IKKß Activation in the Fetal Lung Mesenchyme Alters Lung Vascular Development but Not Airway Morphogenesis.

In the immature lung, inflammation and injury disrupt the epithelial-mesenchymal interactions required for normal development. Innate immune signaling and NF-κB activation disrupt the normal expression of multiple mesenchymal genes that play a key role in airway branching and alveolar formation. To test the role of the NF-κB pathway specifically in lung mesenchyme, we utilized the mesenchymal Twist2-Cre to drive expression of a constitutively active inhibitor of NF-κB kinase subunit ß (IKKßca) mutant in developing mice. Embryonic Twist2-IKKßca mice were generated in expected numbers and appeared grossly normal. Airway branching also appeared normal in Twist2-IKKßca embryos, with airway morphometry, elastin staining, and saccular branching similar to those in control littermates. While Twist2-IKKßca lungs did not contain increased levels of Il1b, we did measure an increased expression of the chemokine-encoding gene Ccl2. Twist2-IKKßca lungs had increased staining for the vascular marker platelet endothelial cell adhesion molecule 1. In addition, type I alveolar epithelial differentiation appeared to be diminished in Twist2-IKKßca lungs. The normal airway branching and lack of Il1b expression may have been due to the inability of the Twist2-IKKßca transgene to induce inflammasome activity. While Twist2-IKKßca lungs had an increased number of macrophages, inflammasome expression remained restricted to macrophages without evidence of spontaneous inflammasome activity. These results emphasize the importance of cellular niche in considering how inflammatory signaling influences fetal lung development.

IL-1ß and Inflammasome Activity Link Inflammation to Abnormal Fetal Airway Development.

Inflammation in the developing preterm lung leads to disrupted airway morphogenesis and chronic lung disease in human neonates. However, the molecular mechanisms linking inflammation and the pathways controlling airway morphogenesis remain unclear. In this article, we show that IL-1ß released by activated fetal lung macrophages is the key inflammatory mediator that disrupts airway morphogenesis. In mouse lung explants, blocking IL-1ß expression, posttranslational processing, and signaling protected the formation of new airways from the inhibitory effects ofEscherichia coliLPS. Consistent with a critical role for IL-1ß, mice expressing a gain-of-functionNlrp3allele and subsequent overactive inflammasome activity displayed abnormal saccular-stage lung morphogenesis and died soon after birth. Although the early-stage fetal lung appeared capable of mounting an NF-κB-mediated immune response, airway formation became more sensitive to inflammation later in development. This period of susceptibility coincided with higher expression of multiple inflammasome components that could increase the ability to release bioactive IL-1ß. Macrophages fromNlrp3gain-of-function mice also expressed higher levels of more mature cell surface markers, additionally linking inflammasome activation with macrophage maturation. These data identify developmental expression of the inflammasome and IL-1ß release by fetal lung macrophages as key mechanisms and potential therapeutic targets for neonatal lung disease.

IκB kinase activity drives fetal lung macrophage maturation along a non-M1/M2 paradigm.

In preterm infants, exposure to inflammation increases the risk of bronchopulmonary dysplasia, a chronic, developmental lung disease. Although macrophages are the key cells that initiate lung inflammation, less is known about lung macrophage phenotype and maturation. We hypothesized that fetal lung macrophages mature into distinct subpopulations during mouse development, and that activation could influence macrophage maturation. Expression of the fetal macrophage markers CD68, CD86, CD206, Ym1, fibrinogen-like protein 2, and indolamine-2, 3-dioxygenase was developmentally regulated, with each marker having different temporal patterns. Flow cytometry analysis showed macrophages within the fetal lung were less diverse than the distinctly separate subpopulations in newborn and adult lungs. Similar to adult alveolar macrophages, fetal lung macrophages responded to the TLR4 agonist LPS and the alternative activation cytokines IL-4 and IL-13. Using a macrophage-specific constitutively active IκB Kinase transgenic model (IKFM), we demonstrated that macrophage activation increased proinflammatory gene expression and reduced the response of fetal lung macrophages to IL-4 and IL-13. Activation also increased fetal lung macrophage proliferation. Fetal IKFM lungs contained increased percentages of more mature, CD11b(low)F4/80(high) cells that also expressed higher levels of the alternative activation markers CD204 and CD206. Development of fetal lung macrophages into mature alveolar macrophages may therefore include features of both proinflammatory and alternative activation paradigms.

Transcriptional profiling of lung macrophages identifies a predictive signature for inflammatory lung disease in preterm infants.

Lung macrophages mature after birth, placing newborn infants, particularly those born preterm, within a unique window of susceptibility to disease. We hypothesized that in preterm infants, lung macrophage immaturity contributes to the development of bronchopulmonary dysplasia (BPD), the most common serious complication of prematurity. By measuring changes in lung macrophage gene expression in preterm patients at risk of BPD, we show here that patients eventually developing BPD had higher inflammatory mediator expression even on the first day of life. Surprisingly, the ex vivo response to LPS was similar across all samples. Our analysis did however uncover macrophage signature genes whose expression increased in the first week of life specifically in patients resilient to disease. We propose that these changes describe the dynamics of human lung macrophage differentiation. Our study therefore provides new mechanistic insight into both neonatal lung disease and human developmental immunology.