Savory Signaling: T1R Umami Receptor Modulates Endoplasmic Reticulum Calcium Store Content and Release Dynamics in Airway Epithelial Cells.

T1Rs are expressed in solitary chemosensory cells of the upper airway where they detect apical glucose levels and repress bitter taste receptor Ca2+ signaling pathways. Microbial growth leads to a decrease in apical glucose levels. T1Rs detect this change and liberate bitter taste receptor signaling, initiating an innate immune response to both kill and expel pathogens through releasing antimicrobial peptides and increasing nitric oxide production and ciliary beat frequency. However, chronic inflammation due to disease, smoking, or viral infections causes a remodeling of the epithelial airway. The resulting squamous metaplasia causes a loss of multi-ciliated cells and solitary chemosensory cells, replaced by basal epithelial cells. To understand how T1R function is altered during disease, we used basal epithelial cells as a model to study the function of T1R3 on Ca2+ signaling dynamics. We found that both T1R1 and T1R3 detect amino acids and signal via cAMP, increasing the responsiveness of the cells to Ca2+ signaling stimuli. Either knocking down T1R1/3 or treating wild-type cells with MEM amino acids caused a reduction in ER Ca2+ content through a non-cAMP signaled pathway. Treatment with amino acids led to a reduction in downstream denatonium-induced Ca2+-signaled caspase activity. Thus, amino acids may be used to reduce unwanted apoptosis signaling in treatments containing bitter compounds.

Utilizing the Off-Target Effects of T1R3 Antagonist Lactisole to Enhance Nitric Oxide Production in Basal Airway Epithelial Cells.

Human airway sweet (T1R2 + T1R3), umami (T1R1 + T1R3), and bitter taste receptors (T2Rs) are critical components of the innate immune system, acting as sensors to monitor pathogenic growth. T2Rs detect bacterial products or bitter compounds to drive nitric oxide (NO) production in both healthy and diseased epithelial cell models. The NO enhances ciliary beating and also directly kills pathogens. Both sweet and umami receptors have been characterized to repress bitter taste receptor signaling in healthy and disease models. We hypothesized that the sweet/umami T1R3 antagonist lactisole may be used to alleviate bitter taste receptor repression in airway basal epithelial cells and enhance NO production. Here, we show that lactisole activates cAMP generation, though this occurs through a pathway independent of T1R3. This cAMP most likely signals through EPAC to increase ER Ca2+ efflux. Stimulation with denatonium benzoate, a bitter taste receptor agonist which activates largely nuclear and mitochondrial Ca2+ responses, resulted in a dramatically increased cytosolic Ca2+ response in cells treated with lactisole. This cytosolic Ca2+ signaling activated NO production in the presence of lactisole. Thus, lactisole may be useful coupled with bitter compounds as a therapeutic nasal rinse or spray to enhance beneficial antibacterial NO production in patients suffering from chronic inflammatory diseases such as chronic rhinosinusitis.

Cilia Stimulatory and Antibacterial Activities of T2R Bitter Taste Receptor Agonist Diphenhydramine: Insights into Repurposing Bitter Drugs for Nasal Infections.

T2R bitter taste receptors in airway motile cilia increase ciliary beat frequency (CBF) and nitric oxide (NO) production. Polymorphisms in some T2Rs are linked to disease outcomes in chronic rhinosinusitis (CRS) and cystic fibrosis (CF). We examined the expression of cilia T2Rs during the differentiation of human nasal epithelial cells grown at air-liquid interface (ALI). The T2R expression increased with differentiation but did not vary between CF and non-CF cultures. Treatment with Pseudomonas aeruginosa flagellin decreased the expression of diphenhydramine-responsive T2R14 and 40, among others. Diphenhydramine increased both NO production, measured by fluorescent dye DAF-FM, and CBF, measured via high-speed imaging. Increases in CBF were disrupted after flagellin treatment. Diphenhydramine impaired the growth of lab and clinical strains of P. aeruginosa, a major pathogen in CF and CF-related CRS. Diphenhydramine impaired biofilm formation of P. aeruginosa, measured via crystal violet staining, as well as the surface attachment of P. aeruginosa to CF airway epithelial cells, measured using colony-forming unit counting. Because the T2R agonist diphenhydramine increases NO production and CBF while also decreasing bacterial growth and biofilm production, diphenhydramine-derived compounds may have potential clinical usefulness in CF-related CRS as a topical therapy. However, utilizing T2R agonists as therapeutics within the context of P. aeruginosa infection may require co-treatment with anti-inflammatories to enhance T2R expression.

The bitter end: T2R bitter receptor agonists elevate nuclear calcium and induce apoptosis in non-ciliated airway epithelial cells.

Bitter taste receptors (T2Rs) localize to airway motile cilia and initiate innate immune responses in retaliation to bacterial quorum sensing molecules. Activation of cilia T2Rs leads to calcium-driven NO production that increases cilia beating and directly kills bacteria. Several diseases, including chronic rhinosinusitis, COPD, and cystic fibrosis, are characterized by loss of motile cilia and/or squamous metaplasia. To understand T2R function within the altered landscape of airway disease, we studied T2Rs in non-ciliated airway cell lines and primary cells. Several T2Rs localize to the nucleus in de-differentiated cells that typically localize to cilia in differentiated cells. As cilia and nuclear import utilize shared proteins, some T2Rs may target to the nucleus in the absence of motile cilia. T2R agonists selectively elevated nuclear and mitochondrial calcium through a G-protein-coupled receptor phospholipase C mechanism. Additionally, T2R agonists decreased nuclear cAMP, increased nitric oxide, and increased cGMP, consistent with T2R signaling. Furthermore, exposure to T2R agonists led to nuclear calcium-induced mitochondrial depolarization and caspase activation. T2R agonists induced apoptosis in primary bronchial and nasal cells differentiated at air-liquid interface but then induced to a squamous phenotype by apical submersion. Air-exposed well-differentiated cells did not die. This may be a last-resort defense against bacterial infection. However, it may also increase susceptibility of de-differentiated or remodeled epithelia to damage by bacterial metabolites. Moreover, the T2R-activated apoptosis pathway occurs in airway cancer cells. T2Rs may thus contribute to microbiome-tumor cell crosstalk in airway cancers. Targeting T2Rs may be useful for activating cancer cell apoptosis while sparing surrounding tissue.

Small-molecule Akt-activation in airway cells induces NO production and reduces IL-8 transcription through Nrf-2.

BACKGROUND: The non-cancerous functions of Akt in the airway are understudied. In some tissues, Akt phosphorylates and activates endothelial nitric oxide synthase (eNOS) to produce nitric oxide (NO) that has anti-inflammatory effects. NO production has antibacterial and antiviral effects in the airway, and increasing NO may be a useful anti-pathogen strategy. Akt also stimulates the nuclear factor erythroid 2-related factor 2 (Nrf-2) transcription factor, which transcribes antioxidant genes. Therefore, we hypothesized that activation of the Akt/eNOS pathway, which also activates Nrf-2, may have protective effects in human airway cells against injury. METHODS: To directly test the effects of Akt signaling in the airway, we treated A549 and 16HBE cells as well as primary bronchial, nasal, and type II alveolar epithelial cells with small molecule Akt activator SC79. We examined the effects of SC79 on eNOS activation, NO production, Nrf-2 target levels, and interleukin-8 (IL-8) transcription during exposure to TNF-α or Pseudomonas flagellin (TLR5 agonist). Additionally, air-liquid interface bronchial cultures were treated with cadmium, an oxidative stressor that causes airway barrier breakdown. RESULTS: SC79 induced a ~ twofold induction of p-eNOS and Nrf-2 protein levels blocked by PI3K inhibitor LY294002. Live cell imaging revealed SC79 increased acute NO production. Quantitative RT-PCR showed a ~ twofold increase in Nrf-2 target gene transcription. TNF-α or flagellin-induced IL-8 levels were also significantly reduced with SC79 treatment. Moreover, the transepithelial electrical resistance decrease observed with cadmium was ameliorated by SC79, likely by an acute increase in tight junction protein ZO-1 levels. CONCLUSIONS: Together, the data presented here demonstrate SC79 activation of Akt induces potentially anti-pathogenic NO production, antioxidant gene transcription, reduces IL-8 transcription, and may protect against oxidative barrier dysfunction in a wide range of airway epithelial cells.

First contact: the role of respiratory cilia in host-pathogen interactions in the airways.

Respiratory cilia are the driving force of the mucociliary escalator, working in conjunction with secreted airway mucus to clear inhaled debris and pathogens from the conducting airways. Respiratory cilia are also one of the first contact points between host and inhaled pathogens. Impaired ciliary function is a common pathological feature in patients with chronic airway diseases, increasing susceptibility to respiratory infections. Common respiratory pathogens, including viruses, bacteria, and fungi, have been shown to target cilia and/or ciliated airway epithelial cells, resulting in a disruption of mucociliary clearance that may facilitate host infection. Despite being an integral component of airway innate immunity, the role of respiratory cilia and their clinical significance during airway infections are still poorly understood. This review examines the expression, structure, and function of respiratory cilia during pathogenic infection of the airways. This review also discusses specific known points of interaction of bacteria, fungi, and viruses with respiratory cilia function. The emerging biological functions of motile cilia relating to intracellular signaling and their potential immunoregulatory roles during infection will also be discussed.

Identification of an Immortalized Human Airway Epithelial Cell Line with Dyskinetic Cilia.

Primary ciliary dyskinesia is an inherited, currently incurable condition. In the respiratory system, primary ciliary dyskinesia causes impaired functioning of the mucociliary escalator, leading to nasal congestion, cough, and recurrent otitis media, and commonly progresses to cause more serious and permanent damage, including hearing deficits, chronic sinusitis, and bronchiectasis. New treatment options for the condition are thus necessary. In characterizing an immortalized human bronchial epithelial cell line (BCi-NS1.1) grown at an air-liquid interface to permit differentiation, we have identified that these cells have dyskinetic motile cilia. The cells had a normal male karyotype, and phenotypic markers of epithelial cell differentiation emerged, as previously shown. Ciliary beat frequency (CBF) as assessed by high-speed videomicroscopy was lower than normal (4.4 Hz). Although changes in CBF induced by known modulators were as expected, the cilia displayed a dyskinetic, circular beat pattern characteristic of central microtubular agenesis with outer doublet transposition. This ultrastructural defect was confirmed by electron microscopy. We propose that the BCi-NS1.1 cell line is a useful model system for examination of modulators of CBF and more specifically could be used to screen for novel drugs with the ability to enhance CBF and perhaps repair a dyskinetic ciliary beat pattern.

Human mast cell line-1 (HMC-1) cells transfected with FcεRIα are sensitive to IgE/antigen-mediated stimulation demonstrating selectivity towards cytokine production.

Mast cells play important roles in allergic and inflammatory diseases. Efforts to better understand human mast cell activation and develop novel inhibitory agents have been hampered by the lack of suitable human mast cell lines. The HMC-1 mast cell line has been extensively used, but lacks native expression of the human high-affinity IgE receptor FcεRI limiting its applications. We have stably transfected HMC-1 cells with the IgE-binding α-subunit of FcεRI to generate HMCα cells that are antigen-responsive. We have used flow cytometry, cell signaling assays, pharmacological pathway inhibitors and cell functional assays to characterize the properties of HMCα cells. IgE/antigen responses were compared with those of the adenosine receptor agonist NECA. Surface expression of FcεRI in HMCα cells was demonstrated and was enhanced by prior sensitization with IgE. Activation of HMCα cells with IgE/antigen did not produce degranulation, but did lead to release of numerous cytokines. Whilst there was no measurable increase of intracellular Ca(2+) or marked general changes in protein tyrosine phosphorylation, IgE/antigen stimulation of HMCα cells enhanced phosphorylation of p38(MAPK) and Erk. Inhibitors of these pathways, as well as the src kinase inhibitor PP2, attenuated IgE/antigen-induced cytokine release. In summary, we have generated and characterized HMCα cells and show that they are a useful and relevant human mast cell model to examine FcεRI stabilization, signaling and mediator release. We envisage that HMCα cells will have utility in understanding the importance of mast cells in human allergic disease and in assessing the activity of novel anti-allergic compounds.