Lung and gut microbiota are altered by hyperoxia and contribute to oxygen-induced lung injury in mice

Abstract

Inhaled oxygen, although commonly administered to patients with respiratory disease, causes severe lung injury in animals and is associated with poor clinical outcomes in humans. The relationship between hyperoxia, lung and gut microbiota, and lung injury is unknown. Here, we show that hyperoxia conferred a selective relative growth advantage on oxygen-tolerant respiratory microbial species (e.g., Staphylococcus aureus) as demonstrated by an observational study of critically ill patients receiving mechanical ventilation and experiments using neonatal and adult mouse models. During exposure of mice to hyperoxia, both lung and gut bacterial communities were altered, and these communities contributed to oxygen-induced lung injury. Disruption of lung and gut microbiota preceded lung injury, and variation in microbial communities correlated with variation in lung inflammation. Germ-free mice were protected from oxygen-induced lung injury, and systemic antibiotic treatment selectively modulated the severity of oxygen-induced lung injury in conventionally housed animals. These results suggest that inhaled oxygen may alter lung and gut microbial communities and that these communities could contribute to lung injury.

Fig. 1.. Hyperoxia alters lung microbiota in humans and in neonatal and adult mice.

(A) Comparison of early exposure to inhaled oxygen (FiO₂) in a cohort of 1523 patients receiving mechanical ventilation for greater than 24 hours and subsequent culture of respiratory specimens. Patients were stratified into tertiles by their mean FiO₂ during their first 24 hours of mechanical ventilation: low (FiO₂ 21 to 46%; 265 isolates from 507 patients), intermediate (FiO₂ 46 to 60%; 231 isolates from 508 patients), and high (FiO₂ 60 to 100%; 181 isolates from 508 patients). (Left) The relationship between early hyperoxia (mean FiO₂ exposure during the first 24 hours) and the identity of bacterial species cultured from respiratory specimens in the week after collection is shown. (Right) The relationship between early hyperoxia and the rate of isolation of S. aureus and P. aeruginosa from all respiratory specimens sent for culture in the week after collection is shown. (B) (Left) Lung microbial community richness (measured as unique OTUs per 1000 sequences) compared across neonatal mice exposed to 2 weeks of normoxia (FiO₂ 21%, n = 8) or hyperoxia (FiO₂ 75%, n = 9) is shown. (Middle and right) Comparison of community composition of lung bacterial communities in neonatal mice exposed for 2 weeks to hyperoxia (FiO₂ 75%, n = 9) or normoxia (FiO₂ 25%, n = 8). (C) Relative abundance (percentage of total bacterial sequences) and bacterial burden (16S rRNA gene copies per lung) of lung microbiota of adult mice exposed to hyperoxia (FiO₂ 95%, n = 20) for different time periods. Significance was determined by multivariable logistic regression (A), Mann-Whitney test (B), PERMANOVA (B), and ANOVA with Dunnet’s test for multiple comparisons (C). Values represent means ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. NS, not significant.

Fig. 2.. Lung microbial dysbiosis precedes lung injury during hyperoxia in mice.

Genetically identical adult mice were exposed to various durations of hyperoxia (FiO₂ 95%, n = 20), and the temporal dynamics of lung injury and lung microbiota disruption were compared. (A) Alveolar protein concentration, a measure of lung injury, was compared to duration of hyperoxia. (B) Disruption of lung microbiota was measured using dissimilarity of lung microbial communities (Bray-Curtis similarity index) from those of mice unexposed to hyperoxia (time 0). Significance was determined using ANOVA with Dunnet’s test of multiple comparisons. Values represent means ± SEM. **P ≤ 0.01; ***P ≤ 0.001.

Fig. 3.. Variation in lung inflammation correlates with variation in lung microbiota in hyperoxia-exposed mice.

Healthy adult C57BL/6 mice were exposed to hyperoxia for 0, 24, and 48 hours (FiO₂ 95%, n = 42 per time point). Variation in lung bacterial diversity (Shannon diversity index) was compared to concentrations of the cytokines IL-1α, TNF-α, and IL-17 at each time point. Significance was determined by linear regression using log-transformed cytokine data.

Fig. 4.. Correlations among hyperoxia, cecal microbiota composition, and alveolar inflammation.

Healthy, adult C57BL/6 mice were exposed to hyperoxia for 0, 24, 48, or 72 hours (FiO₂ 95%). (A) Changes in cecal bacterial communities after exposure of mice to hyperoxia (FiO₂ 95%, n = 10 per time point) for 72 hours reveal depletion of the Firmicutes phylum and enrichment of the Bacteroidetes phylum. PC1, principal component 1; PC2, principal component 2. Values represent means ± SEM. (B) Changes in cecal bacterial communities (Shannon diversity index) were compared with variations in lung inflammation measured by changes in the concentrations of the cytokines IL-1α, TNF-α, and IL-17 after 0, 24, or 48 hours of exposure to hyperoxia (FiO₂ 95%, n = 42 mice per time point). Significance was determined via PERMANOVA (A), mvabund (B), and linear regression using log-transformed cytokine data (B).

Fig. 5.. Germ-free mice are protected from oxygen-induced lung injury.

Conventionally housed (conventional) and germ-free C57Bl/6 adult mice were exposed to normoxia or hyperoxia (FiO₂ 21 or 95%, respectively) for 72 hours, and oxygen-induced lung injury and alveolar inflammation were quantified. (A) Lung injury was quantified by measuring concentrations of alveolar protein and IgM in the lungs, which indicate alveolar capillary permeability (n = 10 per exposure per time point). (B) Representative histological images of lung tissue stained with hematoxylin and eosin from conventional (n = 20) or germ-free (n = 12) C57Bl/6 adult mice exposed to hyperoxia (FiO₂ 95%) for 96 hours are shown. Left insets show epithelial necrosis and the formation of hyaline membranes; right insets show minimal epithelial injury and an intact normal alveolar architecture. (C) Alveolar inflammatory cells (neutrophils) and the neutrophil chemoattractant CXCL1 were quantified in bronchoalveolar lavage fluid from germ-free or conventional C57Bl/6 adult mice exposed to normoxia or hyperoxia (FiO₂ 21 or 95%, respectively) for 72 hours. Significance was determined using Student’s t test in (A) and (C). Values represent means ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Fig. 6.. The effects of antibiotic treatment on lung injury and inflammation in hyperoxia-exposed mice.

(A) C57BL/6 conventionally housed adult mice were treated orally with broad-spectrum enteric antibiotics for 3 weeks (ampicillin, neomycin, metronidazole, and vancomycin) and then were exposed to normoxia (FiO₂ 21%) or hyperoxia (FiO₂ 95%) for 72 hours. Lung injury was quantified by measuring concentrations of alveolar protein and IgM as well as neutrophils in bronchoalveolar lavage fluid from antibiotic-treated and untreated mice. (B and C) C57BL/6 conventionally housed adult mice were treated daily with intraperitoneal vancomycin (B) or intraperitoneal ceftriaxone (C) during a 72-hour exposure to normoxia (FiO₂ 21%) or hyperoxia (FiO₂ 95%). Lung injury was quantified by measuring concentrations of alveolar protein and IgM in bronchoalveolar lavage fluid from antibiotic-treated and untreated mice. Significance was determined using Student’s t test. Values represent means ± SEM. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Fig. 7.. Hyperoxia alters microbial growth conditions in the mouse lung microenvironment.

(A) Conventionally housed C57Bl/6 adult mice were exposed to hyperoxia (95% FiO₂) for 0, 24, or 48 hours (n = 20 mice) and then were administered intranasal S. aureus (10⁷ CFU). Thereafter, mice were observed for 24 hours at normoxia (21% FiO₂), after which their lungs were harvested, and S. aureus CFU were counted. (B) The mouse lung microenvironment was analyzed ex vivo by inoculating 10⁴ CFU of S. aureus into saline as a control or sterilized bronchoalveolar lavage fluid from mice exposed to normoxia or hyperoxia for 72 hours. S. aureus CFU were then quantified after 6 hours of growth (n = 7 per exposure). Significance was determined using ANOVA with Bonferroni correction for multiple comparisons. Values represent means ± SEM. ***P ≤ 0.001; ****P ≤ 0.0001.