TREM-1 activation is a potential key regulator in driving severe pathogenesis of enterovirus A71 infection

Figures

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  Figure 1. EV-A71 viral isolates from 2006 outbreak in Sarawak, Malaysia, and its viral kinetics in human rhabdomyosarcoma (RD) and neuroblastoma (SH-SY5Y) cells. (a) Clinical information about the viruses isolated including genotype, symptoms and area virus was isolated from. (b–e) RD and SH-SY5Y cells were infected with mild, moderate and severe isolates of EV-A71 at MOI 10 and 1 respectively, and harvested at 0, 3, 6, 12 and 24 hours post-infection (hpi). (b,c) Infection kinetics in RD cells via (b) quantification of VP1 antigen by flow cytometry and (c) viral load quantification of VP1 RNA by qRT-PCR. (d,e) Infection kinetics in SH-SY5Y cells via (d) quantification of VP1 antigen by flow cytometry and (e) viral load quantification of VP1 RNA by qRT-PCR. Data are presented as mean ± SEM and representative of three independent experiments. Statistical analysis was carried out with Kruskal-Wallis with Dunn’s multiple comparisons test to compare among EV-A71 isolates at the respective time-points (**p<0.01; ***p<0.001) [Mild vs Severe EV-A71 (*); Moderate vs Severe EV-A71 (+)].

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  Figure 2. EV-A71 infection in primary human peripheral blood mononuclear cells (PBMCs). Human primary PBMCs (n = 5–9) were infected with mild, moderate, severe and heat-inactivated EV-A71 isolates at MOI 5, and harvested at 0, 6, 12 and 24 hpi for viral load quantification and flow cytometry. (a) Viral load levels were determined by qRT-PCR. (b,c) Percentage of EV-A71 Ag+ cells from total CD45+, CD3+ T cells, CD19+ B cells and CD14+ monocytes were determined via flow cytometry. (b) Bar graphs showing the levels of EV-A71 Ag+ cells from the different cell subsets. Data are presented as mean ± SEM. (c) Illustration of representative contour plots from one donor at 12 hpi. Statistical analysis was carried out with Kruskal-Wallis with Dunn’s multiple comparisons test to compare among EV-A71 isolates at the respective time-points (*p < 0.05; **p < 0.01; ***p < 0.001).

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  Figure 3. Transcriptomic profiling of human PBMCs during EV-A71 infection. Primary human PBMCs (n = 4) were infected with mild, moderate, severe and heat-inactivated EV-A71 isolates at MOI 5, and harvested at 0, 6, 12 and 24 hpi for transcriptomic analysis by RNA-seq. Results of infected conditions were compared to heat-inactivated control, and the log2-(fold change) values were analysed in Ingenuity Pathway Analysis (IPA). (a,b) Overall changes of significant (a) differentially expressed genes (DEGs) and (b) canonical pathways identified by IPA over time and in different isolates. (c) Heat-map showing normalised log2-(fold change) values of the common DEGs in all infected conditions of 6, 12 and 24 hpi. (d) Significant canonical pathways determined by IPA from the common DEGs in (c) (*p < 0.05; ***p < 0.001). (e) Heat-map showing the activation z-score values of the top four canonical pathways as determined by IPA.

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  Figure 4. Differences in transcriptomic profiles of human PBMCs infected with different EV-A71 isolates. Primary human PBMCs (n = 4) were infected with mild, moderate, severe and heat-inactivated EV-A71 isolates at MOI 5, and harvested at 0, 6, 12 and 24 hpi for transcriptomic analysis by RNA-seq. Results of infected conditions were compared to heat-inactivated control, and the log2-(fold change) values were analysed in Ingenuity Pathway Analysis (IPA). (a,b) Venn diagrams illustrating the number of significant (a) differentially expressed genes (DEGs) and (b) canonical pathways of the different EV-A71 isolates at 6, 12 and 24 hpi. (c) Heat-map showing the activation z-scores of the top 10 canonical pathways at 24 hpi as determined by IPA. Asterisks indicate the significance of the pathway in respective isolates. (d) Heat-map showing normalised log2-(fold change) values of the genes involved in TREM-1 signalling pathway at 24 hpi. Grey boxes indicate no values. (*p < 0.05; **p < 0.01; ***p < 0.001).

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  Figure 5. Blocking of TREM-1 by peptide LP17 decreased the expression levels of pro-inflammatory genes during moderate and severe EV-A71 infection. Human primary PBMCs (n = 7) were pre-treated with 100 ng/ml of LP17 or the control sLP17 peptide, before infection with mild, moderate, or severe EV71 isolates at MOI 5. Mock-infected PBMCs were used as negative control. Cells were replenished with fresh media containing respective peptides and harvested at 24 hpi for gene expression and viral load quantifications. (a) Total RNA samples were extracted and gene expression studies were performed via qRT-PCR using 10 ng/µl of RNA. Relative expression levels of genes were calculated by comparing the infected conditions to the respective mock. Bar graphs illustrating the expression levels of genes involved in the TREM-1 pathway. Dotted line indicates the baseline levels from mock. (b) Viral load levels were quantified by qRT-PCR of the VP1 RNA copies. Data are presented as mean ± SD. Statistical analysis was carried out with Wilcoxon matched-pairs signed rank test (*p < 0.05).