Lung Fibroblasts Recruit Inflammatory Macrophages That Alter Saccular Stage Elastic Fiber Organization

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Abstract Rationale: Lung fibroblasts produce extracellular matrix components that contribute to the formation of distal airspaces during the saccular stage of lung development. Inflammation in preterm infants disrupts this process, leading to reduced alveolar formation and bronchopulmonary dysplasia (BPD). We previously developed a mouse model to test the effect of mesenchymal-derived inflammation in the saccular stage lung. In this transgenic model, an activated form of human IKKB was expressed in TBx4-lung enhancer-positive cells in a doxycycline (Dox) inducible manner (IKKβTbx4). IKKβTbx4 mice exhibited marked inflammatory cell recruitment to the lung. Further, IKKβTbx4 mice developed a BPD lung phenotype with interstitial thickening and reduced distal airspaces that progressed to emphysematous changes in adulthood. The mechanisms driving the abnormal phenotype in IKKβTbx4 lungs remain unclear. Thus, we examined the contribution of recruited inflammatory cells in this model. Methods: We activated the transgene in neonatal IKKβTbx4 mice during the saccular stage (postnatal [PN] day 0-5) by placing lactating dams on Dox (2g/L). We evaluated lungs at PN5 and 2 months. A small molecule CCR2 antagonist (or vehicle DMSO) was administered intraperitoneally (IP) daily to neonatal IKKβTbx4 mice for macrophage depletion experiments. Primary lung fibroblasts were isolated from IKKβTbx4 or control mice to generate conditioned media. Chemotaxis was evaluated using a modified Boyden chamber assay. Results: Transgene activation recruited inflammatory macrophages and monocytes to the lungs of saccular stage IKKβTbx4 mice (inflammatory macrophages and monocytes [% of viable cells], control: 5.93%, ± 0.88, IKKβTbx4: 35.74%, ± 4.79; P < 0.05). Conditioned media from Dox-treated IKKβTbx4 lung fibroblasts stimulated chemotaxis of macrophages in vitro. Multiplex analysis of chemokines in lung lysate and fibroblast conditioned media from IKKβTbx4 mice demonstrated significantly increased macrophage chemoattractants CCL2 and CCL7, ligands that bind to the monocyte chemotactic receptor CCR2. Lungs of saccular stage IKKβTbx4 mice exhibited abnormal elastin organization, with fewer elastic fiber bands around developing saccules in the lung interstitium. mRNA expression of important elastin assembly components, Elastin and Fibulin-5 were reduced in IKKβTbx4 lungs. Further, both mRNA expression and enzymatic activity of macrophage elastase matrix metalloprotease-9 was increased in IKKβTbx4 lungs. Administration of a small molecule CCR2 antagonist to saccular stage IKKβTbx4 mice restored elastic fiber organization and rescued the phenotype of saccular stage IKKβTbx4 lungs. Conclusions: Our findings emphasize the significant role of CCR2-positive macrophages in disrupting organization of the elastic fiber scaffold of the lung, thus contributing to abnormal saccular stage development.

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