The Significance And Functional Analysis Of Notch Ligand DLL4 Expression In The Peripheral Blood Of Children With HFMD

Background: Hand, foot and mouth disease(HFMD) is a common acute infectious disease worldwide, which usually affects infants and children. In China, there were millions of HFMD cases diagnosed with hundreds of death every year. The major pathogens of HFMD are enterovirus 71(EV71) and coxsackie A virus A16(Cox A 16), presented with notable immune disorders such as abnormal proliferation and differentiation of inflammatory T cells and cytokines. Notch signal is crucial to many physiological and pathological programs such as cell proliferation, differentiation and cell death. It has been shown that the Notch signal pathway regulates the differentiation programs through cytokines independent pathways. The study attempted to investigate the role of the Notch signaling pathway in the pathogenesis of HFMD.Part One The significance of Notch ligand DLL4 expression in theperipheral blood of children with HFMDObjective: To understand the role of the Notch signaling pathway in the pathogenesis of HFMD.Methods: Eight-two children diagnosed with HFMD were enrolled into our study from Jun. 2012 to Dec. 2012. The HFMD group was further divided into uncomplicated HFMD and encephalitis groups. The control group included 40 children who underwent elective surgery for inguinal henias.Results:1. The expression levels of m RNA of Notch ligands DLL1 and DLL4 in peripheral blood of HFMD group were significantly higher than those in the control group(p < 0.05). However, there were no significant differences in Jagged 1 and Jagged 2 expression between the two groups. There were also no significant differences in DLL1, DLL4, Jagged 1 and Jagged 2 expression found between the uncomplicated HFMD and encephalitis group.2. The expression levels of DLL4 on the dendritic cells in peripheral blood of HFMD group were significantly higher than those in the control group though FACScan analysis(p < 0.05).3. Children with HFMD displayed significantly reduced CD3+, CD3+CD4+ and CD3+CD8+ cell subsets, but substantially enhanced CD3-CD19+ cell subsets(p< 0.05). The levels of peripheral CD3+ and CD3+CD4+ cells in the uncomplicated HFMD group were substantially higher than those in the HFMD with encephalitis group(p< 0.05), whereas the number of peripheral CD3-CD19+ cells was significantly lower in uncomplicated HFMD group compared to the encephalitis group(p< 0.05). There is no significant differences in the numbers of peripheral CD3+CD8+ and CD3-CD16+CD56+ cells found between the uncomplicated HFMD and encephalitis groups.4. The expression of Notch ligand DLL4 in the peripheral blood of the HFMD group correlated negatively with the levels of peripheral CD3+(r=-0.23,P=0.00)and CD3+CD8+(r=-0.23,P=0.02), but correlated positively with the levels of peripheral CD3-CD19+ cells(r=0.36,P=0.00). There were no correlations found between DLL4 levels and peripheral CD3+CD4+ and CD3-CD16+56+.5. Positive correlation were found in the HFMD with encephalitis group between DLL4 expression levels in the peripheral blood and total WBC counts in CSF(r=0.45,P=0.01)and total protein in CSF(S-TP)(r=0.37,P=0.01).Conclusion: The Notch ligand DLL4 may be involved in the pathogenesis of HFMD; The Notch ligand DLL4 exhibits a strong correlation with the CD3+, CD3+CD8+ and CD3-CD19+ cell subsets in children with HFMD, indicating that the Notch signaling may have impact on the prognosis by affecting the number and status of peripheral lymphocytes. DLL4 exhibits a strong correlation with WBC count and total protein level in CSF, indicating that DLL4 may be the predicting index of HFMD complicated with encephalitis and the inflammation level of brain. Part Two DLL4 is critical for human DCs induce the differentiation of CD4+ T cell to Th1 and Th17Objective:to understand whether DLL4+DCs may induce the differentiation of CD4+ T cell to Th1 and Th17 in the in inflammatory condition in vitro.Methods: Stimulated PBMCs from healthy donors with different TLRs agonists to simulate the inflammatory setting in vivo, then measured the expression of DLL4 on DCs and the activated state of DCs. Stimulated DCs with R848 then co-cultured with Na?ve CD4+T cells in a ration of 1:10 in U bottom 96 well plate, measured the expression of IFN-a and IL-17 by FACScan analysis.Results:1. DLL4 expressed(18.2%-24.7%)on peripheral DCs from healthy donors after 24 hours of stimulating with different TLRs agonists such as Pam3CSK4, LPS and R8481.2. It has been shown that HLA-DR, CD40, CD80, CD83 and CD86 expression on DCs were up-regulated after the stimulation of R848 by FACScan analysis; The expression levels of m RNA of IFN-α, IFN-β, IFN-γ, TNF-α and IL-6 increased with the induction of R848.3. The higher level of IFN-γ and IL-17 produced by Th cell were found in observation group compared to control group after co-culture DCs stimulated with R848 and CD4+T cells in a ration of 1:10 for 7 days. Blocking DLL4 with neutralizing Ab resulted in decreasing the production of IFN-γ and IL-17.Conclusion: DLL4 can be induced to express on the DCs from human peripheral blood by the stimulation of TLR agonists. DLL4 is critical for human DCs induce the differentiation of CD4+ T cell to Th1 and Th17.