Host Genetics, Innate Immune Responses, and Cellular Death Pathways in Poliomyelitis Patients

Figures

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  FIGURE 1. Whole-exome sequencing (WES) filtering diagram. In brief, we included exonic variants that were predicted to be rare (present in <0.1% of the reference genomes), excluded variants with CADD score <15 (Kircher et al., 2014), and included variants with frameshift, in-frame insertions or deletions (indels) or stop codon change (No CADD score available for these types of variants). Lastly, we included genes with high evolutionary conservation with a phyloP p-value less or equal to 0.01. The variant filtering was verified by random sampling, and all BAM files of the variants identified in Ingenuity Variant Analysis (IVA) were manually examined in order to include only variants with high sequencing quality. BAM files were evaluated by use of the UCSC genome browser or IGV.

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  FIGURE 2. STRING protein-protein interaction network for proteins described in the PPM cohort. Each circle represents the protein affected by at least one variant in the cohort. The thickness of the gray lines represents strength of data supporting a protein-protein interaction. The gray circles include clusters of proteins involved in common pathways/biological functions. The PPI enrichment p-value for the number of identified edges (30) compared to expected (9) in a group of 36 proteins was 4.13 × 10-8, thus significantly more than expected, with a minimum interaction score a 0.4.

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  FIGURE 3. Innate immune responses at cohort level. PBMCs (A–D) or MdMs (E) from patients and controls were infected with PV at an MOI of 100 (PBMCs) or an MOI of 10 (MdMs). Total RNA was harvested 6 h following infection and subjected to RT-qPCR for measurement of IFNβ, CXCL10, TNFα, or IL6. Cytokine mRNA levels were normalized to the housekeeping gene TBP (MdMs) or TBP+18S (PBMCs) and compared to the pooled results of a total of nine controls. Data are shown as box plots with the 5–95% population, and outliers shown as independent dots. Non-parametric Mann-Whitney ranked sum test was used for statistical analysis. ∗∗P ≤ 0.01 and ∗∗∗∗P ≤ 0.0001. PV; poliovirus, PBMCs; peripheral blood mononuclear cells, MdMs; monocyte-derived macrophages.

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  FIGURE 4. Viral replication in MdMs. MdMs were stimulated with PV at an MOI of 10 for 1 h. The inoculum was removed and cells were washed seven times before adding fresh media. The supernatants were harvested at 24 h (total infection time) following infection.(A) ID50/mL is shown as dots representing mean of triplicates or more from one or two independent experiments, box-line represent median. (B) Correlation of IFNβ mRNA response and ID50/mL. Non-parametric Mann-Whitney ranked sum test and Spearman correlation was used for statistical analysis. (C) Top left, uninfected MdMs. Black arrows, M1 morphology; blue arrows, M2 morphology. Top right, MdMs 24 h following infection. Red arrows, cells have started clustering and appear rounded as a sign of infection. Bottom left, a confluent monolayer of uninfected HeLa cells. Bottom right, infected HeLa cells. Cells have rounded and detached from the surface which indicate infection and/or cell death. PV, poliovirus; MdMs, monocyte-derived macrophages.