Role of NLRP3 Inflammasome in Pathogenesis of Pulpal Disease

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DScD (Doctor of Science in Dentistry)
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NLRP3
Caspase-1
IL-1β
inflammasome
macrophages
DPSCs
I-DPSCs
immunomodulation
Dentistry
Endodontics and Endodontology
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Abstract

Pulpal disease, specifically dental pulpitis, is triggered by exogeneous stimuli from cariogenic bacteria or traumatic injury. The specific host defense in the dental pulp is regulated by the innate and adaptive immunity, of which the pulp-dentin complex odontoblasts play an essential role in the early stage of the dental immune response. It has been reported that the NLRP3 inflammasome is expressed in human dental pulp cells and tissues of inflamed pulp. However whether the activation of NLRP3 contributes to dental immune defense remains unclear. Inflammasome activation is important for antimicrobial defense because it induces cell death and regulates the secretion of IL-1 family cytokines, which play a critical role in innate inflammatory responses. IL-1β production is regulated by the intracellular multi-protein structure, NLRP3/caspase-1 inflammasome complex. It is unknown whether NLRP3/caspase-1/IL-1β activation in macrophages constitutes the host immune response in pulpal disease. In this study, normal healthy teeth and teeth diagnosed with reversible or irreversible pulpitis were collected and processed to evaluate the expression of activated NLRP3 and caspase-1 using IHC, western blotting and ELISA. Although a baseline of NLRP3 activity was detected in normal pulp, specifically at the rimming odontoblast layer, a significant elevation of activated NLRP3/caspase-1/IL-1b signals was detected in pulp tissues, diagnosed as pulpitis, particularly in areas adjacent to the caries front as well as the rimming odontoblasts, fibroblasts, and inflammatory cells. Interestingly, NLRP3 expression was abundant in M1 macrophages and resident macrophages/monocytes and the expression pattern and cellular distribution shifted from the pulp chamber to the root apex as pulpitis progresses. To confirm these findings, pulpitis was experimentally induced in rat molars that allowed a closer investigation of the progress of dental pulp inflammation from reversible to irreversible pulpitis. Histological and multicolor immunofluorescence studies showed co-expression of activated NLRP3/caspase-1 signals in ED1 positive macrophages in areas of incipient coronal injuries at early stage of pulpitis. The zone of dense NLRP3/caspase-1/ED1 positive cells migrated from the pulp chamber to the root apex as the inflamed pulp converted to tissue necrosis. These findings suggest that the activated NLRP3/caspase-1 signals and cellular distribution potentially serve as biomarkers to distinguish the transition of inflamed pulp to necrosis with clinical application for pulpal diagnosis in conjunction with existing clinical and radiographic assessments. Next, to determine whether mesenchymal stem cells derived from inflamed human pulp (I-DPSCs) are capable of similar immunomodulatory functions as those from normal healthy pulp (DPSCs), co-culture with differentiated THP-1 cells with/without stimulation of LPS and/or nigericin was carried out and secreted TNF-α and IL-1β were detected by ELISA. Co-culture of DPSC/I-DPSC and differentiated THP-1 macrophages markedly suppressed not IL-1β but TNF-α secretion in response to stimulation with LPS and/or nigericin. Treatment with IDO inhibitor abolished DPSC-mediated suppression of TNF-α secretion by macrophages, suggesting the role of IDO as a mediator. Furthermore, IDO expression was abundant in both macrophages and mesenchymal stromal cells in pulp tissues diagnosed as irreversible pulpitis. In conclusion, the study suggests that NLRP3/caspase-1 inflammasome activation plays a pivotal role in pulpal inflammation and its expression correlates with the progress of pulpal disease. I-DPSCs, similar to DPSCs, preserve stem cell properties and immunosuppressive effects on the production of TNF-α by macrophages. IDO activity partly contributes to DPSC-mediated inhibitory effects on macrophage, suggesting a role in stem cell immunomodulatory function in dental immune defense.

Advisor
Anh D. Le, DDS, PhD
Date of degree
2015-08-28
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