oral lichen planus ( olp ) is a chronic inflammatory disease affecting the oral mucosa , with a prevalence of about 1~2% of the population [ 1 , 2 ] . once established , the lesions rarely undergo self - remission and , in some cases , have a malignant potency . histologically , olp is characterized by a dense subepithelial infiltration of lymphocytes , increased numbers of intraepithelial lymphocytes , and the degeneration of basal keratinocytes . the lymphocytic infiltrate consists predominantly of t cells [ 2 , 4 , 5 ] . 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 [ 2 , 4 , 6 ] . previous studies have identified that a variety of proinflammatory molecules are aberrantly synthesized and secreted by both infiltrated t cells and altered keratinocytes in the lesions of olp , which contribute to the onset and persistence of the inflammatory responses [ 712 ] . these findings indicated that olp lesion development is closely associated with the activation , production , and functions of these 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 [ 13 , 14 ] . il-23 is a heterodimeric proinflammatory cytokine , which is composed of a unique p19 subunit and a common p40 subunit shared with il-12 . secreted by various cell types , such as activated dentritic cells , macrophages , and epithelium , il-23 functions as an important driving factor in the immune response . notably , il-23 serves an essential role in induction and maintenance of a novel subset of cd4+th cells , namely , th17 [ 16 , 17 ] . in addition , il-23 also induces th17 to produce its distinctive cytokine il-17 , which is composed of two il-17a subunits [ 18 , 19 ] . subsequently , il-17 functions as a proinflammatory cytokine , which can activate different cells such as epithelial , endothelial , fibroblast , chondrocyte , and osteoblast to produce numerous inflammatory molecules including cytokines , chemokines , defensins , and mmps [ 20 , 21 ] . together with these downstream proinflammatory products , the il-23/il-17 axis is extensively involved in the processes of chronic inflammation within various pathological states . the il-23/il-17 axis has recently been described to play a major role in the pathogenesis of different chronic inflammatory and immune diseases , such as rheumatoid arthritis ( ra ) , psoriasis , atopic dermatitis , inflammatory bowel disease ( ibd ) , and periodontitis [ 2229 ] . 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 . in others ' investigation , the presence of il-17 has been found in the lesions of olp , suggesting its role in the local environments . however , till now , the role of il-23/il-17 axis in the pathogenesis of olp is still unclear . the aim of the present study was to investigate the expression patterns and regulatory roles of il-23/il-17 axis in olp . 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 . subjects with other oral or systematic diseases or taking corticosteroids or immunosuppressive drugs within 3 months prior to the specimen collection were excluded from the recruitment . 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 , 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 4c . 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 . the expression of il-23p19 subunit was assessed using an arbitrary scoring system as follows : 0 , no staining ; 1 , very weak staining ( 15 cells per section ) ; 2 , weak staining ( 530 cells per section ) ; 3 , moderate staining ( 30100 cells per section ) ; 4 , strong staining ( 100400 cells per section ) ; and 5 , strong staining ( > 400 cells per section ) . the expression activities were assessed as previously described [ 33 , 34 ] . to evaluate the expression of il-17 , five high power fields ( hpf ) at a magnification of 400 were randomly picked up and the numbers of il-17 + cells were counted separately . the average number of il-17 + cells per field was then calculated and compared . five milliliters of heparinized blood was obtained from 10 olp patients , respectively . the peripheral blood mononuclear cells ( pbmcs ) 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  10 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  g / ml of purified mouse anti - human cd3 antibody ( bd pharmingen , usa ) and 5  g / ml of purified mouse anti - human cd28 antibody ( bd pharmingen , usa ) were added . to study the effect of il-23 on the cd4+th cells , the cells were cultured with or without recombinant human il-23 ( ril-23 , 20  ng / ml ; r&d systems , minneapolis , mn , usa ) for 36 hours . subsequently , cells and culture supernatant were collected separately for the following intracellular cytokine staining and elisa detection . for intracellular cytokine staining , collected peripheral blood cd4+th cells were resuspended at a density of 1  10 cells / ml and stimulated in rpml 1640 medium containing phorbol myristate acetate ( ( pma ) , 50  ng / ml , sigma ) , ionomycin ( 1  g / ml , sigma ) , and monensin ( 0.67  g / ml , goldistop , bd pharmingen , usa ) for 4 hours . after that , cells were stained using a cytofix / cytoperm fixation / permeabilization solution kit ( bd pharmingen , usa ) , anti - human cd4 percp - cy5.5 antibody ( bd pharmingen , usa ) , and anti - human il-17 ape antibody ( bd pharmingen , usa ) , successively , according to the manufacturer 's instructions . stained cells were analyzed using the facsaria flow cytometer and bd facsdiva software ( bd biosciences , san diego , ca , usa ) . 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  10/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 , 020  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 70c 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 2 . to avoid the amplification of genome dna , all the primers the real - time quantitative pcr on 10  ng of cdna was performed using the sybr green detection assay on an applied biosystems 7300 real - time pcr system . the amplification procedure consisted of 10  s at 95c , followed by 40 cycles of the amplification procedure composed of 95c for 5  s and 62c for 40  s. each sample was run in triplicate . the relative gene expression normalized to the expression of gapdh housekeeping gene and control sample were analyzed by the ct method . kruskal - wallis test and mann - whitney test were used to determine differences between groups , paired t - test was used for the pair comparison , and spearman 's test was used to analyze the correlations . 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 1(a)1(d ) ) . in contrast , only a few keratinocytes in the epidermis layer of the nom tissues showed weak stain of il-23p19 ( figures 1(e ) and 1(f ) ) . moreover , we found abundant il-17 positive stainings on the cytoplasm of the infiltrated lymphocytes in the lesions of both erosive and reticular olp , but only a few sporadic il-17 + cells in the normal oral mucosa ( figures 1(g)1(l ) ) . the statistical data showed that both the erosive and reticular olp lesions had significantly increased immunostaining scores of il-23p19 , as well as the numbers of il-17 + cells , compared to the normal oral mucosa . in addition , erosive olp lesions contained a significantly increased number of il-17 + cells compared to the reticular olp lesions . however , there is no significant difference in il-23p19 staining score between erosive and the reticular olp lesions ( figures 2(a ) and 2(b ) ) . 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 2(c ) and 2(d ) ) . 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 3(a ) ) . however , in reticular olp subgroup , there was a positive correlation between the il-23p19 staining scores and the numbers of il-17 + cells ( figure 3(c ) ) , whereas no correlation was found in erosive olp group ( figure 3(b ) ) . moreover , we also found that the mrna expressions of both il-23 subunits , il-23p19 ( figure 3(d ) ) and il-12p40 ( figure 3(e ) ) , are positively correlated with mrna expression of il-17 in reticular olp samples . these results showed that overexpressions of il-23 and il-17 are positively correlated in the reticular olp lesion , indicating a potential regulatory role of il-23 to the expression of il-17 in the early stage of olp lesion . 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 , 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 4(a)4(c ) ) . in addition , the il-17 content in the culture supernatant of cd4+th cells also increased under the stimulation of il-23 than the control group ( figure 4(d ) ) . these data suggest that the overexpression of il-23 in olp lesions probably contributes to the induction of th17 and the production of il-17 . 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 -defensins ( hbd ) in hok cells . we observed that il-17 significantly increased mrna expressions of hbd-2 and -3 in a dose - dependent manner , but not hbd-1 ( figure 5(a ) ) . we next detected the mrna expression of 6 chemokines and found that il-17 could significantly increase the mrna expressions of il-8 , ccl-20 , but not cxcl-9 , -10 , -11 or ccl-5 , in hok cells ( figure 5(b ) ) . furthermore , we found that il-17 significantly increased mrna expressions of tnf- , an important proinflammatory cytokine in the pathogenesis of olp , in hok . however , mrna expression of il-6 exhibited no significant difference with or without the effect of il-17 ( figure 5(c ) ) . the data revealed that the il-17 could selectively regulate the expressions of some , but not all , inflammatory mediators in oral keratinocytes , indicating the selectively regulatory role of il-23/il-17 axis in the immune network in olp lesions . 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 . however , study about the expression and regulatory role of this new axis in olp is just at its beginning . in a recent study of our group , we found no difference in the serum levels of il-23 and il-17 between the olp patients and controls . the present study is focused on the expression and regulatory role of il-23/il-17 axis in the local environment of olp lesions . il-23 is the upstream driving cytokine in the il-23/il-17 axis ; its importance in inflammation and autoimmunity has been widely demonstrated . to determine whether il-23 is involved in the development of olp , we first detected its expression in the olp lesions compared with the nom tissues . structurally , il-23 is composed of a unique p19 subunit and a common p40 subunit shared with il-12 . to avoid confusion however , in the quantitative pcr assay , we examined the mrna expressions of both the il-23p19 and il-12p40 , for the expression of each gene could influence the amount of the il-23 . our results revealed an upregulation of il-23p19 in both erosive and reticular olp lesions compared to the nom , indicating its involvement in the disease progress . as a driving factor of immune response , il-23 is predominantly produced by the antigen presenting cells ( apcs ) including dentritic cells and macrophages , but its expression was also found in the keratinocytes from normal and psoriatic skin . in addition , the epidermis in lesional psoriatic skin revealed markedly stronger il-23p19 stain than the epidermis in normal skin . similarly , here we also observed a diffuse and strong expression pattern of il-23p19 in the epithelial layer of olp lesions , where keratinocytes are the major cell type . these findings suggested that keratinocytes may be a major source of il-23 production under the chronic inflammatory condition in the mucocutaneous system . although both the erosive and reticular olp lesions had significantly increased immunostaining scores of il-23p19 than the normal oral mucosa , the expressions of il-23p19 between the erosive and reticular olp lesions are similar . this observation suggests that the overexpression of il-23 may be an early event in the pathogenesis of olp lesion and be maintained at a high level in the later progress . 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 [ 20 , 35 ] . although il-17 can be secreted by a variety of innate and adaptive immune cells , t cells are still its major sources , especially the new subset of cd4+th cells , namely , th17 . considering the involvement of a t - cell - mediated immune response in the pathogenesis of olp , it is not surprising that il-17 and th17 cells may be present and play a regulatory role in the local environment of the disease . indeed , we observed a large number of il-17 + cells located in the subepithelial lymphocytic infiltrate in the olp lesions . besides , our data showed increased numbers of il-17 + cells and higher mrna expressions of il-17 in the olp lesions compared to the nom tissues . in addition , compared to the reticular olp lesions , erosive olp lesions contained much more il-17 + cells . these findings are consistent with the recent published study by piccinni and his colleagues , who also found an elevated mrna expression of il-17 , together with other th17 type molecules in the olp lesions compared to the healthy mucosa . moreover , the presence of th17 was also identified in another recent study conducted by xie et al . . we must admit that the overexpression of il-17 in olp lesions is attributed partly to the infiltration of lymphocytes in the local environment . however , the large amount of il-17 cytokine in the local lesions of olp can not be overlooked , for its potent proinflammatory properties may induce profound biological effects and play an important role in the formation and progress of the disease . on the other hand , piccinni et al . also found that the cd4+t cell clones generated from olp lesions produced significant higher levels of il-17 than those generated from the healthy oral mucosa . 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 needed to be further explored . 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 . in addition , cd4+t cell clones generated from olp lesions exhibited an elevated activity in the il-17 production . thus , we investigated the effect of il-23 on the cd4+t cells from olp patients . our results showed that the stimulation of il-23 could significantly increase the percentage of th17 and the il-17 production in cd4+t cells from olp patients . recent immunological findings have supported the opinion that , although il-23 has no effect on the initiation of th17 differentiation , it is crucially required for the proliferation and stability of precommitted th17 cells , their further migration into the pathological tissues , and the production of il-17 , in which way to promote the pathogenic function of th17 cells [ 37 , 38 ] . based on these findings and our data in the present study , it can be suggested that the overexpression of il-23 may be functional and contributes , at least in part , to the accumulation of th17 cells and the increased level of il-17 in the olp lesion . therefore , the il-23/il-17 axis may represent a new signaling pathway in the crosstalk between the keratinocytes and cd4+t cells , which may be involved in the immunopathogenesis of olp . 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 [ 712 , 3941 ] . however , the mechanism of the functional alterations in the keratinocyte from olp lesions is still unclear . our results showed that the stimulation of il-17 could significantly increase the mrna expressions of hbd-2 , 3 , il-8 , ccl-20 , and tnf- , but not the expression of hbd-1 , cxcl-9 , -10 , -11 , ccl-5 , and il-6 in hok cells . these data revealed that the effects of il-17 on the oral keratinocyte are selective but not extensive , suggesting a unique regulatory role of il-23/il-17 axis in the local environment of olp lesions . 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 6 ) . firstly , keratinocytes in olp lesion produce a large amount of il-23 via an unknown mechanism . next , keratinocyte - derived il-23 may contribute to the accumulation of th17 cells and the overproduction of il-17 in the local lesion of olp . subsequently , 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 . our results warrant further explorations on the intrinsic mechanisms of the overexpression and the regulatory effects of il-23/il-17 axis , as well as its interaction with other signaling pathways , in the olp lesions . notably , il-23/il-17 axis has been recently considered as a relevant therapeutic target in chronic inflammatory and autoimmune diseases , and several biological agents blocking il-23 or il-17 have been currently developed [ 22 , 42 ] . therefore , further understanding of the role of il-23/il-17 axis in the pathogenesis may contribute to the development of novel therapeutic strategies for the prevention and management of olp in the future .
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 -defensin-2 , -3 , ccl-20 , il-8 , and tnf- , but not -defensin-1 , cxcl-9 , -10 , -11 , ccl-5 , and il-6 in human oral keratinocytes . taken together 
 , our results revealed an overexpression pattern and selectively regulatory roles of il-23/il-17 axis in the olp lesions , suggesting that it may be a pivotal regulatory pathway in the complex immune network of olp lesions .