Effects of hyperoxia on alveolar and pulmonary vascular development in germ-free mice

Abstract

Airway microbial dysbiosis is associated with subsequent bronchopulmonary dysplasia (BPD) development in very preterm infants. However, the relationship of airway microbiome in normal pulmonary development has not been defined. To better understand the role of the airway microbiome, we compared normal and abnormal alveolar and pulmonary vascular development in mice with or without a microbiome. We hypothesized that the lungs of germ-free (GF) mice would have an exaggerated phenotypic response to hyperoxia compared with non-germ-free (NGF) mice. With the use of a novel gnotobiotic hyperoxia chamber, GF and NGF mice were exposed to either normoxia or hyperoxia. Alveolar morphometry, pulmonary mechanics, echocardiograms, inflammatory markers, and measures of pulmonary hypertension were studied. GF and NGF mice in normoxia showed no difference, whereas GF mice in hyperoxia showed protected lung structure and mechanics and decreased markers of inflammation compared with NGF mice. We speculate that an increase in abundance of pathogenic bacteria in NGF mice may play a role in BPD pathogenesis by regulating the proinflammatory signaling and neutrophilic inflammation in lungs. Manipulation of the airway microbiome may be a potential therapeutic intervention in BPD and other lung diseases.

Keywords: bronchopulmonary dysplasia; germ free; gnotobiotic; hyperoxia; lung microbiome.

Fig. 1.

Germ-free (GF) animals have decreased lung injury in hyperoxia condition. A–C: representative photomicrographs of hematoxylin-eosin-stained sections of lungs from Swiss Webster [non–germ-free (NGF) and GF] mouse pups [A: postnatal age 7 (PN7); B: postnatal age 14 (PN14); and C: postnatal age 28 (PN28)] in normoxia (21% FiO₂) or hyperoxia (85% FiO₂) exposure from PN3–PN14 (magnification, ×100). Hyperoxia exposure was associated with marked alveolar hypoplasia in both NGF and GF mice at all postnatal ages compared with their respective normoxia group. D–F: radial alveolar counts (RACs) of both NGF and GF hyperoxia-treated pups were significantly lower than their respective normoxia groups at PN7, 14, and 28. However, GF hyperoxia-exposed pups have decreased alveolar simplification and alveolar hypoplasia compared with NGF hyperoxia group at PN7 (A), PN14 (B), and PN28 (C). The RACs of GF hyperoxia-treated groups were increased compared with NGF hyperoxia-exposed pups at PN7 (D), PN14 (E), and PN26 (F), indicating better alveorization in the GF hyperoxia group compared with the NGF hyperoxia group. G–I: alveolar size was comparatively larger in the NGF hyperoxia group compared with the GF hyperoxia group. No difference in RACs or mean linear intercepts (MLIs) was seen between NGF and GF normoxia groups. Data are means ± SE; n = 5–7 animals per group. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 2.

Germ-free (GF) animals have better lung mechanics than non–germ-free (NGF) animals in hyperoxia. A: total lung resistance was similar between NGF and GF normoxia pups. Hyperoxia significantly increased lung resistance in NGF pups compared with normoxia NGF pups. However, lung resistance was not increased in hyperoxia GF pups compared with GF normoxia pups. GF hyperoxia pups had decreased lung resistance compared with NGF hyperoxia pups. B: lung compliance was decreased in the hyperoxia NGF group; however, GF pups did not show reduction in lung compliance after hyperoxia exposure. GF hyperoxia pups had increased lung compliance compared with NGF hyperoxia pups. Data are means ± SE; n = 5–7 animals per group. *P < 0.05, **P < 0.01, ***P < 0.001. PN14, postnatal day 14.

Fig. 3.

Neonatal hyperoxia increases right ventricular systolic pressure in both germ-free (GF) and non–germ-free (NGF) mice. A: representative echocardiogram of Swiss Webster NGF and GF mice at postnatal day (PN) 28 following normoxia and hyperoxia exposure. B: echocardiographic assessment of right ventricle (RV) systolic pressure at PN28 mice exposed to normoxia and hyperoxia. RV systolic pressure was increased in hyperoxia NGF mice compared with normoxia NGF mice. No differences in RV systolic pressure were seen between normoxia and hyperoxia GF mice. Also, no differences in RV systolic pressure were noted between NGF and GF mice in both normoxia and hyperoxia. C: right ventricle-to-left ventricle free wall thickness ratio (RV/LV) was measured at PN14 for right ventricular hypertrophy. Postnatal hyperoxia increased RV/LV in both NGF and GF mice compared with their respective normoxia group. No difference in RV/LV was seen between NGF and GF in either nomoxia or hyperoxia. Data are means ± SE; n = 5–10 animals per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.

No difference in microvascular density is noticed between germ-free (GF) and non–germ-free (NGF) animals. A–C: representative photomicrographs of endomucin-stained sections of lungs from Swiss Webster (NGF and GF) mouse pups [postnatal age 7 (PN7; A), postnatal age 14 (PN14; B), and postnatal age 28 (PN28; C)] in normoxia (21% FiO₂) or hyperoxia (85% FiO₂) exposure from PN3–PN14 (magnification, ×200). D–F: hyperoxia exposure resulted in sparse microvasculature in both NGF and GF mice at all postnatal ages compared with their respective normoxia group on analysis using endomucin stained area (%). No difference was seen between NGF and GF groups in microvascular density in either normoxia or hyperoxia conditions. Data are means ± SE; n = 3 animals per group.

Fig. 5.

Germ-free (GF) animals mount decreased inflammation in hyperoxia condition. A–C: myeloperoxidase (MPO) activity, as a measure of neutrophil activity, measured in bronchoalveolar lavage fluid after normoxia or hyperoxia exposure. Hyperoxia-induced increments in MPO activity are less dramatic in GF mice at both postnatal day 7 (PN7) and PN14 compared with NGF mice. This difference was not seen at PN28. D–I: inflammatory cytokine levels using multiplex assays showed decreased interleukin-1β (IL-1β; D) and interferon γ (IFN-γ; G) in GF hyperoxia mice at PN7 compared with NGF hyperoxia group; however, results are more scattered. Data are means ± SE; n = 2–5 animals per group. *P < 0.05, **P < 0.01, ****P < 0.0001.