Single-cell Transcriptome Analysis Reveals Periodontal Ligament Fibroblast Heterogeneity with Distinct IL-1β and RANKL Expression in Periodontitis

Periodontitis is an inflammatory disease with alveolar bone destruction by osteoclasts. In periodontitis, both inflammation and osteoclast 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 periodontitis, utilizing single-cell RNA sequencing (scRNA-seq) data from human periodontitis patients. We identified distinct subpopulations of PDL-Fib: one expressing IL-1β and another expressing RANKL, both crucial in osteoclast differentiation and bone resorption. In periodontal tissues of mice with periodontitis, active IL-1β, cleaved caspase 1, and NLRP3 proteins were significantly elevated, implicating the NLRP3 inflammasome in IL-1β production. Upon stimulation of PDL-Fib with LPS from Porphyromonas gingivalis (pg), the mostly well characterized periodontal bacteria, a more rapid increase in IL-1β, followed by RANKL induction, was observed. IL-1β and TNF-α, another LPS-responsive cytokine, effectively increased RANKL in PDL-Fib, suggesting an indirect effect of pgLPS through IL-1β and TNF-α on RANKL induction. Immunohistological analyses of mouse periodontal tissues also showed markedly elevated levels of IL-1β- and RANKL upon periodontitis induction and displayed separate locations of IL-1β-expressing PDL-Fib and RANKL-expressing PDL-Fib in periodontitis. The heterogenic feature of fibroblasts expressing IL-1β and RANKL was also mirrored in our combined cross-tissue scRNA-seq datasets analysis. In summary, our study elucidates the heterogeneity of PDL-Fib, highlighting distinct functional groups for producing RANKL and IL-1β, which collectively promote osteoclast generation and bone destruction in periodontitis.