In Vitro And In Vivo Effect Of PD-1/PD-L1 Blockade On Microglia/macrophage Activation And T Cell Subset Balance In Cryptococcal Meningitis

Cryptococcus neoformans meningitis(CM)is a kind of severe central nervous system infection caused by cryptococcus neoformans infection.It is showed that cryptococcus neoformans meningitis is a result of tissue immune damage,CM patients tend to have disorder of macrophages and inhibition of Th1 immune response,Th2 immune response level higher abnormal phenomenon.And the key mechamism to fight agaist cryptococcus neoformans infection is the macrophage activation condition.When the cryptococcus neofoumans invade into the body,macrophages not only participate in the innate and adaptive immune response to protection against cryptococcosis,but also can devour cryptococcus neoformans,as the body diffusion carrier caused systemic infection.Macrophages have dynamic plasticity,under the effect of different cytokines can change its activation phenotype.Th-1 type cell-mediated immunity(CMI)can induce lymphocyte and macrophage recruitment and activation to development of Ag-specific delayed-type hypersensitivity to protect agaist cryptococcosis.Th-2 type CMI and the alternatively activated macrophages can cause fugal overgrowth and deterioration.Co-stimulatory molecule is a kind of proteins expressed on immune cells and immune related cells,which mediated signal is the costimulatory signal.Costimulatory signal regulate immune response is bidirectional,through the positive and negative costimulatory signals to maintain the immune balance.The negative costimulatory molecules such as programmed death factor 1(PD-1),Ligand of Programmed death 1(PD-L1)inhibited T cell proliferation and cytokine production,inhibition of immune response and prevent excessive immune injury and autoimmune diseases,help maintain the body's immune homeostasis.In recent years,research have shown that macrophages also express costimulatory molecules,suggesting that macrophages express costimulatory molecules may impact anti-CM immunity.This study aimed to investigate tha PD-1/PD-L1 signaling pathway and its effects the activation of microglia/macrophage and balancing T cell subsets in cryptococcal meningitis(CM).A total of 126 CM patients and 126 healthy individuals were recruited for the study.The CM patients were treated with amphotericin B(AmB).Seventy fiveC57BL/6 mice were grouped intl the normal control,CM model,CM+AmB,sham and CM+PD-1 antibodies(Ab)groups.CD4~+and CD8~+T cells as well as microglia/macrophages were analyzed by means of flow cytometry.Ionized calcium-binding adaptor molecule 1(Ibal)expression was detected using immunohistochemistry techniques.And the expression of Rab5 and Rab11 were detected using an immunofluorescence assay.Both PD-1 and PD-L1 mRNA and protein expression among the mice in the study were evaluated by q RT-PCR and western blotting methods.Part?Part?The function and expression characteristics ofPD-1/PD-L1 of PMBC on the peripheral blood of cryptococcus meningitispatientsObjective:To analysisi the level of PD-1/PD-L1 and function of CM patientsPBMC,and explore the relationship between the abnormal immune function and co-stimulatory molecule of CM patients.Methods:A total of 126 CM patients and 126 healthy individuals were recruited for the study.PD-1 and PD-L1 mRNA and protein expression in the study were evaluated by qRT-PCR and Elisa methods respectively.The CM patients were treated with amphotericin B(Am B).Both PD-1 and PD-L1 mRNA and protein expression in the study were evaluated by q RT-PCR and Elisa methods respectively before and after the therapy of amphotericin B.Results:(1)In comparison to the control group,mRNA and protein expressions of PD-1and PD-L1 were significantly elevated in the PB of the CM group.(P<0.001,P<0.001).(2)The CM group after treatment with AmB presented an obvious decrease in mRNA and protein expressions of PD-1 and PD-L1 compared to those before treatment with AmB(both P<0.05)Conclusion:Cryptococcal meningitis patients have the abnormalities of the expression of PD-1/PD-L1 on PBMC in the peripheral blood,suggesting the PD-1/PD-L1signal pathway can affect the immune function in patients with cryptpcoccus meningitis.And with the effective treatment,the disorder can partly reversed.Part?The effect of PD-1/PD-L1 blckade on microglia activation and balance of T cell subset in cryptococcal meningitis in vitroObjective:To block PD-1/PD-L1 pathway in vitro,explore the negative co-stimulation signal regulate the immune function of CM.(1)To investigate the death rates and meningeal structures of mice among groups.(2)To analyze the cytokine changes in brain tissues of mice among different groups.(3)To analyze the fuction characteristic of microglia/macrophages in mice brain tissue among different groups.(4)To analyze the CD4+T?CD8+T cell purity and viabilyt in brain tissues of mice among groups.(5)To analyze the mRNA and protein expressions of PD-1 and PD-L1 in CD4+and CD8+T cells and microglia/macrophages among groups.Methods:(1)CM mouse models were established according to the literature.A total of 75wild-type C57BL/6 mice were divided on random into the normal control,CM model,AmB,sham and PD-1 antibodies(Ab)groups(n=15).(2)Count the death rates and investigate meningeal structures with HE staining of mice among groups.(3)Detect the the expressions ofcytokines,such as interferon-?(IFN-?),tumor necrosis factor-?(TNF-?),interleukin-6(IL-6),interleukin-10(IL-10),CXC chemokine ligand 9(CXCL9)and CXC chemokine ligand 10(CXCL10)in brain tissues of mouse models with CM with ELISA.(4)Separation and purification of microglia with adherent and shaken culture method,separation of CD4+and CD8+T cellsfrom brain tissueswith immune magnetic bead separation and count the CD4+and CD8+T cells with flow cytometry.(5)Detect the expression of Iba1,a kind of marker on macrophage activation,with IHC and detect the Rab5 and Rab11 with immunofluorescence staining.(6)Both PD-1 and PD-L1 m RNA and protein expression among the mice in the study were evaluated by q RT-PCR and western blotting methods.Results:(1)no mice died in the normal control group.After 12 days of transfection,7micedied in the CM model group.Death rates of mice of the CM model group had evidently increasedcompared to the rate in the normal control group(P<0.05),and only 1 mouse died intraoperativelyin the sham group,which was not significantly different from the normal control group(P>0.05).Meanwhile,2 mice and 1 mouse died in the in the AmB and the PD-1 Ab groups respectively,which showed an obvious reduction in death rate in comparison to the CM model group(both P<0.05).(2)Meningeal structures were intact and normal in mice of the normal control and sham group.In thebrain tissues in the CM model group heaps of inflammatory exudate,soft tissue necrosis,andvascular congestion of meninges,obvious"the sleeve"phenomenon(arrows indicated"the sleeve"phenomenon)and dilated subarachnoid space were observed.In comparison to the CM model group,brain tissues of the AmB group had decreased inflammatory exudation,improved perivascularcuffing,and reduced dilated subarachnoid space with no obvious soft tissue necrosis.Brain tissuesof the PD-1 Ab group showed little inflammatory exudation,almost normal subarachnoid space,noperivascular cuffing or soft tissue necrosis.(3)In comparison to mice in the normal control group,brain tissues of mice in the CM model groupshowed decreased expressions of Th1 cytokines such as IFN-?and TNF-?and chemokines such asCXCL9 and CXCL10,while the showed increased expressions of Th2 cytokines such as IL-1?,IL-6,IL-10 and IL-17(all P<0.05).No significant difference in cytokine expression was observedbetween the sham and the normal control groups.Expressions of CSF Th1 cytokines(IFN-?andTNF-?)and chemokines(CXCL9 and CXCL10)were significantly elevated whereas theexpressions of Th2 cytokines(IL-1?,IL-6,IL-10 and IL-17)were significantly reduced in the AmBand the PD-1 Ab groups in comparison to the CM model group(all P<0.05).(4)In comparison to the normal control group,decreased purity and viability of CD4+T cells whileincreased purity and viability of CD8+T cells,decreased CD4+/CD8+T cell ratio and increasedviability of microglia were detected in the CM model group(all P<0.05).Cell purity and viabilitydifferences were not significant between the sham and the normal control groups(P>0.05).TheAmB group,compared to the CM model group,had elevated purity and viability of CD4+T cells,reduced purity and viability of CD8+T cells,elevated CD4+/CD8+T cell ratio and reduced viabilityof microglia(all P<0.05).When it came to the PD-1 Ab group,the differences,compared to theCM model group,were more significant(all P<0.05)and the cell purity and viability almostapproached normal samples.(5)Immunohistochemical analysis of brain tissues indicated that Ibal was expressed in cerebral cortexof mice in the normal control,the CM model,the sham,the AmB and the PD-1 Ab groups.Ibal waslocated in the cytoplasm and nucleus.Immunohistochemical staining in manifested thatmicroglia/macrophages were sparsely distributed with smaller soma and many thin synapses in thenormal control and sham groups;microglia/macrophages in the CM model group,compared to thenormal control group,were densely distributed with an increase in cell count and cell soma andenlarged synapses;The AmB and the PD-1 Ab groups,compared to the CM model group,suggested reduced number of microglia/macrophages,and the tendency was more obvious in the PD-1 Abgroup.Western blotting analysis demonstrated that expressions of Ibal in the CM modelgroup,compared to the normal control and sham groups,raised significantly;expressions of Ibal inthe AmB and PD-1 Ab groups,compared to the CM model group,significantly depleted,and thePD-1 Ab group had an even lower expression of Ibal(all P<0.05).(6)Immunofluorescence staining indicated that expressions of markers of microglia earlyphagosomes,Rab5 and Rab11,decreased in the CM model group,compared to the normal controlgroup;expressions of Rab5 and Rab11 in the AmB and the PD-1 Ab groups significantly increased,and those in the PD-1 Ab group were elevated more(all P<0.05).Rab5 and Rab11 expressions were not significantly different between the normal control and the sham groups(both P>0.05).(7)Expressions of PD-1 and PD-L1 in CD4+and CD8+T cells and microglia/macrophages of the CMmodel group evidently increased compared to the normal control group(all P<0.05).Nosignificant differences were observed in the expressions of PD-1 and PD-L1 in CD4+and CD8+Tcells and microglia/macrophages in the sham and normal control groups(all P>0.05).Compared tothe CM model group,the AmB and the PD-1 Ab groups presented significantly reduced expressionsof PD-1 and PD-L1 in CD4+and CD8+T cells and microglia/macrophages,whereas the PD-1 Abgroup had more significant differences(all P<0.05)Conclusion:(1)The inhibit mRNA andprotein expressions of PD-1 and PD-L1 in mice models of CM by AmB and PD-1 antibodies couldaid in reducing the death rates,improved meningeal structure.(2)There is Th1/TH2 inbalance in CM.By inhibiting thePD-1/PD-L1 signaling pathway,the inbalance can partly reversed.(3)The activation and function characteristicsof microglia/macrophages is abnormality in CM models.(4)The inhibit mRNA andprotein expressions of PD-1 and PD-L1 in mice models of CM by Am B and PD-1 antibodies couldelevated CD4+/CD8+T cell ratio.(5)The blocking the PD-1/PD-L1 signaling pathway by PD-1 antibodies or AmB significantlyalleviated CM by restoring T cell subset.