The Suppressive Effects Of TGF-β-induced CD4~+CD25~+Foxp3~+ Regulatory T Cells On Established Collagen-Induced Arthritis In Mice

Rheumatoid Arthritis (RA) is one of autoimmune diseases with chronic, progressive and aggressive arthritis, which end is bone destruction, joint disability and high deformity. Its therapy is mainly focus on immune suppressive agents that have severe side effects.It is reported that diminished frequency and/or dysfunction of CD4+CD25+Foxp3+ regulatory T cells (natural regulatory T cells, nTregs) may contribute to the pathogenesis of rheumatoid arthritis (RA). However, adaptive transfer of nTregs to the mouse model of RA, collagen induced arthritis (CIA) failed to protect established arthritis. Recently, our group found that induced CD4+CD25+Foxp3+regulatory T cells (iTregs) have the same phenotypes and suppressive function as nTregs, but show more stability and maintain suppressive function on T effector cells in the presence of IL-6 (Interleukin-6) in vitro. In the first part of our research, we transfer both Tregs to arthritic mice and investigate their preventive and therapeutic effects on the development of disease.Joint damage is the commonest consequence of RA patients. It's well-known that osteoclasts play a crucial role in bone remodeling in RA. Osteoclasts are multinucleated giant cells and responsible for the bone resorption. The balance between osteoclasts and osteoblasts maintains bone metabolism homeostasis. Osteoclast activation and function are thought to be the key causer of bone erosion in inflammatory arthritic patients. And the elevated number of osteoclasts precursors (OCPs) can also be found in RA's murine model CIA. Osteoclasts come from bone marrows and OCPs are able to convert into mature osteoclasts governed by the differentiation factor receptor activator of NF-kB ligand (RANKL) with the assistance of macrophage colony stimulating factor (M-CSF) that supports cell survival during differentiation.These two cytokines are crucially necessary for osteoclastogenesis. Lack of any of them or blockage of these signal pathways can impede the differentiation of osteoclasts. Therefore, osteoclasts formation has been considered as a crucial therapeutic target for RA.Previous study has demonstrated that activated nTregs can inhibit osteoclastogenesis. Since we and other groups have identified that iTregs have the similar phenotypic and functional characteristics as nTregs, here we try to learn if iTregs also have the suppressive capacity on osteoclastogenesis and CIA based on the results of the first part.ResultsPart I. The preparation and identification of TGF-βinduced CD4+CD25+Foxp3+ regulatory T cells and their effects on CIA1. The preparation and identification of TGF-βinduced CD4+CD25+Foxp3+ regulatory T cellsNaive CD4+cells isolated from spleens of normal DBA1/J mice were stimulated with collagen II (CII) peptide, IL-2 and TGF-βfor four days. Tcon (IL-2 stimulated only), which means effector T lymphocytes, are as control of iTregs. nTregs were isolated from thymus of the same mice and expanded with IL-2 for five days. Detect the expression of Foxp3 with Flow Cytometry, Western Blotting and PCR after culture. Meanwhile, co-culture nTregs and iTregs with T effector cells respectively and identify the suppressive function of both Tregs on the proliferation of T effector cells (CFSE labeled method or proliferation assay). The experiment is set up four groups: baseline, nTregs group, Tcon group and iTregs group.1.1 The expression of Foxp3 in TGF-βinduced CD4+CD25+Foxp3+regulatory T cells and nTregs after inductionThe expression of Foxp3 among cultured cells in nTregs and iTregs is almost the same. The percentage is always above sixty percent while that of Tcon is only ten percent. The total number of CD25+Foxp3+ in Tcon wells is obviously lower than in iTregs wells. Then we detected the expression of Foxp3 protein in CD4+CD25- cells, nTregs, Tcon and iTregs group with western blotting. The expression of Foxp3 protein in both nTregs and iTregs is much higher than CD4+CD25- and Tcon group. The similar result was found in level of Foxp3 mRNA.1.2 The suppressive function of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells on the proliferation of T effector cellsBoth Tregs had the similar suppressive function on the proliferation of effector T lymphocytes compared by baseline and Tcon group.2. The effects of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells on CIACIA was induced by subcutaneous injection of 50μl of emulsion containing bovine collagen II (4mg/mL) and complete Freund's adjuvant (1:1 ratio).3×106 of nTregs and iTregs were adaptively transferred to DBA1/J mice on d0, d14 or d28 after immunization. Mice were divided into four groups including CIA model, nTregs group, Tcon group and iTregs group at random. Investigate the prevalence and clinic scores every other day after onset of arthritis. Detect the level of anti-CII specific antibodies and do HE staining of involved joints to evaluate the severity of arthritis.2.1 The preventive effects of TGF-P induced CD4+CD25+Foxp3+regulatory T cells on CIABoth Tregs can suppress the onset and development of CIA when adaptively transferred to mice on day0. When transferred on d14, only iTregs can decrease the prevalence of arthritis, but both can decrease the clinic scores and anti-CII specific IgG2b significantly.2.2 The therapeutic effects of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells on CIAWhen adaptively transferred to DBAl/J mice on d28 after immunization, only iTregs but not nTregs can significantly suppress the prevalence, severity of arthritis and the level of anti-CII specific IgG2a and IgG2b. HE staining of involved joints showed that iTregs can dramatically suppress inflammatory cells infiltration and bone destruction. The proliferation assay of lymphocytes from each group showed that iTregs could suppress the proliferation of lymphocytes and also the production of pro-inflammatory factors. However, nTregs lost their suppressive function.SummaryAll the results verified that iTregs owned the same phenotypes and suppressive function as nTregs. Meanwhile, iTregs have the similar preventive effects on CIA, but only iTregs maintain their therapeutic function on established arthritis while nTregs lost their function completely.Part II. The mechanism of TGF-βinduced CD4+CD25+Foxp3+ regulatory T cells therapeutic effects on CIABased on the results mentioned above, iTregs are much stable and functional than nTregs in established arthritic mice and can suppress the development of bone destruction. We will study further to clarify its mechanism.3. The stability of TGF-P induced CD4+CD25+Foxp3+regulatory T cells in vitro and in vivoTo expand nTregs and induce iTregs as mentioned in part I.3.1 Investigate the stability of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells in vitroDetect both Tregs's suppressive functions on effector T lymphocytes with or without IL-6 (10ng/mL). Naive CD4+cells were induced to Th17 cells with IL-6 and TGF-P, and nTregs or iTregs were added to some wells (Tregs:naive CD4+cells=1:4) to compare the production of IL-17A.Results showed that nTregs dramatically lost their suppressive function on the proliferation of effector T lymphocytes, the differentiation of Th17 cells and the generation of IL-17A. But iTregs maintained ail these functions.3.2 Investigate the stability of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells in vivoBoth Tregs from GFP-Foxp3 transgenic mice were adaptively transferred to immune deficient(CD3-/-)mice and detect the changes of Foxp3 expression after four and eight weeks transfer. CFSE labeled Tregs were transferred to normal DBA1/J mice and the expression of Foxp3 in both Tregs was detected after one or three weeks transfer. These CFSE labeled Tregs were also injected into established arthritic mice to investigate their fate in inflammatory milieu.3.2.1 The stability of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells in immune deficient and normal miceiTregs maintained the same level of Foxp3 expression as nTregs in immune deficient mice even after eight weeks transfer. The percentage is about fifty percent (GFP+cells among CD3+cells). The same results can be found in normal mice.3.2.2 The stability of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells in established arthritic mice (inflammatory milieu)Compared to iTregs, nTregs were prone to apoptosis, lost Foxp3 expression and convert to Th2 and Th17 cells in the draining lymph nodes in established arthritic mice. Conversely, iTregs can maintain Foxp3 expression and resistant to the conversion to other helper T lymphocytes. At the same time, we found that injection of iTregs but not nTregs altered the balance of Foxp3+/Th17 cells in arthritis mice. The number of Th17 cells in lymph nodes in CIA mice received nTregs were 10-fold greater than those seen in mice received iTregs. However, the number of CD4+Foxp3+ cells in LNs 8-fold less in mice received nTregs than in mice received iTregs. This phenomenon revealed that iTregs can induce more Foxp3+regulatory T cells in inflammatory milieu.4. TGF-βinduced CD4+CD25+Foxp3+regulatory T cells protect CIA from bone destruction through osteoclastogenesis suppressionnTregs can suppress osteoclastogenesis and over generated osteoclasts are the key factors for bone destruction in inflammatory arthritis. Here, we will investigate if iTregs can protect arthritis through osteoclastogenesis suppression.CD11C+ cells as Osteoclast Precursors (OCPs) were isolated from bone marrows of normal DBA1/J mice and stimulated with Receptor Activator of NF-κB Ligand and Mcrophage Colony-stimulating Factor to induce osteoclasts. CD4+CD25-cells. nTregs, Tcon and iTregs were added to some wells respectively and did Tartrate-risistant acid phosphatase (TRAP) staining to investigate their effects on osteoclasts generation.nTregs and iTregs were adaptively transferred to CIA mice on d14 after immunization. The involved joints from each group were accepted CT scanning after observation to evaluate bone erosion and destruction.4.1 The suppressive effects of TGF-P induced CD4+CD25+Foxp3+ regulatory T cells on osteoclastogenesis in vitroThe results showed that both nTregs and iTregs can significantly suppress osteoclasts generation compared to baseline, CD4+ CD25-cells and Tcon. The generation of osteoclasts elevated with decrease of the number of iTregs added to OCPs, but osteoclastogenesis was also suppressed compared with Tcon. This phenomenon revealed that the suppressive function of iTregs was dose dependent.4.1 The suppressive effects of TGF-0 induced CD4+CD25+Foxp3+regulatory T cells on bone erosion and destruction in vivoWe can find from the results of CT scanning that The CIA mice showed the chronic destructive phase of polyarthritis in the feet compared to normal mice. The joint surface showed clear bone erosion, joint integration, ankylosis and loss of bone. However, joints from iTregs group only showed milder joint space narrow without bone erosion compared to normal ones with normal joint space and bone volume.5. The mechanism of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells suppression on osteoclastogenesisWe have five groups as follows, OCPs group as positive control, iTregs+OCPs group, iTregs+ OCPs+ isotype control cIgG group, iTregs+ OCPs+ anti-TGF-P antibody group, iTregs+ OCPs+ anti-IL-10R antibody group. This experiment was set up to detect the role of TGF-βand IL-10 in iTregs suppressive function. Then transwell experiments were done to detect that if iTregs function on osteoclastogenesis is cell-contact dependent. After that, collecting cells from OCPs, Tcon+OCPs and iTregs+OCPs to extract total proteins and doing western blotting to evaluate the level of NF-kB subunits P65/P50. 5.1 TGF-βinduced CD4+CD25+Foxp3+regulatory T cells suppressed osteoclastogenesis in a cell-contact but not cytokine dependent way.We observed that addition of anti-TGF-βand anti-IL-10 receptor did not alter the suppressive effects of iTregs on osteoclast formation compared to addition of similar istotypes of control IgG. Conversely, transwell experiments that saperate iTregs from OCPs completely abolished the suppression of CD4+ iTregs against osteoclast formation.5.2 TGF-βinduced CD4+CD25+Foxp3+regulatory T cells blocked osteoclastogenesis via a NF-kB-P65/P50 pathway.The express of NF-kB/P65 and NF-kB/P50 was significantly decreased in non-T cells that had been treated with iTregs compared with those cells treated with Tcon cells or no CD4+ T cells (OCPs).SummaryBased on the results in Part II, iTregs maintain good suppressive functions as nTregs in immune deficient and normal mice. Conversely, nTregs are prone to apoptosis, convert to Th17 cells and lose their functions on T effector cells proliferation while iTregs are stable and sustain their suppressive functions in inflammatory milieu in established arthritic mice.The suppressive functions of iTregs on CIA mice are partially through their work on osteoclasts formation. Therefore, iTregs can protect CIA mice from bone erosion and destruction. iTregs suppress osteoclastogenesis via a cell-contact way but not cytokine dependent way, a NF-κB-P65/P50 signal pathway and also a dose-dependent way.In brief, we got conclusions as follows,1. iTregs have the same Foxp3 expression and suppressive function on the proliferation of T lymphocytes as nTregs in vitro.2. iTregs have the same preventive effects on CIA mice as nTregs in vivo.3. iTregs but not nTregs can maintain the therapeutic effects on established arthritic mice.4. The possible mechanism of iTregs suppressive effects can be concluded as follows,4.1 iTregs can maintain suppressive function in the presence of pro-inflammatory factor IL-6 in vitro, but nTregs lost their function.4.2 iTregs but not nTregs can suppress Th17 cells differentiation in vitro.4.3 iTregs are as stable as nTregs in immune deficient and normal mice.4.4 iTregs are stable and can decrease the production of IL-17A in inflammatory milieu in vivo but nTregs cannot.4.5 iTregs protect arthritic mice from bone destruction by their suppression on osteoclasts formation. This effect is via a cell-contact and NF-kB-P65/P50 signal pathway and also a dose-dependent way. 5. This research offered theoretical supports for iTregs cellular treatment ofrheumatoid arthritis.