Interaction of CpG-oligodeoxynucleotides with toll like receptor 9 induces apoptosis and modulates metaloproteinase-2 activity in human intestinal epithelium

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 the 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 micro g/ml and 1, 5, 10 micro g/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 micro g/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 micro g/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 TLR-9 interactions in HT-29 cells could provide new approaches in malignant tumor therapeutic strategies