Single-cell RNA-seq of germ-free and specific pathogen-free small intestinal epithelium
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ABSTRACT: The small intestinal epithelial barrier inputs signals from the gut microbiota in order to balance physiological inflammation and tolerance, and to promote homeostasis. Understanding the dynamic relationship between microbes and intestinal epithelial cells has been a challenge given the cellular heterogeneity associated with the epithelium and the inherent difficulty of isolating and identifying individual cell types. Here, we used single-cell RNA sequencing of small intestinal epithelial cells from germ-free and specific pathogen-free mice to study microbe-epithelium crosstalk at the single cell resolution.
Project description:We developed a compartmental model of the small intestinal epithelium that describes stem and progenitor cell proliferation and differentiation and cell migration onto the villus. The model includes a negative feedback loop from villus cells to regulate crypt proliferation and integrates heterogeneous epithelial-related processes, such as the transcriptional profile, citrulline kinetics and probability of diarrhea.
Project description:The small intestinal epithelium is composed of several defined epithelial cell lineages, which in turn exhibit marked transcriptional and functional diversity along the crypt-villus and proximal-to-distal length of the intestinal tract. While epithelial cell type and functional heterogeneity have been characterized in the adult intestinal epithelium, the temporal and spatial changes during the postnatal period, which accompany the transition from placental energy supply to enteral feeding and facilitate the establishment of the enteric microbiota and postnatal immune maturation, have not been systematically investigated. Here we analyzed the total small intestinal epithelium of 1-, 5-, 10-, and 25-day-old specific pathogen-free (SPF) and germ-free (GF) mice by bulk RNA-Seq and used differential gene expression and pathway analysis to identify age-specific expression patterns. In addition, the influence of enteric infection on the neonatal epithelium was investigated by bulk RNA-Seq. We identify gene clusters temporally expressed in the neonatal intestine and correlate their expression with the functional changes during postnatal tissue maturation and the neonate to adult transition.
Project description:To assess the role of LSD1 in mouse small intestinal epithelium, we grew small intestinal organoids in vitro from mice with an epithelial specific deletion of LSD1 (Villin-Cre+; Lsd1f/f) and from wild type (Villin-Cre-; Lsd1f/f) mice. This experiment uses a new Cre strain with 100% recombination efficiency. Similar to intestinal epithelium from mice with an intestinal epithelium specific LSD1-KO, Paneth cells are not present in LSD1-KO small intestinal organoids. We used these sequencing data to show intrinsic epithelial changes in the intestinal epithelium caused by LSD1 deletion in the absence of microbiota and surrounding in vivo cell types.
Project description:Significant progress has been recently achieved in generating in vitro human small intestine models. Nonetheless, there remains ample opportunity for enhancement, both in terms of structure and function, within the current model. In this study, our objective is to construct a multilayered small intestinal tissue composed of an intestinal epithelium, supportive mesenchymal cells, and an extracellular matrix. We developed a multilayered small intestinal tissue by differentiating human induced pluripotent stem cells on a microfluidic device capable of replicating interstitial flow. Under the interstitial flow exposure, a three-dimensional villus-like epithelium and mature intestinal epithelial cells, including polarized enterocytes or goblet cells, were observed in the small intestine tissue-on-a-chips. Further analysis revealed that intestinal fibroblasts and collagen fiber are localized to the basolateral side of the intestinal epithelium. Moreover, we confirmed that small intestine tissue-on-a-chips are useful for pharmaceutical and infectious disease research. Our small intestine tissue-on-a-chips not only overcome the limitations of conventional small intestine models but also offer a unique opportunity to understand the mechanisms underlying intestinal tissue development.
Project description:When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as host-pathogen intreaction shows great promise for modelling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modelling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a dramatic reduction in parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection.
Project description:The intestinal epithelium is our first line of defense against infections of the gut and the plasticity in cellular differentiation of the intestinal epithelium is an important part of this response. Here we sequenced the small intestinal epithelium from mice infected with Nippostrongylus brasiliensis to determine how the intestinal epithelium adapts in the context of an infection. By comparing these data to small intestinal organoids treated with cytokines (see related accessions) we determine that the intestinal epithelial response to N. brasiliensis infection correspond to a type II infection driven by IL-13.
Project description:We report that adhesion of microbes to intestinal epithelial cells is a critical cue for Th17 induction. SFB colonized in the intestine of mice can adhere to mouse small intestinal epithelial cells and induce intestinal Th17 cells. However, SFB colonized in rats cannot adhere to mouse intestinal epithelial cells and induce Th17 cells. Likewise, Citrobacter rodentium (WT) can adhere to mouse colonic epithelial cells and induce Th17 cells, but non-adherent mutant of C. rodentium (Δeae) cannot induce Th17 cells. To examine the influence of adherent bacteria on intestinal epithelial cells, we performed RNA seq. Germ free mice were orally inoculated with M-SFB or R-SFB and total RNA was isolated from small intestinal epithelial cells 1 week after inoculation. Alternatively, germ free mice were orally inoculated with C. rodentium WT or eae mutant and total RNA was isolated from colonic epithelial cells 5 days after inoculation. The gene expression of small intestinal epithelial cells isolated from small intestine of germ free mice (2 mice), mice monocolonized with M-SFB (2 mice) or R-SFB (3 mice), and colon of germ free mice (3 mice), mice monocolonized C. rodentium WT (3 mice) or eae mutant (3 mice).
Project description:Commensal microbiota contribute to gut homeostasis and influence gene expression. Intestinal organoid culture closely represent intestinal epithelium and retain intestinal stem cells and dynamic recovery capabilities as well as all major cell types of the intestinal epithelium. We established organoid culture using colon crypts isolated from germ-free (GF), and gnotobiotic mice monocolonized either with the E.coli strain O6K13 (O) or Nissle 1917 strain (N). The expression profiles of these organoids were compared to the organoid culture isolated from conventionally reared (CR) mice in order to disclose genes differentially expressed in response to the change in the intestinal microflora composition.