The Role And Mechanism Of Nrf2 In The Development Of Acute Graft-versus-host Disease

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The role of Nrf2 activation in acute graft-versus-host diseaseObjective: To investigate the role of Nrf2 activation in the development of acute graft-versus-host disease.Methods: Mouse sys-HSCT and allo-HSCT model were established.The tissues from a GVHD target organs spleen,liver,lung and intestine was isolated and subjected to RNA extraction,the m RNA expression of Nrf2 in a GVHD target organs was examined by real time PCR.Responder T cells were isolated from spleen of C57BL/6 mice by mouse T cell enrichment kit.Stimulators were DCs from BALB/c cells.BM-derived DCs were generated and expanded from BALB/c mice with GM-CSF(10 ng/ml)and IL-4(10 ng/ml)for 7 days.DCs were pretreated with Nrf2 agonist Dimethyl fumarate(DMF)or DMSO for 24 h,then washed twice with PBS.1 × 10~4 DCs were irradiated(30 Gy)and cocultured with 1 × 10~5 allogeneic T cells in U-bottom microwell plates to establish mixed lymphocyte reaction assay.Cell proliferation was measured by ~3H-Td R and CFSE dilution.Cell apoptosis was measured by ANNXINV/PI by Flow cytometry.DCs were treated with DMF or LPS,the levels of CD80,CD86 and CD40 on DCs were measured by Flow cytometry.The levels of IL-6,IFN-? and TNF-? in the culture supernatants were examined by ELISA kit.BALB/c mice were given lethally 650 c Gy(one dose)total body irradiation from X-ray,irradiated BALB/c mice was transplanted with 1 × 10~7 C57BL/6 bone marrow cells and 5 × 10~6 C57BL/6 spleen cells via the tail vein.DMF(30 mg/kg body weight)was administrated to the recipient mice by gavage once daily starting from day-3 to day 3 after bone marrow transplantation(BMT).0.8% methocel at the same volume was used as vehicle control.The recipients were monitored daily for survival and every three days for body weight changes and clinical signs of GVHD.The severity of GVHD was assessed using a GVHD scoring system based on the H&E of a GVHD target organs.Results:(1)The results of Realtime PCR showed that Nrf2 m RNA levels were significantly decreased in the spleen,liver,lung and intestine of allogeneic BMT mice compared to those of syngeneic control animals,suggesting that Nrf2 suppression could be involved in the pathogenesis of a GVHD.(2)DMF significantly inhibited the proliferation of alloreactive T cells in a dose-dependent manner on day 5 determined by ~3H-Td R and CFSE dye dilution.Consistent with the T cell proliferation results,cytokine analysis showed that proinflammatory cytokines IL-2,IL-6,IFN-?,and TNF-? production were significantly decreased in a dose-dependent manner upon DMF treatment.In addition,cell apoptosis assay showed that the apoptosis of alloreactive CD4+ T and CD8+ T cells were significantly increased in the presence of DMF.More interesting,the apoptotic cells were mainly CFSE low populations.(3)To address whether DMF has a direct effect on T cells,we performed a T cell activation assay with anti-CD3/CD28 stimulation,the result showed that DMF could significantly inhibit T cells proliferation directly.(4)we examined the effect of DMF on antigen presenting cells(APC).We cultured bone marrow DCs and then treated with DMF.The data showed that DMF had no effect on DC maturation,however,when DCs were preactivated by LPS,DMF could reduce CD80,CD86,and CD40 expression in a dose-dependent manner.Similar results were observed in IL-6 and TNF-? expression.(5)DMF treatment significantly prolonged survival of the hosts following allo-HSCT.In addition,a GVHD scores were reduced in DMF-treated mice compared with vehicle control recipients.Histological analysis revealed that there was decreased pathological damage in the liver,lung,intestine and skin of recipients receiving DMF 14 days after allo-HSCT.Conclusion: Our data suggested that Nrf2 signaling was down-regulated in allo-HSCT.Activation of Nrf2 by DMF could inhibit alloreactive T cells proliferation and proinflammatory cytokines production,as well as DCs activation.DMF treatment in vivo significantly improved a GVHD outcomes as evidenced by prolonged survival and reduced a GVHD scores following allo-HSCT.Part ? The mechanism of Nrf2 activation in alleviating acute graft-versus-host diseaseObjective: To investigate the immune mechanism of Nrf2 activation by DMF in alleviating acute graft-versus-host disease.Methods: Splenocytes isolated from transplanted recipients 14 days after BMT were as responders,irradiated splenocytes from BALB/c mice were as stimulators.Ex-vivo MLR was established,cell proliferation was measured by ~3H-Td R.Splenocytes were isolated from transplanted recipients 14 days after BMT,the frequencies of CD3+T,CD4+T,CD8+T cells,as well as cell activation in the spleen was examined by FACS.Intracellular staining were performed to examine the IFN-? expression by CD4+T and CD8+T cells after PMA/Ionomycin/BFA stimulation in vitro.The serum levels of IFN-?,IL-6 and TNF-? were measured by ELISA assay.CD3+T cells were isolated from spleen of C57BL/6 mice and activated by plate bound anti-CD3(5 ?g/ml)and anti-CD28(1 ?g/ml)in the presence of DMF or DMSO for 24 h.the expression of CD69,CD25,CD44 and PD-1 were examined by FACS.ROS levels were examined by Reactive Oxygen Species Assay Kit according to the manufacturer's instructions.Total RNA were extracted with TRIzol reagent from spleen14 days after BMT.Transcription levels of Nrf2,Keap1,HO-1,GST-?1 genes were analyzed by real-time PCR.Nrf2 nuclear translocation in T cells was examined by immunofluorescent microscopy.Results:(1)The ex vivo MLR assay demonstrated that DMF treatment significantly reduced donor T cell alloreactivity.(2)On day 14 after allo-HSCT,decreased infiltration of donor CD3+ T and CD4+ T cells was observed in spleen of DMF-treated recipients compared with vehicle control recipients.In addition,both donor CD4+ T and CD8+ T cells had a reduced activation phenotype indicated by CD69 levels in the hosts treated with DMF.(3)Purified CD4+ T cells were activated in vitro by anti-CD3/CD28,and DMF treatment significantly inhibited T cell activation and downregulated CD69,CD44,as well as PD-1 expressions.The CD25 levels,however,were slightly upregulated by DMF treatment.(4)Intracellular staining revealed that the frequencies of IFN-?-producing donor CD4+ T and CD8+ T cells in spleen were significantly decreased in recipients given DMF.Analysis of serum samples on day 14 following allo-HSCT showed that the levels of proinflammatory cytokines IL-6,IFN-? as well as TNF-? were significantly downregulated in DMF-treated recipients compared with vehicle control recipients.(5)Since DMF is a potent activator of Nrf2 and can induce the Nrf2-dependent antioxidant response,we then examined the ROS production and antioxidant genes expression.We found that DMF could significantly inhibit ROS production by activated T cells in a dose-dependent manner in vitro.Moreover,the Nrf2 levels and antioxidant defense enzymes HO-1 and GST-?1 expressions were upregulated in recipients treated with DMF.We then explored the effect of DMF on nuclear translocation of Nrf2,which has been demonstrated as a major mechanism of function for DMF.We observed a dose dependent effect of DMF on nuclear translocation of Nrf2 in the CD3+ T cells by immunofluorescent staining.Conclusion: Our data suggested that DMF could inhibit donor T cell alloreactivity,and production of proinflammatory cytokines,as well as upregulate antioxidant enzymes,which led to the alleviation of a GVHD severity.Part ? Nrf2 activation promoted Treg cell developmentObjective: To investigate Nrf2 activation in regulateing Treg cells development,and whether DMF alleviated acute graft-versus-host disease was Treg dependent.Methods: Splenocytes were isolated from transplanted recipients 14 days after BMT,the frequencies of Treg cells in the spleen was examined by FACS.CD4+T cells were isolated from spleen of C57BL/6 mice and activated by plate bound anti-CD3(5 ?g/ml)and anti-CD28(1 ?g/ml)with TGF-? and IL-2 induction,in the presence of DMF or DMSO for 4d.Treg cell differentiation was examined by FACS.lethally irradiated BALB/c mice were transplanted with 5 × 10~6 TCD-BM plus 1 × 10~6 total spleen T cells or CD25-depleted T cells from B6 mice.CD25 depletion was performed by using mouse CD25 regulatory T cell positive selection kit.Unlabeled CD25 negative cells were collected.The levels of TGF-? in serum and MLR culture supernatants were measured by ELISA.Results:(1)The frequencies,as well as the numbers of Treg cells in spleen were significantly increased in DMF-treated compared with vehicle control recipients,suggesting that DMF could increase Treg cells in vivo.(2)In vitro Treg polarization assay showed that DMF promoted Treg cell differentiation in a dose-dependent manner.(3)To further confirm that the effect of DMF on a GVHD was partially dependent on the promotion of Treg cells,mice were injected with anti-CD25 antibody to deplete Treg cells in vivo.The results demonstrated that depletion of CD25+ cells in DMF recipients aggravated a GVHD mortality compared with Ig G control recipients,while it had no effect on vehicle treatment recipients.(4)TGF-? has been known to promote Treg generation,we therefore examined the TGF-? levels in serum of GVHD mice and in MLR culture supernatants.Date shown that TGF-? was increased in DMF recipients and upregulated upon DMF treatment in vitro.Conclusion: Our data suggested that the protective effect of DMF treatment on a GVHD was at least partially dependent on the promotion of Treg cells.DMF could promote Treg cells differentiation by inducing TGF-? expression.Part ? The role and mechanism of Nrf2 activation in the GVL effect after allo-HSCTObjective: To investigate the role and mechanism of Nrf2 activation in the GVL effect after allo-HSCT.Methods: BALB/c mice were given lethally 650 c Gy(one dose)total body irradiation from X-ray,irradiated BALB/c mice was transplanted with 1 × 10~7 C57BL/6 bone marrow cells and 5 × 10~6 C57BL/6 spleen cells via the tail vein.1 × 10~6 A20-luc cells were added to bone marrow graft as mentioned above,and injected into lethally irradiated BALB/c mice.mice were given an intraperitoneal injection of 200 ?g firefly luciferin and then anesthetized and imaged using Xenogen,IVIS 100 Bioluminescent Imaging System.Splenocytes from transplanted recipients 14 days after BMT were used as killing cells,and their killing ability of A20 targets was measured using Cyto Tox 96 nonradioactive cytotoxicity assay kit.1× 10~4 A20 cells were seeded into 96-well plates and treated with DMF or DMSO for 48 h.Then 10 ?l of CCK-8 solution was added to each well and incubated for additional 2 h,the absorbance was measured at 450 nm.Cell apoptosis was evaluated using a Annexin V Apoptosis Detection Kit.Results:(1)To evaluate the impact of DMF administration on GVL effects,a GVHD mice were challenged with A20-luc leukemia cells post-allo-HSCT.The results showed that mice transplanted with allo-BM alone plus A20-luc leukemia cells all died from leukemia within 40 days after transplant,regardless of whether DMF was administered,suggested that DMF may not directly affect A20 cell growth in vivo.DMF administration to mice receiving allo-BM and splenocytes,plus A20-luc showed prolonged survival compared with mice receiving vehicle control,and low tumor burden was observed in DMF recipients as shown in bioluminescence imaging,indicating the presence of GVL effect in DMF treated mice.(2)The cell proliferation and apoptosis assay showed that low concentrations of DMF could not affect tumor growth and apoptosis in vitro,suggesting that DMF treatment inhibits A20 cell growth only at high concentrations,which may not be reached in vivo.We also observed that donor T cells from DMF-treated recipients showed comparable,or even increased CTL killing activity against A20 leukemia cells compared to vehicle controls.Conclusion: Our data suggested that DMF treatment could preserve GVL effect after allo-HSCT.DMF could not affect tumor cells proliferation and apoptosis,but promote CTL killing activity against A20 leukemia cells.