Upper respiratory tract bacterial-immune interactions during respiratory syncytial virus infection in infancy

Abstract

Background: The risk factors determining short- and long-term morbidity following acute respiratory infection (ARI) due to respiratory syncytial virus (RSV) in infancy remain poorly understood.

Objectives: Our aim was to examine the associations of the upper respiratory tract (URT) microbiome during RSV ARI in infancy with the acute local immune response and short- and long-term clinical outcomes.

Methods: We characterized the URT microbiome by 16S ribosomal RNA sequencing and assessed the acute local immune response by measuring 53 immune mediators with high-throughput immunoassays in 357 RSV-infected infants. Our short- and long-term clinical outcomes included several markers of disease severity and the number of wheezing episodes in the fourth year of life, respectively.

Results: We found several specific URT bacterial-immune mediator associations. In addition, the Shannon ⍺-diversity index of the URT microbiome was associated with a higher respiratory severity score (β =.50 [95% CI = 0.13-0.86]), greater odds of a lower ARI (odds ratio = 1.63 [95% CI = 1.10-2.43]), and higher number of wheezing episodes in the fourth year of life (β = 0.89 [95% CI = 0.37-1.40]). The Jaccard β-diversity index of the URT microbiome differed by level of care required (P = .04). Furthermore, we found an interaction between the Shannon ⍺-diversity index of the URT microbiome and the first principal component of the acute local immune response on the respiratory severity score (P = .048).

Conclusions: The URT microbiome during RSV ARI in infancy is associated with the acute local immune response, disease severity, and number of wheezing episodes in the fourth year of life. Our results also suggest complex URT bacterial-immune interactions that can affect the severity of the RSV ARI.

Keywords: Airway; bronchiolitis; chemokines; cytokines; growth factors; immune response; infancy; mediation; microbiome; nasopharynx; respiratory syncytial virus; severity; wheezing.

Figure 1.

Flow chart diagram of infants with RSV ARI included in the study. The total sample size at each step of the flow chart diagram (n) is shown. *For infants with multiple RSV ARIs, only data from the first RSV ARI available were included in statistical analyses. Definition of abbreviations: ARI = Acute respiratory infection, INSPIRE = Infant Susceptibility to Pulmonary Infections and Asthma Following RSV Exposure Study, rRNA = Ribosomal ribonucleic acid, RSV = Respiratory syncytial virus, URT = Upper respiratory tract.

Figure 2.

Graphical representation of the study design. First, we examined the total effect of the URT microbiome during RSV ARI in infancy on the acute local immune response (path X→M) and on short- and long-term clinical outcomes (all paths X→Y). Based on our results and those of our prior studies showing an effect of the acute local immune on short- and long-term clinical outcomes (path M→Y), we the used a causal mediation framework to assess whether the effect of the URT microbiome during RSV ARI on short- and long-term clinical outcomes (all paths X→Y) could be explained —at least in part— by the acute local immune response (path X→M→Y [indirect effect]). Our causal mediation models also took into account the interaction between the exposure and the mediator (X and M, respectively). Definition of abbreviations: ARI = Acute respiratory infection, RSV = Respiratory syncytial virus, URT = Upper respiratory tract.

Figure 3:

The association of upper respiratory tract microbiome genera during an acute respiratory infection due to respiratory syncytial virus in infancy with the acute local immune response. To identify relevant associations, we used a variable selection method. For each immune mediator, we built a separate model including its median fluorescence intensity as the dependent variable and the log-transformed relative abundances of the top 30 most abundant genera as the independent variables. Every model also included infant- and respiratory syncytial virus-related (see main text for more details). Only genera that were selected for ≥1 immune mediator and immune mediators for which ≥1 genera were selected are shown. The color of each cell represents the regression coefficient from the models (blue = negative association, red = positive association, dark grey = no association). The heatmap columns (genera) and rows (immune mediators) were hierarchically clustered. The corresponding phyla are also shown.

Figure 4:

The association of the α-diversity of the upper respiratory tract microbiome during RSV ARI in infancy with short- and long-term clinical outcomes, including the respiratory severity score (A), type of ARI (B), level of care required (C), and number of wheezing episodes in the 4^th year of life (D). The box-and-whisker plots show the mean (diamond), median (middle bar), 1^st quartile (lower bar), 3^rd quartile (upper bar), minimum observation above the lowest fence (lower whisker), and maximum observation below the upper fence (upper whisker) of the Shannon index for each outcome. The total sample size for each outcome (n) and the estimates from corresponding linear or logistic regression models (regression coefficient [β] or OR, 95% CI, and p-value) are also shown, as appropriate. Every model also included infant- and RSV-related covariates (see main text for more details). The Benjamini-Hochberg procedure was used to control for multiple comparisons by adjusting the p-values for the number of clinical outcomes assessed (i.e., controlling for a total of 4 tests). Definition of abbreviations: ARI = Acute respiratory infection, RSV = Respiratory syncytial virus.

Figure 5:

The association of the β-diversity of the URT microbiome during an acute respiratory infection due to respiratory syncytial virus in infancy with level of care required. The scatter plots show each infant’s microbial community composition (small circles) by level of care required, as well as each group’s centroid (large circles) and 95% CI ellipses. The scatter plots were generated using NMDS ordination based on Bray-Curtis (A) or Jaccard (B) indices. For ease of visualization, only 2 dimensions were used; however, the stress values of ≥0.2 suggest that a higher number of dimensions may be needed to accurately ordinate and represent the multidimensional data. The p-values for the comparison between groups from PERMANOVA tests are also shown. Every model also included infant- and respiratory syncytial virus-related covariates (see main text for more details). For each β-diversity index, the Benjamini-Hochberg procedure was used to control for multiple comparisons by adjusting the p-values for the number of clinical outcomes assessed (i.e., controlling for a total of 4 tests). Definition of abbreviations: NMDS = Non-metric multidimensional scaling.

Figure 6.

The modification of the effect of the acute local immune response on the respiratory severity score by the α-diversity of the upper respiratory tract microbiome during an acute respiratory infection due to respiratory syncytial virus in infancy. The box-and-whisker plots show the mean (diamond), median (middle bar), 1^st quartile (lower bar), 3^rd quartile (upper bar), minimum observation above the lowest fence (lower whisker), and maximum observation below the upper fence (upper whisker) of the first PC of the acute local immune response data for each respiratory severity score value and by Shannon index level (defined as high vs. low based on being above or below the median). The fitted regression lines from corresponding linear regression models are also shown. The first PC of the acute local immune response was negatively correlated with all individual immune mediators. Definition of abbreviations: PC = Principal component.