Treg/Th17 Balance And The Regulatory Role Of Notch Signaling In Rheumatoid Arthritis

Aim:Rheumatoid arthritis (RA) is a chronic inflammatory disease of the synovial joints characterized by leukocyte invasion and synoviocyte activation, leading to cartilage and bone destruction. Although the exact mechanism of RA pathogenesis is not well defined, the infiltration of auto-reactive CD4+ T cells into synovium has been thought to be the major instigator of joint inflammation. TypeⅡcollagen (CII), which is expressed exclusively in the articular cartilage of joints, has been considered as one of the major auto-antigens in human RA as well as in the collagen-induced arthritis(CIA) model. T helper cells clearly play a central role in the development of joint inflammation. The various T-cell populations contain Th1 cells, regulatory T cells and newly reported Th17 cells. The objective of the study was to investigate whether the Th17/Treg balance was impaired in the peripheral blood of patients with rheumatoid arthritis (RA) and to measure the expression profile of Notch receptors and down-stream molecules in peripheral lymphocyte from RA patients. We also want to investigate the role of distinct Notch receptors and ligands in the activation and differentiation of collagenⅡ(CII)-reactive Th cells upon Ag-specific re-stimulation. Such study will demonstrates the Treg/Th17 imbalance and different expression of Notch-related molecules in T helper cells between RA patients and healthy controls, which may offer new areas for treatment of RA using Notch signaling as therapeutic target.Methods1. Regulatory T cells and Th17 cells were determined by using flow cytometric intracellular staining. Peripheral blood mononuclear cells (PBMCs) were surface-labeled followed by fixation and permeabilization and intracellular staining for FoxP3 and IL-17. The effect of different stimulators on Th17 cells was also analyzed.2. The percentage of Treg and Th17 cells and mRNA expression of FoxP3 and RORyt were determined by flow cytometry and real-time PCR, respectively.3. The significance of decreased Treg cells and increased Th17 cells was further explored by calculation of Th17/Treg percentage ratio of individual RA patients and healthy controls. Eleven T helper cell-related cytokines as components in serum cytokine environment of RA patients and healthy controls were measured using flow cytometric bead-based technology. This new technology allows for evaluation of multiple analytes in a single sample and utilization of minimal sample volumes to glean data. We also examined the possible correlations between Th17/Treg ratio and Th1-related cytokine (IFN-γ) and Th17-related cytokines (IL-6, IL-1βand TNF-α) in active RA patients.4. The expression of Notch receptors in peripheral lymphocytes of RA patients was assessed by both flow cytometry and real-time polymerase chain reaction (PCR). The expression of representative Notch target gene HES-1, Notch-ICD in purified T helper cells from RA patients was determined by real-time PCR and immunoblot, respectively.5. Spleen mononuclear cells (SMNCs) from CII-immunized DBA/1J mice were re-stimulated by culturing with CII. CII-specific proliferation and differentiation of T cells were determined by H3-TdR incorporation and flow cytometric analysis, respectively. The mRNA expression of Notch receptors and Hes1 was assessed by real-time PCR. The effect of Notch inhibition, including DAPT, mAb of Notch receptor and fusion protein of Notch ligand, on the CII-specific proliferation and differentiation of T cells was also explored.Results:1. FoxP3 was mainly expressed in CD4+ T cells, especially in CD4+T cells with high level CD25 (about 97%). However, CD4+ T cell with mediate expression of CD25 also expressed low level FoxP3 protein. Furthermore, FoxP3 expression could be induced following the activation of T cells. Such result was consistent with the result from real-time PCR. 2. There are similar frequencies of CD4+CD25+T cells in peripheral blood of RA patients and healthy subjects. However, the percentage of CD4+CD25high and CD4+CD25+FoxP3+ in active RA group is lower than non-active group and healthy subjects. The percentage of Th17 cells in active group was higher than that of stable group and both of them were higher than that of heathy controls (P<0.05). The distribution profile of TH1 cells was similar to that of TH17 cells.3. Inactive RA group exhibited higher Th17/Treg ratio (0.29±0.13) than that of healthy controls (0.11±0.4). Moreover, there was a sharp elevation of Th17/Treg ratio in active RA patients (1.31±0.5). inactive RA group exhibited higher levels of serum IL-12, IFN-γ, IL-2, IL-6, IL-1βand TNF-αthan those of healthy controls (all P<0.05), but lower than those of active RA group (all P<0.05). Both inactive RA and active RA group had higher levels of serum IL-4, IL-5, IL-10 when compared with healthy controls (all P<0.05), while no difference was observed between inactive and active RA group (all P>0.05). There was no obvious difference of serum IL-8, TNF-βprotein among three groups (all P>0.05). Th17/Treg ratios were positively correlated with serum concentrations of (r=0.650, P<0.05), IL-6 (r=0.813, P<0.01), IL-1β(r=0.713, P<0.01), and TNF-α(r=0.6263, P<0.05) of active RA patients.4. Compared with healthy control, T cells from RA patients expressed higher level of Notch 2, Notch 3 and Notch 4, and Notch 3 was mainly expressed by activated T cells. There was no difference of Notch-ICD expression observed in whole T cell fraction between active, inactive RA patients and healthy controls. However, nuclear fraction of T helper cells from active RA patients shown higher levels of Notch-ICD than that of inactive RA patients and healthy controls, which indicated that there was an increased nuclear translocation of Notch-ICD in active RA disease.5. There was a clear increase in the percentage of Thl cells and Th17 cells after CII re-stimulation. No significant difference was observed in the percentage of Treg in SMNCs with or without CII re-stimulation. CII re-stimulation induced up-regulated transcript levels of Hes1 in CII-reactive CD4+ T cells. The mRNA level of Notch3 were also significantly up-regulated while the levels other three Notch receptors were not increased. Inhibition of Notch signaling by DAPT and Notch3 antibody decreased the collagen-specific T cell proliferation and attenuate Th1- and Th17-type responses while treatment with Notch ligand Delta-like 1 promoted such response.Conclusions:1. There would be a Th17/Treg imbalance in peripheral blood from rheumatoid arthritis patients. The frequency of regulatory T cells was decreased while the percentage of Th17 cells was increased which exhibited Th17 cells polarization.2. Flow cytometric intra-cellular staining could be used to analyze the regulatory T cells and Th17 cells on single-cell level. Transcript factor FoxP3 was a useful marker for the determination of regulatory T cells.3. Th1- and Th17-related cytokines were increased in peripheral blood from rheumatoid arthritis patients which may provide an important micro-environment for Th17 cell polarization.4. T helper cells from RA patients display significantly altered expression profile of Notch receptors and enhanced activation of Notch signaling compared with those from healthy controls5. Notch signaling is engaged in CII-specific Th1- and Th17- type differentiation in which Notch3 and Delta-like 1 were involved. Selective inhibition of Notch signaling mediated by Notch3 or Delta-like 1 may offer a new strategy for the treatment of RA.