion system, as well as the translocated effectors EspF and Map. Here we show that a third type III translocated bacterial effector protein, NleA, is also involved in tight junction disruption during EPEC infection. Using the drug Brefeldin A, we demonstrate that the effect of NleA on tight junction integrity is related to its inhibition of host cell protein trafficking through COPII-dependent pathways. These results suggest that NleA's striking effect on virulence is mediated, at least in part, via its role in disruption of intestinal barrier function.
Asunto(s)
Escherichia coli Enteropatógena/fisiología , Escherichia coli Enteropatógena/patogenicidad , Proteínas de Escherichia coli/fisiología , Intestinos/microbiología , Uniones Estrechas/metabolismo , Factores de Virulencia/fisiología , Virulencia/fisiología , Brefeldino A/farmacología , Células CACO-2 , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Línea Celular Tumoral , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Prueba de Complementación Genética , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Microscopía Fluorescente , Técnicas del Sistema de Dos Híbridos , Virulencia/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
As we learn more about chronic lung diseases, we are seeing that an unbalanced immune system plays a key role in disease pathogenesis. Innate immune cells, particularly tissue-resident macrophages, are important navigators of immunity, both during infection and in non-communicable lung disease. In the lung, alveolar macrophages are considered some of the most critical and diverse immune cells, yet despite an array of studies over the years, alveolar macrophages remain poorly understood. In this review, we highlight the importance of alveolar macrophages in health and disease, and discuss how proteomics can be used to elucidate mechanistic information and identify potential targets for therapy development.
Asunto(s)
Inflamación/inmunología , Enfermedades Pulmonares/inmunología , Pulmón/inmunología , Macrófagos Alveolares/inmunología , Proteoma/inmunología , Proteómica/métodos , Animales , Biomarcadores/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/patología , Pulmón/metabolismo , Pulmón/patología , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/patología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Proteoma/metabolismoRESUMEN
The underlying mechanisms contributing to injury-induced infection susceptibility remain poorly understood. Here, we describe a rapid increase in neutrophil cell numbers in the lungs following induction of thermal injury. These neutrophils expressed elevated levels of programmed death ligand 1 (PD-L1) and exhibited altered gene expression profiles indicative of a reparative population. Upon injury, neutrophils migrate from the bone marrow to the skin but transiently arrest in the lung vasculature. Arrested neutrophils interact with programmed cell death protein 1 (PD-1) on lung endothelial cells. A period of susceptibility to infection is linked to PD-L1+ neutrophil accumulation in the lung. Systemic treatment of injured animals with an anti-PD-L1 antibody prevented neutrophil accumulation in the lung and reduced susceptibility to infection but augmented skin healing, resulting in increased epidermal growth. This work provides evidence that injury promotes changes to neutrophils that are important for wound healing but contribute to infection susceptibility.
RESUMEN
Discussion on neutrophil extracellular traps (NETs) clearing inflammation from the closed eye, argues the role of NETs as an anti-inflammatory component of immunity.
Asunto(s)
Trampas Extracelulares , Antiinflamatorios , Humanos , Inflamación , NeutrófilosRESUMEN
It has long been appreciated that understanding the interactions between the host and the pathogens that make us sick is critical for the prevention and treatment of disease. As antibiotics become increasingly ineffective, targeting the host and specific bacterial evasion mechanisms are becoming novel therapeutic approaches. The technology used to understand host-pathogen interactions has dramatically advanced over the last century. We have moved away from using simple in vitro assays focused on single-cell events to technologies that allow us to observe complex multicellular interactions in real time in live animals. Specifically, intravital microscopy (IVM) has improved our understanding of infection, from viral to bacterial to parasitic, and how the host immune system responds to these infections. Yet, at the same time it has allowed us to appreciate just how complex these interactions are and that current experimental models still have a number of limitations. In this review, we will discuss the advances in vivo IVM has brought to the study of host-pathogen interactions, focusing primarily on bacterial infections and innate immunity.
Asunto(s)
Enfermedades Transmisibles/etiología , Interacciones Huésped-Patógeno/inmunología , Inmunidad , Animales , Enfermedades Transmisibles/diagnóstico , Enfermedades Transmisibles/metabolismo , Diagnóstico por Imagen/métodos , Susceptibilidad a Enfermedades , Humanos , Inmunidad Innata , Microscopía Intravital , Especificidad de Órganos , Índice de Severidad de la EnfermedadRESUMEN
Bacterial biofilm infections are difficult to eradicate because of antibiotic insusceptibility and high recurrence rates. Biofilm formation by Pseudomonas aeruginosa, a leading cause of bacterial keratitis, is facilitated by the bacterial Psl exopolysaccharide and associated with heightened virulence. Using intravital microscopy, we observed that neutrophilic recruitment to corneal infections limits P. aeruginosa biofilms to the outer eye surface, preventing bacterial dissemination. Neutrophils moved to the base of forming biofilms, where they underwent neutrophil extracellular trap formation (NETosis) in response to high expression of the bacterial type-3 secretion system (T3SS). NETs formed a barrier "dead zone," confining bacteria to the external corneal environment and inhibiting bacterial dissemination into the brain. Once formed, ocular biofilms were resistant to antibiotics and neutrophil killing, advancing eye pathology. However, blocking both Psl and T3SS together with antibiotic treatment broke down the biofilm and reversed keratitis, suggesting future therapeutic strategies for this intractable infection.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Córnea/microbiología , Trampas Extracelulares/metabolismo , Meningoencefalitis/prevención & control , Neutrófilos/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Modelos Animales de Enfermedad , Ratones , Infecciones por Pseudomonas/complicaciones , Pseudomonas aeruginosa/crecimiento & desarrolloRESUMEN
Enterohemorrhagic Escherichia coli (EHEC) is a water- and food-borne pathogen that causes hemorrhagic colitis. EHEC uses a type III secretion system (T3SS) to translocate effector proteins that subvert host cell function. T3SS-substrates encoded outside of the locus of enterocyte effacement are important to E. coli pathogenesis. We discovered an EHEC secreted protein, NleF, encoded by z6020 in O-island 71 of E. coli EDL933 that we hypothesized to be a T3SS substrate. Experiments are presented that probe the function of NleF and its role in virulence. Immunoblotting of secreted and translocated proteins suggest that NleF is secreted by the T3SS and is translocated into host cells in vitro where it localizes to the host cytoplasm. Infection of HeLa cells with E. coli possessing or lacking nleF and transient expression of NleF-GFP via transfection did not reveal a significant role for NleF in several assays of bacterial adherence, host cytoskeletal remodeling, or host protein secretion. However, competitive coinfection of mice with Citrobacter rodentium strains possessing or lacking nleF suggested a contribution of NleF to bacterial colonization. Challenge of gnotobiotic piglets also revealed a role for NleF in colonization of the piglet colon and rectoanal junction.
Asunto(s)
Citrobacter rodentium/genética , Citrobacter rodentium/patogenicidad , Escherichia coli O157/genética , Escherichia coli O157/patogenicidad , Proteínas de Escherichia coli/metabolismo , Factores de Virulencia/metabolismo , Animales , Células COS , Chlorocebus aethiops , Citoplasma/química , Infecciones por Enterobacteriaceae/microbiología , Células Epiteliales/química , Células Epiteliales/microbiología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Femenino , Tracto Gastrointestinal/microbiología , Células HeLa , Humanos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Porcinos , Virulencia , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
During sepsis, small blood vessels can become occluded by large platelet aggregates of poorly understood etiology. During Staphylococcal aureus infection, sepsis severity is linked to the bacterial α-toxin (α-hemolysin, AT) through unclear mechanisms. In this study, we visualized intravascular events in the microcirculation and found that intravenous AT injection induces rapid platelet aggregation, forming dynamic micro-thrombi in the microcirculation. These aggregates are retained in the liver sinusoids and kidney glomeruli, causing multi-organ dysfunction. Acute staphylococcal infection results in sequestration of most bacteria by liver macrophages. Platelets are initially recruited to these macrophages and help eradicate S. aureus. However, at later time points, AT causes aberrant and damaging thrombosis throughout the liver. Treatment with an AT neutralizing antibody (MEDI4893∗) prevents platelet aggregation and subsequent liver damage, without affecting the initial and beneficial platelet recruitment. Thus, AT neutralization may represent a promising approach to combat staphylococcal-induced intravascular coagulation and organ dysfunction.
Asunto(s)
Bacteriemia/fisiopatología , Toxinas Bacterianas/toxicidad , Proteínas Hemolisinas/toxicidad , Hígado/patología , Agregación Plaquetaria/efectos de los fármacos , Infecciones Estafilocócicas/fisiopatología , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Anticuerpos Neutralizantes/farmacología , Toxinas Bacterianas/inmunología , Anticuerpos ampliamente neutralizantes , Proteínas Hemolisinas/inmunología , Interacciones Huésped-Patógeno/fisiología , Humanos , Microscopía Intravital/métodos , Hígado/efectos de los fármacos , Hígado/microbiología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Mutantes , Agregación Plaquetaria/fisiología , Staphylococcus aureus/patogenicidadRESUMEN
Neutrophils have been implicated as harmful cells in a variety of inappropriate inflammatory conditions where they injure the host, leading to the death of the neutrophils and their subsequent phagocytosis by monocytes and macrophages. Here we show that in a fully repairing sterile thermal hepatic injury, neutrophils also penetrate the injury site and perform the critical tasks of dismantling injured vessels and creating channels for new vascular regrowth. Upon completion of these tasks, they neither die at the injury site nor are phagocytosed. Instead, many of these neutrophils reenter the vasculature and have a preprogrammed journey that entails a sojourn in the lungs to up-regulate CXCR4 (C-X-C motif chemokine receptor 4) before entering the bone marrow, where they undergo apoptosis.
Asunto(s)
Neovascularización Fisiológica/inmunología , Neutrófilos/inmunología , Cicatrización de Heridas/inmunología , Animales , Apoptosis/inmunología , Médula Ósea/inmunología , Movimiento Celular/inmunología , Hígado/irrigación sanguínea , Hígado/lesiones , Pulmón/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores CXCR4/metabolismo , Regulación hacia ArribaRESUMEN
Pseudomonas aeruginosa is a major cause of severe infections that lead to bacteremia and high patient mortality. P. aeruginosa has evolved numerous evasion and subversion mechanisms that work in concert to overcome immune recognition and effector functions in hospitalized and immunosuppressed individuals. Here, we have used multilaser spinning-disk intravital microscopy to monitor the blood-borne stage in a murine bacteremic model of P. aeruginosa infection. P. aeruginosa adhered avidly to lung vasculature, where patrolling neutrophils and other immune cells were virtually blind to the pathogen's presence. This cloaking phenomenon was attributed to expression of Psl exopolysaccharide. Although an anti-Psl mAb activated complement and enhanced neutrophil recognition of P. aeruginosa, neutrophil-mediated clearance of the pathogen was suboptimal owing to a second subversion mechanism, namely the type 3 secretion (T3S) injectisome. Indeed, T3S prevented phagosome acidification and resisted killing inside these compartments. Antibody-mediated inhibition of the T3S protein PcrV did not enhance bacterial phagocytosis but did enhance killing of the few bacteria ingested by neutrophils. A bispecific mAb targeting both Psl and PcrV enhanced neutrophil uptake of P. aeruginosa and also greatly increased inhibition of T3S function, allowing for phagosome acidification and bacterial killing. These data highlight the need to block multiple evasion and subversion mechanisms in tandem to kill P. aeruginosa.
Asunto(s)
Antibacterianos/farmacología , Anticuerpos Monoclonales/farmacología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/inmunología , Animales , Anticuerpos Biespecíficos , Antígenos Bacterianos/inmunología , Carga Bacteriana , Proteínas Bacterianas/inmunología , Toxinas Bacterianas/inmunología , Proteínas del Sistema Complemento/metabolismo , Evaluación Preclínica de Medicamentos , Femenino , Macrófagos del Hígado/microbiología , Pulmón/irrigación sanguínea , Pulmón/microbiología , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Microvasos/microbiología , Neutrófilos/inmunología , Neutrófilos/microbiología , Fagocitosis , Proteínas Citotóxicas Formadoras de Poros/inmunología , Infecciones por Pseudomonas/inmunología , Receptores Fc/metabolismoRESUMEN
iNKT cells are a subset of innate T cells that recognize glycolipids presented on CD1d molecules and protect against bacterial infections, including S. pneumoniae. Using lung intravital imaging, we examined the behavior and mechanism of pulmonary iNKT cell activation in response to the specific iNKT cell ligand α-galactosylceramide or S. pneumoniae infection. In untreated mice, the major fraction of iNKT cells resided in the vasculature, but a small critical population resided in the extravascular space in proximity to monocyte-derived DCs. Administration of either α-GalCer or S. pneumoniae induced CD1d-dependent rapid recruitment of neutrophils out of the vasculature. The neutrophils guided iNKT cells from the lung vasculature via CCL17. Depletion of monocyte-derived DCs abrogated both the neutrophil and subsequent iNKT cell extravasation. Moreover, impairing iNKT cell recruitment by blocking CCL17 increased susceptibility to S. pneumoniae infection, suggesting a critical role for the influx of iNKT cells in host defense.
Asunto(s)
Células Dendríticas/inmunología , Inflamación/inmunología , Células T Asesinas Naturales/inmunología , Neutrófilos/inmunología , Infecciones Neumocócicas/inmunología , Animales , Quimiotaxis de Leucocito/inmunología , Pulmón/inmunología , Pulmón/microbiología , Ratones , Ratones Transgénicos , Monocitos/citología , Monocitos/inmunologíaRESUMEN
Neutrophil extracellular traps (NETs) composed of DNA decorated with histones and proteases trap and kill bacteria but also injure host tissue. Here we show that during a bloodstream infection with methicillin-resistant Staphylococcus aureus, the majority of bacteria are sequestered immediately by hepatic Kupffer cells, resulting in transient increases in liver enzymes, focal ischaemic areas and a robust neutrophil infiltration into the liver. The neutrophils release NETs into the liver vasculature, which remain anchored to the vascular wall via von Willebrand factor and reveal significant neutrophil elastase (NE) proteolytic activity. Importantly, DNase although very effective at DNA removal, and somewhat effective at inhibiting NE proteolytic activity, fails to remove the majority of histones from the vessel wall and only partly reduces injury. By contrast, inhibition of NET production as modelled by PAD4-deficiency, or prevention of NET formation and proteolytic activity as modelled in NE(-/-) mice prevent collateral host tissue damage.