The renewed interest in macrophages has stemmed from an increased understanding of monocyte/macrophage heterogeneity and how it relates to functional diversity [35–38]. Differential activation states have been broadly classified as M1, which encompasses macrophages involved in host defence and inflammation, and M2, which represent a more wound healing or tissue remodelling phenotype. Despite the M1/M2 activation dichotomy arising from studies of tissue disease and repair, understanding macrophage phenotype and function has implications for discerning and potentially enhancing their contribution to organ development. Functions of M2 macrophages, such as extracellular matrix (ECM) production, release of trophic factors and promotion of angiogenesis, are fundamental to organogenesis. Furthermore, our microarray expression profiling has revealed that embryonic macrophages in developing lungs, kidneys and brains show a comparable gene expression profile consistent with an M2 activation state . In addition, CSF-1 can also promote an ‘M2’ macrophage activation state, which is increasingly being linked to tissue repair and regeneration [2, 39, 40].
During embryonic development, macrophages are located abundantly within the embryo and are present in virtually all developing organs . Macrophage functions that support organogenesis include clearance of apoptotic cellular debris associated with tissue remodelling  and the provision of trophic support by producing a range of regulatory mediators [5, 42]. Macrophages also contribute to appropriate cellular differentiation [8, 21, 22] and angiogenic regulation , through both the production of angiogenic factors  and by physically directing angiogenic positioning . As the lung buds form, macrophages surround the elongating primary bronchi . The use of the Csf1r-EGFP reporter mice has been an important tool in demarcating the developmental role of macrophages in the embryo and has demonstrated a significant population of CSF-1R-expressing macrophages within the lungs at E13.5 . Eliciting its effect through binding with the CSF-1R , CSF-1 is a pleiotropic growth factor also important in the regulation of pregnancy, fetal development and tissue regeneration [Reviewed in [33, 34, 40]].
Macrophages in the lung have been well described for their functions in host defence and inflammatory diseases, however the importance of CSF-1R+ macrophages in contributing to lung development has not been elucidated. The present study demonstrates the localisation of CSF-1R+EGFP+ alveolar and interstitial macrophages, which co-express the mature macrophage marker F4/80, in developing lungs during postnatal development. In addition, CSF-1R+ macrophages were identified in embryonic lung explants using wholemount immunofluorescence microscopy, where they were found to accumulate at branch points during lung branching morphogenesis.
The proposal that key organogenic periods are accompanied by an M2 macrophage phenotype was examined in the postnatal lung. During alveolarisation from P14 to P21, the expression of the M2 markers examined (Arg1, Ccl17 and Mrc1) showed a significantly increased expression. The remodelling functions of M2 macrophages are in accordance with the structural changes occurring within the lung at this time. Mrc1 provides an important mechanism for cellular clearance associated with homeostasis and tissue reorganisation . Arg1 is associated with collagen formation and ECM production . This study thus highlights the importance of macrophages in the alveolarisation stage of lung development, and in particular the association with an M2 activation state. Furthermore in the adult lung, an upregulation of M2 genes was also observed, supporting the homeostatic and immunomodulatory functions of resident pulmonary macrophages.
We have previously reported that CSF-1-responsive developmental macrophages are associated with growth and organ development, with delivery of recombinant protein to neonatal mice resulting in increased body and organ weight . The present study showed that administration of CSF-1 to neonatal mice was also increased the number of macrophages in the developing lung and promoted an increase in Fizz1 (Retnla) and Igf1 expression. Fizz1 is an important mediator of lung development and maturation, and is upregulated during the saccular and alveolar stages, where its angiogenic and proliferative functions are suggested to promote alveolar development . Fizz1 is also reported to participate in lung maturation by modulating surfactant production . Expressed on lung cells such as mesenchymal and alveolar type II cells, the function of macrophage-derived Fizz1 is under-examined in previous reports, and its upregulation with CSF-1 supplementation indicates it may provide beneficial effects in regulating lung development.
The increase in the key growth regulator IGF-1 in response to CSF-1 administration provides insight regarding the potential mechanism of trophic macrophage function in organogenesis, and also supports an emerging link between CSF-1, macrophages and the IGF-1 growth axis . Interestingly, many of the growth and developmental deficiencies observed in CSF-1-deficient mice are common to IGF-1-deficient animals . Moreover, an interaction between CSF-1 and the IGF-1 growth axis is supported by the finding that CSF-1-deficient rats fail to produce the postnatal spike in IGF-1 . Furthermore, IGF-1 production as a key mechanism of trophic macrophage function is supported by a previous study which demonstrated that kidney regeneration in an experimental model of acute kidney disease is mediated by CSF-1-responsive macrophages and an upregulation of IGF-1 . This has important parallels for normal lung development as IGF-1 increases during alveolarisation, and the promotion of lung maturation using retinoic acid and dexamethasone has been shown to correlate positively with increased levels of IGF-1 .
Identification of M2-polarised macrophages as an important component of the organogenic milieu during alveolar development has important potential clinical implications, not only for understanding normal developmental processes, but also for addressing lung immaturity and the impact of neonatal inflammation of developmental perturbation. Inflammatory activation of macrophages not only contributes to tissue damage and perturbation of organ development through pro-inflammatory injury, but also disrupts morphogenesis in the lung and alters the expression of key genes important in lung development [23, 52]. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling in fetal macrophages upregulates pro-inflammatory mediators such as interleukin-1β and alters expression of Wnt7b, bone morphogenic protein-4  and fibroblast growth factor-10 . Given the correlation between the timing of lung structural establishment and lung immunological maturity , it is plausible that the developmental deficits associated with inflammation may also result from skewing of macrophages prematurely away from their organogenic activities toward pro-inflammatory mediation roles.
Understanding the regulation of alveolar development has particular relevance with regard to clinical implications of developmental perturbation, and especially in the setting of preterm birth. The neonatal mouse provides an excellent model for studying these aspects of lung development as mice are born at a stage where earlier aspects of organ development are still ongoing. The period of postnatal development in the mouse was characterised; correlating structural and functional maturation with macrophage localisation. The histological time course of postnatal lung development demonstrates the processes of structural maturation, whereby large saccules evident at the end of the saccular stage at P5 begin to subdivide through secondary septation to form definitive alveoli clearly evident at P21. This period of alveolarisation is critical in establishment of the gas exchange units required for proper function of the lung. This is evidenced by the chronic lung dysfunction associated with disruptions of alveolar development that often result from preterm birth and injurious therapeutic interventions that are required to keep the neonate alive [54, 55]. Indeed, Mund et al. reported that murine alveolarisation occurs in two stages; phase 1 from P4-21 where alveoli arise from immature septae, and phase 2 from P21-36 where alveoli lift off from existing mature alveoli .
The histological time course of postnatal lung development was complemented by analysis of functional maturation over this time. Changes in breath cycle parameters were examined throughout postnatal lung development using unrestrained barometric whole-body plethysmography. This technique has been utilised in adult models of lung injury [28, 29] and was optimised for use in neonatal mice in our study. Modifications including the adjustment of the Perspex chamber size and increased sensitivity settings for pressure transduction recording enabled the measurement of lung function from as early as P5. Trace recordings showed discernable changes in breath patterns from P5 to adulthood, which when analysed provided quantitative changes in lung function. As the number of alveoli increased and the gas exchange compartment expanded, there was a correlation with changes in lung function parameters including an 8- and 10-fold increase in tidal and minute volume from the beginning of alveolarisation at P5 to adulthood. Normalisation of inspiratory flow rate and expiratory time were also observed over this time period of postnatal development as the chest wall and diaphragm mature.
The unique saccular architecture of the mature lung is associated with a different developmental cellularity pattern than that of solid organs. Overall cellularity increases rapidly in the alveolarisation phase and peaks at P14. From this stage, development is associated with significant remodelling and apoptosis as alveolar sacs form and mature, resulting in a decrease in cellularity in the adult mature lung. By digesting whole lungs, both interstitial as well as alveolar macrophages were able to be included for analysis, as opposed to the commonly used bronchoalveolar lavage-based collection method which restricts analysis to alveolar macrophages only. The proportion of macrophages in the lung is highest during the alveolarisation stage of lung development. A significant resident population is also maintained in the adult lung post completion of development, indicative of the unique air-exposed environment and the importance of pulmonary macrophages in clearing inhaled debris and modulating immune responses. The increased proportion of macrophages observed during alveolarisation – a time that lacks immunological relevance in this normal setting - therefore suggests that macrophages are associated with developmental functions.