GLUT1 inhibitor BAY-876 induces apoptosis and enhances anti-cancer effects of bitter receptor agonists in head and neck squamous carcinoma cells.

Head and neck squamous cell carcinomas (HNSCCs) are cancers that arise in the mucosa of the upper aerodigestive tract. The five-year patient survival rate is ~50%. Treatment includes surgery, radiation, and/or chemotherapy and is associated with lasting effects even when successful in irradicating the disease. New molecular targets and therapies must be identified to improve outcomes for HNSCC patients. We recently identified bitter taste receptors (taste family 2 receptors, or T2Rs) as a novel candidate family of receptors that activate apoptosis in HNSCC cells through mitochondrial Ca2+ overload and depolarization. We hypothesized that targeting another component of tumor cell metabolism, namely glycolysis, may increase the efficacy of T2R-directed therapies. GLUT1 (SLC2A1) is a facilitated-diffusion glucose transporter expressed by many cancer cells to fuel their increased rates of glycolysis. GLUT1 is already being investigated as a possible cancer target, but studies in HNSCCs are limited. Examination of immortalized HNSCC cells, patient samples, and The Cancer Genome Atlas revealed high expression of GLUT1 and upregulation in some patient tumor samples. HNSCC cells and tumor tissue express GLUT1 on the plasma membrane and within the cytoplasm (perinuclear, likely co-localized with the Golgi apparatus). We investigated the effects of a recently developed small molecule inhibitor of GLUT1, BAY-876. This compound decreased HNSCC glucose uptake, viability, and metabolism and induced apoptosis. Moreover, BAY-876 had enhanced effects on apoptosis when combined at low concentrations with T2R bitter taste receptor agonists. Notably, BAY-876 also decreased TNFα-induced IL-8 production, indicating an additional mechanism of possible tumor-suppressive effects. Our study demonstrates that targeting GLUT1 via BAY-876 to kill HNSCC cells, particularly in combination with T2R agonists, is a potential novel treatment strategy worth exploring further in future translational studies.

Albumin Nanoparticle-Based Drug Delivery Systems.

Nanoparticle-based systems are extensively investigated for drug delivery. Among others, with superior biocompatibility and enhanced targeting capacity, albumin appears to be a promising carrier for drug delivery. Albumin nanoparticles are highly favored in many disease therapies, as they have the proper chemical groups for modification, cell-binding sites for cell adhesion, and affinity to protein drugs for nanocomplex generation. Herein, this review summarizes the recent fabrication techniques, modification strategies, and application of albumin nanoparticles. We first discuss various albumin nanoparticle fabrication methods, from both pros and cons. Then, we provide a comprehensive introduction to the modification section, including organic albumin nanoparticles, metal albumin nanoparticles, inorganic albumin nanoparticles, and albumin nanoparticle-based hybrids. We finally bring further perspectives on albumin nanoparticles used for various critical diseases.

Albumin appears to be a promising carrier for drug delivery with superior biocompatibility and enhanced targeting capacity. This review focuses on the importance of albumin nanoparticles in drug delivery and concludes the recent fabrication techniques to prepare albumin nanoparticles, the modification strategies to require functional albumin nanoparticles, and critical applications of albumin nanoparticles in various diseases. The aim of this review is to help readers understand the significant potential of albumin nanoparticles in drug delivery.

Effects of Akt Activator SC79 on Human M0 Macrophage Phagocytosis and Cytokine Production.

Akt is an important kinase in metabolism. Akt also phosphorylates and activates endothelial and neuronal nitric oxide (NO) synthases (eNOS and nNOS, respectively) expressed in M0 (unpolarized) macrophages. We showed that e/nNOS NO production downstream of bitter taste receptors enhances macrophage phagocytosis. In airway epithelial cells, we also showed that the activation of Akt by a small molecule (SC79) enhances NO production and increases levels of nuclear Nrf2, which reduces IL-8 transcription during concomitant stimulation with Toll-like receptor (TLR) 5 agonist flagellin. We hypothesized that SC79's production of NO in macrophages might likewise enhance phagocytosis and reduce the transcription of some pro-inflammatory cytokines. Using live cell imaging of fluorescent biosensors and indicator dyes, we found that SC79 induces Akt activation, NO production, and downstream cGMP production in primary human M0 macrophages. This was accompanied by a reduction in IL-6, IL-8, and IL-12 production during concomitant stimulation with bacterial lipopolysaccharide, an agonist of pattern recognition receptors including TLR4. Pharmacological inhibitors suggested that this effect was dependent on Akt and Nrf2. Together, these data suggest that several macrophage immune pathways are regulated by SC79 via Akt. A small-molecule Akt activator may be useful in some infection settings, warranting future in vivo studies.

Microglia-targeted inhibition of miR-17 via mannose-coated lipid nanoparticles improves pathology and behavior in a mouse model of Alzheimer's disease.

Neuroinflammation and accumulation of Amyloid Beta (Aß) accompanied by deterioration of special memory are hallmarks of Alzheimer's disease (AD). Effective preventative and treatment options for AD are still needed. Microglia in AD brains are characterized by elevated levels of microRNA-17 (miR-17), which is accompanied by defective autophagy, Aß accumulation, and increased inflammatory cytokine production. However, the effect of targeting miR-17 on AD pathology and memory loss is not clear. To specifically inhibit miR-17 in microglia, we generated mannose-coated lipid nanoparticles (MLNPs) enclosing miR-17 antagomir (Anti-17 MLNPs), which are targeted to mannose receptors readily expressed on microglia. We used a 5XFAD mouse model (AD) that recapitulates many AD-related phenotypes observed in humans. Our results show that Anti-17 MLNPs, delivered to 5XFAD mice by intra-cisterna magna injection, specifically deliver Anti-17 to microglia. Anti-17 MLNPs downregulated miR-17 expression in microglia but not in neurons, astrocytes, and oligodendrocytes. Anti-17 MLNPs attenuated inflammation, improved autophagy, and reduced Aß burdens in the brains. Additionally, Anti-17 MLNPs reduced the deterioration in spatial memory and decreased anxiety-like behavior in 5XFAD mice. Therefore, targeting miR-17 using MLNPs is a viable strategy to prevent several AD pathologies. This selective targeting strategy delivers specific agents to microglia without the adverse off-target effects on other cell types. Additionally, this approach can be used to deliver other molecules to microglia and other immune cells in other organs.

Enhanced dissolution rates of glibenclamide through solid dispersions on microcrystalline cellulose and mannitol, combined with phosphatidylcholine.

OBJECTIVE: This study aimed to investigate the impact of physical solid dispersions of spray-dried glibenclamide (SG) on the surface of microcrystalline cellulose (MC) and mannitol (M) surfaces, as well as their combination with phosphatidylcholine (P), on enhancing the dissolution rate of glibenclamide (G). METHODS: Solid dispersions were prepared using varying proportions of 1:1, 1:4, and 1:10 for SG on the surface of MC (SGA) and M (SGM), and then combined with P, in a proportion of 1:4:0.02 using spray drying. The particle size, specific surface area, scanning electron microscopy (SEM), X-ray diffraction (XRD), and dissolution rate of SGA and SGM were characterized. RESULTS: SEM analysis revealed successful adhesion of SG onto the surface of the carrier surfaces. XRD showed reduced crystalline characteristic peaks for SGA, while SGM exhibited a sharp peaks pattern. Both SGA and SGM demonstrated higher dissolution rates compared to SG and G alone. Furthermore, the dissolution rates of the solid dispersions of SG, MC and P (SGAP), and SG, M, and P (SGMP) were sequentially higher than that of SGA and SGM. CONCLUSIONS: The study suggests that physical solid dispersions of SG on MC and M, along with their combination with P, can effectively enhance the dissolution rate of G. These findings may be valuable in developing of oral solid drug dosage forms utilizing SGA, SGM, SGAP, and SGMP.

Improved Targeting and Safety of Doxorubicin through a Novel Albumin Binding Prodrug Approach.

Human serum albumin (HSA) improves the pharmacokinetic profile of drugs attached to it, making it an attractive carrier with proven clinical success. In our previous studies, we have shown that Caveolin-1 (Cav-1) and caveolae-mediated endocytosis play important roles in the uptake of HSA and albumin-bound drugs. Doxorubicin is an FDA-approved chemotherapeutic agent that is effective against multiple cancers, but its clinical applicability has been hampered by its high toxicity levels. In this study, a doxorubicin-prodrug was developed that could independently and avidly bind HSA in circulation, called IPBA-Dox. We first developed and characterized IPBA-Dox and confirmed that it can bind albumin in vitro while retaining a potent cytotoxic effect. We then verified that it efficiently binds to HSA in circulation, leading to an improvement in the pharmacokinetic profile of the drug. In addition, we tested our prodrug for Cav-1 selectivity and found that it preferentially affects cells that express relatively higher levels of Cav-1 in vitro and in vivo. Moreover, we found that our compound was well tolerated in vivo at concentrations at which doxorubicin was lethal. Altogether, we have developed a doxorubicin-prodrug that can successfully bind HSA, retaining a strong cytotoxic effect that preferentially targets Cav-1 positive cells while improving the general tolerability of the drug.

Akt activator SC79 stimulates antibacterial nitric oxide generation in human nasal epithelial cells in vitro.

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.

Aerosolized Particulate Matter and Blunting of Ciliary Dynamic Responses: Implications for Veterans and Active Duty Military in Southwest Asia.

INTRODUCTION: Respiratory diseases such as chronic rhinosinusitis and asthma are observed at increased rates in active duty and veteran military members, and they are especially prevalent in individuals who have been deployed in Southwest Asia during Operation Iraqi Freedom and Operation Enduring Freedom. Particulate matter, specifically the fine-grain desert sand found in the Middle East, may be a key source of this pathology because of deleterious effects on mucociliary clearance. MATERIALS AND METHODS: With IRB approval, human sinonasal tissue was grown at an air-liquid interface and cultures were exposed to different types and sizes of particulate matter, including sand from Afghanistan and Kuwait. Ciliary dynamic responses to mechanical stimulation and ATP application were assessed following particulate exposure. RESULTS: Particle size of the commercial sand was substantially larger than that of the sand of Afghan or Kuwaiti origin. Following exposure to particulate matter, normal dynamic ciliary responses to mechanical stimulation and ATP application were significantly decreased (P < .01), with corresponding decreases in ATP-induced calcium flux (P < .05). These changes were partially reversible with apical washing after a 16-h period of exposure. After 36 h of exposure to Middle Eastern sand, ciliary responses to purinergic stimulation were completely abolished. CONCLUSIONS: There is a neutralization of the dynamic ciliary response following chronic particulate matter exposure, similar to ciliary pathologies observed in patients with chronic rhinosinusitis. Aerosolized particulate matter endured by military personnel in the Southwest Asia may cause dysfunctional mucociliary clearance; these data help to explain the increased prevalence of respiratory pathology in individuals who are or have been deployed in this region.

Transition-Metal-Free Continuous-Flow Synthesis of 2,5-Diaryl Furans: Access to Medicinal Building Blocks and Optoelectronic Materials.

The direct transformation of 1,3-dienes into valuable 2,5-diarylfurans using transition-metal-free conditions is presented. By employing a simple oxidation─dehydration sequence on readily accessible 1,3-dienes, important 2,5-diarylfuran building blocks frequently used in medicinal and material chemistry are prepared. The oxidation step is realized using singlet oxygen, and the intermediate endoperoxide is dehydrated under metal-free conditions and at ambient temperature using the Appel reagent. Notably, this sequence can be streamlined into continuous flow, thereby eliminating the isolation of the intermediate, often unstable endoperoxide. This leads to a significant improvement in isolated yields (ca. 27% average increase) of the 2,5-diarylfurans while also increasing safety and reducing waste. Our transition-metal-free synthetic approach to 2,5-diarylfurans delivers several important furan building blocks used commonly in medicinal chemistry and as optoelectronic materials, including short-chain linearly conjugated furan oligomers. Consequently, we also complete a short study of the optical and electrochemical properties of a selection of these novel materials.

Optimized Liposomal Delivery of Bortezomib for Advancing Treatment of Multiple Myeloma.

Bortezomib (BTZ), a boronic acid-derived proteasome inhibitor, is commonly employed in treating multiple myeloma (MM). However, the applications of BTZ are limited due to its poor stability and low bioavailability. Herein, we develop an optimized liposomal formulation of BTZ (L-BTZ) by employing a remote-loading strategy. This formulation uses Tiron, a divalent anionic catechol derivative, as the internal complexing agent. Compared to earlier BTZ-related formulations, this alternative formulation showed significantly greater stability due to the Tiron-BTZ complex's higher pH stability and negative charges, compared to the meglumine-BTZ complex. Significantly, the plasma AUC of L-BTZ was found to be 30 times greater than that of free BTZ, suggesting an extended blood circulation duration. In subsequent therapeutic evaluations using two murine xenograft tumor models of MM, the NCI-H929 and OPM2 models showed tumor growth inhibition (TGI) values of 37% and 57%, respectively. In contrast, free BTZ demonstrated TGI values of 17% and 11% in these models. Further, L-BTZ presented enhanced antitumor efficacy in the Hepa1-6 HCC syngeneic model, indicating its potential broader applicability as an antineoplastic agent. These findings suggest that the optimized L-BTZ formulation offers a significant advancement in BTZ delivery, holding substantial promise for clinical investigation in not merely MM, but other cancer types.

Lidocaine induces apoptosis in head and neck squamous cell carcinoma through activation of bitter taste receptor T2R14.

Head and neck squamous cell carcinomas (HNSCCs) have high mortality and significant treatment-related morbidity. It is vital to discover effective, minimally invasive therapies that improve survival and quality of life. Bitter taste receptors (T2Rs) are expressed in HNSCCs, and T2R activation can induce apoptosis. Lidocaine is a local anesthetic that also activates bitter taste receptor 14 (T2R14). Lidocaine has some anti-cancer effects, but the mechanisms are unclear. Here, we find that lidocaine causes intracellular Ca2+ mobilization through activation of T2R14 in HNSCC cells. T2R14 activation with lidocaine depolarizes mitochondria, inhibits proliferation, and induces apoptosis. Concomitant with mitochondrial Ca2+ influx, ROS production causes T2R14-dependent accumulation of poly-ubiquitinated proteins, suggesting that proteasome inhibition contributes to T2R14-induced apoptosis. Lidocaine may have therapeutic potential in HNSCCs as a topical gel or intratumor injection. In addition, we find that HPV-associated (HPV+) HNSCCs are associated with increased TAS2R14 expression. Lidocaine treatment may benefit these patients, warranting future clinical studies.

Macrophage-Targeted Lipid Nanoparticle Delivery of microRNA-146a to Mitigate Hemorrhagic Shock-Induced Acute Respiratory Distress Syndrome.

The pro-inflammatory response of alveolar macrophages to injurious physical forces during mechanical ventilation is regulated by the anti-inflammatory microRNA, miR-146a. Increasing miR-146a expression to supraphysiologic levels using untargeted lipid nanoparticles reduces ventilator-induced lung injury but requires a high initial dose of miR-146a making it less clinically applicable. In this study, we developed mannosylated lipid nanoparticles that can effectively mitigate lung injury at the initiation of mechanical ventilation with lower doses of miR-146a. We used a physiologically relevant humanized in vitro coculture system to evaluate the cell-specific targeting efficiency of the mannosylated lipid nanoparticle. We discovered that mannosylated lipid nanoparticles preferentially deliver miR-146a to alveolar macrophages and reduce force-induced inflammation in vitro. Our in vivo study using a clinically relevant mouse model of hemorrhagic shock-induced acute respiratory distress syndrome demonstrated that delivery of a low dose of miR-146a (0.1 nmol) using mannosylated lipid nanoparticles dramatically increases miR-146a levels in mouse alveolar macrophages and decreases lung inflammation. These data suggest that mannosylated lipid nanoparticles may have the therapeutic potential to mitigate lung injury during mechanical ventilation.

Interkingdom Detection of Bacterial Quorum-Sensing Molecules by Mammalian Taste Receptors.

Bitter and sweet taste G protein-coupled receptors (known as T2Rs and T1Rs, respectively) were originally identified in type II taste cells on the tongue, where they signal perception of bitter and sweet tastes, respectively. Over the past ~15 years, taste receptors have been identified in cells all over the body, demonstrating a more general chemosensory role beyond taste. Bitter and sweet taste receptors regulate gut epithelial function, pancreatic ß cell secretion, thyroid hormone secretion, adipocyte function, and many other processes. Emerging data from a variety of tissues suggest that taste receptors are also used by mammalian cells to "eavesdrop" on bacterial communications. These receptors are activated by several quorum-sensing molecules, including acyl-homoserine lactones and quinolones from Gram-negative bacteria such as Pseudomonas aeruginosa, competence stimulating peptides from Streptococcus mutans, and D-amino acids from Staphylococcus aureus. Taste receptors are an arm of immune surveillance similar to Toll-like receptors and other pattern recognition receptors. Because they are activated by quorum-sensing molecules, taste receptors report information about microbial population density based on the chemical composition of the extracellular environment. This review summarizes current knowledge of bacterial activation of taste receptors and identifies important questions remaining in this field.

Coronary Atherosclerotic Plaque Activity and Future Coronary Events.

Importance: Recurrent coronary events in patients with recent myocardial infarction remain a major clinical problem. Noninvasive measures of coronary atherosclerotic disease activity have the potential to identify individuals at greatest risk. Objective: To assess whether coronary atherosclerotic plaque activity as assessed by noninvasive imaging is associated with recurrent coronary events in patients with myocardial infarction. Design, Setting, and Participants: This prospective, longitudinal, international multicenter cohort study recruited participants aged 50 years or older with multivessel coronary artery disease and recent (within 21 days) myocardial infarction between September 2015 and February 2020, with a minimum 2 years' follow-up. Intervention: Coronary 18F-sodium fluoride positron emission tomography and coronary computed tomography angiography. Main Outcomes and Measures: Total coronary atherosclerotic plaque activity was assessed by 18F-sodium fluoride uptake. The primary end point was cardiac death or nonfatal myocardial infarction but was expanded during study conduct to include unscheduled coronary revascularization due to lower than anticipated primary event rates. Results: Among 2684 patients screened, 995 were eligible, 712 attended for imaging, and 704 completed an interpretable scan and comprised the study population. The mean (SD) age of participants was 63.8 (8.2) years, and most were male (601 [85%]). Total coronary atherosclerotic plaque activity was identified in 421 participants (60%). After a median follow-up of 4 years (IQR, 3-5 years), 141 participants (20%) experienced the primary end point: 9 had cardiac death, 49 had nonfatal myocardial infarction, and 83 had unscheduled coronary revascularizations. Increased coronary plaque activity was not associated with the primary end point (hazard ratio [HR], 1.25; 95% CI, 0.89-1.76; P = .20) or unscheduled revascularization (HR, 0.98; 95% CI, 0.64-1.49; P = .91) but was associated with the secondary end point of cardiac death or nonfatal myocardial infarction (47 of 421 patients with high plaque activity [11.2%] vs 19 of 283 with low plaque activity [6.7%]; HR, 1.82; 95% CI, 1.07-3.10; P = .03) and all-cause mortality (30 of 421 patients with high plaque activity [7.1%] vs 9 of 283 with low plaque activity [3.2%]; HR, 2.43; 95% CI, 1.15-5.12; P = .02). After adjustment for differences in baseline clinical characteristics, coronary angiography findings, and Global Registry of Acute Coronary Events score, high coronary plaque activity was associated with cardiac death or nonfatal myocardial infarction (HR, 1.76; 95% CI, 1.00-3.10; P = .05) but not with all-cause mortality (HR, 2.01; 95% CI, 0.90-4.49; P = .09). Conclusions and Relevance: In this cohort study of patients with recent myocardial infarction, coronary atherosclerotic plaque activity was not associated with the primary composite end point. The findings suggest that risk of cardiovascular death or myocardial infarction in patients with elevated plaque activity warrants further research to explore its incremental prognostic implications.

The ion channel CALHM6 controls bacterial infection-induced cellular cross-talk at the immunological synapse.

Membrane ion channels of the calcium homeostasis modulator (CALHM) family promote cell-cell crosstalk at neuronal synapses via ATP release, where ATP acts as a neurotransmitter. CALHM6, the only CALHM highly expressed in immune cells, has been linked to the induction of natural killer (NK) cell anti-tumour activity. However, its mechanism of action and broader functions in the immune system remain unclear. Here, we generated Calhm6-/- mice and report that CALHM6 is important for the regulation of the early innate control of Listeria monocytogenes infection in vivo. We find that CALHM6 is upregulated in macrophages by pathogen-derived signals and that it relocates from the intracellular compartment to the macrophage-NK cell synapse, facilitating ATP release and controlling the kinetics of NK cell activation. Anti-inflammatory cytokines terminate CALHM6 expression. CALHM6 forms an ion channel when expressed in the plasma membrane of Xenopus oocytes, where channel opening is controlled by a conserved acidic residue, E119. In mammalian cells, CALHM6 is localised to intracellular compartments. Our results contribute to the understanding of neurotransmitter-like signal exchange between immune cells that fine-tunes the timing of innate immune responses.

Dual-Ligand-Functionalized Liposomes Based on Glycyrrhetinic Acid and cRGD for Hepatocellular Carcinoma Targeting and Therapy.

Hepatocellular carcinoma (HCC) is one of the most common cancers, with high mortality. Chemotherapy is one of the main treatment options for HCC. However, the high toxicity and poor specificity of chemotherapeutic drugs have limited their clinical application. In this study, dual-ligand liposomes modified with glycyrrhetinic acid (GA) and cyclic arginine-glycine-aspartic acid (cRGD) (GA/cRGD-LP) were designed to target the GA receptor and αvß3 integrin, respectively. The aim was to develop a highly selective targeted drug delivery system and further enhance the antitumor efficiency of drugs by targeting both hepatic tumor cells and vasculature. A novel lipid conjugate (mGA-DOPE) by coupling dioleoylphosphatidyl ethanolamine (DOPE) with methyl glycyrrhetinic acid (mGA) was synthesized, and its structure was confirmed. The targeting efficiency of GA/cRGD-LP by in vitro cellular uptake and ex vivo imaging was assessed. GA- and cRGD-modified doxorubicin-loaded liposomes (GA/cRGD-LP-DOX) were prepared, and their cytotoxicity in HepG2 and antitumor activity were evaluated. The results showed that the average particle size of the GA/cRGD-LP-DOX was 114 ± 4.3 nm, and the zeta potential was -32.9 ± 2.0 mV. The transmission electron microscopy images showed that the shapes of our liposomes were spherical. cGA/cRGD-LP-DOX displayed an excellent cellular uptake in both HepG2 and human umbilical vein endothelial cells. In the in vivo study, pharmacokinetic parameters indicated that cGA/cRGD-LP can prolong the circulation time of DOX in the blood. GA/cRGD-LP-DOX showed greater inhibition of tumor growth for HepG2-bearing mice than either the single-ligand-modified liposomes or nontargeted liposomes. GA/cRGD-LP-DOX displayed higher liver tumor localization than that of single-ligand-modified liposomes or free DOX. GA/cRGD-LP is a promising drug delivery system for liver cancer targeting and therapy and is worthy of further study.

Loss of CFTR function is associated with reduced bitter taste receptor-stimulated nitric oxide innate immune responses in nasal epithelial cells and macrophages.

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.

Macrophage-targeted lipid nanoparticle delivery of microRNA-146a to mitigate hemorrhagic shock-induced acute respiratory distress syndrome.

The pro-inflammatory response of alveolar macrophages to injurious physical forces during mechanical ventilation is regulated by the anti-inflammatory microRNA, miR-146a. Increasing miR-146a expression to supraphysiologic levels using untargeted lipid nanoparticles reduces ventilator-induced lung injury, but requires a high initial dose of miR-146a making it less clinically applicable. In this study, we developed mannosylated lipid nanoparticles that can effectively mitigate lung injury at the initiation of mechanical ventilation with lower doses of miR-146a. We used a physiologically relevant humanized in vitro co-culture system to evaluate the cell-specific targeting efficiency of the mannosylated lipid nanoparticle. We discovered that mannosylated lipid nanoparticles preferentially deliver miR-146a to alveolar macrophages and reduce force-induced inflammation in vitro . Our in vivo study using a clinically relevant mouse model of hemorrhagic shock-induced acute respiratory distress syndrome demonstrated that delivery of a low dose miR-146a (0.1 nmol) using mannosylated lipid nanoparticles dramatically increases miR-146a in mouse alveolar macrophages and decreases lung inflammation. These data suggest that mannosylated lipid nanoparticles may have therapeutic potential to mitigate lung injury during mechanical ventilation.

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.