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| Title: | Role of TLR4 and TLR9 Signaling in the Innate vs. Adaptive B10 Cells Activation |
| Authors: | Gallucci, German Sima, Corneliu Yu, Qing |
| Description: | Introduction: Periodontal disease is an inflammatory disease that is mainly caused by periopathogenic microogranisms in the jaw area of the human body, which, if left un treated, results in the destruction of tooth supporting tissue and possible tooth loss. The unbalance host immune system due to virulent microorganisms leads to the continuation of periodontal inflammation and alveolar bone resorption. Although attempts to minimize the destruction of periodontal disease that included mechanical and pharmacological approaches have improved periodondtal disease outcome, yet not all do respond favorably to theses approaches. Therefore, new clinical approaches such as autotherapies are needed to further enhance the immunological responses of the body in the event of periodontal inflammation and improve periodontal disease outcomes.
Objective: B10 cells, a regulatory B cell subset that is expressed in the event of inflammatory condition and known by its ability to express a potent anti-inflammatory mediator known as Interluekin-10 (IL-10) have been shown to carry protective and therapeutic effects. Only recently has it been reported that local injection of B10 cells through Toll Like Receptor 4 (TLR4) and Toll Like Receptor 9 (TLR9) ligands resulted in alleviation of periodontal inflammation and alveolar bone resorption in vitro. Accordingly, this study aims to investigate the role of TLR 4 and TLR 9 signaling on B10 cells activation, expression and secretion and in the primed vs. naïve spleen cells and B cells responses in vitro.
Methods: Sixty C57BL mice (male:female = 1:1; 10 to 20 weeks old) Wild-type (WT), TLR4 knockout (TLR4KO) and TLR9 knockout (TLR9KO) mice (10 each) were divided into 2 groups (n=5). Pre-immunized mice were injected with formalin-fixed P. gingivalis (108) on day 0 and boost with 107 on day 7. Mice Spleens were harvested on day 11, Spleen cells and B cells were isolated. The Isolated spleen and B cells from each mouse were treated with 1x108 formalin-fixed P. gingivalis in culture and spleen cells and B cells without treatment were used as control. Cultured spleen cells and B cells were collected at 0, 24 and 48 hrs. Flow cytometry was performed to measure the frequency of B10 cells population (CD19+IL-10+ B cells) in all B cells and the differences of population of antigen-specific B cells (CD19highPIlow) in total B cell. Immunocytochemistry was performed to detect CD45+ IL-10+ cells. Images of five randomly chosen fields for each cell spot were obtained, and the double positive cells among all counted cells was calculated. levels of Pro-inflammatory and anti- inflammatory mediators expression and secretion were performed using qRT-PCR and ELISA respectively. Statistical analysis was performed using T test of P ≤ 0.05.
Results: Our investigations showed that the populations of B10 cells and P. gingivalis-binding B cells increased after treatment with LPS and CpG in-vitro (P<0.05). Stimulation with fixed P. gingivalis significantly increased B10 cell activation (P<0.01) of purified spleen and B cells ICC- stained CD45+IL-10+ after 24 and 48 hours in immunized purified spleen cells and B cells of WT and TLR4KO mice. Also, fixed P. gingivalis significantly up-regulated m-RNA gene expressions of IL-10 and TNF-α levels (P<0.01) after 24 and 48 hours in immunized purified spleen cells of WT and TLR9KO mice and purified B cells of WT mice. The secretion levels of IL-10 after stimulation with fixed P. gingivalis were examined via Elisa testing and showed significant increase of IL-10 levels after 24 hours in immunized purified spleen cells and B cells of WT mice and after 24 and 48 hours in immunized purified spleen cells of TLR9KO mice. Moreover, the innate immune responses after stimulation with fixed P. gingivalis showed significant up-regulated gene expressions of IL-10 levels (P<0.01) after 24 hours of purified B cells in TLR4KO mice, after 48 hours of purified B cells in TLR9KO and after 24 and 48 hours of purified spleen cells in WT mice. Also, TLR9KO mice showed significant down-regulated gene expressions of IL-10 levels (P<0.01) after 24 hours in purified spleen cells of TLR9KO mice. In addition, The IL-10 secretion levels were increased (P<0.01) after 24 and 48 hours of purified spleen cells in WT, TLR4KO and TLR9KO mice.
Conclusion: P. gingivalis-induced B10 cell activation and expansion is achieved mostly by antigen-specific adaptive immune responses. While TLR4 and TLR9 signaling are important in the in innate vs. adaptive activation of B10 responses and IL-10 production, yet they play different roles in that aspect. Antigen presentation and immune cell-cell interaction are prerequisites for IL-10 secretion and selective activation of TLR4/9 signaling in B cells may potentiate B10 function without promoting pro-inflammatory cytokine responses. Oral Biology |
| URI: | http://lib.yhn.edu.vn/handle/YHN/200 |
| Other Identifiers: | Alaqla, Ali. 2020. Role of TLR4 and TLR9 Signaling in the Innate vs. Adaptive B10 Cells Activation. Doctoral dissertation, Harvard School of Dental Medicine. https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37365593 |
| Appears in Collections | Y học |
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