and K.L.V. and IgA transcytosis. We used wild-type and knockout cells to establish that amongst these cytokines, IL-17 was the most potent inducer of pIgR expression/IgA transcytosis. IFN however did not induce pIgR expression, and instead led to cell death. This new method will allow the use of primary cells for studies of intestinal physiology. studies. Most recently, the ability to propagate primary intestinal epithelial cells has greatly advanced the field1,2. Prior to the ability to grow primary intestinal epithelial cells, colon cancer cell lines have been widely used to model physiologic and cell biologic intestinal processes studies have shown that stimulation of HT-29 cells with microbial factors (such as LPS, butyrate, and dsRNA) or heat-killed bacteria can upregulate pIgR expression20,21. Pro-inflammatory cytokines produced in response to microbial stimuli, such as IFN, TNF, IL-1, and most recently IL-17 have also been shown to increase pIgR expression using primary cells. We chose to focus on the process of IgA transcytosis using this system. We were able to adapt the previously established three dimensional (3D) primary intestinal epithelial stem cell culture system into a 2D monolayer in a Transwell. These cells are able to express pIgR after stimulation with LPS, and transcytose IgA across the monolayer. TNF, IL-1, and IL-17 were able to induce pIgR expression and IgA transcytosis in a dose-dependent manner. Importantly, perhaps demonstrating a distinction with previous methodologies using immortalized cell lines, IFN did not enhance pIgR expression. Heat-killed bacteria were also able to stimulate these processes to differing extents. Finally, this system will be readily adaptable for the use with available genetically modified mice to study different genes of interest: primary intestinal epithelial cells from experimental system that allowed for significant expansion of intestinal epithelial stem/progenitor cells2. To obtain cells for a single Transwell, 1,2-Dipalmitoyl-sn-glycerol 3-phosphate we harvested colonic spheroids from three wells (400C500 spheroids/well) of a 24-well plate that were cultured as spheroids for three days in Matrigel using 50% L-WRN (L-cells expressing Wnt3a, R-spondin3, and Noggin) conditioned media (CM). This produced ~5105 cells that were seeded onto a single 0.33 cm2 Transwell insert of a 24-well plate. Typically this cell input created a monolayer of ~2.5105 cells. At the time of seeding cells in Transwells, we used 50% L-WRN CM that also contained 10 M of the ROCK inhibitor Y-27632 (Figure 1a). The media was maintained for one day post-seeding. Open in a separate window Figure 1 Developing a transwell system using mouse primary intestinal epithelial cells(a) Schematic of timeline for Transwell experiments. Wild type cells were treated with +/? 10 M DAPT +/? 1 g/ml LPS and were analyzed on day three post-seeding. (b) Cells were fixed and paraffin-embedded on the transwell membranes. Sections were cut and stained with the following: hematoxylin and eosin, anti-ZO-1, anti-villin, and anti-pIgR. Bars = 50 m. Gene expression analysis was performed by qRT-PCR for pIgR (c), Reg3g 3 per condition). One-way ANOVA: (c) = 96.02, 0.0001; (d) = 3.441, 0.0376; (e) = 1.085, 0.3762. *** 1,2-Dipalmitoyl-sn-glycerol 3-phosphate 0.001 by Bonferroni’s multiple comparison test. (f) Transepithelial electrical resistance was measured on day three. The (resistance Rabbit Polyclonal to OVOL1 area) is shown for each 1,2-Dipalmitoyl-sn-glycerol 3-phosphate condition (mean s.e.m., = 6 per group). Statistical analysis by Student’s 0.4362). On day one after seeding, the 50% CM was replaced with 0% CM supplemented with or without specific treatments that were designed to facilitate the study of IgA transcytosis. The treatment included a combination of the -secretase inhibitor DAPT to differentiate the cells27,28, and LPS to induce the expression of pIgR (which is known to be regulated by microbial and/or cytokine signaling)19. The cells were treated for two days in this media prior to evaluation for differentiation by.