New Strategies And Mechanisms For Anti-GVHD By A Novel MyD88Signaling Inhibitor

Part I Correlation between overexpression of MyD88signaling and acute graft-versus-host disease[Objective] To explore the expression and significance of TLR4/MyD88/NF-κB signaling in murine model with acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation.[Methods] Recipient BALB/c female mice were lethally irradiated and given transplants of bone marrow cells (1*107) and different amounts of splenocytes (1*107, n=12or2*107, n=12) from MHC-mismatched C57BL/6donors to induce GVHD. Those healthy BALB/c mice without any intervention as control. A globe survey observation of GVHD by survival, clinical manifestation, histological detection was performed. RT-PCR, immunohistochemistry, and western blot technology were used to detect the mRNA and protein levels of TLR4, MyD88and NF-KBp65of small intestine tissue.[Results] The exacerbation of GVHD was associated with the increasing dose of allogeneic spleen lymphocytes. The mRNA expression of TLR4, MyD88and NF-icBp65appeared to be rising as the GVHD progresses. All of them in severe GVHD model were significantly increased than that in healthy control (P<0.05). The expression of corresponding protein had the same tendency as mRNA. All of the three genes expression was not only positively correlated with each other, but also positively correlated with the clinical GVHD score:TLR4, R=0.814, P<0.001; MyD88, R=0.828, P<0.001; NF-κBp65, R=0.568, P=0.034).[Conclusion] Excessive activation of TLR4/MyD88/NF-κB signaling pathway do exist in acute GVHD and was positively correlated with the deterioration of GVHD, which could be the new targets of drug development. Part II Novel MyD88inhibitor TJ-5protects against acute GVHD in mice and its mechanism[Objective] To investigate the role of MyD88inhibitor TJ-M2010-5(TJ-5) in murine GVHD after allogeneic bone marrow transplantation and the possible mechanism, and provide the innovation for GVHD treatment based on innate immunity.[Methods](1) The effect of TJ-5on MyD88homodimerization was detected by co-immunoprecipitation assay; activation and maturation of DC by TJ-5were dected by flow cytometry and ELISA; the changes of MyD88signaling in DC after TJ-5treatment were detected by western blot; the allo-responses of lymphocytes were detected by mixed lymphocyte culture test.(2) A fully allogenic C57BL/6(H-2Kb) to BALB/c (H-2Kd) GVHD murine model was established. The experiment was grouped as follows:GVHD control group; TJ-5treatment group; anti-CD40L group; TJ-5+anti-CD40L group. GVHD scores, survival, pathology, inflammatory cytokines, tissue necrosis and repair, the formation of chimera and donor specific tolerance were used to evaluate the efficacy of different anti-GVHD protocols. [Results] Homodimerization of MyD88and LPS-stimulated DC maturation were inhibited by TJ-5in a dose-dependent manner, reducing the secretion of inflammatory cytokines, such as IL-la, IL-10, IL-12and IL-18in DC supernatant. Increasing expression of TLR4and decreasing expressions of the downstream molecules, IRAK4and NF-Bp65, were observed in line with the increasing dose of TJ-5. Proliferation of CD4+T and CD8+T cells in MLR were obviously suppressed by TJ-5. During60days after BMT, the improved survival and GVHD scores were observed in TJ-5-or MR1-treated group compared with control (P<0.05); the best efficacy was observed in combination-treated group, showing the100%survival rate, significantly higher than MR1group (70%), TJ-5group (57%) and the control group (0%). Thomas GVHD pathological grade was higher in control group (Ⅲ Ⅳ) than that in other groups (Ⅰ-Ⅱ). Accelerated formation of allo-chimeras, improved inflammatory cytokines, reduced cell apoptosis and enhanced tissue repair were also observed in TJ-treated group,[Conclusion] Novel MyD88inhibitor TJ-5can alleviate GVHD progression after bone marrow transplantation in mice. Protection against GVHD was more easily achieved with a combined inhibition of innate and adaptive immunity. Targeting MyD88for inhibition to improve GVHD was an effective protocol Part Ⅲ TJ-5prevents the development of multiple myeloma relapse in a GVT effect mouse model[Objective] To text whether TJ-5’s immunosuppressive properties had any impact on the GVT effect in a myeloma relapse mouse model and provide the further experimental evidence for the clinical application of TJ-5.[Methods] A fully allogenic C57BL/6(H-2Kb) to BALB/c (H-2Kd) mice GVHD model was established. For the purpose of induction of GVT effect, BALB/c recipients received BALB/c-susceptible SP2/0myeloma cells on the day of transplantation in addition to donor BM with or without donor T cells. The experiment was grouped as follows:BM reconstruction group, tumor relapse group, T cell-treated group, T cells+TJ-5treated group. Recipients were monitored for tumor-or GVHD-related morbidity and mortality. Treatment was withdrawn at day60after BMT. GVHD morbidity was assessed using a standard scoring system and death from tumor was established according to the histopathology showing tumor infiltration in the BM or spleen. The effects of TJ-5on apoptosis, necrosis and cell cycle of SP2/0, expression of chemokine CCR7on DC, and the balance of Treg in vivo were evaluated by flow cytometry, to further clarify the possible mechanism of TJ-5on the retention of GVT effect.[Results] During the60days after BMT, survival rate in TJ-5-treated group and GVT control group was50%and12.6%, respectively (P-0.04). Alleviated tissue lesions and tumor cell invasion in TJ-5group were observed, in which no obviously weaken GVT effect was observed. In vitro, TJ-5can induce the apoptosis and necrosis of SP2/0cells via G1arrest, and optimize LPS-induced CCR7expression on DCs. Splenic Treg level in TJ-5treated mice was significantly increased compared with GVHD control (P<0.05).[Conclusion] TJ-5preserved GVT activity against myeloma relapse in the progress of anti-GVHD. These anti-relapse effects are likely related by the direct cytotoxicity of TJ-5on SP2/0, the inhibition of CCR7signaling and the up-regulation of Treg. Part IV Abrogation of host MyD88signaling cannot protect against acute graft-versus-host disease[Objective] To delineate the role of host MyD88in the development of acute GvHD following allogeneic bone marrow transplantation and provide the experimental evidence for the time window of TJ-5clinical use.[Methods] BALB/c wild-type (wt) or MyD88-/-mice were used as transplant recipients and B6mice as donors to induce acute GVHD. During30days after BMT, survival, body weight change, GVHD score, pathology, inflammatory cytokine level were used to evaluate the severity of GVHD. The proliferation of donor-derived cells, cell type and function were detected by Confocal assay; LPS level in vivo was detected by ELISA; proliferation and apoptosis of intestinal epithelial cell (IECs) were detected by immunohistochemistry; MyD88-dependent Cox-2signals in IECs was detected by western blot; tight junction proteins (ZO-1, claudin-3and occludin) expression in IECs were detected by real-time PCR to evaluate the intestinal barrier function; the allo-responses of lymphocytes were detected by mixed lymphocyte culture test.[Results] When myeloablated BALB/c MyD88knockout recipients were transplanted with C57BL/6(B6) donor cells as wt recipients, they developed significantly more severe GvHD than wt hosts. The increased morbidity and mortality in MyD88-/-mice correlated with more severe pathological damage, increased serum levels of lipopolysaccharide and elevated inflammatory cytokines in GvHD target organs. Additionally, MyD88deficiency in BMT recipients led to increased donor T-cell expansion and more donor CDllc+cell intestinal infiltration with apoptotic cells but reduced proliferation of intestinal epithelial cells compared with that in wt BMT recipients. Cox-2expression in the GvHD-targeted organs of wt mice is increased upon GvHD induction, but the enhanced expression was obviously inhibited by MyD88deficiency. Decreased expression of tight junction mRNA (ZO-1, claudin-3and occludin) in epithelial cells of MyD88-/-mice (P<0.05) suggested that MyD88contributes to intestinal integrity. MyD88deficiency did not significantly reduce the proliferation of allogeneic lymphocyte in mixed culture system.[Conclusion] Host MyD88signals play a protective role in GVHD progression. The possible mechanisms may be associated with the reduction of donor-derived effector cell infiltration, the maintainance of intestinal barrier integrity and the induction of Cox-2expression. Our data suggest that blockade of host MyD88at the time of transplantation or before BMT are unlikely to prevent GVHD completely.