Immune And Metabolic Predictors And Pathogenesis Of Severe Hand,Foot And Mouth Disease

Objective:1 To analyze immune,metabolic predictors,and the metabolic profile and mechanism during the development of severe hand,foot and mouth disease(HFMD)to better inform public health and improve clinical practice.2 To establish an Enterovirus 71(EV71)-infected animal model with central nervous system symptoms and pulmonary lesions,and a homo sapiens scavenger receptor class B(hSCARB2)knock in(KI)mice for future study of EV71 infection.3 To study the local organ immune response in EV71 infection-induced severe symptoms,which will be useful for the development of drugs and vaccines.Methods:1 HFMD cases were enrolled in this study when they were admitted in the Children’s Hospital of Zhengzhou and the First Affiliated Hospital of Xinxiang Medical University during April to September from 2013 to 2017.The cases were divided into mild(n=178)and severe group(n=178).A nested serial case-control study design was used to analyze changes of immune and metabolic indicators,and multivariable logistic regression model was used to find out potential predictors of severe HFMD.Liquid chromatography-mass spectrometry(LC-MS)was used to analyze the metabolic indicators in serum of EV71 positive cases obtained in 2017.The healthy controls(n=45)under 5-year-old were collected from the Central Hospital of Zhengzhou City.VIP values in the Orthogonal Projections to Latent Structures Discriminant Analysis(OPLS-DA)model combined with Student’s t-test(between two groups)or one-way ANOVA(between three groups)and fold changes(≥1.5 or≤0.667)were applied to find out differential metabolites associated with severe HFMD.Biological functional analysis was used to reveal the metabolic mechanisms during the development of severe HFMD.2 The phylogenetic tree based on VP1 conserved sequence was used to define the subtype of EV71 strain.The cytotoxicity of EV71 strain was measured by MTT,flow cytometry and Western blot.3-day-old BALB/c neonatal mice(2×10~6pfu/mouse)were inoculated with EV71 by intraperitoneal,intracranial and intramuscular injection,and the clinical score and survival rate were recorded.Pathological changes were observed by Hematoxylin-Eosin(H&E)staining and Nissl’s staining.The viral titteroforgansortissueswasdeterminedby VP1 expressionwith immunohistochemistry and TCID50.Mitochondrial damage and ultramicroscopic pathology were analyzed using JC-1 probe and transmission electron microscopy.The age susceptibility of EV71 was analyzed by intraperitoneal infection of neonatal mice with different ages.Spearman correlation analysis was applied to study the relationship between central nervous system injury and pulmonary lesions.At the same time,the hSCARB2 KI mouse model was established by using CRISPR/Cas9system,and the gene and protein expression levels of hSCARB2 were identified by qPCR,immunohistochemistry and western blot.The susceptibility of KI mice to EV71 infection was evaluated by using 5-day-old and 1-week-old mice,and the clinical score and survival were recorded.3 3-day-old BALB/c mice were challenged with EV71(2×10~6pfu/mouse)by intraperitoneal injection.The phenotypic changes of peripheral blood and lung immune cells were detected by flow cytometry at 7 days post infection(dpi).The cytokines in the lungs from infected mice were analyzed by Mouse Inflammation Array I kit.The inflammatory cytokines and relative active substances in tissue lysates were detected by enzyme linked immunosorbent assay(ELISA)and Griess method.Pathological changes in lung tissues were observed by H&E staining,Masson and PAS staining.Mast cells in brain,lung and skeletal muscle tissue were detected by toluidine blue staining and tryptase expression.Spearman rank correlation was used to study the association of mast cells with central nervous system injury and pulmonary edema induced by EV71 infection.The changes of immune molecules in the brains and lungs of infected mice were also analyzed by proteomics and Western blot.Results:1 Univariable analysis showed that rural living(OR=1.89,P=0.005),hyperpyrexia(body temperature>39℃)(OR=2.40,P=0.014)were predictors of severe HFMD.Multivariable regression analysis of clinical data from all HFMD cases showed that eosinophils(OR=0.91,P<0.001),Na~+(OR=0.74,P=0.0002)and alkaline phosphatase(ALP)(OR=1.06,P=0.02)were independent influencing factors for severe HFMD.Multivariable regression analysis of clinical data of HFMD cases by a nested serial case-control study showed that eosinophils(OR=0.80,P=0.02),Na~+(OR=0.53,P=0.02)and CK(OR=0.82,P=0.008)were independent influencing factors for severe HFMD on the 1st after onset.On the 2nd after onset,eosinophils(OR=0.69,P=0.04)and Na~+(OR=0.52,P=0.03)were independent influencing factors for severe HFMD.On the 3rd day after onset,Na~+(OR=0.68,P=0.03)was independent influencing factors for severe HFMD.On the 4th day after onset,eosinophils(OR=0.85,P=0.004),globulin(OR=1.54,P=0.002))were independent influencing factors for severe HFMD.On the≥5th day after onset,globulin(OR=1.20,P=0.02)was independent influencing factors for severe HFMD.The development of HFMD was accompanied by a change in serum metabolic profiles,and a total of 62 differential metabolites was obtained.Biofunctional analysis showed that the metabolic pathways involved in these differential metabolites were:alanine,aspartic acid and glutamic acid metabolism;tricarboxylic acid(TCA)cycle;D-glutamine,D-glutamate metabolism;glyoxylic acid and dicarboxylic acid metabolism;valine,leucine and isoleucine biosynthesis;pantothenic acid and coenzyme A(CoA)biosynthesis;glutathione metabolism.A receiver operating characteristic curve(ROC)was performed on the differential metabolites between mild and severe cases,and the results showed that Docosahexaenoyl PAF C-16(AUC=0.85,95%CI:0.77~0.94,P<0.0001),Eicosapentaenoyl PAF C-16(AUC=0.72,95%CI:0.62~0.83,P=0.0004),Alpha-Ketoisovaleric acid(AUC=0.72,95%CI:0.61~0.83,P=0.0006),L-Phenylalanine(AUC=0.70,95%CI:0.59~0.82,P=0.001)and Sphingosine-1-Phosphate(AUC=0.70,95%CI:0.58~0.80,P=0.003)were potential diagnostic indicators for severe HFMD.Addtionally,the above five indicators exhibited a higher combined diagnostic efficacy for severe HFMD(AUC=0.90,95%CI:0.83~0.97,P<0.0001).Serum Docosahexaenoyl PAF C-16(r=0.20,P=0.027)and Eicosapentaenoyl PAF C-16(r=0.24,P=0.008)exhibited positive correlation with the number of white blood cells.2 Obvious central nervous system lesions and pulmonary lesions were observed in EV71-infected neonatal mice,and all tissues and organs also exhibited severe pathological damage.Correlation analysis showed that central nervous system damage caused by EV71 infection was associated with the occurrence of pulmonary lesions.EV71 infection caused mitochondrial damage both in vitro and in vivo.The EV71 strain posed an age-dependent susceptibility,and the 3-day-old BALB/c neonatal mouse model was suitable for studying the pathogenesis of severe HFMD.For the establishment of hSCARB2 KI mice,a total of 77 Founder mice was obtained in this study.10 mice showed a target band after electrophoresis,indicating that the hSCARB2 gene DNA sequence was successfully inserted into mouse genomic DNA.The positive expression of hSCARB2 mRNA in tissues of 1,4 and 7-week-old KI mice,and the positive expression of hSCARB2 protein were confirmed.Compared to WT mice,KI mice exhibited higher susceptibility to EV71 infection.3 3-day-old BALB/c mice with EV71 infection exhibited obvious pulmonary edema at 7 dpi.The proportion of CD4+T lymphocytes and CD8+CD69+T lymphocytes in peripheral blood was significantly increased(P<0.01),and the proportion of CD8+T lymphocytes was significantly decreased(P<0.01)at 7 dpi.At the same time,the proportion of dendritic cells,CD11b+MHCII+dendritic cells and CD11b-MHCII-dendritic cells were significantly decreased(P<0.05),while the proportion of CD11b+MHCII-dendritic cells was significantly increased at 7dpi(P<0.01).Compared with the controls,the levels of eosinophil chemotactic factor(Eotaxin)-1,Eotaxin-2,intercellular adhesion molecule(ICAM-1),interleukin(IL)-7,IL-13,IL-17 in lung lysates were significantly increased(P<0.05),and granulocyte colony stimulating factor(G-CSF),interferon(IFN)-γand IL-10 were significantly decreased(P<0.05)at 7 dpi.EV71 infection caused mast cell degranulation in the brain,lung and skeletal muscle of infected mice.EV71-infected lungs showed severe inflammation.EV71 infection resulted in a decrease in the number of T lymphocytes,dendritic cells and monocytes,and an increase in the number of eosinophils,regulatory T lymphocytes and mast cells in the lung section(P<0.05).At the same time,T lymphocytes posed a trend towards Th2 differentiation.Proteomic analysis showed that EV71 infection caused activation of the complement system and platelet,innate immune response,high expression of glucocorticoid receptor and angiotensin in the brain section.In addition,EV71infection also induced innate immune response,hypoxia-inducible factor(HIF-1)signal,PI3K/Akt signal activation and T lymphocyte activation in the lung section.Compared to control brains,the expression levels of RIG-I,MDA5,IRAK1,p65,pp38 and Caspase-3 in EV71-infected brains were significantly increased(P<0.05),while the expression levels of MAVS,pTBK1,pIRF7 and pIRF3 posed a trend towards an increase.Compared with control brains,the expression level of pERK1/2in EV71-infected brains was significantly decreased(P<0.05),and the expression level of IRF7 was decreased with no statistical difference(P>0.05).Compared to control lungs,the expression levels of RIG-I,pIKKεand Cleaved Caspase-3 in EV71-infected lungs were significantly increased(P<0.05),while the expression levels of MDA5 and MAVS posed a trend towards an increase.Compared with control lungs,the expression levels of pTBK1,TRAF6,IRF7,pIRF3,pIκBα,pp65and Caspase-3 in the lungs from EV71-infected mice were significantly decreased(P<0.05),and the expression level of MyD88 was decreased with no statistical difference(P>0.05).Conclusions:1 Rural living and hyperpyrexia,immune changes including lower number of eosinophils,increased level of globulin,metabolic alterations including increased level of ALP,and decreased levels of Na~+and CK in peripheral blood are predictors of severe HFMD.The development of severe HFMD is associated with the change of serum metabolic profiles and energy metabolism disorders.Docosahexaenoyl PAF C-16,Eicosapentaenoyl PAF C-16,Alpha-Ketoisovaleric acid,L-Phenylalanine and Sphingosine-1-Phosphate are potential diagnostic indicators for severe HFMD.Docosahexaenoyl PAF C-16 and Eicosapentaenoyl PAF C-16 may be involved in the development of severe HFMD.2 EV71 infection causes central nervous system and lung lesions in neonatal mice.The occurrence of lung lesions may be related to central nervous system injury.The KI mouse model established in this study is efficient,economical and stable,which can be applied to the research areas of EV71 infection-induced pathogenesis and vaccines development.3 T lymphocytes immune response and dendritic cells reduction in peripheral blood and lung sections,mast cell activation,inflammation,innate immune response in brain section and innate immune evasion in lung section induced by EV71infection contribute to the central nervous system damage and pulmonary edema.