294 TLR 9 INHIBITION: A NOVEL TARGET OF THERAPY FOR PRIMARY LIVER CANCER

Abstract

Introduction Toll like receptor 9 (TLR9) is a member of the nucleotide-sensing endosomal TLR family which is critical to the innate immune defense against invading pathogens. TLR9 is activated by unmethylated CpG which is highly specific for bacterial DNA. Upon activation, TLR9 traffics from the endoplasmic reticulum (ER) to endosomes TLR9 signalling is inhibited by the aminoquinolone drug chloroquine. Aims (1) assess changes in TLR9 subcellular distribution. (2) Detect any changes in the endolysosomal system. (3) Determine the effects on cell proliferation in hepatocellular carcinoma (HCC) and cholangio carcinoma cell (CC) lines upon stimulation and inhibition of TLR9 signalling in each case. Methods Huh7D and HUCCT cells were treated with unmethylated CpG (ODN 2006) to stimulate, or chloroquine and Dynavax; IRS compound to inhibit TLR9 signalling. Cells were also treated with the TLR9 antagonist iODN. Cell growth was assessed and confocal immunofluorescence microscopy was used to determine TLR9 subcellular localisation using EEA1 and LAMP1, markers of the endolysosomal system. Results Confocal microscopy indicated a marked nuclear translocation of TLR9 in HUCCT and Huh7D when stimulated with CpG, while unstimulated controls showed cytoplasmic TLR9 localisation. TLR9 inhibition by iODN and chloroquine resulted in decreased cytoplasmic TLR9 meanwhile Dynavax treatment caused translocation of TLR9 to the perinuclear membranes. Dramatic changes were also observed in the distribution of LAMP1 and EEA1, which were found to be localise to juxtanuclear punctae on TLR9 stimulation. While following inhibition they translocated to perinuclear membranes. Huh7D cell counts the CpG treated cells, iODN, chloroquine and Dynavax compound were 4.5×10 5 2.1×10 5 , 1.5×10 5 and 1.7×10 5 per ml respectively, compared with the untreated cells 3×10 5 per ml which indicate a significant increase in proliferation with increased TLR9 stimulation and a significant decrease with TLR9 inhibition (p CpG treated cells, iODN, chloroquine and Dynavax were respectively 3.3×10 5 , 1.8×10 5 , 1.4×10 5 and 1.5×10 5 per ml compared with the untreated cells at 1.7×10 5 per ml. Conclusion Our study indicates that TLR9 activation increases cell proliferation whereas inhibition reduces it. Our data suggest that TLR9 may be associated with tumour proliferation and may provide a potential target for therapy in liver tumours. Competing interests None declared.