Parasites elicit a wide range of gastrointestinal symptoms, often attributed to tissue damage or alterations in the gut microbiota. A recent study by Touhara et al. pioneers the functional understanding of a collaboration between chemosensory epithelial cells and sensory neurons to induce behavioral changes during immune responses against parasitic infections.

Stanbery et al. show that tuft cells, previously defined as epithelial activators of innate type 2 immunity, are dispensable for memory T helper 2 (Th2) cell establishment but required for protective immunity during secondary helminth infection. This involves tuft cell secretion of interleukin-25 and leukotriene C4 to promote memory Th2 effector function.

Acetylcholine (ACh) is a small-molecule neurotransmitter that regulates a wide range of neuronal and non-neuronal physiological functions. The non-neuronal roles of ACh are highly tissue specific, requiring precise measurements of its concentration in situ. Here, we present a protocol for measuring secreted ACh in the lumen of the mouse intestine. We describe the procedures for sample collection, preparation, and metabolite purification. Finally, we detail the use of liquid chromatography-mass spectrometry (LC-MS) to quantify ACh levels. For complete details on the use and execution of this protocol, please refer to Ndjim et al.1.

Induced volatolomics is an emerging field of research that offers new opportunities in biology by detecting volatile reporters released by activatable probes, enabling the exploration of oncogenic processes. Building on its proven efficiency in exploring the evolution of implanted tumours, we hypothesized that induced volatolomics could extend its application to detect precancerous conditions. As a proof of concept, we performed a longitudinal study and investigated glycosidase activity during the early stages of gastric carcinogenesis development induced by Helicobacter felis infection in mice, mimicking the gastric carcinogenesis cascade induced by chronic Helicobacter pylori infection in humans. We identified upregulated exoglycosidases linked to acute infections or inflammatory processes in tissues infected by Helicobacter. Specifically, α-mannosidase and β-galactosidase activities in stomach tissue were found to be strongly associated with the initial stages of Helicobacter infection. Additionally, the activities of β-Glucuronidase and β-N-acetyl-glucosaminidase increased during the progression to preneoplastic stages, potentially signalling the transition from infection to inflammation-driven carcinogenesis. These enzymes may serve as early biomarkers for detecting gastric carcinogenesis. Our study highlights the potential of VOC-based probes for real-time monitoring of gastric cancer progression through tissue biopsies. Therefore, this study demonstrates the potential of induced volatolomics for investigating biological processes and uncovering new therapeutic strategies.

Pathogens have developed multiple strategies to modulate host immune defense mechanisms. Understanding how this is achieved has potential to inform novel therapeutics for diseases caused by immune dysfunction. Parasitic helminths are masters of immune evasion, via release of secreted products, resulting in chronic infection. Helminths secrete small regulatory microRNA (miRNAs), which can interact with host cells. Here we show that a single parasite miRNA (miR-5352), conserved across gastrointestinal (GI) nematodes, suppresses IL-13-induced GI epithelial cell differentiation and cytokine responses, and promotes stem cell maintenance. Mechanistically, this is achieved through targeted repression of critical host factors, including Klf-4 and the IL-22 receptor, together with modulation of Wnt and Notch signalling pathways. Nematode miR-5352 shows seed sequence conservation with mammalian miR-92a family members, indicating that through convergent evolution, GI nematodes exploit a host miRNA regulatory network to suppress host innate responses, promote tissue regeneration and establish a favourable environment for chronic infection.

Intestinal helminth parasites trigger the host immune response through epithelial sensory tuft cells, but helminth-derived molecules that may activate tuft cells are poorly characterized. The study aimed to identify small molecules released in vitro by two nematode parasites, that infect rodents (Nippostrongylus brasiliensis) and ruminants (Haemonchus contortus), and to test candidate ligands in an in vivo model of tuft cell differentiation. Small molecules were analyzed by hydrophilic interaction liquid chromatography (HILIC) of material released by adult parasites incubated in serum-free media, followed by mass spectrometry; selected molecules were administered to mice and tuft cell expansion enumerated after 5 days. A range of different conditions (culture media, timing, oxygenation) were tested, and comparisons made between the conditions, and between the two nematode species at selected points. Common products across the conditions and species included carboxylic acids (malate, succinate), medium chain fatty acids (such as decanoic and undecanoic acids), purines (guanine, xanthine and their derivatives), and phosphocholine compounds. We selected 19 of the prominent molecules for in vivo testing by oral administration, including succinate, a known activator of tuft cell differentiation. Malate elicited a low but significant level of tuft cell expansion, while undecanoic acids with or without a bromine substitution were also able to induce significant differentiation comparable to succinate. Other molecules including phosphorylcholine had no effect. Multiple molecular species including decanoic and undecanoic acids released by helminths may contribute to activation of tuft cells in vivo.

The tumor microenvironment promotes cancer progression in part by supporting cancer stem cells (CSC). In colorectal cancer (CRC), progastrin (PG), an orphan growth factor secreted by tumor cells within the tumor and its microenvironment, maintains CSCs by unidentified mechanisms. Here, the orphan receptor Protein Zero-Related protein (PZR) is identified as an essential component of PG activity and demonstrated its utility as a therapeutic target. PZR is essential for growth of PG-expressing tumors, while genetic inactivation in mice of Mpzl1, which encodes PZR, disrupted chemically-induced colon transformation. Mechanistically, PG binds cellular glycosylated and dimeric PZR and promotes SHP2/SRC/β-catenin-dependent CSC-like signaling. Blocking PZR by monoclonal antibodies inhibited PG-dependent expansion of tumoroids derived from murine intestinal tumors and patient-derived CRC cell lines, while in mice, it reduced adenoma formation triggered by Apc loss in stem cells and disrupted the tumor-initiating capacity of PG-expressing CRC cells. High GAST (which encodes PG) and MPZL1 transcript levels in primary colon cancer patients is predictive of worse prognosis. Collectively, these findings support the inhibition of PZR as a potential targeted treatment of PG-expressing CRC.

Symbiosis between the host and intestinal microbial communities is essential for human health. Disruption in this symbiosis is linked to gastrointestinal diseases, including inflammatory bowel diseases, as well as extra-gastrointestinal diseases. Unbalanced gut microbiome or gut dysbiosis contributes in multiple ways to disease frequency, severity and progression. Microbiome taxonomic profiling and metabolomics approaches greatly improved our understanding of gut dysbiosis features; however, the precise mechanisms involved in gut dysbiosis establishment still need to be clarified. The aim of this review is to present new actors and mechanisms underlying gut dysbiosis formation following parasitic infection or in a context of altered Paneth cells, revealing the existence of a critical crosstalk between Paneth and tuft cells to control microbiome composition.

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.

This file contains supplementary data figures and methods. Supplementary figure S1 shows accumulation of Lgr5+-GFP+ ISCs following Apc inactivation. Supplementary figure S2 describes the aberrant gene expression program triggered by the loss of Apc function in Lgr5+ ISCs and their immediate progenitor daughter cells Supplementary figure S3 identifies the focal alterations in DNA methylation that are produced upon Apc loss of function in GFP+ cells. Supplementary figure S4 demonstrates that de novo DNA methylation critically contributes to homeostatic rupture upon Apc loss. Supplementary figure S5 shows the role of the Alk-pSmad1/5/8 axis in the responsiveness to BMP signalling in ApcKO miniguts.

This file contains all RNAseq results for gene expression comparisons between FACS sorted ApcWT-, ApcHET-, and ApcKO-Lgr5-GFP profiles.

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.

This file contains GSEA results from ApcWT- and ApcKO-Lgr5 cells for collections of signatures related to chemical and genetic perturbations.

Detailed legends for Supplementary Figures S1-S6.

Combined file including 6 supplementary figures. Figure S1 shows clustering analysis of TCGA breast cancer data, related to Fig.2. Figure S2 shows additional bioinformatics analysis data related to Fig 2. Figure S3 shows single cell analysis of cell cycle regulation of Ki-67 expression, related to Fig 3. Figure S4 shows controls for cell cycle arrest for experiments shown in Fig. 5. Figure S5 shows time course of beta-galactosidase activity upon senescence induction in experiment shown in Fig 5. Fig. S6 shows further bioinformatics analysis of CCLE data to correlate Ki-67 expression with drug sensitivity, related to Fig. 6.

This file contains user-defined GSEA signatures related to ISC, transit-amplifying and differentiated cell lineages used in this study.

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.