Dissecting The Role Of Sphingosine 1-Phosphate Receptor 1 In Apical Periodontitis

Apical periodontitis (periapical lesions), one of the most common dental diseases, results in lesion formation around the apex of the root accompanied by alveolar bone resorption. Since these lesions represent the immune response to pathogen invasion, figuring out the immune mechanisms of this disease will help us improve its therapy. Sphingosine-1-phosphate (S1P) is a bioactive lipid molecule; Sphingosine-1-phosphate receptor 1 (S1P1) is one of the five members of G protein-coupled S1P receptors that are expressed at the plasma membrane. S1P binds to S1P1 in the ways of autocrine or paracrine to mediate diverse cellular inflammatory and immune responses. The S1P/S1P1 axis, which playing a crucial role in regulating immune cell movement and function, is essential in inducing inflammatory response and autoimmune diseases, which is partially achieved by down-regulating the expression and function of immune suppressive T regulatory cells (Treg cells) while up-regulating those of immune inductive T helper cells 17 (Th17 cells). S1P1 signalling not only controls the migratory behavior of osteoclast precursors, but also promotes RANKL (which is crucial in osteoclastogenesis) production, therefore play a key role in osteoclasts differentiation and function. The immunomodulating drug Fingolimod (FTY720) is phosphorylated in vivo and then down-regulating the S1P/S1P1 axis in the way of inducing the internalization of S1P1, which subsequently suppressing S1P1-induced immune reaction and bone resorption; therefore FTY720 has a therapeutic effect on diseases with inflammatory bone destruction. The current study aims to investigate the expression of S1P1 in rat Periapical lesions and its relationship with RANKL, Treg cells and other related factors, finding out the possible role of S1P1 in the pathogenesis of apical periodontitis, also assessing the therapeutic effect and mechanisms of modulating S1P1 by FTY720 systemic administration in induced rat periapical lesions model, therefore dissecting the role of S1P1 in apical periodontitis.PART I Dissecting the role of Sphingosine-1-phosphate receptor 1 in rat apical periodontitisExperiment 1:Induction and identification of rat periapical lesions modelObjective:The purpose of the present study was to build up the induced rat periapical lesions model, harvesting samples, identifying the model by using High-resolution X-ray Imaging, ?CT Analysis and histological analysis.Methods:Periapical lesions were induced by pulp exposure under anesthesia in the first lower molars of 55 Wistar rats. Thirty rats were sacrificed on day 0,7,14,21, 28, and 35, and their mandibles harvested for X-ray imaging, micro-computed tomography (?CT) scanning, histological observation and the following experiments (experiment 1 and 2). The remaining 25 rats were sacrificed on days 0,14,21,28, and 35, mandibles and the draining lymph nodes were harvested for flow cytometry (experiment 3).Results:Results From X-ray imaging, ?CT scanning and histological observation showed that the volume and area of the periapical lesions increased from day 0 to day 21, and then remained comparably stable after day 28. The inflammatory cells infiltration started at day 7, peaking on day 14 to day 21 and becoming comparably stable after day 28.Conclusions:The results were according with precious studies, which meaning this model was successful and reliable. It could be concluded that acute phase was from day 7 to day 21, and chronic phase was after day 28. The X-ray imaging, which was firstly used, is an effective way in analyzing periapical lesions.Experiment 2:Identifying the expression and mechanisms of SIP 1 in periapical lesionsObjective:The purpose of the present study was to investigate the expression of S1P1 in rat periapical lesions and its relationship with receptor activator of nuclear factor kappa B ligand (RANKL) and osteoclasts.Methods:Periapical lesions were induced and rats were sacrificed on day 0,7, 14,21,28, and 35, and their mandibles were harvested (experiment 1) for immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis.Results:S1P1-positive cells were observed in the inflammatory periapical regions; the number of S1P1-positive cells peaked at day 14, and then decreased from day 21 to day 35. The distribution of S1P1-positive cells was positively correlated with the dynamics of RANKL-positive cells and osteoclasts. S1P1 and RANKL double-positive cells were found in the acute (higher) and chronic (lower) phases of periapical lesions.Conclusions:S1P1 expression was confirmed in rat periapical lesions; it was positively correlated with RANKL and osteoclasts expression and is therefore a contributing factor to the pathogenesis of periapical lesions.Experiment 3:S1P1 inducing the imbalance of Th17/Treg in periapical lesionsObjective:The purpose of the present study was to investigate the expression of Treg cells, Th17 cells and CD4+Foxp3+IL-17+cells (Tr17 cells) in rat periapical lesions and the relationship with S1P1.Methods:Sections from experiment 1 were get for double and triple immunofluorescence analysis. Primary cells were harvested from lesions tissue and lymph nodes (experiment 1) for FACS analyzing.Results:Tr17 cells were observed in periapical lesions and the draining lymph nodes. The distribution of Th17 cells was positively correlated with the dynamics of S1P1-positive cells, while the distribution of Treg cells was negatively correlated with S1P1 expression after day 14. S1P1 and IL-17 double-positive cells were found in periapical lesions.Conclusions:In the pathogenesis of periapical lesions, S1P1 up-regulated Th17 cells differentiation while down-regulated that of Treg cells, which inducing the imbalance of Thl7/Treg, therefore should be a contributing factor of periapical lesions.PART ? Protective effect of modulating S1P1 on rat periapical lesionsObjective:The purpose of the present study was to investigate the effect and mechanisms of modulating S1P1 by Fingolimod (FTY720) administration on rat periapical lesionsMethods:80 Wistar rats were divided into two groups (the control group and the FTY720 group). Periapical lesions were induced by pulp exposure under anesthesia in the first lower molars of these rats. Rats were sacrificed on day 0,7,14,21,28, and 35, and their mandibles were harvested for histological observation, immunohistochemistry, enzyme histochemistry and western blot analysis.Results:FTY720 administration effectively inhibited the alveolar bone destruction, which was accompanied by down-regulated S1P1, RANKL and IL-17 expression but up-regulated Treg cells expression (no effects on OPG expression), then subsequently impeded osteoclasts differentiation and function.Conclusions:Modulating S1P1 by Fingolimod was effective on rat periapical lesions, which was achieved via down-regulating the S1P1 signaling and therefore modulating the imbalance of RANKL/OPG and up-regulating Treg expression but down-regulating IL-17 expression, suggesting S1P1 could be a potential target for treatment of periapical lesions.