Succinate Produced by Intestinal Microbes Promotes Specification of Tuft Cells to Suppress Ileal Inflammation

Abstract

Background & aims: Countries endemic for parasitic infestations have a lower incidence of Crohn's disease (CD) than nonendemic countries, and there have been anecdotal reports of the beneficial effects of helminths in CD patients. Tuft cells in the small intestine sense and direct the immune response against eukaryotic parasites. We investigated the activities of tuft cells in patients with CD and mouse models of intestinal inflammation.

Methods: We used microscopy to quantify tuft cells in intestinal specimens from patients with ileal CD (n = 19), healthy individuals (n = 14), and TNF^ΔARE/+ mice, which develop Crohn's-like ileitis. We performed single-cell RNA sequencing, mass spectrometry, and microbiome profiling of intestinal tissues from wild-type and Atoh1-knockout mice, which have expansion of tuft cells, to study interactions between microbes and tuft cell populations. We assessed microbe dependence of tuft cell populations using microbiome depletion, organoids, and microbe transplant experiments. We used multiplex imaging and cytokine assays to assess alterations in inflammatory response following expansion of tuft cells with succinate administration in TNF^ΔARE/+ and anti-CD3E CD mouse models.

Results: Inflamed ileal tissues from patients and mice had reduced numbers of tuft cells, compared with healthy individuals or wild-type mice. Expansion of tuft cells was associated with increased expression of genes that regulate the tricarboxylic acid cycle, which resulted from microbe production of the metabolite succinate. Experiments in which we manipulated the intestinal microbiota of mice revealed the existence of an ATOH1-independent population of tuft cells that was sensitive to metabolites produced by microbes. Administration of succinate to mice expanded tuft cells and reduced intestinal inflammation in TNF^ΔARE/+ mice and anti-CD3E-treated mice, increased GATA3⁺ cells and type 2 cytokines (IL22, IL25, IL13), and decreased RORGT⁺ cells and type 17 cytokines (IL23) in a tuft cell-dependent manner.

Conclusions: We found that tuft cell expansion reduced chronic intestinal inflammation in mice. Strategies to expand tuft cells might be developed for treatment of CD.

Keywords: IBD; epithelium; heterogeneity; metabolism.

Figure 1.. Tuft cell number is decreased in inflamed tissue with ileal inflammatory disease.

IF of tuft cell staining in human ilea of (A) healthy and (B) CD patients. Arrows denote co-expression. (C) IF quantification for tuft cells per crypt/villus. SEM for n = 11 normal and n = 14 CD patients. H&E (D) and (E-G) IF of cell markers of inflamed and uninflamed distal ilea from mice. (H-I) IF quantification for marker area normalized to Hoechst area per crypt. SEM for n = 4 mice for LYZ and 3 for MUC2. (J) IF quantification of DCLK1+ tuft cells in wildtype (n = 146 villi) and TNF^ΔARE/+ villi, by low- (n = 11), mid- (n = 125), and high-grade (n = 62) inflammation. p-value * < 0.05, ** < 0.01, *** < 0.001.

Figure 2.. ATOH1-independent tuft cell expansion is microbiome-dependent.

IF of cell markers in (A-B) AtohKO ilea under increasing doses of antibiotics (-, +, ++, +++), and (C-D) wildtype ilea under antibiotics and germ-free conditions. (E) IF of tuft cells in Lrig1^CreERT2/+;Atoh1^fl/fl small intestinal enteroids with or without 4-OHT to abate Atoh1, and with or without exogenous IL-13. (F) Fraction of enteroids with 0, 1–3, 4–10, 11+ tuft cells under different conditions.

Figure 3.. Trajectory analysis of scRNA-seq data supports alternative origins for ATOH1-dependent and -independent tuft cells.

(A-D) t-SNE analysis of scRNA-seq data generated from different murine models annotated with cell type cluster. n = 6 wildtype, 3 TNF^ΔARE/+, 2 antibiotic-treated wildtype, 3 AtohKO. (E) Tuft cell percentage quantification from scRNA-seq. SEM for replicates plotted. (F-I) Top scoring p-Creode topologies of scRNA-seq data with lineage annotation. Node size represents cell state density. (J-M) Quantification of n = 100 p-Creode topology maps for either non-secretory or secretory tuft cell placement. p-value * < 0.05, ** < 0.01.

Figure 4.. Analysis of AtohKO tuft cell gene expression identified upregulation in metabolic pathways.

(A) Heatmap of gene expression trends grouped into different dynamic trends as denoted in Supplementary Figure 11. (B) KEGG enrichment of genes that class switched from lower order to higher order in AtohKO, ordered by NES. (C-H) Representative TCA cycle-related gene trends over the tuft cell lineage pseudotime. Solid lines represent mean expression trends and dashed lines represent confidence intervals fitted to raw data from 10 top-scoring p-Creode topologies. Datapoints are scaled expression data. (I) Top 20 GSEA gene sets enriched in AtohKO tuft cell transcriptomes, along with (J-M) positive enrichment plots with highest NES. (N-S) Scaled TCA cycle gene expression in tuft cells. p-value * < 0.05, ** < 0.01, **** < 0.0001.

Figure 5.. Succinate production in the AtohKO small intestine drives ATOH1-independent tuft cell expansion.

(A-E) Mass spectrometric measurements of metabolites from the cecal lumen and tissue. SEM for n = 5 wildtype, 3 AtohKO, and 3 antibiotic-treated AtohKO animals. (F) Heatmap of z-score normalized PICRUSt category scores between wild type and AtohKO. Categories p-values are all < 0.05. (G) Relative abundance of genus contributing to “Chlorocyclohexane and chlorobenzene degradation” category. SEM for n = 4 wildtype and 3 AtohKO. (H) IF for cell markers of GF animals gavaged with wildtype or AtohKO contents, at 3-days or 7-days. (I) Raw and (J) normalized (to nucleus count) tuft cell number in untreated and oral-gavaged animals. Datapoints represent FOVs and SEM across multiple biological replicates calculated. p-value ** < 0.01, **** < 0.0001.

Figure 6.. Succinate administration enhances anti-parasitic immune response to counteract inflammation in TNF^ΔARE/+ animals.

(A) H&E of TNF^ΔARE/+ ilea with succinate. (B-C) Histopathological scoring of control (n = 5) and long-term succinate-treated (n = 7) TNF^ΔARE/+ mice. SEM plotted. (D-E) IF of immune cell markers. (F-G) IF quantification of immune cell types normalized to nuclei area. Each dot represents a FOV and SEM across n = 4 animals. (H-N) Luminex cytokine measurements from ileal tissues. SEM across multiple biological replicates calculated (circles: males, triangles: females). (O-P) IF of type 2 immune markers. (Q-S) IF quantification of nuclear GATA3+ cells in the lamina propria. (T-U) IF of type 17 immune markers. (V-X) IF quantification of nuclear RORGT+ cells in the lamina propria. Datapoints represent FOVs and SEM from multiple biological replicates calculated. p-value * < 0.05, ** < 0.01, *** < 0.001. **** < 0.0001.

Figure 7.. Tuft cells are necessary for succinate-mediated inflammation suppression.

(AB) IF of cell markers of TNF^ΔARE/+ ilea with succinate. White arrows mark DCLK1+ tuft cells. (C) Percentage body weight change in succinate treated animals in the anti-CD3E model. SEM across multiple biological replicates calculated. (D-G) H&E and IF of cell markers in anti-CD3E-treated mice with or without tuft cells and succinate. (H) IF quantification of MPO+ cells normalized to nuclei area. Datapoints represent FOVs and SEM across multiple biological replicates calculated. (I) Summary diagram. p-value ** < 0.01, *** < 0.001.