Ultrasensitive multiplex salivary cytokine profiling reveals distinct immune signatures in dental caries

Dental caries is a prevalent chronic disease characterized by complex inflammatory responses in the oral environment. This case-control pilot study evaluated the analytical performance of an ultra-sensitive electrochemiluminescence multiplex assay (MSD S-PLEX^®) for salivary cytokine profiling and explored whether combined cytokine signatures can discriminate dental caries lesions (CL) from healthy (H) and non-carious gingival inflammation (GI).

Unstimulated whole saliva from 65 individuals (H, CL, GI) was analyzed using the S-PLEX assay to quantify nine salivary cytokines (IL-1β, TNF-α, IL-6, IL-4, IL-10, IL-12p70, IFN-γ, IL-17, and IL-2). Group comparisons were performed using Mann-Whitney tests, and diagnostic performance was assessed by logistic regression and the area under the receiver operating characteristic curve.

The assay showed high sensitivity and reproducibility. Cytokine profiling revealed selective elevation of certain cytokines in saliva from CL. Among the models tested, TNF-α + IL-17 provided the best discrimination between CL and H (AUC = 0.901, p < 0.001) and between CL and H + GI (AUC = 0.795, p = 0.0003). Inclusion of additional cytokines did not improve performance. However, discrimination between CL and GI was limited with TNF-α + IL-17, while IL-1β + IL-17 provided a modest but significant discrimination (AUC = 0.737, p = 0.004).

This proof-of-concept study demonstrated the feasibility of ultrasensitive multiplex salivary cytokine profiling using the S-PLEX platform and revealed distinct cytokine signatures associated with clinically detected carious lesions. While preliminary, these findings support further investigation of salivary host-response biomarkers in caries research. Future longitudinal studies integrating comprehensive clinical, microbial, and immunological data together with detailed clinical characterization, including radiographic assessment and lesion severity evaluation, will be necessary to validate these associations and clarify their potential biological and clinical significance in caries research and their potential role in personalized caries management.