Experimental Study On The Effects Of Porphyromonas Gingivalis Lipopolysaccharide On The Maturation And Antigen-presenting Functions Of Dendritic Cells

BACKGROUNDDendritic cells(DCs)are the most powerful antigen presenting cells in the immune system,and they have special status in the immune system.DCs efficiently uptake the antigen,process it into peptides and express it with the MHCH molecules on the cells’ surfaces.DCs migrate from the peripheral non-lymphoid tissues through the lymphatic tube and(or)blood circulation to secondary lymphoid organs and during their migration process,DCs become matured.DCs could prime naive T cells activation and proliferation,thus,DCs are considered the start and regulator of the body’s immune process.DCs can promote the innate immunity and start the acquired immunity,therefore DCs are an indispensable bridge between innate immunity and acquired immunity.At present,a lot of studies have pointed out that the inflammation of periodontal tissues is the immune response of pathogen infection,and the host immune system plays a key role in the pathogenesis of periodontitis.In recent years,with the progress of immunology,the role of dendritic cells in the pathogenesis of periodontal diseases is increasingly valued.Understanding of DCs in the pathological environment in periodontal disease,especially the effects of periodontal pathological environment on DCs maturation and antigen-presenting function could further elucidate the role of DCs in the occurrence and development of periodontitis.As cellular immunity entry point,DCs play an important role in the pathogenesis of periodontal disease.Our study will systematically explore the mechanism of P.gingivalis-LPS promoting DCs maturation and antigen presentation,aiming to study the functional status of DCs in periodontal pathological microenvironment.The results of this study will provides experimental basis for studying the possible mechanism of DCs in the occurrence and development of periodontitis,and lay a foundation for the study of regulating DCs immune function to improve or treat periodontal diseases.Section One Experimental periodontitis model establishment in Sprague-Dawley rats and Immunohistochemical identification of DCs in their gingival tissues and submandibular lymph nodesObjective To establish experimental periodontitis model in Sprague-Dawley(SD)rats by thread ligation plus coating P.gingivalis and identificate DCs in the gums and submandibular lymph nodes of these rats by means of IHC.Methods 12 male SD rats were randomly divided into 3 groups,A:control group,B:thread ligation group and C:thread ligation with P.gingivalis coating group.In groups B and C,the necks of the first,second and third molars in bilateral mandibular of each rat were ligated by 4-0 surgical thread.In group C,the teeth necks were also coated with P.gingivalis around the thread.After 4 weeks,the animals were killed and the establishment of periodontitis model was examined.Anti-Langerin and anti-CD11c monoclonal IHC antibodies were used to identify DCs existence in the gingiva of these periodontitis SD rats.Anti-CD11c and anti-CD80 monoclonal IHC antibodies were used to identify DCs existence in the submandibular lymph nodes of these periodontitis SD rats.Results Model of periodontitis was successfully established in the SD rats in group C after 4 weeks.CD11c+ and Langerin DCs could be found in gingiva of these SD rats.CD11c+and CD80+DCs could be found in the submandibular lymph nodes of these SD rats.Conclusion The establishment of experimental periodontitis in SD rats is reliable and it could be used as the model for the occurrence and development of periodontal disease.The presence of DCs could be detected in the gingiva and the submandibular lymph nodes of these SD rats.Section Two Establishment of culture method and detection characteristics of rats DCs in vitroObjective To investigate the establishment and optimization of the culture system for bone marrow derived rats DCs in vitro.Methods Bone marrow cells of SD rats were induced and differentiated in vitro by rrGM-CSF(10ng/ml)and rrIL-4(lng/ml).On day 6,E.coli-LPS(100ng/ml)was added and the cells were cultured for another 48h to generate mature DCs.The cells morphological features were observed by inverted phase contrast microscope.The cell organelles morphological features were observed by TEM.Phenotypes of CD11c,MHCII,CD80,CD86 and CD40 of DCs were detected by FCM.IL-12,IFN-y,IL-10 and IL-13 in the DCs culture supernatant were detected by ELISA.MLR were performed to observe the proliferation of allogenic CD4+T lymphocyte cells co-cultured with the DCs.Results The cells cultured by this means were above 90%CD11c+.After E.coli-LPS stimulation,the cultured cells displayed the typical morphology of DCs.Compared with immature DCs,mature DCs expressed higher MHCII,CD80,CD86 and CD40.They produced significantly more IL-12,IFN-y,IL-10 and IL-13 and they stimulated allogeneic T lymphocyte cells proliferation more effectively in MLR.Conclusion The culture system from bone marrow cells of rats can induce plenty of DCs through rrGM-CSF,rrIL-4 induced and E.coli-LPS stimulated.Section Three Experiments in vitro about the effects of P.gingivalis-LPS on the maturation and antigen-presenting functions of DCsObjective Research the effects of P.gingivalis-LPS on maturation and antigen-presenting functions of DCs.Methods①Observe the morphology of DCs derived from SD rats marrow which were stimulated by P.gingivalis-LPS through inverted phase contrast microscope and TEM.②FCM was used to detect CD11c,MHC Ⅱ,CD80,CD86 and CD40 expressions on DCs which were stimulated by P.gingivalis-LPS and ELISA was used to detect IL-12,IFN-y,IL-10 and IL-13 secreted by DCs.③CCK8 was used to assay CD4+T cells proliferation after co-cultured with DCs stimulated by P.gingivalis-LPS and ELISA was used to detect IL-2,IFN-y,IL-10 and IL-13 secreted by T cells.④TLR4 inhibitor(polymyxin B)or TLR2 and TLR4 inhibitor(OxPAPC)was added to P.gingivalis-LPS group and E.coli-LPS group to observe the effects of these two TLR inhibitors on the maturation and antigen-presenting functions of DCs.Results:①Inverted phase contrast microscope and TEM showed mature state of DCs stimulated by P.gingivalis-LPS as there were long dendrons on cell surfaces.②FCM results showed that the capacity of P.gingivalis-LPS to stimulate DCs maturation was similar to that of E.coli-LPS.The amount of IL-12 and IFN-y secreted by DCs in P.gingivalis-LPS group was significantly lower than that of E.coli-LPS group,but in the amount of IL-10 and IL-13,P.gingivalis-LPS group was higher than E.coli-LPS group.③DCs stimulated by both P.gingivalis-LPS and E.coli-LPS could promote the proliferation of CD4+T cells.The amount of IL-2 and IFN-y secreted by T cells cocultured with DCs in P.gingivalis-LPS group was significantly lower than that of E.coli-LPS group,IL-10 in P.gingivalis-LPS group was higher than E.coli-LPS group and there was no significant difference in the amount of IL-13 between these two groups.④ When TLR4 inhibitor was added to E.coli-LPS group,maturation and antigen-presenting functions of DCs were significantly inhibited.When TLR4 inhibitor was added to P.gingivalis-LPS group,maturation and antigen-presenting functions of DCs were not significantly inhibited.When TLR2 and TLR4 inhibitor was added to P.gingivalis-LPS group,maturation and antigen-presenting functions of DCs were significantly inhibited.Conclusion ① P.gingivalis-LPS could promote DCs maturation and antigen-presenting functions.②DCs stimulated by P.gingivalis-LPS are prone to induce a stronger Th2 cell responses and DCs stimulated by E.coli-LPS are prone to induce a stronger Th1 cell responses.③P.gingivalis-LPS triggers DCs through TLR2 pathway and E.coli-LPS triggers DCs through TLR4 pathway.Section Four Experiments in vitro about the synergism effects of NOD2 agonist and P.gingivalis-LPS on the maturation and antigen-presenting functions of DCsObjective To study the synergy effect of NOD2 agonist(MDP)and P.gingivalis-LPS on maturation and antigen-presenting functions of DCs to provide experimental evidences to explore the possible mechanism of DCs in the occurrence and development of periodontitis.Methods ①Different concentrations of MDP and gingivalis-LPS,separately or synergistically,were used to stimulate SD rats bone marrow-derived DCs.②CDllc,MHCII,CD80,CD86 and CD40 expressions on DCs surface were detected by FCM;DCs TLR2,TLR4 and NOD2 mRNA expressions were detected by real-time PCR;IL-12,IFN-γ,IL-10 and IL-13 secreted by DCs were assayed by ELISA.③CD4+T cells proliferation primed by DCs in each group was assayed by CCK8;IL-2,IL-10,IFN-γ and IL-13 secreted by T cells were assayed by ELISA.Results ①FCM results showed that MDP had weak ability to prime DCs,but the synergistic effect of MDP and P.gingivalis-LPS could obviously promote DCs maturation.PCR results showed that P.gingivalis-LPS could improve TLR2 mRNA expression of DCs,but not TLR4.Synergistic effect of MDP and P.gingivalis-LPS could improve the expression of TLR2 mRNA in DCS.ELISA results showed that the synergistic effect of MDP and P.gingivalis-LPS could improve the amount of IL-10 and IL-13 secreted by DCs.②MLR results showed that the synergistic effect of MDP and P.gingivalis-LPS could promote the proliferation of CD4 T cells,and promoted the secretion of IL-10 and IL-13 by T cells.Conclusion①P.gingivalis-LPS primed DCs through TLR2 signaling pathway.②The ability of MDP to promote the maturation and antigen-presenting functions of DCs was weak.③The synergism of MDP and P.gingivalis-LPS could promote DCs maturation and antigen-presenting functions.④DCs primed by the synergistic effect of MDP and P.gingivalis-LPS could promote the differentiation of Th0 cells to Th2 cells.