Inflammation induced by influenza virus impairs human innate immune control of pneumococcus

Abstract

Colonization of the upper respiratory tract by pneumococcus is important both as a determinant of disease and for transmission into the population. The immunological mechanisms that contain pneumococcus during colonization are well studied in mice but remain unclear in humans. Loss of this control of pneumococcus following infection with influenza virus is associated with secondary bacterial pneumonia. We used a human challenge model with type 6B pneumococcus to show that acquisition of pneumococcus induced early degranulation of resident neutrophils and recruitment of monocytes to the nose. Monocyte function was associated with the clearance of pneumococcus. Prior nasal infection with live attenuated influenza virus induced inflammation, impaired innate immune function and altered genome-wide nasal gene responses to the carriage of pneumococcus. Levels of the cytokine CXCL10, promoted by viral infection, at the time pneumococcus was encountered were positively associated with bacterial load.

Fig. 1. LAIV-pneumococcus co-infection leads to excessive proinflammatory responses that are associated with increased pneumococcal load and impaired monocyte recruitment.

a, Experimental design of the study. Analyzed time points are indicated by black circles. b, Heat map showing for each cytokine the median log₂[fold change] compared with baseline for the time points 0, 2,7 and 9 days; n = 19 per group. c, The delta in median log₂[fold change] following LAIV vaccination just prior to inoculation with S. pneumoniae for subjects becoming carriage⁺ or carriage⁻ (excluding subjects becoming positive by PCR only, who resemble subjects that become carriage⁺ by culture as well). The color of each bar represents the median induction in the entire LAIV group. **P = 0.0097 by two-tailed Wilcoxon test for LAIV carriage⁻ subjects comparing IL-10 day 0 with baseline; P = 0.073 for the LAIV carriage⁺ group. ***P = 0.0008 by two-tailed Wilcoxon test for LAIV carriage+ subjects comparing CXCL10 day 0 with baseline; P = 0.051 for the LAIV carriage⁻ group. d, Pneumococcal load (median and interquartile range of CFU ml⁻¹ in nasal wash shown) for all carriage⁺ subjects with high (top quartile, n = 9) or low (all subjects below top quartile, n = 28) CXCL10 concentrations at day 0. P = 0.019 by two-tailed Mann-Whitney test of area under the curve of log-transformed load over time. e, Scatter plot showing correlation of CXCL10 concentration at baseline with S. pneumoniae load for a second validation cohort (n = 52) with an asymptomatic upper respiratory tract virus infection (n = 15) or not. Spearman correlation test results and linear regression line with 95% confidence interval (gray shading) are shown.

Fig. 2. Neutrophil function is impaired following LAIV administration.

a, Concentrations of myeloperoxidase in nasal wash of volunteers before or 2 days after S. pneumoniae inoculation. Median and interquartile range are shown (for n = 9 LAIV carriage⁻ and LAIV carriage⁺ and for n = 10 control carriage⁻ and control carriage⁺ subjects). *P = 0.014 by two-tailed Wilcoxon paired test. b, S. pneumoniae OPK capacity of blood neutrophils before and 3 days following LAIV (n = 6) or control (tetravalent inactivated influenza vaccine or no, n = 7) vaccination. Individual subjects are shown and connected by lines. *P = 0.031 by two-tailed Wilcoxon paired test. NS, not significant. c,d, Effect of exogenous TNF (c; n = 10) and CXCL10 (d; n = 8) on OPK activity of blood neutrophils of healthy volunteers. **P = 1.15 × 10⁻⁵ by Friedman test. Neutrophils from six subjects were used in three independent experiments. Individual samples are depicted and connected by dashed lines. e, Normalized MAP4K2 and TIGIT counts on sorted neutrophils before LAIV or in control arm (n = 6, red) and following LAIV (n = 4, blue). Individual samples are shown, and paired samples are connected by black lines. **P = 0.008 and ***P = 3.2 × 10⁻⁵ two-tailed unadjusted P values using a negative binomial generalized linear model (DESeq2). f, Correlation between OPK activity and TIGIT counts (n = 10). Spearman rho (r) and P value are shown. Regression line and 95% confidence intervals (shaded area) are shown. g, Levels of TIGIT on blood neutrophil surface measured by flow cytometry after a 30-min incubation without or with 1 ng ml⁻¹ TNF or 100 ng ml⁻¹ TNF (n = 4). *P = 0.042 by Friedman test. Individual subjects are depicted by dots and connected by lines.

Fig. 3. Monocyte recruitment following pneumococcal colonization is impaired during LAIV co-infection.

a, Median and interquartile range of nasal monocyte numbers normalized to epithelial cell numbers are shown for control carriage⁺ (n = 24), control carriage⁻ (n = 37), LAIV carriage⁺ (n = 25) and LAIV carriage⁻ (n = 30) groups. The dashed green line shows the baseline level in the control carriage⁺ group. *P = 0.038 at day 2 and P = 0.030 at day 29; **P = 0.002 by two-tailed Wilcoxon paired non-parametric test. b,c, Levels of maximum pneumococcal (S. pneumoniae) load in nasal wash are shown for the control group (b; n = 22) and LAIV group (c; n = 23) and are correlated with the maximum monocyte recruitment (fold change to baseline). Individual subjects are shown, and Spearman correlation analysis is shown.

Fig. 4. Pneumococcus-specific responses are induced following colonization, which is impaired by LAIV co-infection.

a, Whole nasal cells were collected from 48 subjects 28 days post-inoculation and stimulated for 18 h with heat-killed S. pneumoniae. Supernatant was collected, and concentrations of 30 cytokines were measured by multiplex enzyme-linked immunosorbent assay. The median and interquartile range for cytokines induced at least twofold in at least one condition are displayed. b, Correlations between cytokine production following S. pneumoniae stimulation and pneumococcal load are shown (n = 22). Spearman non-parametric correlation test results and regression lines with shaded 95% confidence intervals are shown per cytokine. c, The cytokine profile from alveolar macrophages (median for six volunteers shown) exposed to S. pneumoniae for 18 h was compared with that of stimulated whole nasal cells (median of control carriage⁺ group shown). Spearman non-parametric correlation test results and regression lines with shaded 95% confidence intervals are shown.

Fig. 5. Nasal transcriptomics following LAIV–S. pneumoniae co-infection (n = 35).

a, The number of differentially expressed genes (DEGs) between each time point and the baseline for each group are shown. Upregulated and downregulated genes are depicted in red and blue, respectively. Connections between bars show the number of common genes between LAIV and control conditions, where colors reflect distinct pathways. b, Circular representation of DEGs and gene set enrichment analysis for LAIV carriage⁺ and control carriage+ groups at days 2 and 9 after S. pneumoniae inoculation. The individual log₂[fold change] values (baseline-normalized) were used as ranks in a single sample gene set enrichment analysis to identify consistently enriched pathways among subjects. Genes and pathways are connected by lines.

Fig. 6. CEMiTool applied to control cohort: module M1.

Raw counts were normalized using log[CPM], and log₂[fold change] values were calculated for each time point against the baseline after which coexpression modules were extracted. a, Gene set enrichment analyses showing the module activity at each time point for carriage⁺ and carriage⁻ groups. Symbol size and color reflect normalized enrichment score (NES). b, Correlation with average fold change counts of all M1 genes at day 9 with paired numbers of monocytes from the volunteer’s other nostril. Individual subjects are shown, along with regression line with 95% confidence interval and Spearman correlation analysis (n = 13). c, The genes of M1 present with genes highly expressed in CD14⁺CD16⁻ (578 genes), CD14⁺CD16⁺ (108 genes) and CD14⁻CD16⁺ (162 genes) , showing the overlapping number of genes between M1 and monocyte subsets in parentheses. The overlap for significance was analyzed using the χ² test. d, Over-representation analysis of module M1 using gene sets from the Reactome Pathway database. e, Interaction plot for M1, with gene nodes highlighted.

Fig. 7. CEMiTool applied to control group: module M3.

Raw counts were normalized using log[CPM], and log₂[fold change] values were calculated for each time point against the baseline after which coexpression modules were extracted. a, Over-representation analysis of module M3 of the control group using gene sets from the Reactome Pathway database. b, Interaction plot for M3, with gene nodes highlighted.

Fig. 8. CEMiTool applied to LAIV.

Raw counts were normalized using log[CPM], and log₂[fold change] values were calculated for each time point against the baseline after which coexpression modules were extracted. a, Gene set enrichment analyses showing the module activity at each time point for carriage⁺ and carriage⁻ LAIV groups. b, Over-representation analysis of module M5 of the LAIV group using gene sets from the Reactome Pathway database. c, Interaction plot for M5, with gene nodes highlighted.