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Regulatory T-cells and IL17A+ cells infiltrate oral lichen planus lesions
Pathology (October 2016) 48(6), pp. 564–573
A N ATO M I C A L PAT H O L O G Y
Regulatory T-cells and IL17A+ cells infiltrate oral lichen planus lesions L. R. JAVVADI, V. P. B. PARACHURU, T. J. MILNE, G. J. SEYMOUR A. M. RICH
AND
Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand
Summary Oral lichen planus (OLP) is a complex immunological disorder, mediated in part by the release of cytokines from activated T-cells. Of late, two closely related T-helper (Th) cell subsets; regulatory T-cells (Tregs; FoxP3+) and Th17 cells (IL17+) have been described in various chronic inflammatory diseases. The aim of this study was to determine the expression of FoxP3 and IL17 in OLP using immunohistochemistry (IHC) and quantitative real-time reverse transcriptase polymerase chain reaction (qPCR). For IHC, formalin fixed, paraffin embedded archival specimens, an OLP group (n = 10) and a non-specific inflammatory (NSI) control group (n = 9) were used. In addition, 12 fresh tissue samples were used to determine gene expression of FoxP3 and IL17. Significantly more FoxP3+ cells were present in OLP than in NSI. IL17+ cells were significantly more frequent in the control tissues than in OLP. The gene expression experiments revealed a significantly higher expression of FoxP3 in OLP when compared to the controls. IL17 gene expression was not different between the groups. Double labelling immunofluorescence indicated co-localisation of IL17 with tryptase+ mast cells. These findings suggest FoxP3+ Tregs have a more prominent role in the pathogenesis of OLP when compared to IL17+cells. Key words: Lichen planus; immunoregulation; regulatory T-cells; interleukin-17. Received 25 November 2015, revised 11 May, accepted 9 June 2016 Available online 2 September 2016
INTRODUCTION Oral lichen planus (OLP) is a chronic inflammatory disease which affects 0.1–4% of the general adult population.1 This disease is characterised histologically by a dense subepithelial lymphocyte-rich infiltrate, degeneration of basal keratinocytes and basement membrane disruption. The exact aetiology of OLP is unclear but accumulating evidence supports the role of immune dysregulation, mediated by the release of cytokines by activated CD4+ T-cells leading to the attraction of inflammatory cells and then to the destruction of basal keratinocytes by cell mediated cytotoxicity via activated CD8+ cytotoxic T-cells (Tc). Studies that investigated
the T-helper (Th)1 and Th2 cytokine profile in tissues and serum from patients with OLP have demonstrated a Th1 cytokine bias2,3 although a Th2-predominant profile has been found in the saliva of patients with OLP.4 In addition to Th1 and Th2 cell subsets, two further Th subsets with opposing functions, namely regulatory T-cells (Tregs) and Th17 cells (IL17+), have been identified.5 Tregs express transcription factor forkhead box P3 (FoxP3) and are believed to have a pivotal role in the regulatory networks that control the immune response and peripheral tolerance to self and non-self.5 These cells differentiate from naïve T-cells under the influence of differentiation factors such as interleukin (IL)2, transforming growth factor (TGF)b, and IL10.6 Tregs regulate the immune response by contact-dependent and contact-independent mechanisms by releasing antiinflammatory cytokines such as TGFb, IL10, and IL35 to suppress the activation of both Th and Tc cells.6,7 Studies have demonstrated elevated numbers of FoxP3+ Tregs in OLP lesions compared to the healthy control tissues.8–11 Furthermore, gene expression studies on OLP tissues and mononuclear cells derived from lichen planus lesions exhibited greater expression of FoxP3 compared to the healthy controls.8,10,12 These studies attribute an immunoregulatory role to Tregs in the pathogenesis of OLP. Further, a CD4 T-cell subset, named Th17, has been described. Th17 cells play critical roles in the development of autoimmunity, allergic reactions and host defense by producing the powerful pro-inflammatory cytokine IL-17 and, to a lesser extent, tumour necrosis factor-a and IL6.13 These cells differentiate from naïve/memory T-cells when cultured in the presence of differentiation factors such as TGFb, IL6, IL1b, IL21 and IL23.14 Th17 cell differentiation and function has been demonstrated to be linked to the expression of specific transcription factor known as retinoic acid-related orphan receptor C.15 Studies using immunohistochemistry (IHC) and ELISA, as well as gene expression methods, have demonstrated more IL17 protein and mRNA expression in OLP than in healthy controls.11,16–20 Furthermore, increased numbers of CD4+ T-cell clones derived from the OLP expressed IL17 when compared to CD4+ T-cell clones of healthy controls.17,19 The gathered evidence suggests a possible role for IL17 in the pathogenesis of OLP. Tregs and Th17 cells share a close relationship with other Th subsets in regard to their differentiation, migration and plasticity. Interestingly, Th17 cells can be converted to Th1 or Th2 cells under the influence of cytokines IL12 or IL4, respectively. Similarly, Tregs co-cultured with dectin-1
Print ISSN 0031-3025/Online ISSN 1465-3931 © 2016 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved. DOI: http://dx.doi.org/10.1016/j.pathol.2016.06.002
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activated dendritic cells expressed IL17.21 This highlights the ‘plastic’ nature of these committed T-cells and their complex inter-relationships with other T-cell subsets when operating in the microenvironment of chronic inflammatory tissues such as OLP. The contrasting role of Tregs and Th17 cells in influencing the immune response has led us to hypothesise that both Tregs (FoxP3+) and Th17 (IL17A+) cells may regulate the immune response in OLP in a ying and yang fashion. An understanding of how these cell types act together to regulate the immune response in OLP is of importance. The aims of this study were (1) to determine if FoxP3+ Tregs and IL17A+ Th17 cells are present in OLP lesions, (2) if present, to determine the numbers of FoxP3+ Tregs and IL17A+ Th17 cells in OLP and to describe their topographical relationship and (3) to determine the expression of FoxP3 and IL17A at a gene level.
MATERIALS AND METHODS The University of Otago Institutional Ethics Committee and Ngai Tahu Research Consultation Committee approved this experimental protocol. Immunohistochemistry Sample selection and tissue preparation
Initially, a total of 30 formalin fixed, paraffin embedded (FFPE) oral mucosal tissue specimens histologically diagnosed as oral lichen planus (OLP; n = 15) and non-specific mucosal inflammation (NSI; n = 15) were retrieved from the archives of Medlab Dental Oral Pathology Diagnostic Services, Faculty of Dentistry, University of Otago. The NSI group were cases where biopsies had been taken from white oral mucosal lesions and histologically had an inflammatory infiltrate within the connective tissue, but which lacked features pathognomonic to OLP or any other specific disease; these lesions were likely to have been caused by mechanical trauma due to cheek chewing and the like. The objective of this study was to compare the immune/inflammatory response characteristic of OLP with inflammation not associated with OLP, and hence uninflamed healthy positive controls were not included. Tissue specimen information, such as age and gender of the patient, affected site, clinical details of the lesion and medical history, were recorded (Table 1). To ensure the results were not confounded by site variation, only buccal mucosal specimens were included in the final analysis (OLP n = 10, Table 1 study Case no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Clinical data of patients participating in the immunohistochemistry
Sex
Age, years
M M F F M M F F F F M M M F F M F F F
70 70 86 60 61 61 44 44 75 69 70 41 79 91 68 56 49 66 26
Affected site Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal
mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa
Clinical form
Diagnosis
White lesion White lesion White lesion White lesion Atrophic Atrophic White lesion White lesion White lesion Atrophic White lesion White lesion White lesion White lesion White lesion White lesion White lesion White lesion White lesion
OLP OLP OLP OLP OLP OLP OLP OLP OLP OLP NSI NSI NSI NSI NSI NSI NSI NSI NSI
Archival FFPE mucosal tissue samples (immunohistochemistry). F, female; FFPE, formalin fixed, paraffin embedded; M, male; NSI, nonspecific mucosal inflammation; OLP, oral lichen planus.
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NSI n = 9). All specimens fulfilled the following selection criteria: (i) diagnosed as OLP or NSI by registered oral pathologists at the Faculty of Dentistry, University of Otago; (ii) consent had been obtained from the patient for residual tissue, after diagnostic investigations had been completed, to be stored in the Histopathology Archive and used in experimental studies; (iii) factors that might modify the immune response (e.g., medically compromised, taking medications such as immunosuppressants), were not present; (iv) specimens which were very small in size and/or lacked significant connective tissue were excluded from the study. All patients in the study were from New Zealand, a low prevalence geographical area for hepatitis C (HCV). There is no evidence of an association between OLP and HCV in areas of low HCV prevalence22 and hence patients in our clinics with OLP are not routinely screened for HCV infection. No patient reported a known diagnosis of hepatitis C. Human tonsil tissue was used as positive control for all IHC antibodies. The antibodies and parameters used to perform IHC, together with optimised working conditions, are presented in Table 2. Immunohistochemistry staining for CD4, CD8 and FoxP3 was performed on OLP and NSI tissue sections using an automated slide stainer, BenchMark XT (Ventana Medical Systems, USA), which performed the deparaffinisation, cell conditioning, primary antibody incubation, labelled secondary antibody incubation, and 3,30 -diaminobenzidine (DAB) application using an ultraView Universal DAB Detection Kit (Ventana Medical Systems) with washing between each step. Primary antibody titration and the end stage haematoxylin counterstaining were performed manually. All slides were manually mounted with DePeX (Merck, Germany). IL17A immunostaining was performed using an already established manual IHC protocol at the MedLab Dental Oral Pathology Diagnostic Services Laboratory. Cell analysis
Each slide was viewed at up to 1000× magnification and six representative areas of the specimen were selected for analysis. Three of them were at the epithelial-connective tissue interface, where distinct basement membrane degeneration was observed, along with intra-epithelial lymphocytes in the OLP specimens. The remaining three sites were deeper in the inflammatory infiltrate, subjacent to the sites selected at the epithelial connective tissue interface (Fig. 1). The selected sites were photographed using QCapture (Q Imaging MicroPublisher 5.0 RTV, Canada) at 400×. Staining characteristics, specifically staining pattern, nature and intensity of staining, location of stained cells, and topographical relationship of stained cells were described. The number of CD4+, CD8+, FoxP3+ and IL17A+ cells and the total number of round mononuclear inflammatory cells were counted using Image J software and recorded. GraphPad Prism Software version 5 (GraphPad Software, USA) was used to perform the data entry and statistical analysis. The unpaired student t-test was used to analyse the difference in the proportion of CD4+, CD8+, FoxP3+ and IL17A+ cells between the groups. Within each disease group, the percentage of FoxP3+ cells were compared with the percentage of IL17A+ cells using paired student t-test. A statistical significance risk rate was set at p < 0.05. Immunofluorescence An indirect double immunofluorescence technique was used to determine the cell types expressing IL17A in the FFPE tissue sections. Using purified antibodies against CD3, IL17A and tryptase, 5 mm FFPE tissue sections were double-immunostained for CD3/IL17A (2.5 mg/mL; 10 mg/mL) and tryptase/ IL17A (2 mg/mL/10 mg/mL) along with concentration/species-matched IgG controls. Blocking serum (20% heat-treated serum in 1% BSA/PBS) was used for 30 min and then the sections were incubated overnight at 4 C. After three PBS washes of 10 min each, the secondary antibodies were conjugated with different fluorochromes (Alexa Fluor488-conjugated goat anti-rabbit and Alexa Fluor594-conjugated donkey anti-mouse; Invitrogen, USA) and all sections were incubated in the dark for 60 min. After washing, the slides were mounted with Vectshield Hardset mounting media with/without 40 ,6diamidino-2-phenylindole (DAPI; Vector Laboratories, USA). All sections were viewed under a fluorescent microscope and digital images captured under different wavelengths using a G0-3 camera (QImaging) and QCapture software (Macintosh QCapture Suite; QImaging). Images were superimposed and analysed using NIH Image J software.
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Primary antibodies and parameters used to perform IHC staining
Antibody/clone/concentration
Anti-Human CD3 rabbit monoclonal, 2GV6; 0.4 mg/mL Anti-Human CD4 rabbit monoclonal, SP35; 2.5 mg/mL Anti-Human CD8 rabbit monoclonal, SP57; 2.5 mg/mL Anti-Human FoxP3 mouse monoclonal; 259D; 500 mg/mL Anti-human IL17A goat polyclonal; 200 mg/mL Rabbit F(ab0 )2 polyclonal secondary antibody to goat IgG (Biotin) Purified goat IgG;5 mg/mL Negative control mouse IgG1; 100 mg/L Negative control rabbit IgG1; 400 mg/mL
Company and catalogue no.
Target cell type
IHC
Antibody incubation Optimal time and concentration temperature (mg/mL)
Ventana Medical Systems; 7904341
T-cells
BenchMark XT Mild CC1 30 min immunostainer
Ready-to-use
45 min at room temperature
Ventana Medical Systems; 7904423
T-cells
BenchMark XT Mild CC1 30 min immunostainer
Ready-to-use
45 min at room temperature
Ventana Medical Systems; 7904460
T-cells
BenchMark XT Mild CC1 30 min immunostainer
Ready-to-use
45 min at room temperature
Biolegend; 320102
Regulatory T-cells
BenchMark XT Mild CC1 30 min immunostainer
5
45 min at room temperature
R&D Systems; AF-317-NA
Th17 cells
Manual
Abcam; ab5753
–
–
3.5 Heat-induced antigen retrieval using citrate buffer (pH 6.0) – 1
Invitrogen; 02-6202 Dako; X0931
– –
– –
Same as respective primary antibody IHC parameters Same as respective primary antibody IHC parameters
SantaCruz Biotechnology, USA
–
–
Same as respective primary antibody IHC parameters
Gene expression Sample selection
Fresh tissue samples from patients with clinical features of OLP (n = 6) and patients with non-specifically inflamed oral mucosa (n = 6) and who were having a biopsy as a part of their diagnostic work-up were used for this part of the study. This group was additional to those described earlier where FFPE archival material was used. Patient clinical information, such as age, gender, past medical history and clinical site, were recorded (Table 3). These biopsy specimens were sectioned into two; the major part was sent for histological diagnosis and the remainder immediately placed in a nuclease free tube containing RNAlater (Ambion, USA) for downstream gene expression experiments. The inclusion and exclusion criteria for the specimens was the same as those for the IHC tissue specimens. Total RNA extraction and cDNA synthesis
Total RNA was isolated from the fresh tissue specimens and fresh human tonsil tissue, using a phenol-chloroform extraction technique and purified using Purelink RNA Mini Kit (Ambion, USA) with TRIzol Reagent (Applied Biosystems, USA). Genomic DNA was removed using on-column PureLink Dnase treatment (Ambion). The purified total RNA quality was assessed
45 min at room temperature
Quantitative real-time PCR (qRT2-PCR)
TaqMan gene expression assays for FoxP3 (Hs01085834_m1), IL17A (Hs00174383_m1), and Hypoxanthine phosphoribosyltransferase 1 (HPRT1, Hs02800695_m1 VIC-MGB) were analysed in the study (Applied Biosystems). HPRT1 acted as the reference gene. Duplex-gene assays were initially conducted for FoxP3/HPRT1 and IL17/HPRT1 with TaqMan Universal Master Mix (Applied Biosystems) on human tonsil tissue to determine the duplex qRT2-PCR efficiency. The calculated linear dynamic range of the assay demonstrated the input RNA (cDNA) concentration. After confirming the efficiency of the duplex qRT2-PCR and the input RNA amount, duplex qRT2-PCR gene expression experiments were performed on OLP and NSI samples using duplicate wells per sample. The standard PCR cycling parameters were 20 s at 95 C, and 40 cycles of 3 s at 95 C and 30 s at 60 C. Data analysis
The data from the raw Cq values of the tested genes normalised against the Cq of the HPRT1 reference gene were analysed using GraphPad Prism software
Case no. 1 2 3 4 5 6 7 8 9 10 11 12
Six representative sites for cell counting in an oral lichen planus tissue specimen.
Overnight at 4 C
spectrometrically using the NanoVue Plus (GE Healthcare, UK). Total RNA (500 ng) was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).
Table 3
Fig. 1
Antigen-retrieval method used
Clinical data of patients participating in the gene expression study Sex
Age, years
M M F F F F M M F F F F
66 69 42 52 66 69 61 61 44 44 75 69
Affected site Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal
mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa
Clinical form
Diagnosis
White lesion Atrophic White lesion White lesion White lesion White lesion White lesion White lesion White lesion White lesion White lesion White lesion
OLP OLP OLP OLP OLP OLP NSI NSI NSI NSI NSI NSI
Archival FFPE mucosal tissue samples (immunohistochemistry). F, female; FFPE, formalin fixed, paraffin embedded; M, male; NSI, nonspecific mucosal inflammation; OLP, oral lichen planus.
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(GraphPad, USA). All Cq values 35 were considered to be beyond the detection limit of the system and were not included in the analysis. Fold changes in the gene expression were determined by comparing the mean normalised gene expression levels between OLP and NSI tissues using DDCq method. The unpaired student t-test analysed the difference in the expression level of the tested genes between the groups. Genes with a statistical significance of ±2 and a p value < 0.05 were considered as significantly regulated.
RESULTS In the OLP lesions a diffuse T-cell (CD4+ and CD8+) infiltrate was seen at the epithelial-connective tissue interface and scattered within the epithelium (Fig. 2A,B) with only scattered CD4+ and CD8+ positivity in the deeper connective tissue. CD4+ and CD8+ cells were diffusely scattered through the connective tissue of the inflamed control tissues (Fig. 2E,F). In both OLP and NSI, FoxP3 staining was predominantly nuclear and FoxP3+ cell morphology was consistent with that of a lymphocyte (Fig. 2C,G). Within OLP lesions, FoxP3+ cells were evenly distributed in the inflammatory infiltrate at the epithelial-connective tissue interface and in the superficial connective tissue. Alternatively, IL17A staining was predominantly cytoplasmic, with a small amount of extracellular staining (Fig. 2D,H). The majority of the IL17A+ cells were located within and deep to the main band of inflammatory cells and very few were observed at the epithelial-connective tissue interface. Interestingly, all IL17A+ cells had an ovoid/plasmacytoid morphology (Fig. 3A,B).
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mean (±SD) of CD4+, CD8+, FoxP3+ and IL17A+ cells in the infiltrate at the epithelial-connective tissue interface was 65.32 ± 6.42, 56.62 ± 8.23, 20.64 ± 4.38 and 0.83 ± 0.65, respectively. There was no difference observed between the % mean (±SD) of CD4+ and CD8+ cells. There were significantly more FoxP3+ cells than IL17A+ cells (p < 0.0001). Similarly, 30 sites were examined at the deeper aspects of the superficial connective tissue infiltrate in the OLP specimens. All sites showed CD4+ (26.45 ± 4.42), CD8+ (24.23 ± 6.32), FoxP3+ (19.38 ± 6.32) cells, 27 sites showed a few IL17A+ cells (2.14 ± 1.57). There was no difference observed between the % mean (±SD) of CD4+ cells and CD8+ cells. Significantly more FoxP3+ cells were present in the superficial connective tissue infiltrate in OLP than IL17A+ cells (p < 0.0001). There were significantly more CD4+ and CD8+ cells in the epithelial-connective tissue interface region than the superficial connective tissue infiltrate region in OLP (p < 0.0001). While there was no difference in the mean percentage of FoxP3+ cells in the interface area compared to deeper within the inflammatory band (p = 0.494), significantly more IL17A+ cells were present in the deeper infiltrate than at the epithelial-connective tissue interface p = 0.044 (Fig. 5). Phenotypic characterisation of IL17A+ cells
Quantification of immune cells: CD4+, CD8+, FoxP3+ and IL17+ cells
All OLP specimens were evaluated for the presence of CD3+/ IL17A+ and IL17A+/tryptase+ cells in the inflammatory infiltrate. Double immunofluorescence labelling revealed that IL17A expression was restricted to tryptase+ mast cells (Fig. 6) and was not found in CD3+ T-cells (Fig. 7).
Oral lichen planus versus non-specific inflammation
Gene expression
Ten OLP cases were examined. All cases showed the presence of CD4+, CD8+, FoxP3+ and few IL17A+ cells. The % mean [± standard deviation (SD)] of CD4+ cells, CD8+ cells, FoxP3+ cells and IL17A+ cells of the total inflammatory cells in the infiltrate was 62.12 ± 10.43, 54.32 ± 8.43, 20.34 ± 4.33 and 1.18 ± 0.64, respectively. There was no significant difference in the % mean of CD4+ and CD8+ cells in OLP. However, there were significantly more FoxP3+ cells than IL17A+ cells in OLP (p < 0.0001). All the nine NSI cases showed CD4+, CD8+, FoxP3+ cells and few IL17A+ cells. The % mean ± SD of CD4+ and CD8+ cells of the total inflammatory cells in the infiltrate was 43.21 ± 6.43 and 32.84 ± 7.44, respectively. There was no significant difference observed between the % mean of the CD4+ and CD8+ cells. Similarly, comparison of % mean ± SD of FoxP3+ cells (5.52 ± 5.22) and IL17A+ cells (3.39 ± 2.69) in the infiltrate in the inflamed controls showed no significant difference. There were significantly more FoxP3+ cells in OLP lesions compared with NSI tissues (p < 0.0001); unlike the IL17A+ cells, which were significantly more common in the NSI tissues (p = 0.021) (Fig. 4).
Relative qRT2-PCR determination of FoxP3 and IL17A gene expression
Epithelial-connective tissue interface region versus superficial connective tissue infiltrate region of OLP A total of 30 sites (six sites/specimen) were examined at the epithelial-connective tissue interface of OLP (n = 10). All sites examined showed CD4+, CD8+, FoxP3+ cells. Twentyseven sites showed a small number of IL17A+ cells. The %
FoxP3 gene was significantly up-regulated in OLP compared with NSI, with a fold regulation of 6.42 (p = 0.001) (Fig. 8A). However, there was no difference in IL17A expression between the groups (Fig. 8B). Within the OLP tissues there was a higher expression of FoxP3 than IL17, which was statistically significant (p = 0.005) (Fig. 8C).
DISCUSSION The present study confirmed the presence of FoxP3+ Tregs and IL17A+ cells in the inflammatory infiltrate of archival OLP tissues. More FoxP3+ Tregs were present in OLP than in the control NSI tissues and FoxP3 genes were up-regulated. The band-like mononuclear cell infiltrate of OLP predominantly consists of macrophages and T-cells, specifically mature CD4+ and CD8+ cells.23–26 In line with this, the results of our study also demonstrated a greater number of CD4+ and CD8+ T cells in the epithelial-connective tissue interface region of OLP tissues. Furthermore, there was no difference observed between the percentage of CD4+ and CD8+ T-cells in any of the OLP lesions investigated. This result is in contrast with earlier studies,27,28 which showed a higher proportion of CD4+ cells than CD8+ cells in both hyperplastic and atrophic OLP lesions. These contrasting results can be attributed to the different clinical forms and stage of the disease. In the initial lesion of an OLP, CD4+ Tcells appear to be abundant in the connective tissue infiltrate,
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Results of the IHC studies. (A) CD4+ T cells in OLP, (B) CD8+ T cells in OLP, (C) FoxP3+ Tregs in OLP, (D) IL17A+ cells in OLP, (E) CD4+ T cells in NSI, (F) CD8+ T cells in NSI, (G) FoxP3+ Tregs in NSI, and (H) IL17A+ cells in NSI. Scale bar = 50 mm.
Fig. 2
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Fig. 3
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Large ovoid/plasmacytoid IL17A+ cells in the deep connective tissue inflammatory infiltrate in OLP tissue. Scale bar = 50 mm.
while in the advanced lesion, CD8+ T-cells predominate.25,26,29 In this context, it is quite possible that OLP tissues investigated in this study may be in a stage between the initial and advanced form. Tregs play a central role in inducing and maintaining immunological tolerance along with suppressing the immune
response, and the up-regulation of FoxP3 in OLP may indicate a greater attempt to regulate the immune response in this condition. There were fewer FoxP3+ cells in the NSI tissues, where an inflammatory response is also present. There have been reports demonstrating higher FoxP3 expression in OLP when compared to healthy uninflamed controls,10,12 but there
Distribution of %FoxP3+ and %IL17A+ cells between OLP tissue and NSl tissue groups. The mean %FoxP3+ cells was significantly higher in OLP tissues compared with NSI tissues (p < 0.0001). The mean %IL17A+ cells was significantly higher in NSI tissues compared with OLP (p = 0.02).
Fig. 4
Distribution of %FoxP3+ and %IL17A+ cells at the epithelial-connective interface and deeper in the superficial connective tissue infiltrate in OLP tissues. Comparison of mean %FoxP3+ cells between the regions was not significant, but significantly more IL17A+ cells were present in the deeper aspects of the infiltrate compared with epithelial connective tissue interface region (p = 0.04)
Fig. 5
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Fig. 6 Immunofluorescence double labelling of IL17A/tryptase in OLP tissue. Note that IL17A expression was localised within tryptase+ mast cells. Scale bar = 50 mm.
are no previous studies comparing their presence in OLP tissues with other inflammatory processes. The immune response that is observed in some inflammatory tissues may appear similar to OLP histologically; however, it may be of a different immune character. Therefore, it is crucial to identify the difference between both the groups, given the antigenic load and inflammatory infiltrate are not similar. The current study demonstrated FoxP3+ Tregs intra-epithelially, as well as in the infiltrate in the superficial connective tissue of OLP
tissues. The presence of FoxP3+ Tregs in proximity to the basal keratinocytes suggests a role in the pathogenesis of the disease. However, there was no significant difference in their expression at the epithelial-connective tissue interface and within the deeper levels of the infiltrate. Of late, IL17 has been reported to play a prime role in the pathogenesis of various chronic inflammatory lesions, and exploring its role in OLP is just beginning.11,16,19 In the current study a small number of IL17A+ cells were identified
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Fig. 7
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Immunofluorescence double labelling of IL17A/CD3 in OLP tissue. Note that IL17A expression was not localised within CD3+ T-cells. Scale bar = 50 mm.
in OLP as well as in controls, with fewer IL17A+ cells in OLP when compared to the NSI tissues. The overall expression of IL17A+ cells in OLP was much lower in the current study than in previous reports.11,19 The reasons for these contrasting results cannot be entirely established. The majority of cases (7/10) in the current study were histologically classified as hyperplastic lichen planus (showing hyperkeratosis/parakeratosis and acanthosis along with the characteristic features
of OLP) with the remaining three cases showing evidence of epithelial atrophy, without ulceration. IL17 has been associated with increased tissue damage in OLP in atrophic lichen planus by comparison with the hyperplastic form,19 but this is unlikely to explain the overall low IL17A+ cell count. In the current study we evaluated the presence of IL17A+ cells in various regions of OLP; they were observed in greater density in the deeper regions of the infiltrate than at the
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Fig. 8 Relative expression level of FoxP3 and IL17A in OLP tissues and NSI tissues. (A) FoxP3 levels were significantly higher in OLP tissues compared with NSI
tissues. (B) IL17A levels were no different between the groups. (C) In OLP, FoxP3 expression levels were significantly higher than IL17A. **p < 0.05.
epithelial-connective tissue interface. Morphologically these cells had an ovoid or plasmacytoid appearance with abundant cytoplasm. Double labelling immunofluorescence attributes IL17A positivity to mast cells rather T-cell clones. These results concur with previous work in our laboratory on periodontitis tissues and OLP where IL17+ cells were found to be mast cells.30,31 Taken together, these data establish the fact that these two T-cell subsets may play a role in the pathogenesis of OLP. Increased numbers of FoxP3+ cells and up-regulation of FoxP3+ genes in OLP demonstrates greater activity of Tregs in OLP lesions, potentially trying to modulate and suppress the chronicity of the lesion. It can be speculated that FoxP3+ Tregs might be attempting to control the immune response to self-antigen and prevent tissue destruction. They are thought to play a pivotal role in the central tolerance to self-antigens.32 The interplay between FoxP3+ cells and IL17A+ cells must be in a proper balance to ensure an efficient immune response rather than pathological damage.33 The increased number of FoxP3+ cells in OLP may reflect their significant role in the disease process. Fewer IL17A+ cells may be attributed to concomitant recruitment of Tregs over the prolonged course of this chronic condition, which may inhibit IL17-induced inflammation at lesional sites. In summary, this study demonstrates the presence of a greater number of FoxP3+ Tregs when compared to IL17A+ cells (Th17) and up-regulation of FoxP3 genes in the inflammatory infiltrate of OLP lesions. These results lead us to speculate that FoxP3+ cells have a more prominent role in
the pathogenesis of OLP when compared to IL17A+ cells. Future studies are required to further characterise these cells and understand their interplay in the pathophysiology of OLP. Conflicts of interest and sources of funding: This project was funded by a New Zealand Dental Association Research Foundation Grant (NZDA 7.07 2012). The authors state there are no conflicts of interest to disclose. Address for correspondence: Prof A. M. Rich, Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand. E-mail: [email protected]
References 1. Lodi G, Scully C, Carrozzo M, et al. Current controversies in oral lichen planus: report of an international consensus meeting. Part 1. Viral infections and etiopathogenesis. Oral Surg Oral Med Oral Pathol 2005; 100: 40–51. 2. Sugerman PB, Savage NW, Walsh LJ, et al. The pathogenesis of oral lichen planus. Crit Rev Oral Biol Med 2002; 13: 350–65. 3. Rhodus NL, Cheng B, Ondrey F. Th1/Th2 cytokine ratio in tissue transudates from patients with oral lichen planus. Mediators Inflamm 2007; 2007: 19854. 4. Liu W, Dan H, Wang Z, et al. IFN-gamma and IL-4 in saliva of patients with oral lichen planus: a study in an ethnic Chinese Population. Inflammation 2009; 32: 176–81. 5. Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 2004; 22: 531–62. 6. Sakaguchi S, Yamaguchi T, Nomura T, et al. Regulatory T cells and immune tolerance. Cell 2008; 133: 775–87.
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7. Collison LW, Workman CJ, Kuo TT, et al. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature 2007; 450: 566–9. 8. Tao XA, Xia J, Chen XB, et al. FOXP3 T regulatory cells in lesions of oral lichen planus correlated with disease activity. Oral Dis 2010; 16: 76–82. 9. Pereira JS, Monteiro BV, Nonaka CF, et al. FoxP3(+) T regulatory cells in oral lichen planus and its correlation with the distinct clinical appearance of the lesions. Int J Exp Pathol 2012; 93: 287– 94. 10. Lei L, Zhan L, Tan W, et al. Foxp3 gene expression in oral lichen planus: a clinicopathological study. Mol Med Rep 2014; 9: 928–34. 11. Shen Z, Gao X, Ma L, et al. Expression of Foxp3 and interleukin-17 in lichen planus lesions with emphasis on difference in oral and cutaneous variants. Arch Dermatol Res 2014; 306: 441–6. 12. Tao XA, Li CY, Xia J, et al. Differential gene expression profiles of whole lesions from patients with oral lichen planus. J Oral Pathol Med 2009; 38: 427–33. 13. Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006; 441: 235–8. 14. Stockinger B, Veldhoen M, Martin B. Th17 T cells: linking innate and adaptive immunity. Sem Immunol 2007; 19: 353–61. 15. Ivanov II, McKenzie BS, Zhou L, et al. The orphan nuclear receptor RORgamma directs the differentiation program of proinflammatory IL17+ T helper cells. Cell 2006; 126: 1121–33. 16. Shaker O, Hassan AS. Possible role of interleukin-17 in the pathogenesis of lichen planus. Br J Dermatol 2012; 166: 1367–8. 17. Lu R, Zeng X, Han Q, et al. Overexpression and selectively regulatory roles of IL-23/IL-17 axis in the lesions of oral lichen planus. Mediators Inflamm 2014; 2014: 701094. 18. Wang H, Han Q, Luo Z, et al. Oral lichen planus may enhance the expression of Th17-associated cytokines in local lesions of chronic periodontitis. Clin Oral Invest 2014; 18: 1647–54. 19. Piccinni MP, Lombardelli L, Logiodice F, et al. Potential pathogenetic role of Th17, Th0, and Th2 cells in erosive and reticular oral lichen planus. Oral Dis 2014; 20: 212–8.
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20. Pouralibaba F, Babaloo Z, Pakdel F, et al. Serum level of interleukin 17 in patients with erosive and non erosive oral lichen planus. J Dent Res Dent Clin Dent Pros 2013; 7: 91–4. 21. Lexberg MH, Taubner A, Forster A, et al. Th memory for interleukin-17 expression is stable in vivo. Eur J Immunol 2008; 38: 2654–64. 22. Zhou Y, Jiang L, Liu J, et al. The prevalence of hepatitis C virus infection in oral lichen planus in an ethnic Chinese cohort of 232 patients. Int J Oral Sci 2010; 2: 90–7. 23. Jungell P, Konttinen YT, Malmstrom M. Basement membrane changes in oral lichen planus. Proc Finn Dent Soc 1989; 85: 119–24. 24. Hirota J, Osaki T, Tatemoto Y. Immunohistochemical staining of infiltrates in oral lichen planus. Pathol Res Pract 1990; 186: 625–32. 25. Walsh LJ, Savage NW, Ishii T, et al. Immunopathogenesis of oral lichen planus. J Oral Pathol Med 1990; 19: 389–96. 26. Porter SR, Kirby A, Olsen I, et al. Immunologic aspects of dermal and oral lichen planus: a review. Oral Surg Oral Med Oral Pathol 1997; 83: 358–66. 27. Bhan AK, Harrist TJ, Murphy GF, et al. T cell subsets and Langerhans cells in lichen planus: in situ characterization using monoclonal antibodies. Br J Dermatol 1981; 105: 617–22. 28. Boisnic S, Frances C, Branchet MC, et al. Immunohistochemical study of oral lesions of lichen planus: diagnostic and pathophysiologic aspects. Oral Surg Oral Med Oral Pathol 1990; 70: 462–5. 29. Zhou XJ, Sugerman PB, Savage NW, et al. Intra-epithelial CD8+ T cells and basement membrane disruption in oral lichen planus. J Oral Pathol Med 2002; 31: 23–7. 30. Parachuru VP, Coates DE, Milne TJ, et al. Forkhead box P3-positive regulatory T-cells and interleukin 17-positive T-helper 17 cells in chronic inflammatory periodontal disease. J Periodontal Res 2014; 49: 817–26. 31. Firth FA, Friedlander LT, Parachuru VP, et al. Regulation of immune cells in oral lichen planus. Arch Dermatol Res 2015; 307: 333–9. 32. Lan Q, Fan H, Quesniaux V. Induced Foxp3(+) regulatory T cells: a potential new weapon to treat autoimmune and inflammatory diseases? J Mol Cell Biol 2012; 4: 22–8. 33. Mai J, Wang H, Yang XF. Th 17 cells interplay with Foxp3+ Tregs in regulation of inflammation and autoimmunity. Front Biosci 2010; 15: 986–1006.
A N ATO M I C A L PAT H O L O G Y
Regulatory T-cells and IL17A+ cells infiltrate oral lichen planus lesions L. R. JAVVADI, V. P. B. PARACHURU, T. J. MILNE, G. J. SEYMOUR A. M. RICH
AND
Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand
Summary Oral lichen planus (OLP) is a complex immunological disorder, mediated in part by the release of cytokines from activated T-cells. Of late, two closely related T-helper (Th) cell subsets; regulatory T-cells (Tregs; FoxP3+) and Th17 cells (IL17+) have been described in various chronic inflammatory diseases. The aim of this study was to determine the expression of FoxP3 and IL17 in OLP using immunohistochemistry (IHC) and quantitative real-time reverse transcriptase polymerase chain reaction (qPCR). For IHC, formalin fixed, paraffin embedded archival specimens, an OLP group (n = 10) and a non-specific inflammatory (NSI) control group (n = 9) were used. In addition, 12 fresh tissue samples were used to determine gene expression of FoxP3 and IL17. Significantly more FoxP3+ cells were present in OLP than in NSI. IL17+ cells were significantly more frequent in the control tissues than in OLP. The gene expression experiments revealed a significantly higher expression of FoxP3 in OLP when compared to the controls. IL17 gene expression was not different between the groups. Double labelling immunofluorescence indicated co-localisation of IL17 with tryptase+ mast cells. These findings suggest FoxP3+ Tregs have a more prominent role in the pathogenesis of OLP when compared to IL17+cells. Key words: Lichen planus; immunoregulation; regulatory T-cells; interleukin-17. Received 25 November 2015, revised 11 May, accepted 9 June 2016 Available online 2 September 2016
INTRODUCTION Oral lichen planus (OLP) is a chronic inflammatory disease which affects 0.1–4% of the general adult population.1 This disease is characterised histologically by a dense subepithelial lymphocyte-rich infiltrate, degeneration of basal keratinocytes and basement membrane disruption. The exact aetiology of OLP is unclear but accumulating evidence supports the role of immune dysregulation, mediated by the release of cytokines by activated CD4+ T-cells leading to the attraction of inflammatory cells and then to the destruction of basal keratinocytes by cell mediated cytotoxicity via activated CD8+ cytotoxic T-cells (Tc). Studies that investigated
the T-helper (Th)1 and Th2 cytokine profile in tissues and serum from patients with OLP have demonstrated a Th1 cytokine bias2,3 although a Th2-predominant profile has been found in the saliva of patients with OLP.4 In addition to Th1 and Th2 cell subsets, two further Th subsets with opposing functions, namely regulatory T-cells (Tregs) and Th17 cells (IL17+), have been identified.5 Tregs express transcription factor forkhead box P3 (FoxP3) and are believed to have a pivotal role in the regulatory networks that control the immune response and peripheral tolerance to self and non-self.5 These cells differentiate from naïve T-cells under the influence of differentiation factors such as interleukin (IL)2, transforming growth factor (TGF)b, and IL10.6 Tregs regulate the immune response by contact-dependent and contact-independent mechanisms by releasing antiinflammatory cytokines such as TGFb, IL10, and IL35 to suppress the activation of both Th and Tc cells.6,7 Studies have demonstrated elevated numbers of FoxP3+ Tregs in OLP lesions compared to the healthy control tissues.8–11 Furthermore, gene expression studies on OLP tissues and mononuclear cells derived from lichen planus lesions exhibited greater expression of FoxP3 compared to the healthy controls.8,10,12 These studies attribute an immunoregulatory role to Tregs in the pathogenesis of OLP. Further, a CD4 T-cell subset, named Th17, has been described. Th17 cells play critical roles in the development of autoimmunity, allergic reactions and host defense by producing the powerful pro-inflammatory cytokine IL-17 and, to a lesser extent, tumour necrosis factor-a and IL6.13 These cells differentiate from naïve/memory T-cells when cultured in the presence of differentiation factors such as TGFb, IL6, IL1b, IL21 and IL23.14 Th17 cell differentiation and function has been demonstrated to be linked to the expression of specific transcription factor known as retinoic acid-related orphan receptor C.15 Studies using immunohistochemistry (IHC) and ELISA, as well as gene expression methods, have demonstrated more IL17 protein and mRNA expression in OLP than in healthy controls.11,16–20 Furthermore, increased numbers of CD4+ T-cell clones derived from the OLP expressed IL17 when compared to CD4+ T-cell clones of healthy controls.17,19 The gathered evidence suggests a possible role for IL17 in the pathogenesis of OLP. Tregs and Th17 cells share a close relationship with other Th subsets in regard to their differentiation, migration and plasticity. Interestingly, Th17 cells can be converted to Th1 or Th2 cells under the influence of cytokines IL12 or IL4, respectively. Similarly, Tregs co-cultured with dectin-1
Print ISSN 0031-3025/Online ISSN 1465-3931 © 2016 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved. DOI: http://dx.doi.org/10.1016/j.pathol.2016.06.002
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activated dendritic cells expressed IL17.21 This highlights the ‘plastic’ nature of these committed T-cells and their complex inter-relationships with other T-cell subsets when operating in the microenvironment of chronic inflammatory tissues such as OLP. The contrasting role of Tregs and Th17 cells in influencing the immune response has led us to hypothesise that both Tregs (FoxP3+) and Th17 (IL17A+) cells may regulate the immune response in OLP in a ying and yang fashion. An understanding of how these cell types act together to regulate the immune response in OLP is of importance. The aims of this study were (1) to determine if FoxP3+ Tregs and IL17A+ Th17 cells are present in OLP lesions, (2) if present, to determine the numbers of FoxP3+ Tregs and IL17A+ Th17 cells in OLP and to describe their topographical relationship and (3) to determine the expression of FoxP3 and IL17A at a gene level.
MATERIALS AND METHODS The University of Otago Institutional Ethics Committee and Ngai Tahu Research Consultation Committee approved this experimental protocol. Immunohistochemistry Sample selection and tissue preparation
Initially, a total of 30 formalin fixed, paraffin embedded (FFPE) oral mucosal tissue specimens histologically diagnosed as oral lichen planus (OLP; n = 15) and non-specific mucosal inflammation (NSI; n = 15) were retrieved from the archives of Medlab Dental Oral Pathology Diagnostic Services, Faculty of Dentistry, University of Otago. The NSI group were cases where biopsies had been taken from white oral mucosal lesions and histologically had an inflammatory infiltrate within the connective tissue, but which lacked features pathognomonic to OLP or any other specific disease; these lesions were likely to have been caused by mechanical trauma due to cheek chewing and the like. The objective of this study was to compare the immune/inflammatory response characteristic of OLP with inflammation not associated with OLP, and hence uninflamed healthy positive controls were not included. Tissue specimen information, such as age and gender of the patient, affected site, clinical details of the lesion and medical history, were recorded (Table 1). To ensure the results were not confounded by site variation, only buccal mucosal specimens were included in the final analysis (OLP n = 10, Table 1 study Case no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Clinical data of patients participating in the immunohistochemistry
Sex
Age, years
M M F F M M F F F F M M M F F M F F F
70 70 86 60 61 61 44 44 75 69 70 41 79 91 68 56 49 66 26
Affected site Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal
mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa
Clinical form
Diagnosis
White lesion White lesion White lesion White lesion Atrophic Atrophic White lesion White lesion White lesion Atrophic White lesion White lesion White lesion White lesion White lesion White lesion White lesion White lesion White lesion
OLP OLP OLP OLP OLP OLP OLP OLP OLP OLP NSI NSI NSI NSI NSI NSI NSI NSI NSI
Archival FFPE mucosal tissue samples (immunohistochemistry). F, female; FFPE, formalin fixed, paraffin embedded; M, male; NSI, nonspecific mucosal inflammation; OLP, oral lichen planus.
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NSI n = 9). All specimens fulfilled the following selection criteria: (i) diagnosed as OLP or NSI by registered oral pathologists at the Faculty of Dentistry, University of Otago; (ii) consent had been obtained from the patient for residual tissue, after diagnostic investigations had been completed, to be stored in the Histopathology Archive and used in experimental studies; (iii) factors that might modify the immune response (e.g., medically compromised, taking medications such as immunosuppressants), were not present; (iv) specimens which were very small in size and/or lacked significant connective tissue were excluded from the study. All patients in the study were from New Zealand, a low prevalence geographical area for hepatitis C (HCV). There is no evidence of an association between OLP and HCV in areas of low HCV prevalence22 and hence patients in our clinics with OLP are not routinely screened for HCV infection. No patient reported a known diagnosis of hepatitis C. Human tonsil tissue was used as positive control for all IHC antibodies. The antibodies and parameters used to perform IHC, together with optimised working conditions, are presented in Table 2. Immunohistochemistry staining for CD4, CD8 and FoxP3 was performed on OLP and NSI tissue sections using an automated slide stainer, BenchMark XT (Ventana Medical Systems, USA), which performed the deparaffinisation, cell conditioning, primary antibody incubation, labelled secondary antibody incubation, and 3,30 -diaminobenzidine (DAB) application using an ultraView Universal DAB Detection Kit (Ventana Medical Systems) with washing between each step. Primary antibody titration and the end stage haematoxylin counterstaining were performed manually. All slides were manually mounted with DePeX (Merck, Germany). IL17A immunostaining was performed using an already established manual IHC protocol at the MedLab Dental Oral Pathology Diagnostic Services Laboratory. Cell analysis
Each slide was viewed at up to 1000× magnification and six representative areas of the specimen were selected for analysis. Three of them were at the epithelial-connective tissue interface, where distinct basement membrane degeneration was observed, along with intra-epithelial lymphocytes in the OLP specimens. The remaining three sites were deeper in the inflammatory infiltrate, subjacent to the sites selected at the epithelial connective tissue interface (Fig. 1). The selected sites were photographed using QCapture (Q Imaging MicroPublisher 5.0 RTV, Canada) at 400×. Staining characteristics, specifically staining pattern, nature and intensity of staining, location of stained cells, and topographical relationship of stained cells were described. The number of CD4+, CD8+, FoxP3+ and IL17A+ cells and the total number of round mononuclear inflammatory cells were counted using Image J software and recorded. GraphPad Prism Software version 5 (GraphPad Software, USA) was used to perform the data entry and statistical analysis. The unpaired student t-test was used to analyse the difference in the proportion of CD4+, CD8+, FoxP3+ and IL17A+ cells between the groups. Within each disease group, the percentage of FoxP3+ cells were compared with the percentage of IL17A+ cells using paired student t-test. A statistical significance risk rate was set at p < 0.05. Immunofluorescence An indirect double immunofluorescence technique was used to determine the cell types expressing IL17A in the FFPE tissue sections. Using purified antibodies against CD3, IL17A and tryptase, 5 mm FFPE tissue sections were double-immunostained for CD3/IL17A (2.5 mg/mL; 10 mg/mL) and tryptase/ IL17A (2 mg/mL/10 mg/mL) along with concentration/species-matched IgG controls. Blocking serum (20% heat-treated serum in 1% BSA/PBS) was used for 30 min and then the sections were incubated overnight at 4 C. After three PBS washes of 10 min each, the secondary antibodies were conjugated with different fluorochromes (Alexa Fluor488-conjugated goat anti-rabbit and Alexa Fluor594-conjugated donkey anti-mouse; Invitrogen, USA) and all sections were incubated in the dark for 60 min. After washing, the slides were mounted with Vectshield Hardset mounting media with/without 40 ,6diamidino-2-phenylindole (DAPI; Vector Laboratories, USA). All sections were viewed under a fluorescent microscope and digital images captured under different wavelengths using a G0-3 camera (QImaging) and QCapture software (Macintosh QCapture Suite; QImaging). Images were superimposed and analysed using NIH Image J software.
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Primary antibodies and parameters used to perform IHC staining
Antibody/clone/concentration
Anti-Human CD3 rabbit monoclonal, 2GV6; 0.4 mg/mL Anti-Human CD4 rabbit monoclonal, SP35; 2.5 mg/mL Anti-Human CD8 rabbit monoclonal, SP57; 2.5 mg/mL Anti-Human FoxP3 mouse monoclonal; 259D; 500 mg/mL Anti-human IL17A goat polyclonal; 200 mg/mL Rabbit F(ab0 )2 polyclonal secondary antibody to goat IgG (Biotin) Purified goat IgG;5 mg/mL Negative control mouse IgG1; 100 mg/L Negative control rabbit IgG1; 400 mg/mL
Company and catalogue no.
Target cell type
IHC
Antibody incubation Optimal time and concentration temperature (mg/mL)
Ventana Medical Systems; 7904341
T-cells
BenchMark XT Mild CC1 30 min immunostainer
Ready-to-use
45 min at room temperature
Ventana Medical Systems; 7904423
T-cells
BenchMark XT Mild CC1 30 min immunostainer
Ready-to-use
45 min at room temperature
Ventana Medical Systems; 7904460
T-cells
BenchMark XT Mild CC1 30 min immunostainer
Ready-to-use
45 min at room temperature
Biolegend; 320102
Regulatory T-cells
BenchMark XT Mild CC1 30 min immunostainer
5
45 min at room temperature
R&D Systems; AF-317-NA
Th17 cells
Manual
Abcam; ab5753
–
–
3.5 Heat-induced antigen retrieval using citrate buffer (pH 6.0) – 1
Invitrogen; 02-6202 Dako; X0931
– –
– –
Same as respective primary antibody IHC parameters Same as respective primary antibody IHC parameters
SantaCruz Biotechnology, USA
–
–
Same as respective primary antibody IHC parameters
Gene expression Sample selection
Fresh tissue samples from patients with clinical features of OLP (n = 6) and patients with non-specifically inflamed oral mucosa (n = 6) and who were having a biopsy as a part of their diagnostic work-up were used for this part of the study. This group was additional to those described earlier where FFPE archival material was used. Patient clinical information, such as age, gender, past medical history and clinical site, were recorded (Table 3). These biopsy specimens were sectioned into two; the major part was sent for histological diagnosis and the remainder immediately placed in a nuclease free tube containing RNAlater (Ambion, USA) for downstream gene expression experiments. The inclusion and exclusion criteria for the specimens was the same as those for the IHC tissue specimens. Total RNA extraction and cDNA synthesis
Total RNA was isolated from the fresh tissue specimens and fresh human tonsil tissue, using a phenol-chloroform extraction technique and purified using Purelink RNA Mini Kit (Ambion, USA) with TRIzol Reagent (Applied Biosystems, USA). Genomic DNA was removed using on-column PureLink Dnase treatment (Ambion). The purified total RNA quality was assessed
45 min at room temperature
Quantitative real-time PCR (qRT2-PCR)
TaqMan gene expression assays for FoxP3 (Hs01085834_m1), IL17A (Hs00174383_m1), and Hypoxanthine phosphoribosyltransferase 1 (HPRT1, Hs02800695_m1 VIC-MGB) were analysed in the study (Applied Biosystems). HPRT1 acted as the reference gene. Duplex-gene assays were initially conducted for FoxP3/HPRT1 and IL17/HPRT1 with TaqMan Universal Master Mix (Applied Biosystems) on human tonsil tissue to determine the duplex qRT2-PCR efficiency. The calculated linear dynamic range of the assay demonstrated the input RNA (cDNA) concentration. After confirming the efficiency of the duplex qRT2-PCR and the input RNA amount, duplex qRT2-PCR gene expression experiments were performed on OLP and NSI samples using duplicate wells per sample. The standard PCR cycling parameters were 20 s at 95 C, and 40 cycles of 3 s at 95 C and 30 s at 60 C. Data analysis
The data from the raw Cq values of the tested genes normalised against the Cq of the HPRT1 reference gene were analysed using GraphPad Prism software
Case no. 1 2 3 4 5 6 7 8 9 10 11 12
Six representative sites for cell counting in an oral lichen planus tissue specimen.
Overnight at 4 C
spectrometrically using the NanoVue Plus (GE Healthcare, UK). Total RNA (500 ng) was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).
Table 3
Fig. 1
Antigen-retrieval method used
Clinical data of patients participating in the gene expression study Sex
Age, years
M M F F F F M M F F F F
66 69 42 52 66 69 61 61 44 44 75 69
Affected site Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal Buccal
mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa
Clinical form
Diagnosis
White lesion Atrophic White lesion White lesion White lesion White lesion White lesion White lesion White lesion White lesion White lesion White lesion
OLP OLP OLP OLP OLP OLP NSI NSI NSI NSI NSI NSI
Archival FFPE mucosal tissue samples (immunohistochemistry). F, female; FFPE, formalin fixed, paraffin embedded; M, male; NSI, nonspecific mucosal inflammation; OLP, oral lichen planus.
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(GraphPad, USA). All Cq values 35 were considered to be beyond the detection limit of the system and were not included in the analysis. Fold changes in the gene expression were determined by comparing the mean normalised gene expression levels between OLP and NSI tissues using DDCq method. The unpaired student t-test analysed the difference in the expression level of the tested genes between the groups. Genes with a statistical significance of ±2 and a p value < 0.05 were considered as significantly regulated.
RESULTS In the OLP lesions a diffuse T-cell (CD4+ and CD8+) infiltrate was seen at the epithelial-connective tissue interface and scattered within the epithelium (Fig. 2A,B) with only scattered CD4+ and CD8+ positivity in the deeper connective tissue. CD4+ and CD8+ cells were diffusely scattered through the connective tissue of the inflamed control tissues (Fig. 2E,F). In both OLP and NSI, FoxP3 staining was predominantly nuclear and FoxP3+ cell morphology was consistent with that of a lymphocyte (Fig. 2C,G). Within OLP lesions, FoxP3+ cells were evenly distributed in the inflammatory infiltrate at the epithelial-connective tissue interface and in the superficial connective tissue. Alternatively, IL17A staining was predominantly cytoplasmic, with a small amount of extracellular staining (Fig. 2D,H). The majority of the IL17A+ cells were located within and deep to the main band of inflammatory cells and very few were observed at the epithelial-connective tissue interface. Interestingly, all IL17A+ cells had an ovoid/plasmacytoid morphology (Fig. 3A,B).
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mean (±SD) of CD4+, CD8+, FoxP3+ and IL17A+ cells in the infiltrate at the epithelial-connective tissue interface was 65.32 ± 6.42, 56.62 ± 8.23, 20.64 ± 4.38 and 0.83 ± 0.65, respectively. There was no difference observed between the % mean (±SD) of CD4+ and CD8+ cells. There were significantly more FoxP3+ cells than IL17A+ cells (p < 0.0001). Similarly, 30 sites were examined at the deeper aspects of the superficial connective tissue infiltrate in the OLP specimens. All sites showed CD4+ (26.45 ± 4.42), CD8+ (24.23 ± 6.32), FoxP3+ (19.38 ± 6.32) cells, 27 sites showed a few IL17A+ cells (2.14 ± 1.57). There was no difference observed between the % mean (±SD) of CD4+ cells and CD8+ cells. Significantly more FoxP3+ cells were present in the superficial connective tissue infiltrate in OLP than IL17A+ cells (p < 0.0001). There were significantly more CD4+ and CD8+ cells in the epithelial-connective tissue interface region than the superficial connective tissue infiltrate region in OLP (p < 0.0001). While there was no difference in the mean percentage of FoxP3+ cells in the interface area compared to deeper within the inflammatory band (p = 0.494), significantly more IL17A+ cells were present in the deeper infiltrate than at the epithelial-connective tissue interface p = 0.044 (Fig. 5). Phenotypic characterisation of IL17A+ cells
Quantification of immune cells: CD4+, CD8+, FoxP3+ and IL17+ cells
All OLP specimens were evaluated for the presence of CD3+/ IL17A+ and IL17A+/tryptase+ cells in the inflammatory infiltrate. Double immunofluorescence labelling revealed that IL17A expression was restricted to tryptase+ mast cells (Fig. 6) and was not found in CD3+ T-cells (Fig. 7).
Oral lichen planus versus non-specific inflammation
Gene expression
Ten OLP cases were examined. All cases showed the presence of CD4+, CD8+, FoxP3+ and few IL17A+ cells. The % mean [± standard deviation (SD)] of CD4+ cells, CD8+ cells, FoxP3+ cells and IL17A+ cells of the total inflammatory cells in the infiltrate was 62.12 ± 10.43, 54.32 ± 8.43, 20.34 ± 4.33 and 1.18 ± 0.64, respectively. There was no significant difference in the % mean of CD4+ and CD8+ cells in OLP. However, there were significantly more FoxP3+ cells than IL17A+ cells in OLP (p < 0.0001). All the nine NSI cases showed CD4+, CD8+, FoxP3+ cells and few IL17A+ cells. The % mean ± SD of CD4+ and CD8+ cells of the total inflammatory cells in the infiltrate was 43.21 ± 6.43 and 32.84 ± 7.44, respectively. There was no significant difference observed between the % mean of the CD4+ and CD8+ cells. Similarly, comparison of % mean ± SD of FoxP3+ cells (5.52 ± 5.22) and IL17A+ cells (3.39 ± 2.69) in the infiltrate in the inflamed controls showed no significant difference. There were significantly more FoxP3+ cells in OLP lesions compared with NSI tissues (p < 0.0001); unlike the IL17A+ cells, which were significantly more common in the NSI tissues (p = 0.021) (Fig. 4).
Relative qRT2-PCR determination of FoxP3 and IL17A gene expression
Epithelial-connective tissue interface region versus superficial connective tissue infiltrate region of OLP A total of 30 sites (six sites/specimen) were examined at the epithelial-connective tissue interface of OLP (n = 10). All sites examined showed CD4+, CD8+, FoxP3+ cells. Twentyseven sites showed a small number of IL17A+ cells. The %
FoxP3 gene was significantly up-regulated in OLP compared with NSI, with a fold regulation of 6.42 (p = 0.001) (Fig. 8A). However, there was no difference in IL17A expression between the groups (Fig. 8B). Within the OLP tissues there was a higher expression of FoxP3 than IL17, which was statistically significant (p = 0.005) (Fig. 8C).
DISCUSSION The present study confirmed the presence of FoxP3+ Tregs and IL17A+ cells in the inflammatory infiltrate of archival OLP tissues. More FoxP3+ Tregs were present in OLP than in the control NSI tissues and FoxP3 genes were up-regulated. The band-like mononuclear cell infiltrate of OLP predominantly consists of macrophages and T-cells, specifically mature CD4+ and CD8+ cells.23–26 In line with this, the results of our study also demonstrated a greater number of CD4+ and CD8+ T cells in the epithelial-connective tissue interface region of OLP tissues. Furthermore, there was no difference observed between the percentage of CD4+ and CD8+ T-cells in any of the OLP lesions investigated. This result is in contrast with earlier studies,27,28 which showed a higher proportion of CD4+ cells than CD8+ cells in both hyperplastic and atrophic OLP lesions. These contrasting results can be attributed to the different clinical forms and stage of the disease. In the initial lesion of an OLP, CD4+ Tcells appear to be abundant in the connective tissue infiltrate,
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Results of the IHC studies. (A) CD4+ T cells in OLP, (B) CD8+ T cells in OLP, (C) FoxP3+ Tregs in OLP, (D) IL17A+ cells in OLP, (E) CD4+ T cells in NSI, (F) CD8+ T cells in NSI, (G) FoxP3+ Tregs in NSI, and (H) IL17A+ cells in NSI. Scale bar = 50 mm.
Fig. 2
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Fig. 3
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Large ovoid/plasmacytoid IL17A+ cells in the deep connective tissue inflammatory infiltrate in OLP tissue. Scale bar = 50 mm.
while in the advanced lesion, CD8+ T-cells predominate.25,26,29 In this context, it is quite possible that OLP tissues investigated in this study may be in a stage between the initial and advanced form. Tregs play a central role in inducing and maintaining immunological tolerance along with suppressing the immune
response, and the up-regulation of FoxP3 in OLP may indicate a greater attempt to regulate the immune response in this condition. There were fewer FoxP3+ cells in the NSI tissues, where an inflammatory response is also present. There have been reports demonstrating higher FoxP3 expression in OLP when compared to healthy uninflamed controls,10,12 but there
Distribution of %FoxP3+ and %IL17A+ cells between OLP tissue and NSl tissue groups. The mean %FoxP3+ cells was significantly higher in OLP tissues compared with NSI tissues (p < 0.0001). The mean %IL17A+ cells was significantly higher in NSI tissues compared with OLP (p = 0.02).
Fig. 4
Distribution of %FoxP3+ and %IL17A+ cells at the epithelial-connective interface and deeper in the superficial connective tissue infiltrate in OLP tissues. Comparison of mean %FoxP3+ cells between the regions was not significant, but significantly more IL17A+ cells were present in the deeper aspects of the infiltrate compared with epithelial connective tissue interface region (p = 0.04)
Fig. 5
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Fig. 6 Immunofluorescence double labelling of IL17A/tryptase in OLP tissue. Note that IL17A expression was localised within tryptase+ mast cells. Scale bar = 50 mm.
are no previous studies comparing their presence in OLP tissues with other inflammatory processes. The immune response that is observed in some inflammatory tissues may appear similar to OLP histologically; however, it may be of a different immune character. Therefore, it is crucial to identify the difference between both the groups, given the antigenic load and inflammatory infiltrate are not similar. The current study demonstrated FoxP3+ Tregs intra-epithelially, as well as in the infiltrate in the superficial connective tissue of OLP
tissues. The presence of FoxP3+ Tregs in proximity to the basal keratinocytes suggests a role in the pathogenesis of the disease. However, there was no significant difference in their expression at the epithelial-connective tissue interface and within the deeper levels of the infiltrate. Of late, IL17 has been reported to play a prime role in the pathogenesis of various chronic inflammatory lesions, and exploring its role in OLP is just beginning.11,16,19 In the current study a small number of IL17A+ cells were identified
IMMUNOREGULATION IN ORAL LICHEN PLANUS
Fig. 7
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Immunofluorescence double labelling of IL17A/CD3 in OLP tissue. Note that IL17A expression was not localised within CD3+ T-cells. Scale bar = 50 mm.
in OLP as well as in controls, with fewer IL17A+ cells in OLP when compared to the NSI tissues. The overall expression of IL17A+ cells in OLP was much lower in the current study than in previous reports.11,19 The reasons for these contrasting results cannot be entirely established. The majority of cases (7/10) in the current study were histologically classified as hyperplastic lichen planus (showing hyperkeratosis/parakeratosis and acanthosis along with the characteristic features
of OLP) with the remaining three cases showing evidence of epithelial atrophy, without ulceration. IL17 has been associated with increased tissue damage in OLP in atrophic lichen planus by comparison with the hyperplastic form,19 but this is unlikely to explain the overall low IL17A+ cell count. In the current study we evaluated the presence of IL17A+ cells in various regions of OLP; they were observed in greater density in the deeper regions of the infiltrate than at the
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Pathology (2016), 48(6), October
Fig. 8 Relative expression level of FoxP3 and IL17A in OLP tissues and NSI tissues. (A) FoxP3 levels were significantly higher in OLP tissues compared with NSI
tissues. (B) IL17A levels were no different between the groups. (C) In OLP, FoxP3 expression levels were significantly higher than IL17A. **p < 0.05.
epithelial-connective tissue interface. Morphologically these cells had an ovoid or plasmacytoid appearance with abundant cytoplasm. Double labelling immunofluorescence attributes IL17A positivity to mast cells rather T-cell clones. These results concur with previous work in our laboratory on periodontitis tissues and OLP where IL17+ cells were found to be mast cells.30,31 Taken together, these data establish the fact that these two T-cell subsets may play a role in the pathogenesis of OLP. Increased numbers of FoxP3+ cells and up-regulation of FoxP3+ genes in OLP demonstrates greater activity of Tregs in OLP lesions, potentially trying to modulate and suppress the chronicity of the lesion. It can be speculated that FoxP3+ Tregs might be attempting to control the immune response to self-antigen and prevent tissue destruction. They are thought to play a pivotal role in the central tolerance to self-antigens.32 The interplay between FoxP3+ cells and IL17A+ cells must be in a proper balance to ensure an efficient immune response rather than pathological damage.33 The increased number of FoxP3+ cells in OLP may reflect their significant role in the disease process. Fewer IL17A+ cells may be attributed to concomitant recruitment of Tregs over the prolonged course of this chronic condition, which may inhibit IL17-induced inflammation at lesional sites. In summary, this study demonstrates the presence of a greater number of FoxP3+ Tregs when compared to IL17A+ cells (Th17) and up-regulation of FoxP3 genes in the inflammatory infiltrate of OLP lesions. These results lead us to speculate that FoxP3+ cells have a more prominent role in
the pathogenesis of OLP when compared to IL17A+ cells. Future studies are required to further characterise these cells and understand their interplay in the pathophysiology of OLP. Conflicts of interest and sources of funding: This project was funded by a New Zealand Dental Association Research Foundation Grant (NZDA 7.07 2012). The authors state there are no conflicts of interest to disclose. Address for correspondence: Prof A. M. Rich, Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand. E-mail: [email protected]
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