The Effect Of Memory B Cells Regulating RANKL On Periodontal Osteoimmunology

Objective:Periodontitis,the sixth-most prevalent disease in the world,is a periodontopathogenic bacteria-induced disease characterized by periodontal immune cells infiltration,inflammatory factors chemotaxis and continual destruction of periodontal supporting tissue.As the disease continues to advance,periodontal ligament and alveolar bone start to dissolve,absorb and destroy,leading to tooth loosened and even fallen off.Apart from oral disfunction,periodontitis has also proven to be closely related to some systemic disease include cardiovascular and cerebrovascular diseases,diabetes and maxillofacial tumors.Therefore,it is become important for dentist to study the occurrence,prevention and therapeutic measures of periodontitis.Notwithstanding periodontitis initiates by the imbalance of subgingival plaque ecosystem,only microflora disorder could not lead to alveolar bone damage.According to newly reported research,the key reason of periodontal tissue destruction is host adaptive immune response which against periodontopathogenic bacteria.As the executor of humoral immunity,B cells are abundant in periodontal lesions.In addition to its well-known function of antibody secreting,B cells have been illustrated to express some pro or pre-inflammatory cytokines which regulated immune response and bone homeostasis.The interaction of skeletal system and immune system is called osteoimmunology.RANKL,a kind of typeⅡtransmembrane protein,is one of the main ligand of tumor necrosis factor receptor.RANKL could combine with RANK which located on the membrane of osteoclast precursor,subsequently initiating the differentiation of osteoclast.Accordingly,RANKL is considered to be an indispensable factor of bone homeostasis regulation.Given the tightly relationship of B cell and RANKL,it is important for studying the effect of B cells on alveolar bone homeostasis to disclose how B cells affect RANKL level during periodontitis.The present study elucidated the functional B cell subpopulation which participates in periodontal osteoimmunology,and discovered its working mechanisms by animal and cellular experiments.The presented study furtherly clarified the pathogenesis of periodontitis.Moreover,considering that B cells exist in gingiva in a long-term and stable mode,the result of this study provide a novel idea for studying physical periodontal bone homeostasis,alveolar bone remodeling and peri-implant inflammation.Methods:The present study is consisted of three parts.1.Memory B cells regulate RANKL expressionWe first establish experimental periodontitis animal models by ligation.Animals were sacrificed after 4 weeks,their lymphocytes were purified and divided into B cells(CD20~+),T cells(CD3~+),and double negative lymphocytes(CD3~-CD20~-).Furtherly,we purified B cells from total lymphocytes and then divided them into different subsets as follows:naive B cells(CD27~-),antibody-secretingB cells(CD27~+CD38~+),memory B cells(CD27~+CD38~-).After a 48 hours culture,the expression of RANKL by each cell subpopulation is detected by flow cytometry,ELISA and Real time PCR.Their results will be compared to illustrated the effect of periodontitis on the process.We also detected the expression of ERK,p38 and JNK in different subsets to illustrate the potential mechanism of change of RANKL production by each lymphocytes subsets.2.The effect and mechanisms of memory B cells regulating the differentiation of osteoclast.Rat bone marrow derived macrophages were extracted from their femur and chosen as osteoclast precursor.We culture the osteoclast precursor by the culture medium of different from the last step in 96 well plates,10~4/well.Half of the culture medium well be renewed every 3 days.In the blocking experiment,an RANKL neutralizing antibody was added to the culture medium.After a 7-day culture,the cells were stained using TRAP kits to detect the formation of osteoclasts.The mRNA level of osteoclast differentiation related gene RANK,NFATc1,c-Src were evaluated by Real time PCR.3.The effect of memory B cells regulating RANKL on bone homeostasis during periodontitisWe established adoptive transfer animal models by into the tail vein injecting of pathogen sensitized memory B cells to the recipients.Equal number of healthy animals and adoptive models were chosen and then divided into 4 groups:healthy group;experimental periodontitis group;adoptive transfer group;adoptive transfer and block group.We induced experimental periodontitis on all animals except the one of healthy group.Animals from adoptive transfer and block group were treated by anti-RANKL after periodontal ligation.4 weeks later,animals were sacrificed,their GCF,peripheral blood sera,left maxilla and gingiva were separated.We gauged ther CEJ-ABC distance and the osteoclasts coverage length on the surface of the alveolar bone within the coronal0.5mm.Also,RANKL mRNA level of their gingiva was detected by Real time PCR.The concentration of RANKL,IL-1β,IL-6,IL-17A and TNF-αin their GCF and blood sera were analyzed by ELISA.Results:1.All lymphocyte subpopulations from healthy animals produce RANKL,however,T and B cells express significantly more RANKL than double negative lymphocytes.During periodontitis,the expression of RANKL by B cells improved significantly and became higher than other lymphocyte subpopulations.Similarly,memory B cells increased its RANKL expression during periodontitis while antibody secreting cells and naive B cells not.These effects are detected at both protein and gene levels.RANKL expression were closely related to p38 expression.2.Soluble RANKL secreted by different subpopulations of all lymphocytes and B cells had the ability to induce osteoclast differentiation.The effect could be blocked by anti-RANKL.In healthy condition,T and B cells induce more osteoclast differentiation than double negative lymphocytes.During periodontitis,this ability of B cells improved significantly and became significantly higher than that of T cells and double negative lymphocytes.Among B cell subpopulations,memory B cells and antibody secreting cells from healthy animals induce more osteoclast differentiation than that of immature B cells.During periodontitis,memory B cells increase their ability of inducing osteoclast differentiation,and become higher than antibody secreting cells and naive B cells.3.In comparison with normal experimental periodontitis model,adoptive transfer animals showed more alveolar bone resorption and longer periodontal osteoclasts coverage length.Similarly,there was more RANKL mRNA expression in their gingiva than normal models.Additionally,levels of RANKL,IL-1β,IL-6,and IL-17A in gingival crevicular fluid of adoptive transfer model were significantly higher than those in normal experimental periodontitis model.anti-RANKL could effectively reduce alveolar bone resorption and periodontal osteoclast formation in adoptive transfer model animals,and significantly down-regulate the level of RANKL in gingival crevicular fluid at the same time.Conclusion:1.Periodontal inflammation could increase the expression of RANKL by memory B cells,however,this effect are not significantly on other B cell subpoplulations.p38/MAPK signal pathway played a probable role in promoting RANKL expression.2.Memory B cells affect the process of periodontitis by regulating local osteoclastogenesis via RANKL manner.