Lipopolysaccharide-induced inflammation attenuates taste progenitor cell proliferation and shortens the life span of taste bud cells

Abstract

Background: The mammalian taste bud, a complex collection of taste sensory cells, supporting cells, and immature basal cells, is the structural unit for detecting taste stimuli in the oral cavity. Even though the cells of the taste bud undergo constant turnover, the structural homeostasis of the bud is maintained by balancing cell proliferation and cell death. Compared with nongustatory lingual epithelial cells, taste cells express higher levels of several inflammatory receptors and signalling proteins. Whether inflammation, an underlying condition in some diseases associated with taste disorders, interferes with taste cell renewal and turnover is unknown. Here we report the effects of lipopolysaccharide (LPS)-induced inflammation on taste progenitor cell proliferation and taste bud cell turnover in mouse taste tissues.

Results: Intraperitoneal injection of LPS rapidly induced expression of several inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-6, in mouse circumvallate and foliate papillae. TNF-alpha and IFN-gamma immunoreactivities were preferentially localized to subsets of cells in taste buds. LPS-induced inflammation significantly reduced the number of 5-bromo-2'-deoxyuridine (BrdU)-labeled newborn taste bud cells 1-3 days after LPS injection, suggesting an inhibition of taste bud cell renewal. BrdU pulse-chase experiments showed that BrdU-labeled taste cells had a shorter average life span in LPS-treated mice than in controls. To investigate whether LPS inhibits taste cell renewal by suppressing taste progenitor cell proliferation, we studied the expression of Ki67, a cell proliferation marker. Quantitative real-time RT-PCR revealed that LPS markedly reduced Ki67 mRNA levels in circumvallate and foliate epithelia. Immunofluorescent staining using anti-Ki67 antibodies showed that LPS decreased the number of Ki67-positive cells in the basal regions surrounding circumvallate taste buds, the niche for taste progenitor cells. PCR array experiments showed that the expression of cyclin B2 and E2F1, two key cell cycle regulators, was markedly downregulated by LPS in the circumvallate and foliate epithelia.

Conclusions: Our results show that LPS-induced inflammation inhibits taste progenitor cell proliferation and interferes with taste cell renewal. LPS accelerates cell turnover and modestly shortens the average life span of taste cells. These effects of inflammation may contribute to the development of taste disorders associated with infections.

Figure 1

Systemic inflammation induced by LPS stimulates the expression of inflammatory cytokines and the chemokine MCP-1 in circumvallate and foliate epithelia. Quantitative real-time RT-PCR analysis of the expression of TNF-α, IFN-γ, IL-6, IL-12, MCP-1, and IL-1β in nontaste (NT) and circumvallate and foliate-containing (CV+F) lingual epithelia 6 h after intraperitoneal injection of PBS or LPS. Relative gene expression levels were shown (fold). Expression in nontaste epithelium from the PBS group was arbitrarily set to 1. β-actin was used as the endogenous control gene for relative quantification. Error bars represent SEM. * p < 0.05; ** p < 0.005.

Figure 2

TNF-α and IFN-γ immunoreactivity in the mouse circumvallate epithelium. Immunofluorescent staining of mouse circumvallate papillae taken 6 h after LPS-injection. A, Confocal fluorescent images of circumvallate sections stained with control normal rabbit IgG (Rb IgG), affinity-purified rabbit antibody against IFN-γ (IFN-γ), or antibody against IFN-γ pre-incubated with purified recombinant murine IFN-γ (Ag Blocking). Cy3-conjugated anti-rabbit secondary antibody was used. B, Confocal fluorescent images of circumvallate sections stained with control normal goat IgG (Gt IgG), affinity-purified goat antibody against TNF-α (TNF-α), or antibody against TNF-α pre-incubated with purified recombinant murine TNF-α (Ag Blocking). Alexa 488-conjugated anti-goat secondary antibody was used. C, D, Colocalization of IFN-γ (C) or TNF-α (D) immunoreactivity (red) with TrpM5-GFP-positive (green) taste bud cells (arrows). Cy3-conjugated anti-rabbit and Cy5-conjugated anti-goat secondary antibodies were used for IFN-γ and TNF-α immunostaining, respectively. Scale bars, 25 μm.

Figure 3

Turnover of BrdU-labeled cells in circumvallate epithelium of PBS- or LPS-treated mice. Circumvallate sections from mice injected with BrdU and either PBS or LPS were processed for immunofluorescent staining with antibodies against BrdU (green) and KCNQ1 (red). Representative confocal images (merged red and green channels) from both PBS and LPS groups are shown for selected time points. Scale bars, 75 μm.

Figure 4

LPS inhibits renewal and shortens average turnover period of taste bud cells. A, Time course of the number of BrdU-labeled taste cells per circumvallate taste bud profile of PBS- or LPS-treated mice. Solid lines indicate the time course curves. Dashed lines show where the numbers of BrdU-labeled taste cells reached half-maximum values for each group. The left and right numbers above the dashed lines are the average times for cells to enter the taste buds (2.5 days for the PBS group and 3 days for the LPS group) and the average taste bud cell turnover periods (12 days for the PBS group and 10 days for the LPS group). LPS treatment slowed the entry of taste bud cells and shortened their turnover period. B, Entry of BrdU-labeled cells into taste buds on days 1-3 after BrdU injection. LPS treatment decreased the number of BrdU-labeled cells per taste bud profile compared with controls. Error bars indicate SEM. * p < 0.05; ** p < 0.005.

Figure 5

The effects of LPS on perigemmal keratinocyte turnover. A, Time course of the number of BrdU-labeled perigemmal cells/mm² in circumvallate epithelium of PBS- or LPS-treated mice. Solid lines indicate the time course curves. Dashed lines show where the numbers of BrdU-labeled perigemmal cells reached half-maximum values for each group. The numbers above the dashed lines are the average turnover periods for the perigemmal keratinocytes in the circumvallate epithelium (4.2 days for the PBS group and 3.9 days for the LPS group). B, Average number of BrdU-labeled cells/mm² in the perigemmal region of circumvallate epithelium 1 day after BrdU injection. LPS treatment significantly decreased the number of BrdU-labeled perigemmal cells. Error bars indicate SEM. ** p < 0.005.

Figure 6

LPS inhibits taste progenitor cell proliferation. A, Quantitative real-time RT-PCR analysis of Ki67 expression in nontaste (NT) and circumvallate and foliate-containing (CV+F) lingual epithelia 24 h after intraperitoneal injection of PBS or LPS. β-actin was used as the endogenous control for relative quantification, and the expression level of Ki67 in nontaste epithelium of PBS-treated mice was arbitrarily set to 1. LPS strongly inhibited the expression of Ki67 in the circumvallate and foliate epithelia. B, The number of Ki67-labeled cells per taste bud profile in the circumvallate epithelium. Only the Ki67-labeled cells that were adjacent to a taste bud were counted (a few examples are indicated by arrows in C). LPS treatment significantly reduced the number of Ki67-labeled cells in the circumvallate epithelium. Error bars indicate SEM. * p < 0.05; ** p < 0.005. C, Confocal fluorescent images of circumvallate papillae stained with antibodies against KCNQ1 (red) and Ki67 (green). LPS treatment strongly reduced the staining intensity and the number of cells labeled with Ki67 antibody. D, Representative confocal images of Ki67 immunostaining of the nontaste lingual epithelium from PBS- or LPS-treated mice. Scale bars, 50 μm.

Figure 7

LPS-induced inflammation downregulates the expression of cyclin B2 and E2F1 in the circumvallate epithelium. A, Scatter plot summarizing gene expression analysis using the Mouse Cell Cycle RT² Profiler PCR Array. The expression of 84 genes involved in cell cycle regulation was analyzed by real-time RT-PCR in circumvallate and foliate epithelial samples prepared 24 h after PBS (x-axis) or LPS (y-axis) injection. Each circle represents a gene in the PCR array. The middle diagonal gray line indicates fold change of 1 (no change of gene expression between PBS and LPS groups). Circles above this gray line indicate increased gene expression, and circles below, decreased expression, in LPS samples versus PBS samples. The two outside gray lines indicate a fold change of 4. The five green circles represent genes that showed >4-fold decreased expression in LPS samples. B, C, Independent real-time RT-PCR expression analyses of cyclin B2 (B) and E2F1 (C) in circumvallate and foliate epithelia prepared 24 h after PBS or LPS injection. LPS significantly decreased the expression of both genes in circumvallate and foliate epithelia. Error bars indicate SEM. ** p < 0.005.