Defects in the Peripheral Taste Structure and Function in the MRL/lpr Mouse Model of Autoimmune Disease

<div><p>While our understanding of the molecular and cellular aspects of taste reception and signaling continues to improve, the aberrations in these processes that lead to taste dysfunction remain largely unexplored. Abnormalities in taste can develop in a variety of diseases, including infections and autoimmune disorders. In this study, we used a mouse model of autoimmune disease to investigate the underlying mechanisms of taste disorders. MRL/MpJ-Fas^lpr/J (MRL/lpr) mice develop a systemic autoimmunity with phenotypic similarities to human systemic lupus erythematosus and Sjögren's syndrome. Our results show that the taste tissues of MRL/lpr mice exhibit characteristics of inflammation, including infiltration of T lymphocytes and elevated levels of some inflammatory cytokines. Histological studies reveal that the taste buds of MRL/lpr mice are smaller than those of wild-type congenic control (MRL/+/+) mice. 5-Bromo-2′-deoxyuridine (BrdU) pulse-chase experiments show that fewer BrdU-labeled cells enter the taste buds of MRL/lpr mice, suggesting an inhibition of taste cell renewal. Real-time RT-PCR analyses show that mRNA levels of several type II taste cell markers are lower in MRL/lpr mice. Immunohistochemical analyses confirm a significant reduction in the number of gustducin-positive taste receptor cells in the taste buds of MRL/lpr mice. Furthermore, MRL/lpr mice exhibit reduced gustatory nerve responses to the bitter compound quinine and the sweet compound saccharin and reduced behavioral responses to bitter, sweet, and umami taste substances compared with controls. In contrast, their responses to salty and sour compounds are comparable to those of control mice in both nerve recording and behavioral experiments. Together, our results suggest that type II taste receptor cells, which are essential for bitter, sweet, and umami taste reception and signaling, are selectively affected in MRL/lpr mice, a model for autoimmune disease with chronic inflammation.</p> </div

Taste bud cell renewal is inhibited in MRL/lpr mice.

<p>(<b>A</b>) BrdU (green) and KCNQ1 (red) double immunostaining of circumvallate papillae from MRL/+/+ and MRL/lpr mice injected with BrdU. Tissues were collected 1 and 5 days after BrdU injection (Day 1 and Day 5, respectively). Arrows denote some BrdU-labeled cells inside taste buds. (<b>B</b> and <b>C</b>) Quantitative analyses of BrdU-labeled cells in the circumvallate (CV) epithelium collected 1 day (<b>B</b>) and 5 days (<b>C</b>) after BrdU injection. Left, number of BrdU-positive taste cells per taste bud profile; right, number of BrdU-positive perigemmal cells per square millimeter (mm²) of circumvallate epithelium. (<b>D</b>) Ki67 (green) and KCNQ1 (red) double immunostaining: merged confocal fluorescent images of circumvallate papillae from MRL/+/+ and MRL/lpr mice. (<b>E</b>) Average number of Ki67-labeled cells in the basal region of taste bud profile. Ki67-labeled cells in the basal regions of circumvallate taste buds were counted, and the average numbers of Ki67-labeled cells per taste bud profile are shown for MRL/+/+ and MRL/lpr mice. (<b>F</b>) Total number of BrdU-positive cells (including cells in the perigemmal regions and inside the taste buds) per square millimeter (mm²) of circumvallate epithelium 1 day after BrdU injection. Four mice per group per time point were included in the experiments. Six circumvallate sections from each animal were included in the analyses. Data are means ± SEM. Student's <i>t</i> tests were used for statistical analysis. * <i>p</i><0.05; ** <i>p</i><0.01. Scale bars, 20 µm.</p

Number of gustducin-positive taste receptor cells is reduced in circumvallate taste buds of MRL/lpr mice.

<p>(<b>A</b>) Confocal images of immunofluorescent staining with antibodies against gustducin and NCAM. Circumvallate sections from MRL/+/+ and MRL/lpr mice were processed for immunostaining with anti-gustducin or anti-NCAM antibody as indicated. Scale bars, 40 µm. (<b>B</b> and <b>C</b>) Quantitative analyses of the average number of gustducin-positive (<b>B</b>) or NCAM-positive (<b>C</b>) cells per taste bud profile based on immunostaining data. Five mice per group were included in the experiment. Four circumvallate tissue sections per animal were included. Student's <i>t</i> tests were used. Data are means ± SEM. ** <i>p</i><0.01.</p

mRNA levels of IFN-γ, TNF-α, and IL-10 are elevated in the taste epithelium of MRL/lpr mice.

<p>qRT-PCR analysis of mRNA expression of IFN-γ (<b>A</b>), TNF-α (<b>B</b>), IL-10 (<b>C</b>), and IL-6 (<b>D</b>) in the circumvallate- and foliate-containing lingual epithelia of MRL/lpr and MRL/+/+ mice. Gene expression is shown as relative fold levels (mean ± SEM), with the mRNA level of each analyzed gene in MRL/+/+ mice arbitrarily set to 1. β-Actin was used as the endogenous control gene for relative quantification. For each mouse group, taste epithelial tissues from 3–4 mice were pooled for each set of sample preparations. Three independent sets of samples were prepared. Together, 9–12 mice per group were included in this study. Each qPCR reaction was run either in duplicate or triplicate. Student's <i>t</i> tests were performed. ** <i>p</i><0.01; *** <i>p</i><0.001.</p

TNF-α and IFN-γ are expressed in subsets of taste bud cells.

<p>Confocal images of immunofluorescent staining of circumvallate papillae of MRL/lpr mice. (<b>A</b>) TNF-α is expressed in a subset of PLC-β2-positive cells. Immunoreactivities of TNF-α (green) and PLC-β2 (red) are colocalized in circumvallate taste buds of MRL/lpr mice. (<b>B</b> and <b>C</b>) IFN-γ is expressed in subsets of PLC-β2- or SNAP25-positive cells. Immunoreactivities of IFN-γ (red) are partially colocalized with PLC-β2 (green) and SNAP25 (green) in circumvallate taste buds of MRL/lpr mice. Scale bars, 20 µm.</p

mRNA levels of gustducin, TrpM5, and NeuroD are reduced in the taste epithelium of MRL/lpr mice.

<p>qRT-PCR analysis was performed to quantify mRNA levels of gustducin (<b>A</b>), TrpM5 (<b>B</b>), NeuroD (<b>C</b>), SNAP25 (<b>D</b>), and PKD2L1 (<b>E</b>) in the circumvallate- and foliate-containing lingual epithelium prepared from MRL/+/+ and MRL/lpr mice. Gene expression (mean ± SEM) is shown in relative fold levels, with mRNA level of each analyzed gene in MRL/+/+ mice arbitrarily set to 1. β-Actin was used as the endogenous control gene for relative quantification. Epithelial tissues from 3–4 mice were pooled for each set of sample preparations. Three independent sets of samples were prepared. Each qPCR reaction was run either in duplicate or triplicate. Student's <i>t</i> tests were used for analysis. * <i>p</i><0.05; ** <i>p</i><0.01.</p

RT-PCR primers.

*<p>GenBank accession number used for primer design.</p>**<p>Size of predicted PCR product in base pairs (bp).</p

T-cell infiltration significantly increases in the circumvallate papilla and epithelium of MRL/lpr mice.

<p>Immunohistochemistry was performed using an antibody against CD3, a T-cell marker. (<b>A–C</b>) Images of the circumvallate papillae from MRL/lpr (<b>A</b> and <b>B</b>) and MRL/+/+ mice (<b>C</b>). T cells, either clustered or dispersed, are stained dark brown. The boxed region in <b>A</b>, which contains a large cluster of T cells in close proximity to the circumvallate epithelium, is shown at higher magnification in <b>B</b>. Scale bars, 40 µm. (<b>D</b>) T-cell densities (mean ± SEM) as percentage of anti-CD3-stained area against the total area of the observed circumvallate sections from MRL/+/+ and MRL/lpr mice. Eight MRL/lpr and nine MRL/+/+ mice were included in this study. Student's <i>t</i> tests were used for analysis. * <i>p</i><0.05.</p

MRL/lpr mice show decreased CT nerve responses to quinine and saccharin.

<p>(<b>A</b>) Representative CT nerve responses to taste compounds. CT nerve responses to 0.1 M NH₄Cl are shown as reference. (<b>B</b>) CT nerve responses (mean ± SEM) to several concentrations of QHCl, saccharin, sucrose, MSG, NaCl, citric acid, and HCl. Nerve responses to taste compounds were normalized against responses to 0.1 M NH₄Cl. Nerve recordings were done with 8–12 MRL/+/+ mice and 7–10 MRL/lpr mice. Data were analyzed with two-way ANOVA with <i>post hoc</i> Fisher LSD tests. Responses to QHCl were significantly affected by strain (F_1,20 = 4.7, <i>p</i> = 0.04) and strain×concentration interaction (F_3,60 = 4.6, <i>p</i> = 0.006). Responses to saccharin were significantly affected by strain×concentration interaction (F_4,52 = 2.7, <i>p</i> = 0.04). * <i>p</i><0.05.</p

MRL/lpr mice exhibit reduced responses to bitter, sweet, and umami taste compounds in brief-access tests.

<p>Five-second brief-access tests were performed with MRL/lpr and MRL/+/+ mice to QHCl (<b>A</b>), sucrose (<b>B</b>), IMP (<b>C</b>), NaCl (<b>D</b>), and citric acid (<b>E</b>). Taste compounds and the tested concentrations are as indicated in the graphs. Lick ratios (mean ± SEM) were calculated as the number of licks of taste stimuli divided by the number of licks of water in each test session. N = 8 for MRL/+/+ mice; N = 7 for MRL/lpr mice. Data were analyzed with two-way ANOVA with <i>post hoc t</i> tests. * <i>p</i><0.05.</p