Study On The Adjuvant Therapy Of Experiment Cerebral Malaria By PDL1 Fusion Protein And Melatonin Via Protecting Blood-brain Barrier

Background and ObjectiveMalaria is still the most serious insect-borne infectious disease worldwide.Cerebral malaria(CM)is one severe complication of Plasmodium falciparum(P.f.)infection.Except the artemisinin-based combination therapies(ACT),it is recommended to combine with hormone treatment to inhibit the over-activated host inflammation when necessary.However,although effective anti-malaria treatment has been administrated,the mortality of CM still reaches up to 18%,and even survivors may suffer from long-term neurological sequelae.Besides,large doses hormone treatment may also lead to serious side-effects.As a result,there is an urgent clinical need for novel adjuvant therapy which is more effective and safer.Blood-brain barrier(BBB)disruption,which is caused by cerebral microcirculation obstruction and vascular immunopathology,is the central pathological change of CM.One of the main pathogenesis is the targeted killing from activated parasite-specific CD8~+T cells to BBB endothelial cells;on the other hand,it is also reported that macrophage as the primary effector cells to phagocytose P.f.during host innate immunity,has an important role in the development of CM.According to the pathogenesis,the novel CM adjuvant therapy may focus on moderate downregulation of over-activated host immune reponse to reduce immunopathology by some ways,while providing BBB protection through small molecule drugs.In recent years,the remarkable achievements of programmed death-1(PD-1)immunotherapy in cancers give us significant inspiration,as the binding of PD-1 and its ligand PDL1 can induce important inhibitory signal to host immune.In our previous investigations,we constructed a fusion protein linking the mouse extracellular domains of PDL1 to the Fc fragment of IgG1 to enhance the PD-1/PDL1 signaling pathway.It has been preliminarily demonstrated that PDL1 fusion protein could inhibit the cytolyticactivity of CD8~+T cell by reducing its activation,thus alleviate BBB disruption of experimental cerebral malaria(ECM)mice and increase the survival rate;however,whether the PDL1 fusion protein could influence the process of ECM through regulating the polarization or antigen presentation capability of the macrophages is still unknown.On the other hand,as melatonin is an endogenous amine which is reported to inhibit inflammation and protect vascular endothelial cells,its potential protective function to BBB in ECM models is also unclear.Hence,in this project,we try to explore the possible regulation fuctions of PDL1 fusion protein on the polarization and antigen presentation capability of macrophage,and the potential BBB protection from melatonin.This project may provide novel directions for further investigation on CM adjuvant therapy.Materials and Methods1.The influence of PDL1 fusion protein on the polarization and antigen presentation capability of macrophage1.1.The PD-1 levels of macrophages in different polarizationsThe bone marrow derived macrophages(BMDM)were separated and purified from wild type C57BL/6 mice,and then planted in 6-well plates.The 6-well plates were divided into 4 groups:M0 group,M1 group,M2 group and control group.The M1 polarization was induced by 20 ng/ml IFN-γand 100 ng/ml LPS,while the M2 polarization was induced by 20 ng/ml IL-4.After culture for 24 h,the PD-1 levels of macrophages in different polarizations were analyzed by flow cytometry(FCM).1.2.Construction of ECM modelPlasmodium berghei ANKA strain(PbA)was maintained in our laboratory.The C57BL/6 male mice(6 weeks old)were infected intraperitoneally(i.p.)with 1×10~7 PbA parasitized-RBCs(pRBCs)to construct ECM model.1.3.The influence of PDL1 fusion protein on the levels of host immune and macrophage cytokine release in the early stage of infectionThe ECM mice were treated with PDL1 fusion protein(100μl,1 mg/ml)by intravenous injection(i.v.)at day 0.At 4 day post infection(dpi),the serum samples of these mice were collected;meanwhile,the splenic macrophages of these mice were separated and purified,then the cultural supernatant samples were also collected.15 important cytokines(such as IFN-γ,IL-1β,TNF-α,etc.)in these serum and cultural supernatant samples were analyzed by MILLIPLEX~?Map Kit.1.4.The influence of PDL1 fusion protein on macrophages polarization and its ability to present malarial antigens to CD8~+T cellsThe BMDMs were separated and purified from wild type C57BL/6 mice,and then induced by 0.5 ng/ml IFN-γand 1×10~7 pRBCs;meanwhile,20μg/ml PDL1 fusion protein was also administrated.After culture for 24 h,the levels of IL-6,IL-10,IL-12p70 and TNF-αin cultural supernatant were analyzed by ELISA.Then,these 6-well plates were washed 3 times by PBS before co-culture with mice splenic CD8~+T cells.After culture for24 h,the CD8~+T cells were separated and purified.The mRNA levels of perforin and granzyme B in these CD8~+T cells were analyzed by quantitative real time PCR(qPCR).2.Investigation on protective function of melatonin on BBB endothelial cells in ECM mice and the potential mechanisms2.1.The influence of melatonin on ECM symptomThe ECM mice were treated with melatonin(10 mg/kg,daily,i.p.)from 0 dpi to death.The survival time,weight and parasitemia were detected daily.2.2.The influence of melatonin on BBB disruption in ECM mice The evans blue(EB)leakage assay,brain water content(BWC)assay,H&E staining,CD31 immunofluorescence staining and TUNEL staining were administrated to evaluate the BBB leakage,encephaledema and BBB endothelial cell apoptosis in ECM mice.According to these results,the portective function of melatonin was assessed.2.3.The influence of melatonin on inflammation and apoptosis of brain vascular endothelial cellsThe bEnd.3 endothelial cells were induced by 8 ng/ml IFN-γand 1×10~8 pRBCs and treated by 20μg/ml melatonin.After 24 h,bEnd.3 cells were collected for western blot(NF-κB,Bax and Bcl-2)and qPCR(IL-1β,IL-6,TNF-α,NF-κB,Bax and Bcl-2).Results1.The influence of PDL1 fusion protein on the polarization and antigen presentation capability of macrophage1.1.The PD-1 levels of macrophages in different polarizationsMacrophages could express PD-1 in different polarizations and the PD-1 levels in different polarizations exhibited no difference.1.2.The influence of PDL1 fusion protein on the levels of host immune and macrophage cytokine release in the early stage of infectionIn serum samples,PDL1 fusion protein notably reduced the level of IL-1βbut failed to influence other cytokines.Besides,PDL1 fusion protein also exhibited no effect on cytokine release of splenic macrophages at this time point.1.3.The influence of PDL1 fusion protein on macrophages polarization and its ability to present malarial antigens to CD8~+T cellsIn BMDMs,PDL1 fusion protein notably reduced the levels of IL-6,IL-12p70 and TNF-α,while enhanced the level of IL-10.The CD8~+T cells,wich were co-cultured with PDL1 fusion protein-treated BMDMs,showed weaker expression in perforin and granzyme B.2.Investigation on protective function of melatonin on BBB endothelial cells in ECM mice and the potential mechanisms2.1.The influence of melatonin on ECM symptom The morbidity of melatonin-treated ECM mice was notably lower than untreated ECM mice,while the weight and parasitemia exhibited no difference.2.2.The influence of melatonin on BBB disruption in ECM miceCompared with untreated ECM mice,the EB leakage,BWC,pRBCs sequestration and lymphocytes infiltration in vascular,and endothelial cell apoptosis of melatonin-treated ECM mice were all al eviated.2.3.The influence of melatonin on inflammation and apoptosis of brain vascular endothelial cellsThe qPCR results showed that the mRNA levels of IL-6,TNF-α,NF-κB and Bax in melatonin-treated bEnd.3 cells were significantly reduced,while the Bcl-2 was enhanced and the IL-1βshowed no difference.The western blot results showed that the protein level of NF-κB and Bax/Bcl-2 rate in melatonin-treated bEnd.3 cel s were both reduced.ConclusionBased on our previous investigations,we further indicated that in ECM model,PDL1 fusion protein could inhibited the M1 polarization of marophages in a certain extent,and this effect could reduce the antigen presentation capability of macrophage to CD8~+T cells,thus indirectly restrained CD8~+T cell activation,contributed to BBB integrity and protected ECM mice.On the other hand,we preliminarily demonstrated that melatonin has a protective role in ECM,and the protection mechenism may related to the anti-inflammation and anti-apoptosis functions of melatonin on BBB endothelial cells.In summary,this project provides novel direction and methods for the investigation on CM adjuvant therapy,and suggesting a possibility to combine with ACT to improve the CM cure rate.