Figure 3

V. cholerae derived OMVs, but not VCC, induce increased expression levels of miR-146a in T84 polarized tight monolayer cells. (a,b) Polarized tight monolayers of T84 cells were challenged at the apical side with 100 µg OMV protein derived from the V. cholerae strain V:5/04 and its VCC deletion mutant before (wt-OMV-c and Δvcc-OMV-c) and after (wt-OMV-p and Δvcc-OMV-p) density gradient centrifugation and with 80 ng soluble VCC (VCC) for 5 hours. Challenged monolayers and sham-treated controls (Ctrl) were thereafter monitored for changes in miR-146a (a) and miR-155 (b) levels. n = 8 challenged monolayers for each OMV-type, 4 monolayers challenged with VCC and 12 sham-treated monolayers. (c) Expression levels of miR-146a in monolayers challenged with 100 µg (100 µg wt-OMV) and 235 µg (235 µg wt-OMV) of the same density gradient centrifugation purified OMV sample from wildtype bacteria of the V. cholerae strain V:5/04, and 100 µg of density gradient centrifugation purified OMV of the VCC deletion mutant for 5 hours and their sham-treated control monolayers (Ctrl). n = 4 challenged monolayers per dose and OMV-type and 4 sham-treated monolayers. (d) Expression levels of miR-146a in monolayers challenged with 100 µg of one density gradient centrifugation purified OMV sample from the wildtype V. cholerae strain V:5/04 (100 µg wt-OMV) for 2, 5 and 12 hours and their respective sham-treated control monolayers (Ctrl). n = 4 challenged and 4 sham-treated monolayers per time-point. (a–d) Amounts of miR-146a were determined by real-time qRT-PCR and normalized to the content of RNU48 in the sample. Results are expressed as relative quantity (RQ) relative to the median of sham-treated tight monolayers incubated in parallel with monolayers challenged with OMV or VCC. Bars indicate mean RQ + 1 SD. Statistically significant differences are shown, ***P-value < 0.001, **P-value < 0.01 and *P-value < 0.05.