Cystic Fibrosis Mice Are Highly Susceptible to Repeated Acute Pseudomonas aeruginosa Pneumonia after Intranasal Inoculation

Abstract

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that controls chloride current. A number of different CFTR transgenic mouse lines have been developed and subjected to both acute and chronic infection models. However, prior studies showed no substantial differences in bacterial clearance between CF and non-CF mice after single inoculations. Here, using F508del transgenic CF mice, we examined the role of repeated acute Pseudomonas aeruginosa (PA) infection, with the second inoculation 7 days after the first. We found that CF mice were more susceptible to PA infection than non-CF mice following the second inoculation, with non-CF mice showing better neutrophil recruitment and effector functions. We further investigated the characteristics of lung immune cells using single-cell RNA sequencing, finding that non-CF lung neutrophils had more prominent upregulation of adhesion molecules including intercellular adhesion molecule-1 (ICAM-1) compared to CF lung neutrophils. Although people with CF are often colonized with bacteria and have high numbers of neutrophils in the airways during chronic infection, these data suggest that CF neutrophils have deficient effector functions in the setting of repeated acute infection.

Figure 1

Lung bacterial loads following Pseudomonas aeruginosa intranasal instillation. (a) CF and non-CF mice were anesthetized and subjected to intranasal instillation of Pseudomonas aeruginosa (PA) strain PAK (2.5 × 10⁶ CFU). At six hours after the instillation, mice were euthanized, and the lungs were removed to assess lung PA loads. Data are shown as mean ± S.D. (n = 6 per group) in log₁₀ scale. Student t test was performed on log₁₀-transformed CFU values. ns = not significant. (b) CF and non-CF mice were subjected to intranasal instillation of PA strain PAK (2.5 × 10⁶ CFU) with seven days apart. At six hours after the second instillation, mice were euthanized, and the lungs were removed to assess lung PA loads. Data are shown as mean ± S.D. (n = 6 per group) in log₁₀ scale. Student t test was performed on log₁₀ transformed CFU values. ^∗p < 0.05.

Figure 2

Lung leukocyte counts in CF and non-CF mice. Lung leukocyte counts were examined at baseline, at 4 hours after the one-time instillation, at 6 days after the instillation, and at 4 hours after the second instillation of Pseudomonas aeruginosa (PA) in CF and non-CF mice using flow cytometry analysis. (a) Neutrophils, (b) macrophages, (c) NK cells, (d) B cells, (e) CD4 T cells, and (f) CD8 T cells were probed using Ly6G, F4/80, NK1.1, B220, CD4, and CD8 antibodies, respectively. Data are shown as mean ± S.D. (n = 3 per arm). One-way ANOVA with the Bonferroni post hoc analysis was performed. ^∗p < 0.05.

Figure 3

Histological analysis of lungs in CF and non-CF mice. H&E-stained sections from (a) naïve CF lung, (b) naïve non-CF lung, (c) CF lung post one-time instillation (PAx1, at 4 hours after the instillation), (d) non-CF lung post one-time instillation (PAx1, at 4 hours after the instillation), (e) CF lung post two-time instillation (PAx2, at 4 hours after the second instillation), and (f) non-CF lung post two-time instillation (PAx2, at 4 hours after the second instillation). Black bar represents 100 μm, and brown bar represents 20 μm. More leukocyte infiltration was observed in non-CF lung after the second instillation (f).

Figure 4

Lung cytokine transcript profiles in CF and non-CF mice. We performed real-time qPCR analysis of a set of cytokine transcripts of the lungs from CF and non-CF mice at baseline, at 4 hours after the one-time instillation, at 6 days after the one-time, and at 4 hours after the second instillation of PA. Data are shown as mean ± S.D. (n = 4). Statistical analysis was done using one-way ANOVA with Bonferroni post hoc analysis. ^∗∗p < 0.01.

Figure 5

Neutrophil effector functions in CF and non-CF mice. Reactive oxygen species (ROS) formation and phagocytosis were examined as neutrophil effector functions in (a) lung neutrophils post one-time PA instillation (PAx1), (b) lung neutrophils post two-time instillation (PAx2), and (c) blood naïve neutrophils. Data are presented as mean ± S.D. (n = 3 per group). One-way ANOVA with Bonferroni post hoc analysis was performed to examine statistical significance. ns = not significant. ^∗p < 0.05. (d) The role of blood in bacterial killing was examined by coincubating 100 μL blood and 1 × 10⁹ CFU PA for four hours. Data are shown as mean ± S.D. (n = 6 per group) in log scale. Student t test was performed on log₁₀-transformed CFU values. ns = not significant.

Figure 6

Single-cell RNA sequencing analysis of lung cells and neutrophil DEG/ontology. (a) Naïve CF and non-CF lung cells and CF and non-CF lung cells post two-time instillation were subjected to single-cell RNA sequencing analysis. Annotation results are shown. (b) Heatmap of lung neutrophil differentially expressed genes (DEGs). (c) The number of up- and downregulated DEGs. (d) Upregulated DEGs in CF and non-CF lung neutrophils were subjected to KEGG pathway analysis and presented as bubble enrichment maps. (e) Downregulated DEGs in CF and non-CF lung neutrophils were subjected to KEGG pathway analysis and presented as bubble enrichment maps. (f) Adhesion molecule expression pattern was shown on the heatmap. (g) tlr4 expression level was shown in violin plots.

Figure 7

Single-cell RNA sequencing analysis of lung macrophage cells. (a) Heatmap of lung macrophage differentially expressed genes (DEGs). (b) The number of up- and downregulated DEGs. (c) Upregulated DEGs in CF and non-CF lung macrophages were subjected to KEGG pathway analysis and presented as bubble enrichment maps. (d) Downregulated DEGs in CF and non-CF lung macrophages were subjected to KEGG pathway analysis and presented as bubble enrichment maps.