Salivary proteomics suggests persistent inflammatory signatures after clinical healing in recurrent aphthous stomatitis: a hypothesis-generating case-control study

BACKGROUND: Recurrent aphthous stomatitis is a common inflammatory condition of the oral mucosa characterized by painful ulcers that heal spontaneously but recur unpredictably. Although lesions resolve clinically, the biological processes occurring during remission remain poorly understood. This exploratory study investigated whether salivary molecular profiles persist after clinical healing and may be associated with the recurrent nature of the disease. METHODS: In this hypothesis-generating case-control study, saliva samples were collected from patients with recurrent aphthous stomatitis during ulcerative and remission stages and from healthy controls. The salivary pellet was analyzed using discovery-based mass spectrometry proteomics. Complement components and dipeptidyl peptidase-4 (DPP4) were evaluated using immunological assays. Microbial identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and quantitative polymerase chain reaction. The functional interaction between bacteria and oral keratinocytes was explored in vitro using a high-dose infection model to probe epithelial responsiveness. RESULTS: Proteomic analysis identified stage-dependent molecular differences across the clinical course of recurrent aphthous stomatitis. Complement-related proteins and Mammaglobin-B (SCGB2A1) were found to be enriched during remission compared with healthy controls, suggesting persistent molecular signatures of epithelial and immune activity after clinical healing. Microbial profiling revealed enrichment of Streptococcus pneumoniae and Clostridium species in disease stages. In a controlled experimental setting, exposure of oral keratinocytes to Streptococcus pneumoniae was associated with increased DPP4 expression and activation of pathways linked to epithelial stress and immune signaling. Collectively, these exploratory findings suggest that remission is characterized by molecular features distinct from the healthy state. CONCLUSIONS: This exploratory proteomic study suggests that persistent molecular features associated with inflammation and epithelial stress can be detected in saliva after clinical healing in recurrent aphthous stomatitis. Complement-related protein enrichment, epithelial stress markers, and microbial associations were observed during remission, pointing to incomplete molecular recovery of the oral mucosa. While causality cannot be inferred, these hypothesis-generating findings offer a basis for future mechanistic studies and suggest that salivary molecular features warrant investigation as potential candidates for monitoring disease activity.