Activation of intestinal tuft cell-expressed Sucnr1 triggers type 2 immunity in the mouse small intestine Weiwei Leia,1, Wenwen Rena,1, Makoto Ohmotoa, Joseph F. Urban Jr.b, Ichiro Matsumotoa, Robert F. Margolskeea, and Peihua Jianga,2 aMonell Chemical Senses Center, Philadelphia, PA 19104; and bDiet, Genomics and Immunology Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, US Department of Agriculture, Beltsville, MD 20705 Edited by Robert J. Lefkowitz, Howard Hughes Medical Institute and Duke University Medical Center, Durham, NC, and approved April 19, 2018 (received for review November 28, 2017) The hallmark features of type 2 mucosal immunity include in- Expulsion of the intestinal worm parasite Nippostrongylus brasi- − − testinal tuft and goblet cell expansion initiated by tuft cell liensis from the gut is significantly delayed in Pou2f3 / mice activation. How infectious agents that induce type 2 mucosal compared with wild-type mice (8). The current model suggests tuft immunity are detected by tuft cells is unknown. Published micro- cells detect parasitic infection via taste-signaling elements and se- array analysis suggested that succinate receptor 1 (Sucnr1) is spe- crete the proinflammatory cytokine IL-25, which subsequently cifically expressed in tuft cells. Thus, we hypothesized that the triggers mucosal type 2 responses via IL-13–producing ILC2s and succinate–Sucnr1 axis may be utilized by tuft cells to detect certain IL-13 receptor alpha-expressing intestinal epithelial progenitor cells infectious agents. Here we confirmed that Sucnr1 is specifically (6–8, 17, 18). Given the importance of gustducin and Trpm5 for expressed in intestinal tuft cells but not in other types of intestinal parasite type 2 immunity, which are downstream transduction epithelial cells, and demonstrated that dietary succinate induces elements for G protein-coupled taste receptors (GPCRs) (19, tuft and goblet cell hyperplasia via Sucnr1 and the tuft cell- 20), we reasoned that activation of tuft cell-expressed GPCRs expressed chemosensory signaling elements gustducin and Trpm5. – −/− may cascade on the gustducin Trpm5 pathway and trigger type Conventional mice with a genetic Sucnr1 deficiency (Sucnr1 ) 2 immunity, resembling the response to parasitic helminth or MEDICAL SCIENCES showed diminished immune responses to treatment with polyeth- protozoan infections. ylene glycol and streptomycin, which are known to enhance Previous microarray analysis of tuft cells suggested that succi- microbiota-derived succinate, but responded normally to inocula- nate receptor 1 (Sucnr1), a GPCR with succinate as its cognate tion with the parasitic worm Nippostrongylus brasiliensis that also agonist (21), is expressed in tuft cells (11). As a metabolite of the produces succinate. Thus, Sucnr1 is required for microbiota-induced Krebs cycle and microbial propionate synthesis, succinate is known butnotforageneralizedworm-inducedtype2immunity. to be secreted by certain parasites, bacteria, and inflamed tis- sues in pathological conditions (22–29). Thus, we hypothesized Sucnr1 | type 2 immunity | tuft cells | Trpm5 | gustducin that the succinate–Sucnr1 axis is utilized by tuft cells to detect infectious agents. he intestinal epithelium comprises mature cells of two major lineages: absorptive enterocytes and secretory cells (1). Most T Significance mature differentiated cells in the intestinal epithelium are enterocytes; secretory cells constitute only a minor fraction. Se- cretory cells belong to four different classes: enteroendocrine Tuft cells in the intestine are known to act as sentinels for in- cells, goblet cells, Paneth cells, and tuft cells (also known as fectious agents [e.g., helminths (worms) and bacterial micro- brush cells) (1). Enteroendocrine cells function as hormone- biota] and express taste-signaling elements. In this work, the G protein-coupled receptor Sucnr1 was shown to be expressed secreting cells in response to macronutrients, goblet cells pro- specifically in tuft cells but not in other intestinal epithelial duce a protective mucus layer, and Paneth cells secrete niche cells. Dietary succinate and perturbations in the microbiota factors to maintain intestinal stem cell homeostasis and also activate tuft cells, and subsequently type 2 immunity, via tuft produce antimicrobial peptides (1). Despite being discovered – cell-expressed Sucnr1. Modulating this pathway using dietary decades ago (2 5), the function of tuft cells in the small intestine succinate or specific Sucnr1 agonists may be a strategy for was only recently uncovered: They mediate host defense against fighting bacterial and parasitic infections or other type 2 immune- parasitic infection or other pathogens by regulating an intestinal related metabolic disorders such as obesity. type 2 innate lymphoid cell (ILC2)–epithelial response circuit, and hence are involved in immune function (6–8). Author contributions: W.L., R.F.M., and P.J. designed research; W.L., W.R., and M.O. per- Tuft cells express a number of taste-signaling elements, in- formed research; W.L., W.R., M.O., J.F.U., I.M., R.F.M., and P.J. contributed new reagents/ cluding transient receptor potential cation channel subfamily M analytic tools; W.L., W.R., M.O., J.F.U., I.M., and P.J. analyzed data; and W.L. and P.J. wrote member 5 (Trpm5), gustducin, and phospholipase β2(Plcβ2) (7, the paper. 9–13). These signaling elements play critical roles in mediating the Conflict of interest statement: W.L. and P.J. have filed a patent application related to the responses of tuft cells to parasitic infections (7). A hallmark fea- effect of succinate on type 2 immunity. ture of such infection is tuft and goblet cell hyperplasia (6–8, 14, This article is a PNAS Direct Submission. 15). In wild-type mice, infections by helminth parasites or proto- Published under the PNAS license. zoans can induce tuft and goblet cell hyperplasia (6–8). In mice Data deposition: The sequence reported in this paper has been deposited in the National lacking gustducin or Trpm5, these responses are not observed Center for Biotechnology Information (accession no. SRP134009). (7). The transcription factor Pou2f3 is required for develop- 1W.L. and W.R. contributed equally to this work. ment of type II taste cells as well as tuft cells in the gut (8, 16). 2To whom correspondence should be addressed. Email: [email protected]. Mice deficient in Pou2f3, and therefore lacking tuft cells, can- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. not mount a tuft cell-mediated type 2 immune response toward 1073/pnas.1720758115/-/DCSupplemental. − − − − infecting pathogens, similar to Trpm5 / and gustducin / mice (8). www.pnas.org/cgi/doi/10.1073/pnas.1720758115 PNAS Latest Articles | 1of6 Downloaded by guest on September 24, 2021 Results to activate mucosal type 2 immunity, resulting in tuft and goblet The Succinate Receptor Sucnr1 Is Specifically Expressed in Intestinal cell expansion, goblet cell enlargement (production of mucus), – Tuft Cells. We began to search for the putative tuft cell-expressed and an increase in IL-13 expressing ILC2s. Dietary succinate receptor(s) responsible for detecting infectious agents by mining (100 mM) was added to the drinking water of wild-type mice for the microarray data of intestinal tuft cells (11). The succinate 7 d, a time consistent with the turnover rate of gut epithelial cells (∼5 d) (31) and the peak of tuft cell hyperplasia in response to receptor Sucnr1 (also referred to as Gpr91) is expressed in tuft ∼ cells as a GPCR. To validate the expression of Sucnr1 in intestinal parasitic infection (day 7 of infection) (6). Mice drank normally tuft cells, Trpm5-GFP–positive cells [the expression of green (SI Appendix, Fig. S1) and had no apparent changes in body fluorescent protein (GFP) is driven by the Trpm5 promoter, and weight or behavior after consuming succinate. Immunostaining GFP is used as a surrogate marker for tuft cells] were isolated by was performed with the antibody against the tuft cell marker fluorescence-activated cell sorting (7, 11) (Fig. 1A), and quanti- Dclk1 to determine if feeding succinate induced tuft cell hyper- tative real-time PCR (qPCR) was performed using primer sets for plasia (32). There was a significant increase in the number of tuft established tuft cell markers as well as a Sucnr1 primer set (Fig. cells in the small intestine of mice drinking water supplemented 1B). Nontuft intestinal epithelial cells (GFP-negative cells) were with succinate compared with plain water (Fig. 2 A and B). Par- asitic helminth infections lead to tuft cell as well as goblet cell used as control. Sucnr1 and other established tuft cell markers – [e.g., Dclk1, Gnat3 (encoding gustducin), Il25, Plcb2, Trpm5]were hyperplasia (6 8). To determine if succinate treatment, like par- robustly expressed in tuft cells compared with nontuft intestinal asitic helminth infection, also impacts goblet cell homeostasis, Alcian blue staining of the intestine of succinate-treated mice was epithelial cells. To directly visualize the expression of Sucnr1 in used to detect goblet cells. As expected, the number and en- tuft cells and determine if Sucnr1 is found in other intestinal ep- largement of goblet cells, features of a type 2 response, signifi- ithelial cells, we performed in situ hybridization of the jejunum cantly increased after succinate treatment (Fig. 2 C–E and SI tissue of wild-type mice using a Sucnr1 RNA probe (Fig. 1C). A Appendix,Fig.S2A). To further validate our immunostaining re- few cells in the jejunum tissue of wild-type mice were stained sults, we performed qPCR analyses of the jejunum of mice treated positively with the Sucnr1 probe, but none were detected in mice with succinate using primers for intestinal epithelial cell markers. deficient for Pou2f3, which results in the specific absence of tuft qPCR data revealed a significant increase in the expression of cells in the intestinal epithelium without affecting other types of genes encoding tuft cell markers (e.g., Sucnr1, Dclk1, Gnat3, intestinal epithelial cells (Fig. 1C) (8). Together, these data show Il25, Plcb2, Trpm5)(6–8) (Fig.