Regulation Of TLR8 Agonists On Dendritic Cells And Its Role In Tumor Immunity

Objective:To investigate the expression of TLR8and the production of cytokines such as IL-12and TNF-a in DC2.4cells after stimulation with human TLR8agonist CL075(a thiazoloquinoline compound) and Poly dT(a17-mer phosphorothioate poly T ODN, TLR7/8modulator);. To investigate cancer immunotherapeutic effects of CL075and Poly dT on murine models of Lewis lung carcinoma; To investigate the expression of TLR8and its relationship with Bcl-2and VEGF in cervical cancer cells.Methods:DC2.4cells were cultured in RPMI1640medium supplemented with streptomycin (100μg/ml), and penicillin (100U/ml) and10%fetal calf serum(FCS). Morphological characteristics of DC2.4were observed under the optical inverted microscope. RT-PCR/qRT-PCR, western blot and immunofluorescence staining were employed to detect the expression of TLR8in DC2.4. After stimulation with PBS,4μM CL075,5μM Poly dT and4μM CL075and5μM Poly dT, quantitative real-time PCR was performed to measure TNF-α, IL-12and IL-10mRNA. The relative mRNA levels were normalized to the β-actin gene. The DC2.4culture supernatants were measured for expression of mouse IL-10, IL-12, and TNF-a. Quantitation of secreted cytokines was done in an enzyme-linked immunosorbent assay (ELISA) using the respective cytokine ELISA kits. The results were normalized to the known standard curves. An one-way mixed lymphocyte culture (MLC) system was established, including DC2.4cells as stimulator and C57BL/6mouse spleen cells (H-2b) as responder. DC2.4cells were treated with CL075(4μM) and Poly dT (5μM) for72h. Spleen cells from C57BL/6mouse were cocultured with stimulated or unstimulated DC2.4at a ratio of5:1or10:1. After3days, proliferation was detected by MTT. The stimulation index(SI) was determined by OD value of proliferating cells to assess the response of T cells to DC2.4stimulation. T cells without DC2.4stimulation severd as a negative control.The murine Lewis lung carcinoma cells(LLC) were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with10%streptomycin (100μg/ml), and penicillin (100U/ml) and10%heat-inactivated FCS. Male C57BL/6mice at6to8weeks old and22to24g body weight were used. Tumors were established by subcutaneous injection of3×106LLC cells in100μl Ca+/Mg+-free PBS Dulbecco’s medium into the right flank of the mice. Immunotherapy treatment was initiated on the same day after tumor implantation. C57BL/6mice were randomised into three experimental groups of10mice each and received the following treatment; control group received PBS; low-dose group received CL0750.2μg and Poly dT5μg; high-dose group received CL0752μg and Poly dT50μg. All drugs were administered by intraperitoneal(i.p.) route. Low-dose group mice were administered once a day. High-dose group and control group were administered once every other day. Mouse body weight was measured once every other day using an electronic balance. Depilatories were used for hair removal in each experimental group of mice to observe tumor growth. After7days, tumor growth was measured every2days, and tumor volume was estimated as length×width2×0.5. After21days from initial treatment, mice were sacrificed under anesthesia by cervical dislocation and samples of tumor mass were removed and weighed. Draining lymph nodes and spleens were taken from each experimental group mice and prepared for the next assay. Quantitative real-time PCR was performed to assess the expression of TLR8, TNF-a, IL-12, IL-10, TGF-β and Foxp3mRNA in spleens and draining lymph nodes. FCM was applied to analyze the changes of TLR8expression in CD11c+cells and the percentages of CD3+CD8-IFN-γ+, CD3+CD8+IFN-γ+and CD4+CD25+Foxp3+T cells in spleens and draining lymph nodes.Hela cells, a kind of human cervical carcinoma cell line, were cultured in DMEM medium supplemented with10%streptomycin (100μg/ml) and penicillin (100U/ml) and10%heat-inactivated FCS. RT-PCR/qRT-PCR and immunofluorescence staining were employed to detect TLR8expression in Hela cells. The mRNA levels of COX-2, Bcl-2, VEGF and TLR-7,-8,-9in CL075-treated Hela cells or in cervical cancer tissues were also detected by quantitative real-time PCR (qRT-PCR). The efficacy of CL075on cell cycle and apoptosis was analyzed by FCM and MTT assay respectively. The correlation between TLR7/TLR8and Bcl-2/VEGF mRNA level in cervical cancer tissue was analyzed with Pearson method.Results:Observed under the optical microscope, DC2.4cells were small and spindle-, star-or polygonal-shaped, with short dendrites compared with the mature dendritic cells. After stimulated by PBS,4μM CL075,5μM Poly dT and4μM CL075and5μM PolydT for24hows, DC2.4cells were activated and their dendrites became more and long. In contrast, unstimulated or CL075/Poly dT alone stimulated DC2.4cells kept naive with a few and short dendrites. Expression of TLR8in DC2.4cells was confirmed by RT-PCR/qRT-PCR, western blot and immunofluorescence staining assay. Combination of Poly dT and CL075induces TNF-a and IL-12release while reduces IL-10release in murine DC2.4(*p<0.05, compared with control). There is no significant changes of cytokine release in CL075or Poly dT alone stimulated-DC2.4cells. After stimulated with CL075(4μM) and Poly dT (5μM) for72h, DC2.4cells induced stronger autologous T cell proliferative reponses in vitro than the negative controls. It is interesting that costimulation with Poly dT and CL075up-regulates TLR8expression in DC2.4.Combination of Poly dT and CL075suppresses tumor growth in mouse Lewis lung carcinoma models. The tumor volumes and tumor weights were significantly decreased in the high-dose group mice received CL0752μg and Poly dT50μg as compared with the PBS group mice(*p<0.05). The potent anti-tumor activity of CL075and Poly dT greatly encouraged us to further explore its profound mechanism. We found that high-dose of CL075and Poly dT significantly increases TNF-a and IL-12mRNA levels in draining lymph nodes and spleens, while the mRNA levels of IL-10, TGF-β and Foxp3were significantly lower than control groups. Consistently, percentages of CD3+CD8-IFN-γ+and CD3+CD8+IFN-γ+T cells in spleens and draining lymph nodes were significantly increased after high-dose of CL075and Poly dT administration, whereas percentages of CD4+CD25+Foxp3+T cells were clearly decreased. Interestingly, similar effects were observed in vivo that TLR8expression in CD11c+cells were upregulated by low dose of CL075and Poly dT. There was no difference in body weight between CL075and Poly dT-treated group mice and control group mice, suggested that as an immune response modifier, CL075or Poly dT has no cytotoxicity.Thirteen human cancer cell lines, including95D, HepG2, NICH446, U266, SGC, HCT-8, BGC, SW620, A549, HT1080, SKOV-3, U937and Hela were examined for TLR8expression. Our data showed that SKOV-3, U937and Hela expressed TLR8mRNA at high levels. Low TLR8levels were found in SGC, HCT-8, BGC, SW620, A549and HT1080; but no TLR8mRNA was detected in95D, HepG2, NICH446or U266. The expression level of TLR8mRNA in Hela cell line was higher than that in the other cell lines. Consistently, immunostaining showed presence of TLR8protein in Hela cells. Treatment of HeLa cells with CL075for48h resulted increases in the percentage of G2/M+S phase, suggesting an increase in cell proliferation. This was confirmed by MTT assay. Unlike DDP, CL075did not induce apoptosis in Hela cells. The levels of COX-2, BCL-2, and VEGF mRNA was significantly increased after CL075treatment for24h, and reached to peak at48h in Hela cells. Quantitative RT-PCR showed higher mRNA levels of TLR7and TLR8in cervical cancer samples from patients than that in cervical tissues from patients without cancer. In contrast, there was no clear difference in TLR9expression in tissue samples from the cancer patients and controls. Bcl-2and VEGF expression levels were significantly increased in cancer tissues from the patients with cervical cancer. Using the method of Spearman analysis, we found that there was a positive correlation between the expression level of TLR8and Bcl-2or VEGF in cervical cancer samples. No correlation was found between the TLR7and Bcl-2or VEGF mRNA.Conclusions:It was shown in our study that poly dT could modulate the imidazoquinoline effects on TLR8. Simultaneous addition of poly dT with humanTLR8agonist CL075activated TLR8signaling events in DC2.4cells and in C57BL/6mouse, demonstrated that murine TLR8is indeed functional. Activation of mouse TLR8could induce TNF-a and IL-12production from DCs and enhance immunostimulatory capacity of DCs, similar to those seen in human. Thus, to use the mouse as an animal model for TLR8activation as described here will help to facilitate such important research.TLR8may be a potential target for lung cancer immunotherapy. TLR8agonists have immune stimulatory effects through the induction of inflammatory cytokines such as TNF-a and IL-12that polarize Thl immune response in DC2.4cells and in Lewis lung carcinoma-bearing mice. On the other hand, TLR8agonists suppress immune tolerance as evidenced by the reduction of several immune suppressive factors including IL-10, TGF-β, Foxp3and T regulatory cells (Treg), all of which could dampen anti-tumor immunity. In general, TLR8agonists possess remarkable properties, particularly with regard to dendritic cell activation, promoting Thl-type cytokine production and optimizing cytotoxic T-cell responses. Importantly, low dose of TLR8agonist could enhance its efficacy by upregulation of TLR8expression.For Hela cells, it was shown that TLR8agonist promotes the proliferation and survival of the malignant cells. TLR8mRNA was upregulated both in cervical cancer tissues and Hela cells, and it consistent with the increased expression of VEGF and Bcl-2which correlated with poor prognosis in human cancers. Our study also indicates that the high TLR8-expressed cervical cancer cells may strongly correlate with carcinogenesis and tumor invasion by inhibiting TLR8positive immune cells to recognize tumor or viral antigen, which influenced anti-tumor immune response.Therefor, TLR8activation can be advantageous for the proliferation, invasiveness, and/or survival of tumor cells. These unwanted effects of TLR8agonists on tumor cells largely depend on the tumor cell type, and need to be carefully taken into account in preclinical studies. In conclusion, TLR8signaling acts as a double-edged sword in cancer therapy.