Screening Of Rare Cytokines In Prion-infected Brain Tissue And Its Relationship With Prion Diseases

Activation of inflammatory cells and upregulations of a number of cytokines in the central nervous system?CNS?of patients with prion diseases are frequently observed,but some low abundance and uncommon cytokines were rarely reported.To evaluate the potential changes of some brain cytokines that were rarely addressed during prion infection,the levels of 17 different cytokines in the brain homogenates of mice infected with different scrapie mouse-adapted agents were screened with Luminex assay.Significant upregulations of interferon gamma-induced protein 10?IP10?,keratinocyte chemoattractant?KC?and macrophage colony stimulating factor?M-CSF?were frequently detected in the brain lysates of many strains of scrapie infected mice.The elevations of those three cytokines in the brains of scrapie infected mice were further validated by individual specific ELISA and immunohistochemical assays.Increased specific mRNAs of IP10,M-CSF and KC in the brains of scrapie infected mice were also detected by the individual specific qRT-PCRs and IP10-specific digital PCR.Dynamic analyses of the brain samples collected at different time points post infection revealed a time-dependent increases of those three cytokines,particularly IP10 during the incubation period of scrapie infection.In addition,we also found that the levels of IP10 in cerebral spinal fluid?CSF?of 45sporadic Creutzfeldt-Jakob disease?sCJD?patients were slightly but significantly higher than those of the cases who were diagnosed with prion diseases.These data give us a better understanding of inflammatory reaction during prion infection and progression of prion diseaseIn order to reveal the relationship between IP10 and prion disease and its distribution characteristics in central nervous system,we used IP10 specific immunofluorescence method to compare the brain tissue sections of mice with scrapie factor 139A and ME7 at terminal stage of infection.It was found that IP10 and the markers of neurons and microglia co-localized in the cortex,hippocampus and thalamus of prion infection.There was no colocalization with astrocyte markers.By using CXCR3 specific immunoblotting,immunofluorescence and RT-PCR,we confirmed that CXCR3,the main receptor of IP10,was abnormally elevated in protein and transcription level in the brain tissue of prion infected mice.CXCR3 was found to be mainly distributed in the cytoplasm of hippocampus,cortex,thalamus and cerebellum,especially in the thalamus,and some granular deposition of CXCR3 would appear around the vacuoles of brain tissue.We also carried out specific immunohistochemistry analysis of IP10,CXCR3 and PrP^Scc on the continuous sections of brain tissue with prion infected mice.The results showed that the common positive signals of PrP^Sc,IP10 and CXCR3 can be observed in the same location of hippocampus,cortex,thalamus and cerebellum of prion infected mice,which indicates that cxcr3-ip10 has obvious correlation with the formation of PrP^Scc plaque.We first found an obvious change of M-CSF as an important cytokine in the central nervous system of prion infection,enriched the content change characteristics of IP10 and KC in the central nervous system of prion infection,and preliminarily explored the correlation between CXCR3-IP10 and prion infection.One step was to clarify the causal relationship between CXCR3-IP10 and prion pathology,and enrich the data of prion pathogenesis.