GAD65Cre Drives Reporter Expression in Multiple Taste Cell Types.

In taste buds, Type I cells represent the majority of cells (50-60%) and primarily have a glial-like function in taste buds. However, recent studies suggest that they have additional sensory and signaling functions including amiloride-sensitive salt transduction, oxytocin modulation of taste, and substance P mediated GABA release. Nonetheless, the overall function of Type I cells in transduction and signaling remains unclear, primarily because of the lack of a reliable reporter for this cell type. GAD65 expression is specific to Type I taste cells and GAD65 has been used as a Cre driver to study Type I cells in salt taste transduction. To test the specificity of transgene-driven expression, we crossed GAD65Cre mice with floxed tdTomato and Channelrhodopsin (ChR2) lines and examined the progeny with immunochemistry, chorda tympani recording, and calcium imaging. We report that while many tdTomato+ taste cells express NTPDase2, a specific marker of Type I cells, we see some expression of tdTomato in both Gustducin and SNAP25-positive taste cells. We also see ChR2 in cells just outside the fungiform taste buds. Chorda tympani recordings in the GAD65Cre/ChR2 mice show large responses to blue light. Furthermore, several isolated tdTomato-positive taste cells responded to KCl depolarization with increases in intracellular calcium, indicating the presence of voltage-gated calcium channels. Taken together, these data suggest that GAD65Cre mice drive expression in multiple taste cell types and thus cannot be considered a reliable reporter of Type I cell function.

A2ML1 and otitis media: novel variants, differential expression, and relevant pathways.

A genetic basis for otitis media is established, however, the role of rare variants in disease etiology is largely unknown. Previously a duplication variant within A2ML1 was identified as a significant risk factor for otitis media in an indigenous Filipino population and in US children. In this report exome and Sanger sequencing was performed using DNA samples from the indigenous Filipino population, Filipino cochlear implantees, US probands, Finnish, and Pakistani families with otitis media. Sixteen novel, damaging A2ML1 variants identified in otitis media patients were rare or low-frequency in population-matched controls. In the indigenous population, both gingivitis and A2ML1 variants including the known duplication variant and the novel splice variant c.4061 + 1 G>C were independently associated with otitis media. Sequencing of salivary RNA samples from indigenous Filipinos demonstrated lower A2ML1 expression according to the carriage of A2ML1 variants. Sequencing of additional salivary RNA samples from US patients with otitis media revealed differentially expressed genes that are highly correlated with A2ML1 expression levels. In particular, RND3 is upregulated in both A2ML1 variant carriers and high-A2ML1 expressors. These findings support a role for A2ML1 in keratinocyte differentiation within the middle ear as part of otitis media pathology and the potential application of ROCK inhibition in otitis media.

Expression of Bitter Taste Receptors and Solitary Chemosensory Cell Markers in the Human Sinonasal Cavity.

Some bitter taste receptors (TAS2R gene products) are expressed in the human sinonasal cavity and may function to detect airborne irritants. The expression of all 25 human bitter taste receptors and their location within the upper airway is not yet clear. The aim of this study is to characterize the presence and distribution of TAS2R transcripts and solitary chemosensory cells (SCCs) in different locations of the human sinonasal cavity. Biopsies were obtained from human subjects at up to 4 different sinonasal anatomic sites. PCR, microarray, and qRT-PCR were used to examine gene transcript expression. The 25 human bitter taste receptors as well as the sweet/umami receptor subunit, TAS1R3, and canonical taste signaling effectors are expressed in sinonasal tissue. All 25 human bitter taste receptors are expressed in the human upper airway, and expression of these gene products was higher in the ethmoid sinus than nasal cavity locations. Fluorescent in situ hybridization demonstrates that epithelial TRPM5 and TAS2R38 are expressed in a rare cell population compared with multiciliated cells, and at times, consistent with SCC morphology. Secondary analysis of published human sinus single-cell RNAseq data did not uncover TAS2R or canonical taste transduction transcripts in multiciliated cells. These findings indicate that the sinus has higher expression of SCC markers than the nasal cavity in chronic rhinosinusitis patients, comprising a rare cell type. Biopsies obtained from the ethmoid sinus may serve as the best location for study of human upper airway taste receptors and SCCs.

Role of the ectonucleotidase NTPDase2 in taste bud function.

Taste buds are unusual in requiring ATP as a transmitter to activate sensory nerve fibers. In response to taste stimuli, taste cells release ATP, activating purinergic receptors containing the P2X2 and P2X3 subunits on taste nerves. In turn, the released ATP is hydrolyzed to ADP by a plasma membrane nucleoside triphosphate previously identified as nucleoside triphosphate diphosphohydrolase-2 (NTPDase2). In this paper we investigate the role of this ectonucleotidase in the function of taste buds by examining gene-targeted Entpd2-null mice globally lacking NTPDase2. RT-PCR confirmed the absence of NTPDase2, and ATPase enzyme histochemistry reveals no reaction product in taste buds of knockout mice, suggesting that NTPDase2 is the dominant form in taste buds. RT-PCR and immunocytochemistry demonstrated that in knockout mice all cell types are present in taste buds, even those cells normally expressing NTPDase2. In addition, the overall number and size of taste buds are normal in Entpd2-null mice. Luciferin/luciferase assays of circumvallate tissue of knockout mice detected elevated levels of extracellular ATP. Electrophysiological recordings from two taste nerves, the chorda tympani and glossopharyngeal, revealed depressed responses to all taste stimuli in Entpd2-null mice. Responses were more depressed in the glossopharyngeal nerve than in the chorda tympani nerve and involved all taste qualities; responses in the chorda tympani were more depressed to sweet and umami stimuli than to other qualities. We suggest that the excessive levels of extracellular ATP in the Entpd2-knockout animals desensitize the P2X receptors associated with nerve fibers, thereby depressing taste responses.

Postsynaptic P2X3-containing receptors in gustatory nerve fibres mediate responses to all taste qualities in mice.

Taste buds release ATP to activate ionotropic purinoceptors composed of P2X2 and P2X3 subunits, present on the taste nerves. Mice with genetic deletion of P2X2 and P2X3 receptors (double knockout mice) lack responses to all taste stimuli presumably due to the absence of ATP-gated receptors on the afferent nerves. Recent experiments on the double knockout mice showed, however, that their taste buds fail to release ATP, suggesting the possibility of pleiotropic deficits in these global knockouts. To test further the role of postsynaptic P2X receptors in afferent signalling, we used AF-353, a selective antagonist of P2X3-containing receptors to inhibit the receptors acutely during taste nerve recording and behaviour. The specificity of AF-353 for P2X3-containing receptors was tested by recording Ca(2+) transients to exogenously applied ATP in fura-2 loaded isolated geniculate ganglion neurons from wild-type and P2X3 knockout mice. ATP responses were completely inhibited by 10 µm or 100 µm AF-353, but neither concentration blocked responses in P2X3 single knockout mice wherein the ganglion cells express only P2X2-containing receptors. Furthermore, AF-353 had no effect on taste-evoked ATP release from taste buds. In wild-type mice, i.p. injection of AF-353 or simple application of the drug directly to the tongue, inhibited taste nerve responses to all taste qualities in a dose-dependent fashion. A brief access behavioural assay confirmed the electrophysiological results and showed that preference for a synthetic sweetener, SC-45647, was abolished following i.p. injection of AF-353. These data indicate that activation of P2X3-containing receptors is required for transmission of all taste qualities.

Mice Lacking Pannexin 1 Release ATP and Respond Normally to All Taste Qualities.

Adenosine triphosphate (ATP) is required for the transmission of all taste qualities from taste cells to afferent nerve fibers. ATP is released from Type II taste cells by a nonvesicular mechanism and activates purinergic receptors containing P2X2 and P2X3 on nerve fibers. Several ATP release channels are expressed in taste cells including CALHM1, Pannexin 1, Connexin 30, and Connexin 43, but whether all are involved in ATP release is not clear. We have used a global Pannexin 1 knock out (Panx1 KO) mouse in a series of in vitro and in vivo experiments. Our results confirm that Panx1 channels are absent in taste buds of the knockout mice and that other known ATP release channels are not upregulated. Using a luciferin/luciferase assay, we show that circumvallate taste buds from Panx1 KO mice normally release ATP upon taste stimulation compared with wild type (WT) mice. Gustatory nerve recordings in response to various tastants applied to the tongue and brief-access behavioral testing with SC45647 also show no difference between Panx1 KO and WT. These results confirm that Panx1 is not required for the taste evoked release of ATP or for neural and behavioral responses to taste stimuli.

Evidence for a role of glutamate as an efferent transmitter in taste buds.

BACKGROUND: Glutamate has been proposed as a transmitter in the peripheral taste system in addition to its well-documented role as an umami taste stimulus. Evidence for a role as a transmitter includes the presence of ionotropic glutamate receptors in nerve fibers and taste cells, as well as the expression of the glutamate transporter GLAST in Type I taste cells. However, the source and targets of glutamate in lingual tissue are unclear. In the present study, we used molecular, physiological and immunohistochemical methods to investigate the origin of glutamate as well as the targeted receptors in taste buds. RESULTS: Using molecular and immunohistochemical techniques, we show that the vesicular transporters for glutamate, VGLUT 1 and 2, but not VGLUT3, are expressed in the nerve fibers surrounding taste buds but likely not in taste cells themselves. Further, we show that P2X2, a specific marker for gustatory but not trigeminal fibers, co-localizes with VGLUT2, suggesting the VGLUT-expressing nerve fibers are of gustatory origin. Calcium imaging indicates that GAD67-GFP Type III taste cells, but not T1R3-GFP Type II cells, respond to glutamate at concentrations expected for a glutamate transmitter, and further, that these responses are partially blocked by NBQX, a specific AMPA/Kainate receptor antagonist. RT-PCR and immunohistochemistry confirm the presence of the Kainate receptor GluR7 in Type III taste cells, suggesting it may be a target of glutamate released from gustatory nerve fibers. CONCLUSIONS: Taken together, the results suggest that glutamate may be released from gustatory nerve fibers using a vesicular mechanism to modulate Type III taste cells via GluR7.

Function, Innervation, and Neurotransmitter Signaling in Mice Lacking Type-II Taste Cells.

The Skn-1a transcription factor (Pou2f3) is required for Type II taste cell differentiation in taste buds. Taste buds in Skn-1a-/- mice lack Type II taste cells but have a concomitant expansion of Type III cells, providing an ideal model to determine the relative role of taste cell types in response specificity. We confirmed that chorda tympani responses to sweet, bitter, and umami stimuli were greatly reduced in the knock-outs (KOs) compared with wild-type (WT) littermates. Skn-1a-/- mice also had reductions to NaCl that were partially amiloride-insensitive, suggesting that both Type II and Type III cells contribute to amiloride-insensitive salt detection in anterior tongue. We also confirmed that responses to sour stimuli are equivalent in the KOs, despite the large increase in the number of Type III taste cells. To examine their innervation, we crossed the Htr3a-GFP (5-HT3A-GFP) reporter mouse with the Skn-1a-/- mice and examined geniculate ganglion neurons for GFP expression and responses to 5-HT. We found no change in the number of 5-HT3A-expressing neurons with KO of Skn-1a Calcium imaging showed that only 5-HT3A-expressing neurons respond to exogenous 5-HT, while most neurons respond to ATP, similar to WT mice. Interestingly, despite loss of all Type II cells, the P2X3 antagonist AF353 blocked all chorda tympani responses. These data collectively raise questions pertaining the source of ATP signaling in the absence of Type II taste cells and whether the additional Type III cells are innervated by fibers that would have normally innervated Type II cells.

RNA sequencing and pathway analysis identify tumor necrosis factor alpha driven small proline-rich protein dysregulation in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is a heterogeneous inflammatory disorder in which many pathways contribute to end-organ disease. Small proline-rich proteins (SPRR) are polypeptides that have recently been shown to contribute to epithelial biomechanical properties relevant in T-helper type 2 inflammation. There is evidence that genetic polymorphism in SPRR genes may predict the development of asthma in children with atopy and, correlatively, that expression of SPRRs is increased under allergic conditions, which leads to epithelial barrier dysfunction in atopic disease. METHODS: RNAs from uncinate tissue specimens from patients with CRS and control subjects were compared by RNA sequencing by using Ingenuity Pathway Analysis (n = 4 each), and quantitative polymerase chain reaction (PCR) (n = 15). A separate cohort of archived sinus tissue was examined by immunohistochemistry (n = 19). RESULTS: A statistically significant increase of SPRR expression in CRS sinus tissue was identified that was not a result of atopic presence. SPRR1 and SPRR2A expressions were markedly increased in patients with CRS (p < 0.01) on RNA sequencing, with confirmation by using real-time PCR. Immunohistochemistry of archived surgical samples demonstrated staining of SPRR proteins within squamous epithelium of both groups. Pathway analysis indicated tumor necrosis factor (TNF) alpha as a master regulator of the SPRR gene products. CONCLUSION: Expression of SPRR1 and of SPRR2A is increased in mucosal samples from patients with CRS and appeared as a downstream result of TNF alpha modulation, which possibly resulted in epithelial barrier dysfunction.

FGF21 Mediates Endocrine Control of Simple Sugar Intake and Sweet Taste Preference by the Liver.

The liver is an important integrator of nutrient metabolism, yet no liver-derived factors regulating nutrient preference or carbohydrate appetite have been identified. Here we show that the liver regulates carbohydrate intake through production of the hepatokine fibroblast growth factor 21 (FGF21), which markedly suppresses consumption of simple sugars, but not complex carbohydrates, proteins, or lipids. Genetic loss of FGF21 in mice increases sucrose consumption, whereas acute administration or overexpression of FGF21 suppresses the intake of both sugar and non-caloric sweeteners. FGF21 does not affect chorda tympani nerve responses to sweet tastants, instead reducing sweet-seeking behavior and meal size via neurons in the hypothalamus. This liver-to-brain hormonal axis likely represents a negative feedback loop as hepatic FGF21 production is elevated by sucrose ingestion. We conclude that the liver functions to regulate macronutrient-specific intake by producing an endocrine satiety signal that acts centrally to suppress the intake of "sweets."

Solitary chemosensory cells and bitter taste receptor signaling in human sinonasal mucosa.

BACKGROUND: Solitary chemosensory cells (SCCs) are specialized cells in the respiratory epithelium that respond to noxious chemicals including bacterial signaling molecules. SCCs express components of bitter taste transduction including the taste receptor type 2 (TAS2R) bitter taste receptors and downstream signaling effectors: α-Gustducin, phospholipase Cß2 (PLCß2), and transient receptor potential cation channel subfamily M member 5 (TRPM5). When activated, SCCs evoke neurogenic reflexes, resulting in local inflammation. The purpose of this study was to test for the presence SCCs in human sinonasal epithelium, and to test for a correlation with inflammatory disease processes such as allergic rhinitis and chronic rhinosinusitis. METHODS: Patient demographics and biopsies of human sinonasal mucosa were obtained from control patients (n = 7) and those with allergic rhinitis and/or chronic rhinosinusitis (n = 15). Reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), and immunohistochemistry were used to determine whether expression of signaling effectors was altered in diseased patients. RESULTS: RT-PCR demonstrated that bitter taste receptors TAS2R4, TAS2R14, and TAS2R46, and downstream signaling effectors α-Gustducin, PLCß2, and TRPM5 are expressed in the inferior turbinate, middle turbinate, septum, and uncinate of both control and diseased patients. PLCß2/TRPM5-immunoreactive SCCs were identified in the sinonasal mucosa of both control and diseased patients. qPCR showed similar expression of α-Gustducin and TRPM5 in the uncinate process of control and diseased groups, and there was no correlation between level of expression and 22-item Sino-Nasal Outcomes Test (SNOT-22) or pain scores. CONCLUSION: SCCs are present in human sinonasal mucosa in functionally relevant areas. Expression level of signaling effectors was similar in control and diseased patients and did not correlate with measures of pain and inflammation. Further study into these pathways may provide insight into nasal inflammatory diseases and may offer potential therapeutic targets.