RESUMEN
The upper respiratory tract (nasopharynx or NP) is the first site of influenza replication, allowing the virus to disseminate to the lower respiratory tract or promoting community transmission. The host response in the NP regulates an intricate balance between viral control and tissue pathology. The hyper-inflammatory responses promote epithelial injury, allowing for increased viral dissemination and susceptibility to secondary bacterial infections. However, the pathologic contributors to influenza upper respiratory tissue pathology are incompletely understood. In this study, we investigated the role of interleukin IL-17 recetor A (IL-17RA) as a modulator of influenza host response and inflammation in the upper respiratory tract. We used a combined experimental approach involving IL-17RA-/- mice and an air-liquid interface (ALI) epithelial culture model to investigate the role of IL-17 response in epithelial inflammation, barrier function, and tissue pathology. Our data show that IL-17RA-/- mice exhibited significantly reduced neutrophilia, epithelial injury, and viral load. The reduced NP inflammation and epithelial injury in IL-17RA-/- mice correlated with increased resistance against co-infection by Streptococcus pneumoniae (Spn). IL-17A treatment, while potentiating the apoptosis of IAV-infected epithelial cells, caused bystander cell death and disrupted the barrier function in ALI epithelial model, supporting the in vivo findings.
Asunto(s)
Gripe Humana , Animales , Ratones , Humanos , Gripe Humana/complicaciones , Interleucina-17/genética , Interleucina-17/metabolismo , Inflamación/complicaciones , Streptococcus pneumoniae/metabolismo , InterleucinasRESUMEN
An exuberant host response contributes to influenza A virus (IAV) (or influenza)-mediated lung injury. However, despite significant information on the host response to IAV, the cellular framework and molecular interactions that dictate the development of acute injury in IAV-infected lungs remain incompletely understood. We performed an unbiased single-cell RNA sequencing (scRNAseq) analysis to examine the cellular heterogeneity and regulation of host responses in the IAV model of acute lung injury. At the cellular level, IAV infection promoted the overwhelming recruitment of monocytes that exhibited the cell differentiation trajectory to monocyte-derived macrophages. Together, monocytes and monocyte-derived myeloid cells constituted over 50% of the total immune cells in IAV-infected lungs. In contrast, IAV infection resulted in a significant loss of nonhematopoietic cells. Molecularly, our data show the multidimensional cell-cell communication dynamics of interferon and chemokine signaling between immune and nonimmune cells and the cell-specific molecular pathways regulating the host responses during IAV-induced lung injury. Our data provide a foundation for further exploring the mechanistic association of the IAV host response with acute lung injury. IMPORTANCE A dysregulated host response develops acute lung injury during IAV infection. However, the pathological immune mechanism(s) associated with acute lung injury during IAV infection is yet to be elucidated. In this study, we performed scRNAseq to examine the dynamics of host responses during the peak of IAV-mediated lung injury. At the cellular level, our data reveal significant myelopoiesis predominated by monocytes and macrophages and the simultaneous disruption of the nonhematopoietic cell framework, crucial for regulating inflammation and barrier integrity in IAV-infected lungs. Molecularly, we observed a complex cellular network involving cell-cell communications and a number of unique regulons dictating the outcome of interferon and chemokine responses during peak lung injury. Our data present a unique atlas of cellular changes and the regulation of global and cell-specific host responses during IAV infection. We expect that this information will open new avenues to identify targets for therapeutic intervention against IAV lung injury.
Asunto(s)
Lesión Pulmonar Aguda , Virus de la Influenza A , Gripe Humana , Infecciones por Orthomyxoviridae , Humanos , Interferones/metabolismo , Pulmón , Lesión Pulmonar Aguda/patología , Antivirales/metabolismoRESUMEN
Allergic asthma (AA) is characterized as a Th2-biased airway inflammation that can develop lung inflammation and remodeling of the respiratory tract. Streptococcus pneumoniae is a major respiratory pathogen, causing noninvasive (otitis media and pneumonia) and invasive diseases (sepsis) in humans. We sought to determine the role of IL-6 in the regulation of lung inflammation in murine AA caused by Aspergillus fumigatus as well as its consequence on the regulation of airway barrier integrity and S. pneumoniae disease. In an AA model, IL-6 deficiency led to increased lung inflammation, eosinophil recruitment, tissue pathology, and collagen deposition. Additionally, IL-6-deficient asthmatic mice exhibited reduced goblet cell hyperplasia and increased TGF-ß production. These key changes in the lungs of IL-6-deficient asthmatic mice resulted in dysregulated tight junction proteins and increased lung permeability. Whereas the host response to AA protected against S. pneumoniae lung disease, the IL-6 deficiency abrogated the protective effect of allergic inflammation against S. pneumoniae pathogenesis. Consistent with in vivo data, IL-6 knockdown by small interfering RNA or the blockade of IL-6R signaling exacerbated the TGF-ß-induced dysregulation of tight junction proteins, E-cadherin and N-cadherin expression, and STAT3 phosphorylation in MLE-12 epithelial cells. Our findings demonstrate a previously unrecognized role of host IL-6 response in the regulation of lung inflammation during AA and the control of S. pneumoniae bacterial disease. A better understanding of the interactions between lung inflammation and barrier framework could lead to the development of therapies to control asthma inflammation and preserve barrier integrity.
Asunto(s)
Asma/inmunología , Células Caliciformes/patología , Hipersensibilidad/inmunología , Interleucina-6/metabolismo , Neumonía Neumocócica/inmunología , Neumonía/inmunología , Mucosa Respiratoria/metabolismo , Streptococcus pneumoniae/fisiología , Uniones Estrechas/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Resistencia a la Enfermedad , Humanos , Hiperplasia , Interleucina-6/genética , Ratones , Ratones Noqueados , ARN Interferente Pequeño/genética , Mucosa Respiratoria/patología , Transducción de Señal , Factor de Crecimiento Transformador beta/sangreRESUMEN
Contamination inspection is an ongoing concern for food distributors, restaurant owners, caterers, and others who handle food. Food contamination must be prevented, and zero tolerance legal requirements and damage to the reputation of institutions or restaurants can be very costly. This paper introduces a new handheld fluorescence-based imaging system that can rapidly detect, disinfect, and document invisible organic residues and biofilms which may host pathogens. The contamination, sanitization inspection, and disinfection (CSI-D) system uses light at two fluorescence excitation wavelengths, ultraviolet C (UVC) at 275 nm and violet at 405 nm, for the detection of organic residues, including saliva and respiratory droplets. The 275 nm light is also utilized to disinfect pathogens commonly found within the contaminated residues. Efficacy testing of the neutralizing effects of the ultraviolet light was conducted for Aspergillus fumigatus, Streptococcus pneumoniae, and the influenza A virus (a fungus, a bacterium, and a virus, respectively, each commonly found in saliva and respiratory droplets). After the exposure to UVC light from the CSI-D, all three pathogens experienced deactivation (> 99.99%) in under ten seconds. Up to five-log reductions have also been shown within 10 s of UVC irradiation from the CSI-D system.
Asunto(s)
Desinfección , Rayos Ultravioleta , Biopelículas , Hongos , Imagen ÓpticaRESUMEN
BACKGROUND: We sought to determine the role of host interleukin 17A (IL-17A) response against colonizing Streptococcus pneumoniae, and its transition to a pathogen during coinfection with an influenza virus, influenza A H1N1 A/Puerto Rico/8/1934 (PR8). METHOD: Wild-type (WT) C57BL/6 mice were intranasally inoculated with S. pneumoniae serotype 6A to establish colonization and later infected with the influenza strain, PR8, resulting in invasive S. pneumoniae disease. The role of the IL-17A response in colonization and coinfection was investigated in WT, RoRγt-/- and RAG1-/- mice with antibody-mediated depletion of IL-17A (WT) and CD90 cells (RAG1-/-). RESULTS: RAG1-/- mice did not clear colonization and IL-17A neutralization impaired 6A clearance in WT mice. RoRγt-/- mice also had reduced clearance. S. pneumoniae-PR8 coinfection elicited a robust IL-17A response in the nasopharynx; IL-17A neutralization reduced S. pneumoniae invasive disease. RoRγt-/- mice also had reduced S. pneumoniae disease in a coinfection model. Depletion of CD90+ cells suppressed the IL-17A response and reduced S. pneumoniae invasion in RAG1-/- mice. CONCLUSION: Our data show that although IL-17A reduces S. pneumoniae colonization, coinfection with influenza virus elicits a robust innate IL-17A response that promotes inflammation and S. pneumoniae disease in the nasopharynx.
Asunto(s)
Coinfección , Gripe Humana/complicaciones , Interleucina-17/farmacología , Infecciones Neumocócicas/inmunología , Streptococcus pneumoniae/efectos de los fármacos , Inmunidad Adaptativa , Animales , Quimiocinas/análisis , Citocinas/análisis , Modelos Animales de Enfermedad , Femenino , Proteínas de Homeodominio/metabolismo , Humanos , Subtipo H1N1 del Virus de la Influenza A , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nasofaringe/microbiología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Infecciones Neumocócicas/patología , Serogrupo , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pneumoniae/patogenicidad , Antígenos Thy-1RESUMEN
The dual role of CD8+ T cells in influenza control and lung pathology is increasingly appreciated. To explore whether protective and pathological functions can be linked to specific subsets, we dissected CD8+ T responses in influenza-infected murine lungs. Our single-cell RNA-sequencing (scRNA-seq) analysis revealed notable diversity in CD8+ T subpopulations during peak viral load and infection-resolved state. While enrichment of a Cxcr3hi CD8+ T effector subset was associated with a more robust cytotoxic response, both CD8+ T effector and central memory exhibited equally potent effector potential. The scRNA-seq analysis identified unique regulons regulating the cytotoxic response in CD8+ T cells. The late-stage CD8+ T blockade in influenza-cleared lungs or continuous CXCR3 blockade mitigated lung injury without affecting viral clearance. Furthermore, adoptive transfer of wild-type CD8+ T cells exacerbated influenza lung pathology in Cxcr3-/- mice. Collectively, our data imply that CXCR3 interception could have a therapeutic effect in preventing influenza-linked lung injury.
Asunto(s)
Gripe Humana , Lesión Pulmonar , Animales , Humanos , Ratones , Linfocitos T CD8-positivos , Pulmón , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de QuimiocinaRESUMEN
Influenza A virus (IAV) infection triggers an exuberant host response that promotes acute lung injury. However, the host response factors that promote the development of a pathologic inflammatory response to IAV remain incompletely understood. In this study, we identify an interferon-γ (IFN-γ)-regulated subset of monocytes, CCR2+ monocytes, as a driver of lung damage during IAV infection. IFN-γ regulates the recruitment and inflammatory phenotype of CCR2+ monocytes, and mice deficient in CCR2 (CCR2-/-) or IFN-γ (IFN-γ-/-) exhibit reduced lung inflammation, pathology, and disease severity. Adoptive transfer of wild-type (WT) (IFN-γR1+/+) but not IFN-γR1-/- CCR2+ monocytes restore the WT-like pathological phenotype of lung damage in IAV-infected CCR2-/- mice. CD8+ T cells are the main source of IFN-γ in IAV-infected lungs. Collectively, our data highlight the requirement of IFN-γ signaling in the regulation of CCR2+ monocyte-mediated lung pathology during IAV infection.
Asunto(s)
Virus de la Influenza A , Gripe Humana , Lesión Pulmonar , Infecciones por Orthomyxoviridae , Animales , Linfocitos T CD8-positivos , Humanos , Interferón gamma , Pulmón , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos , Infecciones por Orthomyxoviridae/complicacionesRESUMEN
Flow cytometry is a popular technique used for both clinical and research purposes. It involves laser-based technology to characterize cells based on size, shape, and complexity. Additionally, flow cytometers are equipped with the ability to take fluorescence measurements at multiple wavelengths. This capability makes the flow cytometer a practical resource in the utilization of fluorescently conjugated antibodies, fluorescent proteins, DNA binding dyes, viability dyes, and ion indicator dyes. As the technology advances, the number of parameters a flow cytometer can measure has increased tremendously, and now some has the capacity to analyze 30-50 or more parameters on a single cell. Here, we describe the basic principles involved in the mechanics and procedures of flow cytometry along with an insight into applications of flow cytometry techniques for biomedical and allergic disease research.
Asunto(s)
Citometría de Flujo/métodos , Hipersensibilidad/inmunología , Inmunofenotipificación/métodos , Análisis de la Célula Individual/métodos , Linfocitos T/citología , Animales , Anticuerpos/análisis , Antígenos CD/genética , Antígenos CD/inmunología , Biomarcadores/análisis , Investigación Biomédica/instrumentación , Investigación Biomédica/métodos , Supervivencia Celular , Citometría de Flujo/instrumentación , Colorantes Fluorescentes/química , Humanos , Hipersensibilidad/diagnóstico , Hipersensibilidad/genética , Hipersensibilidad/patología , Inmunoconjugados/análisis , Rayos Láser , Análisis de la Célula Individual/instrumentación , Linfocitos T/inmunologíaRESUMEN
The use of flow cytometry allows simultaneous measurement and multiparametric analysis of single cells in a heterogenous solution. The purpose of flow cytometry can vary depending on the use of antibodies and dyes targeted for specific cell molecules. The method of immune-phenotyping with fluorescently conjugated antibodies to label cell proteins or DNA works in tandem with fluidic, optic, and electrical systems present in the flow cytometer. Some flow cytometers can detect numerous fluorescent molecules on a single cell, allowing the measurement of more than 30 parameters. This ability to detect, measure, and quantitate multiple fluorescent markers on a single cell makes the flow cytometer a useful tool for analyzing various aspects of cell phenotype and function. Here we describe a standardized protocol for surface and intracellular immune-phenotyping of murine lungs, beginning with the building of an optimal antibody panel and ending with data analysis and representation, including sample gating strategies for innate and adaptive immune responses.
Asunto(s)
Citometría de Flujo/métodos , Colorantes Fluorescentes/química , Inmunofenotipificación/métodos , Pulmón/metabolismo , Coloración y Etiquetado/métodos , Inmunidad Adaptativa , Animales , Anticuerpos/química , Antígenos CD/genética , Antígenos CD/inmunología , Linfocitos B/clasificación , Linfocitos B/citología , Linfocitos B/metabolismo , Biomarcadores/análisis , Carbocianinas/química , Recuento de Células , Supervivencia Celular , Células Dendríticas/citología , Células Dendríticas/metabolismo , Eosinófilos/citología , Eosinófilos/metabolismo , Citometría de Flujo/instrumentación , Humanos , Inmunidad Innata , Pulmón/citología , Macrófagos Alveolares/citología , Macrófagos Alveolares/metabolismo , Ratones , Monocitos/citología , Monocitos/metabolismo , Neutrófilos/citología , Neutrófilos/metabolismo , Cultivo Primario de Células , Linfocitos T/clasificación , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
We evaluated the effectiveness of anti-22F serotype immunity in the prevention of Streptococcus pneumoniae (Spn) bacterial transmission during colonization and influenza virus co-infection. Mice were immunized with 22F formulation and later colonized with Spn or co-infected with Spn and influenza virus. The 22F antisera exhibited strong reactivity to 22F bacteria and promoted the opsonic uptake of Spn by the neutrophils. The 22F vaccination led to a significant reduction of bacterial densities in the nasopharynx and prevented bacterial transmission during colonization and co-infection. The transfer of 22F antisera to infant mice resulted in reduced bacterial transmission in colonization and co-infection models.