Abstract
The histopathology of bronchopulmonary dysplasia (BPD) includes hypoalveolarization and interstitial thickening due to abnormal myofibroblast accumulation. Chorioamnionitis and sepsis are major risk factors for BPD development. The cellular mechanisms leading to these lung structural abnormalities are poorly understood. We used an animal model with repeated lipopolysaccharide (LPS) administration into the airways of immature mice to simulate prolonged airway exposure to gram-negative bacteria, focusing on the role of C-C chemokine receptor type 2-positive (CCR2+) exudative macrophages (ExMf). Repetitive LPS exposure of immature mice induced persistent hypoalveolarization observed at 4 and 18 days after the last LPS administration. LPS upregulated the expression of lung pro-inflammatory cytokines (TNF-α, IL-17a, IL-6, IL-1β) and chemokines (CCL2, CCL7, CXCL1, and CXCL2), while the expression of genes involved in lung alveolar and mesenchymal cell development (PDGFR-α, FGF7, FGF10, and SPRY1) was decreased. LPS induced recruitment of ExMf, including CCR2+ ExMf, as well as other myeloid cells like DCs and neutrophils. Lungs of LPS-exposed CCR2−/− mice showed preserved alveolar structure and normal patterns of α-actin and PDGFRα expression at the tips of the secondary alveolar crests. Compared to wild type mice, a significantly lower number of ExMf, including TNF-α+ ExMf were recruited to the lungs of CCR2−/− mice following repetitive LPS exposure. Further, pharmacological inhibition of TLR4 with TAK-242 also blocked the effect of LPS on alveolarization, α-SMA and PDGFRα expression. TNF-α and IL-17a induced α-smooth muscle actin expression in the distal airspaces of E16 fetal mouse lung explants. In human preterm lung mesenchymal stromal cells, TNF-α reduced mRNA and protein expression of PDGFR-α and decreased mRNA expression of WNT2, FOXF2, and SPRY1. Collectively, our findings demonstrate that in immature mice repetitive LPS exposure, through TLR4 signaling increases lung inflammation and impairs lung alveolar growth in a CCR2-dependent manner.
Highlights
Bronchopulmonary dysplasia (BPD) is the most common chronic pulmonary complication of preterm birth, with over 12,000 new cases annually in the US [1]
LPS upregulated the expression of lung proinflammatory cytokines (TNF-a, IL-17a, IL-6, IL-1b) and chemokines (CCL2, CCL7, CXCL1, and CXCL2), while the expression of genes involved in lung alveolar and mesenchymal cell development (PDGFR-a, FGF7, FGF10, and SPRY1) was decreased
Comparative examination of random Hematoxylin and eosin (H&E) images revealed that following LPS exposure: alveolar chord length was significantly increased both on days of life 14 and 28, indicating long lasting interference with lung alveolar growth (Figure 1)
Summary
Bronchopulmonary dysplasia (BPD) is the most common chronic pulmonary complication of preterm birth, with over 12,000 new cases annually in the US [1]. Infants with BPD have higher complication rates, longer initial hospitalizations [2], and often develop chronic respiratory symptoms and abnormal lung function with airflow obstruction lasting into adulthood [3]. During the first days of life, an influx of polymorphonuclear leukocytes and macrophages is observed in tracheal aspirates of mechanically ventilated premature infants who later develop BPD [17, 18]. Monocytes from tracheal aspirates of premature infants with evolving BPD display a pro-inflammatory phenotype and increased expression of IL-1 cytokines [19]. Tracheal aspirate levels of pro-inflammatory cytokines such as IL-6, IL-1b, TNF-a, and chemokines CCL2, CCL7, CCL8, and IL-8 (CXCL8) are higher in premature infants with respiratory distress syndrome who develop BPD [21,22,23,24,25,26]. Similar findings are present in animal models of BPD [27,28,29]
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