Interleukin- (IL-) 23/IL-17 axis is a newly discovered proinflammatory signaling pathway and has been implicated in the pathogenesis of many chronic inflammatory and immune disorders. Here we investigated whether the IL-23/IL-17 axis was present and functional in the lesions of oral lichen planus (OLP), a chronic inflammatory disease affecting the oral mucosa. Using immunohistochemistry and quantitative PCR, we found that the subunits of IL-23 and IL-17 were overexpressed in OLP lesions than in normal oral mucosa tissues. In addition, the expressions of IL-23 and IL-17 are positively correlated in reticular OLP tissues. Results from in vitro studies revealed that exogenous IL-23 could increase the percentage of Th17 cells and IL-17 production in the CD4+T cells from reticular OLP patients. Furthermore, we also found that exogenous IL-17 could significantly enhance the mRNA expressions of
Oral lichen planus (OLP) is a chronic inflammatory disease affecting the oral mucosa, with a prevalence of about 1~2% of the population [
Although the etiology of OLP remains unclear, accumulating evidence supports a role of immune dysregulation in the pathogenesis of OLP, especially involving a T-cell-mediated immune response and the abnormal production of various inflammatory molecules [
The interleukin- (IL-) 23/IL-17 axis is a newly discovered proinflammatory signaling pathway, in which IL-23 and IL-17 are two pivotal cytokines [
In our previous study, we have detected the serum and saliva levels of IL-17 in OLP patients but found no significant difference compared to healthy groups [
All tissue and blood specimens of OLP patients and healthy volunteers were obtained from West China School and Hospital of Stomatology, Sichuan University. The OLP cases were clinically diagnosed, pathologically confirmed, and subdivided as reticular or erosive forms based on the modified WHO diagnostic criteria of OLP [
For immunohistochemistry (IHC) staining, 27 OLP lesion specimens, including 13 with erosive form and 14 with reticular form, were recruited from the archives of the department of pathology, and 10 normal oral mucosa (NOM) tissues were collected from healthy volunteers receiving orthognathic surgery. For quantitative PCR analyses, 14 reticular OLP and 10 NOM tissues were obtained during the biopsy or orthognathic surgery, respectively, and subsequently snap-frozen in liquid nitrogen for the following experiment procedure. For studying the effect of IL-23 on the CD4+T cells, peripheral blood samples were collected from 10 of 14 reticular OLP patients noted above. The clinical characteristics of these participants were listed in Table
Clinical features of the subjects.
OLP | Control | ||
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Erosive form | Reticular form | ||
Number | 13 | 14 | 10 |
Gender | |||
Male | 3 | 3 | 2 |
Female | 10 | 11 | 8 |
Age (year) | |||
Range | 23~52 | 26~49 | 20~48 |
Mean ± SD |
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Site of biopsy | |||
Buccal | 11 | 12 | 10 |
Tongue | 2 | 2 |
Written informed consent was obtained from each subject, and the whole experiment procedure was conducted in accordance with the Declaration of Helsinki and approved by the Scientific and Ethical Committee Board of Sichuan University.
Paraffin-embedded sections of OLP lesion specimens and NOM tissues were treated in xylene and hydrated in graded ethanol, followed by blocking the activity of endogenous peroxidase with 3% hydrogen peroxide. After antigen retrieval by heat and press, the sections were incubated with goat anti-human IL-17 monoclonal antibody (1 : 100, R&D Systems, USA) or rabbit anti-human IL-23p19 monoclonal antibody (1 : 100, Abcam, UK) overnight at 4°C. Subsequently, sections were incubated with the rabbit anti-goat lgG antibody-HRP polymer or goat anti-rabbit lgG antibody-HRP polymer detection reagent (ZSGB Biotechnology, Beijing, China) for 15 minutes at room temperature and then 3,3′-diaminobenzidine tetrahydrochloride (DAB) for 1-2 minutes. After that, the sections were counterstained with hematoxylin. As negative controls, the nonimmune serum was used instead of the primary antibodies.
Evaluation of immunostaining was preformed independently by 2 observers in a blinded manner. The expression of IL-23p19 subunit was assessed using an arbitrary scoring system as follows: 0, no staining; 1, very weak staining (1–5 cells per section); 2, weak staining (5–30 cells per section); 3, moderate staining (30–100 cells per section); 4, strong staining (100–400 cells per section); and 5, strong staining (>400 cells per section). The average score in each group was calculated and compared. The expression activities were assessed as previously described [
Five milliliters of heparinized blood was obtained from 10 OLP patients, respectively. The peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density-gradient centrifugation. Subsequently, CD4+T helper (Th) cells were purified by anti-human CD4 magnetic particles (BD biosciences, USA) on a cell separation magnet (BD Biosciences, USA) according to the manufacturer’s instructions. The purified CD4+Th cells were resuspended at a density of 1 × 106 cells/mL RPML 1640 medium (Thermo Scientific HyClone, Beijing, China) supplemented with 10% fetal bovine serum (FBS; GIBCO, Grand Island, NY, USA). For activation of CD4+Th cells, 10
For intracellular cytokine staining, collected peripheral blood CD4+Th cells were resuspended at a density of 1 × 106 cells/mL and stimulated in RPML 1640 medium containing phorbol myristate acetate ((PMA), 50 ng/mL, Sigma), ionomycin (1
The concentration of IL-17 in the culture supernatants of peripheral blood CD4+Th cells was measured using a human IL-17 ELISA kit (BOSTER, Wuhan, China). According to the manufacturer’s instructions, the detectable range of IL-17 content was from 31.2 to 2000 pg/mL.
HOK16E6E7, a human immortalized oral keratinocyte cell line, was plated in 6-well plates (2 × 105/well) in keratinocyte serum-free medium (KSFM, Gibco BRL Life Technologies, Grand Island, NY, USA) containing supplemented with epidermal growth factor and calcium. After 24 hours’ growth, medium with different doses of recombinant human IL-17 (rhIL-17, 0–20 ng/mL; R&D Systems, Minneapolis, MN, USA) or medium only was placed. After another 24 hours’ culture, the cells were collected and kept in −70°C until the RNA isolation.
Total RNA was isolated from 14 reticular OLP and 10 NOM tissue specimens or the cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from 400 ng of RNA using the PrimeScript RT reagent Kit (TAKARA Biotechnology, Dalian, China). Primers purchased from TAKARA Biotechnology were listed in Table
Primer design for real-time quantitative PCR.
Gene | GeneBank accession number | Primer sequences | PCR product size (bp) |
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IL-17 | NM_002190.2 | Forward: 5′-atgactcctgggaagacctcat-3′ | 150 |
Reverse: 5′-gttcaggttgaccatcacagtc-3′ | |||
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IL-23p19 | NM_016584.2 | Forward: 5′-ccttctctgctccctgatagc-3′ | 118 |
Reverse: 5′-gactgaggcttggaatctgct-3′ | |||
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IL12p40 | NM_002187.2 | Forward: 5′-ctggagaaatggtggtcctca-3′ | 113 |
Reverse: 5′-gacttggatggtcagggttttg-3′ | |||
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NM_005218.3 | Forward: 5′-ccttctgctgtttactctctgc-3′ | 126 |
Reverse: 5′-gaatagagacattgccctccac-3′ | |||
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NM_004942.2 | Forward: 5′-gggtcttgtatctcctcttctcg-3′ | 130 |
Reverse: 5′-ctagggcaaaagactggatgac-3′ | |||
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NM_001081551.2 | Forward: 5′-ccaggtcatggaggaatcat-3′ | 113 |
Reverse: 5′-gagcacttgccgatctgttc-3′ | |||
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CXCL-9 | NM_002416.1 | Forward: 5′-gggactatccacctacaatcctt-3′ | 127 |
Reverse: 5′-ctgctgaatctgggtttagacat-3′ | |||
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CXCL-10 | NM_001565.3 | Forward: 5′-gctgtacctgcatcagcattagt-3′ | 138 |
Reverse: 5′-gacatctcttctcacccttctttt-3′ | |||
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CXCL-11 | NM_005409.4 | Forward: 5′-ttgtgtgctacagttgttcaagg-3′ | 110 |
Reverse: 5′-atggaggctttctcaatatctgc-3′ | |||
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CCL-5 | NM_002985.2 | Forward: 5′-catattcctcggacaccacac-3′ | 131 |
Reverse: 5′-ctttcgggtgacaaagacgac-3′ | |||
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CCL-20 | NM_004591.2 | Forward: 5′-tgtgctgtaccaagagtttgctc-3′ | 124 |
Reverse: 5′-tgaagaatacggtctgtgtatccaa-3′ | |||
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IL-8 | NM_000584.3 | Forward: 5′-agctctgtgtgaaggtgcagtt-3′ | 126 |
Reverse: 5′-ggtccactctcaatcactctcag-3′ | |||
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IL-6 | NM_000600.3 | Forward: 5′-ggagacttgcctggtgaaaatc-3′ | 140 |
Reverse: 5′-gcaggaactggatcaggactt-3′ | |||
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TNF- |
NM_000594.3 | Forward: 5′-aagcctgtagcccatgttgtag-3′ | 112 |
Reverse: 5′-gctggttatctctcagctccac-3′ | |||
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GAPDH | NM_002046.4 | Forward: 5′-ctttggtatcgtggaaggactc-3′ | 132 |
Reverse: 5′-gtagaggcagggatgatgttct-3′ |
All statistical analysis was performed on GraphPad Prism 5 software. Kruskal-Wallis test and Mann-Whitney test were used to determine differences between groups, paired
To identify whether IL-23/IL-17 is involved in the local pathogenesis of OLP, we firstly detected the expression and distribution of IL-23 p19, a unique subunit of IL-23, and IL-17 in OLP lesions and NOM tissues. Using IHC detection, we observed diffuse and strong expressions of IL-23p19 in both erosive and reticular OLP lesions. The positive staining of IL-23p19 predominantly concentrated on the epithelium of OLP lesions and also on the extracellular matrix of the lamina propria (Figures
Immunohistochemical stainings for IL-23p19 (a–f) and IL-17 (g–l) in erosive (a, b, g, and h) and reticular (c, d, i, and j) OLP lesions and normal oral mucosa tissues (e, f, k, and l). Immunohistochemical staining for IL-23p19 showed diffuse and strong patterns in epithelium and the extracellular matrix of the lamina propria of both erosive ((a) ×100; (b) ×400) and reticular ((c) ×100; (d) ×400) OLP lesions, but weak or absent pattern in normal oral mucosa tissues ((e) ×100; (f) ×400). Abundant IL-17 positive staining was observed on the cytoplasm of the infiltrated lymphocytes in the lesions of both erosive ((g) ×100; (h) ×400) and reticular ((i) ×100; (j) ×400) OLP, but only a few sporadic IL-17+ cells were seen in normal oral mucosa ((k) ×100; (l) ×400).
Expressions of IL-23 and IL-17 in OLP lesions. (a) The average staining scores of IL-23p19 in erosive OLP lesions (
To verify the IHC results, we also detected the mRNA expressions of both subunits of IL-23 (IL-23p19 and IL-12p40) and IL-17 in 14 reticular OLP lesional tissues and 10 NOM tissues and found that the mRNA expressions of all the three genes in OLP lesions were significantly increased compared to NOM tissues (Figures
These data demonstrated overexpression of IL-23 and IL-17 in the OLP lesions, indicating that the IL-23/IL-17 axis may be involved in the local immune network of OLP.
Considering IL-23 as an important upstream inducing cytokine of IL-17, we next investigated whether the upregulation of IL-23 in the progress of OLP lesion is associated with the increased expression of IL-17. Analyzing based on the data above, we found no correlation between the IL-23p19 staining scores and the numbers of IL-17+ cells in the OLP lesions (Figure
Correlation between the expressions of IL-23 and IL-17 in OLP tissue specimens. (a–c) Correlations between the staining scores of IL-23p19 and numbers of IL-17+ cells per hpf in total OLP tissue specimens (
Next, we explored the potential role of IL-23 in the production of IL-17 in OLP. Although IL-17 has been recently reported to be produced by various cell types, a CD4+Th cell subset, namely, Th17 cell, is one of its main sources. On the other hand, in the local lesion of OLP, the dense subepithelial inflammatory infiltrate consists predominantly of CD4+Th cells. Therefore, here we focused on the effect of IL-23 to the IL-17 production in CD4+Th cells from 10 OLP patients. We observed that compared to the control group, the stimulation of rIL-23 significantly increased the percentage of CD4+IL-17+ cells (identified as Th17) in CD4+Th cells from OLP patients (Figures
The effect of recombinant (r) IL-23 on the percentage of Th17 cells and IL-17 production in CD4+T cells from OLP patients. (a) Representative scatter plots of CD4+IL-17+ staining in peripheral blood CD4+T cells from OLP patients (
We next explored the potential biological effects of IL-17, the essence effector of IL-23/IL-17 axis, in OLP lesions. It is well known that oral keratinocyte is an important component in the oral mucosa immunity and plays an important role in the pathogenesis of many chronic inflammatory oral diseases, including OLP, by producing various inflammatory mediators, such as cytokines, chemokines, and defensins. Therefore, we investigated the effect of IL-17 on the expressions of inflammatory mediators by HOK16E6E7, a human oral keratinocyte cell line.
First we detected effect of IL-17 on the mRNA expression of human
The effect of recombinant (r) IL-17 to the mRNA expressions of inflammatory mediators, including
In recent years, the IL-23/IL-17 axis has been widely described to play a pivotal role in the pathogenesis of different chronic inflammatory disorders [
IL-23 is the upstream driving cytokine in the IL-23/IL-17 axis; its importance in inflammation and autoimmunity has been widely demonstrated [
IL-17 is another key component in the IL-23/IL-17 axis and primarily functions as a downstream effector. Overexpressions of IL-17 have been observed in many autoimmune and inflammatory diseases, and its pivotal roles in the pathogenesis have been profoundly identified [
Since IL-23 is a major upstream inducer of IL-17 production, it is reasonable to further explore whether the upregulation of IL-23 has any regulatory role in the IL-17 production in the local environment of OLP. Based on the data, we conducted correlation analysis of the expressions of IL-23 and IL-17. Although no correlation between IL-23p19 staining scores and IL-17+ cell numbers was found in the total OLP group or erosive OLP subgroup, there were positive correlations between the expressions of IL-23 and IL-17 at both protein and mRNA levels in reticular OLP subgroup, indicating that the upregulation of IL-23 is associated with increased levels of IL-17 in the early stage of OLP lesion. On the other hand, the lacking of correlation between IL-23 and IL-17 expressions in erosive OLP lesions may be due to the persistent high levels of IL-23 but various levels of IL-17, indicating the existence of other potentially regulatory mechanisms, other than IL-23, in the IL-17 expressions in the erosive stage of OLP.
According to the recent published data by Xie et al., the IL-17 in the OLP lesion is mainly expressed in CD4+T cells, which is identified as Th17, in the subepithelial lymphocytic infiltration, as observations by double immunofluorescence staining [
To investigate the potential biological effects of IL-23/IL17 in the OLP lesion, we further explored the effects of IL-17, the major effector of IL-23/IL-17 axis, on the production of different inflammatory mediators by the oral keratinocytes. Accumulated studies have demonstrated that oral keratinocyte is one of the major sources of the aberrant expressions of various inflammatory mediators in OLP lesions, including cytokines, chemokines, and defensins, which interact with each other and compose the complex immune network in the OLP environment [
In summary, based on the findings in the present study, we propose a novel model of interaction between the T cells and keratinocytes in the pathogenesis of OLP, in which the IL-23/IL-17 axis is involved (Figure
Schematic model of IL-23/IL-17 axis involved in the pathogenesis of OLP. The whole process is divided into three steps: (1) keratinocytes in OLP lesion produce a large amount of IL-23 via an unknown mechanism; (2) keratinocyte-derived IL-23 may contribute to the accumulation of Th17 cells and the overproduction of IL-17 in the local lesion of OLP; (3) IL-17 reversely induces the keratinocytes to selectively produce various inflammatory mediators, which compose the complex immune network in the inflammatory environment of OLP lesions.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Rui Lu and Xin Zeng contributed equally to this work.
This work was supported by grants from the National Natural Science Foundation of China (nos. 81200791, 81072218, and 81200797), International Science and Technology Cooperation Program of China (ISTCP) (2012DFA31370), and the Doctoral Program of the Ministry of Education of China (no. 20110181110055). The authors also thank Mrs. Min Zhou and Mrs. Sixiu Chen (State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichuan University) for their excellent technical support.