Novel protocol to observe the intestinal tuft cell using transmission electron microscopy

Tuft cell is a chemosensory cell, a specific cell type sharing the taste transduction system with a taste cell on the tongue, of which the existence has been known in various tissues such as gastrointestinal tract, gall bladder, trachea, pancreatic duct, etc. To date, electron microscopic approaches have shown various morphological features of the tuft cell such as long and thick microvilli, tubulovesicular network at the apical side, prominent skeleton structures, etc. Recently, it has been reported that the small intestinal tuft cell functions to initiate type2 immunity in response to helminth infection. However, the mechanisms by which such distinguished structures are involved with the physiological functions are poorly understood. To address this question, the combination of physiological study regarding the tuft cells using genetic models and its morphological study using electron microscopy will be required. However, it is a challenge to observe tuft cells by electron microscopy due to their extremely low frequency on the epithelium. Therefore, in this paper, we suggest the advanced protocol to observe the small intestinal tuft cell efficiently by transmission electron microscopy using serial semi-thin sections on the Aclar film.

RNA binding protein DDX5 directs tuft cell specification and function to regulate microbial repertoire and disease susceptibility in the intestine

ObjectiveTuft cells residing in the intestinal epithelium have diverse functions. In the small intestine, they provide protection against inflammation, combat against helminth and protist infections, and serve as entry portals for enteroviruses. In the colon, they had been implicated in tumourigenesis. Commitment of intestinal progenitor cells to the tuft cell lineage requires Rho GTPase Cell Division Cycle 42 (CDC42), a Rho GTPase that acts downstream of the epidermal growth factor receptor and wingless-related integration site signalling cascades, and the master transcription factor POU class 2 homeobox 3 (POU2F3). This study investigates how this pathway is regulated by the DEAD box containing RNA binding protein DDX5 in vivo.DesignWe assessed the role of DDX5 in tuft cell specification and function in control and epithelial cell-specific Ddx5 knockout mice (DDX5ΔIEC) using transcriptomic approaches.ResultsDDX5ΔIEC mice harboured a loss of intestinal tuft cell populations, modified microbial repertoire, and altered susceptibilities to ileal inflammation and colonic tumourigenesis. Mechanistically, DDX5 promotes CDC42 protein synthesis through a post-transcriptional mechanism to license tuft cell specification. Importantly, the DDX5-CDC42 axis is parallel but distinct from the known interleukin-13 circuit implicated in tuft cell hyperplasia, and both pathways augment Pou2f3 expression in secretory lineage progenitors. In mature tuft cells, DDX5 not only promotes integrin signalling and microbial responses, it also represses gene programmes involved in membrane transport and lipid metabolism.ConclusionRNA binding protein DDX5 directs tuft cell specification and function to regulate microbial repertoire and disease susceptibility in the intestine.

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.

Single-cell sequencing of rotavirus-infected intestinal epithelium reveals cell-type specific epithelial repair and tuft cell infection

Intestinal epithelial damage is associated with most digestive diseases and results in detrimental effects on nutrient absorption and production of hormones and antimicrobial defense molecules. Thus, understanding epithelial repair and regeneration following damage is essential in developing therapeutics that assist in rapid healing and restoration of normal intestinal function. Here we used a well-characterized enteric virus (rotavirus) that damages the epithelium at the villus tip but does not directly damage the intestinal stem cell, to explore the regenerative transcriptional response of the intestinal epithelium at the single-cell level. We found that there are specific Lgr5+ cell subsets that exhibit increased cycling frequency associated with significant expansion of the epithelial crypt. This was accompanied by an increase in the number of immature enterocytes. Unexpectedly, we found rotavirus infects tuft cells. Transcriptional profiling indicates tuft cells respond to viral infection through interferon-related pathways. Together these data provide insights as to how the intestinal epithelium responds to insults by providing evidence of stimulation of a repair program driven by stem cells with involvement of tuft cells that results in the production of immature enterocytes that repair the damaged epithelium.

Tuft cell integration of luminal states and interaction modules in tissues

Chemosensory processes are integral to the physiology of most organisms. This function is typically performed by specialized cells that are able to detect input signals and to convert them to an output dedicated to a particular group of target cells. Tuft cells are cholinergic chemosensory epithelial cells capable of producing immunologically relevant effector molecules. They are scattered throughout endoderm-derived hollow organs and function as sensors of luminal stimuli, which has been best studied in mucosal barrier epithelia. Given their epithelial origin and broad distribution, and based on their interplay with immune pathways, tuft cells can be considered a prototypical example of how complex multicellular organisms engage innate immune mechanisms to modulate and optimize organ physiology. In this review, I provide a concise overview of tuft cells and discuss how these cells influence organ adaptation to dynamic luminal conditions.

CHD8 regulates gut epithelial cell function and affects autism-related behaviours through the gut-brain axis

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by early onset deficits in social behavior and repetitive behavior. Chromodomain helicase DNA binding protein (CHD8) is one of the genes with the strongest association to autism. Alongside with the core symptoms of ASD, individuals with ASD are reported to have gastrointestinal (GI) problems, and a majority of individuals with CHD8 mutations display GI problems. However, the relationship between autism related genes, such as CHD8, gastrointestinal function, and autism related behaviours are yet very unclear. In the current study, we found that mice haploinsufficient for CHD8 have leaky gut, a dysregulated transcriptome in gut epithelial cells, decreased gut tuft cells and goblet cells, and an increase in microbial load. Specific deletion of CHD8 in gut epithelial cells induced an increase in anxiety-related behaviours in, a phenotype that is often observed in autism and full body knockdown of CHD8, in addition to decreased tuft cells. In addition, antibiotic treatment of CHD8 haploinsufficient mice attenuates sociability deficits. Therefore, the current study determines a pathway for autism-related GI deficits, and how these deficits may play a direct role in the development of autism-related behaviours.

Tuft cell-produced cysteinyl leukotrienes and IL-25 synergistically drive lung type 2 inflammation

Aeroallergen sensing by airway epithelial cells can trigger pathogenic immune responses leading to chronic type 2 inflammation, the hallmark of airway diseases such as asthma. Airway tuft cells are specialized chemosensory epithelial cells and the dominant source of the epithelial cytokine IL-25 in the trachea and of cysteinyl leukotrienes (CysLTs) in the naive murine nasal mucosa. The interaction of IL-25 and CysLTs and the contribution of tuft cell-derived CysLTs to the development of allergen-triggered inflammation in the airways has not been clarified. Here we show that inhalation of LTC4 in combination with a subthreshold dose of IL25 leads to dramatic synergistic induction of type 2 inflammation throughout the lungs, causing rapid eosinophilia, dendritic cell (DC) and inflammatory type 2 innate lymphoid cell (ILC2) expansion, and goblet cell metaplasia. While lung eosinophilia is dominantly mediated through the classical CysLT receptor CysLT1R, type 2 cytokines and activation of innate immune cells require signaling through both CysLT1R and CysLT2R. Tuft cell-specific deletion of the terminal enzyme requisite for CysLT production, Ltc4s, was sufficient to reduce both the innate immune response in the lung: eosinophilia, ILC2 activation and DC recruitment, and the systemic immune response in the draining lymph nodes after inhalation of the mold aeroallergen Alternaria. Our findings identify surprisingly potent synergy of CysLTs and IL-25 downstream of aeroallergen-trigged activation of airway tuft cells leading to a highly polarized type 2 immune response and further implicate airway tuft cells as powerful modulators of type 2 immunity in the lungs.

Enteric Tuft Cells in Host-Parasite Interactions

Enteric tuft cells are chemosensory epithelial cells gaining attention in the field of host-parasite interactions. Expressing a repertoire of chemosensing receptors and mediators, these cells have the potential to detect lumen-dwelling helminth and protozoan parasites and coordinate epithelial, immune, and neuronal cell defenses against them. This review highlights the versatility of enteric tuft cells and sub-types thereof, showcasing nuances of tuft cell responses to different parasites, with a focus on helminths reflecting the current state of the field. The role of enteric tuft cells in irritable bowel syndrome, inflammatory bowel disease and intestinal viral infection is assessed in the context of concomitant infection with parasites. Finally, the review presents pertinent questions germane to understanding the enteric tuft cell and its role in enteric parasitic infections. There is much to be done to fully elucidate the response of this intriguing cell type to parasitic-infection and there is negligible data on the biology of the human enteric tuft cell—a glaring gap in knowledge that must be filled.

Interleukin-4 Promotes Tuft Cell Differentiation and Acetylcholine Production in Intestinal Organoids of Non-Human Primate

In the intestine, the innate immune system excludes harmful substances and invading microorganisms. Tuft cells are taste-like chemosensory cells found in the intestinal epithelium involved in the activation of group 2 innate lymphoid cells (ILC2). Although tuft cells in other tissues secrete the neurotransmitter acetylcholine (ACh), their function in the gut remains poorly understood. In this study, we investigated changes in the expression of genes and cell differentiation of the intestinal epithelium by stimulation with interleukin-4 (IL-4) or IL-13 in macaque intestinal organoids. Transcriptome analysis showed that tuft cell marker genes were highly expressed in the IL-4- and IL-13-treated groups compared with the control, and the gene expression of choline acetyltransferase (ChAT), a synthesis enzyme of ACh, was upregulated in IL-4- and IL-13-treated groups. ACh accumulation was observed in IL-4-induced organoids using high-performance liquid chromatography-mass spectrometry (HPLC/MS), and ACh strongly released granules from Paneth cells. This study is the first to demonstrate ACh upregulation by IL-4 induction in primates, suggesting that IL-4 plays a role in Paneth cell granule secretion via paracrine stimulation.