Abstract 17144: miR-146a-5p Induces Pulmonary Barrier Disruption via Macrophage-Derived TNFa

Abstract

Introduction: Sepsis-induced acute lung injury (ALI) has substantial morbidity and mortality. Diffused alveolar damage as a result of increased endothelial cell (EC) permeability is a hallmark of ALI. We have previously demonstrated that certain uridine-rich extracellular (ex) miRNAs such as miR-146a activate innate immune response and possess a potent proinflammatory property, but their effect on EC function is largely unknown. Hypothesis: Pulmonary EC barrier function is impaired by miR-146a-5p. Methods: Conditioned media (CM) were collected from macrophages (Mφ) cultures treated with lipofectamine (lipo) or miR-146a-5p (50 nM) for 24 h. EC barrier function was analyzed in human pulmonary arterial endothelial cells (HPAECs) by transendothelial electric resistance (TER) using an electric cell-substrate impedance sensing system. Results: Direct miR-146a-5p treatment of HPAECs did not cause any change in TER even though it induced robust IL-6 and CXCL2 production in Mφ. To test whether miR-146a-5p affects EC function indirectly, we incubated HPAECs with miR146a-CM and observed a more than 50% reduction in TER ( Fig. A ). Lipo-CM had no such effect. This barrier effect was accompanied by VE-cadherin internalization and disrupted VE-cadherin lining following 8 hours of incubation ( Fig. B) . To determine the potential mediators in the CM responsible for the EC barrier disruption, we performed a cytokine array analysis of 111 cytokines in the CM and identified 15 differentially expressed cytokines. Among them, we found that TNFα neutralizing Ab (1μg/ml), but not control IgG (1μg/ml), partially blocked the miR-146a-CM-induced EC barrier disruption with improved VE-Cadherin integrity ( Fig. B ). Conclusion: Our data demonstrated miR-146a-5p induces HPAEC barrier dysfunction via an indirect mechanism involving Mφ production of TNFα.