title: TNF-α augments RANKL-dependent intestinal M cell differentiation in enteroid cultures.
abstract: Microfold (M) cells are phagocytic intestinal epithelial cells in the follicle-associated epithelium of Peyer's patches that transport particulate antigens from the gut lumen into the subepithelial dome. Differentiation of M cells from epithelial stem cells in intestinal crypts requires the cytokine receptor activator of NF-κB ligand (RANKL) and the transcription factor Spi-B. We used three-dimensional enteroid cultures established with small intestinal crypts from mice as a model system to investigate signaling pathways involved in M cell differentiation and the influence of other cytokines on RANKL-induced M cell differentiation. Addition of RANKL to enteroids induced expression of multiple M cell-associated genes, including Spib, Ccl9 [chemokine (C-C motif) ligand 9], Tnfaip2 (TNF-α-induced protein 2), Anxa5 (annexin A5), and Marcksl1 (myristoylated alanine-rich protein kinase C substrate) in 1 day. The mature M cell marker glycoprotein 2 (Gp2) was strongly induced by 3 days and expressed by 11% of cells in enteroids. The noncanonical NF-κB pathway was required for RANKL-induced M cell differentiation in enteroids, as addition of RANKL to enteroids from mice with a null mutation in the mitogen-activated protein kinase kinase kinase 14 (Map3k14) gene encoding NF-κB-inducing kinase failed to induce M cell-associated genes. While the cytokine TNF-α alone had little, if any, effect on expression of M cell-associated genes, addition of TNF-α to RANKL consistently resulted in three- to sixfold higher levels of multiple M cell-associated genes than RANKL alone. One contributing mechanism is the rapid induction by TNF-α of Relb and Nfkb2 (NF-κB subunit 2), genes encoding the two subunits of the noncanonical NF-κB heterodimer. We conclude that endogenous activators of canonical NF-κB signaling present in the gut-associated lymphoid tissue microenvironment, including TNF-α, can play a supportive role in the RANKL-dependent differentiation of M cells in the follicle-associated epithelium.
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Megan B
authors.initials: MB
authors.lastname: Wood
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Daniel
authors.initials: D
authors.lastname: Rios
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia irwilli@emory.edu.
authors.forename: Ifor R
authors.identifier: 0000-0002-8810-2911
authors.initials: IR
authors.lastname: Williams
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology*; D005260:Female; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000502:physiology*; D007422:Intestines / Q000502:physiology*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism; D053245:RANK Ligand / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013234:Stem Cells / Q000378:metabolism / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D016328:NF-kappa B; D053245:RANK Ligand; C505689:Tnfsf11 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D020930:MAP Kinase Kinase Kinases
keywords: M cells; RANKL; Spi-B; TNF-α; enteroid culture