Intestinal Mucosal Immune Response Assay

Intestinal explants were used to test the reactivity of the mucosal immune system [31 (link),32 (link)]. Adapted from Chatelais et al. [33 (link)], 5 cm segments of jej-PP, jej-LP and il-PP were rinsed with PBS containing 1% dithiothreitol (DTT, Sigma) and 1% FCS (Sigma) and then placed in a solution made with 74% PBS, 25% DMEM (Sigma), 1% FCS, 5 µg/mL gentamicin (Sigma) and 1.25 µg/mL amphotericin B (Sigma) for immediate explant culture. Jejunal and ileal mucosa were then cut in small biopsies of 10–15 mg. Biopsies (two per well, weighing, in total, 20 to 35 mg) were cultured for 2 h under an atmosphere containing 5% CO₂ at 37 °C [33 (link)]. Explants were then transferred into the same solution as above but without FCS (replacement with BSA), and stimulated or not with 50 µg/mL lipopolysaccharides (LPS, TLR4 ligand; Sigma) or 1 µg/mL flagellin (TLR5 ligand; Invivogen, Toulouse, France) and incubated for 20 h under an atmosphere containing 5% CO₂ at 37 °C. Finally, supernatants were collected and frozen at −20 °C for later cytokine analysis, and the biopsies were harvested in 1 mL of Trizol reagent (Fisher Scientific, Illkirch, France) and frozen at −80 °C for RNA extraction.