AIM2 Inflammasome-Mediated Pyroptosis in Enterovirus A71-Infected Neuronal Cells Restricts Viral Replication

Figures

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  Figure 1. Disease and function pathways derived from transcriptome and Ingenuity pathway analysis of infected SK-N-SH cells. The most highly up-regulated disease and function genes involved cellular movement and cell death/survival. The graph shows the category scores where the “threshold” indicates the minimum significance level [calculated as -log (p-value) using the Fisher’s exact test, set at 1.25].

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  Figure 2. EV-A71 strains up-regulated AIM2 expression in infected SK-N-SH cells. AIM2 expression in infected SK-N-SH cells determined by real-time PCR analysis in all three EV-A71 strains (13903, 18431, and SB12736) showed significantly higher AIM2 fold change relative to mock-infected controls at 48 and 72 hpi. All data represent the mean ± standard deviation of a set of triplicates.

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  Figure 3. AIM2, CARD16, caspase-1 and IL-1β up-regulation, caspase-1 activation and viral replication inhibition was observed in infected-SK-N-SH cells but not in SK-N-SH/siAIM2 cells. There was up-regulation of AIM2 (a), caspase-1 (b), CARD16 (d) and IL-1β (e) expressions in infected SK-N-SH cells, and significant reduction (*p < 0.05) in SK-N-SH/siAIM2 cells. Western blot analysis showed a marked reduction/absence of AIM2 protein (a) and cleaved caspase-1 p20 subunits (activated caspase-1). Cropped blots are presented due to different primary and secondary antibody procedures during staining. (b) Viral copy numbers (c) were about 10 fold higher (*p < 0.05) in infected SK-N-SH/siAIM2 cells for all time points, corresponding to Western blot analysis that showed much higher viral proteins in infected cells (c). AIM2 (f) and caspase-1 (g) gene expressions in EV-A71/13903 viral RNA transfected SK-N-SH cells showed significant up-regulation (*p < 0.05) at 12 and 24 hpt, respectively. UV-inactivated EV-A71/13903 RNA transfection showed no up-regulation of AIM2 (f) and caspase-1 (g) gene expression at both time points. Viral copies at 12 to 24 hpt in viral RNA transfected SK-N-SH cells were…

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  Figure 4. Viral replication inhibition did not depend on multiplicity of infection. (a) Viral titration of EV-A71/13903-infected SK-N-SH and SK-N-SH/siAIM2 cells at MOIs of 0.1, 1 and 10. Significantly lower (*p < 0.05) viral titers were observed in infected SK-N-SH cells compared to SK-N-SH/siAIM2 for all time points at all MOIs. In SK-N-SH/siAIM2 cells, a gradual increase in viral titers were observed from 24 to 72 hpi, while in SK-N-SH cells no significant increase was observed after 48 hpi. All data represent the mean ± standard deviation of a set of triplicates.

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  Figure 5. Cell swelling correlated with 7-AAD staining in infected SK-N-SH and SK-N-SH/siAIM2 cells. (a) Cellular morphology of EV-A71/13903-infected SK-N-SH cells at MOI of 10 showed pyroptotic cell swelling (arrows) from 48 hpi. (b) Flow cytometry analysis of percentage of the 7-AAD positive infected SK-N-SH cells showed significant increase from at 24 hpi onwards. (c) In contrast, SK-N-SH/siAIM2 cells showed extensive cell shrinkage (arrow heads) and detachment rather than swelling from 48 hpi onwards. (d) This correlated with the percentage of 7-AAD stained infected SK-N-SH/siAIM2 cells was low at about 6% throughout. All data represent the mean ± standard deviation of a set of triplicates.

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  Figure 6. Viral infection induced AIM2 and pyroptosis in SK-N-SH cells but not in SK-N-SH/siAIM2 cells. EV-A71/13903-infected SK-N-SH cells and SK-N-SH/siAIM2 cells at 24 (a), 48 (b), 72 (c) and 96 hpi (d), stained for viral antigens (green), AIM2 (red) and dapi (blue, nuclear staining). Viral antigen-positive SK-N-SH cells were also positive for AIM2 (a–d, white arrows) for all time points. At 72 and 96 hpi but not at earlier time points, viral antigen-negative cells stained positive for AIM2 (c,d, white arrowheads). SK-N-SH/siAIM2 cells showed extensive viral antigen staining ((a–d), white arrows) without AIM2 expression for all time points. Magnification 20x.

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  Figure 7. AIM2 induced pyroptosis limited viral replication in infected SK-N-SH but not in SK-N-SH/siAIM2 cells. Flow cytometry analysis of viral antigens, AIM2 expression and 7-AAD staining in infected SK-N-SH and SK-N-SH/siAIM2 cells as acquisition plots, at 48 (a,b) and 96 (c,d) hours post-infection (hpi), and as percentage graphs at 24, 48, 72 and 96 hpi (e,f). (a,b) At 48 hpi, viral antigen-positive SK-N-SH cells at about 40% (a,I) was significantly lower (p = 0.0002) compared to SK-N-SH/siAIM2 cells at about 73% (b,I). SK-N-SH cells with viral antigen, AIM2 and 7-AAD positivity at about 53% (a,II) was significantly much higher (p = 0.0002) than in SK-N-SH/siAIM2 cells, which was about 1% (b,II). Among viral antigen-positive SK-N-SH cells, 7-AAD positivity of about 51% (a,III) was significantly much higher (p = 0.0001) than in SK-N-SH/siAIM2 cells which was about 1% (b,III). Similarly, among viral antigen-positive SK-N-SH cells, AIM2 positivity was significantly much higher (p = 0.003) at about 85% (a,IV) than in SK-N-SH/siAIM2 cells, which was about 6% (b,IV). (c,d) A similar pattern was observed in SK-N-SH cells at 96 hpi, in which viral antigen-positivity (about 46%) (c,I) was…

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  Figure 8. AIM2 antigens was expressed in inflamed areas and EV-A71-infected neurons in human encephalomyelitis. AIM2 was positive in inflamed areas of the spinal cord (a,b) and medulla (c) (arrows). In the respective, immediately adjacent spinal cord tissue sections, viral antigens ((e,g,i) arrows) and AIM2 ((f,h,j) arrows) were positive in the same neurons. Some neurons were AIM2 positive but viral antigen negative ((e,f,g,h) arrowheads). The cerebral cortex (d) and other uninflamed areas were negative for AIM2 and viral antigens. Immunohistochemistry using DAP and counter stained with hematoxylin. Magnification 20x (a–d) and 20x (e–j). Scale bar = 100 μm (a–d) and 50 μm (e–j).