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Toll-like receptor 2

a possible role in the pathogenesis of oral lichen planus and hepatitis C virus infection, a case–control study

Nofal, Emana; Nofal, Ahmada; Abdelshafy, Ahmeda; Dosoky, Ahmedb

Journal of the Egyptian Women’s Dermatologic Society: September 2018 - Volume 15 - Issue 3 - p 139–143
doi: 10.1097/01.EWX.0000546170.97639.d9
Original articles
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Background Toll-like receptor 2 (TLR2) is a pathogen recognition pattern that may play a role in the pathogenesis of both oral lichen planus (OLP) and hepatitis C virus (HCV) infection.

Objective To investigate a potential role of TLR2 in the pathogenesis of OLP with or without HCV infection and in HCV infection ‘without OLP’.

Patients and methods This case–control study included three groups: OLP group (20 patients: 12 HCV positive and eight HCV negative patients), HCV positive group ‘without OLP’ (20 patients) and healthy control group (20 subjects). TLR2 expression levels on peripheral blood monocytes (PBMCs) were measured in all participants by flow cytometric analysis.

Results TLR2 expression on PBMCs was significantly higher in both the OLP group and the HCV positive group ‘without OLP’ than in healthy controls (P≤0.001) but was nearly equal in both the OLP and HCV group ‘without OLP’. Although TLR2 expression was higher in HCV positive than in HCV negative OLP cases, the difference did not reach statistical significance.

Conclusion The increased TLR2 expression on PBMCs is probably involved in the pathogenesis of OLP with or without HCV infection, as well as in the pathogenesis of HCV infection ‘without OLP’. The idea that TLR2 can be the gate by which HCV induces OLP cannot be verified by this study.

Departments of aDermatology and Venereology

bClinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt

Correspondence to Ahmed Abdelshafy, MD, Department of Dermatology and Venereology, Faculty of Medicine, Zagazig University, Zagazig 44516, Egypt Tel: +20 100 836 4064; e-mail: ahmed_said3007@yahoo.com

Received May 1, 2018

Accepted August 25, 2018

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Introduction

Oral lichen planus (OLP) is a chronic inflammatory oral condition of unknown etiology. The lesions may result from the induction of epithelial cells apoptosis by cytotoxic CD8+ T cells stimulated by a yet unidentified self antigen on a genetically predisposed patient 1. Under normal conditions, the healthy mucosa is protected by tightly regulated responses mediated through an array of pattern recognition receptors that constantly survey their surroundings for potential threats and invasion. Toll-like receptors (TLRs) are the best characterized pattern recognition receptors that recognize distinct microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) typically shared by a large group of micro-organisms 2. The cutaneous innate immune system selectively alerts the host about the presence of microbial pathogens by sensing PAMPs or endogenous signals of injury through TLRs and responds rapidly by producing cytokines, antimicrobial peptides and antimicrobial intermediates. TLR costimulation by extrinsic TLR ligands and through damage-associated molecular pattern self-ligands holds the potential to activate self-reactive B and T cells and to induce autoimmunity 3.

The association of OLP with hepatitis C virus (HCV) has been more consistently discussed in the Mediterranean region. Although HCV RNA and HCV-specific CD4+ and CD8+ T cells have been retrieved in the mucosal lesions of patients with chronic HCV infection and OLP, the eventual pathophysiology of HCV in OLP lesions remains unclear 1. There is accumulating evidence of a relationship between HCV protein and the TLR-mediated signaling pathways. HCV has been shown to activate TLR2 and trigger inflammatory responses 4.

Because of their ability to respond to most oral commensal micro-organisms, TLR2 and TLR4 are the most widely investigated receptors in oral diseases 2. Few studies have investigated the role of TLR2 in the pathogenesis of OLP by examining its expression in the saliva 5,6, oral epithelium 2,7–9, peripheral blood monocytes (PBMCs) 7 and in the expression of TLR2 gene 7,9. The aim of this study was to investigate a potential role of TLR2 in the pathogenesis of OLP among Egyptian patients with or without HCV infection and in HCV infection ‘without OLP’ through measuring the expression of TLR2 on PBMCs of patients with OLP in comparison with the HCV-infected patients ‘without OLP’ the and healthy control group.

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Patients and methods

This case–control study included 3 groups: the OLP group, the healthy control group and the HCV positive group ‘without OLP’. Patients were enrolled from the outpatient clinics of Dermatology and Tropical medicine of Zagazig University hospitals. The study was carried out in the period between January 2014 and January 2015 after approval of the Institutional Review Board (IRB). A written informed consent was obtained from all subjects after complete description of the study.

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OLP group

It included 20 patients with OLP diagnosed clinically and histopathologically. Exclusion criteria included patients with history of atopic dermatitis, acne vulgaris, psoriasis, wart, malignancy, fever in the last week, skin infection or any other infectious diseases for at least a month. Patients who had received systemic treatment for HCV infection, corticosteroids, immunomodulatory agents or systemic retinoids were also excluded.

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Control group

This group included 20 healthy volunteers, with no systemic, skin or oral diseases.

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HCV positive group ‘without OLP’

This group included 20 patients with HCV infection without OLP. The exclusion criteria were the same as those for the OLP group.

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Methods

  • All patients and controls were subjected to detailed history taking, general and dermatological examination and buccal examination for identification of OLP lesions. Serodiagnosis of HCV was performed for all participants by third generation ELISA (Infinite F50, ELISA Microplate Reader; Tecan, Männedorf, Austria). Accordingly, the OLP group was classified into HCV positive and HCV negative cases.
  • Assay of TLR-2 expression on PBMCs by flow cytometric analysis was performed for all participants of the study. Fresh venous blood (3 ml) added in vacutainers with EDTA was obtained from patients and controls; thereafter, cell surface expression of CD14 and TLR2 were analyzed by cell surface staining and flow cytometry. From each sample of whole blood, 100 μl was incubated with 10 μl FITC (fluorescein isothiocyanate)-conjugated anti-TLR2 monoclonal antibodies (mAb) and 10 μl PE (phycoerythrin)-conjugated anti-CD14 mAb (R&D System Inc., Minneapolis, Minnesota, USA) for 20 min at room temperature, in the dark. This was followed by red cell lysis and washing. Cells were then resuspended in 300 μl of PBS. As a control for this analysis, 100 μl from each sample in a separate tube was incubated with PE-labeled mouse IgG1 antibody and FITC-labeled mouse IgG2b antibody for 20 min at room temperature, in the dark. This was followed by red cell lysis and washing. Cells were then resuspended in 500 μl of PBS. The cells were analyzed on a Becton Dickinson Fac-scan by using Image Quant software (Becton Dickinson, Minneapolis, Minnesota, USA), and monocytes were specifically analyzed by a selective gating based on parameters of forward and side light scatter (Fig. 1). Monocytes were gated according to light scatter properties, and gate was confirmed by CD14 expression. CD14+ cells in patients and control samples were determined, and the mean fluorescence intensity (MFI) of TLR2 expression was estimated on gated CD14+ cells, and shown in histogram for each sample analysis (Fig. 2). The MFI of TLR2 was estimated by substracting the MFI of monocytes labeled with mouse IgG2 antibody from that of monocytes labeled with anti-TLR2 antibodies.
Figure 1

Figure 1

Figure 2

Figure 2

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Statistical analysis

The results were collected, tabulated, and statistically analyzed by IBM personal computer and statistical package SPSS version 18 (SPSS Inc., Chicago, Illinois, USA). Descriptive statistics as mean and SD for continuous data, and frequency and percentage for discrete data were used. Analytic statistics such as t-test was used to study comparison between two continuous parametric data, while analysis of variance test was used to study comparison between more than two continuous data. P values less than 0.05 were considered significant.

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Results

The OLP group included 20 patients, 10 male patients and 10 female patients with a mean age of 51.55±10.53 years. The healthy control group included 11 male individuals and nine female individuals with a mean age of 48.36±9.48 years. The HCV positive group ‘without OLP’ included eight male patients and 12 female patients with a mean age of 50.31±11.24 years. All groups were matched as regards age and sex. The clinical data of the OLP group is summarized in Table 1. According to HCV serodiagnosis, 12 cases of OLP were HCV positive and eight cases were HCV negative.

Table 1

Table 1

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Laboratory data

The MFI of TLR2 on PBMCs in all groups is illustrated in Table 2. It was found that MFI of TLR2 on PMNCs was significantly higher in both the OLP group and the HCV positive group without OLP than in healthy controls (P<0.001). When comparing the OLP group with the HCV positive group ‘without OLP’, the difference was statistically nonsignificant (P=0.60).

Table 2

Table 2

The MFI of expressed TLR2 on PBMCs tended to be higher in the HCV positive OLP cases (28.08±5.64) than in the HCV negative OLP cases (25.02±3.1). However, the difference does not reach statistical significance (P=0.18) (Table 3).

Table 3

Table 3

There were nonsignificant associations between TLR2 expression levels in the OLP group and site, type and duration of the lesions and presence of skin lesions (P=0.33, 0.47, 0.75, and 0.13, respectively). There were also nonsignificant associations between TLR2 expression levels and age and sex of OLP patients (P=0.49, 0.16, respectively).

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Discussion

To our knowledge, this is the first study that compared TLR2 expression levels on PBMCs between HCV positive OLP patients, HCV negative OLP patients, HCV positive patients without OLP and healthy volunteers. We also examined a possible association between OLP and HCV infection and their relationship with the expression levels of TLR2.

TLR2 expression has been found in human cells and tissues in a resting state or after activation 10. Sabroe et al.11 reported positive TLR2 expression on normal PBMCs. In the current study, significant higher levels of TLR2 expression on PBMCs were found in OLP patients and HCV positive ‘patients without OLP’ than in normal subjects, and the intensities of expression were not correlated with different clinical types or sites of OLP, which is consistent with the study of Ohno et al.7.

The mechanism(s) that lead to upregulation of TLR2 expression on PBMCs of OLP patients remains unclear. Various studies have reported that, under certain conditions, PAMPs and cytokines upregulate TLR2 expression in a variety of cells 12,13. The serum cytokine levels were more elevated in OLP patients than in normal individuals 14. Moreover, the PBMCs of OLP patients produce considerably higher amounts of proinflammatory cytokines than that produced by PBMCs of normal individuals 7.

The modulation in TLR2 expression on PBMCs may be involved in the pathogenesis of OLP. Previous data suggested that OLP is a T-cell-mediated autoimmune disease in which autocytotoxic CD8+ T cells trigger apoptosis of oral epithelial cells 15. How are these autocytotoxic CD8+ T cells induced into the lesions of OLP? In cases of OLP, activation of TLR2 on PBMCs leads, via different intracellular signaling pathways, to the production of proinflammatory stimuli, immune-regulatory cytokines, chemokines and costimulatory molecules, and is considered a danger signal that should transform the skin into the functional state of defense 16. Overexpression of TLR2 on PBMCs in patients with OLP results in overproduction of IL12 7. IL12 is an important cytokine that induces Th1 cell differentiation. Th1 cell cytokines promote cytotoxic lymphocyte and NK cell responses and are also linked to the development of autoimmunity 17. It might be proposed that, the shift of the Th1/Th2 balance toward Th1 dominance in OLP patients, probably depends on the upregulation of TLR2 expression 7.

HCV is a global health challenge. Egypt has the highest HCV prevalence in the world, 14.7% 18–20. An association between HCV infection and OLP has been recognized. The pathogenesis of such association is still unclear. It may be due to cell-mediated cytotoxicity to an epitope shared by HCV and damaged keratinocytes. Virus replication in the oral epithelium may also contribute directly to the development of lesions. Moreover, the high mutation rate of the virus may result in repeated activation of immune cells, increasing the probability of cross-reaction with its own tissue and, consequently, the risk of autoimmune disease 21,22.

Whether HCV-infected patients have increased risk of developing OLP or patients with OLP have enhanced risk of developing HCV infection is yet to be answered 23. Early studies 24,25 have suggested that HCV may persist and replicate in the diseased oral mucosa, but later studies 21,26 have shown no HCV transcripts in the epithelium of OLP lesions. Moreover, the value of antiviral treatment effective against HCV has not been proven in OLP patients; there are many reports 27,28 of interferon inducing or aggravating OLP. In the present study, we found no statistical difference between HCV positive OLP patients (60%) and HCV negative OLP patients (40%) that may suggest lack of increased risk of OLP in HCV-infected patients. However, the small size of the study may limit this suggestion.

Various studies have proposed a role for TLR signaling in the pathogenesis of HCV. TLR2 and TLR4 expression have been assessed on monocytes of HCV-infected patients in several studies. HCV may signal directly through TLR2 by core protein and nonstructural protein 3, or indirectly through alternative TLRs, for example, TLR4, 29,30 leading to increased inflammatory cytokines such as TNF-αthat cause hepatocyte damage. Our results were consistent with previous studies that reported increased TLR2 expression on PBMCs during HCV infection ‘without OLP’ 29–31.

In accordance with the immune modulation that occurs in OLP, HCV infection modifies the innate immune system by upregulating the expression of TLR2 that could increase the production of downstream cytokines. Increasing the production of related inflammatory and regulatory cytokines increases the risk of immunopathology and exerts an indirect effect in the pathogenesis of OLP 20,30. However, in our study, we detected a nonsignificant difference between the expression of TLR2 in HCV negative OLP and HCV positive OLP patients. Thus, we believed that, in OLP patients, HCV coincidence may have no additive effect on the expression level of TLR2 on monocytes. To date, there are no published reports about TLR2 expression in OLP associated with HCV infection.

To summarize, oral epithelial cell apoptosis and hepatocyte damage are hallmarks in OLP and HCV infection, respectively. TLR2 expression increased in OLP and HCV infection ‘without OLP’. Overexpression of TLR2 induces Th1 cell differentiation, which promotes cytotoxic lymphocytes and proinflammatory cytokines, which induce oral epithelial cells apoptosis and hepatocyte damage.

We concluded that, the increased TLR2 expression on PBMCs is probably involved in the pathogenesis of OLP with or without HCV infection, as well as in the pathogenesis of HCV infection ‘without OLP’. The idea that TLR2 can be the gate by which HCV induces OLP cannot be verified by this study. Our study supports a critical role for ‘TLR2 – immune‘ interaction in the pathogenesis and maintenance of OLP. Further studies are required to evaluate TLR2 and other TLRs’ expression after treatment of OLP and the possibility to be a therapeutic target in this resistant and progressive disease. Studies to clarify whether HCV infection has a role in OLP or whether it is merely an association are also recommended.

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Conflicts of interest

There are no conflicts of interest.

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References

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Keywords:

flowcytometry; hepatitis C virus infection; oral lichen planus; toll-like receptor 2

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