Figure 3.

Effect of bacteria and immune cell co‐culture on epithelial integrity in non‐inflammatory bowel disease (non‐IBD) versus Crohn's disease (CD) populations. (a) Scheme of intestinal monolayers, with the addition of heat‐killed Faecalibacterium prausnitzii and matched‐patient PBMCs (created in Biorender). Differentiated intestinal monolayers were cultured alone, cultured with 10⁶ PBMCs added to the serosal compartment, cultured with MOI 50 F. prausnitzii added to the mucosal compartment, cultured with both immune cells and MOI 50 F. prausnitzii or cultured with anti‐CD3/28 beads, for a further 72 h. (b) H&E image shows intestinal cells lining a 0.4‐μm Transwell® membrane. Images from two representative donors shown. (c) Change in transepithelial electrical resistance (TEER) from day 0 to day 3 in the presence or absence of PBMCs cultured with anti‐CD3/28 beads (non‐IBD = 9, CD = 6–8). Matched donors are shown by symbols. (d) Change in TEER from day 0 to day 3 in the presence or absence of PBMCs and/or F. prausnitzii as indicated. Left—non‐IBD donors (n = 9); middle—CD donors untreated or treated with nonbiologicals at time of biopsy (n = 6); right—CD donors treated with anti‐TNF at time of biopsy (n = 3). (e) PBMCs recovered from the monolayer co‐culture on day 3 from the monolayer + PBMCs versus monolayer + PBMCs + bacteria cultures were analysed by flow cytometry (non‐IBD donors: n = 6 HC, CD donors: n = 3). Matched donors are shown by symbols. Statistical analyses were calculated using (c) a Kruskal–Wallis test with the Dunn's multiple comparisons test separately within the non‐IBD or CD groups, or using (d) a Friedman test with the Dunn's multiple comparisons test separately within the non‐IBD, untreated CD and treated CD groups (*P < 0.05, **P < 0.01).