Regulation of TLR Signaling in B-Cell Activation and Survival

Objectives: Toll-Like Receptors (TLRs) play a key role in innate and adaptive immune responses in periodontal disease. While B lymphocytes express multiple TLRs, the role of TLR signaling in B cell activation and survival is unclear. In this study we have determined the role of TLR2 and TLR4 signaling in Porphyromonas gingivalis (P. gingivalis) LPS-induced B-cell activation and survival. Methods: Three groups of mice (6-8 weeks old) of C57/BL6 background were used: Wild Type (WT), TLR2 knockout (KO) and TLR4 KO. Splenocytes were extracted from mouse spleen and were stained with FITC-labeled anti-CD19 antibody. B cells were isolated from each splenocytes suspension using flow cytometry cell sorting (FACS, BD FACSAria III). Purified B cells were cultured in 96-well plate at a concentration of 105/well in the presence or absence of P. gingivalis LPS (strain: ATCC 33277). After various time of incubation, Cell Titer 96 AQueous One solution was added to each well and B cell proliferation was determined after 4 hours using microplate reader. Results: After cultured for 48 hours, the percentage of live B cells decreased more sharply in TLR2 KO mice (37.7%) compared to those in WT (21.6%) or TLR4 KO (24.8%) mice. Addition of anti-CD19 antibody did not change the B cell proliferation rate and the percentage of live B cells in culture. B cells from WT or TLR4 KO mice showed increased proliferation after LPS stimulation. However, B cells from TLR2 KO mice did not demonstrate any increase in cell proliferation after LPS stimulation. In the absence of LPS, B cells from TLR4 KO mice showed reduced rate of proliferation as compared to those from WT mice or TLR2 KO mice under normal culture conditions. Conclusions: TLR2 signaling may play a role in B cell proliferation and survival in response to P. gingivalis LPS.