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Lew SQ, Chong SY, Lau GW. Modulation of pulmonary immune functions by the Pseudomonas aeruginosa secondary metabolite pyocyanin. Front Immunol 2025; 16:1550724. [PMID: 40196115 PMCID: PMC11973339 DOI: 10.3389/fimmu.2025.1550724] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2024] [Accepted: 03/05/2025] [Indexed: 04/09/2025] Open
Abstract
Pseudomonas aeruginosa is a prevalent opportunistic Gram-negative bacterial pathogen. One of its key virulence factors is pyocyanin, a redox-active phenazine secondary metabolite that plays a crucial role in the establishment and persistence of chronic infections. This review provides a synopsis of the mechanisms through which pyocyanin exacerbates pulmonary infections. Pyocyanin induces oxidative stress by generating reactive oxygen and nitrogen species which disrupt essential defense mechanisms in respiratory epithelium. Pyocyanin increases airway barrier permeability and facilitates bacterial invasion. Pyocyanin also impairs mucociliary clearance by damaging ciliary function, resulting in mucus accumulation and airway obstruction. Furthermore, it modulates immune responses by promoting the production of pro-inflammatory cytokines, accelerating neutrophil apoptosis, and inducing excessive neutrophil extracellular trap formation, which exacerbates lung tissue damage. Additionally, pyocyanin disrupts macrophage phagocytic function, hindering the clearance of apoptotic cells and perpetuating inflammation. It also triggers mucus hypersecretion by inactivating the transcription factor FOXA2 and enhancing the IL-4/IL-13-STAT6 and EGFR-AKT/ERK1/2 signaling pathways, leading to goblet cell metaplasia and increased mucin production. Insights into the role of pyocyanin in P. aeruginosa infections may reveal potential therapeutic strategies to alleviate the severity of infections in chronic respiratory diseases including cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD).
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Affiliation(s)
| | | | - Gee W. Lau
- Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL, United States
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Lee RJ, Adappa ND, Palmer JN. Akt activator SC79 stimulates antibacterial nitric oxide generation in human nasal epithelial cells in vitro. Int Forum Allergy Rhinol 2024; 14:1147-1162. [PMID: 38197521 PMCID: PMC11219270 DOI: 10.1002/alr.23318] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2023] [Revised: 12/05/2023] [Accepted: 12/26/2023] [Indexed: 01/11/2024]
Abstract
BACKGROUND The role of Akt in nasal immunity is unstudied. Akt phosphorylates and activates endothelial nitric oxide synthase (eNOS) expressed in epithelial ciliated cells. Nitric oxide (NO) production by ciliated cells can have antibacterial and antiviral effects. Increasing nasal NO may be a useful antipathogen strategy in chronic rhinosinusitis (CRS). We previously showed that small-molecule Akt activator SC79 induces nasal cell NO production and suppresses IL-8 via the transcription factor Nrf-2. We hypothesized that SC79 NO production may additionally have antibacterial effects. METHODS NO production was measured using fluorescent dye DAF-FM. We tested effects of SC79 during co-culture of Pseudomonas aeruginosa with primary nasal epithelial cells, using CFU counting and live-dead staining to quantify bacterial killing. Pharmacology determined the mechanism of SC79-induced NO production and tested dependence on Akt. RESULTS SC79 induced dose-dependent, Akt-dependent NO production in nasal epithelial cells. The NO production required eNOS and Akt. The NO released into the airway surface liquid killed P. aeruginosa. No toxicity (LDH release) or inflammatory effects (IL8 transcription) were observed over 24 h. CONCLUSIONS Together, these data suggest multiple immune pathways are stimulated by SC79, with antipathogen effects. This in vitro pilot study suggests that a small-molecule Akt activator may have clinical utility in CRS or respiratory other infection settings, warranting future in vivo studies.
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Affiliation(s)
- Robert J. Lee
- Department of Otorhinolaryngology, University of Pennsylvania Perelman School of Medicine
- Department of Physiology, University of Pennsylvania Perelman School of Medicine
| | - Nithin D. Adappa
- Department of Otorhinolaryngology, University of Pennsylvania Perelman School of Medicine
| | - James N. Palmer
- Department of Otorhinolaryngology, University of Pennsylvania Perelman School of Medicine
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3
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Despotes KA, Zariwala MA, Davis SD, Ferkol TW. Primary Ciliary Dyskinesia: A Clinical Review. Cells 2024; 13:974. [PMID: 38891105 PMCID: PMC11171568 DOI: 10.3390/cells13110974] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2024] [Revised: 05/31/2024] [Accepted: 06/01/2024] [Indexed: 06/21/2024] Open
Abstract
Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous, motile ciliopathy, characterized by neonatal respiratory distress, recurrent upper and lower respiratory tract infections, subfertility, and laterality defects. Diagnosis relies on a combination of tests for confirmation, including nasal nitric oxide (nNO) measurements, high-speed videomicroscopy analysis (HSVMA), immunofluorescent staining, axonemal ultrastructure analysis via transmission electron microscopy (TEM), and genetic testing. Notably, there is no single gold standard confirmatory or exclusionary test. Currently, 54 causative genes involved in cilia assembly, structure, and function have been linked to PCD; this rare disease has a spectrum of clinical manifestations and emerging genotype-phenotype relationships. In this review, we provide an overview of the structure and function of motile cilia, the emerging genetics and pathophysiology of this rare disease, as well as clinical features associated with motile ciliopathies, novel diagnostic tools, and updates on genotype-phenotype relationships in PCD.
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Affiliation(s)
- Katherine A. Despotes
- Department of Pediatrics, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Maimoona A. Zariwala
- Department of Pediatrics, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
- Marsico Lung Institute, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Stephanie D. Davis
- Department of Pediatrics, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Thomas W. Ferkol
- Department of Pediatrics, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
- Marsico Lung Institute, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
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Rochon ER, Xue J, Mohammed MS, Smith C, Hay-Schmidt A, DeMartino AW, Clark A, Xu Q, Lo CW, Tsang M, Tejero J, Gladwin MT, Corti P. Cytoglobin regulates NO-dependent cilia motility and organ laterality during development. Nat Commun 2023; 14:8333. [PMID: 38097556 PMCID: PMC10721929 DOI: 10.1038/s41467-023-43544-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2023] [Accepted: 11/10/2023] [Indexed: 12/17/2023] Open
Abstract
Cytoglobin is a heme protein with unresolved physiological function. Genetic deletion of zebrafish cytoglobin (cygb2) causes developmental defects in left-right cardiac determination, which in humans is associated with defects in ciliary function and low airway epithelial nitric oxide production. Here we show that Cygb2 co-localizes with cilia and with the nitric oxide synthase Nos2b in the zebrafish Kupffer's vesicle, and that cilia structure and function are disrupted in cygb2 mutants. Abnormal ciliary function and organ laterality defects are phenocopied by depletion of nos2b and of gucy1a, the soluble guanylate cyclase homolog in fish. The defects are rescued by exposing cygb2 mutant embryos to a nitric oxide donor or a soluble guanylate cyclase stimulator, or with over-expression of nos2b. Cytoglobin knockout mice also show impaired airway epithelial cilia structure and reduced nitric oxide levels. Altogether, our data suggest that cytoglobin is a positive regulator of a signaling axis composed of nitric oxide synthase-soluble guanylate cyclase-cyclic GMP that is necessary for normal cilia motility and left-right patterning.
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Affiliation(s)
- Elizabeth R Rochon
- Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, 21201, USA
| | - Jianmin Xue
- Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA
| | - Manush Sayd Mohammed
- Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15260, USA
| | - Caroline Smith
- Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA
| | - Anders Hay-Schmidt
- Department of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Anthony W DeMartino
- Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, 21201, USA
| | - Adam Clark
- Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, 21201, USA
| | - Qinzi Xu
- Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, 21201, USA
| | - Cecilia W Lo
- Department of Developmental Biology, Rangos Research Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15201, USA
| | - Michael Tsang
- Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15260, USA
| | - Jesus Tejero
- Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA
- Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261, USA
- Department of Bioengineering, University of Pittsburgh Swanson School of Engineering, Pittsburgh, PA, 15260, USA
- Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, 15261, USA
| | - Mark T Gladwin
- Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.
| | - Paola Corti
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.
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Yang X, Liu X, Nie Y, Zhan F, Zhu B. Oxidative stress and ROS-mediated cellular events in RSV infection: potential protective roles of antioxidants. Virol J 2023; 20:224. [PMID: 37798799 PMCID: PMC10557227 DOI: 10.1186/s12985-023-02194-w] [Citation(s) in RCA: 17] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2023] [Accepted: 09/27/2023] [Indexed: 10/07/2023] Open
Abstract
Respiratory syncytial virus (RSV), a member of the Pneumoviridae family, can cause severe acute lower respiratory tract infection in infants, young children, immunocompromised individuals and elderly people. RSV is associated with an augmented innate immune response, enhanced secretion of inflammatory cytokines, and necrosis of infected cells. Oxidative stress, which is mainly characterized as an imbalance in the production of reactive oxygen species (ROS) and antioxidant responses, interacts with all the pathophysiologic processes above and is receiving increasing attention in RSV infection. A gradual accumulation of evidence indicates that ROS overproduction plays an important role in the pathogenesis of severe RSV infection and serves as a major factor in pulmonary inflammation and tissue damage. Thus, antioxidants seem to be an effective treatment for severe RSV infection. This article mainly reviews the information on oxidative stress and ROS-mediated cellular events during RSV infection for the first time.
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Affiliation(s)
- Xue Yang
- Department of Pediatrics, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, 441021, Hubei, China
| | - Xue Liu
- Department of Pediatrics, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, 441021, Hubei, China
| | - Yujun Nie
- Department of Pediatrics, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, 441021, Hubei, China
| | - Fei Zhan
- Department of Pediatrics, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, 441021, Hubei, China
| | - Bin Zhu
- Department of Pediatrics, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, 441021, Hubei, China.
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Takemoto K, Lomude LS, Takeno S, Kawasumi T, Okamoto Y, Hamamoto T, Ishino T, Ando Y, Ishikawa C, Ueda T. Functional Alteration and Differential Expression of the Bitter Taste Receptor T2R38 in Human Paranasal Sinus in Patients with Chronic Rhinosinusitis. Int J Mol Sci 2023; 24:4499. [PMID: 36901926 PMCID: PMC10002785 DOI: 10.3390/ijms24054499] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2023] [Revised: 01/31/2023] [Accepted: 02/22/2023] [Indexed: 03/02/2023] Open
Abstract
The bitter taste receptors (T2Rs) expressed in human sinonasal mucosae are known to elicit innate immune responses involving the release of nitric oxide (NO). We investigated the expression and distribution of two T2Rs, T2R14 and T2R38, in patients with chronic rhinosinusitis (CRS) and correlated the results with fractional exhaled NO (FeNO) levels and genotype of the T2R38 gene (TAS2R38). Using the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC) phenotypic criteria, we identified CRS patients as either eosinophilic (ECRS, n = 36) or non-eosinophilic (non-ECRS, n = 56) patients and compared these groups with 51 non-CRS subjects. Mucosal specimens from the ethmoid sinus, nasal polyps, and inferior turbinate were collected from all subjects, together with blood samples, for RT-PCR analysis, immunostaining, and single nucleotide polymorphism (SNP) typing. We observed significant downregulation of T2R38 mRNA levels in the ethmoid mucosa of non-ECRS patients and in the nasal polyps of ECRS patients. No significant differences in T2R14 or T2R38 mRNA levels were found among the inferior turbinate mucosae of the three groups. Positive T2R38 immunoreactivity was localized mainly in epithelial ciliated cells, whereas secretary goblet cells generally showed lack of staining. The patients in the non-ECRS group showed significantly lower oral and nasal FeNO levels compared with the control group. There was a trend towards higher CRS prevalence in the PAV/AVI and AVI/AVI genotype groups as compared to the PAV/PAV group. Our findings reveal complex but important roles of T2R38 function in ciliated cells associated with specific CRS phenotypes, suggesting the T2R38 pathway as a potential therapeutic target for promotion of endogenous defense mechanisms.
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Affiliation(s)
| | | | - Sachio Takeno
- Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan
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Carey RM, Palmer JN, Adappa ND, Lee RJ. Loss of CFTR function is associated with reduced bitter taste receptor-stimulated nitric oxide innate immune responses in nasal epithelial cells and macrophages. Front Immunol 2023; 14:1096242. [PMID: 36742335 PMCID: PMC9890060 DOI: 10.3389/fimmu.2023.1096242] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2022] [Accepted: 01/03/2023] [Indexed: 01/19/2023] Open
Abstract
Introduction Bitter taste receptors (T2Rs) are G protein-coupled receptors identified on the tongue but expressed all over the body, including in airway cilia and macrophages, where T2Rs serve an immune role. T2R isoforms detect bitter metabolites (quinolones and acyl-homoserine lactones) secreted by gram negative bacteria, including Pseudomonas aeruginosa, a major pathogen in cystic fibrosis (CF). T2R activation by bitter bacterial products triggers calcium-dependent nitric oxide (NO) production. In airway cells, the NO increases mucociliary clearance and has direct antibacterial properties. In macrophages, the same pathway enhances phagocytosis. Because prior studies linked CF with reduced NO, we hypothesized that CF cells may have reduced T2R/NO responses, possibly contributing to reduced innate immunity in CF. Methods Immunofluorescence, qPCR, and live cell imaging were used to measure T2R localization, calcium and NO signaling, ciliary beating, and antimicrobial responses in air-liquid interface cultures of primary human nasal epithelial cells and immortalized bronchial cell lines. Immunofluorescence and live cell imaging was used to measure T2R signaling and phagocytosis in primary human monocyte-derived macrophages. Results Primary nasal epithelial cells from both CF and non-CF patients exhibited similar T2R expression, localization, and calcium signals. However, CF cells exhibited reduced NO production also observed in immortalized CFBE41o- CF cells and non-CF 16HBE cells CRISPR modified with CF-causing mutations in the CF transmembrane conductance regulator (CFTR). NO was restored by VX-770/VX-809 corrector/potentiator pre-treatment, suggesting reduced NO in CF cells is due to loss of CFTR function. In nasal cells, reduced NO correlated with reduced ciliary and antibacterial responses. In primary human macrophages, inhibition of CFTR reduced NO production and phagocytosis during T2R stimulation. Conclusions Together, these data suggest an intrinsic deficiency in T2R/NO signaling caused by loss of CFTR function that may contribute to intrinsic susceptibilities of CF patients to P. aeruginosa and other gram-negative bacteria that activate T2Rs.
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Affiliation(s)
- Ryan M Carey
- Department of Otorhinolaryngology-Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - James N Palmer
- Department of Otorhinolaryngology-Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - Nithin D Adappa
- Department of Otorhinolaryngology-Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - Robert J Lee
- Department of Otorhinolaryngology-Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.,Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States
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8
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Carey RM, Hariri BM, Adappa ND, Palmer JN, Lee RJ. HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages. Cells 2022; 11:1478. [PMID: 35563784 PMCID: PMC9101439 DOI: 10.3390/cells11091478] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2022] [Revised: 04/22/2022] [Accepted: 04/26/2022] [Indexed: 02/04/2023] Open
Abstract
Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two innate immune cell types are activated by bitter products, including those secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells. NO also increases phagocytosis by macrophages. Using biochemistry and live-cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). Immunofluorescence showed that H441 cells express eNOS and T2Rs and that the bitter agonist denatonium benzoate activates NO production in a Ca2+- and HSP90-dependent manner in cells grown either as submerged cultures or at the air-liquid interface. In primary sinonasal epithelial cells, we determined that HSP90 inhibition reduces T2R-stimulated NO production and ciliary beating, which likely limits pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves as an innate immune modulator by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream Ca2+ signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases such as chronic rhinosinusitis, asthma, or cystic fibrosis.
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Affiliation(s)
- Ryan M. Carey
- Department of Otorhinolaryngology—Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; (B.M.H.); (N.D.A.); (J.N.P.)
| | - Benjamin M. Hariri
- Department of Otorhinolaryngology—Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; (B.M.H.); (N.D.A.); (J.N.P.)
| | - Nithin D. Adappa
- Department of Otorhinolaryngology—Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; (B.M.H.); (N.D.A.); (J.N.P.)
| | - James N. Palmer
- Department of Otorhinolaryngology—Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; (B.M.H.); (N.D.A.); (J.N.P.)
| | - Robert J. Lee
- Department of Otorhinolaryngology—Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; (B.M.H.); (N.D.A.); (J.N.P.)
- Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
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Neuropeptide Y Reduces Nasal Epithelial T2R Bitter Taste Receptor-Stimulated Nitric Oxide Production. Nutrients 2021; 13:nu13103392. [PMID: 34684394 PMCID: PMC8538228 DOI: 10.3390/nu13103392] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2021] [Revised: 09/22/2021] [Accepted: 09/24/2021] [Indexed: 12/30/2022] Open
Abstract
Bitter taste receptors (T2Rs) are G-protein-coupled receptors (GPCRs) expressed on the tongue but also in various locations throughout the body, including on motile cilia within the upper and lower airways. Within the nasal airway, T2Rs detect secreted bacterial ligands and initiate bactericidal nitric oxide (NO) responses, which also increase ciliary beat frequency (CBF) and mucociliary clearance of pathogens. Various neuropeptides, including neuropeptide tyrosine (neuropeptide Y or NPY), control physiological processes in the airway including cytokine release, fluid secretion, and ciliary beating. NPY levels and/or density of NPYergic neurons may be increased in some sinonasal diseases. We hypothesized that NPY modulates cilia-localized T2R responses in nasal epithelia. Using primary sinonasal epithelial cells cultured at air–liquid interface (ALI), we demonstrate that NPY reduces CBF through NPY2R activation of protein kinase C (PKC) and attenuates responses to T2R14 agonist apigenin. We find that NPY does not alter T2R-induced calcium elevation but does reduce T2R-stimulated NO production via a PKC-dependent process. This study extends our understanding of how T2R responses are modulated within the inflammatory environment of sinonasal diseases, which may improve our ability to effectively treat these disorders.
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Bayarri MA, Milara J, Estornut C, Cortijo J. Nitric Oxide System and Bronchial Epithelium: More Than a Barrier. Front Physiol 2021; 12:687381. [PMID: 34276407 PMCID: PMC8279772 DOI: 10.3389/fphys.2021.687381] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2021] [Accepted: 06/07/2021] [Indexed: 12/24/2022] Open
Abstract
Airway epithelium forms a physical barrier that protects the lung from the entrance of inhaled allergens, irritants, or microorganisms. This epithelial structure is maintained by tight junctions, adherens junctions and desmosomes that prevent the diffusion of soluble mediators or proteins between apical and basolateral cell surfaces. This apical junctional complex also participates in several signaling pathways involved in gene expression, cell proliferation and cell differentiation. In addition, the airway epithelium can produce chemokines and cytokines that trigger the activation of the immune response. Disruption of this complex by some inflammatory, profibrotic, and carcinogens agents can provoke epithelial barrier dysfunction that not only contributes to an increase of viral and bacterial infection, but also alters the normal function of epithelial cells provoking several lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) or lung cancer, among others. While nitric oxide (NO) molecular pathway has been linked with endothelial function, less is known about the role of the NO system on the bronchial epithelium and airway epithelial cells function in physiological and different pathologic scenarios. Several data indicate that the fraction of exhaled nitric oxide (FENO) is altered in lung diseases such as asthma, COPD, lung fibrosis, and cancer among others, and that reactive oxygen species mediate uncoupling NO to promote the increase of peroxynitrite levels, thus inducing bronchial epithelial barrier dysfunction. Furthermore, iNOS and the intracellular pathway sGC-cGMP-PKG are dysregulated in bronchial epithelial cells from patients with lung inflammation, fibrosis, and malignancies which represents an attractive drug molecular target. In this review we describe in detail current knowledge of the effect of NOS-NO-GC-cGMP-PKG pathway activation and disruption in bronchial epithelial cells barrier integrity and its contribution in different lung diseases, focusing on bronchial epithelial cell permeability, inflammation, transformation, migration, apoptosis/necrosis, and proliferation, as well as the specific NO molecular pathways involved.
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Affiliation(s)
- María Amparo Bayarri
- Department of Pharmacology, Faculty of Medicine, University of Valencia, Valencia, Spain
| | - Javier Milara
- Department of Pharmacology, Faculty of Medicine, University of Valencia, Valencia, Spain
- Biomedical Research Networking Centre on Respiratory Diseases (CIBERES), Health Institute Carlos III, Madrid, Spain
- Pharmacy Unit, University General Hospital Consortium of Valencia, Valencia, Spain
| | - Cristina Estornut
- Department of Pharmacology, Faculty of Medicine, University of Valencia, Valencia, Spain
| | - Julio Cortijo
- Department of Pharmacology, Faculty of Medicine, University of Valencia, Valencia, Spain
- Biomedical Research Networking Centre on Respiratory Diseases (CIBERES), Health Institute Carlos III, Madrid, Spain
- Research and Teaching Unit, University General Hospital Consortium of Valencia, Valencia, Spain
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11
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Understanding Primary Ciliary Dyskinesia and Other Ciliopathies. J Pediatr 2021; 230:15-22.e1. [PMID: 33242470 PMCID: PMC8690631 DOI: 10.1016/j.jpeds.2020.11.040] [Citation(s) in RCA: 75] [Impact Index Per Article: 18.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/17/2020] [Revised: 11/18/2020] [Accepted: 11/19/2020] [Indexed: 12/12/2022]
Abstract
Ciliopathies are a collection of disorders related to cilia dysfunction. Cilia are specialized organelles that project from the surface of most cells. Motile and primary (sensory) cilia are essential structures and have wide ranging functions. Our understanding of the genetics, pathophysiology, and clinical manifestations of motile ciliopathies, including primary ciliary dyskinesia (PCD), has rapidly advanced since the disease was linked to ciliary ultrastructural defects nearly five decades ago. We will provide an overview of different types of cilia, their role in child health and disease, focusing on motile ciliopathies, and describe recent advances that have led to improved diagnostics and may yield therapeutic targets to restore ciliary structure and function.
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12
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Gopallawa I, Lee RJ. Targeting the phosphoinositide-3-kinase/protein kinase B pathway in airway innate immunity. World J Biol Chem 2020; 11:30-51. [PMID: 33024516 PMCID: PMC7520643 DOI: 10.4331/wjbc.v11.i2.30] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/17/2020] [Revised: 07/24/2020] [Accepted: 08/26/2020] [Indexed: 02/06/2023] Open
Abstract
The airway innate immune system maintains the first line of defense against respiratory infections. The airway epithelium and associated immune cells protect the respiratory system from inhaled foreign organisms. These cells sense pathogens via activation of receptors like toll-like receptors and taste family 2 receptors (T2Rs) and respond by producing antimicrobials, inflammatory cytokines, and chemokines. Coordinated regulation of fluid secretion and ciliary beating facilitates clearance of pathogens via mucociliary transport. Airway cells also secrete antimicrobial peptides and radicals to directly kill microorganisms and inactivate viruses. The phosphoinositide-3-kinase/protein kinase B (Akt) kinase pathway regulates multiple cellular targets that modulate cell survival and proliferation. Akt also regulates proteins involved in innate immune pathways. Akt phosphorylates endothelial nitric oxide synthase (eNOS) enzymes expressed in airway epithelial cells. Activation of eNOS can have anti-inflammatory, anti-bacterial, and anti-viral roles. Moreover, Akt can increase the activity of the transcription factor nuclear factor erythroid 2 related factor-2 that protects cells from oxidative stress and may limit inflammation. In this review, we summarize the recent findings of non-cancerous functions of Akt signaling in airway innate host defense mechanisms, including an overview of several known downstream targets of Akt involved in innate immunity.
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Affiliation(s)
- Indiwari Gopallawa
- Department of Otorhinolaryngology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States
| | - Robert J Lee
- Department of Otorhinolaryngology and Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States
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13
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Joskova M, Mokry J, Franova S. Respiratory Cilia as a Therapeutic Target of Phosphodiesterase Inhibitors. Front Pharmacol 2020; 11:609. [PMID: 32435198 PMCID: PMC7218135 DOI: 10.3389/fphar.2020.00609] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2019] [Accepted: 04/20/2020] [Indexed: 11/30/2022] Open
Abstract
Mucociliary clearance is an essential airway defense mechanism dependent predominantly on the proper ciliary function and mucus rheology. The crucial role of cilia is evident in `a variety of respiratory diseases, as the ciliary dysfunction is associated with a progressive decline in lung function over time. The activity of cilia is under supervision of multiple physiological regulators, including second messengers. Their role is to enable a movement in coordinated metachronal waves at certain beat frequency. Ciliary function can be modulated by various stimuli, including agents from the group of beta2 agonists, cholinergic drugs, and adenosine triphosphate (ATP). They trigger cilia to move faster in response to elevated cytoplasmic Ca2+ originated from intracellular sources or replenished from extracellular space. Well-known cilia-stimulatory effect of Ca2+ ions can be abolished or even reversed by modulating the phosphodiesterase (PDE)-mediated breakdown of cyclic adenosine monophosphate (cAMP) since the overall change in ciliary beating has been dependent on the balance between Ca2+ ions and cAMP. Moreover, in chronic respiratory diseases, high ATP levels may contribute to cAMP hydrolysis and thus to a decrease in the ciliary beat frequency (CBF). The role of PDE inhibitors in airway cilia-driven transport may help in prevention of progressive loss of pulmonary function often observed despite current therapy. Furthermore, administration of selective PDE inhibitors by inhalation lowers the risk of their systemic effects. Based on this review we may conclude that selective (PDE1, PDE4) or dual PDE inhibitors (PDE3/4) increase the intracellular level of cyclic nucleotides in airway epithelial cells and thus may be an important target in the development of new inhaled mucokinetic agents. Further research is required to provide evidence of their effectiveness and feasibility regarding their cilia-modulating properties.
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Affiliation(s)
- Marta Joskova
- Department of Pharmacology, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia
| | - Juraj Mokry
- Department of Pharmacology, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia
| | - Sona Franova
- Department of Pharmacology, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia
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14
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Carey RM, Lee RJ. Taste Receptors in Upper Airway Innate Immunity. Nutrients 2019; 11:nu11092017. [PMID: 31466230 PMCID: PMC6770031 DOI: 10.3390/nu11092017] [Citation(s) in RCA: 59] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2019] [Revised: 08/19/2019] [Accepted: 08/22/2019] [Indexed: 02/07/2023] Open
Abstract
Taste receptors, first identified on the tongue, are best known for their role in guiding our dietary preferences. The expression of taste receptors for umami, sweet, and bitter have been demonstrated in tissues outside of the oral cavity, including in the airway, brain, gastrointestinal tract, and reproductive organs. The extra-oral taste receptor chemosensory pathways and the endogenous taste receptor ligands are generally unknown, but there is increasing data suggesting that taste receptors are involved in regulating some aspects of innate immunity, and may potentially control the composition of the nasal microbiome in healthy individuals or patients with upper respiratory diseases like chronic rhinosinusitis (CRS). For this reason, taste receptors may serve as potential therapeutic targets, providing alternatives to conventional antibiotics. This review focuses on the physiology of sweet (T1R) and bitter (T2R) taste receptors in the airway and their activation by secreted bacterial products. There is particular focus on T2R38 in sinonasal ciliated cells, as well as the sweet and bitter receptors found on specialized sinonasal solitary chemosensory cells. Additionally, this review explores the impact of genetic variations in these receptors on the differential susceptibility of patients to upper airway infections, such as CRS.
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Affiliation(s)
- Ryan M Carey
- Department of Otorhinolaryngology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Robert J Lee
- Department of Otorhinolaryngology and Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
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15
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Price ME, Sisson JH. Redox regulation of motile cilia in airway disease. Redox Biol 2019; 27:101146. [PMID: 30833143 PMCID: PMC6859573 DOI: 10.1016/j.redox.2019.101146] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2019] [Revised: 02/14/2019] [Accepted: 02/15/2019] [Indexed: 02/07/2023] Open
Abstract
Motile cilia on airway cells are necessary for clearance of mucus-trapped particles out of the lung. Ciliated airway epithelial cells are uniquely exposed to oxidants through trapping of particles, debris and pathogens in mucus and the direct exposure to inhaled oxidant gases. Dynein ATPases, the motors driving ciliary motility, are sensitive to the local redox environment within each cilium. Several redox-sensitive cilia-localized proteins modulate dynein activity and include Protein Kinase A, Protein Kinase C, and Protein Phosphatase 1. Moreover, cilia are rich in known redox regulatory proteins and thioredoxin domain-containing proteins that are critical in maintaining a balanced redox environment. Importantly, a nonsense mutation in TXNDC3, which contains a thioredoxin motif, has recently been identified as disease-causing in Primary Ciliary Dyskinesia, a hereditary motile cilia disease resulting in impaired mucociliary clearance. Here we review current understanding of the role(s) oxidant species play in modifying airway ciliary function. We focus on oxidants generated in the airways, cilia redox targets that modulate ciliary beating and imbalances in redox state that impact health and disease. Finally, we review disease models such as smoking, asthma, alcohol drinking, and infections as well as the direct application of oxidants that implicate redox balance as a modulator of cilia motility.
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Affiliation(s)
- Michael E Price
- University of Nebraska Medical Center, Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Omaha, NE, USA; University of Nebraska Medical Center, Department of Cellular & Integrative Physiology, Omaha, NE, USA.
| | - Joseph H Sisson
- University of Nebraska Medical Center, Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Omaha, NE, USA.
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16
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Freund JR, Mansfield CJ, Doghramji LJ, Adappa ND, Palmer JN, Kennedy DW, Reed DR, Jiang P, Lee RJ. Activation of airway epithelial bitter taste receptors by Pseudomonas aeruginosa quinolones modulates calcium, cyclic-AMP, and nitric oxide signaling. J Biol Chem 2018; 293:9824-9840. [PMID: 29748385 DOI: 10.1074/jbc.ra117.001005] [Citation(s) in RCA: 72] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2017] [Revised: 04/17/2018] [Indexed: 12/12/2022] Open
Abstract
Bitter taste receptors (taste family 2 bitter receptor proteins; T2Rs), discovered in many tissues outside the tongue, have recently become potential therapeutic targets. We have shown previously that airway epithelial cells express several T2Rs that activate innate immune responses that may be important for treatment of airway diseases such as chronic rhinosinusitis. It is imperative to more clearly understand what compounds activate airway T2Rs as well as their full range of functions. T2R isoforms in airway motile cilia (T2R4, -14, -16, and -38) produce bactericidal levels of nitric oxide (NO) that also increase ciliary beating, promoting clearance of mucus and trapped pathogens. Bacterial quorum-sensing acyl-homoserine lactones activate T2Rs and stimulate these responses in primary airway cells. Quinolones are another type of quorum-sensing molecule used by Pseudomonas aeruginosa To elucidate whether bacterial quinolones activate airway T2Rs, we analyzed calcium, cAMP, and NO dynamics using a combination of fluorescent indicator dyes and FRET-based protein biosensors. T2R-transfected HEK293T cells, several lung epithelial cell lines, and primary sinonasal cells grown and differentiated at the air-liquid interface were tested with 2-heptyl-3-hydroxy-4-quinolone (known as Pseudomonas quinolone signal; PQS), 2,4-dihydroxyquinolone, and 4-hydroxy-2-heptylquinolone (HHQ). In HEK293T cells, PQS activated T2R4, -16, and -38, whereas HHQ activated T2R14. 2,4-Dihydroxyquinolone had no effect. PQS and HHQ increased calcium and decreased both baseline and stimulated cAMP levels in cultured and primary airway cells. In primary cells, PQS and HHQ activated levels of NO synthesis previously shown to be bactericidal. This study suggests that airway T2R-mediated immune responses are activated by bacterial quinolones as well as acyl-homoserine lactones.
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Affiliation(s)
- Jenna R Freund
- From the Departments of Otorhinolaryngology-Head and Neck Surgery and
| | | | | | - Nithin D Adappa
- From the Departments of Otorhinolaryngology-Head and Neck Surgery and
| | - James N Palmer
- From the Departments of Otorhinolaryngology-Head and Neck Surgery and
| | - David W Kennedy
- From the Departments of Otorhinolaryngology-Head and Neck Surgery and
| | - Danielle R Reed
- the Monell Chemical Senses Center, Philadelphia, Pennsylvania 19104
| | - Peihua Jiang
- the Monell Chemical Senses Center, Philadelphia, Pennsylvania 19104
| | - Robert J Lee
- From the Departments of Otorhinolaryngology-Head and Neck Surgery and .,Physiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104 and
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17
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Freund JR, Lee RJ. Taste receptors in the upper airway. World J Otorhinolaryngol Head Neck Surg 2018; 4:67-76. [PMID: 30035264 PMCID: PMC6051256 DOI: 10.1016/j.wjorl.2018.02.004] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2018] [Accepted: 02/26/2018] [Indexed: 02/08/2023] Open
Abstract
Taste receptors were named for their originally-identified expression on the tongue and role in the sensation of taste (gustation). They are now known to be involved in many chemosensory processes outside the tongue. Expression of the receptors for bitter, sweet, and umami was recently identified in many organs, including the brain, airway, gastrointestinal tract, and reproductive systems. We do not yet know the full roles of these receptors in all of these tissues, nor do we know all of the endogenous ligands that activate them. However, taste receptors are emerging as potentially important therapeutic targets. Moreover, they may mediate some off target effects of drugs, as many medications in common clinical use are known to be bitter. The focus of this review is on recent basic and clinical data describing the expression of bitter (T2R) and sweet (T1R) receptors in the airway and their activation by secreted bacterial compounds. These receptors play important roles in innate immune nitric oxide production and antimicrobial peptide secretion, and may be useful targets for stimulating immune responses in the upper respiratory tract via topical therapies. Moreover, genetic variation in these receptors may play a role in the differential susceptibility of patients to certain types of respiratory infections as well as to differential outcomes in patients with chronic rhinosinusitis (CRS). CRS is a syndrome of chronic upper respiratory infection and inflammation and has a significant detrimental impact on patient quality of life. CRS treatment accounts for approximately 20% of adult antibiotic prescriptions and is thus a large driver of the public health crisis of antibiotic resistance. Taste receptors represent a novel class of therapeutic target to potentially stimulate endogenous immune responses and treat CRS patients without conventional antibiotics.
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Affiliation(s)
- Jenna R Freund
- Department of Otorhinolaryngology-Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Robert J Lee
- Department of Otorhinolaryngology-Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.,Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
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18
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Wyatt TA, Canady K, Heires AJ, Poole JA, Bailey KL, Nordgren TM, Romberger DJ. Alcohol Inhibits Organic Dust-induced ICAM-1 Expression on Bronchial Epithelial Cells. SAFETY 2017; 3:5. [PMID: 29082234 PMCID: PMC5658133 DOI: 10.3390/safety3010005] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Aims: Exposure to dusts/bioaerosols in concentrated animal feeding operations (CAFOs) results in inflammatory lung diseases in workers. Hog CAFOs dust extract (HDE) increases expression of intercellular adhesion molecule-1 (ICAM-1), neutrophil adhesion, and TNFα release in bronchial epithelial cells. Alcohol consumption is increasingly recognized to impair lung immunity. We hypothesized that alcohol impairs HDE-induced TNFα, ICAM-1 expression and neutrophil adhesion by directly inhibiting TNFα converting enzyme (TACE) activity. METHODS Bronchial epithelial cells (BEAS-2B) and primary human bronchial epithelial cells were pretreated with ethanol (EtOH) or TACE inhibitor. ICAM-1 surface expression, TNFα release, and TACE activity were analyzed following HDE stimulation. The effect of alcohol and TACE inhibition on HDE-regulated epithelial cell/neutrophil adhesion interactions was investigated. Finally, utilizing an established animal model, C57BL/6 mice were fed ad libitum ethanol (20%) in drinking water for 8 wk followed by daily intranasal inhalation of HDE or saline during the final two weeks. Mice were sacrificed and lung sections immunostained for ICAM-1. RESULTS Pretreatment with alcohol or TACE inhibitor significantly decreased HDE-induced ICAM-1 expression and TNFα release. HDE augmented neutrophil adhesion to epithelial cells, which was decreased with alcohol (32% decrease) or TACE inhibitor (55% decrease) pretreatment. TACE activity increased following HDE exposure, but TACE activity was inhibited following alcohol pretreatment. Alcohol-fed mice demonstrated decreased HDE-induced airway epithelium ICAM-1 expression. CONCLUSIONS Alcohol diminishes HDE-induced ICAM-1 expression, TNFα release, and neutrophil adhesion via inhibition of TACE activity. These results suggest that alcohol may be an important modulator of lung innate immune responses following CAFO exposure.
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Affiliation(s)
- Todd A. Wyatt
- Pulmonary, Critical Care, Sleep & Allergy Division of the Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198; USA
- Department of Environmental, Agricultural, & Occupational Health, University of Nebraska Medical Center, Omaha, NE, 68198-5910
- Resereach Service, VA Omaha-Western Iowa Health Care System, Omaha, NE, 68105
| | - Kerry Canady
- Pulmonary, Critical Care, Sleep & Allergy Division of the Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198; USA
| | - Art J. Heires
- Pulmonary, Critical Care, Sleep & Allergy Division of the Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198; USA
| | - Jill A. Poole
- Pulmonary, Critical Care, Sleep & Allergy Division of the Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198; USA
| | - Kristina L. Bailey
- Pulmonary, Critical Care, Sleep & Allergy Division of the Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198; USA
- Resereach Service, VA Omaha-Western Iowa Health Care System, Omaha, NE, 68105
| | - Tara M. Nordgren
- Pulmonary, Critical Care, Sleep & Allergy Division of the Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198; USA
| | - Debra J. Romberger
- Pulmonary, Critical Care, Sleep & Allergy Division of the Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198; USA
- Resereach Service, VA Omaha-Western Iowa Health Care System, Omaha, NE, 68105
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19
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Price ME, Pavlik JA, Liu M, Ding SJ, Wyatt TA, Sisson JH. Alcohol drives S-nitrosylation and redox activation of protein phosphatase 1, causing bovine airway cilia dysfunction. Am J Physiol Lung Cell Mol Physiol 2017; 312:L432-L439. [PMID: 28062487 DOI: 10.1152/ajplung.00513.2016] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2016] [Revised: 12/29/2016] [Accepted: 01/05/2017] [Indexed: 11/22/2022] Open
Abstract
Individuals with alcohol (ethanol)-use disorders are at increased risk for lung infections, in part, due to defective mucociliary clearance driven by motile cilia in the airways. We recently reported that isolated, demembranated bovine cilia (axonemes) are capable of producing nitric oxide (∙NO) when exposed to biologically relevant concentrations of alcohol. This increased presence of ∙NO can lead to protein S-nitrosylation, a posttranslational modification signaling mechanism involving reversible adduction of nitrosonium cations or ∙NO to thiolate or thiyl radicals, respectively, of proteins forming S-nitrosothiols (SNOs). We quantified and compared SNO content between isolated, demembranated axonemes extracted from bovine tracheae, with or without in situ alcohol exposure (100 mM × 24 h). We demonstrate that relevant concentrations of alcohol exposure shift the S-nitrosylation status of key cilia regulatory proteins, including 20-fold increases in S-nitrosylation of proteins that include protein phosphatase 1 (PP1). With the use of an ATP-reactivated axoneme motility system, we demonstrate that alcohol-driven S-nitrosylation of PP1 is associated with PP1 activation and dysfunction of axoneme motility. These new data demonstrate that alcohol can shift the S-nitrothiol balance at the level of the cilia organelle and highlight S-nitrosylation as a novel signaling mechanism to regulate PP1 and cilia motility.
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Affiliation(s)
- Michael E Price
- Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska.,Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska
| | - Jacqueline A Pavlik
- Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Miao Liu
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska
| | - Shi-Jian Ding
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska
| | - Todd A Wyatt
- Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska.,Department of Environmental, Agricultural, and Occupational Health, University of Nebraska Medical Center, Omaha, Nebraska; and.,Research Service, Nebraska-Western Iowa VA Healthcare System, Omaha, Nebraska
| | - Joseph H Sisson
- Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska;
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20
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Abstract
Primary ciliary dyskinesia (PCD) is a genetic disease of motile cilia, which belongs to a group of disorders resulting from dysfunction of cilia, collectively known as ciliopathies. Insights into the genetics and phenotypes of PCD have grown over the last decade, in part propagated by the discovery of a number of novel cilia-related genes. These genes encode proteins that segregate into structural axonemal, regulatory, as well as cytoplasmic assembly proteins. Our understanding of primary (sensory) cilia has also expanded, and an ever-growing list of diverse conditions has been linked to defective function and signaling of the sensory cilium. Recent multicenter clinical and genetic studies have uncovered the heterogeneity of motile and sensory ciliopathies, and in some cases, the overlap between these conditions. Here, we will describe the genetics and pathophysiology of ciliopathies in children, focusing on PCD, review emerging genotype-phenotype relationships, and diagnostic tools available for the clinician.
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Affiliation(s)
- Amjad Horani
- a Department of Pediatrics , Washington University School of Medicine , St. Louis , MO , USA
| | - Thomas W Ferkol
- a Department of Pediatrics , Washington University School of Medicine , St. Louis , MO , USA.,b Department of Cell Biology and Physiology , Washington University School of Medicine , St. Louis , MO , USA
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21
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Abstract
Motile cilia of the lungs respond to environmental challenges by increasing their ciliary beat frequency in order to enhance mucociliary clearance as a fundamental tenant of innate defense. One important second messenger in transducing the regulable nature of motile cilia is cyclic guanosine 3′,5′-monophosphate (cGMP). In this review, the history of cGMP action is presented and a survey of the existing data addressing cGMP action in ciliary motility is presented. Nitric oxide (NO)-mediated regulation of cGMP in ciliated cells is presented in the context of alcohol-induced cilia function and dysfunction.
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Affiliation(s)
- Todd A Wyatt
- VA Nebraska-Western Iowa Health Care System, Research Service, Department of Veterans Affairs Medical Center, 4101 Woolworth Avenue, Omaha, NE 68105, USA.
- Department of Environmental, Agricultural, and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, NE 68198-5910, USA.
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, 985910 Nebraska Medical Center, Omaha, NE 68198-5910, USA .
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22
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Price ME, Pavlik JA, Sisson JH, Wyatt TA. Inhibition of protein phosphatase 1 reverses alcohol-induced ciliary dysfunction. Am J Physiol Lung Cell Mol Physiol 2015; 308:L577-85. [PMID: 25575517 DOI: 10.1152/ajplung.00336.2014] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Airway mucociliary clearance is a first-line defense of the lung against inhaled particles and debris. Among individuals with alcohol use disorders, there is an increase in lung diseases. We previously identified that prolonged alcohol exposure impairs mucociliary clearance, known as alcohol-induced ciliary dysfunction (AICD). Cilia-localized enzymes, known as the ciliary metabolon, are key to the pathogenesis of AICD. In AICD, cyclic nucleotide-dependent ciliary kinases, which modulate phosphorylation to regulate cilia beat, are desensitized. We hypothesized that alcohol activates cilia-associated protein phosphatase 1 (PP1) activity, driving phosphorylation changes of cilia motility regulatory proteins. To test this hypothesis we identified the effects of prolonged alcohol exposure on phosphatase activity, cilia beat, and kinase responsiveness and cilia-associated phosphorylation targets when stimulated by β-agonist or cAMP. Prolonged alcohol activated PP1 and blocked cAMP-dependent cilia beat and protein kinase A (PKA) responsiveness and phosphorylation of a 29-kDa substrate of PKA. Importantly, prolonged alcohol-induced phosphatase activation was inhibited by the PP1 specific inhibitor, inhibitor-2 (I-2), restoring cAMP-stimulated cilia beat and PKA responsiveness and phosphorylation of the 29-kDa substrate. The I-2 inhibitory effect persisted in tissue, cell, and isolated cilia-organelle models, highlighting the association of ciliary metabolon-localized enzymes to AICD. Prolonged alcohol exposure drives ciliary metabolon-localized PP1 activation. PP1 activation modifies phosphorylation of a 29-kDa protein related to PKA activity. These data reinforce our previous findings that alcohol is acting at the level of the ciliary metabolon to cause ciliary dysfunction and identifies PP1 as a therapeutic target to prevent or reverse AICD.
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Affiliation(s)
- Michael E Price
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Jacqueline A Pavlik
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Joseph H Sisson
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska;
| | - Todd A Wyatt
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska; Veterans Affairs Nebraska-Western Iowa Healthcare System, Research Service, Omaha, Nebraska; and Department of Environmental, Agricultural, and Occupational Health, University of Nebraska Medical Center, Omaha, Nebraska
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23
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Lee RJ, Cohen NA. Taste receptors in innate immunity. Cell Mol Life Sci 2015; 72:217-36. [PMID: 25323130 PMCID: PMC4286424 DOI: 10.1007/s00018-014-1736-7] [Citation(s) in RCA: 96] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2014] [Revised: 09/11/2014] [Accepted: 09/16/2014] [Indexed: 02/07/2023]
Abstract
Taste receptors were first identified on the tongue, where they initiate a signaling pathway that communicates information to the brain about the nutrient content or potential toxicity of ingested foods. However, recent research has shown that taste receptors are also expressed in a myriad of other tissues, from the airway and gastrointestinal epithelia to the pancreas and brain. The functions of many of these extraoral taste receptors remain unknown, but emerging evidence suggests that bitter and sweet taste receptors in the airway are important sentinels of innate immunity. This review discusses taste receptor signaling, focusing on the G-protein-coupled receptors that detect bitter, sweet, and savory tastes, followed by an overview of extraoral taste receptors and in-depth discussion of studies demonstrating the roles of taste receptors in airway innate immunity. Future research on extraoral taste receptors has significant potential for identification of novel immune mechanisms and insights into host-pathogen interactions.
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Affiliation(s)
- Robert J. Lee
- Department of Otorhinolaryngology, Perelman School of Medicine, University of Pennsylvania, Ravdin Building, 5th floor, Philadelphia, PA 19104 USA
| | - Noam A. Cohen
- Department of Otorhinolaryngology, Perelman School of Medicine, University of Pennsylvania, Ravdin Building, 5th floor, Philadelphia, PA 19104 USA
- Philadelphia Veterans Affairs Medical Center Surgical Services, 3900 Woodland Ave, Philadelphia, PA 19104 USA
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24
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Jackson CL, Lucas JS, Walker WT, Owen H, Premadeva I, Lackie PM. Neuronal NOS localises to human airway cilia. Nitric Oxide 2014; 44:3-7. [PMID: 25460324 DOI: 10.1016/j.niox.2014.11.003] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2014] [Revised: 10/21/2014] [Accepted: 11/03/2014] [Indexed: 01/13/2023]
Abstract
BACKGROUND Airway NO synthase (NOS) isoenzymes are responsible for rapid and localised nitric oxide (NO) production and are expressed in airway epithelium. We sought to determine the localisation of neuronal NOS (nNOS) in airway epithelium due to the paucity of evidence. METHODS AND RESULTS Sections of healthy human bronchial tissue in glycol methacrylate resin and human nasal polyps in paraffin wax were immunohistochemically labelled and reproducibly demonstrated nNOS immunoreactivity, particularly at the proximal portion of cilia; this immunoreactivity was blocked by a specific nNOS peptide fragment. Healthy human epithelial cells differentiated at an air-liquid interface (ALI) confirmed the presence of all three NOS isoenzymes by immunofluorescence labelling. Only nNOS immunoreactivity was specific to the ciliary axonemeand co-localised with the cilia marker β-tubulin in the proximal part of the ciliary axoneme. CONCLUSIONS We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium and conclude that its independent and local regulation of NO levels is crucial for normal cilia function.
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Affiliation(s)
- Claire L Jackson
- Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK; Primary Ciliary Dyskinesia Centre, NIHR Southampton Respiratory Biomedical Research Unit, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK.
| | - Jane S Lucas
- Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK; Primary Ciliary Dyskinesia Centre, NIHR Southampton Respiratory Biomedical Research Unit, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
| | - Woolf T Walker
- Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK; Primary Ciliary Dyskinesia Centre, NIHR Southampton Respiratory Biomedical Research Unit, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
| | - Holly Owen
- Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK
| | - Irnthu Premadeva
- Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK
| | - Peter M Lackie
- Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK; Primary Ciliary Dyskinesia Centre, NIHR Southampton Respiratory Biomedical Research Unit, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
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25
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Mueller KE, Wolf K. C. pneumoniae disrupts eNOS trafficking and impairs NO production in human aortic endothelial cells. Cell Microbiol 2014; 17:119-30. [PMID: 25131610 DOI: 10.1111/cmi.12341] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2014] [Revised: 08/01/2014] [Accepted: 08/05/2014] [Indexed: 11/28/2022]
Abstract
Endothelial nitric oxide synthase (eNOS) generated NO plays a crucial physiological role in the regulation of vascular tone. eNOS is a constitutively expressed synthase whose enzymatic function is regulated by dual acylation, phosphorylation, protein-protein interaction and subcellular localization. In endothelial cells, the enzyme is primarily localized to the Golgi apparatus (GA) and the plasma membrane where it binds to caveolin-1. Upon stimulation, the enzyme is translocated from the plasma membrane to the cytoplasm where it generates NO. When activation of eNOS ceases, the majority of the enzyme is recycled back to the membrane fraction. An inability of eNOS to cycle between the cytosol and the membrane leads to impaired NO production and vascular dysfunction. Chlamydia pneumoniae is a Gram-negative obligate intracellular bacterium that primarily infects epithelial cells of the human respiratory tract, but unlike any other chlamydial species, C. pneumoniae displays tropism toward atherosclerotic tissues. In this study, we demonstrate that C. pneumoniae inclusions colocalize with eNOS, and the microorganism interferes with trafficking of the enzyme from the GA to the plasma membrane in primary human aortic endothelial cells. This mislocation of eNOS results in significant inhibition of NO release by C. pneumoniae-infected cells. Furthermore, we show that the distribution of eNOS in C. pneumoniae-infected cells is altered due to an intimate association of the Golgi complex with chlamydial inclusions rather than by direct interaction of the enzyme with the chlamydial inclusion membrane.
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Affiliation(s)
- Konrad E Mueller
- Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY, 40536, USA
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Picking up speed: advances in the genetics of primary ciliary dyskinesia. Pediatr Res 2014; 75:158-64. [PMID: 24192704 PMCID: PMC3946436 DOI: 10.1038/pr.2013.200] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/16/2013] [Accepted: 08/20/2013] [Indexed: 11/08/2022]
Abstract
Abnormal ciliary axonemal structure and function are linked to the growing class of genetic disorders collectively known as ciliopathies, and our understanding of the complex genetics and functional phenotypes of these conditions has rapidly expanded. While progress in genetics and biology has uncovered numerous cilia-related syndromes, primary ciliary dyskinesia (PCD) remains the sole genetic disorder of motile cilia dysfunction. The first disease-causing mutation was described just 13 y ago, and since that time, the pace of gene discovery has quickened. These mutations separate into genes that encode axonemal motor proteins, structural and regulatory elements, and cytoplasmic proteins that are involved in assembly and preassembly of ciliary elements. These findings have yielded novel insights into the processes involved in ciliary assembly, structure, and function, which will allow us to better understand the clinical manifestations of PCD. Moreover, advances in techniques for genetic screening and sequencing are improving diagnostic approaches. In this article, we will describe the structure, function, and emerging genetics of respiratory cilia, review the genotype-phenotype relationships of motor ciliopathies, and explore the implications of recent discoveries for diagnostic testing for PCD.
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Asymmetric dimethylarginine blocks nitric oxide-mediated alcohol-stimulated cilia beating. Mediators Inflamm 2013; 2013:592892. [PMID: 24307761 PMCID: PMC3836567 DOI: 10.1155/2013/592892] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2013] [Revised: 09/16/2013] [Accepted: 09/18/2013] [Indexed: 01/29/2023] Open
Abstract
The airway epithelium is exposed to alcohol during drinking through direct exhalation of volatized ethanol from the bronchial circulation. Alcohol exposure leads to a rapid increase in the cilia beat frequency (CBF) of bronchial epithelial cells followed by a chronic desensitization of cilia stimulatory responses. This effect is governed in part by the nitric oxide regulation of cyclic guanosine and adenosine monophosphate-dependent protein kinases (PKG and PKA) and is not fully understood. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is implicated in the pathogenesis of several pulmonary disorders. We hypothesized that the inhibition of nitric oxide synthase by ADMA blocks alcohol-stimulated increases in CBF. To test this hypothesis, ciliated primary bovine bronchial epithelial cells (BBEC) were preincubated with ADMA (100 µM) and stimulated with 100 mM ethanol. CBF was measured and PKA assayed. By 1 hr, ethanol activated PKA, resulting in elevated CBF. Both alcohol-induced PKA activation and CBF were inhibited in the presence of ADMA. ADMA alone had no effect on PKA activity or CBF. Using a mouse model overexpressing the ADMA-degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH), we examined PKA and CBF in precision-cut mouse lung slices. Alcohol-stimulated increases in lung slice PKA and CBF were temporally enhanced in the DDAH mice versus control mice.
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Neuronal control of pedal sole cilia in the pond snail Lymnaea stagnalis appressa. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 2012; 199:71-86. [DOI: 10.1007/s00359-012-0770-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2012] [Revised: 10/11/2012] [Accepted: 10/15/2012] [Indexed: 10/27/2022]
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Simet SM, Pavlik JA, Sisson JH. Proteomic analysis of bovine axonemes exposed to acute alcohol: role of endothelial nitric oxide synthase and heat shock protein 90 in cilia stimulation. Alcohol Clin Exp Res 2012; 37:609-15. [PMID: 23078267 DOI: 10.1111/acer.12014] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2012] [Accepted: 08/17/2012] [Indexed: 12/17/2022]
Abstract
BACKGROUND Cilia are finger-like motor-driven organelles, which propel inhaled particles and mucus from the lung and airways. We have previously shown that brief alcohol exposure stimulates ciliary motility through an endothelial nitric oxide synthase (eNOS)-dependent pathway localized in the ciliary metabolon. However, the signaling molecules of the ciliary metabolon involved in alcohol-triggered ciliary beat frequency (CBF) stimulation upstream of eNOS activation remain unknown. METHODS We hypothesized that brief alcohol exposure alters threonine and serine phosphorylation of proteins involved in stimulating CBF. Two-dimensional electrophoresis indicated both increases and decreases in the serine and threonine phosphorylation states of several proteins. One of the proteins identified was heat shock protein 90 (HSP90), which undergoes increased threonine phosphorylation after brief alcohol exposure. Because HSP90 has been shown to associate with eNOS in lung tissue, we hypothesized that HSP90 is a key component in alcohol-triggered eNOS activation and that these 2 proteins co-localize within the ciliary metabolon. RESULTS Immunofluorescence experiments demonstrate that eNOS and HSP90 co-localize within basal bodies of the ciliary metabolon and partially translocate to the axoneme upon brief alcohol exposure. Pretreatment with geldanamycin, which disrupts HSP90 chaperone functions, prevented eNOS-HSP90 association and prevented the translocation of eNOS from the ciliary metabolon to the axoneme. Functional cilia motility studies revealed that geldanamycin blocked alcohol-stimulated ciliary motility in bovine bronchial epithelial cells and mouse tracheal rings. CONCLUSIONS On the basis of the HSP90 localization with eNOS, alcohol activation of HSP90 phosphorylation, and geldanamycin's ability to inhibit HSP90-eNOS association, prevent eNOS translocation to the axoneme, and block alcohol-stimulated ciliary motility, we conclude that alcohol-induced cilia stimulation occurs through the increased association of HSP90 with eNOS. These data help further elucidate the mechanism through which brief alcohol exposure stimulates CBF.
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Affiliation(s)
- Samantha M Simet
- Pulmonary, Critical Care, Sleep, and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA
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Co-exposure to cigarette smoke and alcohol decreases airway epithelial cell cilia beating in a protein kinase Cε-dependent manner. THE AMERICAN JOURNAL OF PATHOLOGY 2012; 181:431-40. [PMID: 22677421 DOI: 10.1016/j.ajpath.2012.04.022] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/18/2011] [Revised: 04/12/2012] [Accepted: 04/24/2012] [Indexed: 11/22/2022]
Abstract
Alcohol use disorders are associated with increased lung infections and exacerbations of chronic lung diseases. Whereas the effects of cigarette smoke are well recognized, the interplay of smoke and alcohol in modulating lung diseases is not clear. Because innate lung defense is mechanically maintained by airway cilia action and protein kinase C (PKC)-activating agents slow ciliary beat frequency (CBF), we hypothesized that the combination of smoke and alcohol would decrease CBF in a PKC-dependent manner. Primary ciliated bronchial epithelial cells were exposed to 5% cigarette smoke extract plus100 mmol/L ethanol for up to 24 hours and assayed for CBF and PKCε. Smoke and alcohol co-exposure activated PKCε by 1 hour and decreased both CBF and total number of beating cilia by 6 hours. A specific activator of PKCε, DCP-LA, slowed CBF after maximal PKCε activation. Interestingly, activation of PKCε by smoke and alcohol was only observed in ciliated cells, not basal bronchial epithelium. In precision-cut mouse lung slices treated with smoke and alcohol, PKCε activation preceded CBF slowing. Correspondingly, increased PKCε activity and cilia slowing were only observed in mice co-exposed to smoke and alcohol, regardless of the sequence of the combination exposure. No decreases in CBF were observed in PKCε knockout mice co-exposed to smoke and alcohol. These data identify PKCε as a key regulator of cilia slowing in response to combined smoke and alcohol-induced lung injury.
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Navarrette CR, Sisson JH, Nance E, Allen-Gipson D, Hanes J, Wyatt TA. Particulate matter in cigarette smoke increases ciliary axoneme beating through mechanical stimulation. J Aerosol Med Pulm Drug Deliv 2012; 25:159-68. [PMID: 22280523 PMCID: PMC3377952 DOI: 10.1089/jamp.2011.0890] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2011] [Accepted: 11/20/2011] [Indexed: 11/13/2022] Open
Abstract
BACKGROUND The lung's ability to trap and clear foreign particles via the mucociliary elevator is an important mechanism for protecting the lung against respirable irritants and microorganisms. Although cigarette smoke (CS) exposure and particulate inhalation are known to alter mucociliary clearance, little is known about how CS and nanoparticles (NPs) modify cilia beating at the cytoskeletal infrastructure, or axonemal, level. METHODS We used a cell-free model to introduce cigarette smoke extract (CSE) and NPs with variant size and surface chemistry to isolated axonemes and measured changes in ciliary motility. We hypothesized that CSE would alter cilia beating and that alterations in ciliary beat frequency (CBF) due to particulate matter would be size- and surface chemistry-dependent. Demembranated axonemes were isolated from ciliated bovine tracheas and exposed to adenosine triphosphate (ATP) to initiate motility. CBF was measured in response to 5% CSE, CSE filtrate, and carboxyl-modified (COOH), sulphate (SO(4))-modified (sulfonated), or PEG-coated polystyrene (PS) latex NPs ranging in size from 40 nm to 500 nm. RESULTS CSE concentrations as low as 5% resulted in rapid, significant stimulation of CBF (p<0.05 vs. baseline control). Filtering CSE through a 0.2-μm filter attenuated this effect. Introduction of sulphate-modified PS beads ~300 nm in diameter resulted in a similar increase in CBF above baseline ATP levels. Uncharged, PEG-coated beads had no effect on CBF regardless of size. Similarly, COOH-coated particles less than 200 nm in diameter did not alter ciliary motility. However, COOH-coated PS particles larger than 300 nm increased CBF significantly and increased the number of motile points. CONCLUSIONS These data show that NPs, including those found in CSE, mechanically stimulate axonemes in a size- and surface chemistry-dependent manner. Alterations in ciliary motility due to physicochemical properties of NPs may be important for inhalational lung injury and efficient drug delivery of respirable particles.
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Affiliation(s)
- Chelsea R. Navarrette
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Omaha, Nebraska
| | - Joseph H. Sisson
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Omaha, Nebraska
| | - Elizabeth Nance
- Department of Chemical & Biomolecular Engineering, Johns Hopkins University School of Engineering, Baltimore, Mayland
| | - Diane Allen-Gipson
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Omaha, Nebraska
| | - Justin Hanes
- Department of Chemical & Biomolecular Engineering, Johns Hopkins University School of Engineering, Baltimore, Mayland
- Center for Nanomedicine and Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Todd A. Wyatt
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Omaha, Nebraska
- VA Research Service, Department of Veterans Affairs Medical Center, Omaha, Nebraska
- Department of Environmental, Agricultural, and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska
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Jiao J, Wang H, Lou W, Jin S, Fan E, Li Y, Han D, Zhang L. Regulation of ciliary beat frequency by the nitric oxide signaling pathway in mouse nasal and tracheal epithelial cells. Exp Cell Res 2011; 317:2548-53. [PMID: 21787770 DOI: 10.1016/j.yexcr.2011.07.007] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2011] [Revised: 07/07/2011] [Accepted: 07/09/2011] [Indexed: 11/30/2022]
Abstract
OBJECTIVES Our purpose was to investigate the role of the nitric oxide (NO) signaling pathway in the regulation of ciliary beat frequency (CBF) in mouse nasal and tracheal epithelial cells. METHODS We studied the effects of the NO donor l-arginine (L-Arg) and specific inhibitors of the NO signaling pathway on CBF of both nasal and tracheal epithelial cells by using high-speed digital microscopy. We also examined eNOS, sGC β, PKG I and acetylated α tubulin expression in native mouse nasal and tracheal epithelium using immunohistochemical methods. RESULTS L-Arg significantly increased CBF of cultured nasal and tracheal epithelial cells, and the effects were blocked by pretreatment with N(G)-nitro-l-arginine methyl ester (L-NAME), a NOS inhibitor, with LY-83583, a sGC inhibitor, or with KT-5823, a PKG inhibitor. Positive immunostaining for NO signaling molecules including eNOS, sGC β and PKG I was observed in either nasal or tracheal ciliated epithelium. CONCLUSION NO plays a role in regulating CBF of mouse respiratory epithelial cells via a eNOS-NO-sGC β-cGMP-PKG I pathway.
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Affiliation(s)
- Jian Jiao
- Key Laboratory of Otolaryngology, Head and Neck Surgery (Ministry of Education), Beijing Institute of Otolaryngology, China
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Miyamoto DM, Ruff K, Beach NM, Stockwell SB, Dorsey-Oresto A, Masters I, Temple LM. Bordetella avium causes induction of apoptosis and nitric oxide synthase in turkey tracheal explant cultures. Microbes Infect 2011; 13:871-9. [PMID: 21609777 DOI: 10.1016/j.micinf.2011.04.011] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2011] [Revised: 04/26/2011] [Accepted: 04/29/2011] [Indexed: 01/18/2023]
Abstract
Bordetellosis is an upper respiratory disease of turkeys caused by Bordetella avium in which the bacteria attach specifically to ciliated respiratory epithelial cells. Little is known about the mechanisms of pathogenesis of this disease, which has a negative impact in the commercial turkey industry. In this study, we produced a novel explant organ culture system that was able to successfully reproduce pathogenesis of B. avium in vitro, using tracheal tissue derived from 26 day-old turkey embryos. Treatment of the explants with whole cells of B. avium virulent strain 197N and culture supernatant, but not lipopolysaccharide (LPS) or tracheal cytotoxin (TCT), specifically induced apoptosis in ciliated cells, as shown by annexin V and TUNEL staining. LPS and TCT are known virulence factors of Bordetella pertussis, the causative agent of whooping cough. Treatment with whole cells of B. avium and LPS specifically induced NO response in ciliated cells, shown by uNOS staining and diaphorase activity. The explant system is being used as a model to elucidate specific molecules responsible for the symptoms of bordetellosis.
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Francis SH, Busch JL, Corbin JD, Sibley D. cGMP-dependent protein kinases and cGMP phosphodiesterases in nitric oxide and cGMP action. Pharmacol Rev 2010; 62:525-63. [PMID: 20716671 PMCID: PMC2964902 DOI: 10.1124/pr.110.002907] [Citation(s) in RCA: 733] [Impact Index Per Article: 48.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
To date, studies suggest that biological signaling by nitric oxide (NO) is primarily mediated by cGMP, which is synthesized by NO-activated guanylyl cyclases and broken down by cyclic nucleotide phosphodiesterases (PDEs). Effects of cGMP occur through three main groups of cellular targets: cGMP-dependent protein kinases (PKGs), cGMP-gated cation channels, and PDEs. cGMP binding activates PKG, which phosphorylates serines and threonines on many cellular proteins, frequently resulting in changes in activity or function, subcellular localization, or regulatory features. The proteins that are so modified by PKG commonly regulate calcium homeostasis, calcium sensitivity of cellular proteins, platelet activation and adhesion, smooth muscle contraction, cardiac function, gene expression, feedback of the NO-signaling pathway, and other processes. Current therapies that have successfully targeted the NO-signaling pathway include nitrovasodilators (nitroglycerin), PDE5 inhibitors [sildenafil (Viagra and Revatio), vardenafil (Levitra), and tadalafil (Cialis and Adcirca)] for treatment of a number of vascular diseases including angina pectoris, erectile dysfunction, and pulmonary hypertension; the PDE3 inhibitors [cilostazol (Pletal) and milrinone (Primacor)] are used for treatment of intermittent claudication and acute heart failure, respectively. Potential for use of these medications in the treatment of other maladies continues to emerge.
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Affiliation(s)
- Sharron H Francis
- Department of Molecular Physiology & Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232-0615, USA.
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Zhou H, Wang X, Brighton L, Hazucha M, Jaspers I, Carson JL. Increased nasal epithelial ciliary beat frequency associated with lifestyle tobacco smoke exposure. Inhal Toxicol 2009; 21:875-81. [PMID: 19555226 DOI: 10.1080/08958370802555898] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
The ciliated epithelium of the respiratory airways is one of the first vital systemic surfaces in contact with the ambient air. Ex vivo nasal epithelial ciliary beat frequency (CBF) at room temperature is on the order of 7-8 Hz but may be stimulated by irritant exposure. The upregulation of CBF in response to acute irritant exposure is generally considered to be a transient event with eventual return to baseline. However, studies of CBF dynamics in response to typical lifestyle exposures are limited. This study assessed nasal epithelial CBF among human subjects as a function of quantifiable lifestyle tobacco smoke exposure. Nasal epithelial biopsies were obtained from human subjects with well documented histories of tobacco smoke exposure. CBF was determined using a digital photometric technique and concurrent assays of nasal nitric oxide and urine cotinine and creatinine were performed. Mean CBF among active smokers and non-smokers exposed to environmental tobacco smoke (ETS) was elevated over non-smokers. Although there were dramatic differences in relative levels of tobacco smoke exposure, CBF values among tobacco smoke-exposed groups were comparable. Parallel in vitro studies of cultured nasal epithelium exposed to cigarette smoke condensate further supported these observations. These studies suggest that persistent elevation in nasal epithelial CBF is an early, subtle, physiologic effect associated with lifestyle tobacco smoke exposure. The molecular mechanisms that upregulate CBF may also create a cell molecular milieu capable of provoking the eventual emergence of more overt adverse health effects and the pathogenesis of chronic airway disease.
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Affiliation(s)
- Haibo Zhou
- Department of Biostatistics, The University of North Carolina at Chapel Hill, 27599-7310, USA
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Simet SM, Sisson JH, Pavlik JA, Devasure JM, Boyer C, Liu X, Kawasaki S, Sharp JG, Rennard SI, Wyatt TA. Long-term cigarette smoke exposure in a mouse model of ciliated epithelial cell function. Am J Respir Cell Mol Biol 2009; 43:635-40. [PMID: 20042711 DOI: 10.1165/rcmb.2009-0297oc] [Citation(s) in RCA: 87] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Exposure to cigarette smoke is associated with airway epithelial mucus cell hyperplasia and a decrease in cilia and ciliated cells. Few models have addressed the long-term effects of chronic cigarette smoke exposure on ciliated epithelial cells. Our previous in vitro studies showed that cigarette smoke decreases ciliary beat frequency (CBF) via the activation of protein kinase C (PKC). We hypothesized that chronic cigarette smoke exposure in an in vivo model would decrease airway epithelial cell ciliary beating in a PKC-dependent manner. We exposed C57BL/6 mice to whole-body cigarette smoke 2 hours/day, 5 days/week for up to 1 year. Tracheal epithelial cell CBF and the number of motile cells were measured after necropsy in cut tracheal rings, using high-speed digital video microscopy. Tracheal epithelial PKC was assayed according to direct kinase activity. At 6 weeks and 3 months of smoke exposure, the baseline CBF was slightly elevated (~1 Hz) versus control mice, with no change in β-agonist-stimulated CBF between control mice and cigarette smoke-exposed mice. By 6 months of smoke exposure, the baseline CBF was significantly decreased (2-3 Hz) versus control mice, and a β-agonist failed to stimulate increased CBF. The loss of β-agonist-increased CBF continued at 9 months and 12 months of smoke exposure, and the baseline CBF was significantly decreased to less than one third of the control rate. In addition to CBF, ciliated cell numbers significantly decreased in response to smoke over time, with a significant loss of tracheal ciliated cells occurring between 6 and 12 months. In parallel with the slowing of CBF, significant PKC activation from cytosol to the membrane of tracheal epithelial cells was detected in mice exposed to smoke for 6-12 months.
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Affiliation(s)
- Samantha M Simet
- Pulmonary, Critical Care, Sleep, and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, 985910 Nebraska Medical Center, Omaha, NE 68198-5910, USA
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Abstract
PURPOSE OF REVIEW The diagnosis of primary ciliary dyskinesia (PCD) has relied on analysis of ciliary motility and ultrastructure; however, these tests are not readily available and have not been standardized. Consequently, the diagnosis of PCD may be delayed or missed or made incorrectly. This review outlines the potential utility of new diagnostic tests, including measurement of nasal nitric oxide production and systematic analysis for mutations in genes encoding ciliary proteins. RECENT FINDINGS Clinical manifestations of PCD have been expanded to include neonatal respiratory distress and heterotaxy. Measurement of nasal nitric oxide has emerged as a useful screening test for PCD based on the very low levels in PCD (approximately 1/10 of normal values). Genetic testing is emerging for PCD and demonstrates extensive genetic heterogeneity. Some genes and gene mutations involved in PCD have been defined. Approximately one-third of PCD cases have identifiable gene mutations in one of six different genes. An international effort is focused on defining PCD-causing defects in other genes. SUMMARY The incorporation of nasal nitric oxide measurement as a screening test to define probable PCD cases and gene mutation analysis to make a definitive diagnosis of PCD should enhance diagnostic evaluation of PCD.
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Sisson JH, Pavlik JA, Wyatt TA. Alcohol stimulates ciliary motility of isolated airway axonemes through a nitric oxide, cyclase, and cyclic nucleotide-dependent kinase mechanism. Alcohol Clin Exp Res 2009; 33:610-6. [PMID: 19183138 DOI: 10.1111/j.1530-0277.2008.00875.x] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND Lung mucociliary clearance provides the first line of defense from lung infections and is impaired in individuals who consume heavy amounts of alcohol. Previous studies have demonstrated that this alcohol-induced ciliary dysfunction occurs through impairment of nitric oxide (NO) and cyclic nucleotide-dependent kinase-signaling pathways in lung airway ciliated epithelial cells. Recent studies have established that all key elements of this alcohol-driven signaling pathway co-localize to the apical surface of the ciliated cells with the basal bodies. These findings led us to hypothesize that alcohol activates the cilia stimulation pathway at the organelle level. To test this hypothesis we performed experiments exposing isolated demembranated cilia (isolated axonemes) to alcohol and studied the effect of alcohol-stimulated ciliary motility on the pathways involved with isolated axoneme activation. METHODS Isolated demembranated cilia were prepared from bovine trachea and activated with adenosine triphosphate. Ciliary beat frequency, NO production, adenylyl and guanylyl cyclase activities, cAMP- and cGMP-dependent kinase activities were measured following exposure to biologically relevant concentrations of alcohol. RESULTS Alcohol rapidly stimulated axoneme beating 40% above baseline at very low concentrations of alcohol (1 to 10 mM). This activation was specific to ethanol, required the synthesis of NO, the activation of soluble adenylyl cyclase (sAC), and the activation of both cAMP- and cGMP-dependent kinases (PKA and PKG), all of which were present in the isolated organelle preparation. CONCLUSIONS Alcohol rapidly and sequentially activates the eNOS-->NO-->GC-->cGMP-->PKG and sAC-->cAMP--> PKA dual signaling pathways in isolated airway axonemes. These findings indicate a direct effect of alcohol on airway cilia organelle function and fully recapitulate the alcohol-driven activation of cilia known to exist in vivo and in intact lung ciliated cells in vitro following brief moderate alcohol exposure. Furthermore, these findings indicate that airway cilia are exquisitely sensitive to the effects of alcohol and substantiate a key role for alcohol in the alterations of mucociliary clearance associated with even low levels of alcohol intake. We speculate that this same axoneme-based alcohol activation pathway is down regulated following long-term high alcohol exposure and that the isolated axoneme preparation provides an excellent model for studying the mechanism of alcohol-mediated cilia dysfunction.
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Affiliation(s)
- Joseph H Sisson
- Department of Internal Medicine, University of Nebraska Medical Center, Pulmonary, Critical Care, Sleep and Allergy Section, Omaha, Nebraska 68198-5300, USA.
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Slager RE, Devasure JM, Pavlik JA, Sisson JH, Wyatt TA. RACK1, a PKC targeting protein, is exclusively localized to basal airway epithelial cells. J Histochem Cytochem 2007; 56:7-14. [PMID: 17875659 PMCID: PMC2323118 DOI: 10.1369/jhc.7a7249.2007] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
The novel isoform of protein kinase C (PKC), PKCepsilon, is an important regulator of ciliated cell function in airway epithelial cells, including cilia motility and detachment of ciliated cells after environmental insult. However, the mechanism of PKCepsilon signaling in the airways and the potential role of the PKCepsilon-interacting protein, receptor for activated C kinase 1 (RACK1), has not been widely explored. We used immunohistochemistry and Western blot analysis to show that RACK1 is localized exclusively to basal, non-ciliated (and non-goblet) bovine and human bronchial epithelial cells. Our immunohistochemistry experiments used the basal body marker pericentrin, a marker for cilia, beta-tubulin, and an airway goblet cell marker, MUC5AC, to confirm that RACK1 was excluded from differentiated airway cell subtypes and is only expressed in the basal cells. These results suggest that PKCepsilon signaling in the basal airway cell may involve RACK1; however, PKCepsilon regulation in ciliated cells uses RACK1-independent pathways.
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Affiliation(s)
- Rebecca E Slager
- Department of Internal Medicine, Pulmonary and Critical Care Medicine Section, University of Nebraska Medical Center, 985300 Nebraska Medical Center, Omaha, NE 68198-5300, USA
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