Neutrophilic inflammation in the respiratory mucosa predisposes to RSV infection

Abstract

The variable outcome of viral exposure is only partially explained by known factors. We administered respiratory syncytial virus (RSV) to 58 volunteers, of whom 57% became infected. Mucosal neutrophil activation before exposure was highly predictive of symptomatic RSV disease. This was associated with a rapid, presymptomatic decline in mucosal interleukin-17A (IL-17A) and other mediators. Conversely, those who resisted infection showed presymptomatic activation of IL-17- and tumor necrosis factor-related pathways. Vulnerability to infection was not associated with baseline microbiome but was reproduced in mice by preinfection chemokine-driven airway recruitment of neutrophils, which caused enhanced disease mediated by pulmonary CD8⁺ T cell infiltration. Thus, mucosal neutrophilic inflammation at the time of RSV exposure enhances susceptibility, revealing dynamic, time-dependent local immune responses before symptom onset and explaining the as-yet unpredictable outcomes of pathogen exposure.

Fig. 1. Kinetics of viral replication and symptoms correlate closely, with no viral replication or symptoms evident until 3 dpi.

(A) Participants were inoculated with RSV at day 0. Nasal wash and nasosorption samples were taken daily during quarantine (up to 10 dpi) and at 14 dpi. Nasal curettage was performed at baseline (7 to 14 days before inoculation) and at 3 dpi. (B) Daily viral load, measured by qPCR of nasal wash, in volunteers who developed symptomatic RSV infections (Cold, n = 23). (C) Daily upper respiratory tract (URT) symptom scores in the Cold (red, n = 23) and No Cold (blue, n = 25) groups. Data in (B) and (C) are shown as the means ± SEM.

Fig. 2. Preexposure airway neutrophil activation is associated with susceptibility to RSV infection.

(A) DEGs (P_adj < 0.05; n = 80) were determined in baseline (7 to 14 days before inoculation) nasal curettage samples between Cold and No Cold groups by RNA-seq. (B) GO molecular functions enrichment analysis of DEGs (P_adj < 0.1, n = 235). (C) GO biological processes enrichment analysis of DEGs (P_adj < 0.1, n = 235). (D) WGCNA cluster of DEGs (P_adj < 0.1, n = 235) enriched for those with annotations of neutrophil association. (E) Day 0 levels of MPO, LCN-2, and IL-17A in nasosorption samples between the No Cold (n = 10-25) and Cold (n = 9-23) groups. DEGs in (A) to (D) were determined by Wald tests using DESeq2. Data in (E) are shown as median and interquartiles with minimum and maximum values and were analyzed by Mann-Whitney U tests. *P < 0.05.

Fig. 3. Early presymptomatic viral clearance is associated with activation of IL-17 signaling.

DEGs were determined by RNA-seq of nasal curettage samples at 3 dpi relative to baseline (7 to 14 days before inoculation) in the No Cold (n = 16) and Cold (n = 16) groups. Heatmaps show (A) DEGs in the No Cold group (n = 87) and (B) DEGs in the Cold group (n = 77). (C) Venn diagram of the number of overlapping DEGs in the Cold and No Cold groups. DEGs from the No Cold group were analyzed for enrichment of (D) GO molecular functions and (E) KEGG pathways. DEGs were defined as transcripts with P_adj < 0.01 and log² fold change >0.5.

Fig. 4. Network analysis reveals a module of correlated DEGs enriched for IL-17 signaling in presymptomatic individuals.

WGCNA of the differentially expressed genes (n = 87) was performed in the No Cold group at 3 dpi relative to baseline. (A) Classification of WGCNA modules. Brown module: (B) network map and (C) molecular function enrichment. Turquoise module: (D) network map and (E) molecular function enrichment. Blue module: (F) network map and (G) molecular function enrichment. DEGs were defined as transcripts with P < 0.01 and log² fold change >0.5.

Fig. 5. Early presymptomatic secretion of intranasal IL-1β, IL-6, and IL-17A is associated with protection from RSV infection.

(A to C) Soluble protein cytokine and chemokine mediator levels from nasosorption samples were determined on each study day and expressed as LOESS plots between Cold (red) and No Cold (blue) groups of log² fold-change levels normalized to day 0. The 95% confidence intervals of LOESS curves are denoted by shaded areas. (D) Logistic regression analysis of mediator responses during 1 to 3 dpi measured at the protein level from nasosorption samples.

Fig. 6. Mice treated with the neutrophil chemoattractant CXCL1 before RSV infection show an early increase in viral load and develop more severe disease.

Mice were treated with mock (PBS) or 10 μg of CXCL1 intranasally. (A) At 12 hpi, BAL and lung neutrophils were quantified by flow cytometry and (B) MMP-9, MPO, and NE levels in the BAL were quantified by ELISA. Mice were treated with mock (PBS) or 5 to 10 μg of CXCL1 intranasally and after 9 to 12 hours were infected with mock (PBS) or 7.5 × 10⁵ FFUs of RSV intranasally. (C) Weight loss as a percentage of original weight. (D) IFN-α and IL-6 quantified in the BAL using ELISA. Levels of (E) Il1b and Tnfa, (F) Cxcl2 and Cxcl10, and (G) Ccl2 were determined in lung tissue using qPCR. (H) Total number of lung monocytes quantified by flow cytometry as previously described (32). (I) RSV L gene copy numbers were quantified at 0.75, 4, and 8 dpi in lung tissue by qPCR. Data in (A) and (B) are shown as the means ± SEM of eight individual mice per group pooled from two independent experiments. Data in (C) are shown as the means ± SEM of 10 (PBS/PBS) or 14 to 16 (RSV) individual mice pooled from two (PBS/PBS) or three (RSV) independent experiments. Data in (D) to (H) are shown as the means ± SEM of 10 individual mice per group pooled from two independent experiments. Data in (I) are shown as the means ± SEM of five individual mice per group representative of at least two independent experiments, with the dotted line representing the limit of detection of the assay. The statistical significance of differences in (A) and (B) was analyzed using unpaired, two-tailed Student’s t test. The data in (C) and (I) were analyzed using two-way ANOVA with Bonferroni’s post hoc test, and only the statistically significant differences between RSV-infected groups are shown. The data in (D) to (H) were analyzed using one-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7. Mice treated with the neutrophil chemoattractant CXCL1 before RSV infection develop CD8⁺ T cell–driven disease.

(A) Correlation between CD8⁺ T cell numbers in the subepithelial layer of endobronchial biopsy samples from human participants infected with RSV at 7 dpi and cumulative symptom scores over the course of infection (n = 11). (B to H) Mice were treated with mock (PBS) or 10 μg of CXCL1 intranasally 12 hours before RSV infection. Lungs were analyzed at 8 dpi. (B) Total number of lung cells, (C) lung CD4⁺ T cells, and (D) lung CD8⁺ T cells assessed by flow cytometry. (E) Representative flow cytometry plots of lung RSV tetramer–positive CD8⁺ T cells; the mean percentage ± SEM is indicated in the upper right quadrant. (F) Total number of lung RSV tetramer–positive CD8⁺ T cells as quantified by flow cytometry. (G) Representative flow cytometry plots of lung granzyme B–positive (GzmB⁺) and IFN-γ⁺ CD8⁺ T cells. (H) Frequency of GzmB⁺ and IFN-γ⁺ CD8⁺ T cells quantified by flow cytometry. (I) Anti-Ly6G or isotype control–treated mice were administered PBS or 10 μg of CXCL1 intranasally on day 0 and at 12 hours after being infected with mock (PBS) or 6 to 7.5 × 10⁵ FFUs of RSV intranasally. Weight loss is shown as the percentage of original weight. (J) Mice treated with anti-CD8 or isotype control antibodies were administered PBS or 10 μg of CXCL1 intranasally on day 0 and at 12 hours after being infected with mock (PBS) or 6 to 7.5 × 10⁵ FFUs of RSV intranasally. Weight loss is shown as the percentage of original weight. Data in (B) to (D), (F), and (H) are shown as the means ± SEM of 10 (PBS/PBS, PBS/RSV) or nine (CXCL1/RSV) mice pooled from two independent experiments; repeat 1, 10 μg of CXCL1 (circles) and repeat 2, 8 μg of CXCL1 (squares). Statistical significance of differences in (A) were analyzed using Spearman’s rank correlation test, where the R score is given as R_S. Data in (B) to (D), (F), and (H) were analyzed using one-way ANOVA with Tukey’s post hoc test. Data in (I) and (J) were analyzed using two-way ANOVA with Bonferroni’s post hoc test. Asterisks indicate statistically significant differences between RSV-infected groups. Hash symbol indicates statistically significant differences between PBS/PBS and CXCL1/PBS groups. *#P < 0.05, **P < 0.01, ***P < 0.001.