An autologous colonic organoid-derived monolayer model to study immune: bacterial interactions in Crohn's disease patients

Abstract

Objectives: Crohn's disease (CD) initiation and pathogenesis are believed to involve an environmental trigger in a genetically susceptible person that results in an immune response against commensal gut bacteria, leading to a compromised intestinal epithelial barrier and a cycle of inflammation. However, it has been difficult to study the contribution of all factors together in a physiologically relevant model and in a heterogenous patient population.

Methods: We developed an autologous colonic monolayer model that incorporated the immune response from the same donor and a commensal bacteria, Faecalibacterium prausnitzii. Two-dimensional monolayers were grown from three-dimensional organoids generated from intestinal biopsies, and the epithelial integrity of the epithelium was measured using transepithelial electrical resistance. We determined the effect of immune cells alone, bacteria alone and the co-culture of immune cells and bacteria on integrity.

Results: Monolayers derived from CD donors had impaired epithelial integrity compared to those from non-inflammatory bowel disease (IBD) donors. This integrity was further impaired by culture with bacteria, but not immune cells, despite a higher frequency of inflammatory phenotype peripheral T cells in CD donors. Variability in epithelial integrity was higher in CD donors than in non-IBD donors.

Conclusion: We have developed a new autologous model to study the complexity of CD, which allows for the comparison of the barrier properties of the colonic epithelium and the ability to study how autologous immune cells directly affect the colonic barrier and whether this is modified by luminal bacteria. This new model allows for the study of individual patients and could inform treatment decisions.

Keywords: Crohn's disease; T cells; bacteria; cytokines; epithelium; organoid.

Figure 1

The integrity of intestinal epithelial monolayers from Crohn's disease (CD) patients is compromised compared to those from non‐inflammatory bowel disease (non‐IBD) donors. (a) Scheme of the generation of intestinal monolayers (created in Biorender). (b) Representative H&E images of non‐IBD (left) and CD donor (right) monolayers (representative of 10 non‐IBD and 8 CD). (c) Non‐IBD and CD donor intestinal monolayers were cultured for 9 days in nondifferentiation media (NDM), then for 3 days in differentiation media (DFM). Transepithelial electrical resistance (TEER) readings were taken at indicated intervals. Vertical dotted line represents the change from NDM to DFM. Non‐IBD (n = 5), CD (n = 6). Statistical analyses were calculated using a mixed‐effects model and Sidak's multiple comparisons test (***P < 0.001, ****P < 0.0001).

Figure 2

Peripheral T cells from Crohn's disease (CD) donors have a more inflammatory phenotype than those from non‐inflammatory bowel disease (non‐IBD) donors. PBMCs were isolated from non‐IBD or CD donors. (a) Frequency of PBMC CD4⁺IL‐17A⁺, CD4⁺IFN‐γ⁺ and regulatory T cells (CD4⁺CD25⁺FOXP3^hi) of CD3⁺ cells. Data are shown as individual patients, and the bar represents the median frequency. Non‐IBD (n = 9), CD (n = 8). (b) 80 000 PBMCs were cultured with anti‐CD3/anti‐CD28 beads at a 1:1 ratio for 48 h. Cytokine supernatants were detected using a LegendPlex Human T Cell Cytokine Panel, then analysed by flow cytometry. Data are shown as individual patients, and the bar represents the median concentration (pg mL⁻¹). Where appropriate, upper or lower limits of detection are shown with the dotted line. Non‐IBD (n = 7), CD (n = 10). (c) Tconvs were stained with Cell‐Trace Violet to allow for tracking of division events. Precursor frequency was calculated by (N/2 ⁱ )/(Total number of events/2 ⁱ ) × 100 = % P, then precursor frequency determined by the sum of % P of all peaks where division occurred (peaks 1–4). Proliferation index was calculated by ((N/2 ⁱ ) × N)/(Total number of events), where N indicates the number of events within a proliferation peak and 2 ⁱ indicates the number progeny per division stage. Non‐IBD (n = 6), CD (n = 6). Statistical analyses were calculated using the Mann–Whitney U‐test (*P < 0.05, **P < 0.01). (d) PBMCs were isolated from non‐IBD and CD donors and cultured for 72 h in the presence or absence of 10⁷ heat‐killed Faecalibacterium prausnitzii, then analysed by flow cytometry. Left top: Frequency of CD4⁺IFN‐γ⁺ T cells. Right top: Frequency of CD4⁺IL‐17A⁺ T cells. Left bottom: Frequency of regulatory T cells (CD4⁺CD25⁺FOXP3^HI). Right bottom: Frequency of CD4⁺CD25^HIFOXP3⁻ T cells. Statistical analyses were calculated using the Wilcoxon matched‐pairs signed‐rank test, (**P < 0.01, ***P < 0.001). Non‐IBD (n = 9), CD (n = 8). Lines represent matched‐patient PBMCs in each condition. Similar results were obtained with culture of 10⁶ bacteria.

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).