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1.
Viruses ; 16(1)2024 01 22.
Article in English | MEDLINE | ID: mdl-38275975

ABSTRACT

BACKGROUND: The inovirus Pf4 is a lysogenic bacteriophage of Pseudomonas aeruginosa (Pa). People with Cystic Fibrosis (pwCF) experience chronic airway infection with Pa and a significant proportion have high numbers of Pf4 in their airway secretions. Given the known severe damage in the airways of Pa-infected pwCF, we hypothesized a high Pf4 burden can affect airway healing and inflammatory responses. In the airway, basal epithelial cells (BCs) are a multipotent stem cell population critical to epithelium homeostasis and repair. We sought to investigate the transcriptional responses of BCs under conditions that emulate infection with Pa and exposure to high Pf4 burden. METHODS: Primary BCs isolated from pwCF and wild-type (WT) donors were cultured in vitro and exposed to Pf4 or bacterial Lipopolysaccharide (LPS) followed by transcriptomic and functional assays. RESULTS: We found that BCs internalized Pf4 and this elicits a strong antiviral response as well as neutrophil chemokine production. Further, we found that BCs that take up Pf4 demonstrate defective migration and proliferation. CONCLUSIONS: Our findings are highly suggestive of Pf4 playing a role in the pathogenicity of Pa in the airways. These findings provide additional evidence for the ability of inoviruses to interact with mammalian cells and disrupt cell function.


Subject(s)
Cystic Fibrosis , Pseudomonas Infections , Animals , Humans , Respiratory System , Epithelial Cells , Epithelium , Cell Proliferation , Antiviral Agents , Pseudomonas aeruginosa/physiology , Pseudomonas Infections/microbiology , Mammals
2.
Sci Rep ; 13(1): 14453, 2023 09 02.
Article in English | MEDLINE | ID: mdl-37660113

ABSTRACT

Primary ciliary dyskinesia (PCD) is a genetic disorder associated with recurrent and chronic respiratory infections due to functional defects of motile cilia. In this study, we aimed to elucidate inflammatory and proliferative responses in PCD respiratory epithelium and evaluate the effect of Azithromycin (AZT) on these responses. Airway basal cells (BCs) were isolated from nasal samples of Wild-type (WT) epitope of healthy donors and PCD donors with bi-allelic mutations in DNAH5, DNAH11 and CCDC39. Cells were expanded in vitro and stimulated with either Lipopolysaccharide (LPS) or vehicle control. Post stimulation, cells were treated with either Azithromycin (AZT) or vehicle control. Cell proliferation was imaged in real-time. Separately, BCs from the same donors were expanded and grown at an air-liquid interface (ALI) to generate a multi-ciliated epithelium (MCE). Once fully mature, cells were stimulated with LPS, AZT, LPS + AZT or vehicle control. Inflammatory profiling was performed on collected media by cytokine Luminex assay. At baseline, there was a significantly higher mean production of pro-inflammatory cytokines by CCDC39 BCs and MCEs when compared to WT, DNAH11 and DNAH5 cells. AZT inhibited production of cytokines induced by LPS in PCD cells. Differences in cell proliferation were noted in PCD and this was also corrected with AZT treatment.


Subject(s)
Azithromycin , Ciliary Motility Disorders , Humans , Azithromycin/pharmacology , Lipopolysaccharides/toxicity , Epithelial Cells , Inflammation/drug therapy , Cell Proliferation , Cytokines
3.
Am J Physiol Lung Cell Mol Physiol ; 324(6): L771-L782, 2023 06 01.
Article in English | MEDLINE | ID: mdl-37039381

ABSTRACT

Multiciliated cell loss is a hallmark of airway epithelial remodeling in chronic inflammatory airway diseases including cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease. It disrupts mucociliary clearance, which fuels disease progression. Effective clearance requires an optimal proportion of multiciliated and secretory cells. This is controlled by Notch signaling such that between two adjacent cells the one that activates Notch becomes a secretory cell and the one that avoids Notch activation becomes a multiciliated cell. Consequently, blocking Notch by a small molecule inhibitor of the γ-secretase enzyme that cleaves the Notch receptor for signal activation directs differentiation toward the multiciliated lineage. Thus, γ-secretase inhibitor (GSI) treatment may alleviate multiciliated cell loss in lung disease. Here, we demonstrate the therapeutic restoration of multiciliated cells by the GSI LY450139 (semagacestat). LY450139 increased multiciliated cell numbers in a dose-dependent manner in healthy primary human nasal epithelial cells (HNECs) during differentiation and in mature cultures, but not when applied during early epithelialization of progenitors. LY450139 did not impact stem cell proliferation. Basal and apical administration were equally effective. In healthy adult mice, LY450139 increased multiciliated cell numbers without detectible toxicity. LY450139 also increased multiciliated cells and decreased excess mucus secretory cells in CF HNECs and IL-13 remodeled healthy HNECs. LY450139 normalized multiciliated cell numbers in CF HNECs without interfering with the activity of CFTR modulator compounds. In summary, we demonstrate that GSI administration is a promising therapeutic to restore multiciliated cells and potentially improve epithelial function in a wide range of chronic lung diseases.NEW & NOTEWORTHY Our findings show that low-dose, short-term topical or systemic γ-secretase inhibitor treatment may lead to restoration of multiciliated cells without toxicity and potentially improve epithelial function in a wide range of chronic lung diseases.


Subject(s)
Asthma , Cystic Fibrosis , Humans , Mice , Animals , Amyloid Precursor Protein Secretases/metabolism , Epithelium/metabolism , Epithelial Cells/metabolism , Signal Transduction/physiology , Receptors, Notch
4.
Cell ; 186(1): 112-130.e20, 2023 01 05.
Article in English | MEDLINE | ID: mdl-36580912

ABSTRACT

How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.


Subject(s)
COVID-19 , Respiratory System , SARS-CoV-2 , Humans , Cilia/physiology , Cilia/virology , COVID-19/virology , Respiratory System/cytology , Respiratory System/virology , SARS-CoV-2/physiology , Microvilli/physiology , Microvilli/virology , Virus Internalization , Epithelial Cells/physiology , Epithelial Cells/virology
5.
Mol Nutr Food Res ; 59(4): 763-72, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25641956

ABSTRACT

SCOPE: High incidence of inflammatory diseases afflicts the increasing aging-population infringing a great health burden. Dietary flavonoids, including the flavone apigenin, are emerging as important anti-inflammatory nutraceuticals due to their health benefits, lack of adverse effects and reduced costs. MicroRNAs (miRs) play a central role in inflammation by regulating gene expression, yet how dietary ingredients affect miRs is poorly understood. The aim of this study was to identify miRs involved in the anti-inflammatory activity of apigenin and apigenin-rich diets and determine their immune regulatory mechanisms in macrophages and in vivo. METHODS AND RESULTS: A high-throughput quantitative reverse transcriptase PCR screen of 312 miRs in macrophages revealed that apigenin reduced LPS-induced miR-155 expression. Analyses of miR-155 precursor and primary transcript indicated that apigenin regulated miR-155 transcriptionally. Apigenin-reduced expression of miR-155 led to the increase of anti-inflammatory regulators forkhead box O3a and smooth-muscle-actin and MAD-related protein 2 in LPS-treated macrophages. In vivo, apigenin or a celery-based apigenin-rich diet reduced LPS-induced expression of miR-155 and decreased tumor necrosis factor α in lungs from LPS-treated mice. CONCLUSION: These results demonstrate that apigenin and apigenin-rich diets exert effective anti-inflammatory activity in vivo by reducing LPS-induced expression of miR-155, thereby restoring immune balance.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Apigenin/pharmacology , Inflammation/drug therapy , MicroRNAs/metabolism , Animals , Apium/chemistry , Cell Line, Tumor , Diet , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Lipopolysaccharides/adverse effects , Macrophages/drug effects , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
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