Inhibition Of Murine Allograft Rejection By Luteolin And Its Immunomodulatory Mechanism

ObjectiveImmune-based allograft rejection is a main reason for graft failure after transplantation.Immunosuppressive regimens are widely used for prevention of both acute and chronic transplantation rejection,but organ survival is still limited by its associated toxicity.However,transplant survival may be compromised due to adverse reactions caused by a conventional immunosuppressant.An ideal immunosuppressive protocol is to induce long-term transplant acceptance or tolerance with minimal adverse reactions,and an imperative research objective is to improve the longevity of transplanted organs and the quality of life in the field of transplantation.Natural ingredients,derived from herbal medicine or edible plants,have been reported to be effective in suppression of allograft rejection with little adverse reactions in experimental animal models.Luteolin,a plant flavonoid,is commonly present in many herbs and vegetables.Luteolin exhibits multiple biological and pharmacological effects,such as anti-inflammatory,anti-cancerous,and neuroprotective properties.Recent studies have reported that luteolin suppresses the expression of proinflammatory cytokines,including TNF-α,IL-6,IFN-γ,and IL-5.Importantly,luteolin has been shown to promote CD4+CD25-T cell differentiation into CD4+CD25+regulatory T cells in the context of airway inflammation.Moreover,a previous study has revealed that renal ischemia/reperfusion injury after kidney transplantation is improved by administration of luteolin.However,it is unknown whether luteolin regulates alloimmunity and suppresses allograft rejection.Given that luteolin has been shown to upregulate Tregs and suppress inflammation,we hypothesized that luteolin could be a new immunosuppressant that might inhibit allograft rejection.In this study,we used a mouse model of skin allotransplantation and islet allotransplantation to determine the effects of luteolin on allograft rejection and to identify its mechanisms of action.Methods1.Effect of luteolin on skin allograft rejection in miceBALB/c mice were skin donors,whereas skin graft recipients were C57BL/6 mice.Mice were randomly divided into four groups,including control group(Control,by mouth,0.5%CMC-Na),rapamycin group(Rapa,i.p.1mg/kg),low dose of luteolin(Lut-low,by mouth,25mg/kg)and high dose of luteolin(Lut-high,by mouth,50mg/kg),and treated daily until graft rejection/sample collection and indicated by median survival time(MST).Skin grafts were removed from euthanized recipient mice,stained with H&E,immunohistochemistry and immunofluorescence to examine cellular infiltration,CD3 and Foxp3 expression 10 days after transplantation.The mRNA expressions of IFN-γ、IL-6、IL-10、TNF-α、IL-17a、Foxp3 in skin allografts were determined by RT-PCR.Draining lymph node cells or splenocytes were isolated from recipient mice,frequencies of CD4+CD44high CD62Llow or CD8+CD44high CD62Llow effector T cells,CD4+Foxp3+Tregs and CD11 c+CD86+or CD 11 c+CD80+mature DCs were analyzed using a flow cytometer.To deplete CD4+CD25+Tregs,recipient mice were treated i.p.with anti-CD25 Ab(PC61)at 0.2mg on days 0,3,and 6 post-transplantation,and divided into four groups,control group(Control,by mouth,0.5%CMC-Na),high dose of luteolin(Lut-high,by mouth,50mg/kg),Lut-high combined with isotype antibody group(Lut-H+Isotype)and luteolin combined with anti-CD25 antibody group(Lut-H+PC61).Skin allograft rejection was monitored daily and frequencies of CD11c+CD86+orCD11c+CD80+mature DCs were analyzed 10 days after transplantation.2.Effect of luteolin on islet allograft rejection in miceSTZ-induced diabetic C57BL/6 mice were transplanted with pancreatic islets from BALB/c mice,and randomly divided into four groups,including control group(Control,by mouth,0.5%CMC-Na),rapamycin group(Rapa,i.p.lmg/kg),low dose of luteolin(Lut-low,by mouth,25mg/kg)and high dose of luteolin(Lut-high,by mouth,50mg/kg),blood glucose levels was monitored daily until rejection occurred and indicated by median survival time(MST).Islet grafts were removed from recipient mice,cellular infiltration and insulin expression were evaluated by immunohistochemistry and H&E staining.Besides,the frequencies of CD4+Foxp3+Tregs in draining lymph nodes and spleen cells were analyzed via a flow cytometer.3.In vitro study on the mechanisms underlying inhibition of allograft rejection by luteolinFACS-sorted CD3+T cells derived from C57BL/6 mice were detected T cell cytotoxicity by CCK-8 assays.T cell proliferation was measured using a FACSCalibur after labeled with 3μM CFSE for 4 days,and the supernatant expression levels of IFN-γ,IL-10 and IL-17a measured by ELISA according to the manufacturer’s protocol with or without Lut-low(2.5μM)or Lut-high(5.0μM)or Rapa(0.1μM).FACS-sorted CD4+CD25-T cells from C57BL/6 mice and cultured with anti-CD3 and anti-CD28 Abs plus rIL-2 in the absence or presence of Lut-low(2.5μM)or Lut-high(5.0μM)or Rapa(0.1 μM)for 4 days,the frequencies of CD4+Foxp3+Tregs were determined using a flow cytometer.The protein expressions of phosphorylated AKT(P-AKT),AKT,P-mTOR and mTOR,P-p70S6K and p70S6K in T cells were determined using western blotting analysis with or without luteolin or Rapa.Results1.Effect of luteolin on skin allograft rejection in miceluteolin,at either low or high doses,prolonged skin allograft survival compared with the control group[MST=23.50±5.34(Lut-low)VS.16.50±2.74(Control),P<0.05;MST=33.00±5.04(Lut-high)VS.16.50±2.74(Control),P<0.01].Interestingly,when compared with Rapa,Lut-high treatment exhibited a nearly equivalent effect on prolongation of skin allograft survival[MST=33.00±5.04(Lut-high)VS.35.00±6.70(Rapa),P>0.05].We found that treatment with either luteolin or Rapa obviously reduced overall cellular infiltration as well as CD3+T-cell infiltration in the skin allografts compared to the control group(P<0.05 or P<0.01)and gene expressions of proinflammatory cytokines IFN-y,TNF-α,IL-6,and IL-17a were downregulated while immunosuppressive cytokines IL-10 and Foxp3 were upregulated.In the secondary lymphatic organs of recipient mice,luteolin significantly decreased the frequencies of CD4+CD44highCD62Llow or CD8+CD44high CD62Llow effector T cells as well as CD11c+CD86+or CD11c+CD80+mature DCs in the spleen and lymph nodes(P<0.05 or P<0.01).In contrast,luteolin or Rapa significantly increased the frequencies of CD4+Foxp3+Tregs in draining lymph nodes compared with the control group(P<0.01).Lut-high significantly prolonged skin allograft survival while depleting CD4+CD25+Tregs largely,although not totally,reversed the luteolin-induced extension of skin allograft survival.However,isotype control for PC61 did not reverse the allograft survival.Depleting CD4+CD25+Tregs also largely reversed the luteolin-induced downregulation of CD11c+CD86+or CD11c+CD80+ mature DCs.2.Effect of luteolin on islet allograft rejection in miceWe found that both low and high doses of luteolin significantly prolonged islet allograft survival,it indicated that luteolin can suppress islet allograft rejection in mice as well.In the islet grafts,luteolin reduced overall cellular infiltration.In the lymph nodes of recipient mice,luteolin upregulated the frequencies of CD4+Foxp3+Treg.3.In vitro study on the mechanisms underlying inhibition of allograft rejection by luteolinLuteolin,at both low and high concentrations,significantly suppressed T cell proliferation and the suppression of T cell proliferation was not attributed to its cytotoxicity.Both low and high concentration of luteolin significantly lowered IFN-y and IL-17a protein levels,whereas it promoted IL-10 secretion in the supernatant compared with the control group(P<0.05 or P<0.01).Further,we found that either Lut-low or Lut-high obviously induced CD4+Foxp3+Tregs compared with control group(P<0.01).Luteolin(at either low or high concentration)effectively inhibited the expressions of P-p70S6K as well as P-mTOR compared with control group,and so did Rapa(P<0.01).However,only Lut-high but not Lut-low reduced unphosphorylated mTOR expression.Importantly,luteolin,but not Rapa,suppressed AKT expression and its phosphorylation.ConclusionsOur data showed that luteolin not only prolonged skin allograft survival,but also extended islet allograft survival,suggesting that luteolin could inhibit the rejection of allogeneic organ transplantation in mice.The possible mechanism may include its direct suppression of the proliferation of effector T cells,indirect inhibition of the activation of T cells by affecting the maturation of DCs,and induction of the differentiation of CD4+Foxp3+Tregs.Furthermore,luteolin inhibited the activation of the AKT/mTOR signaling pathway.Our findings indicate that luteolin,a novel immunosuppressant,modulates alloimmunity and suppresses murine allograft rejection.