pemphigus vulgaris ( pv ) , the most prevalent type of pemphigus , is a life - threatening autoimmune bullous disease characterized by an autoantibody predominant target epitopes to desmoglein 3 ( dsg3 ) , a desmosomal cell adhesion glycoprotein [ 14 ] .
it can be considered as a chronic organ - specific disorder because the autoimmune injury which leads to the formation of intraepidermal blisters and acantholysis is confined to the skin and mucosa . a number of studies about pv , using patient samples or animal models , have been previously reported . however , despite recent advancements , the pathogenesis of pv to date remains to be fully elucidated .
autoreactive t cells are thought to play a central role in the pathogenesis of pv [ 1 , 57 ] .
recently , emerging findings put the spotlight on the contribution of a newly discovered subset of interleukin-17 ( il-17 ) producing t helper ( th ) cells accordingly named th17 cells to autoimmune states [ 812 ] .
furthermore , over the past few years , increasing evidence suggested that the development and maintenance of th17 cells have been linked to interleukin-23 ( il-23 ) , a key initiating cytokine in the development of autoimmunity .
it was reported that il-23 was mainly secreted by macrophages and dendritic cells ( dcs ) and il-23 promoted the expansion of the novel th17 population [ 1315 ] . as a result
, a crucial role was proposed for the il-23/il-17 axis in mediating tissue inflammation and autoimmunity recently , such as psoriasis [ 16 , 17 ] . however , despite the current evidences indicating that il-17 may play an important role in pv [ 18 , 19 ] , few reports have explored the crucial role of the il-23/il-17 axis in the immunopathogenesis of pv .
we thus hypothesized that the il-23/il-17 axis will also be functionally involved in the development and maintenance of pv .
in this study , we examined the immunoexpression of il-23 and il-17 in the lesional biopsy specimens from 10 cases of pv , comparing the results with those of pf patients and normal control skins from 6 healthy individuals , and evaluated the correlation between il-23 + cells and il-17 + cells ; moreover , the sources of il-23 were also evaluated .
in this descriptive - analytical study , the subjects were 10 ( 3 men and 7 women ) unrelated patients with pv and 3 ( 1 man and 2 women ) patients with pemphigus foliaceus ( pf ) diagnosed by clinical and immunohistochemical criteria .
the lesional biopsy specimens of the patients were obtained before treatment during the active phase for the purpose of evaluating the acute state .
the normal control skins ( eyelid skin ) were taken from 6 ( 1 man and 5 women ) healthy people who were selected randomly .
this study was approved by our local ethics committee , and written informed consent was obtained from all participants .
immunofluorescence staining of cryosections from those specimens , both the pv patients and the healthy individuals , was performed with the following primary antibodies : rabbit anti - human il-17 pab ( santa cruz ) , goat anti - human il-23 pab ( santa cruz ) , and mouse anti - human cd163 mab ( santa cruz ) . for negative control preparations ,
the first antibodies were replaced with mouse f(ab)2 igg ( abcam ) , an irrelevant isotype control .
the second antibodies are alexa fluor 488 goat anti - mouse igg ( invitrogen ) , alexa fluor 555 rabbit anti - goat igg ( invitrogen ) , and alexa fluor 555 goat anti - rabbit igg ( invitrogen ) .
the counts of il-17 + cells , possibly th17 cells ( il-17 ) , il-23 + cells ( il-23 ) , and cd163 + cells , possibly macrophages ( cd163 ) , in three sections were quantitatively evaluated .
the counts of positive staining cells were initially reported in the form of descriptive statistics .
unpaired t - test was utilized for comparison of il-17 and il-23 between the three groups and the significance of the correlation was assessed by pearson test .
the numbers of il-23 + cells and il-17 + cells were significantly increased in pv lesions ( figures 1(a)1(d ) and figures 2(a)2(d ) ) , compared to healthy controls ( figures 1(e)1(h ) ; figures 2(e)2(h ) ; figure 3 , p < 0.05 ) .
the counts of il-23 + cells in each 200x field of view in pv lesions and normal control skin were 73.70  5.315 and 29.50  4.448 , respectively ( figure 3(a ) , p < 0.05 ) , while the numbers of il-17 + cells in each 200x field of view in pv lesions and normal control skin were 46.60  5.673 and 9.50  3.354 , respectively ( figure 3(b ) , p < 0.05 ) . besides
, the il-23 + staining was overlapped with the cd163 + macrophages ( figures 1(d ) and 1(h ) ) , which indicated that the il-23 was secreted by macrophages .
as the il-23 + staining was overlapped with the cd163 + macrophages ( figures 1(d ) and 1(h ) ) , we adopted the number of cd163 + cells instead of il-23 + cells ' counts for the correlation analysis ( figure 2 ) . based on pearson test ,
the correlation between il-23 + cells and il-17 + cells in pv lesions was significant ( r = 0.7546 ; p < 0.05 ) ( figure 4 ) .
the counts of il-23 + cells and il-17 + cells in each 200x field of view in pf lesions were 42.67  5.812 and 21.33  4.485 , respectively ( see supplementary figure in the supplementary material available online at http://dx.doi.org/10.1155/2014/463928 ) , where we found a statistically significant decrease in number compared to pv ( p < 0.05 ) .
but no statistically significant differences of these numbers between pf and the control group were identified .
in the current research , we examined the lesions and found that both il-23 and il-17 were overexpressed in pv patients , compared to the healthy controls and pf patients .
more importantly , the results showed a correlation between il-23 + cells and il-17 + cells .
finally , this study demonstrated that the il-23 was secreted by a cell population in dermis expressing cd163 , which was considered as a surface marker of the macrophages . in a previous experimental study conducted by arakawa et al . , the authors presented the possibility of th17 ( il-17 + cells ) that played an important role in the pathogenesis of pv .
they quantified th17 cells in lesional biopsy specimens from pv patients and found a significantly higher expression of il-17 + cells compared to controls , which was also confirmed by our results .
however , the importance of il-23 was not investigated in arakawa 's study . to our knowledge
il-23 was clearly not required for the initial induction of il-17 production in naive t cells either in vitro or in vivo .
however , production of il-17 by memory effector cells was clearly enhanced in the presence of il-23 , and it was shown that il-23 maintained expression of il-17 in activated th17 cells . since its discovery , il-23/il-17 axis has been linked to the pathogenesis of various autoimmunity disorders , such as psoriasis and systemic lupus erythematosus [ 16 , 2325 ] . however
, a limited number of reports have explored the crucial role of the il-23/il-17 axis in the immunopathogenesis of pv . taken together ,
this provided us with the basis for a rising interest in the il-23/il-17 axis in pv .
our results showed overexpression of il-23 and il-17 in the lesion of pv patients and a correlation between il-23 + cells and il-17 + cells , which suggested that the il-23/il-17 axis probably played an important role in the immunopathogenesis of pv .
the first issue which should be considered is which type of immunopathology il-23/il-17 axis may show in pv .
, il-23/il-17 axis may not be a cause but a result of the disease ; in other words , it may possibly appear in a protective response to maintain epithelial homoeostasis .
traditionally , il-23 was found to be expressed mainly by macrophages and dendritic cells ( dcs ) in dermis [ 21 , 26 ] . in an earlier study
, cd163 was recommend to be considered as an alternative marker to identify dermal macrophages for its more specific and more useful in flow cytometry applications .
our findings that the il-23 + staining was overlapped with the cd163 + macrophages in all specimens indicated that the il-23 in dermis was expressed mainly by the cd163 + macrophages . in other words , macrophages , which have been confirmed to play an important role in the pathogenesis of psoriasis , may be also involved in the pathogenesis of pv . in summary ,
the present study suggested the importance of the il-23/il-17 axis in the development of pv , which provided us with some clues for the elucidation of the pathogenesis of pv .
the reason why more il-17 + cells and il-23 + cells were present in lesional specimens from pv than those from pf is not certain . actually , the accurate role of il-23/il-17 axis in the pathogenesis of pv is still unresolved in the present study
. additionally , the current research had some limitations that should be noted , such as the following : ( i ) the results were descriptive mainly and lacked some mechanistic studies , ( ii ) as a retrospective study , the serum levels of il-23 and il-17 were not tested , and a correlation between il-23/il-17 and disease activity or antibody titers was not investigated , and ( iii ) the relatively small number of patients was employed in this study .
prospectively , further study of the il-23/il-17 pathway in the pathogenesis of pv in mice model may be encouraged to further validate our hypothesis . additionally , we hypothesized that targeting the il-23/th17 pathway maybe a highly effective therapeutic approach in the treatment of pv .
future studies are required in order to better explore this pathway as a potential therapeutic target .
in conclusion , our present study provides evidence that the expression of il-23 and il-17 was elevated and correlated in pv patients , which suggested the crucial role of the il-23/il-17 axis in the development of pv and provided us with some clues for the elucidation of the pathogenesis of pv .
 il-23/il-17 axis has been identified as major factor involved in the pathogenesis of several autoimmune diseases ; yet its pathogenetic role in pemphigus vulgaris ( pv ) remains controversial . 
 the aim of this research was to investigate the potential role of il-23/il-17 axis in the immunopathogenesis of pv , and correlation between il-23 + cells and il-17 + cells was also evaluated . for this purpose , 
 ten patients with pv , three patients with pemphigus foliaceus ( pf ) , and six healthy individuals were allocated to this research . 
 the lesional skin biopsy specimens were obtained before treatment . then immunofluorescence staining was performed to analyze the expression of il-23 and il-17 in the pv / pf patients and the healthy individuals . 
 the results showed that the numbers of il-23 + and il-17 + cells were significantly higher in pv lesions , compared to pf lesions and normal control skins , respectively ( all p < 0.05 ) . 
 moreover , the correlation between il-23 + cells and il-17 + cells was significant ( r = 0.7546 ; p < 0.05 ) . taken together , 
 our results provided evidence that the il-23/il-17 axis may play a crucial role in the immunopathogenesis of pv and may serve as novel therapeutic target for pv . 