Detection of Succinate by Intestinal Tuft Cells Triggers a Type 2 Innate Immune Circuit

Abstract

In the small intestine, type 2 responses are regulated by a signaling circuit that involves tuft cells and group 2 innate lymphoid cells (ILC2s). Here, we identified the microbial metabolite succinate as an activating ligand for small intestinal (SI) tuft cells. Sequencing analyses of tuft cells isolated from the small intestine, gall bladder, colon, thymus, and trachea revealed that expression of tuft cell chemosensory receptors is tissue specific. SI tuft cells expressed the succinate receptor (SUCNR1), and providing succinate in drinking water was sufficient to induce a multifaceted type 2 immune response via the tuft-ILC2 circuit. The helminth Nippostrongylus brasiliensis and a tritrichomonad protist both secreted succinate as a metabolite. In vivo sensing of the tritrichomonad required SUCNR1, whereas N. brasiliensis was SUCNR1 independent. These findings define a paradigm wherein tuft cells monitor microbial metabolites to initiate type 2 immunity and suggest the existence of other sensing pathways triggering the response to helminths.

Keywords: ILC2; chemosensing; helminth; nippostrongylus brasiliensis; protist; small intestine; succinate; tritrichomonas; tuft cell; type 2 immunity.

Figure 1. RNA-Seq identifies a transcriptional tuft cell signature and a tissue-specific chemosensory receptor repertoire

(A–I) Tuft cells (CD45^lo EPCAM⁺ Flare25⁺) were sorted from small intestine (SI), gall bladder (Gall), colon (Col), trachea (Tra), and thymus (Thy) of naïve B6.Il25^{Flare25/Flare25} mice for mRNA sequencing. Non-tuft epithelial cells of the small intestine (SI Epi; CD45^lo EPCAM⁺ Flare25⁻) were sorted as a negative control. (A) Principle component analysis of gene expression. (B) Hierarchical clustering of differentially expressed genes (fold change > 8; FDR < 0.01) among tuft cell subsets. (C) Ranked list of a tuft cell transcriptional signature (Log₂ fold-change >4 in all tuft cells relative to SI Epi). (D–F) Normalized read count of indicated genes. G) Heat map of normalized read count of all taste receptors. (H–I) Normalized read count of indicated genes. (J) Indicated genes analyzed by RT-qPCR in small intestinal tuft cells (Flare25⁺) and non-tuft epithelial cells (Flare25⁻). A–I: biological replicates from one mRNA sequencing experiment. J: biological replicates pooled from three experiments. In A–I *, FDR < .05; ***, FDR < .001 by statistical analysis in RNAseq pipeline. In J *, p < 0.05; ***, p < 0.001 by multiple t-tests. n.s., not significant; n.d., not detectable. Graphs show mean + SEM. Also see Figure S1.

Figure 2. Succinate is sufficient to induce a type 2 immune response in the small intestine

(A–J) Unless otherwise indicated, wild-type mice were given 150 mM succinate or control water for 7 d. (A) Representative images of distal (last 10 cm) small intestine (SI). DCLK1 marks tuft cells. Scale bar = 50 μm (B) Quantification of tuft cells in A. (C) Tuft cell quantification by microscopy in the proximal (first 10 cm) and distal SI (D–E) Tuft cell quantification in the distal SI at indicated (D) succinate concetrations and (E) timepoints. (F) Representative images of middle (10–20 cm from cecum) SI stained with periodic acid-Schiff to visualize goblet cells. Scale bar = 50 μm. (G–H) Quantification of goblet cell (G) numbers and (H) hypertrophy. (I–J) Absolute numbers of (I) ILC2s and (J) eosinophils quantified in MLN by flow cytometry. (K) Total worm burden in wild-type mice that received 7 d treatment with 150mM succinate or water control prior to and during infection with N. brasiliensis. Worm burden represented as relative to median of control within each experiment. (L) Tuft cell quantification in distal SI of germ-free mice treated as in A–J. In B–D, G–L each symbol represents an individual mouse from at least three pooled experiments. In E, each symbol represents the average of 3–9 mice pooled from three experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 by one-way ANOVA with comparison to control (B, D), by Mann-Whitney (G–J, L), or by multiple t-test (C, K). Graphs depict mean + SEM. Also see Figure S2.

Figure 3. Succinate signals via the tuft cell-ILC2 circuit in a TRPM5 and SUCNR1-dependent manner

(A) Schematic of cells and proteins in the tuft-ILC2 circuit. (B–J Mice of indicated genotypes were given 150 mM succinate for 7 d. (B) Representative images of distal (last 10 cm) small intestine (SI). DCLK1 marks tuft cells. Scale bar = 50 μm. (C) Quantification of tuft cells in B. (D) Representative images of middle (10–20 cm from cecum) SI stained with Alcian blue to visualize goblet cells. Scale bar = 100 μm. (E–F) Quantification of goblet cell (E) numbers and (F) hypertrophy in D. (G–J) MLN analyzed by flow cytometry to quantify (G) ILC2s, (H) eosinophils, and (I–J) IL-13 production by ILC2s. Smart13: IL-13 reporter. (K–L) Lamina propria cells from mice of indicated genotypes given 150 mM succinate for 36 hours and analyzed as in I–J. In C–H, J–L each symbol represents one mouse from at least two pooled independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 by one-way ANOVA (C, G–H, J, L) with comparison to Wt(B6) or untreated Smart13 control, or by Mann-Whitney (E–F). n.s., not significant. Graphs depict mean + SEM. Also see Figure S3.

Figure 4. Immune sensing of T. rainier but not N. brasiliensis requires SUCNR1

(A) Concentration of indicated molecules measured in N. brasiliensis excretory-secretory product (NES) or media control by NMR. (B) Representative calcium fluxes in wild-type (B6) or SUCNR1-transduced MEFs treated as indicated. (C) Quantification of B. (D) Concentrations of indicated molecules measured in T. rainier conditioned media or media alone by NMR. (E–I) Mice of indicated genotypes were infected with N. brasiliensis. (E) Tuft cell quantification in the distal (last 10 cm) small intestine (SI), and (F) total worm burden at indicated times. (G) Tuft cell quantification in the distal SI 7 d post infection. (H) Tuft cell quantification in the distal SI and (I) total worm burden 8 d post infection. (J–M) Mice of indicated genotypes were colonized with T. rainier for 7 d or left untreated. (J) Representative images of distal SI. DCLK1 marks tuft cells. Scale bar = 50 μm. (K) Quantification of tuft cells in J. (L–M) MLN analyzed by flow cytometry to quantify (L) ILC2s and (M) eosinophils. A–B, D show representative data from at least two independent experiments. In C, each symbol represents one technical replicate. In G–I, K–M each symbol represents one mouse from at least two pooled experiments. In E–F each symbol represents the average of 3–10 mice from three pooled experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 by one-way ANOVA (H–I, K–M) with comparison to Wt(B6) control, by Mann-Whitney (G), or using multiple t-tests (C, E, F). n.s., not significant. Graphs depict mean + SEM. Also see Figure S4.