Priming Of Toll-like Receptor 4 Pathway In Mesenchymal Stem Cells Increases Expression Of B Cell Activating Factor

Mesenchymal stem cells (BMMSC) can be polarized into two distinct populations, MSCl and MSC2, by activation of different Toll-like receptors (TLRs). TLR4-primed MSCl expressed proinflammatory factors whereas TLR3-primed MSC2 expressed suppressive factors. However, little is known about the function of TLRs on B lymphocyte-related immune modulation. In this study, we investigated expression of B cell activating factor (BAFF), a member of the tumor necrosis factor ligand superfamily with notable stimulating activity on B cells, both in human BMMSC (hBMMSC) and in murine BMMSC (mBMMSC) after activation of TLRs. BAFF increasingly expressed in the presence of TLR4 agonist (Lipopolysaccharide, LPS) while TLR2 agonist (Zymosan) and TLR3 agonist (polyinocinic-polycytidykic acid, poly I:C) did’ t show effect on BAFF production. In addition, signaling pathways of NF-k B,p38 MAPK and JNK were demonstrated to be involved in TLR4-primed BAFF expression. Our results suggested that TLR4 and downstream pathway in BMMSC exerted important function in B lymphocyte-related immune regulation. How to define a homogeneous population of BMMSC will play a critical role in BMMSC-based immune-modulating therapy in future.1. Characterization and differentiation of BMMSCObjective:Identification of characteristics of both hBMMSC and mBMMSC.Methods:To stimulate adipogenic differentiation, BMMSC were cultured in the adipogenic medium for 14 days. Adipogenesis was detected by Oil red O staining, and was confirmed by phase contrast microscopic observation. To stimulate osteogenesis, BMMSC were cultured in the osteogenesis medium for three weeks. Alkaline phosphatase (ALP) expression was examined at day 21. Flow cytometry analysis was used to determine its immunophenotype.Results:Non-hematopoietic BM stromal cells were separated based on plastic adhesion, and characterized by immunophenotypes and their differentiation potential. The adherent cells grew as a spindle-shaped fibroblastic morphology after 2-3 passages. To determine their multi-lineage potency, we induced MSC differentiation to osteogenic and adipogenic lineages. After culturing in osteogenic induction medium for 3 weeks, the cells became positive for Alkaline Phosphatase staining, indicating that our cultured MSCs had osteogenic differentiation potential. The adipogenic differentiation of MSCs was confirmed by Oil red O staining after induction in adipogenic medium for 14 days. Murine MSCs were negative for CD45, CD31, or CD34, but mild positive for CD 106 and strong positive for Sca-1 and CD44. While human MSCs were also negative for CD45, CD31, or CD34, they were positive for CD105, CD90, and CD44. These data suggested that the cultured BM stromal cells possessed typical characteristics of MSCs.2. The effect of TLRs on the differentiation of BMMSCObjective:Analysis of the effect of TLRs on the differentiation of mBMMSCMethods:To investigate the effects of TLR2 and TLR4 on mBMMSC differentiation, the cells were induced to differentiate in the presence of TLR2 agonist, zymosan (1μg/mL), and TLR4 agonist, LPS (1μg/ml), respectively. After 14 days of adipogenic differentiation, it was stained Oil red O or Alkaline Phosphatase. After being cultured in osteogenic induction medium for 3 weeks, the cells were detected as positive staining of Alkaline Phosphatase.Results:Murine BMMSC retained the potential to differentiate into osteoclasts and adipocytes when the TLR2 and TLR4 were activated.3. The effect of TLRs on the production of BAFFObjective:Analysis of the effect of TLRs on the production of BAFF in both hBMMSC and mBMMSC.Methods:Firstly, different concentrations of LPS was used to determine the optimal amount of the agonists. Then, TLR2, TLR3 and TLR4 agonists were applied on mBMMSC and hBMMSC for 48h. And then the mRNA and protein expression was examined using real time PCR and western blot. Thirdly, the blocking experiments were used to further detect the function of TLRs. At last, NF-κB, JNK and p38 MAPK pathway inhibitors were used to determine the specific pathways involved. Immunofluorescence was used to detect the translocation of NF-κB.Results:It showed that BAFF was upregulated in a dose-dependent manner and peaked at the concentration of 1μg/ml LPS. Compared with the results of TLR2 activation by zymosan, TLR4 activation was shown to increase BAFF expression by 2-3 folds, whereas TLR2 activation slightly increased BAFF without significant difference. After the blockage of TLR4, BAFF decreased by 53.2+ 12.2%(p<0.05), whereas TLR2 blockage only decreased BAFF expression by 23.5±19.1% which had no significant difference compared to addition of zymosan only. PDTC, SP600125 and SB203580 decreased BAFF expression by 90.4±2.4%,87.0±7.2% and 97.9±4.8%, respectively (p<0.05).indicating that all these three signaling pathways were related to TLR4-regulated BAFF expression. In addition, immunofluorescence detection of NF-κB showed that NF-κB translocated into the nuclear after LPS stimulation. However, specific inhibitor to NF-κB signaling, PDTC, abrogated the transloaction effect.In summary. TLR4 activation was shown to increase BAFF expression by 2-3 folds in both hBMMSC and mBMMSC, whereas TLR2 and TLR3 activation slightly increased BAFF without significant difference. NF-κB, JNK and p38 MAPK pathways were all related to TLR4-regulated BAFF expression. Our results suggested that TLR4 and its downstream pathway in BMMSC exerted important function in B lymphocyte-related immune regulation. How to define a homogeneous population of BMMSC will play a critical role in BMMSC-based immune-modulating therapy in future.