Role Of Cellular Prion Protein In The Inflammation Resolution After Cerebral Ischemia/Reperfusion Injury

Background and purpose:Stroke is the second leading cause of death and disability in the world.The incidence of ischemic stroke gradually increases due to the demographic changes and the prevalence of diabetes and obesity,which puts huge pressure on the society and families.The core treatment of ischemic stroke is vascular recanalization,which restores the blood flow of blocked arteries within the first few hours,thereby salvaging the ischemic penumbra.However,the cerebral damage may not be improved but further aggravated after blood reperfusion,which is called cerebral ischemia-reperfusion injury(CIRI).Inflammation plays an important role in CIRI.During the acute phase of CIRI,an immunologic cascade is caused by the activation of immunocytes and the expression of inflammatory mediators,which leads to inflammation instead of ischemic injury,Inflammation resolution,a series of positive inflammatory responses,occurs when it comes to the subacute phase:apoptotic neutrophils are engulfed by phagocytes,which is mediated by both internal and external inflammatory mediators so that the tissue gradually returns to homeostasis.Inflammation resolution not only relieves the inflammatory damage but also facilitates tissue and function repairment of central nervous system(CNS)during the long-term recovery,and it has been one of the potential theraputic targets for CIRI.Cellular prion protein(PrPC),encoded by Prnp gene,is a highly conserved cell surface glycoprotein,which is also the original structure of the pathogen of prion disease,scrapie prion protein(PrPSc).The presence of PrPC does not alter the physiological phenotype,while the PrPC-dificient animals do not have the potential to develop prion diseases.Therefore,PrPC is the key target in the research of prion diseases,and it is necessary to delve into the functions of PrPC and the cellular events in which it participates.PrPC is closely related to neuroprotection during ischemic stroke.It has been found that PrPC is up-regulated in the ischemic penumbra,promoting angiogenesis and neurogenesis by participating in cell signaling and stem cell homing.However,whether PrPC can induce neuroprotection by regulating the inflammation resolution of CIRI is still unclear.Therefore,this study aims to explore the expression of PrPC on multiple immnuocytes and the effect of PrPC on the recruitment,infiltration and function of neutrophils and phagocytes during the inflammation resolution of CIRI as well as to investigate the regulatory role of PrPC in CIRI-related inflammatory cytokines and mediators.Contents:Part Ⅰ The expression changes of PrPC on neutrophils and phagocytes in peripheral blood and ipsilateral hemisphere during inflammation resolution of CIRI Methods:1.The transient middle cerebral artery occlusion(tMCAO)was operated on wild type(WT)FVB/N male mice.The proportion of neutrophils and PrPC+ subset in peripheral blood and the ipsilateral hemisphere of WT mice were assessed by flow cytometry and compared with sham group 1 d after tMCAO.2.The proportion of monocytes and PrPC+ subset in peripheral blood as well as the proportion of microglia/macrophages and PrPC+ subset in the ipsilateral hemisphere of WT mice were assessed by flow cytometry and compared with sham group 3 d after tMCAO.Results:1.The proportion of neutrophils and PrPC+ subset in the peripheral blood as well as the ipsilateral hemisphere of WT mice remarkably increased 1d after tMCAO.2.In addition,the proportion of monocytes and PrPC+ subset in the peripheral blood,as well as the infiltrating microglia/macrophages together with PrPC+ subset in the ipsilateral hemisphere increased significantly 3 d after tMCAO.Part Ⅱ The possibility of PrPC participating in the inflammation resolution of CIRI Methods:1.tMCAO was operated on Prnp knockout(PRNP K.O.),PRNP overexpressed(tga20)and WT FVB/N male mice.Zea Longa scores were evaluated at 1,3,5 and 7 d after tMCAO in each genotype.The scores were compared to determine the relationship between the change in PrPC expression and the severity of illness at different timepoint after tMCAO.2.The synthesis of LXA4 and RvD1 in the ipsilateral hemisphere of different genotype were detected and compared on 3,5 and 7 d after tMCAO.3.The expression of pro-inflammatory cytokines TNF-α,IL-6,IFN-γ and antiinflammatory cytokines IL-4 and IL-10 in peripheral blood were detected and compared on 3,5 and 7 d after tMCAO.Results:1.The Zea Longa scores of tga20 mice were significantly lower than that of WT mice at each timepoint after tMCAO,while those of Prnp K.O.mice were significantly higher.2.The expression of both LXA4 and RvD1 in the ipsilateral hemisphere of each genotype increased 3-7 d after tMCAO.Compared to WT mice,the expression of LXA4 and RvD1 in tga20 mice significantly increased at all timepoints,and decreased in Prnp K.O.mice.3.The levels of pro-and anti-inflammatory cytokines in the peripheral blood of all genotypes showed an increasing trend.Significant changes occurred in antiinflammatory cytokines(IL-4,IL-10)rather than pro-inflammatory cytokines(TNF-α,IL-6,IFN-γ)in tga20 mice.In Prnp K.O.mice,the expression of pro-inflammatory cytokines raised obviously,while the anti-inflammatory cytokines showed a downtrend without statistical significance.Part Ⅲ The role of PrPC on chemotaxis,infiltration and apoptosis of neutrophils during CIRIMethods:1.The transcription of Cxcl1,Cxcl2 and Cxcl5 mRNA in the ipsilateral hemisphere were detected by RT-qPCR 12 h after tMCAO and compared among each genotype.2.The proportion of neutrophils in peripheral blood and ipsilateral hemisphere of different genotypes were assessed by flow cytometry 1 d after tMCAO.Also,the infiltration of neutrophils in ipsilateral hemisphere was observed by laser scanning confocal microscope(LSCM).The number of infiltrated neutrophils in the ipsilateral hemisphere was calculated and compared among each genotype by ImageJ.3.The neutrophil apoptosis in the ipsilateral hemisphere was assessed by flow cytometry and compared among genotypes 1 d after tMCAO.Results:1.Compared to WT mice,Cxcll and Cxcl2 mRNA transcription in the ipsilateral hemisphere of tga20 mice significantly decreased at 12 h after tMCAO,while those of Prnp K.O.mice significantly increased.There was no significant difference in Cxcl5 mRNA transcription among genotypes.2.The proportion of neutrophils in peripheral blood and the ipsilateral hemisphere in tga20 mice apparently declined 1 d after tMCAO,which significantly increased in Prnp K.O.mice.3.The proportion of apoptotic neutrophils in the ipsilateral hemisphere of tga20 mice apparently increased,and in Prnp K.O.mice,the proportion reduced conversely.Part Ⅳ The role of PrPC on infiltration,differentiation/polarization and function of peripheral blood monocytes and cerebral microglia/macrophages during the inflammation resolution of CIRIMethods:1.The proportion of monocytes and Ly-6Chi subset in peripheral blood of each genotype 3,5 and 7d after tMCAO were assessed by flow cytometry.The dynamics of the proportions along with time were figured and the ratios of each genotype at the same timepoint were compared among genotypes.2.The proportion of infiltrating microglia/macrophages as well as M1 and M2 subtypes in the ipsilateral hemisphere of each genotype 3,5 and 7 d after tMCAO were assessed by flow cytometry.The dynamics of the proportions along with time were figured and the ratios of each genotype at the same timepoint were compared among genotypes.3.The percentage of microglia/macrophages polarization(M1%+M2%)and the ratio of M1/M2(M1%/M2%)in the ipsilateral hemisphere of each genotype were compared 5 d after tMCAO.4.The phagocytosis of apoptotic neutrophils by microglia/macrophages was observed through LSCM and the proportion of engulfed apoptotic neutrophils was calculated in three-dimensional reconstruction images after IF staining.Results:1.The proportion of peripheral monocytes and Ly-6Chi subset of each genotype significantly increased 3-7 d after tMCAO,with a gradual decline along with time.Compared to WT mice,the proportion of peripheral monocytes and Ly-6Chi subset of tga20 mice increased at each timepoint but declined in Prnp K.O.mice.2.The number of microglia/macrophages,M1 and M2 subsets in the ipsilateral hemisphere remarkably increased in each genotype,revealing first increasing and then decreasing dynamics.The proportion of each type all peaked at 5 d.3.The proportion of microglia/macrophages infiltration and M2 subset in the ipsilateral hemisphere of tga20 mice significantly increased while M1 subset obviously decreased at all timepoints.However,the proportion of microglia/macrophages in the ipsilateral hemisphere of Prnp K.O.mice showed an apparent decline only on 5 d,M1 subset increased significantly at all time but M2 subset decreased with statistical significance only on 7 d.4.No significant difference was observed in the total ratio of microglia/macrophages differentiation(M1%+M2%)among each genotype,but M1%/M2%reduction in tga20 mice and increase in Prnp K.O.mice were detected at the peak time(5 d)after tMC AO.5.The proportion of engulfed apoptotic neutrophils by microglia/macrophages showed a significantly rise in tga20 mice but a descend in Prnp K.O.mice.Conclusions:1.PrPC is upregulated in peripheral neutrophils,monocytes and cerebral infiltrating microglia/macrophages participating in inflammation resolution,suggesting that PrPC involves in crucial immunocyte events during inflammation resolution after CIRI.2.PrPC increases the expression of certain SPMs in the ipsilateral hemisphere and anti-i nflammatory cytokines in the peripheral blood,which promotes the transformation towards pro-resolving and anti-inflammatory conditions during the inflammation resolution after CIRI.Also,PrPC alleviates the symptoms during the subacute phase,facilitating the neuronal recovery of ischemia stroke.3.PrPC promotes the inflammation resolution during CIRI by reducing peripheral neutrophils,and neutrophil infiltration in the ipsilateral hemisphere by down-regulating the expression of neutrophil chemokines and enhancing the apoptosis of infiltrating neutrophils in the ipsilateral hemisphere.4.PrPC boosts the inflammation resolution during CIRI by increasing peripheral monocytes and the differentiation of Ly-6Chi subset,enhancing the infiltration of microglia/macrophages and their polarization to M2 subset and improving the engulfment of apoptotic neutrophils by microglia/macrophages.