Fig. 3.

N SCs and healthy intestinal epithelial cells synthesize RA; however, C SCs synthesize less RA than N SCs. (A) Schematic of RA metabolism. (B) RDH10 and ALDH1A1 mRNA expression in normal epithelial cells (n = 9), Crohn’s epithelial cells (n = 2), N SCs (n = 7), and C SCs (n = 6). (C) ALDH activity in normal epithelial cells (n = 7), N SCs (n = 15) and C SCs (n = 15). (D) RA synthesis by normal intestinal epithelial cells (n = 7), N SCs (n = 6), C SCs (n = 6), and dermal cells (n = 6). (E) DHRS3 mRNA expression by N SCs and C SCs (n > 10). (F) Upper panel: Expression pattern of genes in the RA metabolism pathway. Lower panel: Expression level of genes in unstimulated C SCs versus unstimulated N SCs (column 1) and LF82-stimulated C SCs versus LF82-stimulated N SCs (column 2). Values are the activation z-scores; values >2.0 considered significantly activated and values < −2.0 considered significantly inhibited (n = 16). (G, H) C SCs were mock-transfected or transfected with DHRS3 siRNA, cultured in the presence or absence of ALDH enzyme inhibitor (DEAB), and analyzed for ALDH activity (ALDH-FITC) by two pathologists who enumerated FITC-positive SCs in 10 separate, random fields per treatment per experiment in a blinded protocol. Upper panels, bright field, and lower panels, FITC-positive donor-matched SCs. (n = 4). (I) Proliferation (CFSE) and CCR9 expression by anti-CD3/CD28-stimulated CD4⁺ T cells cultured with N SCs, C SCs ± RAI (2 mM), or medium alone (3 days; insets: isotype controls) (n ≥ 5) (mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001). ALDH = aldehyde dehydrogenae-; C SCs = Crohn’s SCs; CD = cluster of differentiation; CFSE = carboxyfluorescein diacetate succinimidyl ester; DEAB = diethylaminobenzaldehyde; FITC = fluorescein isothiocyanate; mRNA = messenger ribonucleic acid; N SCs = normal SCs; RA = retinoic acid; RAI = retinoic acid inhibitor; SEM = standard error of the mean; siRNA = small interfering ribonucleic acid.