| Rheumatoid arthritis (RA) is a systemic autoimmune disease, in which an uncontrolledimmune response against self gave rise to severe joint inflammation. RA primarilytargets the synovial membrane, cartilage, and bone. Dendritic cells (DCs) were apopulation of highly specialized antigen-presenting cells (APCs) recognized for theircrucial role as regulators of innate and adaptive immunity. Recently, more evidenceshave highlighted a critical role of DCs in promoting tolerance, limiting uncontrolledinflammation and maintaining immune cell homeostasis. The diverse functions of DCsin immune regulation depended on the diversity of DCs subsets, such as mature immunogenic DCs (mDCs) and tolerogenic immature DCs (iDCs). iDCs are responsiblefor the formation of peripheral regulatory T cells (Tregs) and play an important role inmaintaining peripheral and central tolerances. CD11b+F4/80+DCs were able to inhibit Tcell proliferation and limit pro-inflammatory cytokine production. Antigen-activatedTh17cells were the main mediators of inflammation and joint damage through thesecretion of IL-17A, which promotes the releases of IL-1and tumor necrosis factor(TNF) by macrophages. Th17cells enter into the joint tissues of RA, and releasepro-inflammatory cytokines and chemokines, which promoted macrophage andneutrophil infiltration and activation. In contrast, Tregs were an anti-inflammatorylineage of T cells that were derived naturally from the thymus and the periphery on thebasis of the local environment. Tregs have been described to be functionally impaired inRA patients and arthritic models.Current pharmacologic therapy for RA included nonsteroidal anti-inflammatory drugs,nonbiologic disease modifying antirheumatic drugs, and biologic agents. Despite ofthese drugs has a good effect, but due to the long-term use of adverse reactions, thepatient is difficult to tolerate. Using cellular immune therapy for RA, rebuild immunetolerance mechanism, has become a new strategy. iDCs control inflammation andimmune cell balance characteristics provide prospects for RA treatment. BMCD11b+F4/80+iDC could be induced in vitro and showed tolerogenic characteristic. It isunclear whether BM CD11b+F4/80+iDC could regulate the balance between Tregs andTh17cells to have immunoregulatory function and attenuate inflammatory arthritis. Themechanism of by which can BM CD11b+F4/80+iDC regulate the function of Tregs andTh17is also unclear. The current study was to induce and purify BM CD11b+F4/80+DCin vitro, and to investigate the therapeutic effect and mechanisms of BM CD11b+F4/80+DC modulating the balance between Treg and Th17cells on collagen induced arthritis(CIA) in DBA/1mice. Aim:This study was undertaken to investigate the therapeutic effect and mechanisms of bonemarrow–derived CD11b+F4/80+immature dendritic cells (BM CD11b+F4/80+iDC) oncollagen induced arthritis (CIA) in DBA/1mice.Methods:BM CD11b+F4/80+iDC were induced by rmGM-CSF and rmIL-4, and were identifiedby the expressions of TLR-2, IDO, IL-10, TGF-β1and mixed leukocyte reaction (MLR).CIA was established in DBA/1mice by the immunization with type II collagen. BMCD11b+F4/80+iDC were injected intravenously into the mice on14days, day21anddays28after immunization. The effect of BM CD11b+F4/80+iDC on CIA was evaluatedby arthritis index, joint and spleen histopathology, body weight, thymus index,thymocytes proliferation, IL-1β, TNF-α, IL-17A, IL-10and TGF-β1levels, and thepercent of Tregs and Th17. In vitro, the regulatory effects of BM CD11b+F4/80+iDC onT lymphocytes were analyzed by the cytokines levels of Tregs and Th17, the percent ofTregs and Th17, and the mRNA expressions of Foxp3, Helios, IL-17and RORγt.The method of reverse transcription PCR (RT-PCR) was utilized to detect the mRNAexpressions of Foxp3, Helios and IL-17, and real-time quantitative PCR (qPCR) wasutilized to detect the mRNA expressions of RORγt in the thymus. The levels of IL-1β,TNF-α, IL-17A, IL-10and TGF-β1in cultrue supernatant and CIA mice were detectedby enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to test thepercentages of the CD11b+F4/80+iDC, Tregs and Th17. The expressions of IDO andCTLA-4were analyzed by western-blot. MTT was utilized to measure the thymocytesproliferation and MLR. The expression levels of TLR-2, IDO and CD83proteins wereobserved by immune-cytochemistrical analysis. Results:1. BM CD11b+F4/80+iDC derived from bone marrow were induced successfullyand displayed a tolerogenic phenotype and functional state1.1BM CD11b+F4/80+iDC were induced successfully and displayed a tolerogenicphenotype and functional stateUsing rmGM-CSF(20μg/L) and rmIL-4(20μg/L) could successfully induce mice femurand tibia bone marrow cells into BM CD11b+F4/80+iDC in vitro. And the resultsshowed that the percent of BM CD11b+F4/80+iDC in bone marrow cells induced byrmGM-CSF and IL-4was approximately26.6%, and the proportion of the CD11b+F4/80+iDC was99.3%by further purification. About1×106BM CD11b+F4/80+iDCcould be collected from each mouse.1.2BM CD11b+F4/80+iDC were displayed a tolerogenic phenotype and functionalstateThe levels of IL-10and TGF-β1in the BM CD11b+F4/80+iDC culture supernatantswere significantly higher than those in the mDCs culture supernatants. The expressionlevels of TLR-2and IDO proteins were higher in BM CD11b+F4/80+iDC than inLPS-treated DCs (mDCs), whereas CD83protein expression levels were lower in BMCD11b+F4/80+iDC than in BM CD11b+F4/80+iDC. In contrast to mDCs whichexhibited potent allo-stimulatory activity, BM CD11b+F4/80+iDC evoked a muchweaker proliferative response. BM CD11b+F4/80+iDC demonstrate a tolerogenicphenotype and functional state.2. Effects of BM CD11b+F4/80+iDC on mice CIA and on Tregs and Th17subsets invivo2.1BM CD11b+F4/80+iDC have therapeutic effect on mice CIACIA mice were injected with BM CD11b+F4/80+iDC on day14, day21and day28after immunization respectively. The swelling peak of CIA mice appeared from day35to day42. The arthritis scores were obviously higher in CIA mice than in normal mice, andinfusion of BM CD11b+F4/80+iDC could significantly reduce the arthritis scores in CIAmice. Synovitis, pannus formation and destruction of bone and cartilage wereapparently observed in untreated CIA mice, however, these pathological changes wereremarkably alleviated in BM CD11b+F4/80+iDC-treated CIA mice. Compared with CIAmice, the scores of synoviocytes hyperplasia, cellular infiltration and cartilages andbone erosion were also reduced in BM CD11b+F4/80+iDC-treated mice.The histological pathology slides of spleens were evaluated and graded in germinalcenter, lymphoid follicular hyperplasia, marginal zone and red pulp by two independentobservers. In CIA model mice, white pulps were hyperplasia, germinal centers emergedand red pulp was congested. The scores of lymphoid follicular hyperplasia, marginalzone and red pulp in mice with CIA were higher than that in normal mice significantly,and the number of germinal center was more than that of normal mice. Injection of BMCD11b+F4/80+iDC could reduce the number of germinal center and the scores of aboveindices.The weights of CIA mice decreased from day35to46after immunization, there wassignificant difference between CIA and normal groups. Weights of mice were increasedin BM CD11b+F4/80+iDC-treated mice. The thymus index was analyzed on day46.Compared with normal mice, CIA mice had an increased thymus index, while treatmentwith BM CD11b+F4/80+iDC could obviously reduce the thymus index in CIA mice.2.2BM CD11b+F4/80+iDC inhibited thymocytes proliferation in CIA miceTo study the effect of BM CD11b+F4/80+iDC on T cell activity in vivo, a thymocyteproliferation experiment was performed. T lymphocyte proliferation was increased in CIA mice compared with that in normal mice, and injection of BM CD11b+F4/80+iDCsignificantly inhibited ConA-induced T lymphocytes proliferation.2.3BM CD11b+F4/80+iDC regulated the balance between Th17and Tregs in CIAmiceResults showed that the percentage of CD4+IL-17+Th17cells was higher than that ofnormal mice. But CD4+CD25+FoxP3+Tregs subset was decreased obviously in CIAmice. Injection of BM CD11b+F4/80+iDC reduced the percentage of CD4+IL-17+Th17cells in CIA mice. In contrast, the percentage of Tregs was enhanced in BM CD11b+F4/80+iDC-treated group. The level of pro-inflammatory cytokine IL-17A in serum washigh, and the levels of IL-10and TGF-β1were lower than that of normal mice. But inBM CD11b+F4/80+iDC-treated mice, IL-17A level was lessen, and IL-10and TGF-β1were up-regulated. Moreover, compared with normal mice, CIA mice had an decreasedexpression of CTLA-4in spleens, while treatment with BM CD11b+F4/80+iDC couldobviously increased the expression of CTLA-4in spleens.2.4BM CD11b+F4/80+iDC decreased levels of pro-inflammatory cytokines andincreased levels of anti-inflammatory cytokines in CIA miceResults showed that there was an remarkable increase in IL-1β, TNF-α and IL-17Alevels and a dramatic decrease in IL-10and TGF-β1levels in the serum of CIA micecompared with that in the serum of normal group; and these changes in cytokinesecretion were efficiently blunted in BM CD11b+F4/80+iDC-treated CIA mice.Furthermore, it was found that the levels of IL-1β and TNF-α in macrophagessupernatants were significantly elevated in CIA mice, and this increase in IL-1β andTNF-α secretion were obviously attenuated in BM CD11b+F4/80+iDC-treated mice.3. BM CD11b+F4/80+iDC on the the differentiation of Tregs and Th17cells in vitro 3.1BM CD11b+F4/80+iDC promoted the differentiation of Tregs in vitroResults showed that the percentage of CD4+CD25+Foxp3+Tregs was significantlyincreased when cultured with BM CD11b+F4/80+iDC in the presence of TGF-β1andOVA. Compared with controls group, the expression of CTLA-4protein in co-culturedT cells and IL-10level in the supernatants of co-culture were significantly increased. Atthe same time, the expression of Foxp3mRNA and Helios mRNA in co-cultured T cellswere significantly up-regulated.3.2BM CD11b+F4/80+iDC reduced the T lymphocytes proliferative and inhibitedthe functions of Th17cells in vitroConA-induced T lymphocyte proliferation was inhibited by BM CD11b+F4/80+iDC.BM CD11b+F4/80+iDC significantly decreased the percentage of CD4+IL-17+Th17cells, decreased the level of IL-17A, down-regulated the expression of IL-17mRNAand RORγt mRNA in vitro.Conclusion:1. BM CD11b+F4/80+iDC could be induced successfully with rmGM-CSF and rmIL-4in vitro.2. BM CD11b+F4/80+iDC have therapeutic effect on mice CIA.3. The therapeutic role of BM CD11b+F4/80+iDC partially depends on the inductionof Foxp3+Treg cells and BM CD11b+F4/80+iDC’ ability to suppress Th17functions. |
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