Single-cell transcriptome analysis reveals periodontal ligament fibroblast heterogeneity with distinct IL-1ß and RANKL expression in periodontitis.

Periodontitis (PD) is an inflammatory disease with alveolar bone destruction by osteoclasts (OCs). In PD, both inflammation and OC activation are significantly influenced by periodontal ligament fibroblasts (PDL-Fib). Yet, whether PDL-Fib has heterogeneity and whether distinct PDL-Fib subsets have specific functions have not been investigated. In this study, we discovered the complexity of PDL-Fib in PD, utilizing single-cell RNA sequencing data from human PD patients. We identified distinct subpopulations of PDL-Fib: one expressing interleukin-1 beta (IL-1ß) and another expressing the receptor activator of nuclear factor-kappa B ligand (RANKL), both crucial in OC differentiation and bone resorption. In periodontal tissues of mice with PD, active IL-1ß, cleaved caspase 1, and nucleotide-binding oligomerization domain-like receptor 3 (NLPR3) were significantly elevated, implicating the NLRP3 inflammasome in IL-1ß production. Upon stimulation of PDL-Fib with LPS from Porphyromonas gingivalis (pg), the most well-characterized periodontal bacteria, a more rapid increase in IL-1ß, followed by RANKL induction, was observed. IL-1ß and tumor necrosis factor alpha (TNF-α), another LPS-responsive cytokine, effectively increased RANKL in PDL-Fib, suggesting an indirect effect of pgLPS through IL-1ß and TNF-α on RANKL induction. Immunohistological analyses of mouse periodontal tissues also showed markedly elevated levels of IL-1ß and RANKL upon PD induction and displayed separate locations of IL-1ß-expressing PDL-Fib and RANKL-expressing PDL-Fib in PD. The heterogenic feature of fibroblasts expressing IL-1ß and RANKL was also mirrored in our combined cross-tissue single-cell RNA sequencing datasets analysis. In summary, our study elucidates the heterogeneity of PDL-Fib, highlighting distinct functional groups for producing RANKL and IL-1ß, which collectively promote OC generation and bone destruction in PD.

PINK1 restrains periodontitis-induced bone loss by preventing osteoclast mitophagy impairment.

The oral colonization of periodontal pathogens onto gingival tissues establishes hypoxic microenvironment, often disrupting periodontal homeostasis in conjunction with oxidative stress. The association between reactive oxygen species (ROS) and osteolytic periodontitis have been suggested by recent studies. PTEN-induced kinase 1 (PINK1), a mitochondrial serine/threonine kinase, is an essential protein for mitochondrial quality control as it protects cells from oxidative stress by promoting degradation of damaged mitochondria through mitophagy. However, the pathophysiological roles of PINK1 in osteoclast-mediated bone loss have not been explored. Here we aimed to determine whether PINK1 plays a role in the regulation of osteoclastogenesis and alveolar bone resorption associated with periodontitis. C57BL/6 wild type (WT) and Pink1 knockout (KO) mice were subjected to ligature-induced periodontitis (LIP), and alveolar bones were evaluated by µCT-analysis and tartrate-resistant acid phosphatase (TRAP) staining. The µCT-analysis showed that bone volume fraction and travecular thickness were lower in Pink1 KO compared to WT mice. The number of TRAP-positive osteoclasts was markedly increased in the periodontal tissues of Pink1 KO mice with LIP. The genetic silencing or deletion of Pink1 promoted excessive osteoclast differentiation and bone resorption in vitro, as respectively indicated by TRAP staining and resorption pits on dentin slices. PINK1 deficiency led to mitochondrial instabilities as indicated by confocal microscopy of mitochondrial ROS, mitochondrial oxygen consumption rate (OCR) analysis, and transmission electron microscopy (TEM). Consequently, a significant increase in Ca2+-nuclear factor of activated T cells 1 (NFATc1) signaling was also found. On the other hand, restoration of mitophagy and autophagy by spermidine (SPD) treatment and the resolution of oxidative stress by N-acetyl-l-cysteine (NAC) treatment protected PINK1 deficiency-induced excessive generation of osteoclasts. Taken together, our findings demonstrate that PINK1 is essential for maintaining mitochondrial homeostasis during osteoclast differentiation. Therefore, targeting PINK1 may provide a novel therapeutic strategy for severe periodontitis with fulminant osteolysis.

PARK2 Induces Osteoclastogenesis through Activation of the NF-κB Pathway.

Osteoclast generation from monocyte/macrophage lineage precursor cells needs to be tightly regulated to maintain bone homeostasis and is frequently over-activated in inflammatory conditions. PARK2, a protein associated with Parkinson's disease, plays an important role in mitophagy via its ubiquitin ligase function. In this study, we investigated whether PARK2 is involved in osteoclastogenesis. PARK2 expression was found to be increased during the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. PARK2 gene silencing with siRNA significantly reduced osteoclastogenesis induced by RANKL, LPS (lipopolysaccharide), TNFα (tumor necrosis factor α), and IL-1ß (interleukin-1ß). On the other hand, overexpression of PARK2 promoted osteoclastogenesis. This regulation of osteoclastogenesis by PARK2 was mediated by IKK (inhibitory κB kinase) and NF-κB activation while MAPK (mitogen-activated protein kinases) activation was not involved. Additionally, administration of PARK2 siRNA significantly reduced osteoclastogenesis and bone loss in an in vivo model of inflammatory bone erosion. Taken together, this study establishes a novel role for PARK2 as a positive regulator in osteoclast differentiation and inflammatory bone destruction.

Compressive force-induced autophagy in periodontal ligament cells downregulates osteoclastogenesis during tooth movement.

BACKGROUND: Autophagy has recently emerged as a protective mechanism in response to compressive force and an important process in maintenance of bone homeostasis. It appears to be involved in the degradation of osteoclasts, osteoblasts, and osteocytes. The aim of this study was to investigate the role of compressive force-induced autophagy in periodontal ligament (PDL) cells in regulating osteoclastogenesis of orthodontic tooth movement (OTM). METHODS: An OTM model and compressive force on PDL cells were employed to investigate the expression of autophagy markers in vivo and in vitro, respectively. Autophagosomes and autolysosomes were observed in PDL cells by transmission electron microscope (TEM) and autophagy LC3 double labelling. 3-Methyladenine (3-MA) and rapamycin were respectively used to inhibit and promote autophagy, and the effect of autophagy on osteoclastogenesis was explored via microcomputed tomography, hematoxylin and eosin (H&E) staining, histochemistry of titrate-resistant acid phosphatase, and real-time polymerase chain reaction (RT-PCR) in vivo. Receptor activator of nuclear factor-kappa B ligand/osteoprotegerin (RANKL/OPG) was investigated by RT-PCR and ELISA in vitro. RESULTS: Orthodontic force-induced autophagy was prominent on the pressured side of PDL tissues. Administration of 3-MA downregulated bone density and upregulated osteoclasts, while rapamycin had reverse results in OTM. The autophagy activity increased initially then decreased in PDL cells during compressive force application and responded to light force. In PDL cells, administration of 3-MA upregulated while rapamycin downregulated the RANKL/OPG ratio. CONCLUSION: Autophagy is activated by compressive force in PDL cells. Besides, it could modulate OTM by negatively regulating osteoclastogenesis and keep bone homeostasis via RANKL/OPG signaling.

Cystathionine gamma lyase-H2S contributes to osteoclastogenesis during bone remodeling induced by mechanical loading.

Hydrogen sulfide (H2S), a gaseous signaling molecule produced by cystathionine γ-lyase (CSE) and cystathionine ß-synthase (CBS), influences bone remodeling in many ways. Osteoclasts play an important role in bone remodeling during tooth movement induced by mechanical loading, which increases the rate of tooth movement. Recently, we found that osteoclasts could produce H2S. However, whether H2S modulates bone remodeling by affecting osteoclasts remains unclear. In this study, we found that CSE-H2S was a dominant H2S generating system in osteoclasts, while CBS did not generate H2S. A significant increase in CSE mRNA expression and H2S production was observed in periodontal ligament (PDL) tissues of wild-type (WT) mice after 3 days of mechanical loading. CSE gene knockout led to a significant reduction in the number of maxillary osteoclasts and in the amount of tooth movement. The number of RANKL-induced TRAP-positive osteoclasts and the mRNA expression of osteoclast markers were downregulated after 6 days of incubation in monocytes extracted from CSE-/- mice. The expression of IL-1, IL-6 and TNF-α, which can stimulate osteoclastogenesis in periodontal tissue and serum samples, was lower in CSE-/- mice after mechanical loading. Application of the H2S donor GYY4137 increased the number of RANKL-induced osteoclasts, the number of osteoclasts in periodontal tissues and tooth movement distance in CSE-/- mice. The results suggested that endogenous H2S and CSE play vital roles in the osteoclastogenesis and alveolar bone resorption induced by mechanical loading.