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Epithelial tuft cells are shown to be the source of intestinal interleukin (IL)-25 that is required for activation of type 2 innate lymphoid cells (ILC2s), ILC2-regulated tuft and goblet cell expansion, and control of parasite infection. Epithelial tuft cells in type 2 immunity The 'weep-and-sweep' response to parasitic helminths and allergens, in which parasites are ejected by increased propulsive activity of the gut combined with fluid and mucus secretion, is a manifestation of type 2 (or allergic) immunity involving the activation of group 2 innate lymphoid cells (ILC2s). The epithelium in the small intestine consists of five or more distinct cellular lineages, including tuft cells, whose functions remain unclear. Two papers in this issue of Nature demonstrate a role for tuft cells in the response to parasites. Richard Locksley and colleagues show that tuft cells are the source of the interleukin 25 (IL-25) that is required for activation of ILC2s, ILC2-regulated tuft and goblet cell expansion, and control of parasite infection. Philippe Jay and colleagues show that tuft cells secrete IL-25 via an IL-13/IL-4R -dependent feedback loop. Parasitic helminths and allergens induce a type 2 immune response leading to profound changes in tissue physiology, including hyperplasia of mucus-secreting goblet cells 1 and smooth muscle hypercontractility 2 . This response, known as ‘weep and sweep’, requires interleukin (IL)-13 production by tissue-resident group 2 innate lymphoid cells (ILC2s) and recruited type 2 helper T cells (T H 2 cells) 3 . Experiments in mice and humans have demonstrated requirements for the epithelial cytokines IL-33, thymic stromal lymphopoietin (TSLP) and IL-25 in the activation of ILC2s 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , but the sources and regulation of these signals remain poorly defined. In the small intestine, the epithelium consists of at least five distinct cellular lineages 12 , including the tuft cell, whose function is unclear. Here we show that tuft cells constitutively express IL-25 to sustain ILC2 homeostasis in the resting lamina propria in mice. After helminth infection, tuft-cell-derived IL-25 further activates ILC2s to secrete IL-13, which acts on epithelial crypt progenitors to promote differentiation of tuft and goblet cells, leading to increased frequencies of both. Tuft cells, ILC2s and epithelial progenitors therefore comprise a response circuit that mediates epithelial remodelling associated with type 2 immunity in the small intestine, and perhaps at other mucosal barriers populated by these cells.

Key Points Intestinal epithelial cells (IECs) provide a physical and biochemical barrier that segregates host tissue and commensal bacteria to maintain intestinal homeostasis. Secretory IECs support this function through the secretion of mucins and antimicrobial peptides. IECs maintain specialized pathways for the delivery of luminal antigens and bacteria to lamina propria-resident antigen-presenting cells. Microbial signals are recognized by IECs to promote intestinal homeostasis. Host–commensal microorganism interactions not only support tissue repair in the setting of injury or acute inflammation but also promote the development of intestinal cancers during chronic inflammation. IECs possess mechanisms for maintaining altered responsiveness to microbial signals that allow for tolerance to continuous exposure to commensal bacteria. IECs convey microbial signals to mucosal immune cells and promote the coordination of appropriate immune responses against commensal bacteria and enteric pathogens. Interactions between antigen-presenting cells and IECs regulate B cell and T cell responses that act on the intestinal barrier. Both innate and adaptive effector cell function and homeostasis are influenced by IEC-derived signals. This allows IECs to locally regulate immune response at the intestinal barrier and promote the maintenance of intestinal homeostasis. Intestinal epithelial cells (IECs) promote gut homeostasis by coordinating the segregation and regulation of commensal microorganisms and the host immune system. This Review highlights the diverse and multifaceted roles of IECs in barrier function, and in their regulation of innate and adaptive immune cell function and homeostasis in response to microbial colonization. The abundance of innate and adaptive immune cells that reside together with trillions of beneficial commensal microorganisms in the mammalian gastrointestinal tract requires barrier and regulatory mechanisms that conserve host–microbial interactions and tissue homeostasis. This homeostasis depends on the diverse functions of intestinal epithelial cells (IECs), which include the physical segregation of commensal bacteria and the integration of microbial signals. Hence, IECs are crucial mediators of intestinal homeostasis that enable the establishment of an immunological environment permissive to colonization by commensal bacteria. In this Review, we provide a comprehensive overview of how IECs maintain host–commensal microbial relationships and immune cell homeostasis in the intestine.

It is generally believed that cells in the gut transduce sensory information through the paracrine action of hormones. Kaelberer et al. found that, in addition to the well-described classical paracrine transduction, enteroendocrine cells also form fast, excitatory synapses with vagal afferents (see the Perspective by Hoffman and Lumpkin). This more direct circuit for gut-brain signaling uses glutamate as a neurotransmitter. Thus, sensory cues that stimulate the gut could potentially be manipulated to influence specific brain functions and behavior, including those linked to food choices. Science , this issue p. eaat5236 ; see also p. 1203 A neuroepithelial circuit that connects the intestinal lumen to the brain stem in one synapse has been identified. The brain is thought to sense gut stimuli only via the passive release of hormones. This is because no connection has been described between the vagus and the putative gut epithelial sensor cell—the enteroendocrine cell. However, these electrically excitable cells contain several features of epithelial transducers. Using a mouse model, we found that enteroendocrine cells synapse with vagal neurons to transduce gut luminal signals in milliseconds by using glutamate as a neurotransmitter. These synaptically connected enteroendocrine cells are referred to henceforth as neuropod cells. The neuroepithelial circuit they form connects the intestinal lumen to the brainstem in one synapse, opening a physical conduit for the brain to sense gut stimuli with the temporal precision and topographical resolution of a synapse.

Satiety and other core physiological functions are modulated by sensory signals arising from the surface of the gut. Luminal nutrients and bacteria stimulate epithelial biosensors called enteroendocrine cells. Despite being electrically excitable, enteroendocrine cells are generally thought to communicate indirectly with nerves through hormone secretion and not through direct cell-nerve contact. However, we recently uncovered in intestinal enteroendocrine cells a cytoplasmic process that we named neuropod. Here, we determined that neuropods provide a direct connection between enteroendocrine cells and neurons innervating the small intestine and colon. Using cell-specific transgenic mice to study neural circuits, we found that enteroendocrine cells have the necessary elements for neurotransmission, including expression of genes that encode pre-, post-, and transsynaptic proteins. This neuroepithelial circuit was reconstituted in vitro by coculturing single enteroendocrine cells with sensory neurons. We used a monosynaptic rabies virus to define the circuit's functional connectivity in vivo and determined that delivery of this neurotropic virus into the colon lumen resulted in the infection of mucosal nerves through enteroendocrine cells. This neuroepithelial circuit can serve as both a sensory conduit for food and gut microbes to interact with the nervous system and a portal for viruses to enter the enteric and central nervous systems.

The glycolytic activity of Paneth cells provides lactate, which is required by self-renewing intestinal stem cells for oxidative metabolism to activate p38 MAP kinase, ensuring regeneration of a mature crypt. Metabolism and gut regeneration Small intestine crypts are regenerated throughout life thanks to self-renewing stem cells located at the bottom of crypts. Differentiated Paneth cells provide the signalling molecules that modulate the regenerative properties of these stem cells. The influence of metabolism on the self-renewal of the crypt has not been studied in great detail. Burgering and colleagues now show that, whereas intestinal stem cells rely on mitochondrial activity for their metabolic needs, Paneth cells use glycolysis, a process that provides the lactate that is required by the stem cells for their oxidative metabolism. This activates the p38 MAP kinase to ensure regeneration of a mature crypt. The findings suggest that the metabolism of certain intestinal cells has an important role in supporting stem cell function. The small intestinal epithelium self-renews every four or five days. Intestinal stem cells (Lgr5 + crypt base columnar cells (CBCs)) sustain this renewal and reside between terminally differentiated Paneth cells at the bottom of the intestinal crypt 1 . Whereas the signalling requirements for maintaining stem cell function and crypt homeostasis have been well studied, little is known about how metabolism contributes to epithelial homeostasis. Here we show that freshly isolated Lgr5 + CBCs and Paneth cells from the mouse small intestine display different metabolic programs. Compared to Paneth cells, Lgr5 + CBCs display high mitochondrial activity. Inhibition of mitochondrial activity in Lgr5 + CBCs or inhibition of glycolysis in Paneth cells strongly affects stem cell function, as indicated by impaired organoid formation. In addition, Paneth cells support stem cell function by providing lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5 + CBCs. Mechanistically, we show that oxidative phosphorylation stimulates p38 MAPK activation by mitochondrial reactive oxygen species signalling, thereby establishing the mature crypt phenotype. Together, our results reveal a critical role for the metabolic identity of Lgr5 + CBCs and Paneth cells in supporting optimal stem cell function, and we identify mitochondria and reactive oxygen species signalling as a driving force of cellular differentiation.

Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract 1 – 4 . The protective effects of IL-2 involve the generation, maintenance and function of regulatory T (T reg ) cells 4 – 8 , and the use of low doses of IL-2 has emerged as a potential therapeutic strategy for patients with inflammatory bowel disease 9 . However, the cellular and molecular pathways that control the production of IL-2 in the context of intestinal health are undefined. Here we show, in a mouse model, that IL-2 is acutely required to maintain T reg cells and immunological homeostasis throughout the gastrointestinal tract. Notably, lineage-specific deletion of IL-2 in T cells did not reduce T reg cells in the small intestine. Unbiased analyses revealed that, in the small intestine, group-3 innate lymphoid cells (ILC3s) are the dominant cellular source of IL-2, which is induced selectively by IL-1β. Macrophages in the small intestine produce IL-1β, and activation of this pathway involves MYD88- and NOD2-dependent sensing of the microbiota. Our loss-of-function studies show that ILC3-derived IL-2 is essential for maintaining T reg cells, immunological homeostasis and oral tolerance to dietary antigens in the small intestine. Furthermore, production of IL-2 by ILC3s was significantly reduced in the small intestine of patients with Crohn’s disease, and this correlated with lower frequencies of T reg cells. Our results reveal a previously unappreciated pathway in which a microbiota- and IL-1β-dependent axis promotes the production of IL-2 by ILC3s to orchestrate immune regulation in the intestine. A microbiota- and IL-1β-dependent axis of IL-2 production by group-3 innate lymphoid cells is shown in a mouse model to be necessary to maintain immunological homeostasis and regulatory T cells in the small intestine.

Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens. Profiling of 53,193 individual epithelial cells from the mouse small intestine identifies previously unknown cell subtypes and corresponding gene markers, providing insight into gut homeostasis and response to pathogens. Surveying the stomach wall Intestinal epithelial cells can sense and respond to microbial stimuli to support their barrier function and coordinate appropriate immune responses, which range from tolerance to active immunity in cases of pathogen infection. In this study, Aviv Regev, Ramnik Xavier and colleagues used single-cell expression profiling to provide a comprehensive analysis of the epithelial cell composition of mouse small intestines when healthy and after infection, providing new markers, transcriptional programs and organizational principles of gut homeostasis and physiology.

Innate lymphoid cells (ILCs) contribute to barrier immunity, tissue homeostasis, and immune regulation at various anatomical sites throughout the body. How ILCs maintain their presence in lymphoid and peripheral tissues thus far has been unclear. We found that in the lymphoid and nonlymphoid organs of adult mice, ILCs are tissue-resident cells that were maintained and expanded locally under physiologic conditions, upon systemic perturbation of immune homeostasis and during acute helminth infection. However, at later time points after infection, cells from hematogenous sources helped to partially replenish the pool of resident ILCs. Thus, ILCs are maintained by self-renewal in broadly different microenvironments and physiological settings. Such an extreme \"sedentary\" lifestyle is consistent with the proposed roles of ILCs as sentinels and local keepers of tissue function.

An algorithm that allows rare cell type identification in a complex population of single cells, based on single-cell mRNA-sequencing, is applied to mouse intestinal cells, revealing novel subtypes of enteroendocrine cells and showing that the Lgr5-expressing population consists of a homogenous stem cell population with a few rare secretory cells, including Paneth cells. Rare cells stand out from the crowd Identifying and tracking rare cell types of physiological importance in a mixed population is a challenge. This study, a collaboration between the labs of Hans Clevers and Alexander van Oudenaarden, tackles the issue by applying single-cell mRNA sequencing to mouse intestinal organoids. The authors develop an algorithm, called RaceID, that allows rare cell type identification in a complex population of single cells. They define a marker for rare enteroendocrine cells of the intestine. Analysis of cells from a primary mouse intestine using this method, reveals that the Lgr5 -expressing population consists of a homogenous stem cell population with a few rare secretory cells, including Paneth cells. Understanding the development and function of an organ requires the characterization of all of its cell types. Traditional methods for visualizing and isolating subpopulations of cells are based on messenger RNA or protein expression of only a few known marker genes. The unequivocal identification of a specific marker gene, however, poses a major challenge, particularly if this cell type is rare. Identifying rare cell types, such as stem cells, short-lived progenitors, cancer stem cells, or circulating tumour cells, is crucial to acquire a better understanding of normal or diseased tissue biology. To address this challenge we first sequenced the transcriptome of hundreds of randomly selected cells from mouse intestinal organoids 1 , cultured self-organizing epithelial structures that contain all cell lineages of the mammalian intestine. Organoid buds, like intestinal crypts, harbour stem cells that continuously differentiate into a variety of cell types, occurring at widely different abundances 2 . Since available computational methods can only resolve more abundant cell types, we developed RaceID, an algorithm for rare cell type identification in complex populations of single cells. We demonstrate that this algorithm can resolve cell types represented by only a single cell in a population of randomly sampled organoid cells. We use this algorithm to identify Reg4 as a novel marker for enteroendocrine cells, a rare population of hormone-producing intestinal cells 3 . Next, we use Reg4 expression to enrich for these rare cells and investigate the heterogeneity within this population. RaceID confirmed the existence of known enteroendocrine lineages, and moreover discovered novel subtypes, which we subsequently validated in vivo . Having validated RaceID we then applied the algorithm to ex vivo -isolated Lgr5 -positive stem cells and their direct progeny. We find that Lgr5 -positive cells represent a homogenous abundant population of stem cells mixed with a rare population of Lgr5 -positive secretory cells. We envision broad applicability of our method for discovering rare cell types and the corresponding marker genes in healthy and diseased organs.

The parasitic helminth Trichinella spiralis, which poses a serious health risk to animals and humans, can be found worldwide. Recent findings indicate that a rare type of gut epithelial cell, tuft cells, can detect the helminth, triggering type 2 immune responses. However, the underlying molecular mechanisms remain to be fully understood. Here we show that both excretory–secretory products (E–S) and extract of T. spiralis can stimulate the release of the cytokine interleukin 25 (IL-25) from the mouse small intestinal villi and evoke calcium responses from tuft cells in the intestinal organoids, which can be blocked by a bitter-taste receptor inhibitor, allyl isothiocyanate. Heterologously expressed mouse Tas2r bittertaste receptors, the expression of which is augmented during tuftcell hyperplasia, can respond to the E–S and extract as well as to the bitter compound salicin whereas salicin in turn can induce IL-25 release from tuft cells. Furthermore, abolishment of the G-protein γ13 subunit, application of the inhibitors for G-protein αo/i, Gβγ subunits, and phospholipase Cβ2 dramatically reduces the IL-25 release. Finally, tuft cells are found to utilize the inositol triphosphate receptor type 2 (Ip₃r2) to regulate cytosolic calcium and thus Trpm5 activity, while potentiation of Trpm5 by a sweet-tasting compound, stevioside, enhances tuft cell IL-25 release and hyperplasia in vivo. Taken together, T. spiralis infection activates a signaling pathway in intestinal tuft cells similar to that of taste-bud cells, but with some key differences, to initiate type 2 immunity.