The role and modulation of the inflammasome in the sickle cell disease oral phenotype in female mice

Abstract Sickle Cell Disease (SCD) is the most common inherited blood disorder affecting millions worldwide. SCD is caused by a genetic mutation which inhibits β-hemoglobin protein folding, causing erythrocyte sickling, vaso-occlusion and hypoxia resulting in damage to tissues and organs including bone. Sickle cell bone disease has been investigated with most studies being conducted in the long bones and very few in the oral cavity. The Hurley lab reported that femur (long bone) loss in SCD mice is associated with phosphaturia and increased Fibroblast Growth Factor 23 (FGF23) and that FGF23 neutralizing antibody (FGF23Ab) reduced phosphate wasting and partially rescued long bone loss in humanized SCD mice that harbor the mutated human β-hemoglobin gene. Among the few published oral studies, the consensus is that it is difficult to pinpoint the exact cause of dental or alveolar bone phenotypes. A common oral phenotype seen in SCD patients is necrosis within the pulp chamber due to chronic inflammation in these patients. Since FGF23 can increase inflammatory cells, we hypothesized that FGF23 mediated inflammation/ activation of the inflammasome play a major role in the SCD dental phenotype. To test the hypothesis,five-month-old female Townes SCD mice were treated subcutaneously for 6 weeks with either IgG or FGF23Ab and then sacrificed. Excised mandibles were subjected to microCT analysis after which they were processed and sectioned for static and dynamic histomorphometry and protein analysis. MicroCT data showed that SCD-IgG mice had significantly smaller pulp chambers as well as decreased enamel, dentin, and alveolar bone volumes. Further, static histomorphometry data showed that SCD-IgG mice had a significantly higher amount of TRAP staining compared to controls, which was significantly decreased after FGF23Ab treatment. Dynamic data showed that SCD-IgG mice exhibited a marked reduction in their interlabel thickness and mineralized surface per bone surface, which were then significantly increased by FGF23Ab treatment. Immunohistochemical staining showed that key factors in the proposed TLR4- inflammasome pathway including TLR4, CD14 and NLRP3 were significantly increased in the pulp tissue and periodontal ligament of SCD-IgG mice compared to controls. Data also showed that FGF23Ab treatment was able to significantly decrease TRL4, NLRP3, TNFα in SCD mice while p38 and CD14 remained unchanged. In conclusion, our data demonstrated that SCD caused a significant impact on dentition and alveolar bone in addition to heightened expression of proinflammatory cytokines leading to increased expression of inflammasome markers. FGF23Ab treatment causes a partial rescue of these modulated markers as well significantly improved the impacted static and dynamic parameters.