Introduction
Immaturity of the lungs due to preterm birth is one of the most significant complications affecting neonatal mortality. As preterm infants with respiratory distress are born with lungs at an anatomical stage not yet conducive to gas exchange, routine care practices include mechanical ventilation and oxygen supplementation. However, these lifesaving interventions can also cause permanent damage to the developing lung, resulting in a chronic lung disease termed bronchopulmonary dysplasia (BPD) [
1],[
2]. Modern therapeutic improvements such as the administration of surfactant and corticosteroids have seen a transformation in BPD pathology away from the fibrotic injury of the past. Instead today, with many extremely preterm babies now surviving, `new’ BPD pathology is characterised by a disruption of alveolar formation [
3],[
4].
Delivery of high concentrations of oxygen to neonatal animals is widely used to investigate the basis of arrested alveolar development associated with BPD in preterm infants [
5],[
6]. In humans, alveolarisation begins
in utero at around 36 weeks gestation, with 85% of alveoli formed after birth [
7]. In the mouse, alveolar formation occurs entirely postnatally, beginning approximately 4 days after birth and proceeding until P36 [
8]. Exposure of the developing lung to high levels of oxygen results in inflammation and oxidant damage, and dysregulation of alveolar development [
9]-[
11]. Understanding the mechanisms of alveolar formation has clinical relevance for the development of therapeutic strategies to improve lung maturation in preterm infants.
Macrophages are key immune cells commonly associated with inflammation and the propagation of tissue injury associated with oxygen toxicity in hyperoxia exposure. However, a greater appreciation of macrophage diversity is revealing that this heterogenous cell type is also essential in regulating organ development and regeneration [
12]-[
15]. In particular, diversity in macrophage subpopulation activation in the lung has been shown to be important in both the induction and resolution of lung injury [
16].
Colony-Stimulating Factor-1 (CSF-1) is a key regulator of macrophage differentiation, proliferation, survival and activation, and acts as a pleiotropic growth factor essential in reproduction and organogenesis [
17]. During pregnancy, uterine production of CSF-1 increases 1000-fold [
18], and macrophages colonise the embryo and are present in large numbers in virtually all developing organs [
19]. The absence of CSF-1 results in severe developmental impairment including reduced growth, neurological and reproductive defects, as well as altered development of the mammary gland, bone and pancreas [
20]-[
22]. We have previously reported that lung CSF-1R+ macrophages increase during normal lung development and peak during the alveolarisation stage [
23]. In the absence of CSF-1, alveolar macrophage populations are severely depleted during postnatal development [
24]-[
26], and in adulthood mice develop spontaneous emphysema associated with deregulated matrix metalloproteinases (MMPs) and abnormal elastin deposition [
26].
CSF-1 administration has been shown to enhance organogenesis. Increased branching morphogenesis was observed in embryonic kidney explants following the addition of CSF-1 [
27], and in embryonic pancreas explants β-cell proliferation and insulin production were increased [
28]. Furthermore, CSF-1 is suggested to preferentially regulate macrophage populations associated with trophic functions such as development [
29]. While abnormalities seen in CSF-1-deficient animals are associated with a virtual absence of tissue macrophages, organogenic enhancement with CSF-1 administration is correlated with increased macrophage numbers [
20]-[
22],[
27],[
28].
CSF-1-responsive macrophages in developing embryonic kidneys and lungs exhibit a gene expression profile characteristic of an M2 or tissue remodelling-type macrophage [
27]. Similarly we have demonstrated the importance of trophic macrophages in alveolarisation, with an upregulation of M2 genes observed during this developmental stage in the mouse [
23]. Furthermore, CSF-1 has important immunomodulatory properties, and in a murine model of neonatal hyperoxia, mesenchymal stem cell (MSC)-mediated amelioration of injury was associated with increased CSF-1 levels [
30]. We hypothesise that the administration of CSF-1 to promote augmentation of developmental macrophages associated with alveolarisation may reduce the need for damaging long-term oxygen therapy in preterm newborns and attenuate the developmental arrest of the lung associated with BPD.
The aim of this study was to determine the impact of CSF-1 administration on lung maturation in a model of hyperoxia-associated lung developmental perturbation, when delivered either as a treatment after hyperoxia or when administered prophylactically with concurrent hyperoxia exposure. In particular, we assessed lung functional maturation and the impact of a CSF-1-mediated increase in macrophages. We found that CSF-1 did not rescue the growth and pulmonary defects associated with hyperoxia in this model; however the concomitant increased infiltration of CSF-1-responsive macrophages was not associated with exacerbation of lung injury.
Discussion
Oxygen supplementation remains a critical and lifesaving intervention for infants with respiratory distress; however, it can result in detrimental alterations to the developing lung. Exposing neonatal mice to high concentrations of oxygen mimics the damage to the lung associated with BPD, providing a relevant model to assess the function of CSF-1 in rescuing and enhancing alveolar formation. This study employed a hyperoxia regime of 65% oxygen for 1 week after birth, providing a milder, more clinically-relevant exposure that more closely resembles the oxygen therapy regime of ventilated premature infants than other previous studies [
5],[
35]. It was also selected to minimize maternal oxygen toxicity and avoid stress and abandonment issues associated with dam rotation. Hallmarks of this hyperoxia model included growth restriction, impaired lung structural development indicative of reduced alveolarisation, and as a consequence measurable perturbations of respiratory function. While body weight was comparable at birth, hyperoxia impacted negatively on neonatal growth as evidenced by the reduction in body weight in the hyperoxia group by the end of exposure. Although there is potential for maternal oxygen toxicity to impact on the growth restriction observed, such growth restriction associated with this model is important to examine the growth promoting functions of CSF-1 as observed previously [
23]. Lung structural alterations characteristic of oxygen toxicity and BPD were also observed. MLI and percentage airspace were increased, while percentage tissue was decreased. This is in agreement with other hyperoxia studies, where oxygen supplementation caused BPD-like pathological changes to the lung parenchyma and impaired alveolarisation resulting in fewer, larger alveoli [
5],[
6].
Functional perturbations were also revealed following hyperoxia. Unrestrained barometric whole-body plethysmography proved a beneficial analytical technique sensitive enough to reveal measurable differences in neonates at 7 days of age. While the accuracy of absolute quantitation in small animals is debatable [
36], this technique provides a qualitative comparative assessment applicable to this study. Furthermore, the non-invasive nature of this technique makes it invaluable for these experiments in assessing the effects of CSF-1 and the rescue of lung function over time. Abnormal breathing patterns were associated with hyperoxia exposure after 7 days. In agreement with previously reported effects of hyperoxia on pulmonary activity [
37], changes included an increase in total cycle time primarily due to lengthened expiratory time. Furthermore, hyperoxia resulted in a reduction in tidal volume, supporting the structural observation of impaired alveolar formation. However, effects on tidal and minute volumes were mitigated when normalised to body weight, suggesting that the overall growth restriction contributes to the reduced lung volumes.
Harnessing the organogenic importance of growth factors involved in alveolar formation has been a fundamental strategy for promoting lung maturation. Factors such as keratinocyte growth factor [
38], hepatocyte growth factor [
39], retinoic acid [
40] and vascular endothelial growth factor [
41] are important in alveolarisation and have shown positive effects in rescuing alveolar development in hyperoxic animal studies. The injury-induced perturbation of development associated with BPD is also being tackled by using anti-inflammatory approaches, such as interleukin (IL)-10 [
42] or inflammatory chemokine blockade [
43], and by optimising ventilation strategies [
44] to mitigate damage and protect alveolar formation.
CSF-1 provides a particularly attractive candidate for use in this setting because it is a growth factor with proven organogenic [
27],[
28], anti-inflammatory [
29],[
45] and regenerative capabilities [
12],[
46]. During development, endogenous CSF-1 preferentially regulates trophic macrophages associated with organogenesis, as evidenced by the widespread developmental defects observed in CSF-1-deficient mice [
20]-[
22]. Functions of trophic macrophages that support organogenesis include apoptotic clearance of cellular debris associated with tissue remodelling [
47], regulation of angiogenesis through the production of angiogenic factors [
48],[
49] and by physically directing vascular development [
50]. Macrophages act as potent effector cells producing a range of important trophic mediators such as insulin-like growth factor-1 (IGF-1) [
51], wingless-type MMTV integration site family, member 7b (Wnt7b) [
52], transforming growth factor-β (TGF-β) [
53] and MMP9 [
54], which are involved in epithelial proliferation and matrix reorganisation. These processes are all essential in lung development, particularly in alveolarisation. Macrophages in both the embryonic lung and the postnatal lung undergoing alveolarisation demonstrate a gene expression profile, indicative of a trophic M2 macrophage activation state [
23],[
27].
The role of CSF-1 in polarising macrophages towards an M2 phenotype may also provide beneficial effects in hyperoxia through its involvement in immune dampening [
29]. Inflammation has a negative impact on lung development [
55],[
56]. Inflammatory cell influx and release of pro-inflammatory mediators promotes apoptotic and necrotic cell death that disrupts lung morphogenesis and impairs function. Furthermore, inflammatory activation disrupts organogenic signalling pathways by altering expression of key genes important in lung development [
57],[
58]. While inflammatory challenges such as lipopolysaccharide (LPS) or IL-1β administration in animal models of chorioamnionitis promote lung maturation, the mechanism is distinct from alveolarisation and is instead a survival adaptation that comes at the expense of proper alveolar formation resulting in a lung pathology associated with BPD [
59].
The infiltration of inflammatory macrophages is associated with the progression of lung injury and pathology [
60],[
61]. However macrophage diversity is revealing the importance of lung macrophages in both the injury and repair stages [
16], and differential activation suggests that a GM-CSF driven M1 pro-inflammatory response may be distinct from CSF-1-mediated stimulation of macrophages to take on a remodelling/anti-inflammatory M2 phenotype [
29]. In a recent study, CSF-1 was associated with repair and rescue of alveolar formation following hyperoxia in the mouse [
30]. It was reported that the administration of MSC–conditioned media into a neonatal murine hyperoxia model reduced inflammation and prevented alveolar and vascular damage [
30]. Interestingly, this correlated with high levels of CSF-1, indicating that the developmentally protective effect of MSCs may be indirectly mediated by the immunomodulatory effect of CSF-1 [
30].
In the present study, CSF-1 was administered to a neonatal murine hyperoxia model and growth, lung structure and respiratory function were assessed. In the first treatment regime, CSF-1 was administered post-hyperoxic injury to investigate whether CSF-1 could rescue developmental perturbation and promote alveolar formation. Results demonstrated limited success of CSF-1 in the mitigation of hyperoxia-induced injury. Deficits in growth were not improved, with the body weights of CSF-1-treated mice comparable to the Hyp + PBS group. With regard to lung function, a negative impact was revealed at P11 at the end of treatment. In parameters such as total cycle time, expiration time and minute volume/body weight ratio the detrimental effect of hyperoxia was exacerbated with CSF-1. Interestingly, these initial defects did normalise over time and by P35 no differences between treatment groups were observed. This normalisation was also seen in the Hyp + PBS group, and therefore CSF-1 treatment did not accelerate or enhance functional recovery, although increased numbers of CSF-1R+ macrophages were evident. Nevertheless, it is noteworthy that CSF-1 did not have a negative impact on the parameters measured in the adult. It is interesting that earlier perturbations observed with hyperoxia resolved by adulthood. Whether this impacts on the lungs capacity to cope with challenges and aging in later life is unknown. Furthermore questions remain about potential negative effects associated with catch up growth. Indeed CSF-1R+ macrophages have been shown to be increased in the alveolarisation stage of lung development [
23], however whether in this setting the increase in CSF-1R+/F4/80+ macrophages is retained and whether it is associated with positive or negative outcomes in lung physiology is also of ongoing interest.
Oxygen supplementation is a critical life saving intervention for babies with respiratory distress, and clinically any maturation-based therapies would need to be delivered concurrently. Therefore, in the second part of this study, CSF-1 was administered from birth in conjunction with hyperoxic exposure, in a setting where a prophylactic effect of CSF-1 treatment was aimed at preventing damage and alveolar loss and enhancing alveolarisation. A shortened exposure time was utilised to enable the 5 daily doses to be administered in conjunction with hyperoxia. The lack of growth restriction is likely due to the decreased oxygen exposure, as pup litters size was consistent amongst regimes. Structurally and functionally, the lungs of both hyperoxia groups were impacted negatively. Morphometric analysis in the adult revealed significant increases in MLI, with fewer, larger alveoli in both the Hyp + PBS and Hyp + CSF-1 groups compared to Norm. Although CSF-1 was not found to improve the structural alterations caused by hyperoxia, this treatment did not exacerbate the negative impact of hyperoxia and no significant difference between PBS and CSF-1-treated mice was observed. There were no differences between any groups at P5, although this time coincides with the beginning of the alveolarisation phase. Of note is that Hyp + CSF-1 values began to approach Norm values in all parameters. Functionally in the adult, CSF-1 did not exacerbate the adverse effects on lung function that were characteristic of hyperoxia exposure, as represented by the Hyp + PBS group. Although no negative effect of CSF-1 was identified, it must be noted that no functionally beneficial effects with regard to preventing hyperoxic damage or further enhancing lung development were observed. The only changes with CSF-1 at P35 were in tidal volume and minute volume. However, these were normalised when body weight was accounted for and, relative to their size, there were no changes in these parameters as a result of CSF-1 treatment.
Overall, CSF-1 treatment showed little effect in promoting alveolar formation in the hyperoxic settings employed in this study. Ongoing studies examining the role of M2 polarisation in the protection and rescue of hyperoxic injury with be important before a definitive statement that CSF-1-responsive macrophages are not effective can be made. Also further experiments utilizing a premature birth model – where animals are prematurely removed from their high uterine CSF-1 environment – will be important to better model this aspect of the clinical situation. However, in this study the finding that CSF-1 treatment did not exacerbate the pathological response to high oxygen levels was encouraging in that macrophage-mediation may provide a novel strategy for the immunomodulation and trophic promotion of alveolar development. In particular in the second regime where potential negative outcomes were a legitimate concern, CSF-1 did not exacerbate damage with concurrent oxygen supplementation. This is a significant finding that is important for any potential clinical use of CSF-1, and supports ongoing studies to improve understanding of the role of CSF-1 in alveolar formation.
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Competing interests
CVJ and SDR hold patents relating to CSF-1 and organ growth.