Interaction of CpG-oligodeoxynucleotides with Toll like receptor 9 induces apoptosis and modulates metaloproteinase-2 activity in human intestinal epithelium.
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 microg/ ml and 1, 5, 10 microg/ 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 microg/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 microg/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.
Khorramizadeh
Mohammad Reza
MR
Department of Pathobiology, School of Public Health, Medical Sciences, University of Tehran, Tehran, Iran. khoramza@sina.tums.ac.ir
Hosseinzadeh
Shahnaz
S
Safavifar
Farnaz
F
Saadat
Farshid
F
Aalizadeh
Nastaran
N
Falak
Reza
R
Jadali
Zohre
Z
Pezeshki
Mohammad
M
D017209:Apoptosis / Q000187:drug effects*; D019073:HT29 Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D008070:Lipopolysaccharides / Q000633:toxicity; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000633:toxicity*; D051217:Toll-Like Receptor 9 / Q000378:metabolism*
C408982:CPG-oligonucleotide; D008070:Lipopolysaccharides; D009838:Oligodeoxyribonucleotides; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; D020778:Matrix Metalloproteinase 2