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.

Unique expression and critical role of metallothionein 3 in the control of osteoclastogenesis and osteoporosis.

Bone homeostasis is maintained by an intricate balance between osteoclasts and osteoblasts, which becomes disturbed in osteoporosis. Metallothioneins (MTs) are major contributors in cellular zinc regulation. However, the role of MTs in bone cell regulation has remained unexplored. Single-cell RNA sequencing analysis discovered that, unlike the expression of other MT members, the expression of MT3 was unique to osteoclasts among various macrophage populations and was highly upregulated during osteoclast differentiation. This unique MT3 upregulation was validated experimentally and supported by ATAC sequencing data analyses. Downregulation of MT3 by gene knockdown or knockout resulted in excessive osteoclastogenesis and exacerbated bone loss in ovariectomy-induced osteoporosis. Transcriptome sequencing of MT3 knockdown osteoclasts and gene set enrichment analysis indicated that the oxidative stress and redox pathways were enriched, which was verified by MT3-dependent regulation of reactive oxygen species (ROS). In addition, MT3 deficiency increased the transcriptional activity of SP1 in a manner dependent on intracellular zinc levels. This MT3-zinc-SP1 axis was crucial for the control of osteoclasts, as zinc chelation and SP1 knockdown abrogated the promotion of SP1 activity and osteoclastogenesis by MT3 deletion. Moreover, SP1 bound to the NFATc1 promoter, and overexpression of an inactive SP1 mutant negated the effects of MT3 deletion on NFATc1 and osteoclastogenesis. In conclusion, MT3 plays a pivotal role in controlling osteoclastogenesis and bone metabolism via dual axes involving ROS and SP1. The present study demonstrated that MT3 elevation is a potential therapeutic strategy for osteolytic bone disorders, and it established for the first time that MT3 is a crucial bone mass regulator.