RES and MAL conceived all studies, with help from DJS and SEM. Most of the experiments were designed by RES and performed by ERC, YHF, and KKB. Additional experiments were conducted by JRD (macrophage and 3T3-L1 ChIP-seq); DJS (H3K27ac and Pol2 ChIP-seq); MJE (cold exposure); MK and PS (primary adipocyte culture); CJM and JFJ (some Ucp1 studies); and ERB, LCP, and RKD (animal husbandry and other assays). Computational analyses of RNA-seq were done by ZL and ChIP-seq by RES, with help from SRR, MD, HWL, and KJW. The manuscript was drafted by RES and MAL and revised and approved by all authors.

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.

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.

Background: Subclinical hypothyroidism is common in older individuals, but the physiologic responses to treatment with levothyroxine (LT4) and liothyronine (LT3) are not well defined in this age group. Methods: We conducted a randomized, double-blind, cross-over study of LT4 and LT3 treatment in men and women aged 70 years and over without anti-thyroid peroxidase antibodies with persistent subclinical hypothyroidism, defined as having a TSH level between 4.5 and 19.9 µIU/mL with a normal free thyroxine (FT4) level at two consecutive time points. Physiologic outcome measures assessed after achieving a TSH level of 0.5-1.5 µIU/mL with each therapy included vital signs, weight and body composition, bone mineral content and bone density, lipids, resting energy expenditure (REE), cognitive function, quality of life, and thyroid symptoms. Results: Thirteen participants [mean (SD) age 77 (5) years], 4 women and 9 men, completed the study. Baseline mean TSH was 4.84 (1.29) µIU/mL. The mean LT4 dose was 105 (36) µg/day [1.4 (0.5) µg/kg/day] and LT4 dose was 34 (9) µg/day [0.4 (0.1) µg/kg/day]. Mean time on LT4 was 200 days and on LT3 was 231 days, with a 28 day washout period. Compared with baseline, participants had an average weight loss of 1.1 kg on LT4 (p<0.02) and 2.5 kg on LT3 (p<0.001), which was significantly different between the two treatments (p=0.01). Fat mass decreased by an average of 0.7 kg on LT4 (p=0.03 vs. baseline) and 1.5 kg on LT3 (p<0.01 vs. baseline) and differed between treatments (p=0.01). There was a significant difference in total cholesterol of 13.3 mg/dL (p<0.001) and in low-density lipoprotein cholesterol (LDL) of 10.8 mg/dL (p<0.001) between LT4 and LT3 treatment arms; for both, the levels were lower on LT3 than LT4. No differences were seen in the other assessed outcomes. Conclusions: In a cross-over study of treatment of LT4 or LT3 in persistent subclinical hypothyroidism, participants lost fat mass and weight after each treatment, with a greater decrease after treatment with LT3. These findings support different physiologic responses to LT4 compared with LT3.

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.