Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells

Abstract

Enteropathogenic bacteria elicit mucosal innate and adaptive immune responses. We investigated whether gut epithelial cells played a role in triggering an adaptive immune response by recruiting dendritic cells (DCs). Immature DCs are selectively attracted by the CCL20 chemokine. The expression of the CCL20 gene in human intestinal epithelial cell lines was up-regulated by pathogenic bacteria, including Salmonella species, but not by indigenous bacteria of the intestinal flora. The Salmonella machinery for epithelial cell invasion was not required for CCL20 gene activation. Flagellin but not the lipopolysaccharide was found to be the Salmonella factor responsible for stimulation of epithelial CCL20 production. CCL20 in turn triggered a specific migration of immature DCs. Our data show that crosstalk between bacterial flagellin and epithelial cells is essential for the recruitment of DCs, a mechanism that could be instrumental to initiate adaptive immune responses in the gut.

Figure 1

S. typhimurium-regulated expression of CCL20 gene in epithelial cells. Caco-2 cells in Transwell cultures were infected apically for 45 min with S. typhimurium ATCC14028 (moi = 100), washed, and incubated for the indicated times in gentamicin-supplemented medium. (a) Transcriptional activation of CCL20 gene: Total RNA was extracted and reverse transcribed. CCL20 mRNA levels were quantified by using real-time PCR and 18S rRNA amplicons as standards. Values were expressed as relative increase of CCL20 mRNA quantity compared with noninfected Caco-2 cells. (b) Secretion of CCL20 chemokine in basal culture medium. CCL20 concentration was measured by CCL20-specific ELISA on cell culture medium of Caco-2 cells.

Figure 2

Pathogen-specific induction of CCL20 transcription in epithelial cells. Monolayers of Caco-2 cells were exposed apically for 45 min to bacterial strains (moi = 100) and incubated for 2.5 h in gentamicin-containing medium. CCL20 expression was quantified by real-time RT-PCR. ATCC14028 was used as a positive control of CCL20 induction. Results are representative of at least 2 independent experiments. CCL20 transcription was analyzed after exposure to laboratory E. coli DH5α (a); bacteria from human colon flora, E. coli EMO, B. vulgatus, and B. bifidum (b); and enteroinvasive bacteria, S. enteritidis SE857 and L. monocytogenes LO28 (c).

Figure 3

Salmonella induction factor for CCL20 expression is a heat-stable secreted protein. Polarized Caco-2 cells were exposed apically for 45 min to bacteria (moi = 100) (a). Then, cells were incubated for 2.5 h in gentamicin-supplemented medium. Alternatively, cells were exposed for 3.25 h to bacterial products (b and c). Activation of CCL20 gene transcription was quantified by real-time RT-PCR. Results are representative of at least 3 independent experiments. (a) Induction of CCL20 transcription does not depend on Salmonella-mediated invasion. (b) LPS-independent CCL20 transcription. Epithelial cells were treated apically or basally with 10 μg/ml of LPS from S. typhimurium. (c) Induction factor is a Salmonella-secreted protein. Cells were exposed apically to 100 μl of supernatants from S. typhimurium, heat-treated supernatant, or trypsin-digested and heat-treated supernatant. LB broth treated in the same conditions was used as control.

Figure 4

Salmonella flagellins are inducing factors of CCL20 and IL-8 transcription in epithelial cells. Polarized Caco-2 cells were treated apically with bacteria (moi = 100) or flagellin. CCL20 and IL-8 gene transcription was quantified by real-time RT-PCR (a and b). Results are representative of at least 3 independent experiments. (a) Cells were infected for 45 min with S. enteritidis, the fliC mutant SEFK32, or SEFK32(pRP2) (complemented with the FliC flagellin of S. typhimurium) and incubated for 2.5 h in gentamicin-containing medium. (b) Dose-dependent induction of CCL20 and IL-8 expression by flagellin. Cells were exposed apically for 3.25 h to purified S. typhimurium FliC flagellin at the indicated concentrations. (c) Flagellin expression in Salmonella strains. Supernatants (0.5 ml) from Salmonella cultures or purified S. typhimurium FliC flagellin (1 μg) were analyzed after SDS/PAGE by Coomassie blue staining (Upper) and by immunoblotting (Lower) with flagellin-specific Ab. Arrow and asterisk indicate the position of flagellins from ATCC14028 (52 kDa) and SE857 (56 kDa), respectively. Agglutination with flagellin-specific Ab was performed on bacteria grown in the same conditions.

Figure 5

Immature DCs migrate in response to medium from flagellin-treated epithelial cells. rhCCL20 (7 ng/ml), control medium, or basal medium of untreated or of flagellin-treated Caco-2 cells (7 ng/ml of CCL20) were used in migration assays of immature DCs. When specified, CCL20-specific mAb was mixed with medium 30 min before assay to neutralize CCL20. Results are representative of 2 independent experiments.