PMC

TNF-α induces Relb and Nfkb2 expression and enhances RANKL-induced Relb and Nfkb2 (NF-κB subunit 2). A: relative expression of Relb and Nfkb2 after 4 h of no stimulation or stimulation with 50 ng/ml TNF-α, 100 ng/ml RANKL, or 50 ng/ml TNF-α + 100 ng/ml RANKL. Values are means ± SE of 3 experiments. B: relative expression of Relb and Nfkb2 after 1 day of no stimulation or stimulation with 50 ng/ml TNF-α, 100 ng/ml RANKL, or 50 ng/ml TNF-α + 100 ng/ml RANKL. Values are means ± SE of 4 experiments. *P < 0.05; **P < 0.01.

RANKL specifically induces Spib in enteroids. A–C: relative expression of Spib, Reg3g (regenerating islet-derived protein 3γ), and Saa1 (serum amyloid A1) in untreated enteroids and enteroids treated with RANKL (100 ng/ml) or IL-22 (50 ng/ml) for 1 day. Values are means ± SE of 3 experiments. D–F: relative expression of Spib and Ccl20 in untreated enteroids or enteroids treated with RANKL (100 ng/ml) or TNF-α (50 ng/ml) at 1 day and 4 h. Values are means ± SE of 4 experiments. *P < 0.05; **P < 0.01; ***P < 0.001. ns, Not significant.

RANKL induces M cell-specific gene expression in the absence of endogenous TNF-α. A and B: relative expression of Spib and Ccl20 by enteroids after 1 day of no stimulation (CTL) or stimulation with 50 ng/ml TNF-α or 100 ng/ml RANKL with or without a neutralizing anti-TNF-α antibody (5 μg/ml). Values are means ± SE of 3 experiments. ns, Not significant.

TNF-α does not induce Spib expression in RANK^ΔIEC enteroids. A and B: relative expression of Spib and Ccl20 by enteroids from mice with conditional deletion of the Tnfrsf11a gene encoding RANK in intestinal epithelial cells (RANK^ΔIEC) and RANK^F/F littermate controls that received no stimulation or enteroids stimulated with 50 ng/ml TNF-α, 100 ng/ml RANKL, or 50 ng/ml TNF-α + 100 ng/ml RANKL. Values are means ± SE of 3 experiments. *P < 0.05; **P < 0.01; ns, Not significant.

Addition of TNF-α does not increase the frequency of GP2⁺ M cells in RANKL-treated enteroids. GP2⁺ cells as a percentage of the total number of DAPI⁺ nuclei was compared in sections of enteroids treated for 3 days with 100 ng/ml RANKL or 50 ng/ml TNF-α + 100 ng/ml RANKL. Results are presented as a scatter plot of data collected from individual enteroids. Means ± SE for each group and number of enteroids are shown above plot. ns, Not significant.

Aly/aly enteroids do not express M cell-specific genes when stimulated with RANKL. A and B: relative expression of Spib and Gp2 following stimulation with RANKL (100 ng/ml) for 3 days in Alymphoplasia control (aly/+) and aly/aly enteroids. Values are means ± SE of 3 experiments. C: relative expression of Reg3g following treatment with IL-22 (50 ng/ml) for 4 h in aly/+ and aly/aly enteroids. Values are means ± SE of 3 experiments. D: relative expression of Ccl20 following treatment of aly/+ and aly/aly enteroids with TNF-α (50 ng/ml) for 1 day. Values are means ± SE of 2 experiments. *P < 0.05; **P < 0.01; ***P < 0.001. ns, Not significant.

TNF-α enhances RANKL-induced M cell-associated gene expression. A: relative expression of M cell-specific and follicle-associated epithelium (FAE)-specific genes after no stimulation or 1 day of stimulation with 50 ng/ml TNF-α, 100 ng/ml RANKL, or 50 ng/ml TNF-α + 100 ng/ml RANKL. Values are means ± SE of 3 experiments. Expression of all genes was increased in TNF-α + RANKL- compared with RANKL-treated enteroids (P < 0.05). B: relative expression of M cell-specific genes after 3 days of stimulation with 50 ng/ml TNF-α, 100 ng/ml RANKL, or 50 ng/ml TNF-α + 100 ng/ml RANKL. Values are means ± SE of 3 experiments. Expression of all genes except Anxa5 was significantly increased in TNF-α + RANKL- compared with RANKL-treated enteroids (P < 0.05). C: average fold induction of M cell- and FAE-specific genes by RANKL and TNF-α + RANKL. ND, not determined.

Enteroids stimulated with receptor activator of NF-κB ligand (RANKL) express M cell-associated genes. A and B: enteroids were stimulated with 100 ng/ml RANKL for 1 or 3 days. Expression of genes was determined by quantitative PCR and reported as relative expression normalized to Gapdh. Numbers above bars indicate fold induction of M cell-associated genes compared with untreated controls. Values are means ± SE of 4 experiments. Relative expression of all genes examined was increased after RANKL treatment compared with controls (P < 0.05 for each gene). Ccl9 and Ccl20, chemokine (C-C motif) ligands 9 and 20; Tnfaip2, TNF-α-induced protein 2; Marcksl1, myristoylated alanine-rich protein kinase C substrate; Anxa5, annexin A5; Tnfrsf11a, TNF-α receptor superfamily member 11A; Gp2, glycoprotein 2. C: immunofluorescence of control enteroids and enteroids treated with 100 ng/ml RANKL for 3 days. Blue, 4′,6-diamidino-2-phenylindole; green, E-cadherin (E-cad); red, GP2. Arrowheads indicate apical GP2 staining on single cells. CTL, control. Scale bars = 50 μm.

RANKL stimulation of enteroids induces the Spib-1 transcript of Spib. A: schematic of mouse chromosome 7 genomic DNA containing the promoters and first 4 exons of Spib. Exon-intron boundaries for the first 4 exons are numbered based on alignment of the National Center for Biotechnology Information Reference Sequence mRNA for mouse Spib (NM_019866.1) with 6,092 bp of C57BL/6 genomic chromosome 7 sequence (NC_000073.6, 44525995..44532086, complement), with nucleotide position 1 assigned based on the predicted 5′ end of a Spib mRNA with the maximum amount of 5′-untranslated sequence. Large arrowheads indicate κB and octamer binding sites located in the 1st and 2nd promoters, respectively. Individual exons are indicated by numbers in white circles. Exons or regions of exons in white boxes are unique to the Spib-1 and Spib-2 transcripts transcribed from promoters 1 and 2, respectively; exons in dark-gray boxes are present in both Spib-1 and Spib-2. Light gray portion at the 5′ end of exon 3 is an alternatively spliced region included in some Spib splice variants. F1, Spib-1-specific forward primer; F2, Spib-2-specific forward primer; CF, Spib common forward primer; CR, common reverse primer. B: agarose gel of PCR products obtained with Spib-1-specific, Spib-2-specific, and common Spib forward primers using cDNA from control enteroids and enteroids stimulated with 100 ng/ml RANKL for 1 day. C: relative expression of total Spib, Spib-1, and Spib-2 after 1 day of stimulation with 100 ng/ml RANKL. Values are means ± SE of 3 experiments. *P < 0.05; **P < 0.01. ns, Not significant.