The Effects Of Bortezomib Administration On The Progression Of AGVHD After Allo-HSCT

Part I Establishment of murine model of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) with different pre-conditioningsObjective: To establish murine model of aGVHD model after Allo-HSCT hematopoietic stem cell transplantation with different pre-conditionings.Methods: Twenty-five SPF grade BALB/c mice were randomly devided into five groups and then received 7, 7.5, 8, 8.5 or 9Gyγray total body irradiation. Adult C57BL/6(H-2~b)males were donor mice and BALB/c(H-2~d,♀)were recipient mice. Host mice were reconstituted with 1×10~6, 2.5×10~6, 5×10~6 or 10×10~6 BMCs via lateral tail vein injection. aGVHD model was established by coinfusion of 1×10~6、2.5×10~6、5×10~6 or 10×10~6 splenocytes via tail vein. Reduced intensity conditioning (RIC) consisted of 200mg/kg per day of fludarabine injected intraperitoneally on day -8 to day -4, 60mg/kg per day of cyclophosphamide injected IP on d-3 to d-2, and 4 Gy of total body irradiation on d-1, then injected 10×10~6 BMCs and 5×10~6 splenocytes via tail vein. Histological analysis including liver, gut, skin and lung were obtained from recipients after transplantation and were detected by HE staining. The relative percentages of host- and donor-origin cells in the recipients were determined by flowcytometry.Results: All mice receiving 7Gy irradiation achieved long-term survival. The median survival time of mice conditioned with 7.5Gy, 8GyTBI was 31 d, 21 d respectively and in above groups 40% or 20% mice survived 40 d after TBI. The mice conditioned with 8.5Gy or 9.0Gy all died within 21 days. The survival time was compared by Log-rank test,withχ2=24.72, P<0.0001. All mice survived 100d post-HSCT when reconstituted with 5×10~6BMCs. No overt aGVHD was observed when mice were reconstituted with BMCs alone. Moderate aGVHD was induced when mice were infused with 5×10~6 splenocytes. Severe aGVHD was induced when mice were infused with 10×10~6 splenocytes. Complete donor chimerism was achieved on +21d after Allo-HSCT. Histopathologic damage of target organs was typical in two different GVHD models.The median survival time of the GVHD model established by reduced intensity conditioning were 18 d. The chimerism was achieved after transplantation..Conclusion : 8.5Gy was myeloablative conditioning in BALB/c mice. Hematopoietic recovery could be achieved when BALB/c mice were reconstituted with 5×10~6 BMCs. aGVHD could be induced when mice were injected with 5×10~6 splenocytes.Part II Early administration of bortezomib could prevent aGVHD by suppressing DC maturation and functionObjective: Early administration of bortezomib (within 2 days after BMT) could protect mice from lethal GVHD. The aim of this study was to explore whether proteasome inhibition with bortezomib early after allo-HSCT could prevent aGVHD through DC suppression.Methods: DCs were prepared from mouse BM progenitor in the presence of GM-CSF/IL-4 and expanded in vitro. To observe the effects of bortezomib on DCs, we performed flow cytometry to study the phenotypic changes of DCs, conducted mixed lymphocyte reaction(MLR) to assay allo-stimulatory capacity of DCs, also performed electrophoretic mobility shift assay to study the NF-κB activity in DCs. DCs were incubated with OVA257–264 (1μg/mL, H-2b-restricted CD8-epitope) with different concentrations of bortezomib and pulsed with OT-I CD8+T cells. Intracellular cytokine staining was performed to analyze the expression of TNF-α, IFN-γ, and IL-2 of CD8+T cells. The secretions of TNF-α, IFN-γ, and IL-2 in the cellular supernatant were detected by ELISA.To evaluate the role of host DCs pretreated with bortezomib in the progression of aGVHD, Mice were divided into three groups: The control group with no DCs infusion, the recipient mice infused with host DCs pretreated with bortezomib and the recipient mice infused with host DCs without bortezomib treatment. Results: Bortezomib prevented maturation of DC with LPS stimulation. Namely, bortezomib inhibited the up-regulation of surface markers CD80, CD86, CD40 as well as MHC-II in DCs. Bortezomib reduced the capacity of DCs to induce allogeneic lymphocytes proliferation in MLR (P<0.05). However, bortezomib with low concentration does not substantially affect the capacity of DCs to prime allogeneic lymphocyte proliferation and TNF-αproduction. Bortezomib treatment inhibited the NF-κB activation upon LPS stimulation in a dose dependent fashion in DCs. The production of IFN-γand TNF-αin CD8+T cells decreased when DCs were treated with bortezomib in a dose dependent manner. Meanwhile, cellular supernatant of TNF-αand IFN-γalso declined. Bortezomib pretreated DCs infusion significantly prolonged survival in the murine aGVHD model when compared with untreated DCs or control groups.Conclusion: Bortezomib could inhibit the maturation of DCs and their function of antigen presentation, and then down-regulate the capacity of stimulating allogeneic lymphocyte proliferation. One of the main mechanisms is inhibition of NF-kappaB activation in host DCs. Hence DC suppression could be one of the mechanisms that early administration of bortezomib could prevent aGVHD.Part III The involvement of TLR4 signaling and IL-1βin promoting aGVHD by delayed administration of bortezomib after Allo-HSCTObject: Delayed bortezomib administration (3 or more days after BMT) could significantly accelerate aGVHD-dependent morbidity. In this study we assessed the effects of TLR4 signaling and IL-1βon promoting aGVHD by delayed administration of bortezomib after Allo-HSCT.Methods: In vitro BM progenitor derived DCs from C57BL/6 background mouse were generated and expanded in the presence of GM-CSF and IL-4. After 7days culture cells were divided four groups: (1) delayed administration of bortezomib group, adding different concentrations of bortezomib at 6 h after LPS treatment. (2) Early administration of bortezomib group, before LPS treatment adding different concentrations of bortezomib at 6 h. (3) LPS control group, with LPS (100 ng/mL) alone. (4) Bortezomib control group, with different concentrations of bortezomib alone. 24 h later, cytokines in the culture supernatants were determined by ELISA. DCs prepared from C57BL/6 mice were treated with as above, and then cultured with allogeneic splencytes from BALB/c, Tritiated thymidine was pulsed to detect the proliferation of allogeneic responder cells. Cytokines in the culture supernatants were determined by ELISA.To test our hypothesis in vivo, we intervened at three critical points to determine of the role of TLR4 pathway and IL-1βin promoting aGVHD by delayed administration of bortezomib.A. TLR4 KO mouse as recipients: We used TLR4 KO mouse as recipients, BALB/c mice as donors and observed the effects of delayed administration of bortezomib without TLR4 signals.B. Reduced intentsity pre-conditioning: We used fludarabine, cyclophosphamide and 4 Gy of total body irradiation to establish reduced intensity conditioning in aGVHD model, detecting the LPS level in serum after BMT +1, +3, +5 day. Up-regulation LPS test were divided four groups: (1) delayed administration group, treated with bortezomib (500ug/kg) at day +3 post-BMT. (2) delayed administration of bortezomib and LPS injection group, treated with LPS (5 mg/kg), then 6h later bortezomib (500ug/kg) at day +3 post-BMT. (3) LPS injection control group (treated with LPS at day +3 post-BMT). (4) GVHD model control group (treated with 100ul PBS at day +3 post-BMT). Serum cytokine levels were determined by ELISA. Histological analysis was performed from recipients target organs after transplantation.C. IL-1βblockade: Murine aGVHD models with mild severity were set up by infusing lethally irradiated BALB/c with doses of 5×10~6 splencytes and 10×10~6 BMs of donor C57BL/6 mice. Detection of serum LPS levels was performed by Tachypleus Limulus Amebocyte Lysate (LAL) assay after allo-HSCT at day+1, +3and +5. Meanwhile, serum cytokine levels were determined by ELISA. We used anakinra to block IL-1βfunction to determinate its effects on the progression of aGVHD by delayed administration of bortezomib. The mice were divided into four groups: (1) early administration group, treated with bortezomib (500ug/kg) daily from day +1 through day +3 post-BMT. (2) delayed administration group, treated with bortezomib on day +3 post-BMT. (3) delayed administration with IL-1βblockade, treated with anakinra (6mg/kg) daily from day 1 through day +3 post-BMT, then bortezomib (500ug/kg). (4) IL-1βblockade alone group, treated with anakinra (6mg/kg) daily from day +1 through day +3 post-BMT. (5) GVHD model control group, (treated with 100ul PBS daily from day +1 through day +3 post-BMT. Serum inflammation cytokines were determined by ELISA. Histological analysis and immunohistochemstry was analyzed from recipients after transplantation in target organs.Results: The IL-1βsecretion in delayed administration group was higher than that of early administration group, LPS control group or bortezomib control group ( P<0.01). [3H]-TdR test showed the capacity of DCs to stimulating allogeneic lymphocytes proliferation in delayed administration group increased comparing with that in early administration group, LPS control group or bortezomib control group ( P<0.01). The IL-4 level of delayed administration group was the highest, compared with that of early administration group, LPS control group or bortezomib control group ( P<0.01).TLR4 KO recipients receiving delayed administration of bortezomib survived longer than C57BL/6 control mice.In RIC GVHD model serum LPS level rarely increased in recipients on day+3 and +5 post- BMT as seen in TBI model. The survival rate was significantly increased in the RIC GVHD model, compared with that of hige doses irradiation group with delayed bortezomib administration. The serum IFN-γand IL-1βlevels were higher in delayed administration group than those in other groups in TBI GVHD model.In IL-1βblockade experiment, anakinra reduced the mortality from GVHD in delayed administration group. The serum TNF-αand IFN-γlevels were also lower than those of anakinra control groups or GVHD control groups. On the contrary, IL-4 increased in anakinra plus delayed administration groups. Histological analysis, clinical scores and immunohistochemistry analysis all demonstrated that the progression of GVHD caused by delayed administration of bortezomib was alleviated by IL-1βblockade. Conclusion: Our study demonstrates that TLR4 pathway plays a critical role in accelerated GVHD-related mortality by delayed administration of bortezomib. Low level of LPS in RIC model, inhibition of TLR4 signal or IL-1βblockade could significantly prevent the accelerated GVHD-related death caused by delayed administration of bortezomib after Allo-HSCT.