Fig 2: HOXA5 stabilization is instrumental for the transcriptional upregulation of Fn14 upon NETs blockade. (A) Known transcription factor (TF)-binding motifs in Fn14 gene promoter predicted by JASPAR database. The name and ID for each TF are shown. (B) Western-blotting analyses comparing Fn14 protein levels in the LPS-primed HK-2 cells with control siRNA (si.Ctrl), SPIB siRNA (si.SPIB), HOXA5 siRNA (si.HOXA5), RXRA siRNA (si.RXRA), ESRRB siRNA (si.ESRBB), RORA_1 siRNA (si. RORA_1) and CREB1 siRNA (si.CREB1) transfection, respectively. (C) RT-qPCR analyses evaluating levels of Fn14 mRNA expression in the LPS-primed HK-2 cells with control siRNA (si.Ctrl), SPIB siRNA (si.SPIB), HOXA5 siRNA (si.HOXA5), RXRA siRNA (si.RXRA), ESRRB siRNA (si.ESRBB), RORA_1 siRNA (si. RORA_1) and CREB1 siRNA (si.CREB1) transfection, respectively. Data are expressed as mean ± s.d. One-way ANOVA with Tukey's test post hoc comparisons was used to calculate the P value. (D) Western-blotting analyses examining Fn14 protein levels in the LPS-primed HK-2 cells with scrambled shRNA (Scr), HOXA5 shRNA (sh.HOXA5) and HOXA1 shRNA (sh.HOXA1) transfection in the presence or absence of the Flag-tagged wild-type HOXA5 re-expression, respectively. (E) RT-qPCR analyses assessing levels of Fn14 mRNA expression in the LPS-primed HK-2 cells with scrambled shRNA (Scr), HOXA5 shRNA (sh.HOXA5) and HOXA1 shRNA (sh.HOXA1) transfection in the presence or absence of the Flag-tagged wild-type HOXA5 re-expression, respectively. Data are expressed as mean ± s.d. One-way ANOVA with Tukey's test post hoc comparisons was used to calculate the P value. ns, no significant. (F) Left panel: Western-blotting analyses determining HOXA5 protein levels in the LPS-primed HK-2 cells with scrambled shRNA (Scr) or HOXA5 shRNA (sh.HOXA5) transfection. Right panel: Western-blotting analyses detecting Fn14 protein levels in the LPS-primed HK-2 cells with scrambled shRNA (Scr) or HOXA5 shRNA (sh.HOXA5) transfection in the presence or absence of SIVE plus Cl-Amidine cotreatment. (G) RT-qPCR analyses measuring levels of Fn14 mRNA expression in the LPS-primed HK-2 cells with scrambled shRNA (Scr), or HOXA5 shRNA (sh.HOXA5) transfection in the presence or absence of SIVE plus Cl-Amidine cotreatment. Data are expressed as mean ± s.d. One-way ANOVA with Tukey's test post hoc comparisons was used to calculate the P value. ns, no significant. (H) Luciferase assays of the LPS-primed HK-2 cells with scrambled shRNA (Scr) or HOXA5 shRNA (sh.HOXA5) transfection in the presence or absence of SIVE plus Cl-Amidine cotreatment. Data are expressed as mean ± s.d. One-way ANOVA with Tukey's test post hoc comparisons was used to calculate the P value. (I) ChIP analysis for HOXA5, HOXA1 and IgG binding to Fn14 gene promoter in the LPS-primed HK-2 cells with SIVE plus Cl-Amidine cotreatment. Enrichment of promoter region was normalized by input and data are expressed as mean ± s.d. of at least four experiments. Unpaired, two-tailed Student's t test was used to calculate the P value. ns, no significant. (J) Western-blotting analyses testing HOXA5 and HOXA1 protein levels in the LPS-primed HK-2 cells with SIVE plus Cl-Amidine cotreatment at the indicated time points. (K) CHX pulse-chase experiments measuring the turnover of HOXA5 protein in the LPS-primed HK-2 cells with SIVE plus Cl-Amidine cotreatment in the presence or absence of 20 µg/mL CHX treatment for the indicated times. (L) Western-blotting analyses comparing HOXA5 protein levels in the LPS-primed HK-2 cells treated with CHX in the presence or absence of MG132 (10 µM) treatment, Bortezomib (1 µM) treatment or SIVE plus Cl-Amidine cotreatment.

Fig 3: Esrrg expression and its relation to other M-cell developmental markers Spi-B and Sox8. (A) Esrrg expression was unaffected by lack of Sox8 expression. qPCR analysis of Esrrg and Gp2 in the FAE and villous epithelium from Sox8+/+ and Sox8-/- mice. (B) Organoids generated from Sox8+/+ and Sox8-/- mice were stimulated with and without RankL for 4 days. Esrrg expression was examined by qPCR analysis. (C) Organoids isolated from Sox8 wild-type and Sox8 KO mice were lysed and analyzed by Western blot for Esrrg expression. (D) Spi-B protein expression in Spi-B KO and Scrambled cells generated by lentiCRISPR v2 genome editing in C57BL/6JRj intestinal organoids. Organoid lysates were analyzed by Western blot. (E) qPCR analysis of Esrrg in a Spi-B KO intestinal organoid by lentiCRISPR v2. (F) Organoid lysates for Esrrg in Scrambled and SpiB KO organoids were analyzed by Western blot. Values in all are presented as the means ± SD. Unpaired 2-tailed Student t test, N = 3. *P < .05, **P < .01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; rRNA, ribosomal RNA.

Fig 4: Esrrg expression is induced by RelB/p52 activation. (A) Lta1ß2 prominently up-regulated the expression of Esrrg in organoids. Organoids from C57BL/6JRj mice were stimulated with LTa1ß2 for 3 days, and the gene expression was analyzed by qPCR. (B) Organoids from wild-type mice were stimulated with RankL for 3 days in the absence or presence of 125 µmol/L SC-514. Gene expression was analyzed by qPCR. (C) Organoids were transduced to express classic and noncanonical NF-?B and the expression of Esrrg and Spi-B (control) are represented relative to glyceraldehyde-3-phosphate dehydrogenase (Gapdh). (D) Organoids expressing p52 and RelB in the presence of SC-514 for 3 days, and the expression of Esrrg and Spi-B was analyzed by qPCR. Values in all are presented as the means ± SD and an unpaired 2-tailed Student t test was performed, N = 3 independent experiments. *P < .05, **P < .01, ***P < .005. Ctrl, control; DMSO, dimethyl sulfoxide.

Fig 5: Esrrg alone is not sufficient for maturation of Gp2+ M cells. (A) Intestinal organoids were dissociated and transduced by lentivirus encoding Esrrg. qPCR analysis of Spi-B, Sox8, and GP2 showed no significant changes. (B) Organoids were grown in the presence and absence of 100 ng/mL Rankl and 1 um tamoxifen-antagonist of Esrrg. Spi-B, Gp2, and Sox8 expression were analyzed with RT-qPCR. (C) Intestinal organoids from mice were grown in the presence and absence of 100 ng/mL Rankl and 10 um GSK 4718-agonist of Esrrg for 3 days. Spi-B, Gp2, and Sox8 were analyzed with RT-qPCR. Values are presented as the means ± SD. Unpaired 2-tailed Student t test, N = 3. *P < .05, **P < .01, ***P < .005, ****P < .0005. Ctrl, control; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; O/E, overexpression.