Single Cell Analysis of Human Colonoids Exposed to Uranium-Bearing Dust.

BACKGROUND: Uranium exposure remains an important environmental legacy and physiological health concern, with hundreds of abandoned uranium mines located in the Southwestern United States largely impacting underserved indigenous communities. The negative effects of heavy metals on barrier permeability and inhibition of intestinal epithelial healing have been described; however, transcriptomic changes within the intestinal epithelial cells and impacts on lineage differentiation are largely unknown. OBJECTIVES: Herein, we sought to determine the molecular and cellular changes that occur in the colon in response to uranium bearing dust (UBD) exposure. METHODS: Human colonoids from three biologically distinct donors were acutely exposed to UBD then digested for single cell RNA sequencing to define the molecular changes that occur to specific identities of colonic epithelial cells. Validation in colonoids was assessed using morphological and imaging techniques. RESULTS: Human colonoids acutely exposed to UBD exhibited disrupted proliferation and hyperplastic differentiation of the secretory lineage cell, enteroendocrine cells (EEC). Single-cell RNA sequencing also showed more EEC subtypes present in UBD-exposed colonoids. DISCUSSION: These findings highlight the significance of crypt-based proliferative cells and secretory cell differentiation using human colonoids to model major colonic responses to uranium-bearing particulate dust exposure. https://doi.org/10.1289/EHP13855.

Uranium-bearing dust induces differentiation and expansion of enteroendocrine cells in human colonoids.

Chronic exposure to environmental toxins and heavy metals has been associated with intestinal inflammation, increased susceptibility to pathogen-induced diseases, and higher incidences of colorectal cancer, all of which have been steadily increasing in prevalence for the past 40 years. The negative effects of heavy metals on barrier permeability and inhibition of intestinal epithelial healing have been described; however, transcriptomic changes within the intestinal epithelial cells and impacts on lineage differentiation are largely unknown. Uranium exposure remains an important environmental legacy and physiological health concern, with hundreds of abandoned uranium mines located in the Southwestern United States largely impacting underserved indigenous communities. Herein, using human colonoids, we defined the molecular and cellular changes that occur in response to uranium bearing dust (UBD) exposure. We used single cell RNA sequencing to define the molecular changes that occur to specific identities of colonic epithelial cells. We demonstrate that this environmental toxicant disrupts proliferation and induces hyperplastic differentiation of secretory lineage cells, particularly enteroendocrine cells (EEC). EECs respond to UBD exposure with increased differentiation into de novo EEC sub-types not found in control colonoids. This UBD-induced EEC differentiation does not occur via canonical transcription factors NEUROG3 or NEUROD1. These findings highlight the significance of crypts-based proliferative cells and secretory cell differentiation as major colonic responses to heavy metal-induced injury.

Inflammatory macrophages prevent colonic goblet and enteroendocrine cell differentiation through Notch signaling.

Inflammatory macrophages in the intestine are a key pathogenic factor driving inflammatory bowel disease (IBD). Here, we report the role of inflammatory macrophage-mediated notch signaling on secretory lineage differentiation in the intestinal epithelium. Utilizing IL-10-deficient (Il10-/-) mice, a model of spontaneous colitis, we found an increase in Notch activity in the colonic epithelium as well as an increase in intestinal macrophages expressing Notch ligands, which are increased in macrophages upon inflammatory stimuli. Furthermore, a co-culture system of inflammatory macrophages and intestinal stem and proliferative cells during differentiation reduced goblet and enteroendocrine cells. This was recapitulated when utilizing a Notch agonist on human colonic organoids (colonoids). In summary, our findings indicate that inflammatory macrophages upregulate notch ligands that activate notch signaling in ISC via cell-cell interactions, which in turn inhibits secretory lineage differentiation in the gastrointestinal (GI) tract.

Intestinal Enteroendocrine Cells: Present and Future Druggable Targets.

Enteroendocrine cells are specialized secretory lineage cells in the small and large intestines that secrete hormones and peptides in response to luminal contents. The various hormones and peptides can act upon neighboring cells and as part of the endocrine system, circulate systemically via immune cells and the enteric nervous system. Locally, enteroendocrine cells have a major role in gastrointestinal motility, nutrient sensing, and glucose metabolism. Targeting the intestinal enteroendocrine cells or mimicking hormone secretion has been an important field of study in obesity and other metabolic diseases. Studies on the importance of these cells in inflammatory and auto-immune diseases have only recently been reported. The rapid global increase in metabolic and inflammatory diseases suggests that increased understanding and novel therapies are needed. This review will focus on the association between enteroendocrine changes and metabolic and inflammatory disease progression and conclude with the future of enteroendocrine cells as potential druggable targets.

Efficient RNA and RNA-protein co-detection in 3D colonoids by whole-mount staining.

Here, we describe a protocol to visualize RNA oligos and proteins independently or together using a combination of fluorescence in situ hybridization (FISH) and immunofluorescence in human colonoids, expanding on previously published research. Whole-mount staining is used to preserve the colonoid structure and fix onto glass slides. We describe procedures for efficient plating, fixation, and preservation of the colonoids. This workflow can be adapted to 3D organoid models from other tissues or organisms. For complete details on the use and execution of this protocol, please refer to In et al. (2020).

Epithelial WNT2B and Desert Hedgehog Are Necessary for Human Colonoid Regeneration after Bacterial Cytotoxin Injury.

Intestinal regeneration and crypt hyperplasia after radiation or pathogen injury relies on Wnt signaling to stimulate stem cell proliferation. Mesenchymal Wnts are essential for homeostasis and regeneration in mice, but the role of epithelial Wnts remains largely uncharacterized. Using the enterohemorrhagic E. coli-secreted cytotoxin EspP to induce injury to human colonoids, we evaluated a simplified, epithelial regeneration model that lacks mesenchymal Wnts. Here, we demonstrate that epithelial-produced WNT2B is upregulated following injury and essential for regeneration. Hedgehog signaling, specifically activation via the ligand Desert Hedgehog (DHH), but not Indian or Sonic Hedgehog, is another driver of regeneration and modulates WNT2B expression. These findings highlight the importance of epithelial WNT2B and DHH in regulating human colonic regeneration after injury.