J Oral Pathol Med (2016) 45: 28–34 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
doi: 10.1111/jop.12319
wileyonlinelibrary.com/journal/jop
Downregulation of toll-like receptor-mediated signalling pathways in oral lichen planus Suraya H. Sinon1, Alison M. Rich2, Venkata P. B. Parachuru2, Fiona A. Firth2, Trudy Milne2, Gregory J. Seymour2 1
Department of Oral Pathology and Oral Medicine, Faculty of Dentistry, Universiti Kebangsaan Malaysia (UKM), Kuala Lumpur, Malaysia; 2The Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand
OBJECTIVE: The objective of this study was to investigate the expression of Toll-like receptors (TLR) and TLRassociated signalling pathway genes in oral lichen planus (OLP). METHODS: Initially, immunohistochemistry was used to determine TLR expression in 12 formalin-fixed archival OLP tissues with 12 non-specifically inflamed oral tissues as controls. RNA was isolated from further fresh samples of OLP and non-specifically inflamed oral tissue controls (n = 6 for both groups) and used in qRT2-PCR focused arrays to determine the expression of TLRs and associated signalling pathway genes. Genes with a statistical significance of two-fold regulation (FR) and a P-value < 0.05 were considered as significantly regulated. RESULTS: Significantly more TLR4+ cells were present in the inflammatory infiltrate in OLP compared with the control tissues (P < 0.05). There was no statistically significant difference in the numbers of TLR2+ and TLR8+ cells between the groups. TLR3 was significantly downregulated in OLP (P < 0.01). TLR8 was upregulated in OLP, but the difference between the groups was not statistically significant. The TLR-mediated signallingassociated protein genes MyD88 and TIRAP were significantly downregulated (P < 0.01 and P < 0.05), as were IRAK1 (P < 0.05), MAPK8 (P < 0.01), MAP3K1 (P < 0.05), MAP4K4 (P < 0.05), REL (P < 0.01) and RELA (P < 0.01). Stress proteins HMGB1 and the heat shock protein D1 were significantly downregulated in OLP (P < 0.01). CONCLUSION: These findings suggest a downregulation of TLR-mediated signalling pathways in OLP lesions. J Oral Pathol Med (2016) 45: 28–34 Keywords: oral lichen planus; Toll-like receptors
Correspondence: Alison Rich, Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, P.O.Box 647, Dunedin 9054, New Zealand. Tel: +64 3 4795686, Fax: +64 3 4797046, E-mail: [email protected] Accepted for publication February 23, 2015
Introduction Oral lichen planus (OLP) is a relatively common condition with severe forms causing persistent mucosal erosions and ulceration. Its pathogenesis is unclear although an immunemediated response is generally agreed. The initiating stimulus is uncertain and, while a range of triggers have been proposed, including exogenous antigens such as viruses, for example hepatitis C virus (1), bacterial products, for example Helicobacter pylori (2), mechanical trauma, systemic drugs, chemical contact and endogenous antigens, for example heat shock proteins (HSP) expressed on basal keratinocytes (3), not all of these are proven. It is proposed that TLRs on antigen-presenting cells (APCs) recognise putative triggers and activate immune responses (4). Following this, signals from both activated APCs and basal keratinocytes lead to the attraction of activated T cells to the epithelial-connective tissue interface (5, 6) and increased production of Th1 cytokines, the end result of which is activation of the keratinocyte caspase cascade and basal keratinocyte apoptosis (6, 7). TLRs are critical innate immune response type 1 transmembrane proteins, which are expressed on various immune cells such as macrophages and dendritic cells and non-immune cells including skin and oral mucosal keratinocytes (8, 9). On these lining epithelia, TLRs act as sensors where they recognise pathogens and are activated when the epithelium is disturbed. They are pattern-recognition receptors whose principal role is to protect tissue by identifying pathogen-associated molecular patterns (PAMPs). Individual TLRs have unique capabilities to recognise PAMP ligands such as lipids and lipopeptides/lipopolysaccharides (TLR1,2,4,6), proteins (TLR5,11) or nucleic acids (TLR3,7,8,9) derived from a wide range of bacteria, viruses, parasites and fungi (10–12). It is increasingly apparent that TLRs also recognise damage/danger-associated molecular patterns (DAMPs), endogenous molecules released from damaged and dying cells. DAMPS include HSP70, fibrinogen, b-defensins and extracellular matrix degradation products that occur in injured or inflamed tissues (11, 13). Once activated, TLRs trigger coordinated expression of genes in specific signalling pathways related to the
Toll-like receptors in oral lichen planus Sinon et al.
regulation of innate and adaptive immunity and tissue repair and regeneration. Their cytoplasmic domain has extensive homology with the interleukin (IL)-1 receptor family and is known as the Toll-IL receptor (TIR) domain (14). With binding of ligand to TLRs, there is activation of signalling transduction pathways involving TIR with coupling to adaptor molecules including myeloid differentiation factor 88 (MyD88), TIR domain-containing protein (TIRAP) and TIR domain-containing adaptor inducing interferon-brelated adaptor molecule (TRAM). This potentially leads to the activation of two main pathways, the MyD88 dependent (used by all TLRs except TLR3) and the MyD88-independent TRAM/TRIF pathway (used by TLR3 and some signals of TLR4) (15). Signalling through the MyD88 pathway leads to the activation of and translocation from the cytoplasm to the nucleus of the transcription factor nuclear factor-jB (NFjB) and the transcription of inflammatory and anti-inflammatory cytokine genes, for example tumour necrosis factor (TNF)-a and IL-6 (16, 17). Activation of the TRAM/TRIF pathway leads to the production of type 1 interferons. In this manner, TLRs regulate the production of cytokines, opsonisation, coagulation cascades, complement activation and upregulation of costimulatory molecules on APCs (4, 18). A further important function of TLRs is the induction of apoptosis through the expression of anti-apoptotic proteins and apoptosis inhibitors (19). There is limited and conflicting evidence relating TLRs to the pathogenesis of OLP. Enhanced expression of TLR2 was found on peripheral blood monocytes and OLP lesional tissue (20). In contrast, reduced expression was reported in skin lichen planus (normal skin as control) and in lesional OLP (fibrous/fibroepithelial hyperplasia controls), respectively (21, 22). Reduced levels of soluble (s) TLR2 were found in the saliva of patients with OLP (23). An increased level of sTLR4 has been described in the saliva of OLP patients, while at the same time, significantly reduced TLR4 mRNA expression was found in epithelial cells of lesional OLP tissue (24). Again, other studies (22, 24, 25) have found TLR4 to be upregulated in OLP tissue in association with upregulation of NF-jb. At the same time, no difference was found in TLR4 expression levels on the subepithelial inflammatory cells in OLP compared with normal controls (26). Various TLRs are expressed on oral keratinocytes (9, 27) and on malignant keratinocytes in oral squamous cell carcinoma (28, 29); however, as noted above, their expression on epithelial cells in OLP is still controversial. The aims of the current study were (i) to determine the presence of TLR2+, TLR4+ and TLR8+ cells in OLP lesions using immunohistochemistry (IHC) and (ii) to investigate the expression of TLRs and associated signalling pathway genes in OLP using quantitative real-time polymerase chain reaction (qRT2-PCR)-focused array technology.
Immunohistochemistry
Methods
Sample selection
29
Sample selection and tissue preparation
Initially, a total of 24 formalin-fixed paraffin-embedded (FFPE) oral mucosal tissue specimens histologically diagnosed as hyperplastic OLP (n = 12) and non-specific inflammatory (NSI) control group (hyperplastic oral mucosa associated with mechanical trauma) (n = 12) were retrieved from the archives of Medlab Dental Oral Pathology Diagnostic Laboratory, Faculty of Dentistry, University of Otago. Information, such as age and gender of the patient and affected sites, was recorded (Table 1). Cases where the patients were using medications capable of causing lichenoid drug reactions were not included, and all samples were initially biopsied prior to diagnosis and management. The original haematoxylin and eosin-stained slides were reexamined under light microscopy to confirm the original diagnosis and to determine the suitability of the specimens for the study. Only cases with an inflammatory infiltrate in the superficial connective tissue were included in the control group. Human tonsil tissue was used as positive control for IHC antibodies against human TLR2, TLR4 and TLR8. Primary antibodies used were anti-human TLR2 mouse monoclonal TL2.1; IgG2a 0.5 lg/ml, anti-human TLR4 mouse monoclonal 76B357.1; IgG2B 1 lg/ml and antihuman TLR8 mouse monoclonal 44C143; and IgG 5 lg/ml (all from Abcam, Melbourne, Vic., Australia). Immunohistochemistry staining was conducted using an automated slide stainer BenchMark XT (Ventanaâ Medical Systems, Inc., Tucson, AZ, USA) which performed the deparaffinisation, cell conditioning, primary antibody incubation, labelled secondary antibody incubation and 3, 30 diaminobenzidine application using an ultraView Universal DAB Detection Kit (Ventanaâ Medical Systems) with washing between each step. Antibody titration (100 ll/ section) and the end-stage counterstaining were performed manually. All slides were manually mounted with DePeX (Merck, Darmstadt, Germany). The images were captured using Leica Firecam software (Version 1.5; Leica Microsystems, Heerbrugg, Switzerland). Cell analysis Each slide was viewed at up to 10009 magnification and representative areas of the specimen including the basement membrane zone and the immediately subjacent connective tissue containing the inflammatory cells of interest were photographed using QCapture (Q Imaging MicroPublisher 5.0 RTV, Surrey, BC, Canada) at 4009. A software package from MacBiophotonics, Image J 1.45 for microscopy (Mac OS X 64-bit version; imagej.en.softonic.com/), was used for digitally superimposing a graticule and quantitative cell counting. The number of positively stained inflammatory cells per area (~25 000 lm2) was recorded. Results were expressed as the total number of immunopositive mononuclear immune cells per area. Gene expression
This experimental protocol was approved by the University of Otago Institutional Ethics Committee.
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 J Oral Pathol Med
Toll-like receptors in oral lichen planus Sinon et al.
30
Table 1 Demographic details of the study participants Group
Mean age (years)
Formalin-fixed paraffin-embedded archival mucosal specimens Oral lichen planus (OLP) (n = 12) 56
Control (n = 12) Fresh mucosal tissue specimens OLP (n = 6) Control (n = 6)
Site (%)
3:2
Buccal mucosa (57.5) Tongue (27.5) Gingiva (7.5) Lower lip (2.5) FOM (2.5) Unspecified (2.5) Buccal mucosa (100)
47
4:1
63
3:2
53
3:1
their diagnostic workup were included. Patient clinical information was recorded (Table 1). The biopsy specimens were divided into two: the major being sent for histological diagnosis and the remainder immediately placed in a nuclease-free tube containing RNAlaterâ (Ambion, Inc., Austin, TX, USA) for downstream gene expression experiments. Cases were only included in the study if the histology confirmed hyperplastic OLP or NSI hyperplasia. Patients with medical conditions that might modify the immune response were excluded from the study. Total RNA isolation and cDNA synthesis Total RNA was isolated using a phenol–chloroform extraction technique with a silica-based spin column using Ambion PurelinkTM RNA Mini Kit with TRIzolâ Reagent (Applied Biosystems, Foster City, CA, USA). Genomic DNA was removed using on-column PureLinkTM Dnase treatment (Ambion). The purified total RNA quality was assessed spectrometrically using the NanoVue PlusTM (GE Healthcare, Little Chalfont, UK). Total RNA (1 lg) was reverse-transcribed into cDNA using the RT2 First Strand Kit (SA Biosciences, Frederick, MD, USA) according to the manufacturers’ protocol. Quantitative real-time polymerase chain reaction A 96-well Human Toll-like Receptor Signalling PCR Array (SA Biosciences) was used for simultaneous quantitative analysis of 84 genes of interest (GOI). The majority of GOI on the plate were TLR signalling genes, but within the 84 genes, there were also chemokines, cytokines and other genes not directly related to these pathways. Briefly, for each plate, an experimental cocktail was prepared, which included 1350 ll 29 SA Biosciences RT2 SYBR Green/ROX qPCR Master Mix, 102 ll cDNA sample (500 ng) and 1248 ll H2O. 25 ll of the experimental cocktail was transferred to each well of the PCR array plate using an 8-channel pipettor. The ABI 7500 Real Fast PCR instrument (Applied Biosystems) was utilised for thermal cycling and detection. The standard PCR cycling parameters were 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. The geNorm Visual Basic application applet for Microsoft Excel was used to determine the most stable reference genes from a panel of five reference genes using methods previously described (30). The three most stable reference genes (HPRT1, RPL13A and B2M) were selected for qRT2PCR normalisation. J Oral Pathol Med
Gender ratio (F:M)
Buccal mucosa (50) Tongue (50) Buccal mucosa (100)
Statistical analysis Data entry and descriptive analysis were performed using GraphPad Prism Software version 5 (GraphPad Software, Inc., La Jolla, CA, USA). The unpaired Student’s t-test was used to analyse the difference in the proportion of TLR2+, TLR4+ and TLR8+ cells between the groups. A statistical significance risk rate was set at P < 0.05. Analysis of the qRT2-PCR data was conducted using raw Cq values of the tested genes normalised against the mean Cq of the selected reference genes. Data were analysed using SA BiosciencesTM PCR Array Data Analysis Web Portal. All Cq values ≥35 were considered as beyond the detection limit of the system and were not included in the analysis. Fold regulation in gene expression was determined by comparison with the mean normalised gene expression levels between OLP and NSI tissues using DDCq method. The data analysis PCR array template used the unpaired Student’s ttest to analyse the difference in the expression level of the 84 genes between the groups. Genes with a statistical significance of two-FR and a P-value < 0.05 were considered as significantly regulated.
Results Immunohistochemistry TLR 2, TLR4 and TLR8 expression
Light to moderately intense cytoplasmic staining of TLR2 and TLR4 was observed on keratinocytes in the basal and lower aspects of the prickle cell layer in both the OLP cases and the controls (Fig. 1A,B,D,E). With TLR2, there was cytoplasmic staining of macrophages and lymphocytes in the subepithelial inflammatory infiltrate with intense staining of larger cells, morphologically consistent with mast cells, in the deeper connective tissue (Fig. 1A). Occasional inflammatory cells in the control group were TLR2+ and endothelial cells showed faint positivity, similar to those in OLP (Fig. 1D). There was no significant difference in the number of TLR2+ cells between the two groups (Table 2). TLR4+ cells were scanty in the connective tissue of the control group, with more obvious TLR4+ cells in the OLP group (Fig. 2B), in a similar distribution as described for TLR2. There were significantly more TLR4+ cells in the inflammatory infiltrate in OLP compared with the control tissues (4.5 vs. 2.9 cells per area, P = 0.036). There was no statistically significant difference in TLR8+ cells between OLP and controls (Table 2); these cells were similarly
Toll-like receptors in oral lichen planus Sinon et al.
A
D
B
E
C
F
31
Figure 1 Immunolocalisation of TLR2, TLR4 and TLR8 in oral lichen planus (OLP) and non-specific inflammatory (NSI) control tissue. A–C represents OLP tissues and D–F represents NSI control tissues. TLR2 and 4 immunostaining showed light cytoplasmic staining of cells in the lower aspect of the epithelium and of cells in the connective tissue. In contrast, TLR8 was not expressed on either the OLP or NSI epithelium. TLR8+ cells were more frequent than TLR2+ and TLR4+ cells in OLP. Scale bar 50 lm.
distributed, although more frequent, compared with TLR2 and TLR4 (Fig. 1C,F). Relative qRT2-PCR determination of gene expression
qRT² qPCR analysis showed that the 84 GOI (i.e. all genes on the plate) 29 genes (34.5%) were differentially expressed (FR >2) in the OLP group compared with the NSI control group Table 2 Comparison of TLR2+, TLR4+ and TLR8+ cells in the stroma of OLP and inflammatory control tissues
Group
TLR2 Mean (SD)
TLR4 Mean (SD)
TLR8 Mean (SD)
OLP Control
3.5 (2.2) 3.9 (5.1)
4.5* (3.3) 2.9* (2.8)
14.6 (11.3) 16.3 (23)
SD, standard deviation; TLR, Toll-like receptors; OLP, oral lichen planus. All results presented as mean number of +ve cells/area *P < 0.05.
(Fig. 2). Six genes [Bruton’s agammaglobulinemia tyrosine kinase (BTK), CD80, chemokine ligand 10 (CXCL10), interferon gamma (IFNG), lymphotoxin alpha (LTA) and lymphocyte antigen 86 (LY86)] were upregulated (>twofold), and 23 downregulated. Of the 23 genes downregulated >twofold, 12 (high-mobility group box 1 (HMGB1), HSP 60 kDa 1a (HSPD1), interleukin-1 receptor-associated kinase 1 (IRAK1), mitogen-activated protein kinase kinase kinase 1 (MAP3K1), mitogen-activated protein kinase 8 (MAPK8), mitogen-activated protein kinase 4 (MAP4K4), MYD88, peroxisome proliferator-activated receptor alpha (PPARA), V-rel reticuloendotheliosis viral oncogene homolog A (RELA), V-rel reticuloendotheliosis viral oncogene homolog (REL), TIRAP and TLR3) were statistically significantly downregulated (FR: >2; P < 0.05). None of the >2-FR upregulated genes were statistically significantly expressed. The array data also revealed a downregulation of TLR2 (FR: 3.02) and TLR4 (FR: 1.34), but this did not reach J Oral Pathol Med
Toll-like receptors in oral lichen planus Sinon et al.
32
Figure 2 Volcano plot presenting the fold change in Toll-like receptors (TLR) signalling pathway genes comparing the two study groups [oral lichen planus (OLP) vs. control]. The vertical axis refers to P-value for t-test differences between the groups and horizontal axis shows expression of log2 fold regulation, with +2 indicating twofold upregulation and 2 indicating twofold downregulation. The volcano plot splays to the left, indicating the downregulation of TLR signalling pathway genes in OLP.
statistical significance. The overall pattern of the ‘volcano plot’ splayed towards the left, indicating a downregulation of TLR signalling pathways in OLP (Fig. 2). The fold change and P-values of the 26 differentially expressed genes directly related to the TLR signalling pathway (i.e. TLR pathway-specific genes) are shown in Fig. 3. In OLP, 12 genes (TIRAP, MYD88, IRAK1, MAPK8, MAP3K1, REL, RELA, HMGB1, HSPD1, PPARA, MAP4K4 and TLR3) were downregulated with FR >2 and with P-value < 0.05; four genes [TLR5, sterile
alpha and TIR motif containing 1 (SARM1), IRAK2 and pellino homolog 1 (PELI1)] were downregulated with FR >2 and with P-values ranging between 0.05 and 0.08. Nine genes (mitogen-activated protein kinase kinase kinase 7 interacting protein 1 (MAP3K7IP1), mitogen-activated protein kinase kinase kinase 1 (MAP3K7), mitogen-activated protein kinase kinase 3 (MAP2K3), protein kinase interferon-inducible double-stranded RNA-dependent activator (PRKRA), ubiquitin-conjugating enzyme E2N (UBE2N), mitogen-activated protein kinase kinase 4 (MAP2K4), mitogen-activated protein kinase 8 interacting protein 3 (MAPK8IP3), heat shock 70 kDa protein 1A (HSPA1A) and ubiquitin-conjugating enzyme E2 variant 1 (UBE2V1) were downregulated with a FR 2 and P-value < 0.05. Yellow arrowhead represents the genes downregulated with FR >2 and P-values ranging between 0.05 and 0.08. Light blue arrowhead represents the genes downregulated with FR
doi: 10.1111/jop.12319
wileyonlinelibrary.com/journal/jop
Downregulation of toll-like receptor-mediated signalling pathways in oral lichen planus Suraya H. Sinon1, Alison M. Rich2, Venkata P. B. Parachuru2, Fiona A. Firth2, Trudy Milne2, Gregory J. Seymour2 1
Department of Oral Pathology and Oral Medicine, Faculty of Dentistry, Universiti Kebangsaan Malaysia (UKM), Kuala Lumpur, Malaysia; 2The Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand
OBJECTIVE: The objective of this study was to investigate the expression of Toll-like receptors (TLR) and TLRassociated signalling pathway genes in oral lichen planus (OLP). METHODS: Initially, immunohistochemistry was used to determine TLR expression in 12 formalin-fixed archival OLP tissues with 12 non-specifically inflamed oral tissues as controls. RNA was isolated from further fresh samples of OLP and non-specifically inflamed oral tissue controls (n = 6 for both groups) and used in qRT2-PCR focused arrays to determine the expression of TLRs and associated signalling pathway genes. Genes with a statistical significance of two-fold regulation (FR) and a P-value < 0.05 were considered as significantly regulated. RESULTS: Significantly more TLR4+ cells were present in the inflammatory infiltrate in OLP compared with the control tissues (P < 0.05). There was no statistically significant difference in the numbers of TLR2+ and TLR8+ cells between the groups. TLR3 was significantly downregulated in OLP (P < 0.01). TLR8 was upregulated in OLP, but the difference between the groups was not statistically significant. The TLR-mediated signallingassociated protein genes MyD88 and TIRAP were significantly downregulated (P < 0.01 and P < 0.05), as were IRAK1 (P < 0.05), MAPK8 (P < 0.01), MAP3K1 (P < 0.05), MAP4K4 (P < 0.05), REL (P < 0.01) and RELA (P < 0.01). Stress proteins HMGB1 and the heat shock protein D1 were significantly downregulated in OLP (P < 0.01). CONCLUSION: These findings suggest a downregulation of TLR-mediated signalling pathways in OLP lesions. J Oral Pathol Med (2016) 45: 28–34 Keywords: oral lichen planus; Toll-like receptors
Correspondence: Alison Rich, Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, P.O.Box 647, Dunedin 9054, New Zealand. Tel: +64 3 4795686, Fax: +64 3 4797046, E-mail: [email protected] Accepted for publication February 23, 2015
Introduction Oral lichen planus (OLP) is a relatively common condition with severe forms causing persistent mucosal erosions and ulceration. Its pathogenesis is unclear although an immunemediated response is generally agreed. The initiating stimulus is uncertain and, while a range of triggers have been proposed, including exogenous antigens such as viruses, for example hepatitis C virus (1), bacterial products, for example Helicobacter pylori (2), mechanical trauma, systemic drugs, chemical contact and endogenous antigens, for example heat shock proteins (HSP) expressed on basal keratinocytes (3), not all of these are proven. It is proposed that TLRs on antigen-presenting cells (APCs) recognise putative triggers and activate immune responses (4). Following this, signals from both activated APCs and basal keratinocytes lead to the attraction of activated T cells to the epithelial-connective tissue interface (5, 6) and increased production of Th1 cytokines, the end result of which is activation of the keratinocyte caspase cascade and basal keratinocyte apoptosis (6, 7). TLRs are critical innate immune response type 1 transmembrane proteins, which are expressed on various immune cells such as macrophages and dendritic cells and non-immune cells including skin and oral mucosal keratinocytes (8, 9). On these lining epithelia, TLRs act as sensors where they recognise pathogens and are activated when the epithelium is disturbed. They are pattern-recognition receptors whose principal role is to protect tissue by identifying pathogen-associated molecular patterns (PAMPs). Individual TLRs have unique capabilities to recognise PAMP ligands such as lipids and lipopeptides/lipopolysaccharides (TLR1,2,4,6), proteins (TLR5,11) or nucleic acids (TLR3,7,8,9) derived from a wide range of bacteria, viruses, parasites and fungi (10–12). It is increasingly apparent that TLRs also recognise damage/danger-associated molecular patterns (DAMPs), endogenous molecules released from damaged and dying cells. DAMPS include HSP70, fibrinogen, b-defensins and extracellular matrix degradation products that occur in injured or inflamed tissues (11, 13). Once activated, TLRs trigger coordinated expression of genes in specific signalling pathways related to the
Toll-like receptors in oral lichen planus Sinon et al.
regulation of innate and adaptive immunity and tissue repair and regeneration. Their cytoplasmic domain has extensive homology with the interleukin (IL)-1 receptor family and is known as the Toll-IL receptor (TIR) domain (14). With binding of ligand to TLRs, there is activation of signalling transduction pathways involving TIR with coupling to adaptor molecules including myeloid differentiation factor 88 (MyD88), TIR domain-containing protein (TIRAP) and TIR domain-containing adaptor inducing interferon-brelated adaptor molecule (TRAM). This potentially leads to the activation of two main pathways, the MyD88 dependent (used by all TLRs except TLR3) and the MyD88-independent TRAM/TRIF pathway (used by TLR3 and some signals of TLR4) (15). Signalling through the MyD88 pathway leads to the activation of and translocation from the cytoplasm to the nucleus of the transcription factor nuclear factor-jB (NFjB) and the transcription of inflammatory and anti-inflammatory cytokine genes, for example tumour necrosis factor (TNF)-a and IL-6 (16, 17). Activation of the TRAM/TRIF pathway leads to the production of type 1 interferons. In this manner, TLRs regulate the production of cytokines, opsonisation, coagulation cascades, complement activation and upregulation of costimulatory molecules on APCs (4, 18). A further important function of TLRs is the induction of apoptosis through the expression of anti-apoptotic proteins and apoptosis inhibitors (19). There is limited and conflicting evidence relating TLRs to the pathogenesis of OLP. Enhanced expression of TLR2 was found on peripheral blood monocytes and OLP lesional tissue (20). In contrast, reduced expression was reported in skin lichen planus (normal skin as control) and in lesional OLP (fibrous/fibroepithelial hyperplasia controls), respectively (21, 22). Reduced levels of soluble (s) TLR2 were found in the saliva of patients with OLP (23). An increased level of sTLR4 has been described in the saliva of OLP patients, while at the same time, significantly reduced TLR4 mRNA expression was found in epithelial cells of lesional OLP tissue (24). Again, other studies (22, 24, 25) have found TLR4 to be upregulated in OLP tissue in association with upregulation of NF-jb. At the same time, no difference was found in TLR4 expression levels on the subepithelial inflammatory cells in OLP compared with normal controls (26). Various TLRs are expressed on oral keratinocytes (9, 27) and on malignant keratinocytes in oral squamous cell carcinoma (28, 29); however, as noted above, their expression on epithelial cells in OLP is still controversial. The aims of the current study were (i) to determine the presence of TLR2+, TLR4+ and TLR8+ cells in OLP lesions using immunohistochemistry (IHC) and (ii) to investigate the expression of TLRs and associated signalling pathway genes in OLP using quantitative real-time polymerase chain reaction (qRT2-PCR)-focused array technology.
Immunohistochemistry
Methods
Sample selection
29
Sample selection and tissue preparation
Initially, a total of 24 formalin-fixed paraffin-embedded (FFPE) oral mucosal tissue specimens histologically diagnosed as hyperplastic OLP (n = 12) and non-specific inflammatory (NSI) control group (hyperplastic oral mucosa associated with mechanical trauma) (n = 12) were retrieved from the archives of Medlab Dental Oral Pathology Diagnostic Laboratory, Faculty of Dentistry, University of Otago. Information, such as age and gender of the patient and affected sites, was recorded (Table 1). Cases where the patients were using medications capable of causing lichenoid drug reactions were not included, and all samples were initially biopsied prior to diagnosis and management. The original haematoxylin and eosin-stained slides were reexamined under light microscopy to confirm the original diagnosis and to determine the suitability of the specimens for the study. Only cases with an inflammatory infiltrate in the superficial connective tissue were included in the control group. Human tonsil tissue was used as positive control for IHC antibodies against human TLR2, TLR4 and TLR8. Primary antibodies used were anti-human TLR2 mouse monoclonal TL2.1; IgG2a 0.5 lg/ml, anti-human TLR4 mouse monoclonal 76B357.1; IgG2B 1 lg/ml and antihuman TLR8 mouse monoclonal 44C143; and IgG 5 lg/ml (all from Abcam, Melbourne, Vic., Australia). Immunohistochemistry staining was conducted using an automated slide stainer BenchMark XT (Ventanaâ Medical Systems, Inc., Tucson, AZ, USA) which performed the deparaffinisation, cell conditioning, primary antibody incubation, labelled secondary antibody incubation and 3, 30 diaminobenzidine application using an ultraView Universal DAB Detection Kit (Ventanaâ Medical Systems) with washing between each step. Antibody titration (100 ll/ section) and the end-stage counterstaining were performed manually. All slides were manually mounted with DePeX (Merck, Darmstadt, Germany). The images were captured using Leica Firecam software (Version 1.5; Leica Microsystems, Heerbrugg, Switzerland). Cell analysis Each slide was viewed at up to 10009 magnification and representative areas of the specimen including the basement membrane zone and the immediately subjacent connective tissue containing the inflammatory cells of interest were photographed using QCapture (Q Imaging MicroPublisher 5.0 RTV, Surrey, BC, Canada) at 4009. A software package from MacBiophotonics, Image J 1.45 for microscopy (Mac OS X 64-bit version; imagej.en.softonic.com/), was used for digitally superimposing a graticule and quantitative cell counting. The number of positively stained inflammatory cells per area (~25 000 lm2) was recorded. Results were expressed as the total number of immunopositive mononuclear immune cells per area. Gene expression
This experimental protocol was approved by the University of Otago Institutional Ethics Committee.
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 J Oral Pathol Med
Toll-like receptors in oral lichen planus Sinon et al.
30
Table 1 Demographic details of the study participants Group
Mean age (years)
Formalin-fixed paraffin-embedded archival mucosal specimens Oral lichen planus (OLP) (n = 12) 56
Control (n = 12) Fresh mucosal tissue specimens OLP (n = 6) Control (n = 6)
Site (%)
3:2
Buccal mucosa (57.5) Tongue (27.5) Gingiva (7.5) Lower lip (2.5) FOM (2.5) Unspecified (2.5) Buccal mucosa (100)
47
4:1
63
3:2
53
3:1
their diagnostic workup were included. Patient clinical information was recorded (Table 1). The biopsy specimens were divided into two: the major being sent for histological diagnosis and the remainder immediately placed in a nuclease-free tube containing RNAlaterâ (Ambion, Inc., Austin, TX, USA) for downstream gene expression experiments. Cases were only included in the study if the histology confirmed hyperplastic OLP or NSI hyperplasia. Patients with medical conditions that might modify the immune response were excluded from the study. Total RNA isolation and cDNA synthesis Total RNA was isolated using a phenol–chloroform extraction technique with a silica-based spin column using Ambion PurelinkTM RNA Mini Kit with TRIzolâ Reagent (Applied Biosystems, Foster City, CA, USA). Genomic DNA was removed using on-column PureLinkTM Dnase treatment (Ambion). The purified total RNA quality was assessed spectrometrically using the NanoVue PlusTM (GE Healthcare, Little Chalfont, UK). Total RNA (1 lg) was reverse-transcribed into cDNA using the RT2 First Strand Kit (SA Biosciences, Frederick, MD, USA) according to the manufacturers’ protocol. Quantitative real-time polymerase chain reaction A 96-well Human Toll-like Receptor Signalling PCR Array (SA Biosciences) was used for simultaneous quantitative analysis of 84 genes of interest (GOI). The majority of GOI on the plate were TLR signalling genes, but within the 84 genes, there were also chemokines, cytokines and other genes not directly related to these pathways. Briefly, for each plate, an experimental cocktail was prepared, which included 1350 ll 29 SA Biosciences RT2 SYBR Green/ROX qPCR Master Mix, 102 ll cDNA sample (500 ng) and 1248 ll H2O. 25 ll of the experimental cocktail was transferred to each well of the PCR array plate using an 8-channel pipettor. The ABI 7500 Real Fast PCR instrument (Applied Biosystems) was utilised for thermal cycling and detection. The standard PCR cycling parameters were 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. The geNorm Visual Basic application applet for Microsoft Excel was used to determine the most stable reference genes from a panel of five reference genes using methods previously described (30). The three most stable reference genes (HPRT1, RPL13A and B2M) were selected for qRT2PCR normalisation. J Oral Pathol Med
Gender ratio (F:M)
Buccal mucosa (50) Tongue (50) Buccal mucosa (100)
Statistical analysis Data entry and descriptive analysis were performed using GraphPad Prism Software version 5 (GraphPad Software, Inc., La Jolla, CA, USA). The unpaired Student’s t-test was used to analyse the difference in the proportion of TLR2+, TLR4+ and TLR8+ cells between the groups. A statistical significance risk rate was set at P < 0.05. Analysis of the qRT2-PCR data was conducted using raw Cq values of the tested genes normalised against the mean Cq of the selected reference genes. Data were analysed using SA BiosciencesTM PCR Array Data Analysis Web Portal. All Cq values ≥35 were considered as beyond the detection limit of the system and were not included in the analysis. Fold regulation in gene expression was determined by comparison with the mean normalised gene expression levels between OLP and NSI tissues using DDCq method. The data analysis PCR array template used the unpaired Student’s ttest to analyse the difference in the expression level of the 84 genes between the groups. Genes with a statistical significance of two-FR and a P-value < 0.05 were considered as significantly regulated.
Results Immunohistochemistry TLR 2, TLR4 and TLR8 expression
Light to moderately intense cytoplasmic staining of TLR2 and TLR4 was observed on keratinocytes in the basal and lower aspects of the prickle cell layer in both the OLP cases and the controls (Fig. 1A,B,D,E). With TLR2, there was cytoplasmic staining of macrophages and lymphocytes in the subepithelial inflammatory infiltrate with intense staining of larger cells, morphologically consistent with mast cells, in the deeper connective tissue (Fig. 1A). Occasional inflammatory cells in the control group were TLR2+ and endothelial cells showed faint positivity, similar to those in OLP (Fig. 1D). There was no significant difference in the number of TLR2+ cells between the two groups (Table 2). TLR4+ cells were scanty in the connective tissue of the control group, with more obvious TLR4+ cells in the OLP group (Fig. 2B), in a similar distribution as described for TLR2. There were significantly more TLR4+ cells in the inflammatory infiltrate in OLP compared with the control tissues (4.5 vs. 2.9 cells per area, P = 0.036). There was no statistically significant difference in TLR8+ cells between OLP and controls (Table 2); these cells were similarly
Toll-like receptors in oral lichen planus Sinon et al.
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D
B
E
C
F
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Figure 1 Immunolocalisation of TLR2, TLR4 and TLR8 in oral lichen planus (OLP) and non-specific inflammatory (NSI) control tissue. A–C represents OLP tissues and D–F represents NSI control tissues. TLR2 and 4 immunostaining showed light cytoplasmic staining of cells in the lower aspect of the epithelium and of cells in the connective tissue. In contrast, TLR8 was not expressed on either the OLP or NSI epithelium. TLR8+ cells were more frequent than TLR2+ and TLR4+ cells in OLP. Scale bar 50 lm.
distributed, although more frequent, compared with TLR2 and TLR4 (Fig. 1C,F). Relative qRT2-PCR determination of gene expression
qRT² qPCR analysis showed that the 84 GOI (i.e. all genes on the plate) 29 genes (34.5%) were differentially expressed (FR >2) in the OLP group compared with the NSI control group Table 2 Comparison of TLR2+, TLR4+ and TLR8+ cells in the stroma of OLP and inflammatory control tissues
Group
TLR2 Mean (SD)
TLR4 Mean (SD)
TLR8 Mean (SD)
OLP Control
3.5 (2.2) 3.9 (5.1)
4.5* (3.3) 2.9* (2.8)
14.6 (11.3) 16.3 (23)
SD, standard deviation; TLR, Toll-like receptors; OLP, oral lichen planus. All results presented as mean number of +ve cells/area *P < 0.05.
(Fig. 2). Six genes [Bruton’s agammaglobulinemia tyrosine kinase (BTK), CD80, chemokine ligand 10 (CXCL10), interferon gamma (IFNG), lymphotoxin alpha (LTA) and lymphocyte antigen 86 (LY86)] were upregulated (>twofold), and 23 downregulated. Of the 23 genes downregulated >twofold, 12 (high-mobility group box 1 (HMGB1), HSP 60 kDa 1a (HSPD1), interleukin-1 receptor-associated kinase 1 (IRAK1), mitogen-activated protein kinase kinase kinase 1 (MAP3K1), mitogen-activated protein kinase 8 (MAPK8), mitogen-activated protein kinase 4 (MAP4K4), MYD88, peroxisome proliferator-activated receptor alpha (PPARA), V-rel reticuloendotheliosis viral oncogene homolog A (RELA), V-rel reticuloendotheliosis viral oncogene homolog (REL), TIRAP and TLR3) were statistically significantly downregulated (FR: >2; P < 0.05). None of the >2-FR upregulated genes were statistically significantly expressed. The array data also revealed a downregulation of TLR2 (FR: 3.02) and TLR4 (FR: 1.34), but this did not reach J Oral Pathol Med
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Figure 2 Volcano plot presenting the fold change in Toll-like receptors (TLR) signalling pathway genes comparing the two study groups [oral lichen planus (OLP) vs. control]. The vertical axis refers to P-value for t-test differences between the groups and horizontal axis shows expression of log2 fold regulation, with +2 indicating twofold upregulation and 2 indicating twofold downregulation. The volcano plot splays to the left, indicating the downregulation of TLR signalling pathway genes in OLP.
statistical significance. The overall pattern of the ‘volcano plot’ splayed towards the left, indicating a downregulation of TLR signalling pathways in OLP (Fig. 2). The fold change and P-values of the 26 differentially expressed genes directly related to the TLR signalling pathway (i.e. TLR pathway-specific genes) are shown in Fig. 3. In OLP, 12 genes (TIRAP, MYD88, IRAK1, MAPK8, MAP3K1, REL, RELA, HMGB1, HSPD1, PPARA, MAP4K4 and TLR3) were downregulated with FR >2 and with P-value < 0.05; four genes [TLR5, sterile
alpha and TIR motif containing 1 (SARM1), IRAK2 and pellino homolog 1 (PELI1)] were downregulated with FR >2 and with P-values ranging between 0.05 and 0.08. Nine genes (mitogen-activated protein kinase kinase kinase 7 interacting protein 1 (MAP3K7IP1), mitogen-activated protein kinase kinase kinase 1 (MAP3K7), mitogen-activated protein kinase kinase 3 (MAP2K3), protein kinase interferon-inducible double-stranded RNA-dependent activator (PRKRA), ubiquitin-conjugating enzyme E2N (UBE2N), mitogen-activated protein kinase kinase 4 (MAP2K4), mitogen-activated protein kinase 8 interacting protein 3 (MAPK8IP3), heat shock 70 kDa protein 1A (HSPA1A) and ubiquitin-conjugating enzyme E2 variant 1 (UBE2V1) were downregulated with a FR 2 and P-value < 0.05. Yellow arrowhead represents the genes downregulated with FR >2 and P-values ranging between 0.05 and 0.08. Light blue arrowhead represents the genes downregulated with FR
