Lactylation‐Driven Macrophage Polarisation Regulates Pulp Inflammation

AIM: This study investigated the effects of lactate-induced lactylation in the inflammatory microenvironment of pulpitis and further explored the mechanism. METHODOLOGY: Lactate levels in pulpitis samples were quantified using a high-sensitivity assay. Histological, immunohistochemical, and immunofluorescence staining were conducted to evaluate lactylation, macrophage marker, pro-inflammatory, and anti-inflammatory markers. A time-course murine experimental pulpitis model (0-72 h) was established to characterise lactylation dynamics during inflammatory progression in pulpitis. An in vitro inflammatory dental pulp microenvironment model of THP-1 macrophages cocultured with LPS-pretreated dental pulp cells derived conditioned media (iCM) was developed to investigate lactate production and macrophage phenotypes. Transcriptomic profiling identified differentially expressed genes, with gene set enrichment analysis (GSEA) employed to assess the functions of differentially expressed genes. Transmission electron microscopy, quantitative real-time PCR (qRT-PCR), concurrent oxygen consumption rate (OCR), and extracellular acidification rate (ECAR) were measured to evaluate the mitochondrial activity of iCM-pretreated macrophages. Mouse experimental pulpitis models treated with iCM were conducted to evaluate anti-inflammation and pro-healing properties by histological, immunohistochemical, and immunofluorescence staining. RESULTS: Histological staining revealed that elevated lactate levels, increased Pan Kla expression, and upregulated extent of M2 phenotype macrophage infiltration in clinical pulpitis specimens. Notably, we identified a positive correlation between Pan Kla levels and M2 macrophage markers. In vitro inflammatory dental pulp microenvironment model, we demonstrated that M1 macrophages actively uptake lactate from iCM, leading to increased lactylation and subsequent M2-like polarisation. Importantly, we found that iCM could regulate polarisation of M1 macrophages via metabolic reprogramming, as evidenced by RNA sequencing. Our integrated analyses revealed significant mitochondrial structural remodelling, while metabolic flux assays demonstrated a characteristic shift from glycolytic metabolism to oxidative phosphorylation. This metabolic reprogramming was functionally linked to M2 polarisation, as evidenced by phenotypic marker analysis. Moreover, iCM treatment significantly downregulated pro-inflammatory cytokine (IL-6) while upregulating anti-inflammatory marker (CD206) in experimental pulpitis models. CONCLUSION: This study revealed that elevated lactate production in the inflammatory microenvironment roles as a mediator of immunometabolic crosstalk, bridging dental pulp cells-macrophage communication. And the mechanism involved in lactylation induced metabolic reprogramming. This helps to better understand the repair potential of inflamed dental pulp, supporting biologically-based preservation approaches.