Mechanisms of M Cell Differentiation: New Insights Acquired with Enteroid Cultures Open Access

Abstract

The primary function of the microfold (M) cell is to take up particulate antigen from the intestinal lumen and present it to the immune cell residing in its basolateral pocket. The M cell is unique to the epithelium overlying the Peyer's patch (PP), isolated lymphoid follicles, and cecal patch of the intestine where the epithelium is exposed to RANKL. M cell differentiation is dependent on RANKL induction of Spi-B and Spi-B dependent genes. RANKL also induces a distinctive gene expression pattern in the follicle-associated epithelium (FAE). M cell specific gene expression and differentiation have been difficult to study in vivo due to low numbers on restricted epithelial sites. RANKL stimulation of intestinal enteroids overcomes this problem by providing a larger number of M cells to study in vitro in a model that replicates M cell specific gene expression and increased transcytosis. The following studies increase the understanding of M cell differentiation in this model by introducing TNF-α to the RANKL stimulation of enteroids. TNF-α activates the canonical pathway of NF-κB leading to increased translation of the precursors for noncanonical NF-κB, Relb and Nfkb2. RANKL can then activate more noncanonical NF-κB heterodimers which results in increased and more rapid M cell specific gene expression. This model is an important tool to continue to study the early steps of M cell differentiation. One of the advantages of the enteroid culture system is that it allows the culture of intestinal epithelium from mice that are not viable past weaning. This advantage was exploited with the study of a conditional knockout of Atoh1 in the small intestinal epithelium. Mice with this mutation lack all secretory cells and rarely live to adulthood. Atoh1 deficient enteroids do not express M cell specific genes upon stimulation with RANKL. The role of Atoh1 in M cell differentiation suggests it belongs to the secretory lineage of intestinal epithelial differentiation. The M cell belonging to the secretory cell type will change the approach to further studies of the M cell as it shares early differentiation steps with a diverse group of specialized epithelial cells.

Table of Contents

ABSTRACT

LIST OF FIGURES

LIST OF TABLES

INTRODUCTION 1

RESULTS

CHAPTER 1: TNF-α Augments RANKL Dependent Intestinal M Cell Differentiation in Enteroid Cultures 13

CHAPTER 2: Intestinal M Cell Differentiation is Compromised in the Absence of Atoh1: Evidence that M Cells Belong to the Secretory Enterocyte Lineage 55

DISCUSSION 86

LITERATURE CITED IN INTRODUCTION AND DISCUSSION 95

LIST OF PUBLICATIONS 107

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Immunology
Keyword	RANKL Enteroid M cell
Committee Chair / Thesis Advisor	Williams, Ifor, Emory University
Committee Members	Weiss, David S, Emory University Boise, Lawrence, Emory University Neish, Andrew S, Emory University Denning, Timothy, Emory University

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