Analysis of immunogenic cell death in periodontitis based on scRNA-seq and bulk RNA-seq data.

Background: Recent studies have suggested that cell death may be involved in bone loss or the resolution of inflammation in periodontitis. Immunogenic cell death (ICD), a recently identified cell death pathway, may be involved in the development of this disease.

Methods: By analyzing single-cell RNA sequencing (scRNA-seq) for periodontitis and scoring gene set activity, we identified cell populations associated with ICD, which were further verified by qPCR, enzyme linked immunosorbent assay (ELISA) and immunofluorescence (IF) staining. By combining the bulk transcriptome and applying machine learning methods, we identified several potential ICD-related hub genes, which were then used to build diagnostic models. Subsequently, consensus clustering analysis was performed to identify ICD-associated subtypes, and multiple bioinformatics algorithms were used to investigate differences in immune cells and pathways between subtypes. Finally, qPCR and immunohistochemical staining were performed to validate the accuracy of the models.

Results: Single-cell gene set activity analysis found that in non-immune cells, fibroblasts had a higher ICD activity score, and KEGG results showed that fibroblasts were enriched in a variety of ICD-related pathways. qPCR, Elisa and IF further verified the accuracy of the results. From the bulk transcriptome, we identified 11 differentially expressed genes (DEGs) associated with ICD, and machine learning methods further identified 5 hub genes associated with ICD. Consensus cluster analysis based on these 5 genes showed that there were differences in immune cells and immune functions among subtypes associated with ICD. Finally, qPCR and immunohistochemistry confirmed the ability of these five genes as biomarkers for the diagnosis of periodontitis.

Conclusion: Fibroblasts may be the main cell source of ICD in periodontitis. Adaptive immune responses driven by ICD may be one of the pathogenesis of periodontitis. Five key genes associated with ICD (ENTPD1, TLR4, LY96, PRF1 and P2RX7) may be diagnostic biomarkers of periodontitis and future therapeutic targets.