Pathway mapping of leukocyte transcriptome in influenza patients reveals distinct pathogenic mechanisms associated with progression to severe infection

Abstract

Background: Influenza infections produce a spectrum of disease severity, ranging from a mild respiratory illness to respiratory failure and death. The host-response pathways associated with the progression to severe influenza disease are not well understood.

Methods: To gain insight into the disease mechanisms associated with progression to severe infection, we analyzed the leukocyte transcriptome in severe and moderate influenza patients and healthy control subjects. Pathway analysis on differentially expressed genes was performed using a topology-based pathway analysis tool that takes into account the interaction between multiple cellular pathways. The pathway profiles between moderate and severe influenza were then compared to delineate the biological mechanisms underpinning the progression from moderate to severe influenza.

Results: 107 patients (44 severe and 63 moderate influenza patients) and 52 healthy control subjects were included in the study. Severe influenza was associated with upregulation in several neutrophil-related pathways, including pathways involved in neutrophil differentiation, migration, degranulation and neutrophil extracellular trap (NET) formation. The degree of upregulation in neutrophil-related pathways were significantly higher in severely infected patients compared to moderately infected patients. Severe influenza was also associated with downregulation in immune response pathways, including pathways involved in antigen presentation such as CD4+ T-cell co-stimulation, CD8+ T cell and Natural Killer (NK) cells effector functions. Apoptosis pathways were also downregulated in severe influenza patients compare to moderate and healthy controls.

Conclusions: These findings showed that there are changes in gene expression profile that may highlight distinct pathogenic mechanisms associated with progression from moderate to severe influenza infection.

Keywords: Influenza; Neutrophil extracellular trap; Neutrophils; Transcriptome.

Fig. 1

Flow chart and study scheme. Flow chart shows study design and analysis workflow

Fig. 2

Differentially expressed genes in moderate and severe influenza. a Break down of statistically significant differentially expressed genes. The table showed the total number of differentially expressed genes in the three comparisons and the number of upregulated or downregulated genes. b Venn diagrams to indicate overlap of up-regulated genes. c Venn diagrams to indicate overlap of down-regulated genes. The Venn diagrams showed evidence that severe and moderate infection share common characteristics, but also have a distinctive gene expression profile

Fig. 3

Top 10 pathways ranked by statistical significance. Top 10 pathways ranked by p-values (vertical bars) in three conditions. Vertical axis denotes statistical significance as measured by minus logarithm of p-values. Blue bars are downregulated pathways; red bars are upregulated pathways. a Moderate influenza patients compared to healthy controls. Upregulated pathways correspond to activation of interferon and neutrophil themes. Downregulated pathway corresponds to the immune response theme. b Severe influenza patients compared to healthy controls. Upregulated pathways correspond to activation of interferon and neutrophil themes. Downregulated pathway corresponds to the immune response theme. c Severe influenza patients compared to moderate influenza patients. Upregulated pathways correspond to activation of neutrophil and cell cycle (delayed apoptosis) themes. Downregulated pathway corresponds to the immune response theme

Fig. 4

Histogram of neutrophil-related significant changes in gene expression between severe influenza, moderate influenza illness and healthy controls. Y-axis shows normalised log₂ expression levels. * indicate p < 0.001, adjusted for multiple testing by Bonferroni method. ns denotes non-significant. HC denotes healthy control. a Genes encoding proteins involved in neutrophil extracellular trap formation. Expression differences are shown for (the strongest regulated) probesets of the individual gene. b Genes encoding proteins involved in neutrophil migration. Expression differences are shown for (the strongest regulated) probesets of the individual gene. c Genes encoding components of neutrophil granules. Expression differences are shown for (the strongest regulated) probesets of the individual gene. Neutrophils-related genes were upregulated in patients with severe influenza illness compared to moderate influenza illness and healthy control subjects

Fig. 5

Histogram of MHC class II significant changes in gene expression between severe influenza compared to moderate influenza illness and healthy controls. Expression differences are shown for (the strongest regulated) probesets of the individual gene. Y-axis shows normalised log₂ expression levels. * indicate p < 0.001, adjusted for multiple testing by Bonferroni method. ns denotes non-significant. HC denotes healthy control MHC class II were downregulated in patients with severe influenza illness compared to moderate influenza illness and healthy control subjects

Fig. 6

Radar chart: gene expression profile in moderate and severe influenza illness. Most representative differentially expressed probesets of the individual gene in the top three principal biological themes were computed in a radar chart. Level of expression was normalized to healthy controls. Expression differences are shown for (the strongest regulated) probesets of the individual gene. In moderate infection, the radar chart showed an upregulation of the interferon and neutrophil genes. In severe infection, besides an upregulation of the interferon and neutrophil genes, we observed a down regulation of the MHC class II genes