Saliva and Serum Cytokine Profiles During Oral Ulceration in Behçet's Disease

Abstract

Behçet's disease (BD) is a chronic, multi-systemic disorder of unknown aetiology typified by recurrent oral and genital mucocutaneous lesions, uveitis and vasculitis. Innate and adaptive immune system dysregulation has been implicated in pathogenesis with alterations in serum cytokine profiles. Few studies have investigated salivary cytokines in BD, despite more than 90% of BD patients first presenting with oral ulceration. The aim of this pilot study was twofold; firstly to investigate whether cytokine levels in matched serum and saliva samples show a differential profile in BD (with and without oral ulcers), recurrent aphthous stomatitis (RAS) and healthy controls (HCs), and secondly, to explore if any differential profiles in serum and/or saliva could provide a panel of cytokines with diagnostic and therapeutic potential for BD. Concentrations of 12 cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IFN-γ, TNF-α, TNF-β) were measured using the Human Th1/Th2 11-Plex FlowCytomix™ kit with IL-17A, in BD (N=20), RAS (N=6) and HCs (N=10). A differential range of cytokines was detected in serum and saliva with the majority of cytokine levels higher in saliva. The most prevalent salivary cytokines were IL-1β, IL-2, IL-8, IL-10 and TNF-α present in all samples in contrast to serum where the most prevalent cytokine detected was IL-8 (91.9%). The least abundant cytokine was IFN-γ in both saliva (43.2%) and serum (2.7%). After normalizing saliva for protein content, BD patients with oral ulcers (BD-MA) had significantly higher levels of salivary IL-1β (p=0.01), IL-8 (p=0.02), TNF-α (p=0.004) and IL-6 (p=0.01) than HCs. Notably, BD patients without oral ulcers (BD-MQ) also had significantly higher salivary IL-1β, IL-8 and TNF-α (p ≤ 0.05) than HCs. During relapsed (BD-RE) and quiet (BD-Q) systemic episodes, salivary IL-β and TNF-α were also significantly increased with IL-8 significantly higher only in BD-Q (p=0.02). BD oral ulcers signify a potential reactivation of systemic inflammation. Identifying cytokines released during asymptomatic episodes and oral ulceration might lead to targeted drug therapy to prevent recurrent oral ulcers and possible disease relapse. This is the first study to report salivary cytokine levels in BD. The detectable levels suggests cytokine profiling of BD saliva may provide an alternative, less invasive, sensitive procedure for frequent monitoring of disease activity and progression.

Keywords: Behçet’s disease; FlowCytoMix; cytokines; immune profiling; inflammation; oral mucosa; saliva; ulceration.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Novak, Hamedi, Bergmeier, Fortune and Hagi-Pavli.

Figures

Figure 1

Levels of cytokines (pg/ml) detected in saliva and serum from BD (N=20), RAS (N=7) and HC (N=10). (A) Serum Th1 cytokines and IL-8 chemokine, (B) Serum Th2 cytokines and IL-17A, (C) Saliva Th1 cytokines and IL-8 chemokine, (D) Saliva Th2 cytokines and IL-17A.

Figure 2

Group median cytokine differences between (A) BD and HC and (B) RAS and HC. For each cytokine, the following was calculated: Group Median BD cytokine concentration (pg/ml) - Group Median HC cytokine concentration (pg/ml) or Group Median RAS cytokine concentration (pg/ml) - Group Median HC cytokine concentration (pg/ml) to reveal the delta (Δ), or difference, of the disease group cytokine levels and the HC. (C) Heat map shows differential expression of 12 cytokines in saliva and serum.

Figure 3

Total protein concentrations in saliva of healthy controls (HC), Behçet’s Disease (BD) and recurrent aphthous stomatitis (RAS) patients. The Tukey box plots show levels in BD-ALL (N=20), RAS-ALL (N=7) and HC (N=10). The BD patients were further grouped into patients with oral ulceration, BD Mouth Active (N=9), and patients with no oral ulceration, BD Mouth Quiet (N=11). Mann-Whitney U test, significance based on two tailed 95% confidence interval (CI). Exact P values are indicated.

Figure 4

Saliva cytokine levels normalised to total protein differentially expressed in BD and RAS patients as compared to HCs. Plots show the cytokine concentrations (pg/ml) from BD-ALL (N=20), RAS-ALL (N=7) and HC (N=10). The BD patients were further grouped into patients with oral ulceration, BD Mouth Active (N=9), and patients with no oral ulceration, BD Mouth Quiet (N=11). (A) IL-1β, (B) IL-8, (C) IL-10, (D) TNF-β, (E) IL-6, (F) TNF-α, (G) IL-2, (H) IL-12p70, (I) IL-4, (J) IL-5, (K) IFN-γ, (L) IL-17A. The median and interquartile range are shown (Median ± IQR). Mann-Whitney U test, significance based on two tailed 95% confidence interval (CI). Exact P values are indicated.

Figure 5

Disease Activity. Normalised cytokine levels differentially expressed in saliva from BD and RAS patients as compared to HC. Graphs show the concentrations of IL-8 (A), IL-1β (B) and TNF-α (C) expressed in pg/ml from BD-ALL (N=20), RAS-ALL (N=7) and HC (N=10). The BD patients were further grouped into disease activity relapsed patients BD-RE (N=7) and disease activity quiet patients BD-Q (N=13). Data is presented as median ± IQR. Significant differences were determined by two tailed Mann-Whitney U test with 95% confidence interval (CI). Exact p values are indicated.

Figure 6

Correlation coefficients between 12 cytokines in Saliva. Correlation of normalised salivary cytokine concentrations in BD saliva (N=20). Spearman correlation was determined, r and p values with 95% CI. (A) IL-12p70 vs IL-5, (B) IL-12p70 vs IL-10, (C) IL-10 vs IL-5, (D) IL-17A vs IL-12p70, (E) TNF-β vs IL-5, (F) TNF-β vs vs IL-12p70, (G) TNF-β vs IL-10, (H) IL-8 vs IL-1β, (I) TNF-α vs IL-5, (J) TNF-α vs IL-4, (K) TNF-α vs IL-1β, (L) IL-5 vs IL-2, (M) IL-4 vs IL-2.