Type II taste cells participate in mucosal immune surveillance

Abstract

The oral microbiome is second only to its intestinal counterpart in diversity and abundance, but its effects on taste cells remains largely unexplored. Using single-cell RNASeq, we found that mouse taste cells, in particular, sweet and umami receptor cells that express taste 1 receptor member 3 (Tas1r3), have a gene expression signature reminiscent of Microfold (M) cells, a central player in immune surveillance in the mucosa-associated lymphoid tissue (MALT) such as those in the Peyer's patch and tonsils. Administration of tumor necrosis factor ligand superfamily member 11 (TNFSF11; also known as RANKL), a growth factor required for differentiation of M cells, dramatically increased M cell proliferation and marker gene expression in the taste papillae and in cultured taste organoids from wild-type (WT) mice. Taste papillae and organoids from knockout mice lacking Spib (SpibKO), a RANKL-regulated transcription factor required for M cell development and regeneration on the other hand, failed to respond to RANKL. Taste papillae from SpibKO mice also showed reduced expression of NF-κB signaling pathway components and proinflammatory cytokines and attracted fewer immune cells. However, lipopolysaccharide-induced expression of cytokines was strongly up-regulated in SpibKO mice compared to their WT counterparts. Like M cells, taste cells from WT but not SpibKO mice readily took up fluorescently labeled microbeads, a proxy for microbial transcytosis. The proportion of taste cell subtypes are unaltered in SpibKO mice; however, they displayed increased attraction to sweet and umami taste stimuli. We propose that taste cells are involved in immune surveillance and may tune their taste responses to microbial signaling and infection.

Fig 1. RNAScope analysis of M cell marker gene expression in taste cells.

RNAscope Hiplex fluorescence assay was used to determine the coexpression of Spib (A-D), Gp2 (E-H), and Tnfrsf11a (I-L) with the taste cell markers Tas1r3, Gnat3, Trpm5, and Ddc in the CVP. Strong coexpression of Spib is observed with Tas1r3 and Trpm5 and less strong coexpression was observed with Gnat3 and Ddc. Gp2 tended to me more correlated with type II taste cells, while Tnfrsf11a expression is evenly distributed among all taste cell types. Scale bars = 10 μm.

Fig 2. SPIB is coexpressed with T1R3.

Double-labeled immunofluorescence confocal microscopy of CVP and FOP sections with antibodies against SPIB and type II taste cell markers TRPM5 (A, F), T1R3 (B, G), and GNAT3 (C, H), the type III taste receptor marker CAR4 (D, I), and the type I taste cell marker ENTPD2 (E, J) show frequent coexpression of SPIB with T1R3 and less frequently with TRPM5 and GNAT3 and negligible coexpression with CAR4 and ENTPD2. Nuclei are counterstained blue with DAPI. Scale bar, 50 μm.

Fig 3. Spib knockout mice have impaired immune responses.

(A-F) Indirect immunofluorescence confocal microscopy of CVP sections from WT and Spib^KO mice stained with antibodies against mouse immune cell markers CD45 (A, D), CD11B (B, E), and CD3 (C, F). Compared to WT mice, Spib^KO mice had fewer immune cells in the CVP. (G-H) Uptake of 200-nm diameter fluorescent beads in taste cells from CVP of WT (G) and Spib^KO mice (H) were observed using confocal microscopy. Taste cells are visualized by staining with a pan-taste cell marker KCNQ1 in panels A-H. (I) Uptake of beads was quantitated by image analysis and normalized so that the average uptake in WT mice was 1.0. (The data underlying the graphs can be found in Data I in S6_Data.) Compared to WT mice, Spib^KO mice took up fewer beads. ***p < .001. Scale bars, 50 μm.

Fig 4. Spib^KO mice show increased behavioral attraction to sweet and umami tastants.

Brief access tests were used to measure behavioral responses to sweet (sucrose and sucralose, A and B), umami (monopotassium glutamate [MPG], C), bitter (denatonium, D), salty (NaCl, E), and sour (citric acid, F) taste stimuli. (The data underlying the graphs can be found in Data A-F in S8_Data.) Compared to littermate control WT mice, Spib^KO mice show increased lick responses to sweet and umami (sucrose and MPG), while the responses to other taste stimuli are unchanged. Lick ratios were calculated by dividing the number of licks to a taste solution by the number of licks to water in each test session. Data are means ± SEM analyzed with two-way ANOVA with post hoc t test. N = 12 (WT mice) and 14 (Spib^KO mice). *p < .05, **p < .01.