We found that flagellin-induced NF��B activation was dramatically inhibited in TLR5-KD cells, whereas it was markedly induced in WT cells (Fig. 4B). These results indicate STA-9090 that the activated signaling by the flagellin preparation is specifically mediated by TLR5. Third, using a NF��B reporter activity assay, we showed that NCM460 cells are potently responsive to TLR5 ligand (flagellin), but nonresponsive to TLR2 (Pam3Cys) or TLR4 (LPS) stimulation (Fig. 4C). Taken together, our data obtained from these three experiments clearly indicate that flagellin-induced responses are specifically mediated by TLR5, but not compounded by TLR4. Therefore, the remaining signaling in flagellin-treated MyD88-KO cells is not attributed to a potential contaminant such as LPS. FIGURE 4.

Activated intracellular signaling by the flagellin preparation is not affected by the potential LPS contaminant, but rather is specifically mediated by TLR5. A, primary mouse intestinal epithelial cells from C3H/HeJ (TLR4-null) and its control C3H/HeOuJ … TRIF Deficiency Inhibits Flagellin-induced Inflammatory Cytokine Expression Having found that TRIF deficiency negatively regulates some of the TLR5-dependent intracellular signaling, we tested whether inflammatory cytokine expression induced by flagellin/TLR5 engagement is altered in the absence of TRIF expression. For this purpose, we stimulated primary intestinal epithelial cells from TRIF-KO, MyD88-KO, and WT mice with flagellin, followed by measuring TLR5-induced cytokine production.

As expected, the levels of KC (mouse ortholog of human IL-8), macrophage inflammatory protein 3��, and IL-6 expression were inhibited in MyD88-KO cells compared with the levels in WT cells (Fig. 5, A�CC). Notably, the cytokine expression was also dramatically suppressed in TRIF-KO cells compared with the expression level in WT cells. These results are in agreement with our findings demonstrating that NF��B and MAPK activation by flagellin is reduced in TRIF-KD and TRIF-KO cells (Figs. 2B and and33F). FIGURE 5. TRIF deficiency inhibits flagellin-induced cytokine expression. A�CC, primary mouse intestinal epithelial cells from WT, MyD88-KO, and TRIF-KO mice were stimulated with flagellin (100 ng/ml, 8 h). KC (A), macrophage inflammatory protein 3�� …

When the primary intestinal epithelial cells are stimulated with IL-1�� mediating its responses via IL-1R in a MyD88-dependent manner, IL-1�� stimulation failed to induce KC cytokine expression in MyD88-KO cells, whereas it strongly induced the cytokine expression in both TRIF-KO cells and WT cells (Fig. 5D). Moreover, TNF��, utilizing non�CTLR-associated signaling pathways, potently elicited the cytokine expression in the primary intestinal epithelial GSK-3 cells from MyD88-KO, TRIF-KO, and WT mice (Fig. 5E).