The Ets transcription factor Spi-B is essential for the differentiation of intestinal microfold cells.

Intestinal microfold cells (M cells) are an enigmatic lineage of intestinal epithelial cells that initiate mucosal immune responses through the uptake and transcytosis of luminal antigens. The mechanisms of M-cell differentiation are poorly understood, as the rarity of these cells has hampered analysis. Exogenous administration of the cytokine RANKL can synchronously activate M-cell differentiation in mice. Here we show the Ets transcription factor Spi-B was induced early during M-cell differentiation. Absence of Spi-B silenced the expression of various M-cell markers and prevented the differentiation of M cells in mice. The activation of T cells via an oral route was substantially impaired in the intestine of Spi-B-deficient (Spib(-/-)) mice. Our study demonstrates that commitment to the intestinal M-cell lineage requires Spi-B as a candidate master regulator.

Distinct developmental requirements for isolated lymphoid follicle formation in the small and large intestine: RANKL is essential only in the small intestine.

Cryptopatches (CPs) and isolated lymphoid follicles (ILFs) are organized intestinal lymphoid tissues that develop postnatally in mice and include stromal cells expressing the receptor activator of nuclear factor kappa-B ligand (RANKL). We investigated how stromal RANKL influences the development and differentiation of CPs and ILFs by analyzing the development of these lymphoid structures in knockout mice lacking RANKL. We found that RANKL(-/-) mice had a fourfold reduction in the overall density of CPs in the small intestine compared to control mice, with the largest decrease in the proximal small intestine. No B cells were present in CPs from the small intestine of RANKL(-/-) mice and ILF formation was completely blocked. In sharp contrast, colonic ILFs containing B cells were present in RANKL(-/-) mice. Stromal cells within CPs in the small intestine of RANKL(-/-) mice did not express CXCL13 (originally called B lymphocyte chemoattractant) and often lacked other normally expressed stromal cell antigens, whereas colonic lymphoid aggregates in RANKL(-/-) mice retained stromal CXCL13 expression. The CXCL13-dependent maturation of precursor CPs into ILFs is differentially regulated in the small intestine and colon, with an absolute requirement for RANKL only in the small intestine.

RANKL is necessary and sufficient to initiate development of antigen-sampling M cells in the intestinal epithelium.

Microfold cells (M cells) are specialized epithelial cells situated over Peyer's patches (PP) and other organized mucosal lymphoid tissues that transport commensal bacteria and other particulate Ags into intraepithelial pockets accessed by APCs. The TNF superfamily member receptor activator of NF-kappaB ligand (RANKL) is selectively expressed by subepithelial stromal cells in PP domes. We found that RANKL null mice have <2% of wild-type levels of PP M cells and markedly diminished uptake of 200 nm diameter fluorescent beads. Ab-mediated neutralization of RANKL in adult wild-type mice also eliminated most PP M cells. The M cell deficit in RANKL null mice was corrected by systemic administration of exogenous RANKL. Treatment with RANKL also induced the differentiation of villous M cells on all small intestinal villi with the capacity for avid uptake of Salmonella and Yersinia organisms and fluorescent beads. The RANK receptor for RANKL is expressed by epithelial cells throughout the small intestine. We conclude that availability of RANKL is the critical factor controlling the differentiation of M cells from RANK-expressing intestinal epithelial precursor cells.