{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T17:08:16Z","timestamp":1792084096064,"version":"4.4.0"},"reference-count":57,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2025,8,22]],"date-time":"2025-08-22T00:00:00Z","timestamp":1755820800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Retinoic acid (RA) plays a key role in mucosal immune regulation and tolerance, with implications for inflammatory bowel disease (IBD). However, its effects have not been extensively studied in humanized in vitro models that recapitulate epithelial\u2013immune interactions. We established a 3D in vitro small intestinal model composed of three epithelial cell types, na\u00efve CD4+ T cells, and monocyte\/dendritic cell (M\/DC) precursors derived from CD34+ umbilical cord blood hematopoietic stem\/progenitor cells. The epithelial microenvironment strongly suppressed monocyte\/DC differentiation and T cell activation, indicating a regulatory role of epithelial-derived signals. Retinoic acid (RA) priming of M\/DC precursors induced CD103+CD11b+Sirp1\u03b1\u2212 regulatory DCs and promoted a shift from naive to memory-type T cells. Upon addition of pro-inflammatory cytokines (TNF-\u03b1, IFN-\u03b3, IL-1\u03b2), the model mimicked an inflamed intestinal state, resulting in CD14+CD16+ inflammatory monocytes and increased T cell activation (CD25+CD69+). RA-primed DCs modestly counterbalanced T cell activation and IBD-like responses, even under inflammatory conditions. Flow cytometry and clustering analysis revealed distinct immune cell phenotypes depending on RA exposure and cytokine context. This model provides a reproducible and physiologically relevant human system to study RA-mediated immune programming in the intestinal mucosa and may support the development of novel therapeutic strategies for IBD and related inflammatory conditions. Statistical differences were evaluated using ANOVA with Tukey\u2019s post-hoc test (n = 4; p &lt; 0.05).<\/jats:p>","DOI":"10.3390\/cells14171300","type":"journal-article","created":{"date-parts":[[2025,8,22]],"date-time":"2025-08-22T11:50:31Z","timestamp":1755863431000},"page":"1300","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Retinoic Acid Modulates Immune Differentiation in a Human Small Intestinal In Vitro Model"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3706-9514","authenticated-orcid":false,"given":"Christa","family":"Schimpel","sequence":"first","affiliation":[{"name":"Division of Immunology, Otto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Medical University of Graz, 8010 Graz, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christina","family":"Passegger","sequence":"additional","affiliation":[{"name":"Division of Immunology, Otto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Medical University of Graz, 8010 Graz, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3123-6342","authenticated-orcid":false,"given":"Carmen","family":"Tam-Amersdorfer","sequence":"additional","affiliation":[{"name":"Division of Immunology, Otto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Medical University of Graz, 8010 Graz, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Herbert","family":"Strobl","sequence":"additional","affiliation":[{"name":"Division of Immunology, Otto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Medical University of Graz, 8010 Graz, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1038\/nrgastro.2015.186","article-title":"Immunopathogenesis of IBD: Current state of the art","volume":"13","author":"Fiocchi","year":"2016","journal-title":"Nat. Rev. Gastroenterol. Hepatol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1627","DOI":"10.1016\/S0140-6736(07)60750-8","article-title":"Inflammatory bowel disease: Cause and immunobiology","volume":"369","author":"Baumgart","year":"2007","journal-title":"Lancet"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Ramos, G.P., and Papadakis, K.A. (2019). Mechanisms of disease: Inflammatory bowel diseases. Mayo Clinic Proceedings, Elsevier.","DOI":"10.1016\/j.mayocp.2018.09.013"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1111\/imr.12192","article-title":"Macrophages in intestinal homeostasis and inflammation","volume":"260","author":"Bain","year":"2014","journal-title":"Immunol. Rev."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/j.cell.2014.03.011","article-title":"Role of the microbiota in immunity and inflammation","volume":"157","author":"Belkaid","year":"2014","journal-title":"Cell"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.coi.2013.12.005","article-title":"Regulatory T cells in cancer immunotherapy","volume":"27","author":"Nishikawa","year":"2014","journal-title":"Curr. Opin. Immunol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1016\/j.it.2011.06.003","article-title":"Intestinal CD103+ dendritic cells: Master regulators of tolerance?","volume":"32","author":"Scott","year":"2011","journal-title":"Trends Immunol."},{"key":"ref_8","first-page":"823710","article-title":"Peyer\u2032 s Patches: The Immune Sensors of the Intestine","volume":"2010","author":"Jung","year":"2010","journal-title":"Int. J. Inflamm."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"113795","DOI":"10.1016\/j.addr.2021.05.005","article-title":"An overview of in vitro, ex vivo and in vivo models for studying the transport of drugs across intestinal barriers","volume":"175","author":"Xu","year":"2021","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1190","DOI":"10.1126\/science.1234852","article-title":"Growing self-organizing mini-guts from a single intestinal stem cell: Mechanism and applications","volume":"340","author":"Sato","year":"2013","journal-title":"Science"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Costa, J., and Ahluwalia, A. (2019). Advances and current challenges in intestinal in vitro model engineering: A digest. Front. Bioeng. Biotechnol., 7.","DOI":"10.3389\/fbioe.2019.00144"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2250131","DOI":"10.1002\/eji.202250131","article-title":"A novel 3D cell culture model to study the human small intestinal immune landscape","volume":"53","author":"Schimpel","year":"2023","journal-title":"Eur. J. Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Yang, Z.-J., Wang, B.-Y., Wang, T.-T., Wang, F.-F., Guo, Y.-X., Hua, R.-X., Shang, H.-W., Lu, X., and Xu, J.-D. (2021). Functions of dendritic cells and its association with intestinal diseases. Cells, 10.","DOI":"10.3390\/cells10030583"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3761","DOI":"10.3748\/wjg.v17.i33.3761","article-title":"Intestinal dendritic cells in the pathogenesis of inflammatory bowel disease","volume":"17","author":"Rutella","year":"2011","journal-title":"World J. Gastroenterol. WJG"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1524","DOI":"10.1097\/MIB.0000000000001204","article-title":"CD103+ dendritic cell function is altered in the colons of patients with ulcerative colitis","volume":"23","author":"Matsuno","year":"2017","journal-title":"Inflamm. Bowel Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1038\/mi.2015.48","article-title":"Macrophage and dendritic cell subsets in IBD: ALDH+ cells are reduced in colon tissue of patients with ulcerative colitis regardless of inflammation","volume":"9","author":"Magnusson","year":"2016","journal-title":"Mucosal Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1111\/imr.12194","article-title":"Intestinal dendritic cells in the regulation of mucosal immunity","volume":"260","author":"Bekiaris","year":"2014","journal-title":"Immunol. Rev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1016\/j.tiv.2017.08.011","article-title":"Development of an in vitro co-culture model to mimic the human intestine in healthy and diseased state","volume":"45","author":"Gioria","year":"2017","journal-title":"Toxicol. In Vitro"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Noel, G., Baetz, N.W., Staab, J.F., Donowitz, M., Kovbasnjuk, O., Pasetti, M.F., and Zachos, N.C. (2017). A primary human macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions. Sci. Rep., 7.","DOI":"10.1038\/srep45270"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"113616","DOI":"10.1016\/j.celrep.2023.113616","article-title":"Developmental role of macrophages modeled in human pluripotent stem cell-derived intestinal tissue","volume":"43","author":"Song","year":"2024","journal-title":"Cell Rep."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"380","DOI":"10.1016\/j.ejps.2006.12.006","article-title":"An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells","volume":"30","author":"Fievez","year":"2007","journal-title":"Eur. J. Pharm. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"771","DOI":"10.1242\/jcs.106.3.771","article-title":"Differential expression of the human mucin genes MUC1 to MUC5 in relation to growth and differentiation of different mucus-secreting HT-29 cell subpopulations","volume":"106","author":"Lesuffleur","year":"1993","journal-title":"J. Cell Sci."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"808","DOI":"10.1021\/mp400507g","article-title":"Development of an advanced intestinal in vitro triple culture permeability model to study transport of nanoparticles","volume":"11","author":"Schimpel","year":"2014","journal-title":"Mol. Pharm."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1097\/00062752-200105000-00004","article-title":"Role of hematopoietic growth factors\/flt3 ligand in expansion and regulation of dendritic cells","volume":"8","author":"McKenna","year":"2001","journal-title":"Curr. Opin. Hematol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1441","DOI":"10.1016\/j.tiv.2010.04.002","article-title":"Inflammatory parameters in Caco-2 cells: Effect of stimuli nature, concentration, combination and cell differentiation","volume":"24","author":"Hendrickx","year":"2010","journal-title":"Toxicol. In Vitro"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1210","DOI":"10.1111\/j.1572-0241.2007.01714.x","article-title":"Adsorptive depletion of elevated proinflammatory CD14+ CD16+ DR++ monocytes in patients with inflammatory bowel disease","volume":"103","author":"Hanai","year":"2008","journal-title":"Off. J. Am. Coll. Gastroenterol. ACG"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1111\/j.1365-2249.2010.04177.x","article-title":"Investigating the role of proinflammatory CD16+ monocytes in the pathogenesis of inflammatory bowel disease","volume":"161","author":"Koch","year":"2010","journal-title":"Clin. Exp. Immunol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Tindemans, I., Joosse, M.E., and Samsom, J.N. (2020). Dissecting the heterogeneity in T-cell mediated inflammation in IBD. Cells, 9.","DOI":"10.3390\/cells9010110"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1038\/mi.2014.64","article-title":"Retinoic acid primes human dendritic cells to induce gut-homing, IL-10-producing regulatory T cells","volume":"8","author":"Bakdash","year":"2015","journal-title":"Mucosal Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"256","DOI":"10.1136\/gutjnl-2014-307916","article-title":"Compartment-specific immunity in the human gut: Properties and functions of dendritic cells in the colon versus the ileum","volume":"65","author":"Mann","year":"2016","journal-title":"Gut"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Snyder, L.M., Arora, J., Kennett, M.J., Weaver, V., and Cantorna, M.T. (2020). Retinoid signaling in intestinal epithelial cells is essential for early survival from gastrointestinal infection. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.559635"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"733","DOI":"10.4049\/jimmunol.1002701","article-title":"Functional specializations of intestinal dendritic cell and macrophage subsets that control Th17 and regulatory T cell responses are dependent on the T cell\/APC ratio, source of mouse strain, and regional localization","volume":"187","author":"Denning","year":"2011","journal-title":"J. Immunol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"767","DOI":"10.1146\/annurev.immunol.18.1.767","article-title":"Immunobiology of dendritic cells","volume":"18","author":"Banchereau","year":"2000","journal-title":"Annu. Rev. Immunol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e37537","DOI":"10.1097\/MD.0000000000037537","article-title":"Causal role of immune cells in inflammatory bowel disease: A Mendelian randomization study","volume":"103","author":"Chen","year":"2024","journal-title":"Medicine"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"458","DOI":"10.1002\/ibd.20344","article-title":"Suppression of colon inflammation by CD80 blockade: Evaluation in two murine models of inflammatory bowel disease","volume":"14","author":"Eri","year":"2008","journal-title":"Inflamm. Bowel Dis."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1111\/j.1365-2567.2005.02317.x","article-title":"Imbalance of regulatory T cells in human autoimmune diseases","volume":"117","author":"Dejaco","year":"2006","journal-title":"Immunology"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1136\/gutjnl-2013-306518","article-title":"Identification of inflammatory mediators in patients with Crohn\u2019s disease unresponsive to anti-TNF\u03b1 therapy","volume":"64","author":"Leal","year":"2015","journal-title":"Gut"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Hasegawa, A., Iwamura, C., Kitajima, M., Hashimoto, K., Otsuyama, K.-i., Ogino, H., Nakayama, T., and Shirai, M. (2013). Crucial role for CD69 in the pathogenesis of dextran sulphate sodium-induced colitis. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0065494"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"497056","DOI":"10.1155\/2015\/497056","article-title":"CD69 is the crucial regulator of intestinal inflammation: A new target molecule for IBD treatment?","volume":"2015","author":"Radulovic","year":"2015","journal-title":"J. Immunol. Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"521","DOI":"10.1016\/1074-7613(95)90180-9","article-title":"Interleukin-2 receptor \u03b1 chain regulates the size and content of the peripheral lymphoid compartment","volume":"3","author":"Willerford","year":"1995","journal-title":"Immunity"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1198","DOI":"10.1007\/s00535-012-0591-2","article-title":"Blocking IL-25 signalling protects against gut inflammation in a type-2 model of colitis by suppressing nuocyte and NKT derived IL-13","volume":"47","author":"Camelo","year":"2012","journal-title":"J. Gastroenterol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"620","DOI":"10.1038\/s41467-017-00658-6","article-title":"TGF\u03b2R signalling controls CD103+ CD11b+ dendritic cell development in the intestine","volume":"8","author":"Bain","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"e1183850","DOI":"10.1080\/2162402X.2016.1183850","article-title":"Inhibition of SIRP\u03b1 in dendritic cells potentiates potent antitumor immunity","volume":"5","author":"Liu","year":"2016","journal-title":"Oncoimmunology"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Ness, S., Lin, S., and Gordon, J.R. (2021). Regulatory dendritic cells, T cell tolerance, and dendritic cell therapy for immunologic disease. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.633436"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1038\/mi.2015.50","article-title":"Generation and transcriptional programming of intestinal dendritic cells: Essential role of retinoic acid","volume":"9","author":"Zeng","year":"2016","journal-title":"Mucosal Immunol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1159\/000353243","article-title":"The mucosal factors retinoic acid and TGF-\u03b21 induce phenotypically and functionally distinct dendritic cell types","volume":"162","author":"Savelkoul","year":"2013","journal-title":"Int. Arch. Allergy Immunol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1038\/ni.2768","article-title":"Comparative transcriptional and functional profiling defines conserved programs of intestinal DC differentiation in humans and mice","volume":"15","author":"Watchmaker","year":"2014","journal-title":"Nat. Immunol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.mam.2011.11.001","article-title":"Vitamin A and immune regulation: Role of retinoic acid in gut-associated dendritic cell education, immune protection and tolerance","volume":"33","author":"Cassani","year":"2012","journal-title":"Mol. Asp. Med."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1757","DOI":"10.1084\/jem.20070590","article-title":"A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-\u03b2\u2013and retinoic acid\u2013dependent mechanism","volume":"204","author":"Coombes","year":"2007","journal-title":"J. Exp. Med."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/j.imlet.2021.02.001","article-title":"Dynamic role of macrophage CX3CR1 expression in inflammatory bowel disease","volume":"232","author":"Li","year":"2021","journal-title":"Immunol. Lett."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"e5","DOI":"10.4110\/in.2018.18.e5","article-title":"Tissue-specific role of CX3CR1 expressing immune cells and their relationships with human disease","volume":"18","author":"Lee","year":"2018","journal-title":"Immune Netw."},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Cai, Z., Wang, S., and Li, J. (2021). Treatment of inflammatory bowel disease: A comprehensive review. Front. Med., 8.","DOI":"10.3389\/fmed.2021.765474"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"1592","DOI":"10.1053\/gast.2002.33426","article-title":"Biologic therapy of inflammatory bowel disease","volume":"122","author":"Sandborn","year":"2002","journal-title":"Gastroenterology"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1007\/s44164-022-00017-w","article-title":"In vitro models and ex vivo systems used in inflammatory bowel disease","volume":"1","author":"Joshi","year":"2022","journal-title":"In Vitro Models"},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Katsandegwaza, B., Horsnell, W., and Smith, K. (2022). Inflammatory bowel disease: A review of pre-clinical murine models of human disease. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23169344"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"1045","DOI":"10.1002\/JLB.3RI0620-405RR","article-title":"Age-related changes in intestinal immunity and the microbiome","volume":"109","author":"Walrath","year":"2021","journal-title":"J. Leucoc. Biol."},{"key":"ref_57","doi-asserted-by":"crossref","unstructured":"Zheng, H., Zhang, C., Wang, Q., Feng, S., Fang, Y., and Zhang, S. (2022). The impact of aging on intestinal mucosal immune function and clinical applications. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.1029948"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/14\/17\/1300\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:33:39Z","timestamp":1760034819000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/14\/17\/1300"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,22]]},"references-count":57,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2025,9]]}},"alternative-id":["cells14171300"],"URL":"https:\/\/doi.org\/10.3390\/cells14171300","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,22]]}}}