| BackgroundRheumatoid arthritis (RA) represents an example of the autoimmune diseases. With a prevalence of1%worldwide, the pathogenesis of RA is not clear yet. Bone and cartilage destruction in the course of persistent inflammation is a serious clinical problem in the pathophysiology of RA. The activation of T cells recognizing autoantigens is implicated in the pathogenesis of RA. It has been well established for many years that Thl cells have been implicated to play an important pathogenetic role in RA. Thl cells are predominant in synovial fluid of RA. However, the situation of Thl in the peripheral blood is more controversial:with either decreased or unchanged Thl profile have been reported in RA patients. In recent years additional effector T cell subsets have been described, including interleukin-17(IL-17)-producing cells (Thl7cells). T-helper (Th)17cells are inflammatory CD4+T cells that produce IL-17A but not IFN-y. These cells and their secreted cytokines are found to be elevated in the peripheral blood of RA patients. In synovial fluid of RA patients, levels of Thl7cells were demonstrated to be much higher than that in peripheral blood, suggesting a pathogenetic role of Th17in RA. The exact mechanism of Th17cells in pathogenesis of RA remains to be elucidated.Th22subset is a more recently identified CD4+T helper subset, which is characterized by secretion of IL-22but not IL-17or IFN-y. Th22cells showed distinct differences in the profile of altered genes compared to other T cells such as Th1, Th2, and Th17cells, confirming an individual signature for the Th22subset. The clonal stability, the selective expression of transcription factors, PDGF receptor and CCR-10, and the fact that naive T cells differentiate toward the Th22phenotype in the presence of TNF-α and IL-6provide strong evidence that Th22cells represent a terminally differentiated and independent T cell subtype. It has been established that Th22cells are increased within psoriasis lesions and peripheral blood in psoriasis patients. Stefanie et al have identified Th22cells that infiltrate the epidermis in individuals with inflammatory skin disorders. The proinflammatory Th22responses were synergistically dependent on IL-22and TNF-a. These mean that Th22cells are probably implicated in the pathophysiology of some autoimmune diseases.The effector cytokine of Th22cells is IL-22, which belongs to the IL-10cytokine family. IL-22signals through a heterodimeric receptor complex consisting of the IL10receptor (IL-10R)β chain and the IL-22R. The precise function of IL-22remains unclear. Recent studies implicated that IL-22was involved in the pathogenesis of some autoimmune diseases such as systemic lupus erythematosus (SLE), systemic sclerosis (SS) and inflammatory bowel disease (IBD) in humans. However, the situation of IL-22was different in different diseases. Decreased plasma IL-22level was found in patients with SLE. Otherwise, there was increased IL-22in the serum samples from psoriasis patients. Consistently, increased IL-22has also been reported in serum samples from Crohn disease patients.IL-17, the main effector cytokine of Th17cells, plays an important role in the acute mechanisms in host defense. And it was involved in the pathogenesis of many autoimmune diseases such as ankylosing spondylitis, multiple sclerosis and Crohn's disease. Although the amount of IL-17in serum of RA patients is hard to detect, increasing IL-17levels have been demonstrated in synovial fluid of patients with RA.Up to date, there is no data about Th22cells (CD4+IFNy-IL17-IL-22+T cells) and pure Thl7cells (CD4+IFNy-IL-22-IL17+T cells) in patients with RA. To investigate their roles in the pathogenesis of RA, we measured the frequencies of Th22, pure Thl7, Thl7, Thl and plasma IL-22or IL-17, and examined their correlations with disease activity.ObjectiveTo examine and compare the frequencies of Th22cells, Th17cells, pure Th17cells and Thl cells in the peripheral blood of RA, OA patients and healthy people; To examine and compare the levels of IL-22and IL-17in the plasma of RA, OA patients and healthy people; To analyze the correlations between each T cell subset or cytokine and the disease activity in RA patients. To investigate the roles of Th22cells and Th17cells in the pathogenesis of RA.Materials and Methods(1) A total of30patients with active RA according to the criteria of the American College of Rheumatology were included in this study. Each patient with active RA was defined by a DAS28score≥2.6. This group consisted of23women and7men, with mean±SD disease duration of10.7±8.6years. The mean age of the patients was53.9±10.9years. The demographic and key clinical information of the RA patients are summarized in Table1. All of the patients did not receive immunosuppressive or immunomodulatory drugs for at least2months when sampling. Eleven osteoarthritis (OA) patients(6females and5males;mean age55±5.3years) as disease controls and twenty-three healthy controls(18females and5males; mean age54±10.8years) were also recruited in the study. Enrollment took place between March,2010and January,2011in two centers:the Department of Orthopedics, Qilu Hospital, Shandong University and the Department of Rheumatology, Shandong Provincial Hospital, Shandong University, China. The study was approved by the Institutional Review Boards of each participating center. Informed consent was obtained from each patient before being included in the study.(2)Intracellular cytokines were studied by flow cytometry to reflex the cytokine-producing cells. Briefly, heparinized peripheral whole blood (400μl) with an equal volume of Roswell Park Memorial Institute1640medium were incubated for4h at37℃,5%CO2in the presence of25ng/mL of phorbol myristate acetate (PMA),1μg/mL of ionomycin, and1.7μg/ml Golgiplug(Monensin; all from Alexis Biochemicals, San Diego, CA, USA). PMA and ionomycin are pharmacological T-cell-activating agents that mimic signals generated by the T-cell receptor (TCR) complex and have the advantage of stimulating T cells of any antigen specificity. Monensin was used to block intracellular transport mechanisms, thereby leading to an accumulation of cytokines in the cells. After incubation, the cells were stained with PE-Cy5-conjugated anti-CD4monoclonal antibodies at room temperature in the dark for20min. The cells were next stained with FITC-conjugated anti-interferon (IFN)-y monoclonal antibodies, PE-conjugated anti-IL-17monoclonal antibodies and APC-conjugated anti-IL22monoclonal antibodies after fixation and permeabilization. All the antibodies were from eBioscience, San Diego, CA, USA. Isotype controls were given to enable correct compensation and confirm antibody specificity. Stained cells were analyzed by flow cytometric analysis using a FACScan cytometer equipped with CellQuest software (BD Bioscience PharMingen).(3)Peripheral blood was collected into heparin-anticoagulant vacetainer tubes. Plasma was obtained from all subjects by centrifugation and stored at-80℃for determination of cytokines. IL-22and IL-17levels were determined with a quantitative sandwich enzyme immunoassay technique in accordance with the manufacturer's recommendations (lower detection limit9pg/ml; eBioscience).(4)Disease activity score in28-joints (DAS28)[26] was calculated in our study. Each patient with active RA was defined by a DAS28score≥2.6. At the time of clinical assessment for disease activity, blood samples were collected for the measurement of levels of C-reactive protein (CRP).Results(1)①We analyzed the frequency of Th22based on cytokine patterns after in vitro activation by PMA/ionomycin in short-term cultures. Th22was defined as CD4+IFNγTL17-IL-22+T cells to exclude Th1or Thl7cells. The percentage of Th22cells was significantly elevated in RA patients (1.56±0.86%) compared to OA patients (0.76±0.26%, P<0.05) or healthy controls (0.74±0.31%, P<0.05).②Plasma IL-22was investigated by ELISA. The level of IL-22was significantly increased in RA patients [median,36.62pg/ml (range,21.89-171.85)] compared with OA [median,23.68pg/ml (range,17.61-29.96pg/ml), P<0.05] and healthy controls [median,22.72pg/ml (range,13.61-32.90), P<0.05].③A positive correlation was found between Th22cells and plasma level of IL-22(r=0.67, P=0.034) in RA patients. However, Thl, pure Th17or Th17cells failed to show a statistical correlation with plasma level of IL-22(P=0.323, P=0.709or P=0.747). (2)①Pure Th17was defined as CD4+IFNγ-IL17+IL-22-T cells to exclude Th1or Th22cells. Similarly, we found significantly increased percentage of pure Th17cells in RA patients (2.14±0.86%) compared with OA patients (1.21±0.43%, P<0.05) and healthy controls (1.18±0.49%, P<0.05).②In addition, the frequency of Th17cells (CD4+IL17+) increased significantly in RA patients (2.96±1.10%) compared to OA patients (1.23±0.36%, P<0.05) or healthy controls(1.28±0.26%, P<0.05).③However, there was no significant difference regarding plasma IL-17between RA patients (15.56±3.77pg/ml, P>0.05)and OA patients (14.55±2.89pg/ml, P>0.05) or healthy controls (14.80±2.92pg/ml, P>0.05). For Th1cells, there was no significant difference between RA (10.86%±3.51%) patients and OA patients (10.39%±2.38%) or healthy controls (10.48%±2.12%) in this study.(3)①In RA patients, a significant positive correlation was found between Th22cells and pure Th17cells (r=0.52, P=0.003) as well as Th17cells(r=0.51, P=0.004).②However, Th1cells failed to show a significant correlation with Th22cells (P=0.751), pure Th17cells (P=0.940) and Th17cells (P=0.884).(4)②In patients with RA, there were positive correlations between the percentage of Th22cells and CRP level or DAS28(r=0.619, P<0.01or r=0.518, P=0.003respectively).②Consistently, positive correlations were also found between the percentage of pure Th17cells (r=0.879, P<0.001or r=0.690, P<0.001respectively) or Th17cells (r=0.897, P<0.001or r=0.697, P<0.001respectively) and CRP level as well as DAS28.③However, the percentage of Th1cells was not correlated with either CRP level or DAS28(P=0.856or P=0.634), and plasma level of IL-22or IL-17failed to show a statistical correlation with CRP level or DAS28(P=0.660and P=0.544or P=0.859and P=0.690).Conclusions(1)Our data demonstrated that the expression of Th22cells,Th17cells and pure Th17were significantly higher in the peripheral blood of RA patients compared with OA patients and healthy controls. Moreover, the percentages of Th22cells, Th17cells and pure Th17were elevated consistently. (2) Our data demonstrated that the level of IL-22was significantly higher in the plasma of RA patients compared with OA patients and healthy controls. In addition, the percentage of Th22cells in peripheral blood and level of IL-22in plasma were elevated consistently.(3) Our data demonstrated that Th22cells, pure Th17cells and Thl7cells were involved in the disease activity of RA.In summary, the increased frequencies of Th22cells, Thl7cells and pure Th17cells in the peripheral blood of RA patients suggested the roles of Th22cells, Th17cells and pure Th17cells in the pathogenesis of RA. Blockade of Th22cells might be of clinical profits in RA patients. Background:Ankylosing spondylitis (AS) is a chronic inflammatory disease that is characterized by mainly involving bilateral sacroiliitis and axial joints, but sometimes peripheral joints and extra-articular organs are also involved. Rheumatoid arthritis (RA), which represents an example of autoimmunity disease, is another form of arthritis. The abnormality of T cells is implicated in the pathogenesis of many autoimmune diseases, and many autoimmune diseases, especially arthritis, were considered to be mainly driven by Thl cells. A new IL-17-producing T cell subset, termed Th17cells, has been described in recent years. It has been established that Thl7cells play critical roles in several animal models of autoimmunity, such as experimental allergic encephalomyelitis (EAE) and murine arthritis models. Besides, Th17cells are considered to be involved in many human inflammatory diseases, including multiple sclerosis, psoriasis and inflammatory arthritis. As to AS and RA, increased Th17cells were found in PBMC from patients with AS and RA. IL-17, sccreted mainly by Th17cells, is a cytokine shown to stimulate RA synovial fibroblast (RASF) to release several mediators of joint inflammation including IL-6, IL-8, GM-CSF and PGE2. Moreover, elevated serum levels of IL-17and IL-23has been reported in AS which is one of the forms of arthritis.IL-22, a member of IL-10cytokine family, exerts its effects via a heterodimeric transmembrane receptor complex consisting of IL-10R2and IL-22R1. IL-22has been believed as an important player in regulating inflammatory responses associated with many inflammatory diseases. Higher expression of IL-22mRNA was observed in psoriatic skin lesion, and elevated serum IL-22levels were found in patients with psoriasis. In addition, the involvement of IL-22in other inflammatory diseases such as inflammatory bowel disease also proves its proinflammatory roles. However, diminishing intestinal inflammatory in a mouse model of ulcerative colitis and providing protection to hepatocytes during acute liver inflammation by IL-22demonstrate its anti-inflammatory properties. The situation of IL-22was not completely consistent in autoimmune diseases. Consistent with psoriasis, increased IL-22has also been found in serum samples from RA and Crohn disease patients. On the contrary, decreased plasma IL-22levels were found in patients with SLE. So, diverse pathogenic mechanisms and tissue microenvironments may result in different contributions of IL-22in autoimmune disease development. The precise pathophysiologic function of IL-22remains unclear, and the involvement of IL-22in AS and RA remains to be established.Th22subset is a more recently identified new human T helper subset, which is characterized by abundant secretion of IL-22but not IL-17or IFN-y. Th22cells express the chemokine receptors CCR4, CCR6and CCR10. Moreover, this newly identified CD4+T cells clones have low or undetectable expression of Thl and Th17transcription factor T-bet and ROR Y t, and arylhydrocarbon receptor (AHR) has been considered to be the key transcription factor of Th22subset. In addition, naive T cells differentiate toward the Th22phneotype in the presence of IL-6and TNF-α. All of above provide strong evidence that Th22cells represent an independent and terminally differentiated T cells subtype. It has been reported that Th22cells were detected in psoriatic skin lesions. Moreover, the increasing circulating Th22cells suggest that Th22cells may be implicated in the pathogenesis of psoriasis which is a chronic inflammatory disease. In addition, elevated Th22cells in peripheral blood of RA patients have also been reported in our previous study. Thus, the involvement of Th22cells in other chronic inflammatory diseases needs to be further investigated.The roles of both Th22cells and IL-22in the pathogenesis of ankylosing spondylitis and rheumatoid arthritis are still unclear and remain to be clarified. Therefore, to investigate their roles in the pathogenesis of AS and RA, we examined the frequencies of Th22cells in peripheral blood as well as the levels of plasma IL-22 of both AS and RA patients, and assayed their correlations with disease activity in this study.Objective:To examine and compare the frequencies of Th22cells, Thl7cells and Thlcells in the peripheral blood of AS, RA, OA patients and healthy people; To examine and compare the levels of IL-22and IL-17in the plasma of AS, RA, OA patients and healthy people; To analyze the correlations between each T cell subset or cytokine and the disease activity in AS and RA patients. To investigate the roles of Th22cells and Th17cells in the pathogenesis of AS and RA.Materials and Methods:(1)A total of32patients with AS according to the modified New York criteria were recruited in this study. The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score were measured for the patients with AS. All patients were HLA-B27-positive. This group consisted of27men and5women, with mean±SD disease duration of8.6±5.9years. The mean age of the patients was36.6±10.2years. A total of20patients with active RA according to the criteria of the American College of Rheumatology were included in this study. The DAS28score were measured for the patients with RA. This group consisted of16women and4men, with mean±SD disease duration of8.9±3.9years. The mean age of the patients was47.5±9.2years. The demographic and key clinical information of AS and RA patients are summarized in Table1and Table2. All of the patients did not receive immunosuppressive or immunomodulatory drugs for at least2months when sampling. The major previous treatment of AS and RA patients were shown in Table S1and Table S2. Ten osteoarthritis (OA) patients (3females and7males; mean age48.9±10.3years) as disease controls and twenty healthy controls (5females and15males; mean age37.9±9.1years) were also recruited in the study, and all of them did not have any rheumatologic conditions.(2)Intracellular cytokines were studied by flow cytometry to reflex the cytokine-producing cells. Briefly, heparinized peripheral whole blood (400μl) with an equal volume of Roswell Park Memorial Institute1640medium were incubated for 4h at37℃,5%C02in the presence of25ng/mL of phorbol myristate acetate (PMA),1μg/mL of ionomycin, and1.7pg/ml Golgiplug(Monensin; all from Alexis Biochemicals, San Diego, CA, USA). PMA and ionomycin are pharmacological T-cell-activating agents that mimic signals generated by the T-cell receptor (TCR) complex and have the advantage of stimulating T cells of any antigen specificity. Monensin was used to block intracellular transport mechanisms, thereby leading to an accumulation of cytokines in the cells. After incubation, the cells were stained with PE-Cy5-conjugated anti-CD4monoclonal antibodies at room temperature in the dark for20min. The cells were next stained with FITC-conjugated anti-interferon (IFN)-y monoclonal antibodies, PE-conjugated anti-IL-17A monoclonal antibodies and APC-conjugated anti-IL22monoclonal antibodies after fixation and permeabilization. All the antibodies were from eBioscience, San Diego, CA, USA. Isotype controls were given to enable correct compensation and confirm antibody specificity. Stained cells were analyzed by flow cytometric analysis using a FACScan cytometer equipped with CellQuest software. Th22, Th17, Th1and Th1/Th17cells were defined as CD4+IFNγ-IL17-IL-22+, CD4+IFNγ-IL17+, CD4+IFNy+and CD4+IFNγ+IL17+T cells respectively.(3)Peripheral blood was collected into heparin-anticoagulant vacetainer tubes. Plasma was obtained from all subjects by centrifugation and stored at-800C for determination of cytokines. Plasma IL-22levels were determined with a quantitative sandwich enzyme immunoassay technique in accordance with the manufacturer's recommendations.(4)BASDAI score of AS patients and disease activity score in28-joints (DAS28) of RA patients was calculated in our study. At the time of clinical assessment for disease activity, blood samples were collected for the measurement of levels of C-reactive protein (CRP) and ESR.Results:(1)①The percentage of Th22cells was significantly elevated in AS (1.2±0.42%) and RA (1.37±0.49%) patients compared to OA patients (0.70±0.19%) or healthy controls (0.68±0.18%).②In addition, we also quantified the number of Th22cells per volume (50μL) of peripheral blood. The number of Th22cells was significantly increased in AS (204±34) and RA (211±43) patients compared with healthy controls (123±23) after stimulation with phorbol myristate acetate, ionomycin, and monensin for4h.③lasma levels of IL-22were examined by ELISA. The levels of IL-22were significantly increased in AS (41.03±16.00pg/ml) and RA (44.53±29.84pg/ml) patients compared to OA patients (24.53±3.45pg/ml) and healthy controls (25.33±3.75pg/ml).④A positive correlation was found between Th22cells and plasma levels of IL-22in AS (r=0.743, P<0.001) and RA (r=0.548, P=0.027) patients. Moreover, positive correlations were also found between IL-22plasma levels and Th17cells (r=0.587, P=0.045) in AS patients. However, no corresponding correlation was found in RA patients (P=0.801).(2)①We found significantly increased percentage of Th17cells in AS (2.58±0.86%) and RA (2.57±0.72%) patients compared with OA patients (1.15±0.31%) and healthy controls (1.07±0.26%).②Consistently, the number of Thl7cells per volume (50μL) of peripheral blood was significantly increased in AS (320±36) and RA (337±39) patients compared with healthy controls (214±26) after stimulation with phorbol myristate acetate, ionomycin, and monensin for4h.③However, there was no significant difference regarding plasma IL-17between each group.(AS:16.28±4.25, P>0.05; RA:15.02±4.60, P>0.05; OA:14.80±2.88, P>0.05; HC:14.39±2.72, P>0.05).④As to Th1cells, there was no significant difference between each group.(AS:11.05±3.41%, P>0.05; RA:11.13±4.09%, P>0.05; OA:10.73±2.50%, P>0.05; HC:10.37±2.00, P>0.05)(3)①Though most Th17cells did not simultaneously express both IL-22and IL-17, the percentage of CD4+IFNγ-IL17+IL-22+T cells was significantly increased in AS (0.63±0.34%) and RA (0.65±0.29%) patients compared with OA patients (0.20±0.04%) and healthy controls (0.19±0.05%).③In addition, ratios of Th1/Th17cells were significantly increased in AS patients (0.63±0.35%) and RA patients (0.66±0.21%) compared to OA patients (0.22±0.07%) and healthy controls (0.22±0.06%). (4)①In AS patients, there was a significant positive correlation between Th22cells and Thl7cells (r=0.676, P<0.001).②Similarly, a positive correlation was also found between Th22cells and Th17cells (r=0.46,P=0.041) in RA patients.③However, Thl cells failed to show a significant correlation with Th22cells and Th17cells.(5)①In patients with RA, there were positive correlations between the percentage of Th22cells and CRP level or DAS28(r=0.576, P=0.008or r=0.544, P=0.013respectively).②Consistently, positive correlations were also found between the percentage of Th17cells (r=0.709, P<0.001or r=0.706, P<0.001respectively) and CRP level as well as DAS28.③However, the percentage of Thl cells was not correlated with either CRP level or DAS28(P=0.105or P=0.205), and plasma level of IL-22or IL-17failed to show a statistical correlation with CRP level or DAS28(P=0.622and P=0.357or P=0.317and P=0.872) in RA patients.④In patients with AS, there was no correlation between the percentage of Th22cells and clinical parameters, including ESR (P=0.964), CRP (P=0.393) and BASDAI score (P=0.226). Consistently, no correlation was found between plasma level of IL-22and clinical parameters in AS patients, including ESR (P=0.918), CRP (P=0.862) and BASDAI score (P=0.320).⑤Correlation analysis between the percentage of Th17cells and clinical parameters also showed no association (P=0.189, P=0.852and P=0.733respectively).Conclusions(1) Our data demonstrated that the expression of Th22cells and Th17cells were significantly higher in the peripheral blood of AS and RA patients compared with OA patients and healthy controls. Moreover, the percentages of Th22cells and Th17cells were elevated consistently.(2) Our data demonstrated that the level of IL-22was significantly higher in the plasma of AS and RA patients compared with OA patients and healthy controls. In addition, the percentage of Th22cells in peripheral blood and level of IL-22in plasma were elevated consistently. (3) Our data demonstrated that the percentage of Th17cells in peripheral blood and level of IL-22in plasma were elevated consistently in AS patients but not in RA patients.(4) Our data demonstrated that Th22cells and Th17cells were involved in the disease activity of RA.In summary, the increasing percentages of Th22cells and Th17cells and the elevation of IL-22may play important roles in the pathogenesis of AS and RA. Thus, Th22cells and IL-22as well as Th17cells may prove to be a promising therapeutic targets for AS and RA.
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