Association of Dynamic Changes in the CD4 T-Cell Transcriptome With Disease Severity During Primary Respiratory Syncytial Virus Infection in Young Infants

Abstract

Background: Nearly all children are infected with respiratory syncytial virus (RSV) within the first 2 years of life, with a minority developing severe disease (1%-3% hospitalized). We hypothesized that an assessment of the adaptive immune system, using CD4+ T-lymphocyte transcriptomics, would identify gene expression correlates of disease severity.

Methods: Infants infected with RSV representing extremes of clinical severity were studied. Mild illness (n = 23) was defined as a respiratory rate (RR) < 55 and room air oxygen saturation (SaO2) ≥ 97%, and severe illness (n = 23) was defined as RR ≥ 65 and SaO2 ≤ 92%. RNA from fresh, sort-purified CD4+ T cells was assessed by RNA sequencing.

Results: Gestational age, age at illness onset, exposure to environmental tobacco smoke, bacterial colonization, and breastfeeding were associated (adjusted P < .05) with disease severity. RNA sequencing analysis reliably measured approximately 60% of the genome. Severity of RSV illness had the greatest effect size upon CD4 T-cell gene expression. Pathway analysis identified correlates of severity, including JAK/STAT, prolactin, and interleukin 9 signaling. We also identified genes and pathways associated with timing of symptoms and RSV group (A/B).

Conclusions: These data suggest fundamental changes in adaptive immune cell phenotypes may be associated with RSV clinical severity.

Keywords: RNA sequencing; T cell; disease severity; gene espression; respiratory syncytial virus.

Figure 1.

Sequencing statistics of respiratory syncytial virus–infected CD4 samples. A, Number of total raw reads. B, Genome mapping rate of overall samples. C, Gene detection rate of 51 samples.

Figure 2.

Heat map and pathway analysis of CD4 T cell expression. A, Gene expression associated with clinical severity. Shown are normalized expression levels for the 140 genes selected by multivariate analysis; rows represent genes, and columns represent samples. Red indicates higher expression; green indicates low/no expression. Samples are grouped by phenotype (mild, severe) and by time when sample was obtained (acute, convalescent). B, Pathways associated with severe phenotype. Ingenuity pathway analysis (IPA) was used to identify canonical pathways represented by genes associated with severity in CD4 lymphocytes from respiratory syncytial virus–infected subjects. The variables used to generate gene sets for IPA were multivariate severity phenotype (a; n = 140) and univariate severity phenotype (b; n = 551). Thirteen pathways are shown where Fisher’s exact test P values were <.05 for at least 1 variable. Orange and blue circles indicate predicted increased or decreased pathway activation (activation z score), respectively. Genes included in each pathway are listed and are colored red if increased in severe subjects or green if decreased in severe subjects. Genes with >1.5-fold increases are bold.

Figure 3.

Canonical pathways and regulators associated with time since onset of RSV infection. A, Pathways associated with stage of infection. Ingenuity pathway analysis (IPA) was used to identify canonical pathways represented by genes associated with multivariate modeling of time since onset of clinical symptoms (a; acute vs convalescent; n = 63) or univariate analysis of time since onset of clinical symptoms (b; acute vs convalescent; n = 35). Eight pathways are shown where Fisher’s exact test P values were <.05 for at least 1 analysis. Orange and blue circles indicate predicted increased or decreased pathway activation (activation z score), respectively. Genes included in each pathway are listed and are colored red if increased in acute stage or green if decreased in acute stage and in bold for genes with >1.5-fold changes in expression. B, Regulators associated with stage of infection. Ingenuity pathway analysis was used to identify putative regulators represented by genes associated with multivariate modeling of time since onset of clinical symptoms (a; acute vs convalescent; blue bars) or univariate analysis of time since onset of illness (b; acute vs convalescent; red bars). Twenty-four upstream regulators are shown where P values were <.05 for at least 1 gene list. Orange and blue circles indicate a predicted activation/inhibition state based upon the expression of targets. Upstream regulator targets are listed in red if upregulated and green if downregulated in acute stage, and in bold for genes with >1.5-fold changes in expression.

Figure 4.

Pathways and regulators associated with RSV group A and B. A, Pathways associated with respiratory syncytial virus (RSV) group infection. Ingenuity pathway analysis (IPA) was used to identify canonical pathways represented by genes associated with infection by RSV group (A/B) in CD4 lymphocytes. The variables used to generate gene sets for IPA were derived from multivariate analyses (a; n = 68) or univariate analysis (b; n = 53). Ten pathways are shown where Fisher’s exact test P values were <.05 for at least 1 gene list. Genes included in each pathway are listed and are colored red if increased in RSV strain A or green if decreased in strain A and in bold for genes with >1.5-fold changes in expression. B, Regulators associated with RSV group infection. Ingenuity pathway analysis was used to identify upstream regulators associated with infection by RSV group (A/B) using gene sets identified with multivariate (blue; n = 68 genes) or univariate (red; n = 53 genes) analysis. Nine upstream regulators are shown where P values were <.05 for at least 1 gene list. Upstraem regulator targets are listed in red if upregulated and green if downregulated in RSV A strain–infected subjects.

Figure 5.

Quantitative polymerase chain reaction validation (qPCR). A, We validated expression of SOCS2, which is associated with illness severity. Left, Gene expression by RNA sequencing (RNA-seq). Right, Gene expression by qPCR. Gene expression is plotted relative to severity. B, We validated expression of OAS1 and RSAD2, which are associated with stage of infection. Left, Gene expression by RNA-seq. Right, Gene expression by qPCR. Gene expression is plotted relative to the day of onset of clinical symptoms, separately for subjects with mild or severe phenotypes. P value indicates significance level for association with stage. Abbreviations: qPCR, quantiative polymerase chain reaction; RNA-seq, RNA sequencing.