Tuft cell acetylcholine is released into the gut lumen to promote anti-helminth immunity.

Upon parasitic helminth infection, activated intestinal tuft cells secrete interleukin-25 (IL-25), which initiates a type 2 immune response during which lamina propria type 2 innate lymphoid cells (ILC2s) produce IL-13. This causes epithelial remodeling, including tuft cell hyperplasia, the function of which is unknown. We identified a cholinergic effector function of tuft cells, which are the only epithelial cells that expressed choline acetyltransferase (ChAT). During parasite infection, mice with epithelial-specific deletion of ChAT had increased worm burden, fitness, and fecal egg counts, even though type 2 immune responses were comparable. Mechanistically, IL-13-amplified tuft cells release acetylcholine (ACh) into the gut lumen. Finally, we demonstrated a direct effect of ACh on worms, which reduced their fecundity via helminth-expressed muscarinic ACh receptors. Thus, tuft cells are sentinels in naive mice, and their amplification upon helminth infection provides an additional type 2 immune response effector function.

Cross talk between Paneth and tuft cells drives dysbiosis and inflammation in the gut mucosa.

Gut microbiota imbalance (dysbiosis) is increasingly associated with pathological conditions, both within and outside the gastrointestinal tract. Intestinal Paneth cells are considered to be guardians of the gut microbiota, but the events linking Paneth cell dysfunction with dysbiosis remain unclear. We report a three-step mechanism for dysbiosis initiation. Initial alterations in Paneth cells, as frequently observed in obese and inflammatorybowel diseases patients, cause a mild remodeling of microbiota, with amplification of succinate-producing species. SucnR1-dependent activation of epithelial tuft cells triggers a type 2 immune response that, in turn, aggravates the Paneth cell defaults, promoting dysbiosis and chronic inflammation. We thus reveal a function of tuft cells in promoting dysbiosis following Paneth cell deficiency and an unappreciated essential role of Paneth cells in maintaining a balanced microbiota to prevent inappropriate activation of tuft cells and deleterious dysbiosis. This succinate-tuft cell inflammation circuit may also contribute to the chronic dysbiosis observed in patients.

Intestinal tuft cells: Sentinels, what else?

The intestinal epithelium plays crucial roles in maintaining gut homeostasis. A key function consists in constituting a physical and chemical barrier between self and non-self-compartments, and, based on its crosstalk with the luminal environment, in controlling activation of the host immune system. Tuft cells are a unique epithelial cell lineage, the function of which remained a mystery even 50 years after their initial discovery. The first function of intestinal tuft cells was recently described, with a central role in initiating type 2 immune responses following infection with helminth parasites. Since then, tuft cells have emerged as sentinel cells recognizing a variety of luminal cues, mediating the host-microorganisms crosstalk with additional pathogens, including viruses and bacteria. Although it can be anticipated that more functions will be discovered for tuft cells in the future, recent discoveries already propelled them at the forefront of gut mucosal homeostasis regulation, with important potential impact in gut physiopathology. This review focuses on intestinal tuft cells, from their initial description to the current understanding of their functions, and their potential impact in diseases.

CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium.

CDK8 and CDK19 form a conserved cyclin-dependent kinase subfamily that interacts with the essential transcription complex, Mediator, and also phosphorylates the C-terminal domain of RNA polymerase II. Cells lacking either CDK8 or CDK19 are viable and have limited transcriptional alterations, but whether the two kinases redundantly control cell proliferation and differentiation is unknown. Here, we find in mice that CDK8 is dispensable for regulation of gene expression, normal intestinal homeostasis, and efficient tumourigenesis, and is largely redundant with CDK19 in the control of gene expression. Their combined deletion in intestinal organoids reduces long-term proliferative capacity but is not lethal and allows differentiation. However, double-mutant organoids show mucus accumulation and increased secretion by goblet cells, as well as downregulation of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) and functionality of the CFTR pathway. Pharmacological inhibition of CDK8/19 kinase activity in organoids and in mice recapitulates several of these phenotypes. Thus, the Mediator kinases are not essential for cell proliferation and differentiation in an adult tissue, but they cooperate to regulate specific transcriptional programmes.

Single-cell mapping of the thymic stroma identifies IL-25-producing tuft epithelial cells.

T cell development and selection are coordinated in the thymus by a specialized niche of diverse stromal populations1-3. Although much progress has been made over the years in identifying the functions of the different cell types of the thymic stromal compartment, there is no comprehensive characterization of their diversity and heterogeneity. Here we combined massively parallel single-cell RNA-sequencing4,5, spatial mapping, chromatin profiling and gene targeting to characterize de novo the entire stromal compartment of the mouse thymus. We identified dozens of cell states, with thymic epithelial cells (TECs) showing the highest degree of heterogeneity. Our analysis highlights four major medullary TEC (mTEC I-IV) populations, with distinct molecular functions, epigenetic landscapes and lineage regulators. Specifically, mTEC IV constitutes a new and highly divergent TEC lineage with molecular characteristics of the gut chemosensory epithelial tuft cells. Mice deficient in Pou2f3, a master regulator of tuft cells, have complete and specific depletion of mTEC IV cells, which results in increased levels of thymus-resident type-2 innate lymphoid cells. Overall, our study provides a comprehensive characterization of the thymic stroma and identifies a new tuft-like TEC population, which is critical for shaping the immune niche in the thymus.

Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites.

Helminth parasitic infections are a major global health and social burden. The host defence against helminths such as Nippostrongylus brasiliensis is orchestrated by type 2 cell-mediated immunity. Induction of type 2 cytokines, including interleukins (IL) IL-4 and IL-13, induce goblet cell hyperplasia with mucus production, ultimately resulting in worm expulsion. However, the mechanisms underlying the initiation of type 2 responses remain incompletely understood. Here we show that tuft cells, a rare epithelial cell type in the steady-state intestinal epithelium, are responsible for initiating type 2 responses to parasites by a cytokine-mediated cellular relay. Tuft cells have a Th2-related gene expression signature and we demonstrate that they undergo a rapid and extensive IL-4Rα-dependent amplification following infection with helminth parasites, owing to direct differentiation of epithelial crypt progenitor cells. We find that the Pou2f3 gene is essential for tuft cell specification. Pou2f3(-/-) mice lack intestinal tuft cells and have defective mucosal type 2 responses to helminth infection; goblet cell hyperplasia is abrogated and worm expulsion is compromised. Notably, IL-4Rα signalling is sufficient to induce expansion of the tuft cell lineage, and ectopic stimulation of this signalling cascade obviates the need for tuft cells in the epithelial cell remodelling of the intestine. Moreover, tuft cells secrete IL-25, thereby regulating type 2 immune responses. Our data reveal a novel function of intestinal epithelial tuft cells and demonstrate a cellular relay required for initiating mucosal type 2 immunity to helminth infection.

Random chromosome segregation in mouse intestinal epithelial stem cells.

The mammalian intestinal epithelium is endowed with a high cell turnover sustained by a few stem cells located in the bottoms of millions of crypts. Until recently, it was generally assumed that the extreme sensitivity to DNA damaging agents leading to cell death and the asymmetric mode of chromosome segregation of intestinal epithelial stem cells prevented the illicit survival of mutated stem cells and guarded against mistakes leading to aneuploidy and neoplastic transformation. Recent evidence points instead to a pool of mutipotent self-renewing stem cells capable of repairing DNA by homologous recombination significantly more efficiently than other crypt cells. Furthermore, the equilibrium between cell division and differentiation is achieved at the level of the cell population obeying to a random mode of chromosome segregation and a predominantly symmetric mode of cell division. This review summarizes the experimental findings on the mode of cell division adopted by intestinal epithelial stem cells.

The intestinal epithelium tuft cells: specification and function.

The intestinal epithelium, composed of at least seven differentiated cell types, represents an extraordinary model to understand the details of multi-lineage differentiation, a question that is highly relevant in developmental biology as well as for clinical applications. This review focuses on intestinal epithelial tuft cells that have been acknowledged as a separate entity for more than 60 years but whose function remains a mystery. We discuss what is currently known about the molecular basis of tuft cell fate and differentiation and why elucidating tuft cell function has been so difficult. Finally, we summarize the current hypotheses on their potential involvement in diseases of the gastro-intestinal tract.

Tuft Cells: Detectors, Amplifiers, Effectors and Targets in Parasite Infection.

Tuft cells have recently emerged as the focus of intense interest following the discovery of their chemosensory role in the intestinal tract, and their ability to activate Type 2 immune responses to helminth parasites. Moreover, they populate a wide range of mucosal tissues and are intimately connected to immune and neuronal cells, either directly or through the release of pharmacologically active mediators. They are now recognised to fulfil both homeostatic roles, in metabolism and tissue integrity, as well as acting as the first sensors of parasite infection, immunity to which is lost in their absence. In this review we focus primarily on the importance of tuft cells in the intestinal niche, but also link to their more generalised physiological role and discuss their potential as targets for the treatment of gastrointestinal disorders.

Type 2 cGMP-dependent protein kinase regulates proliferation and differentiation in the colonic mucosa.

Signaling through cGMP has emerged as an important regulator of tissue homeostasis in the gastrointestinal tract, but the mechanism is not known. Type 2 cGMP-dependent protein kinase (PKG2) is a major cGMP effector in the gut epithelium, and the present studies have tested its importance in the regulation of proliferation and differentiation in the mouse colon and in colon cancer cell lines. Tissue homeostasis was examined in the proximal colon of Prkg2(-/-) mice using histological markers of proliferation and differentiation. The effect of ectopic PKG2 on proliferation and differentiation was tested in vitro using inducible colon cancer cell lines. PCR and luciferase reporter assays were used to determine the importance of Sox9 downstream of PKG2. The colons of Prkg2(-/-) mice exhibited crypt hyperplasia, increased epithelial apoptosis, and reduced numbers of differentiated goblet and enteroendocrine cells. Ectopic PKG2 was able to inhibit proliferation and induce Muc2 and CDX2 expression in colon cancer cells, but did not significantly affect cell death. PKG2 reduced Sox9 levels and signaling, suggesting possible involvement of this pathway downstream of cGMP in the colon. The work presented here demonstrates a novel antiproliferative and prodifferentiation role for PKG2 in the colon. These homeostatic functions of PKG2 were reproducible in colon cancer cells lines where downregulation of Sox9 is a possible mechanism. The similarities in phenotype between PKG2 and GCC knockout mice positions PKG2 as a likely mediator of the homeostatic effects of cGMP signaling in the colon.

Wnt signalling induces maturation of Paneth cells in intestinal crypts.

Wnt signalling, which is transduced through beta-catenin/TCF4, maintains the undifferentiated state of intestinal crypt progenitor cells. Mutational activation of the pathway initiates the adenomacarcinoma sequence. Whereas all other differentiated epithelial cells migrate from the crypt onto the villus, Paneth cells home towards the source of Wnt signals--that is, the crypt bottom. Here, we show that expression of a Paneth gene programme is critically dependent on TCF4 in embryonic intestine. Moreover, conditional deletion of the Wnt receptor Frizzled-5 abrogates expression of these genes in Paneth cells in the adult intestine. Conversely, adenomas in Apc-mutant mice and colorectal cancers in humans inappropriately express these Paneth-cell genes. These observations imply that Wnt signals in the crypt can separately drive a stem-cell/progenitor gene programme and a Paneth-cell maturation programme. In intestinal cancer, both gene programmes are activated simultaneously.

Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium.

The HMG-box transcription factor Sox9 is expressed in the intestinal epithelium, specifically, in stem/progenitor cells and in Paneth cells. Sox9 expression requires an active beta-catenin-Tcf complex, the transcriptional effector of the Wnt pathway. This pathway is critical for numerous aspects of the intestinal epithelium physiopathology, but processes that specify the cell response to such multipotential signals still remain to be identified. We inactivated the Sox9 gene in the intestinal epithelium to analyze its physiological function. Sox9 inactivation affected differentiation throughout the intestinal epithelium, with a disappearance of Paneth cells and a decrease of the goblet cell lineage. Additionally, the morphology of the colon epithelium was severely altered. We detected general hyperplasia and local crypt dysplasia in the intestine, and Wnt pathway target genes were up-regulated. These results highlight the central position of Sox9 as both a transcriptional target and a regulator of the Wnt pathway in the regulation of intestinal epithelium homeostasis.

A 31-plex panel for high-dimensional single-cell analysis of murine preclinical models of solid tumors by imaging mass cytometry.

Currently, the study of resistance mechanisms and disease progression in cancer relies on the capacity to analyze tumors as a complex ecosystem of healthy and malignant cells. Therefore, one of the current challenges is to decipher the intra-tumor heterogeneity and especially the spatial distribution and interactions of the different cellular actors within the tumor. Preclinical mouse models are widely used to extend our understanding of the tumor microenvironment (TME). Such models are becoming more sophisticated and allow investigating questions that cannot be addressed in clinical studies. Indeed, besides studying the tumor cell interactions within their environment, mouse models allow evaluating the efficacy of new drugs and delivery approaches, treatment posology, and toxicity. Spatially resolved analyses of the intra-tumor heterogeneity require global approaches to identify and localize a large number of different cell types. For this purpose, imaging mass cytometry (IMC) is a major asset in the field of human immuno-oncology. However, the paucity of validated IMC panels to study TME in pre-clinical mouse models remains a critical obstacle to translational or basic research in oncology. Here, we validated a panel of 31 markers for studying at the single-cell level the TME and the immune landscape for discovering/characterizing cells with complex phenotypes and the interactions shaping the tumor ecosystem in mouse models.

The IL-25-dependent tuft cell circuit driven by intestinal helminths requires macrophage migration inhibitory factor (MIF).

Macrophage migration inhibitory factor (MIF) is a key innate immune mediator with chemokine- and cytokine-like properties in the inflammatory pathway. While its actions on macrophages are well-studied, its effects on other cell types are less understood. Here we report that MIF is required for expansion of intestinal tuft cells during infection with the helminth Nippostrongylus brasiliensis. MIF-deficient mice show defective innate responses following infection, lacking intestinal epithelial tuft cell hyperplasia or upregulation of goblet cell RELMß, and fail to expand eosinophil, type 2 innate lymphoid cell (ILC2) and macrophage (M2) populations. Similar effects were observed in MIF-sufficient wild-type mice given the MIF inhibitor 4-IPP. MIF had no direct effect on epithelial cells in organoid cultures, and MIF-deficient intestinal stem cells could generate tuft cells in vitro in the presence of type 2 cytokines. In vivo the lack of MIF could be fully compensated by administration of IL-25, restoring tuft cell differentiation and goblet cell expression of RELM-ß, demonstrating its requirement upstream of the ILC2-tuft cell circuit. Both ILC2s and macrophages expressed the MIF receptor CXCR4, indicating that MIF may act as an essential co-factor on both cell types to activate responses to IL-25 in helminth infection.

Intestinal epithelial tuft cell induction is negated by a murine helminth and its secreted products.

Helminth parasites are adept manipulators of the immune system, using multiple strategies to evade the host type 2 response. In the intestinal niche, the epithelium is crucial for initiating type 2 immunity via tuft cells, which together with goblet cells expand dramatically in response to the type 2 cytokines IL-4 and IL-13. However, it is not known whether helminths modulate these epithelial cell populations. In vitro, using small intestinal organoids, we found that excretory/secretory products (HpES) from Heligmosomoides polygyrus blocked the effects of IL-4/13, inhibiting tuft and goblet cell gene expression and expansion, and inducing spheroid growth characteristic of fetal epithelium and homeostatic repair. Similar outcomes were seen in organoids exposed to parasite larvae. In vivo, H. polygyrus infection inhibited tuft cell responses to heterologous Nippostrongylus brasiliensis infection or succinate, and HpES also reduced succinate-stimulated tuft cell expansion. Our results demonstrate that helminth parasites reshape their intestinal environment in a novel strategy for undermining the host protective response.

Tuft cells: sentinels of the intestinal mucosa.

The intestinal epithelium is one of our main interfaces with the outside world, including the intestinal microbiota. This epithelium thus combines the two essential functions of nutrient absorption and barrier. In order to fulfill its different roles, the intestinal epithelium is made up of several specialized cell types. Among these, tuft cells have long remained in the shadows, but the understanding of their function has accelerated dramatically in recent years. The purpose of this review is to outline the characterization of tuft cells and the discovery of their sentinel function in the intestinal mucosa.

L'épithélium intestinal constitue l'une de nos principales interfaces avec le monde extérieur, y compris le microbiote intestinal. Cet épithélium combine ainsi les deux fonctions essentielles d'absorption de nutriments et de barrière. Afin de remplir ses différents rôles, l'épithélium intestinal est constitué de plusieurs types cellulaires spécialisés. Parmi ceux-ci, les cellules tuft sont longtemps restées dans l'ombre, mais la compréhension de leur fonction a connu une accélération spectaculaire ces dernières années. L'objet de cette revue est de retracer les grandes lignes de la caractérisation des cellules tuft et de la découverte de leur fonction de sentinelle dans la muqueuse intestinale.

Tuft Cells Increase Following Ovine Intestinal Parasite Infections and Define Evolutionarily Conserved and Divergent Responses.

Helminth parasite infections of humans and livestock are a global health and economic problem. Resistance of helminths to current drug treatment is an increasing problem and alternative control approaches, including vaccines, are needed. Effective vaccine design requires knowledge of host immune mechanisms and how these are stimulated. Mouse models of helminth infection indicate that tuft cells, an unusual type of epithelial cell, may 'sense' infection in the small intestine and trigger a type 2 immune response. Currently nothing is known of tuft cells in immunity in other host species and in other compartments of the gastrointestinal (GI) tract. Here we address this gap and use immunohistochemistry and single cell RNA-sequencing to detail the presence and gene expression profile of tuft cells in sheep following nematode infections. We identify and characterize tuft cells in the ovine abomasum (true stomach of ruminants) and show that they increase significantly in number following infection with the globally important nematodes Teladorsagia circumcincta and Haemonchus contortus. Ovine abomasal tuft cells show enriched expression of tuft cell markers POU2F3, GFI1B, TRPM5 and genes involved in signaling and inflammatory pathways. However succinate receptor SUCNR1 and free fatty acid receptor FFAR3, proposed as 'sensing' receptors in murine tuft cells, are not expressed, and instead ovine tuft cells are enriched for taste receptor TAS2R16 and mechanosensory receptor ADGRG6. We also identify tuft cell sub-clusters at potentially different stages of maturation, suggesting a dynamic process not apparent from mouse models of infection. Our findings reveal a tuft cell response to economically important parasite infections and show that while tuft cell effector functions have been retained during mammalian evolution, receptor specificity has diverged. Our data advance knowledge of host-parasite interactions in the GI mucosa and identify receptors that may potentiate type 2 immunity for optimized control of parasitic nematodes.

The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium.

The R2TP chaperone cooperates with HSP90 to integrate newly synthesized proteins into multi-subunit complexes, yet its role in tissue homeostasis is unknown. Here, we generated conditional, inducible knock-out mice for Rpap3 to inactivate this core component of R2TP in the intestinal epithelium. In adult mice, Rpap3 invalidation caused destruction of the small intestinal epithelium and death within 10 days. Levels of R2TP substrates decreased, with strong effects on mTOR, ATM and ATR. Proliferative stem cells and progenitors deficient for Rpap3 failed to import RNA polymerase II into the nucleus and they induced p53, cell cycle arrest and apoptosis. Post-mitotic, differentiated cells did not display these alterations, suggesting that R2TP clients are preferentially built in actively proliferating cells. In addition, high RPAP3 levels in colorectal tumors from patients correlate with bad prognosis. Here, we show that, in the intestine, the R2TP chaperone plays essential roles in normal and tumoral proliferation.

Expression of POU2F3 Transcription Factor Control Inflammation, Immunological Recruitment and Metastasis of Pancreatic Cancer in Mice.

TUFT cells have been described as strong modulators of inflammatory cells in several tissues including pancreas. TUFT cells, also known as DCLK1+ cells, are dependent of the transcriptional factor POU2F3. Several works report DCLK1+ cells in early stages of PDAC development suggesting an important role of TUFT cells in PDAC development. Therefore, we developed a mice model (PDX1-Cre;KrasG12D;Ink4afl/fl), known as PKI model, deficient or not of POU2F3. In this animal model, deficiency of POU2F3 results in the absence of TUFT cells in PDAC as expected. Although, tumor development and growth are not significantly influenced, the development of liver metastasis was almost completely inhibited in POU2F3-deficient mice. Surprisingly, the absence of metastasis was associated with a higher expression of epithelial-to-mesenchymal transition markers, but to a lower inflammatory microenvironment suggesting that inflammation influences metastasis production more than epithelial-to-mesenchymal transition in this animal model. We can conclude that POU2F3 could be a new therapeutic target for control PDAC progression.

Loss of Apc Rapidly Impairs DNA Methylation Programs and Cell Fate Decisions in Lgr5+ Intestinal Stem Cells.

Colorectal cancer initiation and progression result from the accumulation of genetic and epigenetic alterations. Although aberrant gene expression and DNA methylation profiles are considered hallmarks of colorectal cancer development, the precise timing at which these are produced during tumor establishment remains elusive. Here we investigated the early transcriptional and epigenetic changes induced by adenomatous polyposis coli (Apc) inactivation in intestinal crypts. Hyperactivation of the Wnt pathway via Apc inactivation in crypt base columnar intestinal stem cells (ISC) led to their rapid accumulation driven by an impaired molecular commitment to differentiation, which was associated with discrete alterations in DNA methylation. Importantly, inhibiting the enzymes responsible for de novo DNA methylation restored the responsiveness of Apc-deficient intestinal organoids to stimuli regulating the proliferation-to-differentiation transition in ISC. This work reveals that early DNA methylation changes play critical roles in the establishment of the impaired fate decision program consecutive to Apc loss of function. SIGNIFICANCE: This study demonstrates the functional impact of changes in DNA methylation to determine the colorectal cancer cell phenotype following loss of Apc function.