القائ آپوپتوز و تعدیل فعالیت متالو پروتئیناز 2 در اثر واکنش الیگو داکسی نوکلئوتید CpG با TLR9

Title

Interaction of CpG-Oligodeoxynucleotides with Toll Like Receptor 9 Induces Apoptosis and Modulates Metaloproteinase-2 Activity in Human Intestinal Epithelium

English Abstract

Recent reports have indicated different effects of immunostimulatory sequences containing CpG-Oligodeoxynucleotides (ODN) on various immune cells. However, the exact role of CpG-ODN in human gut is unclear.In the present study ,we assessed potential effects of CpG –ODN on non lymphoid cell(intestinal epithelial cell line HT-29) on a dose-response and time –course basis. Intestinal epithelial cell line HT-29 was treated with CpG –ODN (CpG 2006) and lipopolysaccharide (LPS) at 5, 10, 25, 50 µg/ml and 1,5,10 µg/ml concentrations, respectively. Following treatments, dose-response and time-course cytotoxicity using a colorimetric method, Metaloproteinase-2(MMP-2)activity (using gelatin zymography)and apoptosis (using annexin-v flowcytometry method)assays were performed. Chloroquine treatment was also used for its inhibitory effect on endosomal acidification process to verify specific CpG –ODN and Toll Like Receptor 9 (TLR9) interactions.Cytotoxicity analysis of CpG –ODN showed that CpG –ODN increased significantly the proliferation of CpG –ODN treated cells ,as compared to untreated cells, at concentrations of 10-25 µg/ml (p<0.05).Overall MMP-2 activity analysis showed significant differences between treated and untreated cells. However, minimal changes were observed when MMP-2 activity was assessed per cell. Moreover, CpG –ODN treated cells demonstrated an increasing apoptosis rate of 0.8%, 6.46% and 14.21% at concentrations of 5,10,25 µg/ml, respectively. Collectively, our data indicated that intestinal epithelial cell line HT-29 is highly responsive to CpG effect in vitro and exhibits modified activities. The direct CpG –ODN and TLR9 interactions in HT-29 cells could provide new approaches in malignant tumor therapeutic strategies.

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