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Am J Physiol Cell Physiol. 2016 Sep 1;311(3):C498-507. doi: 10.1152/ajpcell.00108.2016. Epub 2016 Jul 13.

TNF-α augments RANKL-dependent intestinal M cell differentiation in enteroid cultures.

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Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia


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.


M cells; RANKL; Spi-B; TNF-α; enteroid culture

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