| Part I The effect of MyD88inhibitor TJ-M2010on the biological functions of dendritic cells.[Objective] To the effect of TJ-M2010on the biological functions of mice bone marrow-derived dentritic cells including the activation of NF-κB, the expression of co-stimulatory molecules, phagocytic function, migration ability, apoptosis, the ability to activate T cells proliferation.[Methods] Mice immature bone marrow-derived dendritic cells were induced and cultivated in vitro and the cells were divided to4groups:Normal control group (cells without any treatment), CpG group, CpG+TJ-M2010(low concentration,20μM) group, CpG+TJ-M2010(high concentration,40μM) group. After incubation for24hours, the activation of NF-κB was detected by western blot. The expression of CD80, CD86, MHCⅡand CCR7on DCs were detected by flow cytometry. C57T cells stained with CSFE were co-cultured with BALB/c DCs treated with TJ-M2010of different concentrations in the presence of CpG for3days, and CD3+CFSE+T cells were analyzed by flow cytometry. BMDCs were cultured with TJ-M2010at different concentrations in the presence of FITC-Dextran and the phagocytic function was detected by flow cytometry. Apoptosis and necrosis were detected with the Annexin-V/PI apoptosis kit.[Results] The flow cytometry results demonstrated that CpG could induce high level expression of CD80/CD86/MHCⅡ on DCs (53.1±0.3%/55.6±3.5%/64.6±4.3%). TJ-M2010could reduce the up-regulation of these molecules dose-dependently (40μM:20.7%±.1%/26.3±1.5%/28.1±3.4%VS20μM:38.2%±2.2%/40.4%±1.6%/45.1%±2.4%, p<0.05). TJ-M2010could reduce the up-regulation of CCR7upon stimulation of CpG. TJ-M2010could also reduce the frequency of reactive CD3+CFSE+T cells during the mix lymphocyte culture with BMDCs. There was no significant difference in the phagocytic rate of FITC-Dextran between the normal control group and TJ-M2010groups (63.4%±3.4%vs65.7%±2.6%vs62.6%±2.6%, p>0.05); The frequency of Annexin-V/PI double positive DCs in TJ-M2010groups also showed no significant difference when compared with the normal control group (6.6%±0.9%vs6.5%±1.1%vs6.6±1.1%p>0.05).[Conclusion] TJ-M2010could dose-dependently inhibit DCs maturation, migration ability and activation of NF-κB, as well as the proliferation of reactive T cells indirectly. TJ-M2010did not influence DCs phagocytic function and it also showed no obvious toxicity to DCs. Part Ⅱ MyD88inhibitor TJ-M2010promotes tolerance to cardiac allografts in mice and its mechanism[Objective] To investigate the role of MyD88inhibitor TJ-M2010in the induction of tolerance for mice cardiac transplantation and the possible mechanism.[Methods](1) A fully allogenic BALB/c (H-2d) to C57BL/6(H-2b) mice abdominal cardiac transplantation model was established. Cardiac recipients were left untreated or given either TJ-M2010or control vehicle (0.5%carboxymethyl cellulose, CMC). The day of operation was recorded as day0. TJ-M2010(50mg/kg/day) suspended in0.5%CMC was administrated to experimental recipients by intraperitoneal injection (i.p.) from Day-2to Day7. Graft beating was monitored by daily palpation. The survival of cardiac grafts was observed.(2) Cardiac grafts were obtained7days after the transplantation. HE staining was performed to estimate the severity of rejection. Mononuclear cells in grafts were obtained by grafts digestion with collagenase Ⅱ and CD11c+CD80+double positive cells were detected by flow cytometry. The mRNA expression of IL-1, IL-6and TNF-a in allografts were analyzed by real-time PCR.(3) The frequency of CD4+CD25+Foxp3+Tregs in recipients was detected by flow cytometry. The allo-response of splenocytes were detected by INF-y ELISPOT.(4) For long-term-survived recipients, second cervical cardiac transplantation was carried out and the allografts survival time was observed.[Results] The grafts of normal control group and CMC group were promptly rejected at early period (MST=7.4±0.5days vs MST=7.6±0.5days, p>0.05). However, TJ-M2010monotherapy (50mg/kg/day, from Day-2to Day7) led to%of allografts surviving for more than100days. For second cardiac transplantation, long-term-survival recipients did not reject the BALB/c hearts for more than100days. Pathological examination demonstrated that TJ-M2010attenuated the rejection responses in allografts in the aspect of lymphocytes infiltration and tissue damage. Real-time PCR results showed that the mRNA expression of IL-1, IL-6and TNF-a in allografts of TJ-M2010group was significantly reduced when compared with that of CMC group (p<0.05). The frequency of CD11c+CD80+double positive cells in allografts of TJ-M2010group was also reduced when compared with that of CMC group. The frequency of CD4+CD25+Foxp3+Treg in recipients treated with CMC was6.43%while12.92%in long-term-survived recipients treated with TJ-M2010. INF-y ELISPOT results showed that splenocytes in long-term-survived recipients had a normal response to C3H antigen in vitro (p>0.05) but an attenuated response to BALB/c antigen (p<0.05).[Conclusion] Short-term monotherapy of MyD88inhibitor TJ-M2010could induce donor-specific tolerance for mice cardiac transplantation. Reduced inflammatory cytokines secretion and Treg induction may involved in the mechanism. Part III MyD88inhibitor combined with anti-CD154lead to tolerance in mice skin transplantation[Objective] To investigate the possibility that MyD88inhibitor TJ-M2010combined with anti-CD154can induce tolerance in mice skin transplantation and the mechanisms. [Methods] A fully allogenic BALB/c (H-2d) to C57BL/6(H-2b) mice skin transplantation model was established. Generally, full thickness skin grafts (-1cm2) from tails of donor mice were transplanted to dorsal flank of recipient mice. The experiment was grouped as follows:TJ-M2010treatment group; anti-CD154group; TJ-M2010+anti-CD154group. The day of operation was recorded as day0. TJ-M2010(50mg/kg/day) suspended in0.5%CMC was administrated to experimental recipients by intraperitoneal injection (i.p.) on day-2-day7, day9, day11, day13, day15. Anti-CD154(MR1clone,200μg/dose) was administrated by i.p. on day0to day3, day7and day14. Rejection was defined as necrosis of the entire graft surface by daily inspection. The survival of cardiac grafts was observed. For long-term-survived recipients, second skin or cardiac transplantation were carried out and the survival time was observed. The frequency of CD4+CD25+Foxp3+Tregs in recipients was detected by flow cytometry. The allo-response of splenocytes were detected by INF-γ ELISPOT.[Results] There were no significant differences between TJ-M2010group and anti-CD154group in allografts survival time (9.0±0.71days VS9.6±0.55days, p>0.05) while TJ-M2010combined with anti-CD154led to22%of allografts surviving for more than100days. For second cardiac transplantation, long-term-survived recipients did not reject the BALB/c hearts for more than100days. For second skin transplantation, long-term-survived recipients rejected both the BALB/c and C3H skin allografts at early stage. However, when the recipients were treated with TJ-M2010at the time of second skin transplantation, the allografts survival time prolonged significantly. The frequency of CD4+CD25+Foxp3+Treg in recipients treated with TJ-M2010+anti-CD154was significantly rised. INF-γELISPOT results showed that splenocytes in long-term-survived recipients had a normal response to C3H antigen in vitro (p>0.05) but an attenuated response to BALB/c antigen (p<0.05).[Conclusion] MyD88combined with anti-CD154induced tolerance in mice allogenic skin transplantation. The tolerance was unstable. MyD88played an essential role in the maintenance of this tolerance. |
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