A Metabolite-Triggered Tuft Cell-ILC2 Circuit Drives Small Intestinal Remodeling

Abstract

The small intestinal tuft cell-ILC2 circuit mediates epithelial responses to intestinal helminths and protists by tuft cell chemosensory-like sensing and IL-25-mediated activation of lamina propria ILC2s. Small intestine ILC2s constitutively express the IL-25 receptor, which is negatively regulated by A20 (Tnfaip3). A20 deficiency in ILC2s spontaneously triggers the circuit and, unexpectedly, promotes adaptive small-intestinal lengthening and remodeling. Circuit activation occurs upon weaning and is enabled by dietary polysaccharides that render mice permissive for Tritrichomonas colonization, resulting in luminal accumulation of acetate and succinate, metabolites of the protist hydrogenosome. Tuft cells express GPR91, the succinate receptor, and dietary succinate, but not acetate, activates ILC2s via a tuft-, TRPM5-, and IL-25-dependent pathway. Also induced by parasitic helminths, circuit activation and small intestinal remodeling impairs infestation by new helminths, consistent with the phenomenon of concomitant immunity. We describe a metabolic sensing circuit that may have evolved to facilitate mutualistic responses to luminal pathosymbionts.

Keywords: A20; IL-25; ILC2s; TRPM5; Tritrichomonas; concomitant immunity; helminths; succinate; succinate receptor; tuft cells.

Figure 1. Constitutive high A20 expression in small intestine ILC2s restrains IL-25-mediated expansion of the tuft cell – ILC2 circuit

(A–C) IL-17RB expression measured by qPCR (A) or flow cytometry (B and C) in ILC2s from indicated tissues. (D) A20 expression measured by qPCR in sorted ILC2s from indicated tissues, normalized to BM. (E and F) Presence of ILC2s (E) and tuft cells (F) in the small intestine (SI) of RR and A20^flRR mice, visualized by expression of IL-5 (Red5 reporter) in red (E) and DCLK1 in green (F); DAPI in blue. (G and H) Quantification of tuft cells in SI by flow cytometry. (G) Tuft cell frequencies in proximal and distal SI of RR and A20^flRR mice. (H) Percentages of tuft cells in SI of RR, A20^flRR mice, Il25^−/−A20^flRR mice and Il4ra^−/−A20^flRR mice. (I) Presence of ILC2s and tuft cells in SI as in (E and F) from mice as in (H). (J) Flow cytometry analysis of IL-13 (Sm13 reporter) and IL-5 (Red5 reporter) expression by ILC2s gated on CD45+Lin-IL-17RB+KLRG1+ cells isolated from the SI of R+, Il25^−/−A20^flR+ and Il4ra^−/−A20^flR+ mice. (K) Percentages of IL-13+ ILC2s among CD45+ lamina propria cells as in (J). Data pooled from multiple independent experiments (A–D, G, H, K) or from one experiment representative of at least two independent experiments (E, F, I, J). Scale bars; 100 μm. *p < 0.05, ***p < 0.001, ****p < 0.0001; ns, not significant by Mann-Whitney U or one-way ANOVA.

Figure 2. Energy balance in A20^flRR mice is associated with an elongated small intestine

(A, B) Quantification of secretory cells by immunofluorescence analysis of RR and A20^flRR mice; secretory cell types in red identified by staining for Muc2 (goblet cells), DCLK1 (tuft cells), ChromograninA (ChgA, enteroendocrine cells) and lysozyme (Paneth cells), EpCAM in green, DAPI in blue. (C–F) Metabolic parameters of R+ and A20^flR+ mice. (C) Oxygen consumption rate measured using CLAMS cages. (D) Food intake during a 4 day period. (E) Lean and adipose weight determined by EchoMRI. (F) Fecal energy content measured using bomb calorimetry. (G and H) Small intestine (SI) length (G) and representative image (H) of 8–12 week old R+, A20^flR+, RR and A20^flRR mice. The black bar indicates difference in SI length. Scale bars; 100 μm. Data from one experiment representative of at least three independent experiments (A–E, G, H) or from one experiment (F). (E, mean and s.e.m., n = 10–11). **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant by Mann-Whitney U or one-way ANOVA.

Figure 3. Activation of the tuft cell – ILC2 circuit drives small intestine lengthening

(A) Small intestine (SI) length in RR, A20^flRR, Il25^−/−A20^flRR, Il4ra^−/−A20^flRR and Pou2f3^−/−A20^flRR mice. (B–E) SI length in (B) wild type, (C) C57BL/6 GF, (D) Il4ra^−/− and (E) Pou2f3^−/− mice after 4 weeks of serial treatment with rIL-25. (F) SI length of mice 5 weeks after hydrodynamic gene delivery with IL-13 or hIgG1 control plasmid. (G) SI length of mice treated with rIL-4 complexes for 4 weeks. (H) Cytospins stained with H&E. Micrographs show representative images of Tritrichomonas isolated from cecum of mice housed in UCSF vivarium. Scale bars; 20 μm. (I and J) Wild type (WT) mice were colonized with purified Tritrichomonas at 3 weeks of age. (I) SI length measured 8 weeks later and compared to age-matched Pou2f3^−/− mice naturally colonized with Tritrichomonas. (J) SI length of wild type mice in (I) plotted against tuft cell frequency. (K) SI length in wild type mice 28 days after infection with H. polygyrus (H.p.). (L and M) Crypt fission in the proximal SI in Lgr5-CreERT2-EGFP x R26R-Confetti mice as described in methods. (L) Representive images from mice 28 days after infection with H. polygyrus. Lgr5 stem cells are pseudocolored in dim yellow (Lgr5-CreERT2-EGFP) and clonal crypts are randomly marked with bright yellow (YFP), red (RFP) or blue (CFP), driven from the R26R-Confetti locus. (M) Quantification of crypt fission of mice as in (L), or after 4 weeks of serial treatment with rIL-25. Scale bars; 200 μm. Data pooled from multiple independent experiments (A, C, D, F, M) or from one experiment representative of at least two independent experiments (B, E, G–I, L). (L, mean and s.e.m, n = 6–11). **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant by Mann-Whitney U or one-way ANOVA.

Figure 4. Weaning leads to IL-25-dependent activation of the tuft cell – ILC2 small intestinal circuit

(A and B) Small intestine (SI) length (A) and tuft cell frequencies (B) of RR and A20^flRR mice at 2 and 4 weeks of age. (C) Expression of IL-17RB and IL-5-reporter (Red5) by cells gated on Lin- CD45+ cells isolated from SI of RR and Il25^−/−RR mice at days 12 and 40 post-birth (p). (D) MFI of Red5 in ILC2s gated on Lin-CD45+IL-17RB+ cells of mice as in (C). (E and F) Frequencies of tuft cells (E) based on expression of DCLK1 or IL-25-reporter and of IL-13+ (Sm13 reporter) ILC2s (F) gated on CD45+Lin-IL-17RB+ cells quantified in SI by flow cytometry at the indicated time points post-birth (p). Data pooled from multiple independent experiments (A, B, E, F) or from one experiment representative of at least two independent experiments (C and D). Student’s t test was performed. (D–F, mean and s.e.m, n = 3–5). Statistical significance is indicated by *p < 0.05; ns, not significant.

Figure 5. Dietary fibers facilitate Tritrichomonas colonization after weaning

(A and B) The small intestine (SI) was isolated from GF mice, or wild-type mice that were offspring of JAX mice, which were bred without further manipulation (JAX) or after colonization with Tritrichomonas isolated from cecal content of mice in UCSF vivarium (UCSF). Expression of KLRG1 and IL-17RB by cells gated on Lin- CD45+ cells (A) and frequencies of tuft cells (B) analyzed by flow cytometry. (C) Adult IL-13-reporter (Sm13) mice fed bovine milk as the lone dietary constituent for 2 weeks and IL-13 expression by ILC2s in SI quantified by flow cytometry. (D and E) A20^flRR mice fed bovine milk as the lone dietary constituent following weaning for 4 weeks and frequencies of tuft cells (D) and SI length (E) were measured. (F) Abundance of Tritrichomonas in cecal content of WT mice at 3 and 5 weeks post-birth quantified by qPCR. (G) Adult IL-13-reporter (Sm13) mice treated with metronidazole (MNZ) in drinking water for 2 weeks and IL-13 expression by ILC2s analyzed as in (C) and compared to the same control group. (H and I) A20^flRR mice treated with metronidazole in drinking water starting at weaning and analyzed 6–8 weeks later. Tritrichomonas-free A20^flRR mice established as described in methods. The frequencies of tuft cells (H) and SI length (I) measured as in (D, E) and compared to the same control groups. (J and K) Tritrichomonas-free R+ and A20^flR+ mice colonized with Tritrichomonas after weaning and analyzed 7 days later. Expression of IL-5 (Red5) and IL-13 (Sm13) by ILC2s and CD3⁺CD4⁺ T cells in SI quantified by flow cytometry (J) and the frequencies of IL-13⁺ ILC2s shown (K). (L) WT mice fed chow or purified diets containing no fiber, 10% cellulose or 10% inulin. One day later mice were gavaged with 10⁴ purified Tritrichomonas and the abundance of Tritrichomonas in the cecum analyzed after 14 days by qPCR. (M) A20^flRR mice fed chow or purified diets containing 10% cellulose or 10% inulin after weaning for 6 weeks and the SI length measured. Data from one experiment representative of at least two independent experiments (A, C, G, J–M) or pooled from multiple independent experiments (B, D–F, H, I). F, mean and s.e.m.; n = 5–6. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant by Mann-Whitney U or one-way ANOVA.

Figure 6. Succinate is produced by Tritrichomonas and sufficient to activate the tuft cell – ILC2 circuit

(A and B) GF mice monocolonized with Tritrichomonas and concentrations of acetate (A) and succinate (B) measured in the cecal content after 6 weeks. (C) Metabolite receptor mRNA expression quantified by qPCR in tuft cells versus other epithelial cells sorted from small intestine (SI) and normalized to levels in non-tuft epithelial cells. (D–F) Tritrichomonas-free IL-13-reporter (Sm13) mice were treated with succinate, acetate or a SCFA mix (acetate, butyrate, propionate) in drinking water for 4 days. Expression of Ki-67 and IL-13 by ILC2s in SI quantified by flow cytometry and representative dot plot from wild-type (WT) mice shown (D). Expression of Ki-67 (E) and IL-13 (F) by ILC2s from WT, Pou2f3^−/−, Il25^−/− and Trpm5^−/− mice treated with indicated solutions. (G) Tritrichomonas-free WT mice treated with 100 mM succinate in drinking water for 10 days. Frequencies of tuft cells in SI by flow cytometry. (H and I) Tritrichomonas-free A20^flR+ and Il25^−/−A20^flR+ mice treated with 100 mM succinate in drinking water for 25 days and frequencies of tuft cells (H) and SI length (I) analyzed. Data from one experiment (A, B) or from one experiment representative of at least two independent experiments (C, D, G–I) or pooled from multiple independent experiments (E, F). C, mean and s.e.m.; n = 3. **p < 0.01, ****p < 0.0001; ns, not significant by Mann-Whitney U or one-way ANOVA.

Figure 7. The tuft cell – ILC2 circuit contributes to concomitant immunity

(A) Small intestinal (SI) tuft cell frequencies in indicated mice with presence (+) or absence (−) of Tritrichomonas as indicated. Some data is repeated from previous figures for comparison. Wild-type (WT) mice as in Fig. 5: JAX, offsprings of mice from JAX; UCSF, offsprings of mice colonized with Tritrichomonas. A20^flR pooled from A20^flR+ (open circle) and A20^flRR (closed circle) mice. WT mice analyzed at indicated time points after infection with H. polygyrus (H.p.) or N. brasiliensis (N.b.). (B and C) Adult R+ and A20^flR+ mice treated with metronidazole (MNZ) in drinking water for 2 weeks and frequencies of tuft cells (B) and SI length (C) analyzed. (D) WT mice serially treated with rIL-25 or infected with H. polygyrus (H.p.) for 4 weeks followed by worm clearance (cleared) in indicated mice using pyrantel pamoate. SI length measured 2 months after the last rIL-25 administration or worm clearance. (E and F) Worm burden (E) and Alcian blue/PAS staining (F) in SI of RR and A20^flRR mice 5 days after infection with N. brasiliensis. (G) SI worm counts of indicated mouse strains 13 days after infection with H. polygyrus. Presence (+) or absence (−) of Tritrichomonas indicated. One group of Tritrichomonas-free A20^flRR mice was colonized with Tritrichomonas 2 weeks before the infection. Data pooled from multiple independent experiments (A–E) or from one experiment representative of at least two independent experiments (F, G).*p < 0.05, **p < 0.01, ****p < 0.0001; ns, not significant by Mann-Whitney U or one-way ANOVA.