| BackgroundRheumatoid Arthritis (RA) is involving the joint chronic autoimmune disease, Its pathological characteristics is the main inflammatory cell infiltration and hyperplasia of synovial cells, seriously affecting people’s health status and quality of life. To date, RA etiology and pathogenesis is not very clear, most scholars think and infection and cellular immune status. HMGB1is a high mobility group protein superfamily member, and a rich source of non-histone nuclear proteins. Secreted to the extracellular HMGB1was found to be an important inflammatory cytokines. Recent studies suggest that, HMGB1is also involved in autoimmune diseases, such as RA, SLE etc. Th17as a new found inflammatory cells, mainly involved in the inflammatory and autoimmune diseases. Our previous studies showed that, in patients with rheumatoid arthritis, the existence of Th17cells upregulate and the advantages of HMGB1expression in the microenvironment condition, which have close relationship with rheumatoid arthritis occurrence and development. This study aims is to further explore the HMGBl on Th17regulation of immune cells and the related signal transduction pathways, and research the relationship between them and rheumatoid arthritis in the mechanism of action, for establishing a new way on treatment of rheumatoid arthritis.Toll-like receptor2(TLR2) signaling is thought to be essential for the inflammatory response and for immune disorders. In recent studies, enhanced HMGB1and TLR2expression have been found in rheumatoid arthritis (RA) respectively. The aim of this study is to explore whether HMGB1stimulating can up-regulate the expression of TLR2on CD14+monocytes from RA patients, and to clarify the subsequent events involving Th17cells and Th17cell-associated cytokines changes and find TLR2dependent signaling pathway in CIA models.ObjectiveTo establish chicken type Ⅱ collagen-induced arthritis (CIA) mouse model, analyze TLR distribution on PBMC from mice with CIA and RAW264.7cell line, and its related serum cytokine changes in expression levels. Further in the detection of rheumatoid arthritis patients peripheral blood cytokine changes in monocytes and TLR2, IL-17expression levels. Based on detecting the expression of TLR2in mononuclear cells in patients with rheumatoid arthritis, and the changes of Th17cells levels in patients, the relationship between the expression levels of TLR2on monocytes and Th17cells will be studied.Methods1. Induction of murine collagen induced arthritis model8to10-wk-old DBA/1LacJ male mice (supplied by Shanghai SLAC laboratory animal company) were used for this study. DBA/1LacJ mice were injected intradermally at the base of the tail with100μg of chicken type Ⅱ collagen (Sigma-Aldrich), which was emulsified in equal volumes of Freund’s complete adjuvant (2mg/ml Mycobacterium tuber-culosis) on day0. The mice were examined for the development and severity of arthritis from day1to50. Disease severity was scored on a scale from0to4by visual inspection of paws, and score criteria were as follows:0, No evidence of erythema and swelling.1. Erythema and mild swelling confined to the mid-foot (tarsals) or ankle joint.2. Erythema and mild swelling extending from the ankle to the mid-foot.3. Erythema and moderate swelling extending from the ankle to the metatarsal joints.4. Erythema and severe swelling encompass the ankle, foot, and digits.2. Fluorescence quantitative PCR method (qRT-PCR) was used to detecte the mRNA expression levels of IL-17, IL-6, IL-23, TGF beta and TLR2in the peripheral blood mononuclear cells or mononuclear cellsTo determine of TLR2expression on monocyte cells also stimulated cells with HMGB1at1,2,4,8and12hour time courses after PBMC the cells collected with Trizol (Invitrogen, USA) and after extraction of mRNA, the levels of TLR2were measured with real time PCR. Real time PCR was performed with5μL SYBER Green mix (Bio-Rad, USA); and0.4μL forward and reverse primer and0.06μL Tag polymerase; and2.5μL dd H20; and2μL cDNA templates making a total volume of10μL using a7500fast Real Time PCR system(Applied Biosystem, USA). β-actin was used as an internal control. Temperatures used were940C for2min;940C for10sec;600C for15sec;720C for30min;950C for2min;720C for1min and95 OC for30sec. The primers for TLR2were5/-ttgtcccgtgcaaacttgccggggagga-3/and5/-aagtcccgttattacttgccggttagga-3/, respectively.3. Immunofluorescence staining for analyzing IL-17secreting cellsHuman macrophage cells from RA patients and healthy control groups fixed after PBMC and after24hours stimulation with HMGB1the anti-IL-17antibody were applied for2h at room temperature. After washing PE labeled secondary antibody were added for1h.The slides were viewed with a fluorescence microscope (Olympus, Japan) and analyzed using Image J software.4. FACS analysisPeripheral blood mononuclear cells (PBMCs) were prepared from heparinized blood samples by centrifugation over Ficoll-Hypaque density gradients (Pharmacia, Uppsala, Sweden). Cell surface expression of CD14, CD45, and TLR2was analyzed by cell surface staining and flow cytometric analysis, as described previously. PBMC suspensions were incubated with biotinylated human anti-TLR-2monoclonal antibody (mAb)(TLR2.1; eBioscience, San Diego CA); Cells were washed and then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD14mAb (Leu M3; Becton Dickinson, Franklin Lakes, NJ) and PE-conjugated anti-TLR2mAb (Leull; Becton Dickinson). After washing, cells were resuspended in1%FCS/PBS. Analysis was performed on a FACS flow cytometer (Becton Dickinson, USA), and the monocytes were specifically analyzed by selective gating based on\parameters of forward and side light scatter.5. ELISA for Cytokine measurementsThe concentration of IL-6, IL-23and IL-17from plasma of RA patients and normal group and supernatant from cell culture with HMGB1stimulation were determined using commercially obtained Enzyme-Linked Immunosorbent Assay (ELISA) kits according to the manufactures instructions.(Bender Med Systems, Austria, Europe).6. Western blot analysis for detecting NF-KBHuman macrophage cells from RA patients and healthy control group after stimulation with HMGB1for4-8-12and24hours were lysated then electrophoresed on12%SDS-PAGE gels and transferred onto polyscreen PVDF transfer membranes (PVDF; PerkinElmer, USA). Membranes were blocked with5%(w/v) non-fat dry milk1%(v/v) Tween20in PBS for1h at room temperature and incubated overnight with commercially available anti-NF-kB antibodies (1:1000)(Abcam,USA) at4oC. Detection was performed with electrochemiluminesce (ECL) and the blots were quantified by densitometry using the image analysis program (Amercontrol Biosciences, USA).Results1. The successful establishment of chicken type Ⅱ collagen induced arthritis in mice model, Murine arthritis incidence rate was100%, mean time to onset of35+5days, joint inflammation scores was10+2points.2. In vitro tests show that, the expressions of TLR2, TLR4, TLR5, TLR7, TLR8and TLR9etc. in macrophage lineage (RAW) surface could be found. On the basis of CIA model successful, we found that Th17cells were infiltrated in mice model after CⅡ vaccination45days. The expression of TLR2on CⅡ stimulated peritoneal macrophages was increased. In addition, the TLR2, TLR4, TLR8, TLR9, mRNA expression levels in peritoneal macrophages from mice models were also significant increased, and serum IL-6, IL-17, TGF beta, beta IL-1content were increased significantly. The antibody against TLR2could block the expression levels of IL-6, IL-17, TGF-beta, beta-IL-1.3. The cell surface expression of CD14+, and CD45on peripheral blood monocytes from patients with RA and healthy controls was determined by flow cytometric analysis. The results showed that representative staining patterns of CD14+, CD45expression in the monocyte population from patients with RA. Compared with12healthy control subjects,30patients had significantly increased TLR2expression on freshly isolated monocytes (P<0.005), we also found statistically significant correlations in peripheral monocyte cell counts from mRNA levels and expression of TLR2. QRT-PCR results showed the expression of TLR2in RA patients was higher than healthy control samples (P<0.005).4. Increased TLR2expression on mononuclear cells with HMGB1stimulation. Mononuclear cells from patient blood and healthy control groups cultured and stimulated with HMGB1for1-2-4-8and12hours then mRNA levels of TLR2were measured by qRT-PCR. The mRNAs of TLR2increased in PBMCs from RA patients compared with healthy controls. Also in time course in PBMCs from RA patients mRNA levels increased in12and8hours in RA patients.5. Increased cytokine concentrations in supernanatant of cells and Plasma from Rheumatoid Arthritis patients. We used ELISA to quantify plasma levels of IL-23in rheumatoid arthritis (RA) and healthy controls. Control subjects had decreased plasma levels of IL-23compared with RA subjects, matched for age (p<0.005). For control subjects, the median with inter quartile range IL-23level was45.48±21.7pg/ml and for the RA subjects it was98±6.18pg/ml. IL-17levels also was detected by ELISA. The correlation between IL-17and clinical manifestations as well as laboratory findings were analyzed. The plasma level of IL-17in RA patients was180.8±21.7pg/ml, significantly higher than that of healthy control (100±8.6pg/ml; P<0.05). The results of measurement of IL-6in the plasma of30patients with RA and12controls shown that the levels of IL-6were higher in RA patients than in healthy persons (P<0.005). The cytokine concentrations results in supernatant indicated that the levels of IL-17, IL-6and IL-23in RA patients were more than the healthy control. Also in stimulation time courses just8hour statically increased.6. Increased Protein levels of NF-kB were found in RA patients compare with healthy control and blocking group. The mAb against NF-kB binding with macrophage lysates stimulated by HMGB1showed the protein levels in RA patients are more than the control group. Comparing time courses the results showed that the protein level is increased with time course in the patient group.β-actin was used as a control group.ConclusionsIn this study and our previous studies, we find that Th17cells and HMGB1are increased in patients with RA, these results agree with recently published findings. We hypothesize that the immune dysregulation in RA may potentially be due in part to increased frequency of Th17cells, and the stimulation of HMGB1secreted by activated cells or leaked by injured cells may induce Th17cells differentiation via up-regulate TLR2and Th17cell-associated cytokines. To test this hypothesis, we detected the expression of TLR2on CD14+monocytes in patients with RA and its changes after HMGB1stimulation in vitro, we also examined the levels of IL-17, IL-23and IL-6in plasma of patients or produced by HMGB1stimulated monocytes from patients’ blood. Our results showed that the frequency of CD14+cells in peripheral blood mononuclear cell was obviously increased, and enhanced expression of TLR2on CD14+monocytes was also found in patients with RA, compared with healthy controls. In addition, the levels of IL-17, IL-23and IL-6in supernatants from cultured monocytes and in plasma from patients were increased. NF-κB, the downstream target of TLR2, also showed a marked elevation after monocytes was stimulated by HMGB1. This implies that the enhanced TLR2pathway and Th17cell polarization may due to HMGB1stimulation in rheumatoid arthritis, it may help for further understanding the mechanism of RA and designing HMGB1-targeted therapies. |
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