Differential expression of TLR-2 and TLR-4 in the epithelial cells in ...

archives of oral biology 57 (2012) 495–502

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Differential expression of TLR-2 and TLR-4 in the epithelial cells in oral lichen planus Srihari B. Janardhanam, Sivaraman Prakasam, Venkatesh T. Swaminathan, Krithika N. Kodumudi, Susan L. Zunt, Mythily Srinivasan * Department of Oral Pathology, Medicine and Radiology, Indiana University School of Dentistry, 1121 W. Michigan St., Indianapolis, IN 46202-5186, United States

article info

abstract

Article history:

Objective: Oral lichen planus (OLP) is a chronic inflammatory condition of the mucosa

Accepted 17 October 2011

mediated by a complex signalling network between the keratinocytes and the sub-epithelial lymphocytes. Since OLP occurs in constantly renewing epithelium continuously exposed to

Keywords:

commensals, we hypothesised that the epithelial cell microflora interactions may mediate

Toll-like receptor

the persistent inflammation. By virtue of their ability to respond to most oral commensal

Oral lichen planus

microorganisms, the toll like receptor-2 (TLR-2) and TLR-4 are the most widely investigated

Keratinocytes

receptors in oral diseases. The overall objective of this study was to investigate the role of TLR-2 and TLR-4 in OLP.

Saliva

Design: Systemically healthy OLP and control subjects were recruited after obtaining the institutional review board approval. Expression of TLR-2 and TLR-4 proteins and transcripts in the tissue epithelium and in the epithelial cells isolated from saliva were determined by immunohistochemistry and quantitative real-time polymerase chain reaction respectively. Results: The tissue epithelium and the salivary epithelial cells expressed reduced TLR-2 and increased TLR-4 proteins and transcripts in OLP. The salivary epithelial cells from OLP subjects secreted elevated IL-12. However, upon stimulation with bacterial lipopolysaccharide the epithelial cells from OLP exhibited a mixed Th1 (IL-12) and Th2 (IL-4) response. Presence of dexamethasone significantly reduced inflammatory cytokines in the in vitro stimulated cultures of salivary epithelial cells from OLP subjects. Conclusion: Collectively, our data support a critical role for the host–microbial interactions in the OLP pathogenesis. The potential use of exfoliated oral epithelial cells in saliva for functional analysis exponentially increases its value as biological specimen for clinical research. # 2011 Elsevier Ltd. All rights reserved.

1.

Introduction

Lichen planus (LP) is a common mucocutaneous disease affecting 2–4% of the population worldwide. Oral mucosal involvement occurs frequently presenting varied clinical appearances ranging from reticular/keratotic to erosive/

ulcerative lesions.1,2 Pathogenetically LP is considered as an immune mediated response, wherein the cytokines secreted by activated keratinocytes promote transendothelial migration of mononuclear cells and activation of autoreactive T cells precipitating the chronic inflammatory pathology.1,3 Although the initiating antigen in oral lichen planus (OLP) remains to be established, the physiological process of epithelial turnover

* Corresponding author. Tel.: +1 317 278 9686; fax: +1 317 278 3018. E-mail address: [email protected] (M. Srinivasan). 0003–9969/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2011.10.013

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and the plethora of commensal flora constitute a rich source of antigens capable of stimulating oral keratinocytes.3,4 Amongst the oral microorganisms, the Candida albicans, the Helicobacter pylori and the Hepatitis C virus have been more frequently associated with OLP.5–10 Additionally, a relative preponderance of Gram negative bacteria has been reported at OLP sites.11,12 Furthermore circulating mononuclear cells have been shown to exhibit reduced cell mediated response to specific microbial products in OLP.6,13 These observations suggest that the host recognition and response to the commensal or pathogenic microbe may play a critical role in the pathogenesis of OLP. Under normal conditions, the healthy mucosa is protected by tightly regulated responses mediated through an array of pattern recognition receptors (PRRs) that constantly survey their surroundings for potential threats and invasion. Toll like receptors (TLRs) are the best characterised PRRs that recognise distinct microbe/pathogen associated molecular patterns (MAMPs/PAMPs) typically shared by a large group of microorganisms. To date 11 TLRs and many of their ligands are known.14–17 In addition to PAMPs, TLRs also bind certain endogenous molecules such as heat shock proteins.18 Oral epithelial cells have been shown to express most members of the TLR family.16,19 Differential expression of TLRs in epithelial cells has been reported in many oral mucosal diseases including aphthous ulcers, OLP, squamous cell carcinoma and candidiasis.4,20–22 The TLR mediated signalling plays a significant role in the induction of adaptive immune responses.23 Whilst signalling via TLR-4 promotes Th1 responses, TLR-2 stimulation has been shown to mediate development of Th2 responses.24 In this study we evaluated the epithelial cell expression of TLR-2 and TLR-4 in OLP and explored their functional role in the disease pathogenesis. Unstimulated whole saliva (UWS) is a complex mixture composed of secretions from three major salivary glands and numerous minor salivary glands contaminated with serum constituents, blood corpuscles, exfoliated oral epithelial cells and a plethora of microorganisms. We observed that the tissue epithelium and the epithelial cells isolated from the UWS expressed reduced TLR-2 and increased TLR-4 proteins and transcripts in OLP. Furthermore, cultures of epithelial cells from the OLP UWS exhibited increased IL-12 secretion. Interestingly, upon stimulation with lipopolysaccharides (LPS), the epithelial cell cultures from the OLP UWS exhibited a mixed pattern of elevated Th1 (IL-12) and Th2 (IL-4) secretion.

2.

Materials and methods

2.1.

Study population

The study group consisted of 25 OLP subjects exhibiting characteristic mucosal lesions with radiating Wickham’s striae. Concomitant mucosal erosion was observed in 10 subjects.25 All participants were non-smokers, had no known systemic illness and had healthy periodontium with a minimum of 20 teeth. The control group consisted of age and sex matched subjects with no known systemic or oral disease. All OLP and control subjects were from the Indiana University School of Dentistry Oral Medicine Clinic. Informed

consent was obtained from all subjects in accordance with the Indiana University’s institutional review board.

2.2.

Saliva collection and culture of oral epithelial cells

UWS was collected by the drooling method for 10 min into a 15 ml chilled centrifuge tube as described.26 Saliva flow rate was calculated by the volumetric (ml/min), the gravimetric method (assuming the density of saliva being 1.0 g/ml) and the modified Schirmer test (MST), a strip test to measure UWS flow rate.26 The samples were transported immediately on ice to the laboratory, with a maximum lag phase of 15 min. Each sample was then diluted 1:10 in isotonic diluent (Hematronix, Inc., CA, USA) and 2 drops of Zap-o-Globin lytic reagent (Beckman Coulter, USA) and then centrifuged at 1200–1400 rpm for 10 min. The cell pellet was washed with sterile PBS, resuspended in Hanks’ balanced salt solution (HBSS) (Life Technologies, MI), and passed over a 20 mm sterile nylon membrane (Small Parts Inc., Miami Lakes, FL). The epithelial cell-enriched population was cultured in a 96well flat bottom plate at 1 104 cells/well in 200 ml culture medium containing 3% keratinocyte growth factor supplemented with HEPES 15 mmol/L-glutamine 2 mmol/L, penicillin 100 U/ml, streptomycin 100 mg/ml, amphotericin B 500 mg/ml and b-mercaptoethanol 50 mmol/L in an atmosphere of 5% CO2 at 37 8C. Some cultures were stimulated with 10 mg/ml of Escherichia coli LPS in the presence or absence of 100 mg/ml of dexamethasone. The supernatant and cells collected at 3 h, 6 h, 12 h and 24 h were stored at 80 8C until further analysis.

2.3.

Immunohistochemistry

Tissues exhibiting histological features of fibroma or fibrous/ fibroepithelial hyperplasia/papilloma of the buccal mucosa, lateral surface of the tongue or gingiva with adequate normal oral epithelium and tissues with histological diagnosis consistent with OLP, 10 from each group, were immunostained for TLR-2 and TLR-4 as described.27 Serial 5 mm tissue sections were sequentially incubated briefly in PBS: 3% H2O2 and 3% horse serum albumin to block endogenous peroxidase activity and non specific immunoglobulin binding respectively. The tissue sections were then incubated overnight at 4 8C with mouse monoclonal anti-human TLR-2 (clone: 1030A5.138, Imgenex, San Diego, CA) and mouse monoclonal anti-human TLR-4 (clone: 76B357.1, Imgenex) diluted 1:1000 as the primary antibodies. The sections were then washed and serially incubated with diluted biotinylated secondary antibody solution (Vector Laboratories, Burlingame, CA) and Vectastain elite ABC following manufacturer’s recommendation. The enzyme activity was detected with the chromogen 3-3 diaminobenzidine deposition (DAB, Vector Laboratories, Burlingame, CA). The sections were counterstained with haematoxylin. Negative control consisted of sections incubated with PBS instead of primary antibody. Positive control included cutaneous epithelium, previously shown to express TLR-2 and TLR-4 proteins by immunohistochemistry.

2.4.

Laser capture micro-dissection

The epithelial cells from buccal mucosal tissues exhibiting histological features of OLP or fibroma, ten of each diagnosis,

497


were selectively dissected by using PixCell II Laser capture micro-dissection system (Arcturus Engineering, Mountain View, CA, USA).23

2.5.

Real time polymerase chain reaction (RT-PCR)

Total cellular RNA was isolated from tissue/salivary epithelial cells using Qiagen RNA isolation kit (Invitrogen, Carlsbad, CA) as recommended. 2–4 mg of total RNA was reverse transcribed using iScript cDNA synthesis kit (Biorad, CA). The concentration of the cDNA was measured at 260 nm and 280 nm by the Gensys5 model UV–visible spectrophotometer (Thermoelectronic Corp., CA). Real-time PCR was performed by using the SYBR green/ROX qPCR master mix (SABiosciences, Frederick, MD) according to manufacturer’s recommendations on the ABI Prism 7000 Sequence Detection System (Perkin Elmer Applied Systems, Foster City, CA, USA). Each reaction contains 2 12.5 ml of SYBR green Master Mix, 1 ml of 10 mM of primers, 50 ng of the cDNA, to a total volume of 25 ml. The thermal cycling conditions included an initial denaturation step at 50 8C for 2 min, 95 8C for 10 min; 40 cycles at 95 8C for 15 s, primer specific annealing temperature for 30 s and extension at 72 8C for 30 s. Message for small proline rich protein (SPRR 2a), a gene abundantly expressed in stratified squamous epithelia was amplified as an internal control.24,25 The primers include; CD14F: 50 -CGTGCGCGACAGGGCGTTCT-30 , CD14R: 50 TAAAGG TGGGGCAAAGGGTT-30 ; TLR-2F: 50 -GGCCAGCAAATTACCTGTGT-30 ; TLR-2R: 50 -TTCT CCACCCAGTAGGCATC-30 and TLR-4F: 50 -CCACGTAGGTCTTGGTGTTCA-30 ; TLR-4R: 50 TCCCACTCCAGGTAACGTGTT-30 and SPRR2aF: 50 -AGTGCCAGCAGAAATATCCTCC-30 , SPRR2aR: 50 -GAACGAGGTGAGCCAAATATCC-30 .28,29 Melt curve analysis was performed to confirm that the detected signal was that of SYBR green binding to the expected amplification product and not to the possible primerdimers. The housekeeping gene GAPDH was amplified as the PCR control. CD14, TLR-2 and TLR-4 mRNA from human monocytic cell lines (Thp-1) were amplified as positive controls. Amplification was performed in duplicates. The magnitude of change in the mRNA normalised to that of SPRR was expressed by the method of 2DDCt with untreated controls as the reference samples.

2.6. Enzyme linked immunosorbent assay (ELISA) for cytokines The levels of cytokines interleukin-8 (IL-8), IL-12, IL-4, IL-10 and interferon-g (IFN-g) in culture supernatants were measured using BD OptEIATM ELISA kits. The amount of cytokine was determined with reference to the absorbance of standard of known concentration of each cytokine.

2.7.

Statistical analysis

The differences in the mRNA levels of CD14, TLR-2 and TLR-4 between the groups were determined by Student’s t-test. p values less than 0.05 were considered significant.

3.

Results

3.1.

Clinical characteristics

The study cohort included 25 OLP and 25 control subjects. Burning sensation of the mouth and dry mouth were the chief complaints of the OLP subjects. The average age of the OLP cohort was 61.5 7 years and that of the control group was 49.4 7.8 years. There was a female sex predilection in the OLP cohort with a female to male ratio of 2.5–1. Consistent with previous reports the volume of UWS as determined by rate of flow was reduced in the OLP cohort as compared with the control group.30 The gravimetric and MST assessment also exhibited a trend towards reduced salivary flow rate in the OLP cohort. Qualitatively the UWS was slightly acidic (pH < 7) in both groups (Table 1).25

3.2.

Expression of TLR-2 and TLR-4 in OLP

Variations in the expressions of TLR-2 and TLR-4 have been reported in the cutaneous and mucosal epithelium exhibiting chronic inflammatory conditions such as psoriasis and inflammatory bowel disease respectively.18,31 Initially we investigated the expression of TLR-2 and TLR-4 proteins in OLP tissues. Histologically OLP tissues exhibited features of epithelial atrophy, basement membrane zone degeneration and presence of a dense band of subepithelial inflammatory cell infiltration (Fig. 1A and B).13 Immunohistochemistry showed that the TLR-2 and TLR-4 proteins were expressed predominantly in the lower spinous cell layers in normal oral epithelium (Fig. 1C–F). Whilst the TLR-2 and CD14 expressions were equivalent in the OLP and normal epithelium, the TLR-4 expression was significantly upregulated in the OLP epithelium (Fig. 1G and H). The epithelial cells isolated by laser capture microdissection were assessed for the expression of TLR-2 and TLR-4 mRNA with respect to SPRR2a, a gene linked to keratinocyte terminal differentiation and abundantly expressed in stratified epithelium. Whilst the expression of CD14 mRNA was equivalent, the TLR-2 mRNA was significantly lower and that of the TLR-4 mRNA was significantly higher in the epithelial cells in OLP as compared with that in the normal oral epithelium (Fig. 2A and B).

Table 1 – OLP: clinical and demographic features. MST, modified Schirmer test.

Normal OLP

Age

M:F

Volume (ml/min)

Gravimetric (g/ml)

MST

pH

49.4 7 61.5 7.8

9:16 7:18

2.53 0.76 1.14 0.97

0.48 0.22 0.18 0.19

24.7 11.8 9.93 13.5

6.4 0.66 6.08 0.47

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Fig. 1 – Photomicrographs of immunostained tissues of oral lichen planus: (A) negative control; control mucosa for TLR-2 (B) and TLR-4 (D); OLP tissues for TLR-2 (C) and TLR-4 (E).

3.3. UWS

TLR-2 and TLR-4 expression in the epithelial cells in

Cell exfoliation is a characteristic feature of epithelia covering the skin and the mucosa covering the internal cavities.32 The concentration of epithelial cells in the UWS has been shown to vary between 9.94 103 and 4.3 105 cells/ml.33,34 We investigated the expression of TLR-2 and TLR-4 mRNA in the epithelial isolated from the UWS of OLP and control subjects by quantitative RT-PCR. Equivalent number of epithelial cells was present in the UWS of control and OLP subjects with the percent viability ranging between 21.8 and 33.5 (Fig. 3A). Much like the expression in the tissue epithelium, the epithelial cells in the UWS of OLP subjects expressed significantly higher TLR4 mRNA and reduced TLR-2 mRNA as compared with the epithelial cells from the saliva of healthy subjects (Fig. 3D).

3.4. Epithelial cells from the saliva of OLP subjects are functional Under normal conditions oral micro-organisms adhere to various surfaces in the mouth for survival and multiplication.33,35 Based on salivary flow rate and the microbial division time, it has been suggested that the total number of epithelial cell adherent bacteria may be over twofold higher than the free bacteria in saliva.33 Following exfoliation, whilst some epithelial cells undergo detachment induced apoptosis/anoikis, others maintain structural integrity and viability for extended periods ranging from minutes to hours.32 We investigated the response of the epithelial cells isolated from the UWS following in vitro culture. Whilst the concentration of IL-12 was significantly higher, that of IL-6 was lower in

cultures of epithelial cells from OLP UWS as compared with the cultures of cells from control UWS (Fig. 4A and B). Stimulation with LPS significantly elevated IL-12 and IL-4 secretion by the OLP UWS epithelial cells as compared with that by the cells from control UWS (Fig. 4A). The concentrations of IL-8 and TNF-a did not differ significantly between the LPS stimulated or unstimulated cultures of the epithelial cells from either OLP UWS or control UWS. Furthermore, treatment with dexamethasone significantly decreased secretions of IL12, IL-6, and IL-8 and elevated the IL-4 concentration in the LPS stimulated cultures of epithelial cells from the OLP UWS as compared with similarly treated cultures of cells from control UWS (Fig. 4A). Importantly, the increase in IL-4 and the decrease in IL-6 and IL-8 in the dexamethasone treated cultures of in vitro stimulated epithelial cells from the OLP UWS were significant when compared to the secretions from unstimulated epithelial cell cultures (Fig. 4A). We next investigated the effect of LPS stimulation of the UWS epithelial cells on the expression of TLR-2 and TLR-4 transcripts. Quantitative RT-PCR showed that the epithelial cells from the OLP UWS exhibited significantly higher TLR-4 mRNA in response to LPS stimulation as compared with the cells from control UWS (Fig. 4B). The relative expression of TLR-2 mRNA was equivalent in the LPS stimulated epithelial cells from the OLP and control groups (Fig. 4B). This is consistent with the expression observed in the epithelial cells derived from OLP lesions, although the two cohorts were not the same. Previously oral keratinocytes stimulated with IFN-g have been shown to upregulate TLR-2 and TLR-4 expression.17 Stimulation of gingival epithelial cells with oral microbes/ microbial ligands has been shown to differentially regulate TLR-2 and TLR-4 expression.36


Fig. 2 – TLR-4 transcripts are upregulated in the epithelial cells in OLP. Pure populations of epithelial cells were obtained from histologically normal buccal mucosa or tissues that exhibit features of OLP by laser capture microdissection. Total RNA isolated from the epithelial cells was reverse transcribed. Equal amount of cDNA was amplified for SPRR2a, CD14, TLR-2 and TLR-4 by quantitative RT-PCR using SYBR green master mix. (A) Gel electrophoresis of the PCR products. Relative quantity was estimated with respect to that of SPRR2a. (B) Fold change of CD14, TLR-2 and TLR-4 was determined by the 2SDDCt method. Each experiment was repeated three times. Data are represented as mean W SEM. *p < 0.05 when compared to control group.

4.

Discussion

LP is a disease of the stratified squamous epithelia in constant contact with the normal commensal flora.1,4 Elevated counts predominantly of Gram negative bacteria including Capnocytophaga sputigena, Eikenella corrodens, Lactobacillus crispatus, Mobiluncus curtisii, Neisseria mucosa, Prevotella bivia and Prevotella intermedia have been observed at OLP sites.11,12 The TLR mediated responses of the keratinocytes have been shown to play a critical role in mediating the chronic inflammation in other epithelial pathologies such as colitis and psoriasis.18,31 Our results suggest that the TLR-2 and TLR-4 mediated responses in the epithelial cells potentially contribute to the pathogenesis of OLP. TLR-2 and TLR-4 expressions have been reported in the basal and spinous cell layers of the normal gingival epithelium by immunohistochemistry.27 Recently Ohno et al. reported

499

Fig. 3 – Epithelial cells in the OLP UWS express elevated TLR-4 transcripts. (A) The percentage of viable epithelial cells isolated from the UWS of OLP and control subjects as counted by the Countess (Invitrogen). (B) The relative quantity and fold change in TLR-2 and TLR-4 transcripts were determined as described in Fig. 2. Data are represented as mean W SEM. *p < 0.05 when compared to control group.

moderate elevation in TLR-2 expression and significantly reduced TLR-4 expression in the epithelium of OLP lesions.4 This is in contrast to our observation of reduced TLR-2 and significantly elevated TLR-4 expression in the epithelial cells in OLP. The discrepancy may be attributed to the differences in the antibodies and the methodology used. In their study Ohno et al. reported the ratio of TLR expression in OLP tissues with respect to the expression in normal tissues from different individuals.4 In this study we measured the expressions of TLR-2 and TLR-4 transcripts expression with respect to that of epithelial specific SPRR2a, in both normal and OLP cohorts. The observed increase in the epithelial expression of TLR-4 potentially supports the higher counts of Gram negative bacteria at sites of OLP lesions.12 The histological feature of reduced epithelium in OLP has been largely attributed to the enhanced apoptosis of the keratinocytes mediated by the infiltrating CD8+ T cells.1,3 A higher turnover supported by increased proliferation of the basal and spinous cell layers of the epithelium has been suggested as a mechanism to accommodate the increased apoptosis of the keratinocytes in OLP.37 Cheng et al. reported equivalent proportions of live and apoptotic epithelial cells in the OLP and normal saliva.38 Our observations concur with their report. Interestingly, much like the epithelial cells derived from the OLP tissues, the epithelial cells derived from the OLP UWS exhibited significantly reduced TLR-4 and mildly elevated TLR-2 expression compared with that from normal

500


A Cells Only

Cells+LPS

Cells+LPS+Dex

B

pg/ml

IL-6

IL-8

IL-12

TNF-α

IL-4

Normal

65.88+/-3.9

43.3+/-13.5

81.6+/-4.0

17.5+/-1.1

16.91+/-2.2

OLP

52.52+/-4.0

49.4+/-12.4

141.7+/-9.4

17.1+/-1.3

17.45+/-.07

p-value

0.0264

0.3758

0.00149

0.82334

0.78766

Normal

62.68+/-4.6

53.5+/-15.2

168+/-33

17.8+/-0.7

13.52+/-1.1

OLP

52.28+/-4.1

51.5+/-13.4

283.7+/-44

16.4+/-0.7

26.2+/-8.1

p-value

0.1067

0.4627

0.03873

0.20114

0.0214

Normal

25.32+/-3.4

11.65+/-1

54.7+/-3.3

15.32+/-0.93

35.3+/-1.4

OLP

47+/-2.7

23.15+/-4.3

52.03+/-2.5

18+/-0.55

40.12+/-0.7

p-value

0.000007

0.0436

0.52153

0.02035

0.0251

Relative expression

3 2.5

Normal OLP

2 1.5 1 0.5 0 Cells Only

LPS

Cells Only

TLR-2

LPS

TLR-4

Fig. 4 – Epithelial cells in saliva secrete cytokines and upregulate TLR-4 in response to LPS: epithelial cells (1 T 105/well) isolated from the UWS were cultured in the presence or absence of LPS (10 mg/ml) and dexamethasone (100 mg/ml). Supernatants and cells were collected 24 h later for cytokine and genetic analyses. The experiments were repeated three times. (A) ELISA of the indicated cytokines and the p value as determined by Student’s t-test. (B) The relative quantity and the fold change in TLR-2 and TLR-4 transcripts were determined as described in Fig. 2. Data are represented as mean W SEM. *p < 0.05 when compared to control group.

individuals. It is tempting to speculate that the epithelial cells isolated from saliva could indeed represent effective samples of the affected tissues. The keratinocytes and the infiltrating mononuclear cells express an array of cytokine and cytokine receptors that through paracrine and autocrine processes could potentially perpetuate the inflammatory pathology in OLP.1,3 The increased secretion of IL-12 by the OLP UWS epithelial cells suggests a potential source of contribution to the higher concentration of inflammatory mediators in the saliva of OLP patients. Interestingly, the epithelial cells from the saliva of OLP subjects exhibited increased IL-12 and IL-4 secretion following LPS stimulation suggesting a mixed Th1 and Th2 response. Significantly, the response of the epithelial cells was countered by treatment with dexamethasone, a commonly used steroid in the management of OLP. Previously Yamamoto et al. have shown that the keratinocytes derived from the OLP tissues cultured in hydrocortisone enriched medium secrete elevated level of IL-4, TNF-a, IL-6, and IL-1 b in response to LPS.39 Although the clinical use of exfoliated epithelial cells has long been recognised, most applications are restricted to genetic/DNA analysis.40 Recently epithelial cells isolated from urine have been shown to be viable and functional in primary cultures exhibiting increased renal glucose transporter ex-

pression and activity associated with diabetes.41 To our knowledge this is the first study to report the functional analysis of the epithelial cells derived from the saliva of OLP patients. The similarities in the expression profiles of TLR-2 and TLR-4 in the epithelial cells in OLP tissues and in the LPS stimulated epithelial cells derived from saliva of OLP patients together with the differential Th1/Th2 response supports a critical role for the host–microbial interactions in the pathological process in OLP. The potential use of exfoliated oral epithelial cells in saliva for functional and molecular analyses exponentially increases its value as biological specimen for clinical research and diagnostics.

Funding None.

Ethical approval Saliva samples were collected with a protocol approved by the Indiana University Purdue University at Indianapolis Institutional Review Board.


Conflict of interest statement None declared.

Competing interests None declared.

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