Absence of functional <i>TLR2</i> and <i>TLR4</i> genes attenuates LPS-induced TNF-α expression in taste buds.

<p>LPS (5 mg kg⁻¹ body weight) was given to <i>TLR2^−/−/TLR4^−/−</i> double-knockout mice and wild-type C57BL/6 mice via i.p. injection. The same volume of endotoxin-free PBS (vehicle) was administrated to control animals. At 3 h post-LPS challenge, <i>TNF-α</i> mRNA levels in circumvallate- and foliate-containing taste epithelium (TE) and nontaste lingual epithelium (NT) were determined using qRT-PCR. Relative levels (fold) of <i>TNF-α</i> mRNA expression are shown. The expression level of <i>TNF-α</i> mRNA in nontaste tissue of wild-type mice receiving PBS was arbitrarily defined as 1. β-Actin served as the endogenous control gene for relative quantification. The same sets of 18 C57BL/6 mice described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043140#pone-0043140-g004" target="_blank">Figure 4F</a> were used as wild-type control mice. Parallel samples from <i>TLR2^−/−/TLR4^−/−</i> mice were prepared. Totally 18 <i>TLR2^−/−/TLR4^−/−</i> mice were used in this study. Data are mean ±SD. **<i>P<0.001</i> (ANOVA with <i>post hoc</i> Dunnett tests, Mean squared error  = 21.6, df = 16).</p

Expression of TNF-α in the taste epithelium of control adult mice.

<p>(<b>A</b>) qRT-PCR analysis of TNF-α expression in taste (TE) and nontaste (NT) epithelium: relative expression levels (fold) of the <i>TNF-α</i> gene. β-Actin served as the endogenous control gene for relative quantification. Epithelial tissues from 3 mice were pooled for each set of RNA sample preparation. Four independent sets of samples were established, and totally 12 mice were used in this study. Data are mean ±SD. **<i>P</i><0.01 (t = 7.8, df = 6). (<b>B–D</b>) Confocal images of specific immunofluorescent staining of TNF-α in taste buds of fungiform papillae (FFP) (<b>B</b>), foliate papillae (FP) (<b>C</b>), and circumvallate papillae (CVP) (<b>D</b>). (<b>E</b>) No TNF-α expression was observed in nontaste epithelium (NT) adjacent to foliate papillae (FP). Arrows indicate TNF-α-positive cells in foliate taste buds. (<b>F</b>) Spleen (SP) was used as a positive-tissue control. (<b>G</b>) The specificity of the TNF-α antibody was determined by blocking the antibody with recombinant TNF-α protein before staining (Ag Blocking). (<b>H</b>) The specificity of the secondary antibody was tested by immunostaining with nonspecific goat IgG instead of the TNF-α antibody (Goat IgG). (<b>I–L</b>) Antibodies against CD11b (<b>I, J</b>) and CD3 (<b>K, L</b>) were used to identify macrophages and granulocytes (CD11b), as well as T lymphocytes (CD3), on circumvallate sections. Brown solid staining in connective tissues indicates specifically stained cells. Dotted lines indicate areas of taste epithelium in circumvallate papillae. Boxed regions in <b>I</b> and <b>K</b> are shown in <b>J</b> and <b>L</b>, respectively, as enlarged images. These images show that CD11b- and CD3-positive cells are in connective tissue layers but not in taste buds. Arrows indicate representative CD11b- and CD3-positive cells. Ten mice were used for panels <b>B</b>–<b>F</b> and <b>I</b>–<b>L</b>, and five were used for panels <b>G–H</b>. Scale bars: 10 µm (<b>B</b>), 25 µm (<b>C, D, F</b>), and 50 µm (<b>E, G–L</b>).</p

Production and secretion of TNF-α by taste tissue upon LPS challenge <i>in vitro</i>.

<p>Mouse taste epithelium (TE; circumvallate and foliate) and nontaste lingual epithelium (NT) were isolated and incubated in DMEM culture medium. The samples were treated with 5 µg ml⁻¹ LPS for the time periods indicated. The cultures having no LPS in the medium served as controls. Concentrations of TNF-α in the supernatant of the cultured tissues were measured using ELISA. For each repeat of the experiment, tissues from three mice were used for medium control or LPS treatment. Each collected supernatant was assayed in duplicate for TNF-α concentration. The experiment was repeated three times, and the results were analyzed together. Data are mean ±SD. *<i>P<0.01</i> (t = 4.3, df = 4), <i>**P<0.001</i> (t = 9.8, df = 4), compared with medium control.</p

Systemic administration of microbial LPS increases TNF-α expression in taste cells.

<p>(<b>A–D</b>) Confocal images of double-immunofluorescent staining of TNF-α (green) and the type II taste cell marker PLC-β2 (red) on tissue sections of foliate (<b>A, B</b>) and circumvallate (<b>C, D</b>) papillae collected 3 h after PBS (vehicle control; <b>A</b>, <b>C</b>) or LPS (5 mg kg⁻¹ body weight, i.p.; <b>B</b>, <b>D</b>) injection. Scale bars: 35 µm. (<b>E</b>) Numbers of TNF-α-positive cells and PLC-β2-positive cells from foliate (F) and circumvallate (CV) sections obtained from PBS- or LPS-treated mice. The percentage of TNF-α-positive cells in the population of PLC-β2-positive cells was calculated and plotted (TNF-α/PLC-β2 (%)). Data are mean ±SD (n = 5 mice). *<i>P</i><0.05 (t = 3.2, df = 8), **<i>P</i><0.01 (t = 5.3, df = 8), compared with PBS groups. (<b>F</b>) qRT-PCR analysis of TNF-α expression in taste epithelium (TE) containing circumvallate and foliate taste buds and in nontaste lingual epithelium (NT) of PBS- and LPS-treated mice. Relative expression levels (fold) of the <i>TNF-α</i> gene are shown. <i>TNF-α</i> expression level in nontaste epithelium of control mice (NT-PBS) was set to 1. β-Actin served as the endogenous control gene for relative quantification. For each mouse group, epithelial tissues from 2–3 mice were pooled for each set of RNA sample preparation. Four independent sets of samples were prepared, and totally 18 C57BL/6 mice were used in this study. Data are mean ±SD. **<i>P</i><0.001 (t = 6.1, df = 6), compared with the PBS group.</p

Identification of specific type II taste cells that express TNF-α in mouse taste buds.

<p>Confocal images of immunofluorescent staining of TNF-α and markers for different subtypes of type II taste cells on taste papillae sections of control C57BL/6 mice. (<b>A</b>) Top and middle panels: double immunostaining for TNF-α (green) and T1R3 (red), showing TNF-α expression in T1R3-positive taste cells in foliate (top) and circumvallate (CV; middle) papillae. Bottom panels: TNF-α immunostaining (red) in a fungiform (FF) papilla from a T1R3-GFP (green fluorescent protein) mouse. (<b>B</b>) Double immunostaining of TNF-α (green) and gustducin (red), showing the difference in TNF-α expression in gustducin-positive type II taste cells in foliate, circumvallate (CV), and fungiform (FF) papillae. Five mice were included in each group of the experiment. Scale bars: 35 µm.</p

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

Taste buds of MRL/lpr mice are significantly smaller than those of MRL/+/+ mice.

<p>(<b>A</b>) H&E staining of circumvallate papillae of MRL/+/+ and MRL/lpr mice. The basal layer of the circumvallate epithelium is uneven, and taste buds are less organized in MRL/lpr mice. Scale bars, 40 µm. (<b>B</b>) Average areas of circumvallate taste bud profiles for MRL/+/+ and MRL/lpr mice. Five mice per group were included in the experiment. Areas of taste bud profiles were measured and averaged from at least four circumvallate sections per animal. Student's <i>t</i> tests were used for analysis. Data are means ± SEM. ** <i>p</i><0.01.</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

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