Antiviral Innate Immune Responses Of Human Tonsillar Epithelial Cells And Macrophages Against EV71 Infection

Hand, foot, and mouth disease (HFMD) is a common disease in children less than 5 years which affects children’s health and this disease poses a huge disease bueden and economic burden to the patients and patients’homes. EV71 as a major agent of HFMD, the patient which infected with EV71 could appear the classical syndrome of HFMD, some severe cases would develop central never system (CNS) complications and even death. The recently studies showed EV71 viral RNA and/or antigens were localized to squamous epithelium lining the tonsillar crypts in EV71-caused fatal autopsy cases and most EV71 viral RNA-positive inflammatory cells in the CNS were macrophages, however, these results were only limited in EV71-caused fatal cases, whether human tonsollar epithelial cells and macrophages were susceptive to EV71 still remain unknown. Antiviral innate immunity is the first line against viral infection, which plays an important role in erading the invading viruses. EV71 using itself encoding 2A and 3C proteases cleavaged the adaptors and disturbed the signal transduction pathway of innate immunity to implement the immune evasion. The innate immune responses of human tonsillar epithelial cells and macrophages against EV71 are also not conduct yet, so we choose two human tonsillar epithelial cells UT-SCC-60A and UT-SCC-60B, macrophages U937 as the cell models to conduct the susceptivility to EV71 and innate immunity against EV71.PART One:Innate immune responses of human tonsillar epithelial cells and macrophages against EV71The human tonsillar epithelial cells UT-SCC-60A and UT-SCC-60B had the classical morphologies of epithelial cells and highly expressed keratin-5. Human monocyte U937 was successfully differentiated into macrophages induced by PMA. The significantly cytopathic effects appeared in EV71-infected human tonsillar epithelial cells UT-SCC-60A and UT-SCC-60B. The viral titers of EV71 in human tonsillar epithelial cells UT-SCC-60A and UT-SCC-60B and mcrophages U937 were higher than the known EV71 susceptive cells and the SCARB2 mediated the EV71 infection. The cytokines including IL-8, IL-1β, IL-6 were induced in EV71-infected human tonsillar epithelial cells UT-SCC-60A and UT-SCC-60B and mcrophages U937. The PI3K/AKT, p38, JNK1/2 and ERK1/2 signal pathways were activated and the ERK1/2 and JNK1/2 pathways were essential for the replication of EV71 at early phase of EV71-infected human tonsillar epithelial cells UT-SCC-60A and UT-SCC-60B and mcrophages U937. The NF-κB pathway was also activated and a crosstalk contained between the NF-κB pathway and The PI3K/AKT, p38, JNK1/2 and ERK1/2 signal pathways, this crosstalk regulated the production of cytokines in EV71-infected human tonsillar epithelial cells UT-SCC-60A and UT-SCC-60B and mcrophages U937.PART Two:The TLR2 heterodimers recognizing EV71 and mediating the antiviral innate immunityThetranscriptions of TLR2 and TLR4 were up-regulated induced by EV71, UV-EV71 and EV71 virus-like particle (VLP) in human tonsillar epithelial cells UT-SCC-60A and UT-SCC-60B and mcrophages U937. The immunofluorescence co-locazation of TLR2 and EV71 were occurred at cytoplasm in UT-SCC-60A, cytomembrane in UT-SCC-60B and cytoplasm in U937. The subcellular distriction of TLR2 showed TLR2 was distributed in cytoplasm in UT-SCC-60A, cytomembrane and cytoplasm in UT-SCC-60B and U937. The immunofluorescence co-locazation of TLR4 and EV71 were occurred at cytoplasm in UT-SCC-60A, cytomembrane in UT-SCC-60B and cytoplasm in U937. The subcellular distriction of TLR2 showed TLR2 was distributed in cytoplasm in UT-SCC-60A, cytomembrane in UT-SCC-60B andcytoplasm in U937. The protein levels of TLR2, TLR4, MyD88, IRAK-1, TRAF3 and TRAF6 were increased in EV71-infected human tonsillar epithelial cells and macrophages and the p65 and IRF3 were phosphorylated. The TLR2/TLR1 and novel TLR2/TLR4 heterodimers were formed in EV71 and UV-EV71 infected UT-SCC-60B and U937. The TLR2/TLR1, TLR2/TLR6, TLR2/CD14 heterodimers which transfected into HEK293 could significantly inhibit the CPE of EV71-infected HEK293 cells, this result indicated TLR2/TLR1, TLR2/TLR6, TLR2/CD14 heterodimers had potential role in inhibiting EV71 replication. Excessive IL-1β, IL-6, IL-8, TNF-a were produced in U937 treated with various TLR2 heterodimers ligands. Finally we found proinflammatory cytokine and chemokine responses were activated in EV71-infected mouse macrophage RAW264.7.In conclusion, we first identified two novel findings in this research. (1) Human tonsillar epithelial cells (UT-SCC-60A and UT-SCC-60B) and human mcrophage (U937) were susceptive to EV71 and activated the PI3K/AKT and MAPKs signal pathways. A crosstalk contained between PI3K/AKT, MAPKs and NF-κB pathway and regulated the productions of cytokines; (2) TLR2/TLR1 and TLR2/TLR4 heterodimers formed in UT-SCC-60B and U937 induced by EV71 and UV-EV71, these TLR2 heterodimers may be mediate the activation of MyD88/IRAK-1/TRAF3/IRF3 or MyD88/IRAK-1/TRAF6/NF-κB pathway leading to the production of cytokine. Our results firstly indentified human tonsillar epithelial cells and macrophage were novel susceptive cells of EV71, and also found the novel potential antiviral innate immune pathway, these results will provide a scientific evidence for better knowing the infection and pathogenesis of EV71.