Dual role of N4BP1 in neutrophil-epithelial crosstalk in periodontitis.

Background: Periodontitis is a chronic inflammatory disease driven by immune dysregulation, in which neutrophils play a central but functionally heterogeneous role. The contributions of specific neutrophil subsets to disease pathogenesis remain poorly defined. Methods: We integrated single-cell RNA sequencing (scRNA-seq) and Mendelian randomization analysis to identify disease-associated immune cell subsets and gene regulatory mechanisms. Cell-type-specific interactions and pathway alterations were further analyzed to uncover potential pathogenic mediators. Key findings were validated using a murine ligature-induced periodontitis model, supplemented by targeted modulation of N4BP1 in primary human gingival epithelial cells and neutrophils. Results: We identified a transcriptionally defined neutrophil subset that resembles previously reported low-density neutrophils, which we provisionally designate as LDN-1. This subset is characterized by aberrant interactions with epithelial cells primarily mediated by dysregulation of N4BP1. N4BP1 exhibited a dual, cell-type-specific role: its downregulation in epithelial cells impaired mucosal barrier integrity, leading to increased inflammatory tissue damage. In LDN-1, N4BP1 exhibited a context-dependent regulatory function: it suppresses both pro-inflammatory CXCL1/6-CXCR1/2 signaling and anti-inflammatory ANXA1-FPR1/FPR2 pathways. This duality suggests that N4BP1 may calibrate the balance between pro- and anti-inflammatory signals during periodontitis progression. Conclusions: Our findings identify N4BP1 as a critical cell-type-specific molecular candidate that coordinates epithelial barrier function and neutrophil activity. Modulating N4BP1 or its downstream pathways may represent a potential precision therapeutic strategy for periodontitis and related inflammatory diseases, pending further interventional validation.