A single-cell survey of the small intestinal epithelium

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.

[1]  P. Linsley,et al.  MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data , 2015, Genome Biology.

[2]  J. Gordon,et al.  Examining the Role of Paneth Cells in the Small Intestine by Lineage Ablation in Transgenic Mice* , 1997, The Journal of Biological Chemistry.

[3]  Aleksandra A. Kolodziejczyk,et al.  Accounting for technical noise in single-cell RNA-seq experiments , 2013, Nature Methods.

[4]  T. Freeman,et al.  Identification of Novel Genes Selectively Expressed in the Follicle-Associated Epithelium from the Meta-Analysis of Transcriptomics Data from Multiple Mouse Cell and Tissue Populations , 2012, DNA research : an international journal for rapid publication of reports on genes and genomes.

[5]  J. Tack,et al.  REVIEWS IN BASIC AND CLINICAL GASTROENTEROLOGY The Serotonin Signaling System: From Basic Understanding To Drug Development for Functional GI Disorders , 2007 .

[6]  R. Xavier,et al.  Human leucine-rich repeat proteins: a genome-wide bioinformatic categorization and functional analysis in innate immunity , 2010, Proceedings of the National Academy of Sciences.

[7]  Laurens van der Maaten,et al.  Accelerating t-SNE using tree-based algorithms , 2014, J. Mach. Learn. Res..

[8]  N. Paton,et al.  Identification of Novel Genes in Intestinal Tissue That Are Regulated after Infection with an Intestinal Nematode Parasite , 2005, Infection and Immunity.

[9]  W. Garrett,et al.  Tuft cells, taste-chemosensory cells, orchestrate parasite type 2 immunity in the gut , 2016, Science.

[10]  P. Jay,et al.  Intestinal tuft cells: epithelial sentinels linking luminal cues to the immune system , 2016, Mucosal Immunology.

[11]  A. Buja,et al.  Remarks on Parallel Analysis. , 1992, Multivariate behavioral research.

[12]  R. Locksley,et al.  Tuft-cell-derived IL-25 regulates an intestinal ILC2–epithelial response circuit , 2015, Nature.

[13]  R. Batterham,et al.  The role of peptide YY in appetite regulation and obesity , 2009, The Journal of physiology.

[14]  T. Möröy,et al.  Gfi1b:green fluorescent protein knock-in mice reveal a dynamic expression pattern of Gfi1b during hematopoiesis that is largely complementary to Gfi1. , 2007, Blood.

[15]  Åsa K. Björklund,et al.  Full-length RNA-seq from single cells using Smart-seq2 , 2014, Nature Protocols.

[16]  Nathalie Perreault,et al.  The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon. , 2002, Development.

[17]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[18]  Matthew D. Young,et al.  Gene ontology analysis for RNA-seq: accounting for selection bias , 2010, Genome Biology.

[19]  John B. Furness,et al.  The gut as a sensory organ , 2013, Nature Reviews Gastroenterology &Hepatology.

[20]  A. Fürholz,et al.  Murine intestinal cells expressing Trpm5 are mostly brush cells and express markers of neuronal and inflammatory cells , 2008, The Journal of comparative neurology.

[21]  Hans-Peter Kriegel,et al.  A Density-Based Algorithm for Discovering Clusters in Large Spatial Databases with Noise , 1996, KDD.

[22]  A. van Oudenaarden,et al.  Induced Quiescence of Lgr5+ Stem Cells in Intestinal Organoids Enables Differentiation of Hormone-Producing Enteroendocrine Cells. , 2017, Cell stem cell.

[23]  Y. Taché,et al.  The bile acid TGR5 membrane receptor: from basic research to clinical application. , 2014, Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver.

[24]  H. Clevers,et al.  Identification of stem cells in small intestine and colon by marker gene Lgr5 , 2007, Nature.

[25]  R. Siezen,et al.  Dietary modulation and structure prediction of rat mucosal pentraxin (Mptx) protein and loss of function in humans , 2007, Genes & Nutrition.

[26]  Alessandro Laio,et al.  Clustering by fast search and find of density peaks , 2014, Science.

[27]  S. Linnarsson,et al.  Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq , 2015, Science.

[28]  Hans Clevers,et al.  Single-cell messenger RNA sequencing reveals rare intestinal cell types , 2015, Nature.

[29]  Geoffrey E. Hinton,et al.  Visualizing Data using t-SNE , 2008 .

[30]  W. de Lau,et al.  Peyer's Patch M Cells Derived from Lgr5+ Stem Cells Require SpiB and Are Induced by RankL in Cultured “Miniguts” , 2012, Molecular and Cellular Biology.

[31]  F. Reimann,et al.  Enteroendocrine cells-sensory sentinels of the intestinal environment and orchestrators of mucosal immunity , 2017, Mucosal Immunology.

[32]  Irving L. Weissman,et al.  Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal , 2017, Nature.

[33]  Cheng Li,et al.  Adjusting batch effects in microarray expression data using empirical Bayes methods. , 2007, Biostatistics.

[34]  Andrew E. Jaffe,et al.  Bioinformatics Applications Note Gene Expression the Sva Package for Removing Batch Effects and Other Unwanted Variation in High-throughput Experiments , 2022 .

[35]  T. Avnit-Sagi,et al.  Epithelial microRNAs regulate gut mucosal immunity via epithelium–T cell crosstalk , 2011, Nature Immunology.

[36]  Sean C. Bendall,et al.  viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia , 2013, Nature Biotechnology.

[37]  Raul Cano.  On The Bayesian Bootstrap , 1992 .

[38]  S. Duncan,et al.  GATA factors regulate proliferation, differentiation, and gene expression in small intestine of mature mice. , 2011, Gastroenterology.

[39]  David Artis,et al.  Intestinal epithelial cells: regulators of barrier function and immune homeostasis , 2014, Nature Reviews Immunology.

[40]  T. D. Du Clos.  Pentraxins: Structure, Function, and Role in Inflammation , 2013, ISRN inflammation.

[41]  Hui Liu,et al.  AnimalTFDB: a comprehensive animal transcription factor database , 2011, Nucleic Acids Res..

[42]  Ann B. Lee,et al.  Geometric diffusions as a tool for harmonic analysis and structure definition of data: diffusion maps. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Evan Z. Macosko,et al.  Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics , 2016, Cell.

[44]  H. Kiyono,et al.  Comprehensive Gene Expression Profiling of Peyer’s Patch M Cells, Villous M-Like Cells, and Intestinal Epithelial Cells1 , 2008, The Journal of Immunology.

[45]  Marco Bruschi,et al.  Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites , 2016, Nature.

[46]  M. Lazar,et al.  RELMbeta/FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinal tract. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[47]  Kevin W Eliceiri,et al.  NIH Image to ImageJ: 25 years of image analysis , 2012, Nature Methods.

[48]  Hanlee P. Ji,et al.  Haplotyping germline and cancer genomes using high-throughput linked-read sequencing , 2015, Nature Biotechnology.

[49]  H. Clevers,et al.  Mapping early fate determination in Lgr5+ crypt stem cells using a novel Ki67-RFP allele , 2014, The EMBO journal.

[50]  N A Mabbott,et al.  Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium , 2013, Mucosal Immunology.

[51]  Chien-wen Su,et al.  Coinfection with an Intestinal Helminth Impairs Host Innate Immunity against Salmonella enterica Serovar Typhimurium and Exacerbates Intestinal Inflammation in Mice , 2014, Infection and Immunity.

[52]  R. Desharnais,et al.  Expansion of Paneth Cell Population in Response to Enteric Salmonella enterica Serovar Typhimurium Infection , 2011, Infection and Immunity.

[53]  Hanlee P. Ji,et al.  Intestinal Enteroendocrine Lineage Cells Possess Homeostatic and Injury-Inducible Stem Cell Activity. , 2017, Cell stem cell.

[54]  C. Reynet,et al.  GPR119, a novel G protein‐coupled receptor target for the treatment of type 2 diabetes and obesity , 2008, British journal of pharmacology.

[55]  Sean Hughes,et al.  Clustering by Fast Search and Find of Density Peaks , 2016 .

[56]  R. Arsenescu,et al.  Intestinal Epithelial Serum Amyloid A Modulates Bacterial Growth In Vitro and Pro-Inflammatory Responses in Mouse Experimental Colitis , 2010, BMC gastroenterology.

[57]  Colin N. Dewey,et al.  RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome , 2011, BMC Bioinformatics.

[58]  Fabian J. Theis,et al.  Diffusion maps for high-dimensional single-cell analysis of differentiation data , 2015, Bioinform..

[59]  A. M. Habib,et al.  Co-localisation and secretion of glucagon-like peptide 1 and peptide YY from primary cultured human L cells , 2013, Diabetologia.

[60]  Camille Stephan-Otto Attolini,et al.  Mex3a Marks a Slowly Dividing Subpopulation of Lgr5+ Intestinal Stem Cells. , 2017, Cell stem cell.

[61]  M. Drent,et al.  The role of leptin and ghrelin in the regulation of food intake and body weight in humans: a review , 2007, Obesity reviews : an official journal of the International Association for the Study of Obesity.

[62]  F. Reimann,et al.  Enteroendocrine Cells: Chemosensors in the Intestinal Epithelium. , 2016, Annual review of physiology.

[63]  Takafumi Hara,et al.  Free fatty acid receptors act as nutrient sensors to regulate energy homeostasis. , 2009, Prostaglandins & other lipid mediators.

[64]  J. Dekker,et al.  REG3γ-deficient mice have altered mucus distribution and increased mucosal inflammatory responses to the microbiota and enteric pathogens in the ileum , 2013, Mucosal Immunology.

[65]  Martin Rosvall,et al.  Maps of random walks on complex networks reveal community structure , 2007, Proceedings of the National Academy of Sciences.

[66]  Sean C. Bendall,et al.  Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis , 2015, Cell.

[67]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[68]  Monika S. Kowalczyk,et al.  Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells , 2015, Genome research.