Study On The Mechanism Of Acute Brain Injury Induced By Interaction Between CCL3 And Neutrophils During Severe EV71 Infection

Enterovirus 71(EV71)is a single-stranded,positive-sense RNA,neurotropic virus belonging to the Enterovirus A species of the Enterovirus genus of the family Picornaviridae.Infection with EV71 occasionally causes acute brain injury that was clinically characterized by many central nervous system syndrome including aseptic meningitis,acute generalized flaccid paralysis and encephalitis.Encephalitis may affect the brainstem,resulting in severe neurogenic-induced pulmonary edema and cardiorespiratory symptoms,which often ended by sudden death or severe sequela in all the survivors.Developing efficacious therapy or making a correct diagnosis against EV71 remains an unmet and urgent need,which unfortunately cannot be met until a clearer understanding of the immunopathological mechanisms of acute brain injury is attained.Host neurons infected with EV71 undergo apoptosis-mediated cell death,on which the underlying molecular pathology mechanism is clear.However,EV71-mediated effects on the host immune system,particularly on promotion of inflammation in the central nervous system(CNS),are likely important contributors to acute brain injury that was characterized by central nervous system syndrome.Severe inflammation can occur in various regions of the CNS system upon EV71 infection,including the cerebral cortex,brainstem,and all levels of the spinal cord.Meanwhile,EV71-associated systemic inflammatory responses that were characterized by cytokine storm may also lead to severe EV71-related diseases.However,there remains a lacuna in understanding the immunopathological mechanisms of acute brain injury caused by severe EV71 infection.The undermentioned results preliminarily addressed and resolved the immunopathological mechanism of acute brain injury caused by critical EV71 infection,which represented potential diagnostic and therapeutic targets for treating fatal EV71 infection.(1)78 EV71 strains were isolated and identified by q RT-PCR from 534 HFMD samples,which were collected from patients with clinical HFMD from the different regions in China.To obtain the candidate virulent strain for clarifying the fatal immunopathology mechanism,the virulence of each EV71 isolate to the suckling mice was assayed by i.p.injection of the correspondent virus.Three lethal EV71 viruses were passaged in the 3T3 cells and the suckling mice in order to enhance the viral virulence.Two stable lethal mouse-adapted strains were obtained for establishment of animal models,named EV71-BJ(Gen Bank accession NO.JQ319054)and EV71-TS(Gen Bank accession NO.JQ708210.1).We established an EV71-TS infection model in 14-day-old C57BL/6 mice,in which disease presentation and pathologic features that were similar to severe EV71-infected patients.(2)To investigate which immune cells are involved in the fatal pathogenesis of EV71 infection,we examined the leukocytes in the peripheral blood and the CSF/BIL of EV71-infected humans and our modeled mice by routine blood test,FCM and IF assay.There were markedly increased neutrophils and macrophages in the EV71-infected brain tissues that were s?ggested to act as main effectors in this course.We performed Luminex assay to examine the differential profiles of cytokines and chemokines in sera and CSF/BIL of EV71-infected patients and our modeled mice.The results showed that among all the examined cytokines and chemokines,only the levels of the chemokines CCL2,CCL3,CCL4 and CCL5,and of the cytokines IL6 and G-CSF were significantly increased in all samples of severe EV71 infection as compared with the control.Among the chemokines with significantly altered levels,CCL3 and CCL2 showed the most robust increases.In EV71 acute brain injury,elevated neutrophils,macrophages,CCL3 and CCL2 levels are key markers of poor prognosis.(3)Based on the EV71 infection mouse model,neutrophil depletion by i.p.injection of 1A8 antibody led to significantly enhanced survival,however,macrophage depletion by i.p.injection of gadolinium(III)chloride hexahydrate appeared to have no effect.Meanwhile,compared to the 100% rate of death within 1 week among the isotype-treated control mice,~30% of the mice treated with the neutralizing anti-CCL3 antibody survived thro?gh 2 weeks post-infection,however,the mice treated with the neutralizing anti-CCL2 antibody,which showed no obvious effects on survival.To further verify the roles of CCL3 and CCL2 in fatal immunopathogenesis of EV71,2-week old C57BL/6 mice with CCL3 and CCL2 knocked-out(KO)were infected with EV71.Compared to the 100% rate of death within 1 week among the wild-type(WT)control mice,60% of the CCL3 KO mice survived thro?gh 2 weeks post-infection,however,the CCL2 KO mice showed survival rates that were similar to those observed in the WT mice.FCM assay and IF staining demonstrated that knock-out of CCL3 prevented neutrophil accumulation in the cerebral ventricles following EV71 infection while the knock-out of CCL2 treatment provided no such effects.Transwell assays further indicated that CCL3 was the main attractant for neutrophil accumulation in the brain tissues of mice infected with EV71.MPO activity assay,neutrophil oxidative burst assay and IF analysis demonstrated that CCL3 is capable of direct and efficient activation of those neutrophils.Neutrophil accumulation and activation may be thro?gh CCL3/CCR1/3 axis in humans or CCL3/CCR5 axis in mice.Deep sequencing and data analysis yielded a total of 5 candidate micro RNAs that may target CCL3,including 2 human micro RNAs and 3 mouse micro RNAs.Analysis by western blot and luciferase reporter assay showed that EV71-induced CCL3 expression was up-regulated thro?gh the PI3K/NF-?B pathway at transcriptional level and thro?gh five newly identified mi RNAs at post-transcriptional level.The routine treatment of severe EV71 infection currently relies on non-specific anti-inflammation and cardiotonic treatments including application of glucocorticoid,immunoglobulin,epinephrine and Milrinon.However,there remains a lacuna in the EV71 treatment strategies for those targeting EV71-induced immunopathology in the focal sites.This study of human cases of EV71 infection and an EV71 infection mouse model showed that recruitment of CCL3-mediated neutrophils contributes to the fatal immunopathology during acute brain injury,s?ggesting CCL3 as a potential intervening therapeutic target.Inhibitors of human CCL3 or its cognate receptors are safe in clinical trials for treatment of many autoimmune diseases,based upon their ability to block the recruitment of many different immune cells.It is possible,then,that these antagonists may be effective for the treatment of EV71 immunopathology as well.In addition,the newly observed key roles of PI3 K and NK-?B pathways in activation of neutrophils in the CNS during EV71 infection and the effective inhibitory effects of the identified micro RNAs on CCL3 gene expression s?ggest that these pathways and micro RNAs may also be therapeutic targets for treating fatal EV71 infection,particularly relying on their potential to down-regulate the detrimental roles of neutrophils exerted on host tissues(bystander injury).