Neutrophil extracellular traps degrade fibronectin in a rat model of bronchopulmonary dysplasia induced by perinatal exposure to lipopolysaccharide

Dear Editor, Over the 50 years since its first description, bronchopulmonary dysplasia (BPD) has gained major advances in therapy, including antenatal corticosteroids, continuous positive airway pressure and surfactant replacement.1 However, BPD is still the most common chronic respiratory morbidity in preterm infants, and the challenge of exploring unmasked culprits in BPD pathogenesis remains unresolved. Neutrophils are abundant leucocytes in acute inflammation. Peripheral blood neutrophil counts are increased in infants with moderate-severe BPD, and the prolonged influx of neutrophils into the lung is associated with BPD severity,2 suggesting that neutrophils may contribute to BPD. As a major component of pulmonary ECM, expression of fibronectin coincides with lung development,3 especially fibronectin is indispensable for branch morphogenesis in murine lung explants.4 Moreover, fibronectin expression is directly linked with alveolar septation, a key step in alveologenesis.5 The differentiation of alveolar epithelial cells, the major constituents of the alveolus in the developing lung, is regulated by fibronectin.6 As inadequate fibronectin impairs the airway epithelial branching morphogenesis,7 we hypothesize that fibronectin deficiency may retard lung branching development, thereby promoting BPD initiation. Neutrophil extracellular traps (NETs), first described in the immune defence against bacterial infection, have been linked to diverse pulmonary diseases.8 Recently, it is reported that neutrophil elastase from neutrophils exosome destructs extracellular matrix (ECM) and induces BPD-like disease in mice.9 NETs are quite different from exosomes. However, NETs also contain various proteases. To the best of our knowledge, the roles of NETs on the fibronectin expression have not been investigated in BPD pathogenesis. The rat model of BPD was established by intra-amniotic LPS injection in pregnant rats (see Appendix S1 in Supporting Informaion). Compared with the control group, the number of alveoli in the BPD group was significantly reduced. By contrast, mean linear intercept in the developing lung from LPS control was significantly increased (Figure 1A). As fibronectin played key roles in branching morphogenesis and alveolar epithelial cells differentiation, we found that pulmonary fibronectin 1st day post-birth (P1) was significantly lower in LPS group the than that in PBS control group (Figure 1B,C). Expression of fibronectin from P3 lung tissues from LPS or PBS group, however, was comparable. Similar observation was recorded for P7 lung tissues. LPS down-regulated fibronectin and distal airway branching in the foetal lung.10 To explore whether LPS directly decreased fibronectin in alveolar epithelial cells, we treated MLE-12 cells (alveolar epithelial cell line) or primary alveolar epithelial cells with LPS. Indeed, LPS directly reduced fibronectin in both MLE-12 cells (Figure 1D,E) and primary alveolar epithelial cells (Figure 1F). The TGF-β/Smad signalling pathway was essential for fibronectin expression in bronchial epithelial cells. Similarly, Smad-3 deficiency retarded lung alveolarization.11 As expected, LPS decreased TGF-β and inactivated Smad-3 on alveolar epithelial cells (Figure 1G). By contrast, Smad-3 inhibitor (SIS3) rescued the expression of fibronectin on epithelial cells (Figure 1H). TLR-4 inhibitor (TAK-242), which blocked the roles of LPS, also reversed the reduction in fibronectin on epithelial cells (Figure 1I). Collectively, these results implied that LPS bound to TLR4 down-regulated the ECM protein fibronectin on alveolar epithelial cells, promoting the progression of BPD. Besides the direct effects, LPS was a potent inducer of NETs. We speculated that LPS may trigger the formation of NETs in the rat model of BPD. In the lung tissues from LPS-treated rats, myeloperoxidase (MPO) and citrullinated histone 3 (H3cit) double-stained structures were recorded in the alveoli (Figure 2A), indicating the formation of NETs. NETs, decorated with protein enzymes in the DNA fibrous backbones, are potential to cleave ECM components.12 To investigate whether NETs play roles in the fibronectin expression of alveolar epithelial cells, we stimulated MLE-12 cells with purified NETs in vitro. NETs decreased fibronectin on alveolar epithelial cells in a timeand dose-dependent manner (Figure 2B,C). DNase, destroying the DNA backbones of NETs (Figure 2D), blocked the reduction in fibronectin induced by NETs (Figure 2E), further demonstrating that NETs decreased fibronectin expression in alveolar epithelial cells. NETs cleaved ECM via elastase and MMP-9.12 We asked whether MMP-9 was involved in the fibronectin reduction from NETs-treated alveolar epithelial cells. As shown in Figure 2F, similar to DNase, MMP-9 inhibitor recuperated the expression of fibronectin, suggesting that NETs reduced fibronectin expression


Neutrophil extracellular traps degrade fibronectin in a rat model of bronchopulmonary dysplasia induced by perinatal exposure to lipopolysaccharide
Dear Editor, Over the 50 years since its first description, bronchopulmonary dysplasia (BPD) has gained major advances in therapy, including antenatal corticosteroids, continuous positive airway pressure and surfactant replacement. 1 However, BPD is still the most common chronic respiratory morbidity in preterm infants, and the challenge of exploring unmasked culprits in BPD pathogenesis remains unresolved. Neutrophils are abundant leucocytes in acute inflammation.
Peripheral blood neutrophil counts are increased in infants with moderate-severe BPD, and the prolonged influx of neutrophils into the lung is associated with BPD severity, 2 suggesting that neutrophils may contribute to BPD.
As a major component of pulmonary ECM, expression of fibronectin coincides with lung development, 3 especially fibronectin is indispensable for branch morphogenesis in murine lung explants. 4 Moreover, fibronectin expression is directly linked with alveolar septation, a key step in alveologenesis. 5 The differentiation of alveolar epithelial cells, the major constituents of the alveolus in the developing lung, is regulated by fibronectin. 6 As inadequate fibronectin impairs the airway epithelial branching morphogenesis, 7 we hypothesize that fibronectin deficiency may retard lung branching development, thereby promoting BPD initiation.
Neutrophil extracellular traps (NETs), first described in the immune defence against bacterial infection, have been linked to diverse pulmonary diseases. 8 Recently, it is reported that neutrophil elastase from neutrophils exosome destructs extracellular matrix (ECM) and induces BPD-like disease in mice. 9 NETs are quite different from exosomes. However, NETs also contain various proteases.
To the best of our knowledge, the roles of NETs on the fibronectin expression have not been investigated in BPD pathogenesis.
The rat model of BPD was established by intra-amniotic LPS injection in pregnant rats (see Appendix S1 in Supporting Informaion).
Compared with the control group, the number of alveoli in the BPD group was significantly reduced. By contrast, mean linear intercept in the developing lung from LPS control was significantly increased ( Figure 1A). As fibronectin played key roles in branching morphogenesis and alveolar epithelial cells differentiation, we found that pulmonary fibronectin 1st day post-birth (P1) was significantly lower in LPS group the than that in PBS control group ( Figure 1B DNase, destroying the DNA backbones of NETs ( Figure 2D), blocked the reduction in fibronectin induced by NETs ( Figure 2E), further demonstrating that NETs decreased fibronectin expression in alveolar epithelial cells. NETs cleaved ECM via elastase and MMP-9. 12 We asked whether MMP-9 was involved in the fibronectin reduction from NETs-treated alveolar epithelial cells. As shown in Figure 2F

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.