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[This corrects the article DOI: 10.1371/journal.pone.0260706.].
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Protein kinase D (PKD) is a serine/threonine kinase family with three isoforms (PKD1-3) that are expressed in most cells and implicated in a wide array of signaling pathways, including cell growth, differentiation, transcription, secretion, polarization and actin turnover. Despite growing interest in PKD, relatively little is known about the role of PKD in immune responses. We recently published that inhibiting PKD limits proinflammatory cytokine secretion and leukocyte accumulation in mouse models of viral infection, and that PKD3 is highly expressed in the murine lung and immune cell populations. Here we focus on the immune-related phenotypes of PKD3 knockout mice. We report that PKD3 is necessary for maximal neutrophil accumulation in the lung following challenge with inhaled polyinosinic:polycytidylic acid, a double-stranded RNA, as well as following influenza A virus infection. Using reciprocal bone marrow chimeras, we found that PKD3 is required in the hematopoietic compartment for optimal neutrophil migration to the lung. Ex vivo transwell and chemokinesis assays confirmed that PKD3-/- neutrophils possess an intrinsic motility defect, partly because of reduced surface expression of CD18, which is critical for leukocyte migration. Finally, the peak of neutrophilia was significantly reduced in PKD3-/- mice after lethal influenza A virus infection. Together, these results demonstrate that PKD3 has an essential, and nonredundant, role in promoting neutrophil recruitment to the lung. A better understanding of the isoform-specific and cell type-specific activities of PKD has the potential to lead to novel therapeutics for respiratory illnesses.
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Neutrófilos , Proteína Quinasa C , Virosis , Animales , Ratones , Neutrófilos/metabolismo , Isoformas de Proteínas , Transducción de Señal , Proteína Quinasa C/metabolismoRESUMEN
Airway epithelial barrier dysfunction is increasingly recognized as a key feature of asthma and other lung diseases. Respiratory viruses are responsible for a large fraction of asthma exacerbations, and are particularly potent at disrupting epithelial barrier function through pattern recognition receptor engagement leading to tight junction dysfunction. Although different mechanisms of barrier dysfunction have been described, relatively little is known about whether barrier integrity can be promoted to limit disease. Here, we tested three classes of drugs commonly prescribed to treat asthma for their ability to promote barrier function using a cell culture model of virus-induced airway epithelial barrier disruption. Specifically, we studied the corticosteroid budesonide, the long acting beta-agonist formoterol, and the leukotriene receptor antagonist montelukast for their ability to promote barrier integrity of a monolayer of human bronchial epithelial cells (16HBE) before exposure to the viral mimetic double-stranded RNA. Of the three, only budesonide treatment limited transepithelial electrical resistance and small molecule permeability (4 kDa FITC-dextran flux). Next, we used a mouse model of acute dsRNA challenge that induces transient epithelial barrier disruption in vivo, and studied the effects budesonide when administered prophylactically or therapeutically. We found that budesonide similarly protected against dsRNA-induced airway barrier disruption in the lung, independently of its effects on airway inflammation. Taken together, these data suggest that an under-appreciated effect of inhaled budesonide is to maintain or promote airway epithelial barrier integrity during respiratory viral infections.
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Asma/tratamiento farmacológico , Bronquios/efectos de los fármacos , Broncodilatadores/farmacología , Budesonida/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Poli I-C/antagonistas & inhibidores , Acetatos/farmacología , Administración por Inhalación , Animales , Asma/inducido químicamente , Asma/metabolismo , Asma/patología , Bronquios/metabolismo , Bronquios/patología , Línea Celular , Ciclopropanos/farmacología , Dextranos/metabolismo , Impedancia Eléctrica , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Fumarato de Formoterol/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Imitación Molecular , Poli I-C/farmacología , Quinolinas/farmacología , ARN Bicatenario/antagonistas & inhibidores , ARN Bicatenario/farmacología , ARN Viral/antagonistas & inhibidores , ARN Viral/farmacología , Sulfuros/farmacología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismoRESUMEN
Rationale: Protein kinase D (PKD) is a serine/threonine kinase family that is involved in a wide array of signaling pathways. Although PKD has been implicated in immune responses, relatively little is known about the function of PKD in the lung or during viral infections. Objectives: We investigated the hypothesis that PKD is involved in multiple aspects of host response to viral infection. Methods: The selective PKD inhibitor CRT0010166 was administered to C57BL/6 mice prior to and during challenge with either inhaled double-stranded RNA or Influenza A Virus. PKD signaling pathways were investigated in human bronchial epithelial cells treated with CRT0010166, double-stranded RNA, and/or infected with Influenza A Virus. Measurements: Total protein and albumin accumulation in the bronchoalveolar fluid was used to asses inside/out leak. Clearance of inhaled FITC-dextran out of the airspace was used to assess outside/in leak. Cytokines and neutrophils in bronchoalveolar lavage were assayed with ELISAs and cytospins respectively. Viral RNA level was assessed with RT-PCR and protein level assessed by ELISA. Main Results: PKD inhibition prevented airway barrier dysfunction and pro-inflammatory cytokine release. Epithelial cells express PKD3, and PKD3 siRNA knock-down inhibited polyI:C induced cytokine production. Lung epithelial-specific deletion of PKD3 (CC10-Cre x PKD3-floxed mice) partially attenuated polyI:C-induced barrier disruption in vivo. Mechanistically, we found that PKD promoted cytokine mRNA transcription, not secretion, likely through activating the transcription factor Sp1. Finally, prophylactic CRT treatment of mice promoted barrier integrity during influenza virus infection and reduced viral burden. Conclusions: Inhibiting PKD promotes barrier integrity, limit pathogenic cytokine levels, and restrict Influenza A Virus infection. Therefore, PKD is an attractive target for novel antiviral therapeutics.
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Virus de la Influenza A/fisiología , Gripe Humana/inmunología , Infecciones por Orthomyxoviridae/inmunología , Proteína Quinasa C/metabolismo , Mucosa Respiratoria/metabolismo , Animales , Células Cultivadas , Dextranos , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa C/genética , Inhibidores de Proteínas Quinasas/administración & dosificación , ARN Interferente Pequeño/genética , Mucosa Respiratoria/patología , Transducción de Señal , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismoRESUMEN
The airway epithelial barrier is critical for preventing pathogen invasion and translocation of inhaled particles into the lung. Epithelial cells also serve an important sentinel role after infection and release various pro-inflammatory mediators that recruit and activate immune cells. Airway epithelial barrier disruption has been implicated in a growing number of respiratory diseases including viral infections. It is thought that when a pathogen breaks the barrier and gains access to the host tissue, pro-inflammatory mediators increase, which further disrupts the barrier and initiates a vicious cycle of leak. However, it is difficult to study airway barrier integrity in vivo, and little is known about relationship between epithelial barrier function and airway inflammation. Current assays of pulmonary barrier integrity quantify the leak of macromolecules from the vasculature into the airspaces (or "inside/out" leak). However, it is also important to measure the ease with which inhaled particles, allergens, or pathogens can enter the subepithelial tissues (or "outside/in" leak). We challenged mice with inhaled double stranded RNA (dsRNA) and explored the relationship between inside/out and outside/in barrier function and airway inflammation. Using wild-type and gene-targeted mice, we studied the roles of the dsRNA sensors Toll Like Receptor 3 (TLR3) and Melanoma Differentiation-Associated protein 5 (MDA5). Here we report that after acute challenge with inhaled dsRNA, airway barrier dysfunction occurs in a TLR3-dependent manner, whereas leukocyte accumulation is largely MDA5-dependent. We conclude that airway barrier dysfunction and inflammation are regulated by different mechanisms at early time points after exposure to inhaled dsRNA.