The Experimental Study Of Neuronal PirB As A Novel Po-Tential Theranostic Target For Ischemic Stroke

Cerebral ischemic stroke is characterized by the sudden loss of blood circula-tion to an area of the brain and is caused by thrombotic and embolic occlusion of a cerebral artery,resulting in a corresponding loss of neurologic function.Acute ischemic stroke is more common than hemorrhagic stroke.It's one of the major causes of death and disability worldwide,as well as being a significant socioeco-nomic burden.Given an aging population,ischemic stroke is projected to become even more important in the future.Therefore,it is urgent to investigate more targets for adjutant diagnosis,real-time monitoring and treatment of ischemic stroke.Mouse paired immunoglobulin-like receptor B(PirB)is a type I transmembrane glycoprotein with six extracellular immunoglobulin-like domain,a hydrophobic transmembrane segment and an intracellular polypeptide with four ITIM sequences.PirB originally thought to function exclusively in the immune system,is now also known to be expressed by neurons.More and more studies indicate that PirB plays an inhibitory role in neurite outgrowth and restricts neuronal plasticity.LILRB2,a human ortholog of PirB,may play the same function in humans.In cerebral ische-mia-reperfusion stroke model,germline PirB-/-mice recover more rapidly than WT mice.And PirB protein levels are markedly increased in the damaged hemisphere after MCAO at 7 days.However,the expression and function of PirB in cerebral ischemic brain remains unclear.To date,there is no study in molecular imaging and therapy on the basis of PirB for ischemic stroke.The aim of this study was to investigate the expression and roles of neuronal PirB in the pathogenesis of multiple ischemic stroke model including cerebral ischemia-reperfusion stroke,photothrombotic focal ischemic stroke and neuronal oxygen glucose deprivation model,and to develop anti-PirB vectorized stealth im-munoliposomes containing imaging probes for real-time monitoring of ischemic stroke.Futhermore,we have successfully screened and obtained a novel phage dis-play-derived peptide that targets both PirB and LILRB2 protein.Our results include the following several parts:1.Firstly,we investigated the spatial-temporal expression of PirB and its immediate downstream signaling in cerebral ischemia-reperfusion stroke.In this model,stria-tum,cortex,and hippocampus were the main infarct areas.Real-time quantitative PCR analysis revealed highly increased PirB mRNA in the ischemic hemisphere compared to contralateral side after reperfusion both at 24 hours and at 4 days.The upregulation of PirB in the hippocampus of ischemic hemisphere was the most ob-vious.The results of immunohistochemistry demonstrated increased protein level after ischemia-reperfusion in the damaged hemisphere relative to the undamaged side.The majority of PirB immunostaining colocalized with neuronal marker NeuN in the ischemic hemisphere after ischemia-reperfusion.Flow cytometry analysis showed that proportion of PirB positive neurons in ischemic hemisphere was much more than that in contralateral side.We also found that classical MHC I molecules were upregulated in the ischemic hemisphere.And tyrosine phosphorylation of PirB on cytoplasmic motifs increased significantly in the ischemic hemisphere compared to contralateral side at 24h after reperfusion.It meaned that the activation of PirB-mediated downstream signaling was significantly increased in ischemia.2.The mRNA and protein expression of PirB in photothrombotic focal ischemic stroke were detected by real-time quantitative PCR and Western blot,respectively.In photothrombotic middle cerebral artery occlusion model,PirB was significantly up-regulated in the ischemic hemisphere compared to the undamaged contralateral side at 24h after ischemia.In focal cortical ischemic stroke via photothrombosis,the neuronal expression of PirB was significantly up-regulated in the ischemic cortex compared to the undamaged contralateral cortex from 3d to 14d after ischemia.The peak of upregulation of PirB in the cerebral cortex with ischemia was observed on day3 after stroke.With the increase of ischemic damage in ipsilateral side,the upre-gulation of neuronal PirB was more obvious when compared with contralateral side.3.We have successfully developed anti-PirB vectorized stealth immunoliposome nanoprobe containing near-infrared fluorophore DiR to make them traceable by flu-orescence.Transmission electron microscopy of the immunoliposomes verified the formation of homogeneous unilamellar liposomes of a mean diameter consistent with that obtained with dynamic light scattering around 150nm.The binding speci-ficity of anti-PirB immunoliposome nanoprobe was first demonstrated in the PirB-positive spleen cells.The signal intensities of the cells on NIRF images were significantly higher in the anti-PirB immunoliposome nanoprobe group compared with the control nanoprobe groups.The majority of the signal intensity in the anti-PirB immunoliposome nanoprobe group was blocked by pre-treatment with free anti-PirB antibody(6C1).To investigate the targeting specificity of nanoprobes in vivo,anti-PirB immunoliposome nanoprobe(6C1/PEG/LP/DiR)or rat-IgG im-munoliposome nanoprobe(IgG/PEG/LP/DiR)were administered intravenously in focal cortical ischemic stroke mice at 48h after ischemia.The signal intensities of the ischemic hemispheres on the NIRF images were significantly higher in the 6C1/PEG/LP/DiR group compared with the IgG/PEG/LP/DiR group from 4h to 24h after injection.The results indicated that anti-PirB immunoliposome nanoprobe was accumulated in ischemic hemisphere.4.The oxygen glucose deprivation model was used to study ischemic injury in vitro.Expression of PirB and its ligands classical MHC I molecules in cultured cortical neuron in the OGD group were remarkably higher than those in the control in a time-dependent manner.It suggested that the activation of PirB-mediated down-stream signaling was significantly increased in in vitro model.Soluble PirB ectodo-mains were used to block PirB binding to endogenous ligands.Blockade of PirB li-gand binding significantly alleviated apoptosis of neurons after exposed to OGD.sPirB/PEG/LP improved photothrombotic focal ischemic stroke model recovery.5.We carried out a biopanning by incubating the phage display dodecapeptide li-brary on ectodomain of LILRB2,a human ortholog of PirB.After 3 rounds of pan-ning,the titers of phages recovered from protein were significantly increased by 856 fold when compared with titers in the first round.The panning was very successful.The peptide whose appearance frequency ranking the top was named as LBP1.The LBP1 phage clone showed a significantly higher binding ability to ectodomains of both PirB and its human ortholog.Almost no signals were observed when insertless phage particles were added to each protein,suggesting that LBP1 peptide bound to PirB and its human ortholog with a very high affinity.These results showed that PirB was significantly upregulated after cerebral ischemic injury in multiple ischemic stroke models.It is a significant element in the pathological process of cerebral ischemia.Therefore PirB may act as a novel poten-tial theranostic target for ischemic stroke.Anti-PirB immunoliposome nanoprobe specifically bound to PirB in vitro.There was accumulation of anti-PirB immunoli-posome nanoprobe in ischemic hemisphere in vivo.The novel phage display-derived peptide targeted to PirB and LILRB2 will be a promising moiety for molecular im-aging and treatment of ischemic stroke tissue.Our results provided insights into de-velopment of innovative molecular imaging agents for the adjutant diagnosis,treat-ment and real-time monitoring of ischemic stroke.