CXCR2 + Neutrophils Drive Neutrophil Extracellular Traps Formation and Exacerbate Pulpitis in Rats: An In Vitro and In Vivo Laboratory Investigation

ABSTRACT Aim To characterise neutrophil extracellular traps (NETs) and their regulatory mechanisms in pulpitis, and to evaluate the therapeutic potential of targeting neutrophil subsets: Methodology Transcriptomic analyses of microarray (GSE92681) and single‐cell RNA sequencing datasets (GSE274562, GSE280528) were performed to delineate immune heterogeneity and intercellular communication. HL‐60‐derived neutrophils, THP‐1 macrophages, and human Dental Pulp Cells (hDPCs) were used for in vitro assays of NETs induction, chemotaxis, osteogenic differentiation and immunofluorescence. Healthy male Sprague–Dawley rats (7–8 weeks, N = 5/group) were used to establish pulpitis models treated with the CXCR2 inhibitor AZD5069 (S6645, Selleck), followed by histology, immunostaining, and micro‐CT analysis. Results Transcriptomic profiling revealed significant enrichment of NETs‐related pathways in inflamed pulp. Immunofluorescence confirmed MPO + /CitH3 + NETs in inflamed tissues, while NETs significantly impaired hDPCs osteogenesis in vitro. Single‐cell analysis identified a distinct CXCR2 + neutrophil subset (CXCR2 + Neu), representing a mature pro‐inflammatory population specialised in NETs release. Cell–cell interaction analysis highlighted CXCL8–CXCR2 signalling from pro‐inflammatory macrophages as the major driver of neutrophil recruitment and NETs formation. In vitro, M1 macrophages enhanced neutrophil chemotaxis and NETs release, whereas NETs reciprocally promoted M1‐like polarisation. In vivo, CXCR2 inhibition markedly reduced neutrophil infiltration and NETs release, while promoting reparative mineralization in rat pulpitis. Conclusions CXCR2 + neutrophils act as a pro‐inflammatory subset in pulpitis, driving NETs release through macrophage‐derived signals. Inhibition of CXCR2 attenuates inflammation and enhances dentine repair, supporting CXCR2 as a promising therapeutic target for immune modulation in pulpitis.