| BackgroundRheumatoid arthritis is a chronic inflammatory disease characterized by persistent synovitis and bone destruction in multiple joints. The exact pathogenesis of RA remains unknown. The abnormality of T cells is believed to contribute greatly to joint pathology in RA. CD4+ T helper cell has already been considered to be involved in the process of tissue destruction of RA, and the activated T helper cells could secrete cytokines that regulate the inflammatory process. RA has traditionally deemed to be a Thl-associated disease. Recently, the new effector T cell subsets, named Th17 cell, have been described and has been deemed to be implicated in the pathogenesis of RA. Th17 cell has been demonstrated to be elevated in both peripheral blood and synovial fluid of patients with RA, which suggest that Th17 cell play a crucial role in RA. Moreover, the level of Th17 cell was found to be higher in synovial fluid in comparison to peripheral blood in RA. IL-17A is a representative Th17 cytokine that is implicated in the development of RA. IL-17A is shown to induce the production of TNF-a, which is a main pathogenic cytokine in RA. It has been demonstrated that IL-17A, synergizing with TNF-a and IL-1, contributes to the inflammatory process of RA.Treg cells, which are characterized by expressing Foxp3 in the nuclei, has the ability to suppress CD4+ and CD8+ T cell by means of cell-contact dependent mechanisms. Moreover, Treg cells also play crucial roles in T-cell tolerance by releasing anti-inflammatory cytokines including IL-10 and TGF-p. The mounting evidences strongly indicate that Treg cell is involved in the pathogenesis in RA. The frequency of Treg cell in the PB of RA patients is still conflicting. However, the situation of Treg in synovial fluid is clear, and the Treg percentage in the synovial fluid of RA patients is higher than that in the peripheral blood. Because Treg cells specially express the chemokine receptors CXCR4, CCR4 and CCR8, Treg cells can migrate to and retained in the bone marrow through CXCL12/CXCR4 signal. Otherwise, the situation of Treg cell in bone marrow environments in RA patients remains unclear.It has been demonstrated that the inflammation may target the bone tissue and result in the adverse effects on structure and function in inflammatory bone disease. Association between immune cells and bone tissues has been increasingly revealed in recent years. At the bone level, RA is characterized by focal erosions of marginal and subchondral bone as well as generalized osteoporosis. Bone marrow is believed as a primary lymphoid organ, which is deemed to retain memory T cells, suggesting that this compartment is a site for migration and/or selective retention of memory T cell. The immune and skeletal systems have already been demonstrated to share many similar cytokines, signaling molecules, transcription factors and membrane receptors. Bone marrow abnormality was observed in many inflammatory (autoimmune) diseases. For example, bone marrow dysplasia were reported in SLE patients, which appear to be associated with disease activity. In addition, bone marrow was observed to function as a reservoir for CD4+ memory T cell, which plays a critical role in inducing colitis in murine colitis models. Under physiologic condition conditions, mature CD4+ T cell exhibit extensive migration from the blood to the bone marrow and vice versa. As T helper cells are considered to exist in bone marrow, their contribution in situ is probable.Both local bone erosion and juxta-articular osteoporosis are the common abnormal pathologic changes at the bone level in RA. Therefore, the profiles of related T cell in peripheral blood can not exactly clarify the local condition of disease, while the research of these cells in bone marrow microenvironment were deemed to be better exploring the immune change in situ and illuminate the local pathogenic mechanism. There are plenty of reports about the situation of T cell subset (Th1,Th17 and Treg cells) in the peripheral blood and the synovial fluid environment. However, relatively little is known about the profile of these T cell in the bone marrow environments of RA. In order to explore the abnormality of T cells in bone marrow environment of RA, we examined the frequency of Thl, Th17 and Treg cells in bone marrow of patients with RA and investigated their correlation with disease activity in this study.ObjectiveSeveral reports of the frequency of T-helper (Th) 17, Treg and Thl cells in peripheral blood (PB) of patients with rheumatoid arthritis (RA) have been published. The profiles of T helper subset in peripheral blood can not exactly reflect the local bone condition of RA. In order to exploring the immune change in situ and illuminate the local pathogenic mechanism, we analyzed the frequencies of Th17, Treg and Th1 cells in bone marrow of RA patients and determined their correlation with disease activity in this study. Then we can provide the more comprehensive understanding of disorder of osteoimmunology.Materials and Methods(1)A total of 26 patients 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 DAS28 score≥2.6. This group consisted of 21 women and 5 men, with mean ± SD disease duration of 12.9±7.6 years. The mean age of the patients was 62.4±7.4 years. The demographic and key clinical information of the RA patients are summarized in Table 1. Eleven osteoarthritis (OA) patients(8 females and 3 males; mean age 64.1±3.4 years) as disease controls were also recruited in the study. As healthy controls,10 trauma patients (7 females and 3 males; mean age 62.3±5.1 years) who had no systemic inflammatory disease or immune abnormalities were also studied. Paired samples of bone marrow and PB were obtained from the same patient with RA, OA and healthy controls. Bone marrow samples were obtained from RA and OA patients during total knee arthroplasty. None of OA patients and healthy controls had any systemic inflammatory diseases or immunological background. Enrollment took place between March,2013 and September,2015 in the Department of Orthopedics, Shandong Provincial Qianfoshan Hospital, Shandong University, China. The study was approved by the Institutional Review Boards of the hospital. Informed consent was obtained from each patient before being included in the study.(2) Sample preparations.5 ml bone marrow blood was aspirated from the tibial proximal epiphysis by needle puncture at the time of operation. Simultaneously,5 ml sample of venous blood was collected from the matched patient. In the course of bone marrow blood collection, peripheral blood contamination is considered to be probable. Therefore, we performed a preliminary experiment by examining the T cell subsets of different volume bone marrow blood in the same patients in this study. When the volume obtained from the bone marrow did not exceed 5ml, the frequencies of T cell subsets showed no significant difference and the bone marrow blood was not thought to be contaminated by peripheral blood (data not shown). So the first 5 ml of bone marrow blood was believed to be properly exhibit the profile of T cell subsets in the bone marrow and could be analyzed in our study.(3) Intracellular cytokines were studied by flow cytometry to reflex the cytokine-producing cells. Briefly, heparinized peripheral whole blood (400 pi) and bone marrow blood with an equal volume of Roswell Park Memorial Institute 1640 medium were incubated for 4 h at 37℃,5% CO2 in the presence of 25 ng/mL of phorbol myristate acetate (PMA),1 p.g/mL of ionomycin, and 1.7 μg/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-CD4 monoclonal antibodies at room temperature in the dark for 20 min. The cells were next stained with FITC-conjugated anti-interferon (IFN)-y monoclonal antibodies, PE-conjugated anti-IL-17 monoclonal antibodies and APC-conjugated anti-TNF-a monoclonal 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).(4) We evaluated CD4+CD25+Foxp3+ T cells as Treg cells using the Human Regulatory T-cell Staining Kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s protocol. Before staining, peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMC) were isolated by centrifugation over Ficoll-Hypaque gradients. Then, the single-cell suspension was incubated with a cocktail of anti-CD4-FITCmonoclonal antibody and anti-CD25-APC monoclonal antibody for 30 min in the dark at 4℃ to stain the surface. After being washed with 2 ml cold flow cytometry staining buffer, the PBMCs were incubated with 1 ml freshly prepared eBioscience Foxp3 fixation/permeabilization buffer for 60 min at 4℃ in the dark. The cells were washed with 2 ml freshly prepared 1×permeabilization buffer twice. The cells were then blocked in normal rat serum for 15 min and stained using anti-Foxp3-PEmonoclonal anti-body (PCH101) or PE-conjugated rat IgG2a (used as an isotype control) for 45 min in the dark at 4℃. After washing the cells twice, we added a 300 μl cold flow cytometry staining buffer to resuspend the cells. Stained cells were analyzed by flow cytometric analysis using a FACScan cytometer equipped with CellQuest software (BD Bioscience PharMingen).(5) Disease activity score in 28-joints (DAS28) [21] was calculated in our study. Each patient with active RA was defined by a DAS28 score≥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) The percentage of Th17 cells in peripheral blood from RA patients was significantly elevated compared to that in peripheral blood from OA patients (P＜0.05) or healthy controls (P＜0.05), and the percentage of Th17 cells in bone marrow blood from RA patients was significantly higher compared to that in bone marrow blood from OA patients (P＜0.05) or healthy controls (P＜0.05). However, the percentages of Th17 cells in peripheral blood from OA patients and healthy controls were not significantly different from those in bone marrow from OA patients and healthy controls, respectively. By contrast, the percentage of Th17 cells in bone marrow from RA patients was significantly higher than that in the paired peripheral blood from RA patients (P＜0.05).(2)The frequencies of Thl cells in the peripheral blood of RA patients were not markedly increased compared with those of OA patients or healthy controls, but the frequencies of Thl cells in bone marrow of RA patients were significantly higher than those in OA patients (P< 0.05) or healthy controls (P＜0.05). Consistent with the result of Th17 cells, the percentage of Thl cells in bone marrow was significantly higher than that in paired peripheral blood samples from RA patients (P＜0.05). In addition, the frequencies of CD4 T cells co-expressing IFN-γ and IL-17 (named Th17/Th1 cells) were also analyzed. The percentage of Th17/Th1 cells in peripheral blood of RA patients was significantly elevated compared to that in OA patients (P＜ 0.05) or healthy controls (P＜0.05). Similarly, the percentage of Th17/Th1 cells in bone marrow of RA patients was significantly higher than that in OA patients (P＜ 0.05) or healthy controls (P＜0.05). In addition, the percentage of Th17/Th1 cells in bone marrow blood of RA patients was significantly increased compared to that in peripheral blood of RA patients (P＜0.05). The results indicate that frequencies of Thl cells and Th17/Th1 cells in bone marrow of RA patients are higher than those in peripheral blood of RA patients.(3) The data showed that the percentage of Treg cells in peripheral blood from RA patients was not significantly different from that from OA patients or healthy controls. By contrast, the percentage of Treg cells in bone marrow from RA patients was significantly lower than that from OA patients (P＜0.05) or healthy controls (P＜ 0.05). In addition, the percentage of Treg cells in bone marrow of RA patients was significantly lower than that in peripheral blood of RA patients (P＜0.05). The results suggest that frequencies of Treg cells in peripheral blood of RA patients were higher than those in bone marrow of RA patients.(4) In RA patients, frequencies of Thl (r=0.739, P＜0.05), Thl7 (r=0.752, P ＜0.05) and Th17/Th1 cells (r=0.574, P＜0.05) in peripheral blood were found to have significant positive correlation with those in bone marrow. The frequencies of TNF-a-producing T helper cells (r=0.740, P＜0.05), TNF-a-producing Thl cells (r= 0.697, P＜0.05) and TNF-a-producing Th17 cells (r=0.660, P＜0.05) in peripheral blood were also positively correlated with their frequencies in bone marrow of RA patients. In addition, frequency of Treg cells in bone marrow was positively correlated with that in peripheral blood of RA patients (r=0.792, P＜0.05). These results suggest that frequencies of each T subpopulation of RA patients have positive correlation between bone marrow and peripheral blood samples.(5) The data showed that Thl7 (r=0.611, P＜0.05, Thl (r=0.502, P＜0.05) and Thl7/Thl cells (r=0.424, P＜0.05) were inversely correlated with Treg cells in bone marrow of RA patients. Similarly, Treg cells exhibited negative correlation with TNF-a-producing Thl cells (r=0.602, P＜0.05) and TNF-a-producing Th17 cells (r =0.562, P＜0.05) in bone marrow of RA patients. The result indicates that T helper subset is negatively correlated with Treg subset in bone marrow of RA patients.(6) Th1 and Th17 cells were positively correlated with either CRP level or DAS28 in bone marrow (r=0.526, P＜0.05; r=0.632, P＜0.05 or r=0.447, P＜0.05; r=0.594, P＜0.05,respectively).Consistently, TNF-a-producing T helper cells, TNF-a-producing Thl cells and TNF-a-producing Th17 cells exhibited positive correlations with either CRP level or DAS28 in bone marrow (r=0.560, P＜0.05;r= 0.720, P＜0.05; r=0.529, P＜0.05 or r=0.622, P＜0.05; r=0.506, P＜0.05;r= 0.636, P＜0.05, respectively). On the contrary, Treg cells were inversely correlated with either CRP level or DAS28 in bone marrow (r=-0.630,P＜0.05 or r=-0.797,P ＜0.05, respectively). These results suggest that Th17 cells, Thl cells, TNF-a-producing T helper cells, TNF-a-producing Thl cells and TNF-a-producing Th17 cells have positive correlation with CRP level or DAS28 in bone marrow, while Treg cells have negative correlation with CRP level or DAS28.Conclusions(1) Our data demonstrated that the profiles of Th17, Thl and TNFa-producing Th cells were markedly increased in bone marrow of RA patients.(2) Our data demonstrated that the profiles of Treg cells were markedly decreased in bone marrow of RA patients.(3) Our data demonstrated that Th17, Thl and TNFa-producing Th cells correlated negatively with Treg cell in bone marrow of RA patients.(4) Our data demonstrated that Th17, Thl and TNFa-producing Th cells correlated positively with disease activity, but Treg cells correlated negatively with disease activity in RA.SignificanceOur study provided evidences that abnormal Th17, Treg and Th1 cells may play pathological role in the subchondral bone marrow of RA patients. In addition, our study highlights the local aberrant T helper cells in subchondral region of RA. Finally, pathogenic T helper cells in BM could be a potential target for immunotherapeutic strategies against inflammatory joint destruction in RA in the future.Backround:RA is a chronic inflammatory autoimmune disease characterized by articular cartilage and bone destruction. The interactions between immune cells and bone cells contribute to pathogenesis of inflammation and bone destruction in RA. Among these immune cells, activated CD4+ T cells have been implicated in bone damage associated with chronic inflammation. In autoimmune arthritis, the generation of osteoclasts is directly and indirectly regulated by CD4+ T cells migrating to the lesion of bone, which contributes to bone destruction. Th17 cells are important inflammatory CD4+ T cells that secret IL-17A and not IFN-y Thl7 cells were shown to be elevated in the peripheral blood and synovial fluid of RA patients, suggesting a pathogenic role of Th17 in RA. Additionally, Th17 cells were also shown to act as an osteoclastogenic helper T cells. IL-17, the main effective cytokine of Th17 cells, was associated with increased osteoclastogenesis by inducing RANKL expression on OBLs in RA. Th22 cells are recently identified inflammatory CD4+ T cell subset, which is characterized by production of IL-22 but not IL-17 or IFN-y. IL-22, a main signature cytokine of Th22 subset, was shown to promote osteoclastogenesis and enhance the bone destruction in arthritic mice. Elevated serum IL-22 was observed to be associated with disease activity in RA patients. Furthermore, recently serum levels of IL-22 exhibited to be related to the radiographic progression of RA patients, which suggested a pathogenic role for IL-22 in bone destruction of RA patients. Consistent with IL-17, neutralization of IL-22 could also result in a significant reduction of inflammatory cells and had similar effect on bone erosion. TNF-a, another crucial effective cytokine of Th22 cells, is a main pathogenic cytokine in RA. TNF-a has been considered to have destruction effect on bone. In addition, TNF-a produced by aberrant T helper cells have been shown to be implicated in the pathogenesis of bone loss in RA.Before the discovery of Th17 and Th22 subsets, inflammatory CD4+ T cell researched in RA focused on Thl cells, which secret IFN-y as their main effector cytokine. RA has traditionally believed to be a Thl-associated disease, and Thl cells were observed to be abundant in synovial fluid of RA patients. Activated Thl cells were shown to intensify osteoclastogenesis despite of the anti-osteoclastogenic effect of IFN-y. It is well known that systemic inflammation results in increased circulating inflammatory immune cells. The profiles of Th22, Th17 and Thl cells in peripheral blood of RA patients have already been analyzed in our previous studies. Local bone erosion is generally believed to be driven by the inflammatory synovium in RA. In the past, most of immunological studies were concentrated on T helper cells subset in peripheral blood, synovial fluid and synovium. Recent attention has been focused on the subchondral bone of the joints. According to the results from magnetic resonance imaging (MRI) of RA joints, bone marrow is under attack and associated with bone erosion in the early course of disease when synovitis do not spread to subchondral bone tissue cross the relatively intact cartilage. Therefore, we speculated that the pathologic change of the bone marrow in joint destruction is independent to a certain extent, and BM may play several unexpected roles in the pathogenesis of RA. Despite the obvious destructive process in subchondral bone of RA, relatively little is known about the profiles of CD4+ cells subset in subchondral BM in this disease.The profiles of T helper subset in peripheral blood can not exactly reflect the local bone condition of RA. In order to exploring the immune change in situ and illuminate the local pathogenic mechanism, we analyzed the frequencies of Thl, Th17 and Th22 cells in bone marrow of RA patients and determined their correlation with disease activity in this study.Objective:Th22, Th17 and Thl cells are three main proinflammatory Th cell subsets. Several reports of the frequency of Th 22, Th17 and Thl cells in peripheral blood (PB) of patients with rheumatoid arthritis (RA) have been published. The profiles of T helper subset in peripheral blood can not exactly reflect the local bone condition of RA. In order to exploring the immune change in situ and illuminate the local pathogenic mechanism, we analyzed the frequencies of Th22, Th17 and Thl cells in bone marrow of RA patients and determined their correlation with disease activity in this study. Then we can provide the more comprehensive understanding of disorder of osteoimmunology.Materials and Methods:(1) A total of 40 patients 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 DAS28 score≥2.6. This group consisted of 33 women and 7 men, with mean ± SD disease duration of 12.8±6.5 years. The mean age of the patients was 62.2±7.0 years. The demographic and key clinical information of the RA patients are summarized in Table 1. Nine osteoarthritis (OA) patients(7 females and 2 males; mean age 63.8±3.8 years) as disease controls were also recruited in the study. As healthy controls,9 trauma patients (7 females and 2 males; mean age 62.9±4.7 years) who had no systemic inflammatory disease or immune abnormalities were also studied. Paired samples of bone marrow and PB were obtained from the same patient with RA, OA and healthy controls. Bone marrow samples were obtained from RA and OA patients during total knee arthroplasty. None of OA patients and healthy controls had any systemic inflammatory diseases or immunological background. Enrollment took place between March,2013 and December,2015 in the Department of Orthopedics, Shandong Provincial Qianfoshan Hospital, Shandong University, China. The study was approved by the Institutional Review Boards of the hospital. 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 (400 μl) with an equal volume of Roswell Park Memorial Institute 1640 medium were incubated for 4 h at 37℃,5% CO2 in the presence of 25 ng/mL of phorbol myristate acetate (PMA), 1 μg/mL of ionomycin, and 1.7 μg/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-CD4 monoclonal antibodies at room temperature in the dark for 20 min. The cells were next stained with FITC-conjugated anti-interferon (IFN)-y monoclonal antibodies, PE-conjugated anti-IL-17 monoclonal antibodies and APC-conjugated anti-IL22 monoclonal 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 and bone marrow blood was collected into heparin-anticoagulant vacetainer tubes. Plasma of different sample was obtained from all subjects by centrifugation and stored at -80℃ for determination of cytokines. IL-22, IL-17, IFN-y levels were determined with a quantitative sandwich enzyme immunoassay technique in accordance with the manufacturer’s recommendations respectively (lower detection limit:IL-22,5 pg/ml; IL-17,0.5 pg/ml; IFN-y,0.99 pg/ml, eBioscience).(4)Disease activity score in 28-joints (DAS28) [21] was calculated in our study. Each patient with active RA was defined by a DAS28 score≥2.6.Results(1) The percentage of Th22 cells in peripheral blood from RA patients was significantly elevated compared to that in peripheral blood from OA patients (P＜ 0.05) or healthy controls (P＜0.05), and the percentage of Th22 cells in bone marrow blood from RA patients was significantly higher compared to that in bone marrow blood from OA patients (P＜0.05) or healthy controls (P＜0.05). Meaningfully, the percentage of Th22 cells in bone marrow from RA patients was significantly higher than that in the paired peripheral blood from RA patients (P< 0.05).(2) We found significantly increased percentage of Th17 cells in peripheral blood from RA patients compared to that in OA patients (P＜0.05) or healthy controls (P＜0.05). Consistently, Th17Th1 cells in peripheral blood from RA patients was also significantly elevated compared to that in peripheral blood from OA patients (P< 0.05) or healthy controls (P＜0.05). For Thl cells, however, there was no significant difference in peripheral blood between each group in this study. Similar to the trend of the results in peripheral blood, Th17 and Th17/Th1 cells in bone marrow were significantly increased in RA patients compared with OA patients (P＜0.05 and P＜0.05) and healthy controls (P＜0.05 and P＜0.05). Unlike the situation in peripheral blood, Thl cells were observed to be significantly elevated in bone marrow of RA patients compared to OA patients (P＜0.05) and healthy controls (P＜0.05) in our study. The percentage of Th17, Th1 and Th17/Th1 cells in bone marrow from RA patients was significantly higher than these in the paired peripheral blood from RA patients (P＜0.05; P＜0.05 and P＜0.05,respectively).(3) The levels of IL-22 and IL-17 in peripheral blood plasma were significantly elevating in RA patients compared with OA patients (P＜0.05 and P＜0.05) and healthy controls (P＜0.05 and P＜0.05). Accordingly, the levels of IL-22 and IL-17 in bone marrow blood plasma were also significantly elevating in RA patients compared with OA patients (P＜0.05 and P＜0.05) and healthy controls (P＜0.05 and P＜0.05). By contrast, no significant differences of IFN-y level in both peripheral blood plasma and bone marrow blood plasma were observed among each group in this study.In this study, the levels of IL22 and IL17 in bone marrow plasma from RA patients were significantly higher than these in the paired peripheral blood plasma from RA patients (P＜0.05 and P＜0.05, respectively). On the contrary, IFN-y was observed to be lower in peripheral blood plasma than that in bone marrow blood plasma from RA patients, but the difference was not statistical.(4) In RA patients, positive correlations were found between the frequency of Th22 cells and plasma level of IL-22 in both peripheral blood (r=0.71, P＜0.001) and bone marrow blood (r=0.43, P=0.006) of RA patients. Consistently, Th17 cells also showed a positive correlation with IL-17 in peripheral blood (r=0.542, P< 0.001) and bone marrow (r=0.633, P＜ 0.001) of RA patients. Although there was a positive correlation between Thl cells and IFN-y level in peripheral blood (r=0.67, P＜ 0.001), Thl cells failed to show a statistical correlation with IL-17 level in bone marrow (P= 0.169).(5) In bone marrow of RA patients, significant positive correlations were found between Th22 cells and Th17 cells (r=0.452, P=0.003) or Thl cells (r=0.56, P 0.001). In addition, Th17 cells also show a positive correlation with Thl cells (r=0.451,P=0.003).(6)In patients with RA, there were positive correlations between Th22, Th17 as well as Thl cells of bone marrow and DAS28 score (r= 0.646, P＜0.001; r= 0.572, P ＜0.001 and r=0.459, P=0.003 respectively). Consistently, positive correlations were also found between the level of IL-22 or IL-17 in bone marrow plasma and DAS28 (r= 0.442, P=0.004 or r=0.484, P=0.002 respectively). However, the level of IFN-y in bone marrow plasma was not correlated significantly with DAS28 in this study (P=0.063).Conclusions(1) The profiles of Th22, Th17 and Thl cells were markedly increased in bone marrow of RA patients.(2) The levels of IL-22 and IL-17 were markedly elevated in bone marrow plasma of RA patients.(3) The correlations of Th22, Th17 and Thl cells correlated positively with each other in bone marrow of RA patients. These three Th cells were increased consistently in bone marrow of RA patients. Furthermore, IL-22 and IL-17 levels were elevated correspondingly.(4) In RA, Th22, Th17 and their main effective cytokines IL-22 as well as IL-17 in bone marrow correlated positively with disease activity.SignificanceTh22, Th17 and Thl cells are three main proinflammatory Th cell subsets. Our study provided evidences that aberrant Th22, Th17 and Thl cells as well as their related cytokines may play important roles in the mechanism of the pathogenesis of RA patients. In addition, our study highlights the local aberrant T helper cells and their related cytokines in subchondral region of RA. Finally, the proinflammatory T helper cells and their related cytokines in BM could be a potential target for immunotherapeutic strategies against inflammatory joint destruction in RA. |
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