Sequential Exposure to Antenatal Microbial Triggers Attenuates Alveolar Growth and Pulmonary Vascular Development and Impacts Pulmonary Epithelial Stem/Progenitor Cells

Abstract

Perinatal inflammatory stress is strongly associated with adverse pulmonary outcomes after preterm birth. Antenatal infections are an essential perinatal stress factor and contribute to preterm delivery, induction of lung inflammation and injury, pre-disposing preterm infants to bronchopulmonary dysplasia. Considering the polymicrobial nature of antenatal infection, which was reported to result in diverse effects and outcomes in preterm lungs, the aim was to examine the consequences of sequential inflammatory stimuli on endogenous epithelial stem/progenitor cells and vascular maturation, which are crucial drivers of lung development. Therefore, a translational ovine model of antenatal infection/inflammation with consecutive exposures to chronic and acute stimuli was used. Ovine fetuses were exposed intra-amniotically to Ureaplasma parvum 42 days (chronic stimulus) and/or to lipopolysaccharide 2 or 7 days (acute stimulus) prior to preterm delivery at 125 days of gestation. Pulmonary inflammation, endogenous epithelial stem cell populations, vascular modulators and morphology were investigated in preterm lungs. Pre-exposure to UP attenuated neutrophil infiltration in 7d LPS-exposed lungs and prevented reduction of SOX-9 expression and increased SP-B expression, which could indicate protective responses induced by re-exposure. Sequential exposures did not markedly impact stem/progenitors of the proximal airways (P63+ basal cells) compared to single exposure to LPS. In contrast, the alveolar size was increased solely in the UP+7d LPS group. In line, the most pronounced reduction of AEC2 and proliferating cells (Ki67+) was detected in these sequentially UP + 7d LPS-exposed lambs. A similar sensitization effect of UP pre-exposure was reflected by the vessel density and expression of vascular markers VEGFR-2 and Ang-1 that were significantly reduced after UP exposure prior to 2d LPS, when compared to UP and LPS exposure alone. Strikingly, while morphological changes of alveoli and vessels were seen after sequential microbial exposure, improved lung function was observed in UP, 7d LPS, and UP+7d LPS-exposed lambs. In conclusion, although sequential exposures did not markedly further impact epithelial stem/progenitor cell populations, re-exposure to an inflammatory stimulus resulted in disturbed alveolarization and abnormal pulmonary vascular development. Whether these negative effects on lung development can be rescued by the potentially protective responses observed, should be examined at later time points.

Insights into the Black Box of Intra-Amniotic Infection and Its Impact on the Premature Lung: From Clinical and Preclinical Perspectives

“…Different from rodents but similar to humans, sheep born at term (~E145) are in the alveolar stage [ 3 ] ( Figure 1 ). As such, most experimental models delivered lambs per cesarean section at E125, corresponding to ~31 weeks of human gestation [ 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 ] ( Table 2 ). The performance of sheep experiments was restricted to several research groups in the USA, Australia, and the Netherlands, thereby ensuring a high degree of homogeneity in animals and the route of prenatal infection, which was exclusively i.a.…”

“…Moreover, multiple and sequential infectious stimuli (i.e., LPS, IL1, Ureaplasma spp.) were given at the pseudoglandular and/or canalicular stage, better simulating prolonged and acute IAI of a polymicrobial nature as encountered in real clinical scenarios [ 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 ] ( Table 2 ). There was no maternal mortality or intrauterine fetal death reported in sheep models, and the birth weights of IAI-exposed preterm lambs were similar to their non-exposed counterparts of the same GA [ 94 , 98 , 100 ].…”

“…The influence of IAI on developing sheep lung was dependent on the type, duration, and dose of infectious insults. LPS of higher concentrations (4 and 10 mg/sac vs. 0.1 and 1 mg/sac) and a shorter duration (2 d vs. 7 d) elicited more profound cytokine responses and recruited more inflammatory cells in the lung [ 86 , 87 , 88 , 89 , 91 , 92 , 94 ]. On the contrary, Ureaplasma parvum (UP) provoked none or only mild inflammatory responses, due to their low virulence [ 93 ].…”

“…Similarly, extensive chronic UP infection in the pseudoglandular stage mitigated subsequent LPS-stimulated fetal pulmonary inflammation [ 93 ]. On the contrary, moderately chronic UP infection in the canalicular stage aggravated LPS-induced lung injury, whereas acute UP exposure shortly before delivery did not influence the effect of the subsequent LPS [ 87 , 92 ].…”

“…While chronic UP infection in the canalicular stage (E83) primarily impaired stem/progenitor populations in the distal airways, an acute LPS insult in the saccular stage (E118) suppressed stem/progenitor cells throughout the entire lung [ 86 ]. Depending on the dose and combination of infectious insults, UP and/or LPS could disrupt the expression of vascular endothelial growth factor (VEGF), angiopoietin 1 (ANG1), tunica interna endothelial cell kinase 2 (TIE2), and transforming growth factor beta 1 (TGFB1), predisposing to vascular remodeling and fibrosis [ 86 , 87 , 91 , 93 , 96 ]; meanwhile, the synthesis of SPs and molecules implicated in branching morphogenesis, such as fibroblast growth factor 10 (FGF10) and bone morphogenetic protein 4 (BMP4) were upregulated, resulting in a net beneficial effect as demonstrated by increased lung gas volume [ 86 , 87 , 92 , 93 ]. This indicates differential responses of the three lung compartments upon exposure to IAI, and disruptive alterations in the lung can be counteracted by protective responses, leading to a favorable net effect.…”