EVALUATION OF THE POSSIBLE ROLE OF IL-17 AND ITS RECEPTOR IL-17R IN THE PATHOGENESIS OF ORAL LICHEN PLANUS

LP is a relatively common chronic

inflammatory

muco-cutaneous disease of probable immune based

etiology, and involves oral and genital mucosal

surfaces, skin, scalp and nails. OLP presents oral

mucosal lesions such as white striations, papules,

plaques, erythema, blisters or ulcers.

 

(1) LP is

characterized by a T cell mediated immune response

against epithelial cells, causing epithelial cell

damage and subepithelial persistent accumulation

of T lymphocytes. The mechanism involved in this

chronic inflammatory disease remains unclear.

 

(2-4)

Cell mediated immunity in OLP may be

regulated by various cytokines and their receptors,

 

(5)

so identification of the specificity of T helper

cells(Th) is one of the most important steps to reveal

pathogenesis and etiology of OLP.

* Lecturer at Department of Oral Medicine, Periodontology and Diagnosis, Faculty of Oral and Dental Medicine,

Cairo University.

** Prof. at Department of Medical Biochemistry, Faculty of Medicine, Cairo University.

Al-Azhar Journal of Dental Science

Vol. 19 - No. 1 - January 2016

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Amal A. Hussine, et al. A.J.D.S. Vol. 19, No. 1

Although cytotoic CD8+T lymphocytes are the

majority of intraepithelial lymphocytes and subepithelial

lymphocytes in OLP, most lymphocytes

in the lamina propria are identified as CD4+ T cells.

(5, 6)

 

CD4+ helper cells have been suggested to play

an important role in cytotoxic CD8+T cell activation

via T helper cell related cytokine release.

 

(6)

It is well accepted that two CD4+ helper T cells

subsets namely Th1 and Th2 are associated with

immunopathogenesis of OLP. Th1 and Th2 cells

are identified by their signature cytokines interferon

gamma (IFN γ) and IL-4 respectively.

 

(5, 7)

Recently, Th 17 cells (IL-17 producing CD4 T

cells) have been discovered as a unique subset of

T helper cells that develop along with a pathway

that distinct from the Th1 and Th2 differentiation

pathways. Th17 cells are characterized by the

production of potent pro-inflammatory cytokines

IL-17, IL-17 f, IL-21, IL-22 and IL-26, which

suggests that these cells function as pleiotropic

pro-inflammatory T helper cells.

 

(8, 9) IL-17 (also

referred as IL-17A) is the signature cytokine of T

helper 17 cells, which are considered a key T cell

subset involved in the etiology of autoimmune

and inflammatory disorders such as Lupus

Erythematosis, Multiple Sclerosis, Rheumaroid

Arthritis and tumor microenvironments.

 

(10, 11, 12)

The human IL-17A gene is a protein of 150

amino acids with a molecular weight of 15 KDa and

is secreted as disulfide linked homodimer of 30-35

KDa glyco-proteins.

 

(13)

There were five related cytokines identified (B,

C, D, E, F), that share 20- 50% homology to IL-

17A. IL-17A has been designated A to indicate that

it is the essential member of the IL-17 cytokines

family.

 

(14)

IL-17 has been shown to signal through the IL-

17R molecule and promoting production of tumor

necrosis factor alpha (TNF-α), IL-1ß IL-6, IL-8 and

granulocyte colony stimulating factor (G-CSF).

 

(15-17)

Thus, IL-17A functions as a pro-inflammatory

cytokine which can activate different cells such

as epithelial, endothelial, fibroblast, chondrocyte

and osteoblast producing numerous inflammatory

molecules including cytokines, chemokines,

defensins and MMPs.

 

(14, 18)

The receptor for IL-17A (IL-17R) is a single

pass-transmembrane protein of approximately 130

KDa, while the IL-17A cytokine expressed only

by T cells, its receptor is expressed in all tissues

examined. The activation of the receptor by IL-

17A generally results in the induction of other

proinflammatory cytokines through activation of

NK-Kß.

 

(19)

Although it is accepted that OLP is a localized

disease, an increasing number of studies indicate

that many significant changes in peripheral blood

are implicated in the pathogenesis.

 

(20, 21, 22)Several

previous investigations focused on the alteration

of T lymphocyte subsets in the peripheral blood of

OLP lesions.

 

(22)

Some studies found the presence of IL-17 in

the local lesion of OLP, suggesting its role in local

environments.

 

(23) Other studies detected the serum

and saliva levels of IL-17 in OLP patients, but

found no significant difference compared to healthy

groups.

 

(24)

The aim of this study was to investigate possible

correlation between serum and tissue levels ofIL-

17 as well as the expression of its tissue receptor

in order to detect their possible role in the etiopathogenesisof

OLP.

SUBJECTS AND METHODS

I. Study Population

The entire study sample comprised 30

individuals;

 

20 oral lichen planus (OLP) patients

were selected, in addition, 10 age and sex matched

healthy normal volunteer individuals free from any

systemic diseasewere recruited as control subjects.

A.J.D.S. Vol. 19, No. 1

EVALUATION OF THE POSSIBLE ROLE OF IL-17 AND ITS RECEPTOR 3

II. Inclusion and exclusion criteria

All subjects participated in this study were

selected from the Outpatient Clinic, Department of

Oral Medicine, Oral Diagnosis and Periodontology,

Faculty of Oral and Dental Medicine, Cairo

University, between February 2014 and December

2014.

A detailed medical history of each subject was

obtained according to the detailed questionnaire of

the modified Cornell Medical Index.

 

(20) All subjects

were free from any systemic disease and did not

receive any medication either topical or systemic

that could cause lichenoid reaction during the 3

months prior to the specimen collection. Moreover,

patients with suspected restoration-related reaction

were excluded from this study. All patients had oral

symptoms, and every case was clinically diagnosed

as erosive lichen planus (ELP) or atrophic lichen

planus(ALP). Duration of the disease ranged

from 2 to 3 months with periods of remission and

exacerbation. The lesions were bilaterally selected

and extended to involve the buccal mucosa,

labial mucosa, and tongue which varied from

one patient to another. Diagnosis was confirmed

by histopathologic examination according to the

World Health Organization’s (WHO’s) clinicopathological

diagnostic criteria for LP.

 

(21)

III. Ethical procedures:

All subjects were informed about the detailed

procedure and they were given written approval

consent to sign. Patients were treated after the samples

had been collected. The study was performed

between February 2014 and December 2014.

The thirty selected participants were divided into

two groups as follow:-

Group A:

It included 20 patients suffering from OLP,

3 males and 12 females. Their ages ranged from

36-48 years.

Group B:

It included 10 medically free subjects as controls,

4 males and 11 females. Their ages ranged

from 35-42 years.

VI. Collection of samples

i. Serum sample collection

Peripheral venous blood samples (5 ml) were

obtained by standardvenipuncture from subjects

using plain tubes. Samples were centrifuged. The

clarifying supernatant was filtered and stored at −20

°C until assayed.

Serum IL-17 level was determined using a

special kit (Human IL-17, ELISA Crokit ko13207p,

South Korea) with ELISA assay according to

manufacturer’s instructions.

ii. Tissue samples.

20 OLP lesion specimens were biopsied from selected

patients, and 10 normal oral mucosa (NOM)

tissues were collected from healthy volunteers receiving

ortho-gnathic surgery.

IL-17protein in tissue levels were assessed using

same ELISA kit.

Real-time RT-PCR:

Detection of tissue IL-17 receptor gene

expression by Quantitative real time polymerase

chain reaction (qRT-PCR).

Total RNA was extracted from frozen tissue

samples using the RN easy Mini Kit (Qiagen Inc)

following the manufacturer’s protocol, extracted

RNA was quantified by spectrophotometry. The

RNA integrity was assessed using agarose gel

electrophoresis and ethidium bromide staining.

2 μg of total RNA were reverse transcribed in

0.05 M Tris–HCl pH 8.3, 40 mM KCl, 7 mM MgCl2

buffer containing 0.05 μg of random hexamers,

1 mM dNTPs mix, 0.05 U/l RNase inhibitor and

200 U/l murineleukemia virus reverse transcriptase

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Amal A. Hussine, et al. A.J.D.S. Vol. 19, No. 1

M-MLV. Samples were incubated for 10 min at

70˚C and then 60 min at 37 C. Inactivation of the

reverse transcriptase was achieved by heating the

samples at 95C for 10 min.

Real-time RT-PCR for quantitative assessment

of mRNA expression was performed on step

one plus (Applied Biosystems, USA), reaction

contained SYBR Green Master Mix (Applied

Biosystems), gene-specific forward and reverse

primers (10 μM), cDNA and nuclease-free water.

The sequences of PCR primer pairs used for each

gene are shown in Table 1. With cycling conditions

(10 min at 95°C followed by 40 cycles of 15 s at

95°C and 60 s at 60°C). The level of expression

of each target gene was normalized relative to

the expression of GAPDH mRNA in that sample

using the ΔCt method. Relative differences in gene

expression among groups were determined using the

comparative Ct (ΔΔCt) method and fold expression

was calculated as 2

 

−ΔΔCt, where ΔΔCt represents

ΔCt values normalized relative to the mean ΔCt of

control samples (R).

TABLE (1)

Primer sequences used for RT-PCR(25)

Primer Sequence

IL-17

 

Forward primer :5′-GCTCCAGAAGGCCC TCAGACT-3′

Reverse primer : 5′-CCAGCTTTCCCTCCGCATTGA-3′

IL-17 R A Forward: 5′-AGACACTCCAGAACCAATTCC-3′,

Reverse: 5′-TCTTAGAGTTGCTCTCCACCA-3′,

GAPDH

Forward: 5`-AGA GAT ATC CCT CTG TG ATC-3`

Reverse: 5`-TAC CCC AAA GTT ATC TCA GG-3`′

V. Statistical Analysis:

Quantitative data were presented as mean,

median, standard deviation (SD), range (Minimum

– Maximum) and 95% Confidence interval (95%

CI) for the mean values. Data were explored for

normality by checking the data distribution and

using Kolmogorov-Smirnov and Shapiro-Wilk tests.

IL-17 levels data showed parametric distribution.

Student’s t-test was used to compare between the

two groups. Pearson’s correlation coefficient was

used to determine the correlation between IL-17

levels in serum, tissue and tissue receptor.

ROC (Receiver Operating Characteristic) curve

was constructed to determine the cut-off values of

IL-17 for detection of OLP. Areas under the ROC

curve (AUCs), sensitivity, specificity, predictive

values and diagnostic accuracy was calculated.

The significance level was set at P ≤ 0.05. Sta

 

tistical

analysis was performed with IBM

 

® SPSS®

Statistics Version 20 for Windows.

Comparison between the two groups

In serum, tissues as well as tissue receptor, study

group showed statistically significantly higher mean

IL-17 level than control group ( Table 2, figure 1)).

Correlation between serum and receptor IL-17

levels

There was a statistically significant positive

(direct) correlation between serum and receptor

IL-17 levels (

 

r = 0.940, P-value <0.001) i.e. an

increase in serum level of IL-17 is associated with

an increase in receptor level of IL-17 (Fig. 2).

Correlation between serum and tissue IL-17 levels

There was a statistically significant positive

(direct) correlation between serum and tissue IL-17

levels (

 

r = 0.862, P-value <0.001) i.e. an increase in

serum level of IL-17 is associated with an increase

in tissue level of IL-17 (Fig. 3).

Correlation between receptor and tissue IL-17

levels

There was a statistically significant positive

(direct) correlation between receptor and tissue

IL-17 levels (

 

r = 0.828, P-value <0.001) i.e. an

increase in receptor level of IL-17 is associated with

an increase in tissue level of IL-17 (Fig.4).

®

 

IBM Corporation, NY, USA. ®SPSS, Inc., an IBM Company.

A.J.D.S. Vol. 19, No. 1

EVALUATION OF THE POSSIBLE ROLE OF IL-17 AND ITS RECEPTOR 5

TABLE (2)

Descriptive statistics and results of Student’s t-test for comparison between IL-17 serum, tissue

levels as well as IL-17 tissue receptor levels in the two groups

Group Mean SD Median Minimum Maximum

95% CI

Lower

 

P-value

bound

Upper

bound

Serum

Study

113.3 21.5 107.0 67.4 162.8 103.2 123.4

<0.001*

Control

25.5 6.7 24.1 18.2 40.6 20.8 30.3

Receptor

Study

9.6 2.8 10.1 4.9 13.7 8.3 10.9

<0.001*

Control

1.1 0.1 1.0 0.9 1.3 1.0 1.2

Tissue

Study

142.6 26.5 142.7 97.3 201.3 130.2 155.0

<0.001*

Control

43.4 8.3 41.7 33.4 61.2 37.5 49.4

*: Significant at P ≤ 0.05

FIG (1) Bar chart representing mean and standard deviation

values of IL-17 levels in the two groups

FIG (3) Scatter diagram representing positive correlation between

serum and tissue levels of IL-17

FIG (2) Scatter diagram representing positive correlation between

serum and receptor levels of IL-17

FIG (4) Scatter diagram representing positive correlation between

receptor and tissue levels of IL-17

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Amal A. Hussine, et al. A.J.D.S. Vol. 19, No. 1

ROC curve analysis (Cut-off values)

ROC curve analysis of serum, receptor and tissue

levels of IL-17 in the present study showed cut-off

values of 40.6, 1.3 and 61.2 pg/ml, respectively.

DISCUSSION

The pathogenesis of OLP still remains poorly

understood although it is characterized by a T cell

mediated immune response against epithelial cells

causing epithelial cell damage and subepithelial

infiltration of T lymphocytes.

 

(26, 27)Interleukin-17

family consists of cytokines that are secreted by Th-

17 cells and have an important role in the regulation

of mucous and epithelial immune responses.

 

(28)

There is increasing evidence that IL-17 is

a critical pro-inflammatory cytokine in human

autoimmune diseases, and it has previously been

demonstrated to stimulate the expression of

TNF-α and IL-6 in several cell types including

keratinocytes, fibroblasts, endothelial cells and

macrophages, as well as inducing the production

of several chemokines, such as CCL-20 and

CXCL-8

 

(8). High levels of IL-17 and Th17 cells

can provoke an inflammatory response and are

correlated with autoimmune diseases such as

inflammatory bowel disease, rheumatoid arthritis,

multiple sclerosis and psoriasis.

 

(29,30)

TABLE (3)

Sensitivity, specificity, predictive values, diagnostic accuracy, Area under the ROC curve

(AUC), 95% confidence interval (95% CI) of serum and tissue IL-17 levels

IL-17 (pg/ml) Cut-off Sensitivity %

 

Specificity % +PV% -PV% Diagnostic

accuracy % AUC 95% CI

Serum

40.6 100.0 100.0 100.0 100.0 100.0 1.000 0.884 – 1.000

Receptor

1.3 100.0 100.0 100.0 100.0 100.0 1.000 0.884 – 1.000

Tissue

61.2 100.0 100.0 100.0 100.0 100.0 1.000 0.884 – 1.000

+PV: Positive Predictive Value, -PV: Negative Predictive Value

At these cut-off values, the diagnostic accuracy

of IL-17 as a marker for detecting OLP is 100.0%.

Results of ROC curve analysis are presented in

Table (3)

A previous study demonstrated that Th1 and

Th17 were scattered in lamina propria of local

lesions in OLP, in addition, there was increased

proportions of circulating Th1 and Th17 cells,

 

(31)

which correlates closely with our findings of

increased IL-17 in serum and tissue with increased

expression of IL-17R in the local lesions, indicating

that IL-17 mediates immune response in OLP.

Recent studies have suggested that different

lichenoid tissue reaction/ interface dermatitis

disorder, including lichen planus and psoriasis,

may share a common inflammatory signaling

pathway

 

(32) which supports our research studying

the IL-17 and its receptor expression in local lesions

of OLP correlating them with serum IL-17 levels.

In a study done by Ruilu et al,

 

(33) they observed a

large number of IL-17 + cells located in the sub

 

epithelial

lymphocytic infiltrate in the OLP lesions

and showed increased higher mRNA expression

of IL- 17 in OLP lesions compared to the normal

tissues. In our study we investigated these findings

by measuring IL-17 itself in local environment to

prove whether mRNA completed its transduction

A.J.D.S. Vol. 19, No. 1

EVALUATION OF THE POSSIBLE ROLE OF IL-17 AND ITS RECEPTOR 7

pathway and gave its final product IL-17. In

addition, we measured IL-17R in the local lesions

to be sure that IL-17 will perform its action. In the

present study, it was found that there is a statistically

significant relation between the increases of IL-17

in serum of patient than in normal individual.

These findings are also consistent with the

published study by Piccinni and his colleagues who

also found an elevated mRNA expression of IL-17,

together with other TH-17 type molecules in OLP

lesions compared to healthy mucosa.

 

(33 )Moreover,

the presence of Th17 was also identified in another

study conducted by Xie et al

 

(31)

Overexpression of IL-17 in OLP lesions

highlighted its potent pro-inflammatory properties

which may induce profound biological effects and

play an important role in the formation and progress

of the disease, especially that our study not only

found an increase in the local lesion, but also in

serum of the patients. This phenomenon indicated

that the overexpression of IL-17 in OLP lesions

attributes not only to the lymphocytic infiltration,

but also to other unknown regulatory mechanisms,

which is recommended for further exploration.

Finding possible techniques and materials that

control and decrease IL-17 serum levels may open

new horizons to the treatment of such medical

condition other than corticosteroids with their side

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