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. 2018 May 22;115(21):5552-5557.
doi: 10.1073/pnas.1720758115. Epub 2018 May 7.

Activation of intestinal tuft cell-expressed Sucnr1 triggers type 2 immunity in the mouse small intestine

Weiwei Lei  1 , Wenwen Ren  1 , Makoto Ohmoto  1 , Joseph F Urban Jr  2 , Ichiro Matsumoto  1 , Robert F Margolskee  1 , Peihua Jiang  3
Affiliations

Activation of intestinal tuft cell-expressed Sucnr1 triggers type 2 immunity in the mouse small intestine

Weiwei Lei et al. Proc Natl Acad Sci U S A. .

Abstract

The hallmark features of type 2 mucosal immunity include intestinal tuft and goblet cell expansion initiated by tuft cell activation. How infectious agents that induce type 2 mucosal immunity are detected by tuft cells is unknown. Published microarray analysis suggested that succinate receptor 1 (Sucnr1) is specifically expressed in tuft cells. Thus, we hypothesized that the succinate-Sucnr1 axis may be utilized by tuft cells to detect certain infectious agents. Here we confirmed that Sucnr1 is specifically expressed in intestinal tuft cells but not in other types of intestinal epithelial cells, and demonstrated that dietary succinate induces tuft and goblet cell hyperplasia via Sucnr1 and the tuft cell-expressed chemosensory signaling elements gustducin and Trpm5. Conventional mice with a genetic Sucnr1 deficiency (Sucnr1-/-) showed diminished immune responses to treatment with polyethylene glycol and streptomycin, which are known to enhance microbiota-derived succinate, but responded normally to inoculation with the parasitic worm Nippostrongylus brasiliensis that also produces succinate. Thus, Sucnr1 is required for microbiota-induced but not for a generalized worm-induced type 2 immunity.

Keywords: Sucnr1; Trpm5; gustducin; tuft cells; type 2 immunity.

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Conflict of interest statement

Conflict of interest statement: W.L. and P.J. have filed a patent application related to the effect of succinate on type 2 immunity.

Figures

Fig. 1.
Fig. 1.
Specific expression of the Sucnr1 receptor in intestinal tuft cells. (A) Fluorescence-activated cell-sorting–based isolation of tuft cells (Trpm5-GFP–positive cells; green) and nontuft epithelial cells (GFP-negative cells; red). GFP: excitation, 488 nm; emission, 513 nm. Autofluorescence: excitation, 561 nm; emission, 585 nm. Numbers indicate percentages of cells in that population. (B) Expression of Sucnr1 and other tuft cell marker genes (Dclk1, Gnat3, Il25, Plcb2, Trpm5) in Trpm5-GFP–positive tuft cells and GFP-negative nontuft cells (control) was determined by real-time quantitative PCR. Data (mean ± SEM) are biological replicates (n = 3). **P < 0.01, ***P < 0.001 (Student’s t test). (C) In situ hybridization shows the presence of Sucnr1-expressing cells in the small intestine (jejunum) in wild-type (WT) but not in Pou2f3−/− mice. [Scale bars, 100 µm (low-magnification images) and 20 µm (Inset).]
Fig. 2.
Fig. 2.
Dietary succinate induces type 2 immune responses. Jejunum tissues were collected from wild-type mice after consumption of water (H2O) or water containing 100 mM succinate or 100 mM monomethyl succinate. (A) Tissues were immunostained with anti-Dclk1 (green) and anti–E-cadherin (red) antibody and counterstained with DAPI (blue). (Scale bar, 50 μm.) (B) Dclk1-positive cells per mm villus (n = 12 for H2O, n = 8 for succinate, and n = 6 for MMS treatment). (C) Goblet cell staining with Alcian blue/nuclear red. (Scale bar, 50 μm.) (D) Goblet cells per mm villus (n = 7 for H2O and succinate). (E) Goblet cell size (n = 7 for H2O and n = 8 for succinate). (F) qPCR results show increased expression of tuft cell marker genes (Sucnr1, Dclk1, Gnat3, Il25, Plcβ2, Trpm5) (n = 5 for H2O, n = 6 for succinate, and n = 3 for MMS treatment). (G) qPCR result shows increased expression of Il13 after succinate treatment (n = 14 for H2O and n = 10 for succinate). All data are biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant (Student’s t test). Data are mean ± SEM.
Fig. 3.
Fig. 3.
Sucnr1 is required for succinate-induced type 2 immune responses. Jejunum tissues were collected from Sucnr1+/− and Sucnr1−/− mice after consumption of water or water containing 100 mM succinate. (A) The tissues were immunostained with anti-Dclk1 (green) and anti–E-cadherin (red) antibodies and counterstained with DAPI (blue). (Scale bar, 50 μm.) (B) Dclk1-positive cells per mm of villus (n = 6 for each group). (C) Goblet cell staining with Alcian blue/nuclear red. (Scale bar, 50 μm.) (D) Goblet cell number per mm villus (n = 6 for each group). (E) Goblet cell size (n = 6 for each group). All data are biological replicates. **P < 0.01, ***P < 0.001; NS, not significant (Student’s t test). Data are mean ± SEM.
Fig. 4.
Fig. 4.
Altered microbiome triggers tuft cell expansion in the distal ileum. Littermate or age-matched Sucnr1+/− and Sucnr1−/− mice were either treated with streptomycin (gavage) for 5 consecutive days and then the distal ileum and cecum were collected 2 d later, or provided with a drinking bottle containing 10% PEG3350 for 10 d and the distal ileum and cecum were collected at day 10. (A and B) Representative images of the distal ileum (dashed lines) and cecum of Sucnr1+/− (A) and Sucnr1−/− (B) mice immunostained with anti-Dclk1 antibody (red) and counterstained with DAPI (blue), showing tuft cell expansion in the distal ileum in Sucnr1+/− mice but not in Sucnr1−/− mice after treatment with either streptomycin (Middle) or PEG3350 (Bottom). Tissues from mice drinking only water were used as controls (Top). (A and B, Left) Low magnification. (A and B, Right) High magnification of boxed areas. (Scale bars, 100 μm.) (C) Immunohistochemical quantification of tuft cells (Dclk1+) cells in the distal ileum (n = 7 for Sucnr1+/− with H2O treatment and n = 6 for other groups). ****P < 0.0001; NS, not significant (Student’s t test). All data are biological replicates. Data are mean ± SEM.
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