Intrinsic apoptosis and cytokine induction regulated in human tonsillar epithelial cells infected with enterovirus A71

Figures

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  Fig 1. Cell death induced in human tonsillar epithelial cells infected with EV-A71. (A) Cytopathic Effects of EV-A71-infected UT-SCC-60A and UT-SCC-60B cells. Human tonsillar epithelial cell lines, UT-SCC-60A and UT-SCC-60B, were infected with EV-A71 at an MOI of 5 and morphology of the cells was subjected to observation under light microscopy starting 24 hrs after infection. Magnification, ×100. (B) Cell viabilities of the infected UT-SCC-60A and UT-SCC-60B cells were assessed by the CCK-8 assay as described in Methods. (C) Titration of infectious viral titers in infected UT-SCC-60A and UT-SCC-60B cells. Supernatants from infected cells were collected at various time points p.i. Infectious viral titers were measured by a standard TCID50 assay. (D & E) Chromatin DNA breakage was detected in infected UT-SCC-60A and UT-SCC-60B cells. Uninfected and infected cells were on glass slides, fixed at 48 h p.i. and subjected to a TUNEL assay. The green fluorescence indicates DNA strand breaks generated in cell death. Images were captured using an Olympus confocal laser scanning microscope. Ctl, uninfected cells. Magnification, ×600.

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  Fig 2. Induction of apoptosis in EV-A71-infected human tonsillar epithelial cells. (A & B) Cleavage of executioner pro-caspases and substrates PARP and Lamin A. Cell lysates were prepared from EV-A71-infected and uninfected UT-SCC-60A (A) and UT-SCC-60B (B) cells at indicated time points. Western blot analyses were performed for detection of the caspases as well as substrates with specific antibodies. β-tublin was used as an internal control. (C) Relative caspase-3 activities in UT-SCC-60A and UT-SCC-60B cells infected with EV-A71 detected at indicated time points. Ac-DEVD-pNA was used as the substrate for caspase-3. The data were expressed as mean fold of values at A405 relative to uninfected controls. The assay for each sample was repeated twice (t-test; *p < 0.05, **p < 0.01, ***p < 0.001). (D) Effect of caspase inhibitors on EV-A71-induced cleavage of PARP and Lamin A. UT-SCC-60A and UT-SCC-60B cells were pre-treated with a pan-caspase inhibitor (Z-VAD-FMK), caspase-3-specific inhibitor (Z-DEVD-FMK), or caspase-6-specific inhibitor (Ac-VEID-CMK) for 12 hrs, followed by EV-A71 infection for another 72 hrs. Cell lysates were prepared for western blot analyses with specific antibodies…

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  Fig 3. Intrinsic apoptosis induced in EV-A71-infected human tonsillar epithelial cells. (A & B) Activation of caspase-9, but not caspase-8 or -2, in infected human tonsillar epithelial cells. UT-SCC-60A (A) and UT-SCC-60B (B) cells were infected with EV-A71 at an MOI of 5 and cell lysates were prepared at indicated time points for western blot analyses with specific antibodies. Ctl, control. (C & D) Enhancement of caspase-9 activity, but not caspase-8 activity, induced by EV-A71 infection. The cells were pre-treated with the substrate chemicals, IETD-pNA and LEHD-pNA for caspase-8 and -9, respectively, and the lysates were prepared for measurements from 12 to 72 hrs p.i. The data were expressed as mean relative fold of the A405 values. The assay for each sample was repeated in triplicates. (E) Inhibition of caspase-9 activation blocked the cleavage of caspase-3, -6, and -7 effectively in EV-A71-infected UT-SCC-60A and UT-SCC-60B cells. Both cell lines were pre-treated with a caspase-8 inhibitor (Z-LETD-FMK, 50 μmol/L) or a caspase-9 inhibitor (Z-LEHD-FMK, 50 μmol/L), followed by EV-A71 infection for another 48 hrs. Cell lysates were prepared and analyzed by western blot analyses with…

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  Fig 4. Release of cytochrome c and upregulation of Bax in EV-A71-infected human tonsillar epithelial cells. (A & B) Upregulation of Bax and release of cytochrome c. Cytosolic (Cyto) and mitochondrial (Mito) fractions of the cell lysates were prepared from uninfected (Ctl) and infected cells at various time points p.i. The levels of Bax and cytochrome c were determined by western blot analyses. GAPDH and VDAC1 were detected as internal control for proteins in the cytosolic and mitochondrial fractions, respectively. The assays were repeated at least twice. (C & D) Down-regulation of Bcl-2 and survivin and upregulation of Bax in EV-A71-infected UT-SCC-60A and UT-SCC-60B cells. Uninfected (Ctl) and infected cells were infected with EV-A71 and total cell lysates were collected and analyzed by western blot analyses with specific antibodies. (E & F) Regulation of the apoptotic genes in UT-SCC-60A (E) and UT-SCC-60B (F) cell lines infected with EV-A71. Total RNA was prepared at the indicated time points p.i. for quantitative RT-PCR using primers specific for Bax, Bid, Survivin or FLIP. Values represent mean ± SD from triplicates of independent experiments (t-test; NS, no significance; *p<0.05,…

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  Fig 5. Pyroptosis and necroptosis were not induced in EV-A71-infected human tonsillar epithelial cells. (A & B) UT-SCC-60A and UT-SCC-60B cells were infected with EV-A71 at an MOI of 5 and cell lysates were prepared at indicated time points p.i. for western blot analysis with antibodies specific for proteins involved in pyroptosis (pro/cleaved caspase-1 and pro/cleaved GSDMD) or necroptosis (RIPK1/p-RIPK1, MLKL/p-MLKL, and ZBP1).

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  Fig 6. Induction of cytokine and chemokine gene transcription in EV-A71-infected tonsillar epithelial cells. UT-SCC-60A (A & C) and UT-SCC-60B (B & D) cells were infected with EV-A71 at an MOI of 5 and total RNA was prepared at the indicated time points for quantitative RT-PCR using primers specific for IFN-α, IFN-λ1, IFN-λ3, IL-1β, IL-8, IL-18, ISG-54, ISG-56, or IP-10. Values represent mean ± SD from triplicates of independent experiments (t-test; *p < 0.05, **p < 0.01).

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  Fig 7. Induction of IFN-β and CCL5 in EV-A71-infected human tonsillar epithelial cells. UT-SCC-60A and UT-SCC-60B cells were infected with EV-A71 and total RNA was prepared for quantitative RT-PCR using primers specific for IFN-β (A), IL-6 (C), CCL5 (E), or TNF-α (G). The cultural media of both cell lines infected with EV-A71 were collected at various time points after infection for ELISA to measure the protein levels of IFN-β (B), IL-6 (D), CCL5 (F), and TNF-α (H). Values represent mean ± SD from triplicates of independent experiments (t-test; *p< 0.05, **p < 0.01).

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  Fig 8. Regulation of cytokine induction by MAPKs in EV-A71-infected human tonsillar epithelial cells. UT-SCC-60A and UT-SCC-60B cells were pre-treated with inhibitors for ERK1/2 (U0126), JNK1/2 (SP600125), or p38 (SB203580) at 1 hr prior to EV-A71 infection. The cultural media were collected at 48 and 72 hrs p.i. for ELISA to measure the levels of IFN-β (A), IL-6 (B), CCL5 (C), and TNF-α (D). The experiments for each sample were repeated at least three times (t-test; NS, no significance. *p < 0.05, **p < 0.01).