Figure_3.tif (760.76 kB)

TLR3 is indispensable for iNKT cell activation in EV71 infection.

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posted on 2015-01-23, 03:18 authored by Kai Zhu, Juhao Yang, Kaiming Luo, Chunhui Yang, Na Zhang, Ruifeng Xu, Jianxia Chen, Mingfei Jin, Bin Xu, Nining Guo, Jianrong Wang, Zuolong Chen, Ying Cui, Hui Zhao, Yan Wang, Chaoyang Deng, Li Bai, Baoxue Ge, Cheng-Feng Qin, Hao Shen, Chun-Fu Yang, Qibin Leng

(A) TLR3 signaling is required for IFN-γ production by EV71-infected splenocytes. Splenocytes from WT, TLR3^-/-, TLR7^-/- and MyD88^-/- mice (n = 3–5) were cultured with EV71M or mock for 24 hours, and IFN-γ production was quantified by ELISA. (B) TLR3 deficiency dramatically reduced the IFN-γ production of iNKT cells. Splenocytes from CD1d-deficient or WT C57BL/6 mice (n = 3) were infected with EV71M for 24 hours and then stained with TCRβ, CD1d tetramer and IFN-γ. Splenocytes were stimulated with PMA and ionomycin (PMA/I) or mock treated (mock), which served as positive and negative controls, respectively. IFN-γ-producing cells are shown among CD1d tetramer⁺ TCR⁺-gated cells. (C) iNKT cell activation in EV71 infection is dependent on TLR3 signaling. Macrophages from WT or TLR3^-/- mice were cultured in the presence of EV71M for 24 hours. After extensive washing, macrophages were co-cultured for 48 hours with purified iNKT cells from WT mice, and cytokine production was quantified by ELISA. (D) TLR3 signaling is required for in vivo activation of iNKT cells upon EV71M infection. Six-8 week-old WT or TLR3^-/- mice (n = 3–6) were injected with 1×10⁵ PFU of EV71M or saline intraperitoneally. After 16 hours, splenocytes of mock (PBS) or EV71M-infected WT or TLR3^-/- mice were stained with TCRβ, CD1d tetramer, CD69 and DAPI. The mean fluorescent intensity (MFI) of CD69 expression on iNKT cells is shown. NS, not significant; **, P<0.01. (E) TLR3 triggering in macrophages activated iNKT cells. BMDCs or macrophages were cultured with medium alone or pI:C for 24 hours and then co-cultured with purified iNKT cells from WT mice for 48 hours. Cytokine production was quantified by ELISA. All results represent the mean values of cultures of 5 mice ± SD. *, P<0.05. (F) The survival rates of 7-day-old wild-type (WT) mice and TLR3-deficient (TLR3^-/-) mice (n = 6, per group) after infection by 1×10⁶ PFU of EV71M. Data are representative of three (A, C, E, F) or two (B. D) independent experiments.