ABSTRACT: Pulp cells play a central role in the defence against cariogenic microorganisms, orchestrating key immunological processes. Peripheral pulp cells, located adjacent to dentin, encounter bacteria and toxins earlier than central cells. To understand the specific signalling pathways between pulp cells and their interplay with immune cells, this study aimed to analyse the transcriptomic profiles of two distinct pulp cell subpopulations - dentin-adherent cells (DACs) and central dental pulp cells (DPCs) - in coculture with Streptococcus mutans. Primary cultures of both DACs and DPCs were obtained from healthy third molars of three female and three male donors aged 17 to 18. Cells were cocultured with viable S. mutans (2 × 10⁸ CFU/mL) for 6 hours (n = 6). Controls included γ-inactivated bacteria and unexposed cells. RNA was isolated, subjected to library preparation (Illumina® Stranded mRNA Prep) and transcriptome profiling was performed via paired-end RNA sequencing (Illumina NextSeq2000). Bioinformatic analysis included differential gene expression (DESeq2), gene set enrichment analysis (GSEA), and protein-protein interaction (PPI) network construction using STRING and Cytoscape. Batch effects were corrected, and significantly regulated genes (|log₂FC| > 1.5, padj < 0.05) were identified. Validation of DEGs was performed via reverse transcription quantitative polymerase chain reaction (RT-qPCR). RNA-Seq revealed a dynamic shift in the transcriptome of DACs and DPCs stimulated with S. mutans, while cells exposed to γ-inactivated or no bacteria did not. Although DACs and DPCs shared common DEGs (33 up, 8 down), several regulations were exclusive to DACs (22 up, 9 down) and DPCs (9 up, 25 down) highlighting a donor-independent functional specificity of the pulp subpopulations. Functional enrichment analysis revealed a strong and comparable activation of hypoxia-related pathways in both DPCs and DACs. However, DACs additionally showed enrichment in extracellular matrix organization and cytokine signaling, while DPCs were characterized by intracellular stress responses and protein folding pathways. Additionally, protein-protein-interaction analysis identified IL-6 as a key hub gene in DACs, while ANGPTL4 played a primary role in DPCs. Following exposure to S. mutans, mechanically isolated DACs and DPCs displayed distinct transcriptomic profiles, indicating functional heterogeneity in the pulpal immune response. DACs engaged immunomodulatory pathways, while DPCs were marked by cellular stress responses, suggesting divergent contributions to tissue defense and homeostasis.
