Dissertation
Dissertation > Medicine, health > Pediatrics > Children within the science > Pediatric Infectious Diseases

Studies on Immunity Mechanism of Central Nervous System Injury Caused by EV71Virus

Author GongXun
Tutor JinYu; DuanZhaoJun
School Lanzhou University
Course Traditional Chinese Medicine
Keywords Enterovirus71 Macrophages Cytokine Plasma Cerebrospinal Fluid PRRs
CLC R725.1
Type PhD thesis
Year 2012
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Background:Enterovirus71, a positive-stranded RNA virus, can cause hand-foot-and-mouth disease (HFMD), herpangina, encephalitis, aseptic meningitis, brain stem encephalitis (BE) in infants and young children. Many of patients died from fulminant pulmonary edema (PE) or hemorrhage, which was based on nervous system injury. However, the mechanism of neurological diseases caused by EV71infection is unclear. This study assessed the quantitative relationship between cytokines or chemokines in plasma/cerebrospinal fluid (CSF) and their correlations with clinical severity, and examined their levels in CSF in acute and convalencence phase. In addition, we also tested if there is any change in cytokine profiles in patients with EV71-associated encephalitis administrated by intravenous immunoglobulin (IVIG) or glucocorticoid.Study design:Blood and (or) cerebrospinal fluid samples of children patients with EV71infection were collected in Nanjing Children Hospital, between Apr.2010and Sep.2011. CSF samples were obtained from patients with CNS combinations on admission and (or)7-14days after administration, and blood samples were obtained from all patients on admission.14blood samples from healthy children (age≤5ys) were also included as negative controls. The plasm samples from62children with EV71infection, included18uncomplicated cases,33severe cases and11fatal cases. The CSF samples were obtained from patients with EV71-associated encephalitis (n=18), BE (n=14) and myelitis (n=10) in acute and convalencence phase, and6patients with PE only in acute phase. Concentration of cytokines (IL-1β, IL-6, TNF-α, IL-10, IFN-a, IFN-y, IP-10, IL-8, RANTES, MCP-1, MIG) from plasma and CSF were determined by the assay of cytometric bead array.Results:The plasma concentration of IL-1β, IL-6was significant higher in severe or fatal EV71infection cases as compared with healthy children. The plasma levels of IL-6and IL-8was elevated in fatal EV71infected patients than in patients with uncomplicated or severe EV71infection. Moreover, the significantly lower levels of IL-6, IP-10, IL-8, MIG and (or) IL-1β in CSF were found in patients with CNS combinations after administration as compared to them on admission by Paired-Samples T Test. The concentration of IL-6, IFN-y, IL-8and (or) IL-1β,IP-10in CSF was significant higher in patients with PE or BE than other groups. However, there was no signigicant changes on the concentration of TNF-a, IL-10, IFN-a, RANTES and MCP-1in plasma or CSF between severe cases (BE, Encephalitis, Myelitis) and uncomplicated EV71infection.Conclusions:Inflammatory responses predominated in the central nervous system, and the amount of IL-1β, IL-6, IL-8and IFN-y in CSF of patients might be a good parameter correlating to clinical severity. Systemic inflammatory response together with the local response in CNS may play an important role in the development of EV71-related PE. Glucocorticoid might be considered to have a therapeutic role, through reducing the expression of cytokines, in EV71infection with CNS combinations.Background:The binding of pathogen-associated molecular patterns (PAMPs) to pattern recognition receptors (PRRs), results to the activation of an innate immunity response. Recent studies have identified three major types of PRRs, including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs). In response to virus infections, viral components such as RNA and DNA are recognized by TLRs, RLRs and NLRs, and cell are activated to produce type I interferons (IFNs), proinflammatory cytokines and chemokines. An elevated cytokines in blood and cerebrospinal fluid were found in EV71infection with CNS combinations, yet the signalling mechanism of cytokines production remains undefined. In this study, we focused on the expression of TLRs, RLRs and NLRs in patients with EV71infection.Study design:Peripheral blood mononuclear cells (PBMCs) were isolated from patients with EV71infection. Patients were stratified by clinical severity: uncomplicated cases (n=18), severe cases (n=34) and fatal patients (n=11). And14healthy children were also included as negative controls. We analyzed the mRNA expression of TLR1-10, RIG-I, Mda-5, NLRP1, NLRP3, NLRX1, XLRC5in PBMCs from patients by Real-time RT-PCR, and compared their levels with those in healthy children.Results:The result showed that the mRNA expression of TLR2/4/7/8and Mda-5appeared to be significantly increased in patients with EV71infection as compared with healthy children. Unexpectedly, lower expression of TLR3and RIG-Ⅰ was found in severe cases and fetal cases than healthy children, and it suggested that the gene transcription of TLR3and RIG-Ⅰ could be inhibitted by viral components of EV71. In addition, an elevated NLRP3mRNA expression was found in only two patients with EV71-associated PE. However, there was little change in the mRNA expression of other PPRs such as TLR1, TLR5, TLR6, TLR9, TLR10, NLRX1, NLRC5, NLRP1in EV71patients.Conclusions:Many types of PRRs activated by EV71may be responsible for the induction of proinflammatory cytokines and chemokines. There was no apparent correlation between PRRs expression levels in PBMCs and clinical severity. Further study on the specific role of PRRs in the cytokines production of EV71is needed.Background:The levels of proinflammatory cytokines or chemokines in blood and cerebrospinal fluid are thought to be one of predictors for clinical severity of enterovirus71(EV71) infection, yet the cellular sources or signalling mechanism remain undefined. Here, we focused on the responses of human primary monocyte-derived macrophages (MDMs) to EV71virus and its possible mechanisms.Study design:Human primary MDMs were infected by EV71virus in vitro. Infectivity and viral replication were assayed, and cytokine responses were determined by Cytometric Bead Array(CBA). The relative changes of Toll-like receptors, retinoic acid-inducible gene Ⅰ (RIG-Ⅰ) and melamoma differentiation associated gene5(MDA5) mRNA expression were analyzed by real-time RT-PCR.Results:Effective infection and viral replication were detected in EV71-infected MDMs. Significant higher level of TNF-α was rapidly induced by live EV71infection other than mock or UV-irradiated EV71at the12-h POI., and then gradually decreased over time. Both IL-1β and IL-6from MDMs were triggered by live EV71, and maintained a higher level as compared with that of mock. In addition, we found that EV71infection failed to induce IFN-α and IFN-γ releasing from human MDMs. At early time points (12-h POI.), enhanced production of IL-8and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs, and the concentrations of these chemokines increased along with time. At24-h POI., much higher concentrations of IL-8, RANTES and IP-10were found in EV71-infected MDMs than in those of mock. Moreover, compared with mock-infected MDMs, UV-irradiated EV71can induce significantly high level of IL-8production at12-h and24-h POI. than mock. Upregulation of TLR2, TLR7and TLR8mRNA expression rather than other PRRs was found at different time points in EV71-infected MDMs.Conclusions:In summary, effective viral replication in EV71-infected MDMs and excessive inflammatory cytokine and chemokine responses of MDMs to the virus were demonstrated for the first time in our study. The results indicate that macrophages are an important target for EV71, and they can trigger proinflammatory immunity response against viral infections. However, inordinate macrophages response may be detrimental to the infected host due to exacerbate virus inflammation and pathology. TLR2, TLR7and TLR8may all participate in the induction of inflammation in EV71-infected MDMs. These data suggested that MDMs maybe play an important role in the pathogenesis of EV71infection in vivo though more evidence is needed.

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