Figure 3 |. Activation of TLR4 induces epithelial production of H₂O₂ via DUOX2.

Left panels show kinetic production of H₂O₂ in one representative experiment expressed in relative fluorescence units (RFU) normalized to MTT viability values. Right panels show the H₂O₂ production rate in all experiments. (A) Freshly isolated CECs were assayed for the kinetic production of H₂O₂ in SPF and GF-raised villin-TLR4 mice and littermates. Villin-TLR4 mice had increased H₂O₂ production rates (***P<0.001) when compared to WT littermates (n=9-12 mice). (B) We tested the effects of NADPH oxidase inhibition in freshly isolated CECs by adding DPI or DMSO after 9 minutes of kinetic determination and measuring for 10 additional minutes. DPI markedly reduced H₂O₂ production in WT (**P<0.01) and villin-TLR4 (***P<0.001) CECs (n=7 mice). (C) Production of H₂O₂ was determined in WT and TLR4-KO colonoids stimulated for 24 hours with LPS. Left panel shows H₂O₂ production kinetics in WT organoids. LPS stimulation induced a release of H₂O₂ in WT colonoids (***P<0.001) that was completely abrogated by DPI (***P<0.001; n=4 cultures). (D) and (E) TLR4-KO, DUOXA-KO, and NOX1-KO colonoids were stimulated with LPS (n=4-6 cultures), F. prausnitzii, or AIEC (n=4-9 cultures) and their release of H₂O₂ was determined (***P<0.001). Data in all figures were analyzed by two-way ANOVA followed by Sidak’s post-hoc test.