Inflammation Protein-2 Inhibits TLR4-Mediated Lung Alternatively Spliced Myeloid Differentiation

We previously identified a novel alternatively spliced isoform of human myeloid differentiation protein-2 (MD-2s) that competitively inhibits binding of MD-2 to TLR4 in vitro. In this study, we investigated the protective role of MD-2s in LPS-induced acute lung injury by delivering intratracheally an adenovirus construct that expressed MD-2s (Ad-MD-2s). After adenovirus-mediated gene transfer, MD-2s was strongly expressed in lung epithelial cells and readily detected in bronchoalveolar lavage fluid. Compared to adenovirus serotype 5 containing an empty vector lacking a transgene control mice, Ad-MD-2s delivery resulted in significantly less LPS-induced inflammation in the lungs, including less protein leakage, cell recruitment, and expression of proinflammatory cyto-kines and chemokines, such as IL-6, keratinocyte chemoattractant, and MIP-2. Bronchoalveolar lavage fluid from Ad-MD-2s mice transferred into lungs of naive mice before intratracheal LPS challenge diminished proinflammatory cytokine levels. As house dust mite (HDM) sensitization is dependent on TLR4 and HDM Der p 2, a structural homolog of MD-2, we also investigated the effect of MD-2s on HDM–induced allergic airway inflammation. Ad-MD-2s given before HDM sensitization significantly inhibited subsequent allergic airway inflammation after HDM challenge, including reductions in eosinophils, goblet cell hyperplasia, and IL-5 levels. Our study indicates that the alternatively spliced short isoform of human MD-2 could be a potential therapeutic candidate to treat human diseases induced or exacerbated by TLR4 signaling, such as Gram-negative bacterial endotoxin-induced lung injury and HDM-triggered allergic lung inflammation. A n essential component of the outer membrane of Gram-negative bacteria, LPS induces a powerful inflammatory response that can lead to septic shock and death (1). LPS signals through TLR4 by binding to its coreceptor myeloid differentiation protein-2 (MD-2; LY96) and other accessory molecules , including LPS-binding protein and CD14. Among these molecules, LPS-binding protein and CD14 are important for enhancing LPS binding to MD-2 but are not essential for triggering TLR4 signaling (2, 3). MD-2 is an essential component of the signaling receptor complex that recognizes and initiates the innate immune response to bacterial LPS (4). MD-2 is both a membrane bound and a secreted glycoprotein that binds both LPS and the ectodomain of TLR4, forming the TLR4/MD-2/LPS complex (5, 6). Loss-of-function approaches (knockdown and mutation) demonstrated that MD-2 is indispensable for LPS-induced immune cell activation (7–9) in both mouse and human models (10, 11). Upon LPS binding , a receptor multimer composed of two copies of the TLR4/MD-2/LPS complex is formed (10, 12), which triggers a downstream signaling cascade, …