Biology subjects

Lee, R. J.

Publications and source records attributed to Lee, R. J..

28 records · Page 2Linked to original sources

bioRxiv2022

Taste receptor T1R3 in nasal cilia detects Staphylococcus aureus D-amino acids to enhance apical glucose uptake

Bitter (T2R) and sweet/umami (T1R) taste receptors serve chemosensory roles throughout the body. In airway cilia, T2Rs detect bacterial metabolites to stimulate bactericidal nitric oxide. T1Rs in solitary chemosensory cells detect glucose in airway surface liquid (ASL) and bacterial D-stereoisomer amino acids to regulate antimicrobial peptides. Using differentiated air-liquid interface cultures of primary nasal cells, we show that the T1R3 receptor is also expressed in human and mouse nasal cell cilia. D-amino acids produced at low mM concentrations by Staphylococcus aureus activate T1R3 to decrease ASL glucose and increase apical glucose uptake by increasing GLUT2 and GLUT10 expression through a {beta}-arrestin pathway. Our data suggests that T1R3 localized to cilia functions as an immune detector for D-amino acids to reduce ASL glucose and potentially limit bacterial growth. Given that glucose protected S. aureus against bactericidal NO produced during T2R activation, the reduced ASL glucose with T1R3 activation may also sensitize bacteria to other innate defenses. HIGHLIGHTSO_LINasal motile cilia express the T1R3 subunit of the sweet taste receptor C_LIO_LIS. aureus D-amino acids activate cilia T1R3 to enhance mucosal glucose uptake C_LIO_LIReduced airway surface liquid glucose likely reduces S. aureus growth C_LIO_LIReduced airway glucose also sensitizes S. aureus to epithelial NO production C_LI

physiology↗

bioRxiv2022

Bitter taste receptor-stimulated nitric oxide innate immune responses are reduced by loss of CFTR function in nasal epithelial cells and macrophages

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. Using qPCR, immunofluorescence, and live cell imaging of air-liquid interface cultures, we found 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. 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.

physiology↗

bioRxiv2022

Effect of time spent on active learning on exam performance: A controlled case study on a course with different instructors but identical teaching materials

Active learning, including student thinking and discussion in class, has been shown to increase student learning gains. However, it is less clear how variations in how instructors implement active learning affect student gains. Our study aims to investigate the extent to which the time spent on individual episodes of active learning activities influences student performance. We hypothesized that instructors who let students spend more time on peer discussion and individual thinking on practice problems associated with particular learning objectives will have better student exam scores on exam questions addressing those objectives. To test this hypothesis, we obtained a large data set of classroom recordings and student exam scores from an introductory biology course at a large four-year university, where three instructors shared identical teaching materials and exams for different course offerings. Contrary to our hypothesis, although the three instructors spent significantly different amounts of time on episodes of thinking and peer discussion, there was no correlation between the total time spent on active learning activities and student performance on exam questions. Linear mixed-effects modeling of the effect of length of episodes of student thinking and discussion on exam score found that the amount of course time spent on active learning activities did not reliably predict student performance on associated exam questions. This result held true even when only considering learning objectives with high variations in performance between offerings, difficult exam questions, exam questions requiring higher-order thinking skills, or within-instructor performance. Although our study was only conducted in one course, our results imply that time spent per individual episode of student thinking or peer discussion may not be the primary factor explaining the positive effects of active learning and that it may be worthwhile to explore other factors.

scientific communication and education↗

bioRxiv2022

Cilia stimulatory and antibacterial activities of bitter receptor agonist diphenhydramine: insights into potential complimentary strategies for CF nasal infections

BACKGROUNDBitter compounds increase ciliary beating and nitric oxide (NO) production in nasal epithelial cells through T2Rs in motile cilia. We examined expression of cilia T2Rs and both host and bacterial responses to T2R14 agonist diphenhydramine. METHODUsing cultured human nasal epithelial cells grown at air liquid interface, we measured expression of T2Rs via qPCR. We measured effects of diphenhydramine on ciliary beat frequency via high-speed imaging and nitric oxide production via fluorescent dye DAF-FM. We measured effects of diphenhydramine on growth of lab and clinical strains of Pseudomonas aeruginosa. We measured biofilm formation of P. aeruginosa using crystal violet staining and surface attachment of P. aeruginosa to cystic fibrosis bronchial epithelial (CBFE41o-) cells using CFU counting. RESULTST2R expression increased with mucocilliary differentiation and did not vary between CF and non-CF ALIs. Treatment with P. aeruginosa flagellin decreased expression of diphenhydramine-responsive T2R14 and 40, among other isoforms. Diphenhydramine increased both NO and CBF. Increases in CBF were disrupted after flagellin treatment. Diphenhydramine impaired growth, biofilm production, and surface attachment of P. aeruginosa. CONCLUSIONST2R expression is similar between normal and CF cells but decreases with flagellin treatment. Utilizing T2R agonists as therapeutics within the context of CF, P. aeruginosa infections may require co-treatment with anti-inflammatories to prevent the reduction of T2R expression with TLR activation. T2R agonist diphenhydramine increases NO production and CBF while also decreasing bacterial growth and biofilm production, and thus diphenhydramine or derivate compounds may have potential clinical usefulness in CF infections as a topical therapy. HIGHLIGHTSO_LIT2R14 agonist diphenhydramine increases nitric oxide production and cilia beating C_LIO_LIFlagellin decreases T2R14 expression in primary airway epithelial cells C_LIO_LIT2R14 agonist Diphenhydramine inhibits Pseudomonas growth and biofilm formation C_LI

physiology↗

bioRxiv2021

HSP90 function is required for T2R bitter taste receptor nitric oxide production and innate immune responses in human airway epithelial cells and macrophages

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 airway innate immune cell types are activated by bitter products, including some 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 and 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). We used immunofluorescence to show that H441 cells express eNOS and certain T2Rs and that the bitterant denatonium benzoate activates NO production in an HSP90-dependent manner in cells grown either as submerged cultures and at air liquid interface. In primary sinonasal epithelial cells, we determined that HSP-90 inhibition reduces T2R-stimulated NO production and ciliary beating which are crucial for 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 an innate immune role by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream calcium signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases like chronic rhinosinusitis, asthma, or cystic fibrosis.

physiology↗

bioRxiv2021

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 (acyl-homoserine lactones and quinolones). Activation of T2Rs leads to calcium-driven NO production that increases cilia beating and directly kills bacteria. Several airway diseases, including chronic rhinosinusitis, COPD, and cystic fibrosis, are characterized by epithelial remodeling, including loss of motile cilia and/or squamous metaplasia. To understand the function of T2Rs within the altered landscape of airway disease, we studied T2Rs in non-ciliated airway cell lines and primary cells de-differentiated to a squamous phenotype. In differentiated cells, T2Rs localize to cilia, however in de-differentiated, non-ciliated cells they localize to the nucleus. Cilia and nuclear import utilize many shared proteins, thus in the absence of motile cilia some T2Rs may target to the nucleus. T2R agonists selectively elevated both nuclear and mitochondrial calcium through a G-protein-coupled receptor, phospholipase C, and InsP3 receptor-dependent 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 T2R-induced apoptosis may be a last-resort defense against infection, possibly when bacteria have breached the epithelial barrier and reach non-ciliated cells below. 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. T2R agonists may also be useful topical therapeutics (e.g., delivered by nasal rinse or nebulizer) for activating airway cancer cell apoptosis without killing surrounding differentiated tissue.

physiology↗

bioRxiv2021

T2R bitter taste receptors regulate apoptosis and may be associated with survival in head and neck squamous cell carcinoma

Better management of head and neck squamous cell carcinomas (HNSCCs) requires a clearer understanding of tumor biology and disease risk. Bitter taste receptors (T2Rs) have been studied in several cancers, including thyroid, salivary, and GI, but their role in HNSCC has not been explored. We found that HNSCC patient samples and cell lines expressed functional T2Rs on both the cell and nuclear membranes. Bitter compounds, including bacterial metabolites, activated T2R-mediated nuclear Ca2+ responses leading to mitochondrial depolarization, caspase activation and ultimately apoptosis. Buffering nuclear Ca2+ elevation blocked caspase activation. Furthermore, increased expression of T2Rs in HNSCCs from The Cancer Genome Atlas (TCGA) is associated with improved overall survival. This work suggests that T2Rs are potential biomarkers to predict outcomes and guide treatment selection, may be leveraged as therapeutic targets to stimulate tumor apoptosis, and may mediate tumor-microbiome crosstalk in HNSCC.

cancer biology↗

bioRxiv2020

Altered polarization of PAR-2 signaling during airway epithelial remodeling

BackgroundProtease-activated receptor 2 (PAR-2) is activated by proteases involved in allergy and triggers airway epithelial secretion and inflammation. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells. ObjectiveWe tested if epithelial remodeling during diseases characterized by loss of cilia and squamous metaplasia may alter PAR-2 polarization. MethodsEndogenous PAR-2 responses were measured by live cell calcium and cilia imaging, measurement of fluid secretion, and quantification of cytokines. We utilized airway squamous cell lines, primary differentiated air-liquid interface cultures, and tissue explants. Cells were exposed to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency. We used concentrations and exposure times that altered epithelial morphology without causing breakdown of the epithelial barrier, likely reflecting early disease states. ResultsPAR-2 signaling in airway squamous cells activated calcium and inflammatory responses. Squamous cells cultured at air liquid interface (ALI) responded to PAR-2 agonists applied both apically and basolaterally. Primary well-differentiated nasal epithelial ALI cultures responded only to basolateral PAR-2 stimulation. Primary cultures exposed to IL-13, cigarette smoke condensate, or reduced retinoic acid responded to both apical and basolateral PAR-2 stimulation. Nasal polyp tissue, but not control middle turbinate, exhibited apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyp and turbinate maintained basolateral PAR-2 polarization. ConclusionsSquamous metaplasia and/or loss of cilia enhances apical PAR-2 responses. Altered PAR-2 polarization in dedifferentiated or remodeled epithelia may contribute to increased sensitivity to inhaled protease allergens in inflammatory airway diseases.

physiology↗

bioRxiv2019

Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling

Bitter taste receptors (T2Rs) are GPCRs involved in detection of bitter compounds by type 2 taste cells of the tongue, but are also expressed in other tissues throughout the body, including the airways, gastrointestinal tract, and brain. These T2Rs can be activated by several bacterial products and regulate innate immune responses in several cell types. Expression of T2Rs has been demonstrated in immune cells like neutrophils; however, the molecular details of their signaling are unknown. We examined mechanisms of T2R signaling in primary human monocyte-derived unprimed (M0) macrophages (M{Phi}s) using live cell imaging techniques. Known bitter compounds and bacterial T2R agonists activated low-level calcium signals through a pertussis toxin (PTX)-sensitive, phospholipase C-dependent, and inositol trisphosphate receptor-dependent calcium release pathway. These calcium signals activated low-level nitric oxide (NO) production via endothelial and neuronal NO synthase (NOS) isoforms. NO production increased cellular cGMP and enhanced acute phagocytosis [~]3-fold over 30-60 min via protein kinase G. In parallel with calcium elevation, T2R activation lowered cAMP, also through a PTX-sensitive pathway. The cAMP decrease also contributed to enhanced phagocytosis. Moreover, a co-culture model with airway epithelial cells demonstrated that NO produced by epithelial cells can also acutely enhance M{Phi} phagocytosis. Together, these data define M{Phi} T2R signal transduction and support an immune recognition role for T2Rs in M{Phi} cell physiology.

physiology↗

bioRxiv2019

Inverse regulation of secretion and inflammation in human airway gland serous cells by neuropeptides upregulated in allergy and asthma

Airway submucosal gland serous cells are sites of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) and are important for fluid secretion in conducting airways from the nose down to small bronchi. We tested if serous cells from human nasal turbinate glands secrete bicarbonate (HCO3-), important for mucus polymerization, during stimulation with the cAMP-elevating agonist vasoactive intestinal peptide (VIP) and if this requires CFTR. Isoalted serous cells stimulated with VIP exhibited a ~20% cAMP-dependent decrease in cell volume and a ~0.15 unit decrease in intracellular pH (pHi), reflecting activation of Cl- and HCO3- secretion, respectively. Pharmacology, ion substitution, and studies using cells from CF patients suggest serous cell HCO3- secretion is mediated by conductive efflux directly through CFTR. Interestingly, we found that neuropeptide Y (NPY) reduced VIP-evoked secretion by blunting cAMP increases and reducing CFTR activation through Gi-coupled NPY1R. Culture of primary gland serous cells in a model that maintained a serous phenotype confirmed the activating and inhibiting effects of VIP and NPY, respectively, on fluid and HCO3- secretion. Moreover, VIP enhanced secretion of antimicrobial peptides and antimicrobial efficacy of gland secretions while NPY reduced antimicrobial secretions. In contrast, NPY enhanced the release of cytokines during inflammatory stimuli while VIP reduced cytokine release through a mechanism requiring CFTR conductance. As levels of VIP and NPY are up-regulated in disease like allergy, asthma, and chronic rhinosinusitis, the balance of these two peptides in the airway may control airway mucus rheology and inflammatory responses through gland serous cells.

physiology↗