Expression And Function Of Toll-like Receptor In Multiple Myeloma Patients

1. Expression of Toll-like receptors in Multiple myeloma patientsObjective To investigate the expression profiles of Toll-like Receptors (TLRs) in Bone Marrow mononuclear cells (BMMCs) from Multiple myeloma (MM) patients and healthy dornors,and explore the function of TLRs in MM cells.Methods After informed consent was given, bone marrow from 29 MM patients at diagnosis and 25 healthy donors were collected in this study. Mononuclear cells were obtained by centrifugation on Ficoll-hypaque medium,TLR1-10 mRNA in BMMCs were detected by real-time PCR and TLR protein were analyzed by FACS。The percentage of CD138 positive cells was detected in BMMCs followed treatment with TLR ligands. Primary myeloma cells from 5 MM patients and normal plasma cells from 3 healthy donors were purified using CD138 microbeads according to the instruction of the manufacturer,TLR1-10 mRNA were detected by RT-PCR。Results We measured the TLR1-10 specific transcripts in BMMCs from 29 MM patients at diagnosis and 25 healthy donors by quantitative RT-PCR. It was interesting to observe that the expression of TLR2, TLR4, and TLR9 from MM patients were significantly higher than those from healthy donors (over 2-fold and p<0.05). We further detected TLR2, TLR4 and TLR9 expression in the thawed BMMCs from 10 patients and donors with flow cytometry; and the results showed that expressions of TLR4 and TLR9 in MM patients were significantly higher than those from healthy donors as well; nevertheless the expression of TLR2 was not significantly different between the two groups. The freshly separated BMMCs of MM patients and healthy donors were cultured with specific ligands of TLR 2, 4 and 9, namely Pam3Csk4, LPS, and CpG DNA. The expression of CD138 of their progenies after 72 hour culture was analyzed with FACS. Upon treatments of LPS and CpG DNA the CD138 expressed cells increased by nearly 60%, but the treatment of Pam3Csk4 did not significantly alter the proportion of CD138 expressed cells. To explore whether TLR ligands directly promote MM cells growth, we first purified CD138+ cells from 5 MM patients and 3 healthy donors with microbeads to reach the purity over 90%. The expression of mRNA of TLRs of these 5 primary samples together with two MM cell lines U266 and RPMI-8226 and 3 normal plasma cells were analyzed with RT-PCR TLRs mRNA could be detected in most of these 5 patients, and TLR4, 7, 8 and 9 were detectable in all patients. However, few TLRs mRNA were detected in normal plasma cells. It was interesting that TLR4 and TLR9 were detectable in all 5 patients and the two cell lines; in contrast only TLR9 was detected in one out three healthy donors.Conclusions Compared to healthy dornor, the transcript expression of TLR2, TLR4, and TLR9 were increased in BMMCs from MM patients. Moreover, TLR4 and TLR9 protein were also increased in BMMCs from MM patients. TLR mRNA in primary MM cells was significantly higher than the ones in normal plasma cells. LPS and CpG DNA could promote MM cells proliferation.2. The mechanism of promotion of MM cells proliferation and survival by LPS and CpG DNAObjective To explore the mechanism on promotion of MM cells proliferation and survival by LPS and CpG DNA.Methods MTT assay to evaluate proliferation of primary MM cells, RPMI-8226,XG-7 and normal plasma cells with the treatment of Pam3Csk4,LPS and CpG DNA. Annexin V antibody detection to evaluate MM cells apoptosis and ELISA assay to detect IL-6 levels in the supernatants obtained from purified MM cells and MM cell lines following TLR ligands treatment. After blockage of IL-6 with neutralizing monoclonal anti-human IL-6 antibody,MM cells proliferation were measured by MTT assay. Results LPS and CpG DNA could promote the cell growth compared to control cells with primary cells,RPMI-8226 cells and XG-7 cells, however Pam3Csk4 could not consistently promote the growth with all these cells. Worth of note, normal plasma cells failed to show significant difference in cell proliferation following Pam3Csk4, LPS or CpG DNA treatments. MM cells apoptosis caused by the serum deprivation was dramatically reduced with LPS and CpG treatments, while Pam3Csk4 showed a less effect on protecting these cells from apoptosis.No significant difference was observed in normal plasma cell apoptosis among Pam3Csk4,LPS and CpG DNA group. IL-6 production in supernatants was dramatically increased upon TLR ligands treatment compared to the control cells. Interestingly, the neutralizing IL-6 antibody partially inhibited LPS and CpG DNA induced cell proliferation of MM7, MM8 , RPMI-8226 and XG-7 cells, while the isotype control IgG could not abolish the ligands induced proliferation.Conclusions LPS and CpG DNA could promote the cell growth and survival compared to control cells with primary cells, RPMI-8226 cells and XG-7 cells. IL-6 production in supernatants was dramatically increased upon TLR ligands treatment compared to the control cells. The increased proliferation in MM cells and MM cell line following TLR ligands treatment was partially due to induction of IL-6 production.3. The study of the potential signaling pathway employed by LPS and CpG DNA on the promotion of MM cells growth.Objective To explore the potential signaling pathway employed by LPS and CpG DNA on the promotion of MM cells growth.Methods JNK-I(SP600125), PD169316 and PD9805 were the inhibitor of JNK, p38 and ERK kinase, while plasmid IκBα-SR encoded a superrepressor form of IκBα. We firstly inhibited each of them respectively with either small chemicals or inhibitory plasmid and measured cell proliferation with MTT assay. Western blot analysis was used to detect the activation of signal molecular. Cytoplasm and nuclear fractions were obtained to measure nuclear translocation of p65 subunit took place upon TLR ligands stimulation. Real-time PCR to detect IL-mRNA in MM cells under activation and inhibition of NF-κB.Results JNK-I(SP600125), PD169316 and PD9805 were the inhibitor of JNK, P38 and ERK kinase, while plasmid IκBα-SR encoded a superrepressor form of IκBα. The growth of MM cells were reduced by all these inhibitory agents; LPS and CpG DNA were able to recover the JNK, p38 and ERK inhibitor suppressed cells but not the IκBαsuppressed cells, which indicated a crucial role of activated NF-κB in TLR ligands enhanced MM cell growth. Subcellular fractionation of NF-κB p65 subunit, demonstrated that the nuclear translocation of p65 subunit took place upon TLR ligands stimulation. NF-κB activating plasmids-IKKαand IKKβwere transfected into U266 and RPMI-8226 cells, and they were capable to promote the cell growth in a dose-dependent manner; at the same time, IL-6 transcript was found to be up-regulated in the presence of these two activation plasmids in a dose-dependent manner as well. In the contrast, the inhibitory plasmid IκBα-SR alone was found to suppress the cell proliferation and IL-6 transcript expression.Conclusions LPS and CpG DNA could recover the suppression of MM cells growth caused by blockage of MAPK and JNK signaling pathways but not NF-κB. Activation of Nuclear factor-kappa B by LPS and CpG DNA contributed to MM cells growth.