Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium.
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ABSTRACT:
SUBMITTER: Marozkina N
PROVIDER: S-EPMC8322277 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Nitric oxide (NO) is an important mediator of inflammatory responses in the lung and a key regulator of bronchomotor tone. An airway NO synthase (NOS; EC 1.14.13.39) has been proposed as a source of endogenous NO in the lung but has not been clearly defined. Through molecular cloning, we conclusively demonstrate that NO synthesis in normal human airways is due to the continuous expression of the inducible NOS (iNOS) isoform in airway epithelial cells. Although iNOS mRNA expression is abundant in airway epithelial cells, expression is not detected in other pulmonary cell types, indicating that airway epithelial cells are unique in the continuous pattern of iNOS expression in the lung. In situ analysis reveals all airway epithelial cell types express iNOS. However, removal of epithelial cells from the in vivo airway environment leads to rapid loss of iNOS expression, which suggests expression is dependent upon conditions and/or factors present in the airway. Quantitation of NOS activity in epithelial cell lysates indicates nanomolar levels of NO synthesis occur in vivo. Remarkably, the high-level iNOS expression is constant in airway epithelium of normal individuals over time. However, expression is strikingly decreased by inhaled corticosteroids and beta-adrenergic agonists, medications commonly used in treatment of inflammatory airway diseases. Based upon these findings, we propose that respiratory epithelial cells are key inflammatory cells in the airway, functioning in host defense and potentially playing a role in airway inflammation.
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Project description:Nitric oxide (NO) is generated from arginine and oxygen via NO synthase (NOS). Staphylococcus aureus NOS (saNOS) has previously been shown to affect virulence and resistance to exogenous oxidative stress, yet the exact mechanism is unknown. Herein, a previously undescribed role of saNOS in S. aureus aerobic physiology was reported. Specifically, aerobic S. aureus nos mutant cultures presented with elevated endogenous reactive oxygen species (ROS) and superoxide levels, as well as increased membrane potential, increased respiratory dehydrogenase activity and slightly elevated oxygen consumption. Elevated ROS levels in the nos mutant likely resulted from altered respiratory function, as inhibition of NADH dehydrogenase brought ROS levels back to wild-type levels. These results indicate that, in addition to its recently reported role in regulating the switch to nitrate-based respiration during low-oxygen growth, saNOS also plays a modulatory role during aerobic respiration. Multiple transcriptional changes were also observed in the nos mutant, including elevated expression of genes associated with oxidative/nitrosative stress, anaerobic respiration and lactate metabolism. Targeted metabolomics revealed decreased cellular lactate levels, and altered levels of TCA cycle intermediates, the latter of which may be related to decreased aconitase activity. Collectively, these findings demonstrate a key contribution of saNOS to S. aureus aerobic respiratory metabolism.
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Project description:Human bocavirus 1 (HBoV1) is a small DNA virus that belongs to the Bocaparvovirus genus of the Parvoviridae family. HBoV1 is a common respiratory pathogen that causes mild to life-threatening acute respiratory tract infections in children and immunocompromised individuals, infecting both the upper and lower respiratory tracts. HBoV1 infection causes death of airway epithelial cells, resulting in airway injury and inflammation. In vitro, HBoV1 only infects well-differentiated (polarized) human airway epithelium cultured at an air-liquid interface (HAE-ALI), but not any dividing human cells. A full-length HBoV1 genome of 5543 nucleotides has been cloned from DNA extracted from a human nasopharyngeal swab into a plasmid called HBoV1 infectious clone pIHBoV1. Transfection of pIHBoV1 replicates efficiently in human embryonic kidney 293 (HEK293) cells and produces virions that are highly infectious. This article describes protocols for production of HBoV1 in HEK293 cells, generation of HAE-ALI cultures, and infection with HBoV1 in HAE-ALI. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Human bocavirus 1 production in HEK293 cells Support Protocol 1: HEK293 cell culture and transfection Support Protocol 2: Quantification of human bocavirus 1 using real-time quantitative PCR Basic Protocol 2: Differentiation of human airway cells at an air-liquid interface Support Protocol 3: Expansion of human airway epithelial cell line CuFi-8 Support Protocol 4: Expansion of human airway basal cells Support Protocol 5: Coating of plastic dishes and permeable membranes of inserts Support Protocol 6: Transepithelial electrical resistance measurement Basic Protocol 3: Human bocavirus 1 infection in human airway epithelium cultured at an air-liquid interface Support Protocol 7: Isolation of infected human airway epithelium cells from inserts Basic Protocol 4: Transduction of airway basal cells with lentiviral vector.
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Project description:Basal cells (BC) are the stem/progenitor cells of the human airway epithelium capable of differentiating into secretory and ciliated cells. Notch signaling activation increases BC differentiation into secretory cells, but the role of individual Notch ligands in regulating this process in the human airway epithelium is largely unknown. The objective of this study was to define the role of the Notch ligand JAG1 in regulating human BC differentiation. JAG1 over-expression in BC increased secretory cell differentiation, with no effect on ciliated cell differentiation. Conversely, knockdown of JAG1 decreased expression of secretory cell genes. These data demonstrate JAG1-mediated Notch signaling regulates differentiation of BC into secretory cells.
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