Overexpression and varied clinical significance of Th9 versus Th17 cells in distinct subtypes of oral lichen planus

ObjectiveOral lichen planus (OLP) presents with large numbers of T lymphocytes accumulating beneath the epithelium of the oral mucosa; however, its aetiology remains obscure. A potential role for an emerging novel T cell subset, Th9, in OLP has recently been suggested but remains to be clarified. The current aim was to investigate the expression and potential clinical significance of Th9 cells in distinct subtypes of OLP.Materials and methodsPeripheral blood samples were collected from 41 OLP patients and 18 healthy controls (HCs). Flow cytometric analysis was used to detect the CD4+ T helper subset Th9 (IL-9⁺IL-17⁻CD4⁺ Th cells) and Th17 (IL-9⁻IL-17⁺CD4⁺ Th cells) expression levels.ResultsFlow cytometry results showed significantly elevated levels of Th9 cells in reticular and erosive OLP compared to HCs. Th9 expression in erosive OLP was less than in reticular OLP, indicating that Th9 but not Th17 cells may play a predominant role in reticular disease. However, in erosive OLP patients, we found much higher levels of Th17 cells compared to reticular OLP patients and HCs, indicating that Th17 dominates in erosive OLP. Statistical analysis showed positive correlations of Th9 cells and Th17 cells in patients with reticular or erosive OLP but none in HCs.ConclusionsTh9 and Th17 cells may take the predominant roles in reticular and erosive OLP respectively, and their numbers were positively correlated in reticular and erosive OLP patients. Elevated circulating Th9 cells may help maintain immune balance in OLP immunopathogenesis, which requires further investigation.     

The membrane-associated MUC1 improves adhesion of salivary MUC5B on buccal cells. Application to development of an in vitro cellular model of oral epithelium

ObjectivesThe mucosal pellicle is a thin layer of salivary proteins, mostly MUC5B mucins, anchored to epithelial oral cells. This pellicle is involved in protection of oral mucosae against abrasion, pathogenic microorganisms or chemical xenobiotics. The present study aimed at studying the involvement of MUC1 in mucosal pellicle formation and more specifically in salivary MUC5B binding using a cell-based model of oral epithelium.DesignMUC1 mRNAs were not detected in TR146 cells, and therefore a stable cell line named TR146/MUC1 expressing this protein was developed by transfection. TR146 and TR146/MUC1 were incubated with human saliva in order to evaluate retention of MUC5B by epithelial cells.ResultsThe cell surface of both TR146 and TR146/MUC1 was typical of a squamous non-keratinized epithelium, with the presence of numerous microplicae. After incubation for 2 h with saliva diluted in culture medium (1:1) and two washes with PBS, saliva deposits on cells appeared as a loose filamentous thin network. MUC5B fluorescent immunostaining evidenced a heterogeneous lining of confluent cell cultures by this salivary mucin but with higher fluorescence on TR146/MUC1 cells. Semi-quantification of MUC5B bound to cells confirmed a better retention by TR146/MUC1, evaluated by Dot Blot (+34.1%, p < 0.05) or by immunocytochemistry (+44%, p < 0.001).ConclusionThe membrane-bound mucin MUC1 is a factor enhancing the formation of the mucosal pellicle by increasing the binding of salivary MUC5B to oral epithelial cells. An in vitro model suitable to study specifically the function and properties of the mucosal pellicle is proposed.     

Simultaneous detection of IFN-gamma and IL-4 in lesional tissues and whole unstimulated saliva from patients with oral lichen planus

Background:  Although the roles of interferon (IFN)-gamma and interlekin (IL)-4 in oral lichen planus (OLP) have been described extensively in past decades, the available results are controversial. Moreover, few studies have utilized simultaneous detection of cytokines in local tissues and saliva to determine whether salivary cytokines could reflect the fact of local lesions.
Methods:  IFN-gamma and IL-4 were determined simultaneously in lesions and whole unstimulated saliva (WUS) from OLP patients with various clinical forms.
Results:  In OLP lesions, both IFN-gamma and IL-4 in erythematous/ulcerated OLP were higher significantly than that in control specimens. In WUS, however, only IFN-gamma of erythematous/ulcerated OLP was increased compared with control. Remarkably, IFN-gamma and IL-4 in WUS showed a more significant correlation to those in local tissues of all subjects.
Conclusion:  These results indicate that both IFN-gamma and IL-4 may play more important role in pathogenesis of erythematous/ulcerated OLP, and changes of these proinflammatory cytokines in WUS may reflect the status of the OLP lesion.     

Dynamic changes of Th1/Th2/Th17 cytokines and human beta defensin 2 in HIV-infected patients with oral candidiasis during the first year of highly active anti-retroviral therapy

ObjectiveTo investigate the change of Th1/Th2/Th17 cytokines and human beta defensin 2 (HBD-2) in HIV-infected patients with oral candidiasis (OC) and gather information about OC-specific immunity.DesignDuring the 1st year of highly active anti-retroviral therapy (HAART), 25 HIV-infected patients were followed up at the baseline, 3rd, 6th, 12th month. At each visit, oral manifestations were examined; oral rinses were collected and cultured for Candida; peripheral venous blood was taken to determine CD4 + T cell counts and HIV RNA viral load (VL); both unstimulated whole saliva and peripheral venous blood were taken to determine cytokine (IL-4, IL-17(A/F), IFN-γ) and HBD-2 levels. Twenty-five healthy individuals were enrolled as control.ResultsHIV-infected patients displayed lower levels of IL-17(A/F) and IFN-γ but higher level of IL-4 and HBD-2 compared with healthy controls. During the 1st year of HAART, salivary IL-17(A/F) and IFN-γ were in uptrend, whereas salivary IL-4 and salivary HBD-2 were in downtrend. Serum cytokines all show no significant changes. After 1 year of HAART, serum, salivary IL-4 level and salivary IL-17(A/F) showed no significant difference from healthy controls. HIV-infected patients with OC had a higher IL-4 level but lower IFN-γ and IL-17(A/F) levels than those without OC since the 3rd month of HAART. The occurrence of OC was negatively correlated to IL-17(A/F) and IFN-γ, but positively correlated to IL-4. Salivary HBD-2 expression was up-regulated in HIV and might associate with Candida albicans.ConclusionsIn HIV-infected patients, the decrease of IL-17 and IFN-γ, and the increase of IL-4 in local and systemic level could influence the prevalence of OC. Salivary HBD-2 may also play an important role against OC.     

Expression and possible immune-regulatory function of ghrelin in oral epithelium

Originally found in stomach mucosa, ghrelin is a peptide appetite hormone that has been implicated as an immuno-modulatory factor. Ghrelin has also been found in salivary glands and saliva; however, its expression patterns and biological properties in the oral cavity remain unclear. Therefore, we investigated the expression patterns of ghrelin in saliva, gingival crevicular fluid (GCF), and gingival tissue, as well as its in vitro effects on IL-8 production by TNF-α or LPS-stimulated oral epithelial cells. In the clinical samples obtained from 12 healthy volunteers, the concentration of ghrelin in GCF remarkably exceeded that detected in saliva. The expression of ghrelin mRNAs and growth hormone secretagogue (GHS) receptors could be detected in human oral epithelial cells. Immunohistochemical analysis revealed the expression of ghrelin in gingival epithelium, as well as in fibroblasts in the lamina propria. Ghrelin increased intracellular calcium mobilization and cAMP levels in oral epithelial cells, suggesting that ghrelin acts on epithelial cells to induce cell signaling. Furthermore, synthetic ghrelin inhibited the production of IL-8 from TNF-α or LPS-stimulated oral epithelial cells. These results indicate that ghrelin produced in the oral cavity appears to play a regulatory role in innate immune responses to inflammatory infection.     

Saliva initiates the formation of pro-inflammatory macrophages in vitro

ObjectivesSaliva can support oral wound healing, a process that requires a temporary inflammatory reaction. We have reported previously that saliva provokes a strong inflammatory response in oral fibroblasts. Bone marrow cells also give rise to macrophages, a heterogeneous subset of cell population involved in wound healing. Lipopolysaccharide (LPS) and interleukin 4 (IL-4) induce activation of pro-(M1), and anti-(M2) inflammatory macrophages, respectively. Yet, the impact of saliva on programming bone marrow cells into either M1 or M2 macrophages remains unclear .DesignHerein, we examined whether sterile saliva affects the in vitro process of macrophage polarization based on murine bone marrow cultures and RAW264.7 mouse macrophages.ResultsWe report that sterile saliva, similar to lipopolysaccharides, provoked a robust activation of the M1 phenotype which is characterized by a strong increase of the respective genes IL-12 and IL-6, based on a real-time gene expression analysis, and for IL-6 with immunoassay. Arginase-1 and Ym1, both genes characteristic for the M2 phenotype, were not considerably modulated by saliva. Inhibition of TLR4 signaling with TAK-242, blocking NFκB signaling with Bay 11-7085, but also autoclaving saliva greatly reduced the development of the M1 phenotype.ConclusionThese data suggest that saliva activates the TLR4 dependent polarization into pro-inflammatory M1 macrophages in vitro.     

Proinflammatory cytokine levels in saliva before and after treatment of (erosive) oral lichen planus with dexamethasone

OBJECTIVE: To explore the potential of detecting the level of proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin-1-alpha (IL-1-alpha), IL-6, and IL-8 in whole unstimulated saliva (WUS) in monitoring the therapeutic effects of topical dexamethasone on these salivary cytokines in subjects with erosive oral lichen planus (OLP). STUDY DESIGN: Thirteen definitively diagnosed OLP subjects were enrolled in the study as were 13 age- and sex-matched controls. The OLP subjects were treated with 0.1% dexamethasone oral rinse for 6 weeks. Prior to treatment and at the end of clinical trial, the visual analog scale (VAS) for symptoms was recorded, WUS was collected and these proinflammatory cytokines were analyzed by ELISA. RESULTS: Following the dexamethasone treatment, the levels of TNF-alpha, IL-1-alpha, IL-6, and IL-8 were decreased significantly, and IL-1-alpha and IL-8 were detected at a level without a statistically significant difference from controls. VAS value was decreased significantly and was found to significantly correlate with the decrease in IL-1-alpha and IL-8 levels. CONCLUSIONS: These preliminary results indicate that salivary analysis of NF-kappaB-dependent cytokines may be applied to monitoring the therapeutic response of OLP.     

The relationship between levels of salivary and serum interleukin-6 and oral lichen planus: A systematic review and meta-analysis

Background

The relationship between levels of salivary and serum interleukin (IL)-6 and oral lichen planus (OLP) is not understood fully. The authors conducted a systematic review and meta-analysis to compare levels of salivary and serum IL-6 among people with OLP and healthy control participants.

Dysplasia in oral lichen planus: relevance,controversies and challenges. A position paper

Background Patients with oral lichen planus (OLP) have an increased risk of oral cancer. For this reason, OLP is classified as an oral potentially malignant disorder. However, the precise personal (or individual) risk is unknown. Recent meta-analytical studies have reported that dysplastic OLP may transform to cancer in around 6% of cases, while the rate of transformation is lower (<1.5%) in non-dysplastic cases. The presence of epithelial dysplasia has emerged as the most powerful indicator for assessing cancer risk in oral potentially malignant disorders in routine practice. However, the general acceptance of epithelial dysplasia as an accompanying histologic feature in OLP is subject to great controversy. Many pathologists consider the presence of dysplasia as a criterion to exclude OLP when routinely reporting on this disease. This practice, widespread among oral pathology professionals, has resulted in the underestimation of the potential for malignancy of OLP.Material and Methods A review of the literature was carried out in order to critically analyze the relevance, controversies and challenges encountered across the diagnosis of epithelial dysplasia in OLP.Results 12 studies have been published examining dysplastic changes in OLP, reporting Figures ranging from 0.54% to 25% of cases with dysplasia in the first diagnostic biopsy. The diagnosis of dysplasia in the OLP poses an additional difficulty due to the fact that the affected oral epithelium per se develops changes related to autoimmune aggression. Among the most frequent histological features of OLP that develops dysplasia are basal cell hyperplasia with basaloid appearance, loss of basal cells polarity, cellular and nuclear pleomorphism and irregular stratification.Conclusions Epithelial dysplasia should not be considered an exclusion criterion for OLP; its evaluation requires experienced pathologists in this field. Key words:Oral lichen planus, epithelial dysplasia, oral cancer.     

Unstimulated whole salivary flow rate and anxiolytics intake are independently associated with oral Candida infection in patients with oral lichen planus

Many factors have been proposed to influence oral infection with yeast. The aim of this study was to determine the prevalence of oral yeasts in oral lichen planus (OLP) patients and control subjects, and to perform a multiple logistic regression analysis to identify factors that influence oral Candida infection in OLP patients. In this cross‐sectional study, 90 new patients with OLP and 90 sex‐ and age‐matched control subjects with no mucosal lesions were interviewed about their health status, use of medication, and smoking and alcohol habits. Swab and unstimulated whole saliva samples were collected and salivary pH was measured. A positive Candida culture was more prevalent among OLP patients (48.9%) than among control subjects (26.7%). Candida albicans was the most frequently isolated species in both groups. By logistic regression analysis, unstimulated whole salivary flow rates of 0.11–0.24 ml min^?1 (OR = 5.90) and 0.25–0.32 ml min^?1 (OR = 3.51) and benzodiazepine anxiolytics intake (OR = 8.30) were independently associated with the presence of Candida among OLP patients. Age, denture wearing, levels of dentition, decreased salivary pH, antihypertensive drugs, and alcohol consumption were not associated with oral Candida infection in OLP patients. The results indicate that data on benzodiazepine anxiolytics intake and evaluation of unstimulated whole salivary flow rate should be considered as part of the clinical evaluation to identify OLP patients at risk for Candida infection.     

糖皮质激素对口腔扁平苔藓辅助性T细胞平衡的影响

目的　明确口腔扁平苔藓 (OLP)患者的Th1 /Th2免疫应答模式,探讨糖皮质激素对OLP患者辅助性T细胞平衡的影响。方法　密度梯度离心法分离OLP患者和对照组外周血单个核细胞,分别用植物血凝素(PHA)和地塞米松刺激OLP患者外周血单个核细胞,采用酶联免疫吸附法,检测培养上清液中干扰素γ(IFN γ)和白细胞介素 4(IL 4)的含量;应用半定量逆转录聚合酶链反应技术,检测培养细胞中IFN γ和IL 4mRNA的表达水平。结果　外周血单个核细胞经PHA诱导培养,OLP患者组IFN γ的水平低于健康对照组 (P<0 05),IL 4的水平高于对照组,但差异无统计学意义(P>0 05),IFN γ/IL 4的比值低于对照组 (P<0 05 )。地塞米松可抑制IFN γ和IL 4的水平(P<0 01),且对IFN γ的抑制作用较IL 4更为显著。结论　OLP患者存在Th1 /Th2的平衡失调,为Th2占优势的免疫应答,糖皮质激素对OLP的Th1 /Th2细胞因子均有抑制作用。     

Role of distinct CD4+ T helper subset in pathogenesis of oral lichen planus

Oral lichen planus (OLP) is one of the most common chronic inflammatory oral mucosal diseases with T‐cell‐mediated immune pathogenesis. In subepithelial and lamina propria of OLP local lesions, the presence of CD4⁺ T helper (CD4⁺ Th) cells appeared as the major lymphocytes. These CD4⁺ T lymphocytes can differentiate into distinct Th cell types such as Th1, Th2, Treg, Th17, Th22, Th9, and Tfh within the context of certain cytokines environment. Growing evidence indicated that Th1/Th2 imbalance may greatly participate into the cytokine network of OLP immunopathology. In addition, Th1/Th2 imbalance can be regulated by the Treg subset and also greatly influenced by the emerging novel CD4⁺ Th subset Th17. Furthermore, the presence of novel subsets Th22, Th9 and Tfh in OLP patients is yet to be clarified. All these Th subsets and their specific cytokines may play a critical role in determining the character, extent and duration of immune responses in OLP pathogenesis. Therefore, we review the roles of distinct CD4⁺ Th subsets and their signature cytokines in determining disease severity and susceptibility of OLP and also reveal the novel therapeutic strategies based on T lymphocytes subsets in OLP treatment.     

Relationships between oral MUC1 expression and salivary hormones in burning mouth syndrome

ObjectivesTo investigate possible relationships among oral mucosal epithelial MUC1 expression, salivary female gonadal hormones and stress markers, and clinical characteristics in patients with burning mouth syndrome (BMS).DesignThirty post-menopausal female patients with BMS (60.0 ± 5.0 years) were included. Clinical and psychological evaluations were performed and the expression level of oral mucosal epithelial MUC1 was analyzed. The levels of cortisol, dehydroepiandrosterone (DHEA), 17β-estradiol, progesterone, chromogranin A, and blood contamination were determined from unstimulated whole saliva (UWS) and stimulated whole saliva (SWS) samples.ResultsSalivary progesterone level had significant positive correlations with oral mucosal epithelial MUC1 expression level and with salivary cortisol and DHEA levels. The salivary level of 17β-estradiol showed significant positive correlations with period of symptom duration, severity of effects of oral complaints on daily life, and results from psychological evaluations. Cortisol level in UWS and cortisol/DHEA ratio in UWS and SWS had negative correlations with severity of oral burning sensation significantly. The severity of taste disturbance had positive correlations with results from psychometry significantly.ConclusionDysregulated psychoendocrinological interactions might affect oral mucosal MUC1 expression and severity of oral burning sensation in post-menopausal BMS patients.     

No detection of TSH or TSHR in oral lichen planus lesions in patients with or without hypothyroidism

Abstract

Objective: An association between hypothyroidism (HT) and oral lichen planus (OLP) has been reported. However, the mechanisms that could explain this association remain unresolved. This study aimed to evaluate the expression of thyroid-stimulating hormone (TSH) and thyroid-stimulating hormone receptor (TSHR) in healthy oral mucosa and in OLP lesions of individuals with and without HT.

Material and methods: Immunohistochemical expression of TSH and TSHR was studied in oral mucosal biopsies obtained from 14 OLP patients with HT, 14 OLP patients without HT and 10 healthy controls without oral mucosal lesions. Gene expression of TSHR was investigated by using three different PCR techniques in oral mucosal samples from 7 OLP patients with HT, 3 OLP patients without HT, 9 healthy controls and in cultured human oral epithelial cells. Gene expression of TSH was examined by employing 2 PCR techniques in oral mucosal samples from 2 OLP patients with HT, 2 OLP patients without HT and 4 healthy controls.

Results: TSH and TSHR stainings were negative in the studied oral mucosal specimens. Gene quantification assays demonstrated negative gene expression of TSH and TSHR in clinical and in vitro samples.

Conclusions: These results suggest that TSH and TSHR may not be commonly involved in the pathogenetic mechanism that could explain the association between OLP and hypothyroidism.     

扁平苔藓患者血清及唾液中IL-1O的表达及相关性研究

目的：探讨口腔扁平苔藓（OLP）患者唾液及血清中白细胞介素-10 （IL-10）的表达情况及其相关性,以进一步了解OLP患者唾液代替血液作为生物学样本来研究OLP中IL-10的可行性.方法：实验分3组：OLP糜烂组15例,OLP非糜烂组15例,正常对照组15例.采用ELISA法分别检测3组对象血清和唾液中IL-10的含量,对结果进行统计分析.结果：OLP糜烂组血清中IL-10水平为（30.11±3.02） pg/mL,明显高于非糜烂组（19.03±3.33） pg/mL和正常对照组（16.43±2.19） pg/mL,差异有统计学意义（P ＜0.05）;OLP糜烂组唾液中IL-10平均水平为（10.05 ±3.12） pg/mL明显高于非糜烂组（5.50±1.1） pg/mL和正常对照组（4.47±1.7） pg/mL,差异有统计学意义（P＜0.05）;两实验组血清和唾液中IL-10水平成正相关,（r=0.731,P＜0.01）.结论：实验组血清和唾液中IL-10水平高度相关,可以通过检查唾液替代检查血液来研究OLP中IL-10的水平.     

Salivary tissue factor concentration and activity in patients with oral lichen planus

Purpose

This study aimed to evaluate the tissue factor (TF) concentration and activity in patients with oral lichen planus (OLP) under oxidative stress.

Langerin-expressing and CD83-expressing cells in oral lichen planus lesions

Objective. Dendritic Langerhans cells (LCs) have been attributed a role in the pathogenesis of lichen planus as autoantigen-presenting cells initiating expansion of autoreactive T cells. Langerin and CD83, which are cell molecules expressed on LCs, are associated with antigen presentation. The present study examined expression of Langerin and CD83 molecules on LCs in patients with oral lichen planus (OLP). Material and methods. Biopsies were obtained from seven patients with OLP. Oral mucosa from seven healthy subjects served as controls. Monoclonal antibodies (mAbs) were used in standard immunohistochemical procedures to visualize CD1a-, Langerin-, and CD83-molecule-expressing cells. Results. CD1a+ and Langerin+ cells were found in significantly higher frequencies in OLP epithelium compared with healthy oral epithelium (p<0.01 and p<0.05, respectively); however, the frequency of CD83+ cells did not differ (p>0.05). The connective tissue in OLP lesions showed significantly higher frequencies of CD1a+, Langerin+, and CD83+ cells compared with healthy connective tissue (p<0.01, p<0.01, and p<0.05). CD1a+ and Langerin+ cells in OLP and healthy epithelium had a dendritic morphology. Conclusions. The study shows increased numbers of CD1a- and Langerin-expressing LCs in OLP compared with healthy controls. In the connective tissue, CD83+ cells with dendritic morphology were localized to regions of lymphocyte clusters. The presence of CD83+ dendritic cells in areas of lymphocyte clusters in the connective tissue of OLP lesions indicates the possibility of ongoing autoantigen presentation.     

Elevated production of salivary nitric oxide in oral mucosal diseases

Nitric oxide (NO) is known to play an important role in biological systems. In this study, we measured levels of NO in the saliva of 39 patients with oral mucosal diseases: 21 had oral lichen planus (OLP) and 18 had recurrent aphthous ulceration (RAU). NO was assayed using the Griess reagent, which measures nitrite (NO2), the byproduct of NO. NO2 was detected in all tested samples, and levels in the saliva of patients were significantly increased relative to those of healthy subjects. We also examined the effect of NO on fibroblasts, keratinocytes and NA cells (an epithelial cancer cell line) in vitro. S-nitroso-N-acetyl-DL-penicillamine (SNAP) and 3-morpholinosydnonimine (SIN-1) were used as NO donating reagents. The results revealed that cell viability was significantly reduced by NO derived from SNAP and SIN-1 in a dose-dependent manner. Although the role of salivary NO in normal physiology is as yet unknown, these findings suggest that excessive salivary NO plays a potential role in modifying oral mucosal diseases as a physiopathological regulator.