Regulation of Neonatal Lung Inflammation by Novel Innate Immune Mechanisms

Abstract Prematurity-related chronic respiratory disease, including bronchopulmonary dysplasia (BPD), is associated with high morbidity, healthcare use, and expenditures. Inflammation in early life increases the risk of BPD develop- ment, but the innate immune mechanisms behind this are poorly understood. Inflammatory stress induces the

expression of ligands for the activating receptor natural-killer group 2, member D (NKG2D), in diverse target cell populations. NKG2D is a C-type lectin-like receptor, expressed by γδ T cells, NK cells, and other lymphocytes that are enriched in fetal lungs. Ligand engagement and activation of NKG2D+ lymphocytes enhance inflamma-

tion and induce cytotoxicity. The goal of this proposal is to determine the mechanisms by which pulmonary inflammation in early life induces NKG2D ligand expression on target cells and activates NKG2D+ lung lympho- cytes (γδ T cells and NK cells), and how this in turn leads to BPD-like lung immunopathology. The hypothesis is

that early-life exposure to bacterial lipopolysaccharide (LPS) or hyperoxia up-regulates the expression of NKG2D ligands (NKG2DLs) that engage and activate lung NKG2D+ lymphocytes leading to pulmonary inflammation, cytotoxicity, and that in turn impairs alveolar and vascular development. Studies have implicated activated lym-

phocytes and γδ T cells, as well as IL-17A and other pro-inflammatory cytokines, as key drivers of inflammation in preterm infants. Cytotoxic Granzyme B (GzmB)+ NKG2D+ cells and CD68+ macrophages that express the human NKG2D ligands MHC class-I-related protein A (MICA) and MICB were found using tracheal aspirates of

premature infants who are mechanically ventilated in the first week of life and later develop BPD. Also, aspirate MICA mRNA levels positively correlate with median FiO2. To investigate the contribution of lung lymphocytes and NKG2DL-NKG2D signaling to lung inflammation, this team used a mouse model of BPD with chronic early-

life exposure to LPS. LPS increased IL-17a and GzmB expression in lung NKG2D+ γδ T cells and, to a lesser extent, in NK cells. NKG2D blockade attenuated the effects of LPS on NKG2D+ γδ T cells, pro-inflammatory cytokine and cytotoxic responses, and preserved alveolar growth. LPS also induced cellular stress pathways

and increased the expression of NKG2DLs (retinoic acid early transcript 1 (Rae-1) and murine UL16-binding protein-like transcript 1 (MULT1)) in lung macrophages and epithelial cells. Rae-1 blockade attenuated LPS- induced lung immunopathology. In a second BPD model, neonatal hyperoxia induced similar responses. These

findings point to a role for activated NKG2D+ lung lymphocytes and NKG2DLs as key inflammatory mediators in BPD pathogenesis. The aims of this proposal are: 1. Map the cell fate and define the role of NKG2D+ lympho- cytes (γδ T cells and NK cells) in early-life LPS- and hyperoxia-induced lung immunopathology. 2. Determine

the cellular sources for NKG2DLs and define the immunogenicity and regenerative potential of ligand-expressing cells in neonatal LPS- and hyperoxia-induced lung immunopathology. These studies will significantly impact the mechanistic understanding of the inflammatory pathways that lead to chronic lung disease in preterm infants.