Osteopontin interacts with dendritic cells and macrophages in pulp inflammation: Comprehensive transcriptomic analysis and laboratory investigations

Abstract

Aim: To investigate novel diagnostic markers for pulpitis and validate by clinical samples from normal and inflamed pulp. To explore the relationship between diagnostic markers and immune cells or their phenotypes during pulp inflammation.

Methodology: Two microarray datasets, GSE77459 and GSE92681, and identified differential expression genes were integrated. To understand immune features, gene functions, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Disease Ontology (DO) and ImmuneSigDB Gene Set Enrichment Analysis (GSEA) were analysed. For predictive purposes, machine learning techniques were applied to detect diagnostic markers. Immune infiltration in inflamed pulp was studied using CIBERSORT. The relationship between diagnostic markers and immune cells was investigated and validated their gene expression in clinical samples from the normal or inflamed pulp by qRT-PCR. Finally, the correlation between one marker, secreted phosphoprotein 1 (SPP1), encoding osteopontin (OPN), and dendritic cells (DCs)/macrophages was identified via HE staining and multiplex immunohistochemistry. An in vitro inflammatory dental pulp microenvironment model of THP-1 macrophages cocultured with dental pulp cells derived conditioned media (DPCs-CM) to investigate OPN production and macrophage phenotypes was established.

Results: Analysis revealed unique immunologic features in inflamed pulp. Three diagnostic markers for pulpitis: endothelin-1 (EDN1), SPP1, and purine nucleoside phosphorylase (PNP), and validated them using qRT-PCR were predicted. Multiplex immunohistochemistry demonstrated OPN co-localized with activated DCs and M2 macrophages during pulp inflammation. In vitro experiments showed that THP-1 macrophages produced the highest levels of OPN when stimulated with DPCs-CM derived from the 20 μg/mL LPS pre-conditioned group, suggesting an M2b-like phenotype by increasing surface marker CD86 and expression of IL6, TNFα, IL10, and CCL1 but not CCL17 and MerTK. Levels of CCL1 and IL10 elevated significantly in the macrophages' supernatant from the 20 μg/mL LPS pre-conditioned CM group. OPN was proven co-localizing with CD86 in the inflamed pulp by immunofluorescence.

Conclusions: The current findings suggest that OPN can serve as a promising biomarker for pulpitis, correlated with DCs and macrophages. OPN⁺ macrophages in the inflamed pulp are associated with M2b-like phenotypes. These insights offer the potential for improved diagnosis and targeted therapy.

Keywords: bioinformatics; biomarkers; immunity; inflammation; pulpitis.