Periodontal ligament stem cells inhibit THP-1 macrophage pyroptosis via modulating the NF-κB/NLRP3/GSDMD signaling axis

Abstract:Background: To investigate the pathological crosstalk between pyroptosis and periodontitis by addressing the following questions: 1) Does pyroptosis play any pathobiological roles in the onset and progression of periodontitis?; 2) Are the localized periodontal macrophages major cell type in pyroptosis?; 3) Can in vitro stimulation by Porphyromonas gingivalis-derived lipopolysaccharide (P.g-LPS) and adenosine triphosphate (ATP) in THP-1 macrophages induce pyroptosis via activation of the NF-κB/NLRP3/GSDMD signaling axis?; and 4) Is there any paracrine effect of periodontal ligament stem cells (PDLSCs) on the NF-κB/NLRP3/GSDMD pathway in pyroptosis?Methods: Clinical periodontitis tissue samples were collected from voluntarily enrolled participants who visited the Dental Clinic of the Affiliated Hospital of Qingdao University. In vitro, THP-1 macrophage pyroptosis was induced by treating with P.g-LPS in combination with ATP. The qRT-PCR, western blotting, and ELISA methods were used to determine the expression levels of NF-κB, NLRP3, caspase-1, ASC, GSDMD, IL-1β, and IL-18. We used the 2ʹ,7ʹ‑dichlorodihydrofluorescein diacetate fluorescence probe to measure ROS levels in THP-1 cells. The caspase-1 activity assay, cytotoxicity assay, and double staining with Hoechst 33342 and propidium iodide were performed in pyroptotic THP-1 macrophages to test the paracrine effect of human PDLSCs on pyroptosis. Results: High expressions of the canonical pyroptosis-associated factors were detected in periodontitis gingival tissues from patients compared to the healthy controls. P.g-LPS treatment exhibited significant stimulation of the canonical pyroptotic effectors, including NF-κB, NLRP3, caspase-1, GSDMD-N, IL-1β, and IL-18 at both mRNA and protein levels. Furthermore, induction of pyroptosis drastically increased the caspase-1 activity, the release of lactate dehydrogenase, and the percentage of cells with damaged membranes, but these pathological effects could be ameliorated via paracrine effects of PDLSCs. Conclusions: Macrophage pyroptosis is an important contributor to the development of periodontitis, while PDLSCs can significantly attenuate the pyroptosis process induced by P.g-LPS/ATP by inhibiting the NF-κB/NLRP3/GSDMD signaling through its paracrine effects in vitro. These evidences cumulatively confirm the immunomodulatory roles of PDLSCs in periodontitis and may be exploited as an effective therapeutic target.