The Regulation Function And Mechanism Of PD-L1 On Neuronal Injury In Experimental Cerebral Malaria Mice

Background:Cerebral malaria（CM）represents the most severe neurological manifestation of plasmodium falciparum infected,however its underlying mechanisms remain elusive.The current study utilizes a mouse model of experimental cerebral malaria accompanied by brain damage（ECM）to investigate the disease.While previous research has primarily focused on the impact of activated CD8⁺T cells on the onset and progression of cerebral malaria,there is a paucity of reports exploring the pathogenesis of neuronal tissue damage in CM,specifically from the interaction between CD8⁺T cells and neurons.Neuroscientific research indicates that CD8⁺T cells have the potential to damage neurons,while neurons may exhibit immunomodulatory functions towards T cells.The immune checkpoint molecule PD-1/PD-L1,acting as a crucial"brake"in immune responses,is widely employed in tumor therapy and can effectively suppress the activation of CD8⁺T cells.Both interferon-β（IFN-β）and interferon-γ（IFN-γ）have the ability to induce the expression of PD-L1,however the specific molecular mechanism impacting this process are contingent upon the neuro-immune microenvironment.Commencing with an examination of the direct interaction between neurons and CD8⁺T cells,this study undertakes an in-depth investigation into the neurobiological mechanisms underlying neuronal injury and the signal pathway associated with neuronal PD-L1 expression.It further postulates a novel hypothesis suggesting that neurons exert a negative immunomodulatory effect on activated CD8⁺T cells within the brain parenchyma during the pathogenesis of cerebral malaria.It is anticipated that this study will provide a novel theoretical framework and investigative paradigm for a deeper understanding of the pathogenesis of cerebral malaria-induced nerve tissue damage as well as the development of immune-assisted therapeutic strategies for cerebral malaria.Objective:In this study,it aims to confirm that the infiltrating CD8⁺T cells within brain tissue exert deleterious effects on neurons and trigger ferroptosis during the progression of ECM through in vitro experiments involving primary neuronal cultures,a mouse model of ECM-induced brain injury,and gene knockout mice infected with plasmodium.Further,the study is to elucidate the regulation of IFN-βand IFN-γ（predominantly secreted by activated CD8⁺T cells in ECM）on the expression of PD-L1 in neurons of ECM mice,as well as the signaling pathway that induces the expression of PD-L1.Additionally,the study is to explore the use of an exogenous adenovirus expressing PD-L1 recombinant protein,and to explicit that intrathecal/cerebral stereotaxic injection of ADV encoding PD-L1 enhanced the protective effects of intracranial PD-L1 signaling against ECM-induced neuronal injury.Methods and Results:1.Confirmation of CD8⁺T cells-mediated neuronal damage and ferroptosis in ECM mice brain（1）Neuronal injury in ECM miceUsing hematoxylin and eosin（HE）staining,immunohistochemical staining,and Nissl staining,we confirmed the presence of cerebral hemorrhage,the reduction of neuronal markers,and depletion of Nissl bodies in the brain of ECM mice.Electron microscopy analysis revealed changes in nuclear shape change of neuron,demyelination,mitochondrial damage,and the emergence of autophagosomes in the brain stem of ECM mice.Immunofluorescence staining further confirmed the up-regulation of TUNEL-positive apoptosis and LC3-positive autophagy in ECM mice.（2）Activation characteristics of CD8⁺T cells in the brain of ECM mice and the damage effect of ECM-activated CD8⁺T cells on mouse primary cortical neuronsSingle-cell sequencing analysis revealed that the T cells infiltrating the ECM mouse brainstem were predominantly CD8⁺T cells,exhibiting characteristics of T cell memory signaling(Cd62^lowCcr7^low),activation(Cd2^highCd69^highCd28^high),and tissue residency(Cxcr6^highLfa1^highItga4^high).These cells expressed the proinflammatory cytokine(Ifng^high)and immune checkpoints molecular(Pdcd1^high).Fluorescence staining and flow cytometry verified that CD8⁺T cells within the brain parenchyma of ECM mice possessed Ki67proliferation characteristics and the ability to secrete IFN-γ.An in vitro co-culture system was established using mouse primary cultured cortical neurons and magnetic bead-sorted CD8⁺T cells isolated from the spleens of ECM mouse（ECM-activated CD8⁺T cells）.It was confirmed that ECM-activated CD8⁺T cells could damage neurons through direct contact,resulting in increased neuronal death as evidenced by LDH and CCK8 assays Flow cytometry using PI,JC1,and Sy Tox green staining further confirmed the enhanced damage to neuronal membranes and mitochondria.Time-lapse imaging revealed that CD8⁺T cells could actively adhere to the cell bodies and synapses of primary cortical neurons,causing axonal damage.Q-PCR analysis confirmed the upregulation of MHCⅠmolecules in neurons.Immunofluorescence assay observed the formation of immune synaptic like structures between CD8⁺T cells and neurons.（3）Ferroptosis occurs in ECM mouse neurons and is induced by ECM-activated CD8⁺T cellsAn increase of lipid peroxidation levels and Fe ion content in the brain tissue of ECM mice was confirmed using lipid and iron detection kit.Q-PCR,western blotting（WB）and immunohistochemical staining revealed that the ferroptosis-related molecules,transferrin receptor 1（Tf R1）and acetyl-coenzyme A synthetase-4（ACSL4）,were upregulated in the brain tissue of ECM mice,while the ferroptosis regulatory molecule glutathione peroxidase 4（GPX4）was downregulated in injured neurons.The downregulation of GPX4 in neurons was correlated with the downregulation of Neu N,a neuronal marker.In vitro experiments using a co-incubation system of primary cultured neurons and ECM-activated CD8⁺T cells,WB analysis confirmed that Tf R1 was upregulated and GPX4 down-regulated in neurons.Furthermore,the ferroptosis inhibitor Fer-1 reduced the downregulation of neuronal marker APP,indicating its protective effect.JC-1 detection showed that Fer-1 alleviated the damage of neuronal mitochondria.In vivo experiments in ECM mice also validated that Fer-1 exhibited a certain protective effect,reducing the decrease in Neu N levels and the increase in malondialdehyde（MDA）content,a marker of oxidative stress.2.ECM brain neurons express the immune checkpoint molecule PD-L1,which can be induced by IFN-βand IFN-γvia the IFN receptor/Stat1/IRF1 pathway（1）PD-L1 expression was up-regulated in neurons of ECM mice and be induced by IFN-β,IFN-γ,or ECM-activated CD8+T cells in vitroA single cell sequencing analysis of the ECM mouse brain revealed that ECM brainstem neurons expressed PD-L1.The upregulation of PD-L1 in various regions of ECM brain tissue was further validated through immunohistochemical staining and WB detection.In vitro experiments involving primary cultured neurons and ECM-activated CD8⁺T cells demonstrated that IFN-β,IFN-γand CD8⁺T cells have the ability to induce neurons to enhance the transcription and translation levels of PD-L1.Notably,WB analysis revealed distinct level of PD-L1 expression in the cell membrane,cytoplasm,and nucleus of neurons.（2）Both primary cultured neurons stimulated by IFN-β/IFN-γand neurons in brains of ECM mice regulate the expression of PD-L1 via upregulated STAT1/IRF1signaling pathwayNeuronal transcriptome sequencing revealed that IFN-β,IFN-γ,and ECM-activated CD8⁺T cells can induce the upregulation of STAT1/IRF1 signaling pathway and subsequently enhance PD-L1 expression in neurons.In vitro co-incubation experiments of primary neurons with ECM-activated CD8⁺T cells,coupled with q-PCR,immunofluorescence staining,and WB detection,confirmed that IFN-βand IFN-γcan induce the upregulation of phosphorylated STAT1（p-STAT1）in neurons.Moreover,the STAT1 inhibitor Flu was shown to attenuate the upregulation of p-STAT,IRF1 and PD-L1.Immunohistochemical/immunofluorescence staining and WB detection further demonstrated that the levels of p-STAT1,IRF1,and PD-L1 were upregulated in the brain tissue of ECM-damaged mice,with this upregulation being intensified by prolonged Pb A infection.（3）The absence of IFN receptor impairs the ability of neurons to express PD-L1 in response to stimulation by IFN-β,IFN-γ,and ECM activated-CD8⁺T cellsBlocking the IFN receptor using antibodies revealed that IFN-βand IFN-γfailed to induce the upregulation of PD-L1 in primary cultured neurons.Upon infecting IFNAR and IFNGR knockout mice with Pb A respectively,blood-brain barrier（BBB）damage was still evident through Evans blue（EB）extravasation detection and HE staining.Immunofluorescence staining indicated that CD8⁺T cells could secrete IFN-γin the brain of knockout mice infected with Pb A.Additionally,immunofluorescence staining and WB detection demonstrated that the expression of p-STAT1,IRF1 and PD-L1 in the brain of infected knockout mice was reduced compared to that in ECM mice.Immunofluorescence detection further revealed decreased expression of PD-L1,p-STAT1,and IRF1 in neurons of gene knockout mice.When IFNAR or IFNGR gene knockout neurons were cultured in vitro.WB and flow cytometry confirmed that the upregulation of p-STAT1,IRF1 and PD-L1 induced by IFN-βor IFN-γwas diminished due to the deletion of IFN receptor genes.3.In vitro and in vivo experiments confirmed that the PDL1-Fc adenovirus exhibits a certain protective effect against neuronal damage induced by ECM-activated CD8⁺T cellsThe expression vector for the fusion protein PDL1-Fc,comprising the extracellular domain of PD-L1 fused with the Fc segment of Ig G1 protein,was constructed using an adenovirus system.In vitro,primary neurons were infected with PDL1-Fc adenovirus and subsequently co-cultured with ECM-activated CD8⁺T cells.Immunofluorescence analysis validated that PDL1-Fc adenovirus upregulated the expression of PD-L1 and Neu N in neurons.Furthermore,PDL1-Fc adenovirus reduced neuronal damage,as evidenced by decreased LDH release and light microscopy examination.Flow cytometry analysis using JC-1 and PI staining revealed that PDL1-Fc adenovirus mitigated neuronal death and mitochondrial damage.In vivo,a mouse model was established by intrathecal injection of PDL1-Fc adenovirus following Pb A infection.The survival rate of mice receiving PDL1-Fc PDL1-Fc injection was improved compared to controls,as demonstrated by EB extravasation detection.Immunofluorescence analysis showed a decrease in TUNEL⁺（apoptotic）and Ki67⁺（proliferating）cells in the brains of mice treated with PDL1-Fc adenovirus.Additionally,a mouse model with brain stereotaxic microinjection of PDL1-Fc adenovirus following Pb A infection was established.Immunohistochemistry and HE staining confirmed a reduction in bleeding and the CD8⁺T cells infiltration in the region of mice brainstem injected with PDL1-Fc adenovirus.Conclusion:This study demonstrated the ECM-activated CD8+T cells inflict damage on neurons via direct contact,potentially leading to neuronal ferroptosis.Crucially,both typeⅠand typeⅡIFN are capable of inducing the expression of the neuronal immune checkpoint molecule PD-L1 via the STAT1/IRF1 signaling pathway,which is mediated by IFN receptors.Our findings demonstrate that the intrathecal injection and brain stereotaxic microinjection of PDL1-Fc recombinant adenovirus exerts a protective effect against the progression of ECM and associated nerve injury.This approach provides a theoretical foundation and novel insights for targeted nerve injury prevention/treatment strategies,aiming to alleviate the burden of neurological injury and sequelae of cerebral malaria on the nervous system.