Pathological Features of Enterovirus 71-Associated Brain and Lung Damage in Mice Based on Quantitative Proteomic Analysis

Figures

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  FIGURE 1. The quantitative proteomic profile of EV71-infected brain and lung tissues. (A) Study design of quantitative proteomic analysis for EV71-infected brain and lung tissues. (B,C) Principal component analysis of the proteome profile. Biological replicates were generated for each sample, represented by different color points in the figure. (D,F) Volcano plots of the −log10 p value vs. the log2 protein abundance comparisons between brains or lungs from controls and those infected with EV71. Proteins outside the significance threshold lines (p value < 0.05 and EV71/control > 1.2 or < 1/1.2) were colored in red (upregulated) or blue (downregulated). (E,G) Schematic of changes in the cell components of EV71-infected brain and lung tissues. Red and blue fonts represent up- and downregulated proteins detected in brain or lung tissues from EV71-infected mice compared to controls.

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  FIGURE 2. Gene Ontology (GO) enrichment bubble plot of differentially expressed proteins in the three categories. (A,C,E) GO enrichment analysis of differentially expressed proteins in the brain. (B,D,F) GO enrichment analysis of differentially expressed proteins in the lung.

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  FIGURE 3. Visualization of changing proteins and their biological pathways. (A) Heat map of differentially expressed proteins in the brain (control: n = 4, EV71: n = 4) that are involved with key biological pathways. (B) Heat map of differentially expressed proteins in the lung (control: n = 4, EV71: n = 4) that are involved with key biological pathways.

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  FIGURE 4. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of significantly dysregulated proteins. (A) KEGG enrichment analysis of significantly dysregulated proteins in the brain. (B) KEGG enrichment analysis of significantly dysregulated proteins in the lung. (C) A hypothetical systems view of the two organs’ responses to EV71 infection. In the brain or lung, the virus and its released RNA could induce an antiviral innate immune response. APCs bind antigens and internalize, process, and express them on their surface for recognition by certain lymphocytes such as CD4 T cells and CD8 T cells. In the brain, the virus infection induces complement activation which contributes to disease by attracting and activating inflammatory cells and by direct damage to endothelial cells through MAC. The endothelial dysfunction leads to the activation of coagulation cascade and platelet through KKS cascade. In the lung, the NADPH oxidase activity was significantly increased in response to EV71 infection, which might activate the HIF-1 signaling pathway, leading to a hypoxic condition. Red boxes, upregulated proteins/pathways; blue boxes, downregulated proteins/pathways.

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  FIGURE 5. Comparison of laboratory measurements and proteome. At 7 dpi, EV71-infected mice (n = 4) and controls (n = 4) were euthanized, and brain and lung tissues were taken out for immunohistochemical (IHC) staining (n = 2) and Western blotting (n = 2). (A) Immunohistochemical analyses of NR3C1, STAT1, TRIM25, Cathepsin S, and ISG15 in EV71-infected brain tissues and control brain tissues. Black arrows represent positively stained areas (brown). (B) Comparison of intensities of IHC staining and proteomic data (TMT reporter ion intensities) in the brain. (C) Immunohistochemical analyses of CDKN1B, STAT1, Arginase-1, Cathepsin S, and NOX2 in EV71-infected lung tissues and control lung tissues. (D) Comparison of intensities of IHC staining and proteomic data (TMT reporter ion intensities) in the lung. (E) Western blotting analyses of NR3C1, TRIM25, STAT1, and Cathepsin S in EV71-infected brain tissues and control brain tissues. (F) Western blotting analyses of CDKN1B, Cathepsin S, Arginase-1, and STAT1 in EV71-infected lung tissues and control lung tissues. (G) Comparison of intensities of immunoblots and proteomic data (TMT reporter ion intensities) in the brain and lung. All laboratory…

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  FIGURE 6. Protein–protein interaction analysis of dysregulated proteins in the brain. The size of the nodes corresponds to the number of interacting proteins, whereas the different colors represent the regulation type and magnitude of protein changes. Red color represents upregulation, and blue color represents downregulation. The full list of key driver proteins can be found in Supplementary Table 3.

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  FIGURE 7. Protein–protein interaction analysis of dysregulated proteins in the lung. The size of the nodes corresponds to the number of interacting proteins, whereas the different colors represent the regulation type and magnitude of protein changes. Red color represents upregulation, and blue color represents downregulation. The full list of key driver proteins can be found in Supplementary Table 4.