Interleukin-10 is produced by a specific subset of taste receptor cells and critical for maintaining structural integrity of mouse taste buds

Abstract

Although inflammatory responses are a critical component in defense against pathogens, too much inflammation is harmful. Mechanisms have evolved to regulate inflammation, including modulation by the anti-inflammatory cytokine interleukin-10 (IL-10). Previously we have shown that taste buds express various molecules involved in innate immune responses, including the proinflammatory cytokine tumor necrosis factor (TNF). Here, using a reporter mouse strain, we show that taste cells also express the anti-inflammatory cytokine IL-10. Remarkably, IL-10 is produced by only a specific subset of taste cells, which are different from the TNF-producing cells in mouse circumvallate and foliate taste buds: IL-10 expression was found exclusively in the G-protein gustducin-expressing bitter receptor cells, while TNF was found in sweet and umami receptor cells as reported previously. In contrast, IL-10R1, the ligand-binding subunit of the IL-10 receptor, is predominantly expressed by TNF-producing cells, suggesting a novel cellular hierarchy for regulating TNF production and effects in taste buds. In response to inflammatory challenges, taste cells can increase IL-10 expression both in vivo and in vitro. These findings suggest that taste buds use separate populations of taste receptor cells that coincide with sweet/umami and bitter taste reception to modulate local inflammatory responses, a phenomenon that has not been previously reported. Furthermore, IL-10 deficiency in mice leads to significant reductions in the number and size of taste buds, as well as in the number of taste receptor cells per taste bud, suggesting that IL-10 plays critical roles in maintaining structural integrity of the peripheral gustatory system.

Keywords: cytokines; inflammation; taste buds.

Figure 1.

Expression of IL-10 in the taste buds and other tissues. A, qRT-PCR analysis of IL-10 expression in taste (TE) and nontaste (NT) lingual epithelia of C57BL/6 mice: relative expression levels (fold) of IL-10 mRNA. β-Actin served as an endogenous control gene for relative quantification, and IL-10 expression level in nontaste samples was designated as 1. Taste and nontaste epithelial tissues from three mice were pooled for each set of RNA sample preparations. Data are mean ± SD; **p = 0.001 (unpaired two-tailed t test, df = 4, n = 3 experiments). B, C, IL-10 mRNA in situ hybridization. Mouse circumvallate sections were processed for in situ hybridization with antisense (B) and sense (C) probes to mouse IL-10. D, E, qRT-PCR analysis of IL-10 and GFP mRNA expression in IL-10-GFP mice. Relative expression levels (fold) in taste (TE) and nontaste (NT) lingual epithelia were analyzed using β-actin as the endogenous control gene. Expression levels in nontaste samples were designated as 1. Data are mean ± SD; **p = 0.0002 in D; **p = 0.001 in E (unpaired two-tailed t test, df = 4, n = 3 experiments). F, Colocalization of GFP fluorescent signal (green) with IL-10 antisense probe in situ hybridization (pseudocolored red) on a circumvallate section from IL-10-GFP mice. Two taste buds are shown (circled areas). G–O, IL-10 expression was detected by intrinsic fluorescence of GFP (white) in IL-10-GFP mice in taste papillae (G–I) and other tissues (J–O). Strong GFP signals were observed in circumvallate papillae (CV; G), foliate papillae (Fo; H), and fungiform papillae (FF; I), as well as in the spleen, an immune organ (J), which was used as a positive control. Weak but detectable levels of GFP signals were also observed in some cells of the colon epithelium (K, arrows). No GFP expression was observed in the kidney (L), heart (M), liver (N), or brain (O). Scale bars: G–K, 20 μm; L–O, 40 μm.

Figure 2.

Identification of IL-10-producing cells in taste buds. Confocal images of intrinsic fluorescence of GFP (green) and immunofluorescent staining of different taste cell-type markers (red) on circumvallate papillae sections from IL-10-GFP mice. A, Immunostaining of type I taste cell marker ENTPDase2 (red), showing no GFP expression in type I taste cells. B, Immunostaining of type II taste cell marker PLC-β2 (red), showing GFP expression in type II taste cells. Note that all GFP-positive cells express PLC-β2, but some PLC-β2-positive cells do not express IL-10. C, D, Immunostaining of type III taste cell markers CA4 (red) and PKD2L1 (red) showing no GFP expression in type III taste cells. Five mice were included in each experimental group. Scale bars: 30 μm.

Figure 3.

Identification of a subpopulation of type II taste cells that express IL-10 in mouse taste buds. Confocal images of intrinsic fluorescence of GFP (green) and immunofluorescent staining of different type II taste cell markers (red) on circumvallate papillae sections from IL-10-GFP mice. A, Immunostaining of gustducin (Gust; red), showing GFP is coexpressed with gustducin in type II taste cells. B, Immunostaining of T1R3 (red), showing no coexpression of GFP and T1R3 in taste cells. Scale bars: 35 μm. C, Venn diagrams illustrate colocalization of IL-10 (represented by GFP signals) with gustducin (Gust), but not with T1R3 in circumvallate sections of IL-10-GFP mice.

Figure 4.

IL-10 and TNF are produced by different subsets of type II taste cells. Confocal images of intrinsic fluorescence of GFP (green) and immunofluorescent staining of TNF (red) on tissue sections of taste papillae (A, B) and spleen (C) of IL-10-GFP mice. Both TNF and IL-10 (represented by GFP expression) were highly expressed in circumvallate (A) and foliate papillae (B), but no clear coexpression of IL-10 and TNF was observed, indicating separate cellular origins of these two cytokines in the taste bud. In contrast to taste tissues, most GFP-positive cells in the spleen coexpressed TNF (C), and only a small proportion of cells in the spleen expressed either IL-10 or TNF. Scale bars: A, B, 25 μm; C, 40 μm.

Figure 5.

Increased expression of IL-10 following repeated administration of SEA. IL-10-GFP mice were injected with SEA or PBS three times with 2 d intervals. IL-10 and GFP mRNA expression in taste tissues was determined by qRT-PCR. GFP fluorescence in circumvallate papillae was detected using confocal microscopy. The amount of IL-10 in the blood was measured by ELISA. A, B, qRT-PCR analysis of IL-10 and GFP mRNA expression in taste epithelium containing circumvallate and foliate taste buds from IL-10-GFP mice injected with SEA or PBS. β-Actin served as the endogenous control gene for relative quantification. Data are mean ± SD; **p = 0.002 in A; **p = 0.007 in B (unpaired two-tailed t test, df = 4, n = 3 experiments). C, D, GFP expression in circumvallate taste buds of PBS-injected control mice (C) and SEA-injected mice (D) detected by intrinsic fluorescence of GFP (green). Scale bars: 40 μm. E, F, Changes in GFP- and gustducin-positive (Gust) cell counts in circumvallate taste buds of IL-10-GFP mice after PBS (E) or SEA (F) injections, expressed as the percentage of each cell type. G, Average intensities of intrinsic fluorescence of GFP of circumvallate taste buds from SEA- or PBS-treated mice. *p = 0.029 (unpaired two-tailed t test, df = 5, n = 3–4 animals per group). H, Concentrations of IL-10 in the serum of PBS- and SEA-injected mice. Data are mean ± SD; *p = 0.041 (unpaired two-tailed t test, df = 5, n = 3–4 animals per group).

Figure 6.

LPS induces IL-10 expression and secretion in taste buds. A, Relative levels (fold) of IL-10 and GFP mRNA expression in taste epithelia of IL-10-GFP mice. LPS or PBS (vehicle control) was intraperitoneally injected into IL-10-GFP mice, and 3 h later IL-10 and GFP mRNA levels in taste epithelia containing circumvallate and foliate taste buds were determined using qRT-PCR. The expression level of mRNA in taste epithelia of PBS-injected mice was defined as 1. β-Actin served as the endogenous control gene for relative quantification. Data are mean ± SD; **p = 0.004 for IL-10; **p = 0.0002 for GFP (unpaired two-tailed t test, df = 4, n = 3 experiments). B, Production and secretion of IL-10 by taste epithelia upon LPS challenge in vitro. Taste epithelia (TE; containing circumvallate and foliate taste buds) and nontaste lingual epithelia (NT) were isolated from C57BL/6 mice and incubated in complete DMEM, with or without 5 μg ml⁻¹ LPS, for the time periods indicated. Cultures with no LPS in the medium served as controls. Concentrations of IL-10 in the supernatant of the cultured tissues were measured using ELISA. IL-10 was detected in the supernatant of cultured taste epithelium treated with LPS, but was below the detection limit in all other samples. Three mice were used for each treatment, and all the collected samples were assayed in duplicate for each experiment. The results are representative of three experiments. Data are mean ± SD; *one-way ANOVA: F = 6.88, p = 0.018; post hoc t test: p = 0.04 (TE LPS vs NT LPS).

Figure 7.

Identification of IL-10R1-expressing cells in taste buds. A, Confocal fluorescent images of circumvallate sections stained with no specific primary antibody (No Primary), affinity-purified rabbit antibody against IL-10R1 (IL-10R1), or antibody against IL-10R1 pre-incubated with the peptide used for antibody production (Ag Blocking). A DyLight 649-conjugated donkey anti-rabbit secondary antibody was used for the experiment. Specific staining of taste bud cells was seen only in IL-10R1 antibody staining (middle). B–E, Confocal images of double immunofluorescent staining of IL-10R1 (red) with different taste cell-type markers (green) on foliate papillae sections of C57BL/6 mice. B, Immunostaining of IL-10R1 (red) and type II taste cell marker PLC-β2 (green), showing IL-10R1 expression in type II taste cells. C, D, Immunostaining of IL-10R1 (red) and taste cell marker gustducin (Gust) or T1R3 (green), showing more selective IL-10R1 expression in T1R3-positive type II taste cells. E, Immunostaining of IL-10R1 (red) and type III taste cell marker CA4 (green), showing no IL-10R1 expression in type III taste cells. F, Confocal images of intrinsic fluorescence of GFP (green) and immunofluorescent staining of IL-10R1 (red) in IL-10-GFP mice, showing largely nonoverlapping expression of IL-10R1 and GFP in foliate taste buds. G, IL-10R2 expression in taste and nontaste lingual epithelia. Left, DNA products from RT-PCR experiments showing RNA expression of IL-10R2 and β-actin in nontaste lingual epithelium (NT) and in taste epithelia containing circumvallate and foliate taste buds (CV-F). Right, Western blots using antibodies against IL-10R2 and β-actin showing protein expression of IL-10R2 and β-actin in nontaste lingual epithelium (NT), in taste epithelium containing fungiform taste buds (FF), and in taste epithelia containing circumvallate and foliate taste buds (CV-F). Five mice per group were included in the immunostaining experiments. Scale bars: 30 μm.

Figure 8.

Structural defects in taste buds of IL-10-knock-out mice. A–C, Reduction in the number and size of taste buds in IL-10-knock-out (IL-10^−/−) mice compared with wild-type mice. A, Immunofluorescent staining of KCNQ1 (red) showing taste buds in typical circumvallate sections from wild-type and IL-10^−/− mice. DAPI staining (blue) shows nuclei. Scale bars: 100 μm. B, The number of circumvallate taste buds (TB) in IL-10^−/− mice was significantly reduced compared with that in wild-type (WT) control mice; *p = 0.034 (df = 7, N = 4–5 animals per group). C, The average area of TB profiles from IL-10^−/− mice was significantly decreased; **p = 0.0038 (df = 7, N = 4–5 animals per group). D, The average number of taste cells per taste bud profile was significantly reduced in IL-10^−/− mice; *p = 0.0241 (df = 9, n = 5–6 animals). E–G. Decreased numbers of type II (gustducin labeled or T1R3 labeled) and type III (CA4 labeled) taste receptor cells in IL-10^−/− mice compared with those in WT control mice. The circumvallate taste tissues were immunostained with antibodies to gustducin, T1R3, and CA4, and the numbers of specifically stained cells in each taste bud with typical morphology were counted and averaged. At least five taste buds were selected from each section; **p = 0.001 (E, the number of gustducin-expressing cells); *p = 0.047 (F, the number of T1R3-expressing taste cells); **p = 0.002 (G, the number of CA4-expressing taste cells). Data were analyzed using unpaired two-tailed t test; df = 7, N = 4–5 animals per group. Data are mean ± SD.

Figure 9.

T-cell infiltration and TNF responses in taste tissues of IL-10-knock-out mice. A, CD3⁺ T-cells in the taste epithelium (TE) and lamina propria (LP) of circumvallate papillae from wild-type and IL-10-knock-out (IL-10^−/−) mice. T-cells were labeled by an anti-CD3 antibody (brown). B, Quantitative analysis of T-cell populations in the TE and LP of wild-type (WT) and IL-10^−/− mice. T-cell populations in TE and LP were significantly increased in IL-10^−/− mice compared with WT mice. Data are mean ± SD; **one-way ANOVA multiple-comparisons test: F = 14.5, p = 0.0001; post hoc t test: p = 0.004 (IL-10^−/− TE vs WT TE); p = 0.0018 (IL-10^−/− LP vs WT LP, df = 8, n = 5 animals). C, D, qRT-PCR analyses of TNF mRNA expression in taste epithelia of WT and IL-10^−/− mice after LPS stimulation. LPS (0.5 mg/kg body weight) or PBS (vehicle control) was injected intraperitoneally into WT and IL-10^−/− mice, and circumvallate- and foliate-containing taste epithelia were collected 16 h (C) or 5 d (D) later for TNF mRNA analysis. β-Actin served as the endogenous control gene for relative quantification. Data are mean ± SD; **one-way ANOVA multiple-comparisons test: F = 148.2, p = 0.0001 (C); F = 38.36, p = 0.007 (D); post hoc t test: p = 0.0035 (C, WT LPS vs WT PBS); p < 0.0001 (C, IL-10^−/− LPS vs IL-10^−/− PBS); p = 0.0016 (C, IL-10^−/− LPS vs WT LPS); p = 0.001 (D, IL-10^−/− LPS vs IL-10^−/− PBS, df = 4, n = 3 experiments).

Figure 10.

Proposed model of cell–cell interactions through IL-10 and TNF signaling among different types of taste cells. A, Circumvallate and foliate taste buds have two subsets of type II cells, based on the differential expression of TNF and IL-10, which also coincide with the differential expression of T1R3 and gustducin. The first subset (type IIa) is characterized by the expression of TNF and T1R3, and the second subset (type IIb) features the expression of IL-10 and gustducin. In addition, TNFRs are ubiquitously expressed in taste buds, and their expression has been detected in type I, IIa, IIb, and III cells. IL-10 receptor (IL-10R), however, is predominantly expressed in type IIa cells. These expression patterns suggest that type IIa cells (sweet and umami receptor cells) produce TNF, which can act on all taste cells via TNFR, and type IIb cells (mostly bitter receptor cells) produce IL-10, which selectively acts on type IIa cells to downregulate TNF production. This signaling paradigm indicates a novel mechanism of cell–cell communication in taste buds and also explains the general protective effects of IL-10 in taste buds. B, Summary of expression of IL-10, IL-10R, TNF, TNFR, T1R3, and gustducin (Gust) in different types of circumvallate and foliate taste cells. +, expressed; −, no detectable expression; −*, not expressed in most of the cells; ND, not determined.