| Background and ObjectivesUntil now, allogeneic organ transplantation has been wildly used in treating organ failure, blood diseases or cancers. However, acute rejection, which occurs within the first few weeks or months, remains a major cause of treatment failure. The post-transplantant immunosuppressive drugs routinely used in clinic seriously affect patients’ immunologic function along with side and toxic effects. To overcome this issue, great efforts have been paid to induce specific immune-tolerance in recipients and it is a hotspot to study how to induce immune-tolerance via augmenting frequency of regulatory T cells (Tregs) or enhance their functions. Treg is an important kind of cell subset for maintaining peripheral tolerance. It has many advantages of using Tregs for inducing immuno-tolerance; therefore Tregs maybe developed into a significant tool for inducing allotransplantation immune tolerance and transplantation rejection therapy.At present, the cell therapy utilization of Tregs augmented in vitro is seriously limited by sterilization, cell amounts and so on, however, the method that using drugs to augment frequency of recipients’ Tregs or enhance their suppressive functions showed a great implication prospects. Treg cells in human body mainly existed in CD4+CD25hi (highly-expressed) cell subset, which approximately occupied1%-2%of CD4+T cells in peripheral blood. As an important kind of cell subset which possessed function of negative-regulating immune response in vitro and in vivo, Treg could be classified into naturally occurring CD4+CD25+Treg (nTreg) and induced CD4+CD25+Treg (iTreg) according to their surface molecules, cytokine-produced and mechanism of action. It was confirmed in a large number of transplantation animal models that Tregs played a key role in keeping living condition of grafts; and augmented frequency of Tregs could induce immune tolerance and alleviate rejection reaction. In solid-organ transplantation, compared with equivalent nTregs, iTregs specifically induced by allogeneic antigens ex vivo and in vitro were more useful in prolonging allograft survival and inducing immune tolerance. Mechanisms of Tregs’immunoregulation mainly include the following aspects: suppressing antigen presenting cells (APCs), mediating target cell dissolution and killing target cells, secreting TGF-β and IL-10for immuno-suppression, et al. The process of Tregs inhibiting CD4+effector T cells was by means of immediate contaction, secreting cytokine or combined to APCs and then changing their metabolic pathway or secrete way, et al. Treg cells highly expressed CD4and CD25and, in addition, a kind of characteristic marker-Foxp3, which was pivotal for phenotype, development and functional maintain of Tregs. It was reported that frequency of Tregs and expression level of Foxp3could act as an indicator for improving recipients’ immunologic condition and inducing immune tolerance. However, research on how to augment frequency of Tregs, to improve expression of Foxp3or to enhance immunosuppressive function of Tregs was still inadequate. So, it is urgent to find a safe agent to augment frequency of Tregs in recipients or enhance their suppressive functions.It was reported that co-culture of human CD4+T cells and recombinant flagellin (rFliC) in vitro significantly enhanced immunosuppressive ability of CD4+CD25+Treg cells and increased expression of Foxp3. Flagellin was also reported to be effective in protecting body from chemical, bacterial, viral and radioactive injury. Therefore, flagellin may act as a new agent to strengthen function of Tregs and thus to induce body’s immune tolerance. Flagellin was a major ingredient of bacterial flagella, and the specific ligand of Toll-like receptor5(TLR5). It was found that TLR5was expressed both in human T cells and murine Treg cells. Toll-like receptor (TLR) family was a key factor in mediating innate immunity, and it activated innate immune response at the early time when pathogens invaded body; in addition, flagellin played an important role in pathogen-induced immuno-pathological injuries. Immune response mediated by flagellin was dependent on classic TLR signaling pathway. Flagellin could activate TLR5signaling specifically; the downstream signaling was MyD88-dependent and associated with activation of NF-kB or MAPK. The MAPK pathway was regulated by numerous factors.In conclusion, we proposed a scientific hypothesis in this paper that rFliC was effective in inducing allotransplantation immune tolerance by means of augmenting frequency of recipient CD4+CD25+Tregs, increasing expression of Foxp3and enhancing functions and suppressive ability of Treg cells, et al. The objectives of this research were to clarify the impact of rFliC on allografts’ living condition and survival time, to explore the undergoing mechanism and molecular regulation of rFliC’s effects, and thus, to provide theoretical and experimental basis for studying flagellin-induced-transplantation tolerance and offer a new candidate agent for clinical transplantation rejection therapy. So this research was of theoretical and practical significance.Part One Impact of rFliC on murine skin allograft and relationship with TLR5expressionMethods1. Preparation of purified recombinant flagellin (rFliC) Firstly, BL21E.coli-GST-FliC bacterial strain was established by transferring GST-FliC-Burkholderia pseudomallei-pGEX4T-2plasmid into competent BL21E.coli. Established bacterial strain was screened according to antibiotics resistance characteristic and then activated and induced to express recombinant flagellin (rFliC). Flagellin was purified by Glutathione Sepharose4B gelatum and the LPS contaminant was cleared away by ToxinEraser endotoxin removal resin.2. Establishment of murine skin allograft models The dorsal skin of C57BL/6mice was transplanted onto the BALB/c mice under sterile conditions. The bandage was removed on day7post-transplantation. rFliC (3mg/kg) was i.p. injected into the models on day-1.3.5.7.9and11of the operation, PBS was used as the control.3. Examining the impact of rFliC on living condition and survival time of allograftsFrom day7post-transplantation, survival condition of transplantation models in two groups was observed; rejection percentage and survival time was recorded. On the day that grafts of control group were totally rejected, two groups of model mice were euthanatized and the grafts were prepared to be paraffin sections for HE staining.4. Examing impact of rFliC on TLR5expression in allografts and recipientsOn the day that grafts of control group totally rejected, two groups of model mice were euthanatized and the grafts were prepared to be paraffin sections for immunohistochemistry (IHC) staining using anti-TLR5antibody.Results1. Allografts of control mice were early rejected characterized by massive epidermal damage, scab formation and a rapid loss of graft volume. Compared with control, living condition of grafts in rFliC-treated group was much better and the survival time was longer. The result indicated that rFliC was effective in prolonging allograft survival and improving living condition.2. HE staining of allografts showed that the skin graft sections from the control mice exhibited a clear border between thehost and grafted skin, epidermal and dermal necrosis and extensive inflammatory cell infiltration. By contrast, the grafts from the rFliC-treated hosts showed healthy growth, which was manifested as an imprecise boundary between the host and grafted skin and less inflammation and skin appendage degeneration.This result proved the effect of rFliC on allograft survival.3. IHC staining of allografts showed that high TLR5expression was induced by rFliC treatment compared with PBS control. These data suggested that rFliC treatment in vivo improved allograft survival and this effect was correlated with TLR5activation.Part Two Effect of rFliC on Treg cells of allotransplanted recipients Methods1. Establishment of murine skin allograft modelsThe dorsal skin of C57BL/6mice was transplanted onto the BALB/c mice under sterile conditions. rFliC (3mg/kg) was i.p. injected into the models on day-1.3.5.7.9and11of the operation, PBS was used for control. One hour before rFliC administered, anti-TLR5antibody was injected into model mice to set up as TLR5-blocking group.2. Frequency of recipient CD4+CD25+Foxp3+TregsOn the day that grafts of control group were totally rejected, three groups of model mice were euthanatized to prepare splenocytes and axillary lymph node cells. Cells were stained by CD4, CD25and Foxp3antibodies, and then, frequency of CD4+CD25+Foxp3+Tregs in CD4+T cells was tested.3. Examining Foxp3expression in recipient spleen sectionOn the day that grafts of control group were totally rejected, three groups of model mice were euthanatized and the spleen was prepared to be paraffin sections for IHC staining using anti-Foxp3antibody.4. Checking expression of Treg-related molecules in recipients On the day that skin grafts of control group were totally rejected, splenocytes were freshly prepared and CD4+CD25+Tregs were sorted. Then, expression of TLR5, Foxp3, TGF-β1and IL-10was tested by qRT-PCR. Expression of TLR5and Foxp3was additionally examined by Western blot.5. Proliferation of recipient Treg cellsWhen skin grafts of control group were totally rejected, CD4+CD25+Tregs were sorted using freshly prepared splenocytes. Three groups of Tregs were cultured for4d, then3H-TdR was incorporated and dpm was tested after16h.6. Suppression of recipient Treg cellsWhen skin grafts of control group were totally rejected, CD4+CD25+Tregs and CD4+CD25" effector T cells (Teffs) were sorted using freshly prepared splenocytes. Teffs from naive BALB/c mice were also prepared. Firstly, CD4+CD25+Tregs from rFliC-treated, PBS-control and TLR5-blocking groups were cultured for3d. Then, CD4+CD25" Teffs from rFliC-treated, PBS-control and naive BALB/c groups were added into Treg cultures for another24h. At last,3H-TdR was incorporated and dpm was tested after16h.7. Preparation of antigen presenting cells (APCs) and allogeneic antigenAPCs were prepared by treating splenocytes from naive B6mice with40μg/ml Mitomycin C for40min at37℃in5%CO2.Naive B6mice splenocytes were sonic disrupted and centrifuged to get supernatants as allogeneic antigen.8. Examining molecule expression of alloantigen-stimulated Tregs in vitroCD4+CD25+Tregs were prepared from naive BALB/c mice and stimulated by alloantigen and rFliC. Expression of TLR5, Foxp3, TGF-β1and IL-10was examined by semi-quantitative RT-PCR. Expression of TLR5and Foxp3was additionally tested by Western blot.9. Proliferation of alloantigen-stimulated Tregs in vitroCD4+CD25+Tregs were prepared from naive BALB/c mice firstly. Then, APC, APC+rFliC or rFliC was added into the culture to stimulate Tregs. At last,3H-TdR was incorporated and dpm was tested. TLR5-blocking group was set up by pre-treating Tregs with anti-TLR5antibody.10. Suppression of alloantigen-stimulated Tregs in vitroCD4+CD25+Tregs and CD4+CD25" Teffs were prepared from naive BALB/c mice. Firstly, Tregs were stimulated by APC or APC+rFliC. Then, Teffs were added for co-culture. At last,3H-TdR was incorporated and dpm was tested. TLR5-blocking group was set up by pre-treating Tregs with anti-TLR5antibody.Results1. For both of splenocytes and axillary lymph node cells in rFliC-treated mice, frequencyof CD4+CD25+Foxp3+cells in CD4+T cells was significantly high than control. However, frequency of triple positive cells in TLR5-blocked mice was obviously lower than that in rFliC-treated mice. This result showed that rFliC administration could augment frequency of recipient Treg cells and this effect was associated with TLR5signaling pathway.2. IHC staining of spleen section showed that expression of Foxp3in rFliC-treated mice was significantly higher than that in PBS-control mice; Foxp3expression in TLR5-blocked mice was much lower than that in rFliC-treated group. The result indicated that rFliC might activate allotransplantation recipient Treg cells and enhance Foxp3expression via a TLR5-dependent manner.3. The mRNA expression of TLR5, Foxp3, TGF-βl and IL-10in rFliC-treated recipient Tregs was significantly higher than that in control. In TLR5-blocked mice, expression of these four molecules was attenuated, compared with rFliC-treated mice. The expression condition of TLR5and Foxp3was confirmed by Western blot. The data suggested that treatment of rFliC increased expression of Treg-related molecules in a TLR5-correlated way.4. Proliferation of rFliC-treated recipient Tregs was obviously stronger than that of control mice. Proliferation of TLR5-blocked recipient Tregs was inhibited compared with rFliC-treated group. This result indicated that rFliC up-regulated proliferation of recipient Tregs in a TLR5-dependent manner.5. Treg cells from rFliC-treated hosts possessed the strongest suppressive ability on Teffs, and the ability was inhibited when TLR5pathway was blocked. This data revealed that rFliC enhanced suppressive function of recipient Tregs and this effect was related with TLR5activation.6. Molecules expression and functions of alloantigen stimulated Tregs in vitro was also up-regulated by rFliC stimulation, which was consistent with results in vivo.Part Three Mechanism of rFliC regulating Foxp3expression in alloantigen stimulated Tregs and the molecular modulation Methods1. Examining activation of JNK and p38MAPK in Tregs under rFliC stimulationCD4+CD25+Tregs from naive BALB/c mice were stimulated with alloantigen. Then, rFliC stimulation was added for gradient times and phosphorylation of JNK and p38MAPK was tested by Western blot.2. Checking the relevance between activated JNK, p38MAPK and Foxp3expressionAlloantigen stimulated Tregs were pre-treated to block JNK and p38MAPK signaling. Then, rFliC was added for stimulation, and expression of Foxp3was tested by Western blot assay.3. Examining activation of PI3K-Akt signaling in Tregs under rFliC stimulationCD4+CD25+Tregs were stimulated by alloantigen. Then, rFliC was added and phosphorylation of Akt was tested by Western blot.4. Checking the impact of activated PI3K-Akt on p38MAPK signalingAlloantigen stimulated Tregs were prepared and PI3K signaling was blocked. Then, rFliC was added for gradient times and phosphorylation of p38MAPK was tested by Western blot assay.5. Testing the relevance between activated PI3K signaling and Foxp3expressionAlloantigen stimulated Tregs were prepared and PI3K signaling was blocked, then, rFliC was added for stimulation. Expression of Foxp3was examined by Western blot and semi-quantitative RT-PCR assay. Alloantigen stimulated splenocytes were prepared and PI3K signaling was blocked, then, rFliC was added for stimulation. Frequency of CD4+CD25+Foxp3+cells in CD4+T cells was tested by flow cytometry (FCM).6. Examining the relationship between activated PI3K and p38MAPK signaling and Foxp3expressionPrepared alloantigen stimulated Tregs was pre-treated to block p38signaling firstly. After that, PI3K signaling was blocked too. Then, rFliC was added for stimulation. Expression of Foxp3was examined by Western blot and RT-PCR. Prepared alloantigen stimulated Tregs was pre-treated to block p38and PI3K signaling and then stimulated by rFliC. Frequency of CD4+CD25+Foxp3+cells in CD4+T cells was tested by FCM.Results1. rFliC could activate JNK and p38MAPK signaling in alloantigen stimulated Tregs. In addition, p38signaling was associated with rFliC-induced up-regulation of Foxp3expression. In other words, effect of rFliC on Foxp3expression in alloantigen stimulated Tregs was achieved via an rFliC-p38-Foxp3pathway in alloantigen stimulated Tregs, and PI3K signaling negatively regulated p38activation.2. PI3K signaling negatively regulated rFliC-p38-Foxp3pathway via inhibiting p38activation.Conclusions1. rFliC administration in vivo significantly prolonged survival time and improved living condition of murine skin allograft, and furthermore, induced allotransplantation immune tolerance. In addition, rFliC activated TLR5expression in allografts, indicating that effect of rFliC in inducing allograft tolerance was associated with TLR5pathway.2. rFliC improved frequency of recipient CD4+CD25+Foxp3+Tregs, enhanced expression of Treg-related molecules and strengthened function of Tregs, in a TLR5dependent manner. It was indicated that rFliC-induced allotransplantation tolerance may be achieved by rFliC up-regulating Tregs in a TLR5-dependent manner.3. Improvement of Foxp3expression in Tregs stimulated by rFliC was mediated by an rFliC-p38MAPK-Foxp3passageway; PI3K signaling negatively regulated this pathway by inhibiting p38MAPK activation.Points of Innovation1. It is the first time to prove the effect of rFliC in inducing allotransplantation immune tolerance in a murine skin allograft model. These results provided an experimental basis for investigating new candidate agent for inducing immune tolerance in solid-organ transplantation.2. It is the first time to verify that rFliC could improve frequency and function of CD4+CD25+Foxp3+Treg cells in allografted recipients. Furthermore, this kind of effect was in a TLR5-dependent manner.3. It is the first time to prove that effect of rFliC in up-regulating expression of Foxp3could be mediated by activating p38MAPK signaling, which means that Foxp3expression in Tregs was improved by rFliC in a rFliC-p38MAPK-Foxp3 passageway. In addition, the negatively regulation of PI3K signaling on this pathway was demonstrated. These results provided a new idea and regulating target for studying signaling pathway in rFliC inducing allotransplantation immune tolerance. |
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