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

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.

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.

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.