Ultrasensitive Salivary Cytokine Profiling Reveals Distinct Immune Signatures in Dental Caries
核心洞察
A case-control pilot study used the S-PLEX electrochemiluminescence multiplex assay to quantify nine salivary cytokines in unstimulated whole saliva from 79 young adults.
Carious lesion samples showed significantly higher IL-17 (搜索) and TNF-alpha (搜索) levels than healthy controls and noncarious gingival inflammation (搜索) samples.
The IL-17 (搜索) plus TNF-alpha (搜索) combination achieved the highest discriminatory accuracy for caries versus health, with an AUC of 0.901 (p<0.0001).
Salivary immune profiling using an ultrasensitive multiplex assay can distinguish dental caries (搜索) from both healthy oral environments and noncarious gingival inflammation (搜索), according to a case-control pilot study published in BDJ Open. The proof-of-concept analysis, conducted within the ORASPORT cohort at Université Côte D'Azur (搜索) in Nice, France, found that combinations of inflammatory cytokines — particularly IL-17 (搜索) with TNF-alpha (搜索) — produced distinct signatures in patients with carious lesions.
Validating an Ultrasensitive Platform in Saliva
The study tested the MSD S-PLEX Proinflammatory Panel 1 (human) kit, an electrochemiluminescence sandwich immunoassay based on MULTI-ARRAY technology, to quantify nine cytokines spanning key immune pathways: innate immunity (IL-1 beta (搜索), TNF-alpha (搜索)), Th1 (IL-12p70, IFN-gamma), Th2 (IL-4), Th17 (IL-17 (搜索)), regulatory responses (IL-10), and general T-cell activation (IL-2). The platform achieves femtogram-per-mL detection limits, up to 1000-fold lower than conventional immunoassays, and had previously been validated in plasma and serum but remained largely underexplored in saliva.
Analytical validation confirmed robust performance. Standard curves for all nine cytokines showed excellent 4-parameter logistic fits, with R-squared values ranging from 0.998 to 0.999, and the assay covered a broad dynamic range exceeding 4 log10 without signal saturation or hook effect at high concentrations. Intra-assay coefficients of variation remained below 10% across all concentration ranges when concentrations were normalized by volume (fg/mL), which proved more robust than protein-based normalization. Median cytokine concentrations typically ranged between 100 and 10,000 fg/mL, and IL-1 beta (搜索) reached very high levels, with two samples exceeding the upper detection limit of greater than 1.70 x 10^5 fg/mL.
Study Design and Cohort
Of the 200 participants planned for the ongoing ORASPORT cohort (NCT05765422), 79 healthy young adults aged 18 to 30 years were included in this ancillary analysis, reflecting an interim analysis based on available data. Participants were recruited from university campuses, the Dental Department of CHU Nice, and local sports events. Exclusion criteria included use of anti-inflammatory medication within 48 hours or antibiotic or probiotic treatment within three weeks prior to sampling.
Caries status was recorded using the Decayed, Missing, and Filled Teeth (DMFT) index following WHO 5th edition criteria and ICDAS guidelines, while gingival status was evaluated with the Loe and Silness Gingival Index (LSI). Participants were classified as healthy controls (n = 31), carious lesions (n = 17), or gingival inflammation (搜索) (n = 17); 14 individuals with multiple conditions, mainly caries plus gingival inflammation, were excluded from cytokine analyses to avoid confounding. Unstimulated whole saliva (1 mL) was collected by passive drooling into tubes containing protease inhibitors and stored at -80 degrees Celsius.
Clinical indices supported the group classifications. The DMFT index was significantly higher in the caries group (4.59 +/- 3.71) compared with both gingival inflammation (搜索) (0.76 +/- 1.64, p < 0.0001) and healthy controls (0.84 +/- 1.53, p < 0.0001). Plaque Index was significantly higher in the gingival inflammation group (0.88 +/- 0.69) relative to healthy controls (0.19 +/- 0.59, p = 0.0002), and LSI was markedly higher in gingival inflammation (1.29 +/- 0.59) compared with caries (0 +/- 0, p < 0.0001) and healthy controls (0.09 +/- 0.53, p < 0.0001). Demographic variables including age, sex, BMI, and stress level were comparable across groups.
Cytokine Signatures and Diagnostic Performance
Among single cytokines, TNF-alpha (搜索) was the most robust biomarker, showing significantly higher levels in caries versus healthy controls (p < 0.001) and versus combined controls (p = 0.0003), but it did not discriminate caries from gingival inflammation (搜索) (p > 0.05). IL-6 was also significantly higher in caries compared with healthy controls (p = 0.01), whereas IL-1 beta (搜索) alone was not significant.
Bi-cytokine models improved discrimination. The IL-1 beta (搜索) plus TNF-alpha (搜索) combination showed the strongest significance for caries versus health (p < 0.0001) and remained highly significant versus combined controls (p = 0.0005). The IL-17 (搜索) plus TNF-alpha pair was significantly higher in caries compared with both healthy controls and combined controls (p < 0.001 and p = 0.0002, respectively), with minimal overlap between groups. The IL-1 beta plus IL-17 pair also discriminated caries from other conditions, including gingival inflammation (搜索) (p = 0.0162).
Receiver operating characteristic analysis identified the IL-17 (搜索) plus TNF-alpha (搜索) combination as the most accurate discriminator of caries versus health, with an area under the curve of 0.901 (p < 0.0001). IL-17 plus IL-1 beta (搜索) showed good accuracy for caries versus combined controls (AUC = 0.795, p = 0.016), as did the tri-cytokine combination of IL-17, TNF-alpha, and IL-1 beta (AUC = 0.798, p = 0.007). Adding a third cytokine did not significantly increase discriminatory power over the best-performing pairs.
The authors interpret these patterns as evidence of both innate immune responses (TNF-alpha (搜索)) and Th17-mediated mucosal immune pathways (IL-17 (搜索)) in the immunopathology of carious lesions. They propose that the IL-1 beta (搜索)–IL-17 axis may reflect interconnected inflammasome-driven and Th17-mediated pathways, citing the NLRP3 inflammasome (搜索) mechanism in pulp tissue, in which activation and pyroptosis promote IL-1 beta release, amplifying downstream mediators such as IL-6, which in turn drives Th17 differentiation and IL-17 secretion. IL-17 can also induce IL-1 beta production, establishing a positive feedback loop that may sustain chronic inflammation. The authors note that the ability of IL-1 beta-containing combinations to discriminate caries from gingival inflammation (搜索) suggests these markers may reflect tissue-invasive inflammation rather than superficial gingival changes.
Limitations and Next Steps
The researchers emphasize that the study was designed as a methodological proof-of-concept rather than a definitive biomarker discovery study. Limitations include the modest sample size, the narrow demographic of young adults, and the cross-sectional design, which restrict generalizability and preclude causal inference. The absence of radiographic examinations and periodontal probing data limited the precision of participant classification, potentially leading to underdetection of proximal or hidden dentinal caries lesions. The exclusion of mixed clinical cases, while intended to maintain clearly defined oral health profiles, may limit representation of overlapping conditions seen in practice. The study also could not stratify carious lesions by severity, and the exploratory cytokine combination analysis — conducted post hoc with false discovery rate control using the Benjamini-Krieger-Yekutieli procedure — increases the possibility of false-positive findings.
Because saliva is an integrated oral fluid, the study was not designed to determine the anatomical origin of cytokine signals, which may arise from the lesion itself, adjacent gingival tissues, gingival crevicular fluid, the oral mucosa, or the dentine-pulp complex. The authors also note that the S-PLEX panel targets a predefined set of nine cytokines, excluding some clinically relevant markers such as IL-8, though customized panels could address this. Specialized instrumentation and proprietary reagents may limit widespread accessibility.
The researchers conclude that future prospective validation studies in larger, independent, and more heterogeneous cohorts will be required to define clinically relevant decision thresholds and determine whether these inflammatory signatures can improve disease identification, risk stratification, and monitoring, with the goal of enhancing individualized care strategies for dental decay.