Increased susceptibility of airway epithelial cells from ataxia-telangiectasia to S. pneumoniae infection due to oxidative damage and impaired innate immunity

Abstract

Respiratory disease is a major cause of morbidity and mortality in patients with ataxia-telangiectasia (A-T) who are prone to recurrent sinopulmonary infections, bronchiectasis, pulmonary fibrosis, and pulmonary failure. Upper airway infections are common in patients and S. pneumoniae is associated with these infections. We demonstrate here that the upper airway microbiome in patients with A-T is different from that to healthy controls, with S. pneumoniae detected largely in patients only. Patient-specific airway epithelial cells and differentiated air-liquid interface cultures derived from these were hypersensitive to infection which was at least in part due to oxidative damage since it was partially reversed by catalase. We also observed increased levels of the pro-inflammatory cytokines IL-8 and TNF-α (inflammasome-independent) and a decreased level of the inflammasome-dependent cytokine IL-β in patient cells. Further investigation revealed that the ASC-Caspase 1 signalling pathway was defective in A-T airway epithelial cells. These data suggest that the heightened susceptibility of these cells to S. pneumoniae infection is due to both increased oxidative damage and a defect in inflammasome activation, and has implications for lung disease in these patients.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Difference in microbial profile of upper respiratory tracts between healthy controls and patients with A-T. 16S rDNA sequencing from nasal swabs obtained from patients with A-T (n = 10) and healthy controls (n = 10). (A) Distribution of the 20 most abundant bacterial families found in A-T and controls. (B) Rank test plot showed no significant differences (Anova p = 0.14) at this level in the Streptococceae family between A-T and controls. (C) PCR analysis revealed the presence of S. pneumoniae in all A-T samples. RFU; relative fluorescence units. (D) Multivariate analysis using canonical correlation analysis (CCA) showed distinct clustering of microbial populations between patients with A-T and healthy controls (p = 0.034).

Figure 2

Increased sensitivity in A-T airway epithelial cells to S. pneumoniae infection. (A) Percentage of cell death induced by S. pneumoniae infection in control and A-T cells in the presence or absence of catalase (cat). Unpaired, two-tailed Mann-Whitney tests were carried out. *p < 0.03 between control and A-T at 8 h, and A-T and A-T + cat at 8 h. (B,C) Increase in inflammasome-independent pro-inflammatory cytokines IL-8 and TNF-α respectively in A-T as compared to control cells. Co-culture with catalase (cat) decreased this response. (D) Decrease in inflammasome-dependent cytokine IL-1β in A-T as compared to control cells, suggesting a defect in inflammasome activation. A-T n = 7, healthy controls n = 7. All data were plotted as the mean ± s.d. Kruskal-Wallis tests were performed and when significant (p < 0.05), Mann-Whitney tests were carried out. ns; not significant, *p < 0.03.

Figure 3

Increased sensitivity in A-T air-liquid interface cultures to S. pneumoniae infection. (A) Immunostaining of individual cell types in differentiated epithelia at ALI. Left panel: goblet cells (MUC5B; green), tight junctions (ZO-1; red); right panel: basal cells (cytokeratin 14; green) and ciliated epithelial cells (Ac α-tubulin; red). (B) Disruption of the respiratory epithelia at ALI by 24 h was observed in A-T but not in controls. (C) Percentage of cell death induced by S. pneumonia infection in control and A-T cells. (D) Transepithelial electrical resistance (TEER) reading performed on control and A-T ALI cultures post-infection. A-T n = 3, healthy controls n = 3. Unpaired, two-tailed Mann-Whitney tests were carried out. p < 0.03 between control and A-T at 48 h. All data were plotted as the mean ± s.d.

Figure 4

Increase in pro-inflammatory cytokines in A-T ALI cultures post-infection. (A,B) Increase in inflammasome-independent pro-inflammatory cytokines IL-8 and TNF-α in A-T as compared to control cells respectively. (C) Decrease in inflammasome-dependent cytokine IL-1β in A-T as compared to control cells, suggesting a defect in inflammasome activation. A-T n = 3, healthy controls n = 3. All data were plotted as the mean ± s.d. Unpaired, two-tailed Mann-Whitney tests were carried out. ns; not significant, *p < 0.03.

Figure 5

Loss of ATM impairs inflammasome activation following S. pneumoniae infection. (A) Immunostaining for caspase-1 (green) and ASC (red) showed evidence of inflammasome activation in control cells following infection with S. pneumoniae. In contrast, reduced levels of Caspase-1 and ASC specks were observed in A-T cells post-infection, suggesting a defect in inflammasome assembly. (B) Quantitation of Caspase-1-ASC foci formation in airway epithelial cells from healthy controls and patients with A-T. A-T n = 3, healthy controls n = 3. Experiment was performed in triplicates with a minimum of 200 cells per experiment counted for Caspase-1 and ASC foci. All data were plotted as the mean ± s.d. Unpaired, two-tailed Mann-Whitney test was performed, *p < 0.05.