Requirements for microbe-induced Ang4 and IL‑23 production by intestinal epithelial cells.

<p>(A) Invasive or non-invasive <i>Salmonella</i> strains (4x10⁷ CFU in PBS) were injected into exteriorised intestinal ligated loops of WT mice. Four hours later mucosal RNA was isolated and Ang4 mRNA expression analysed by qPCR. The data (mean±SEM) are expressed relative to mRNA levels obtained from loops exposed to PBS alone (n=3; ***p<0.001). (B) IL‑23 protein levels assessed by ELISA (mean±SEM) from intestinal epithelial cells of WT mice exposed for 12h to medium alone, (+Medium) or media containing invasive or non‑invasive <i>Salmonella</i> strains at a ratio of 10 bacteria per epithelial cell (n=3; ***p<0.001, *p<0.05). (C) m-ICc12 intestinal epithelial cells were cultured for 4h with medium alone (control) or containing live cells of various intestinal commensal bacteria at a ratio of 10 bacteria per epithelial cell. IL‑23 mRNA expression was assessed by qPCR. Data (mean±SEM) were expressed relatively to mRNA levels in control samples (n=4). (D) IL‑23 protein production measured by ELISA in intestinal epithelial cells from WT mice cultured at 37°C for 12h with medium alone or containing either, live WT (SL1344) or various <i>Salmonella</i> mutant strains that are non-invasive (SL1344ΔSPI1), invasive but non-flagellated (JH3220=SL1344Δ<i>fliC</i>Δ<i>fljB</i>), non-invasive and non-flagellated (JH3515=SL1344∆SPI1Δ<i>fliC</i>Δ<i>fljB</i>) or non-invasive, flagellated but unable to transcytose flagellin (JH3574=SL1344∆SPI1Δ<i>ssrA</i>) (n=3; mean±SEM). (E) IL‑23 mRNA expression assessed by qPCR in m-ICc12 epithelial cells cultured at 37°C for 4h with medium alone or containing live or heat‑killed WT <i>Salmonella</i> SL1344 cells at a ratio of 10 bacteria per epithelial cell. Data (n=4; mean±SEM) are expressed relative to mRNA levels obtained in non‑infected cells. (F) IL‑23 mRNA levels analysed by qPCR in intestinal epithelial cells from WT mice cultured at 37°C for 2h with medium alone or containing lipopolysaccharide (LPS), peptidoglycan (PGN), muramyl dipeptide (MDP) or methylated DNA (CpG). Data (n=3; mean±SEM) are expressed relative to mRNA levels obtained in cells exposed to medium alone.