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A1293 - Eosinophil Binding and Activation Is Regulated Through the Coordinated Expression of miR-1 Endothelial Targets
Author Block: A. Korde1, F. Ahangari2, S. Mehta1, L. E. Cohn3, A. Gonzalez4, S. Takyar5; 1Internal Medicine, Yale school of Medicine, New Haven, CT, United States, 2Yale School of Madicine, New Haven, CT, United States, 3Yale Univ Sch of Medicine, New Haven, CT, United States, 4Internal Medicine, Yale school of Engineering and Applied Science, New Haven, CT, United States, 5Pulm and Crit Care, Yale School of Med, New Haven, CT, United States.
Background: Eosinophils are recruited to the allergic tissue through coordinated expression of cytokines and chemokines. We have previously shown that type 2 immunity downregulates miR-1 in the lung endothelium. Overexpression of miR-1 specifically in the lung endothelium inhibited allergic airway inflammation without significantly altering the secretion of type 2 cytokines. Hypothesis: An endothelial-specific miRNA-controlled gene circuit regulates eosinophil recruitment into the allergic tissues. Methods: Eosinophil binding and activation on the endothelial surface were measured in an adhesion chamber. Interactions between eosinophils and endothelial cells were monitored with video-microscopy and attachment and activation events were quantified in real-time and during follow-up image analysis, respectively. We performed a RISC recruitment analysis through Argonaute2 (Ago2) immunoprecipitation and RNA sequencing (RIP-seq). HUVECs were transduced with a miR-1 overexpression vector. MiR-1 was specifically expressed in the lung endothelium through induction of pri-miR-1 in an inducible vascular-specific transgenic mouse model. Airway biopsies were collected from chronic rhinosinusitis (CRS) patients. Expression of target genes were analyzed through qRT-PCR, ELISA, and immunofluorescence. Results: Eosinophil binding and activation were significantly decreased on HUVECs transduced with miR-1 overexpression vector. MiR-1 overexpression also lowered P-selectin levels on the endothelial surface. RIP-seq analysis on these cells showed that CCL-26 (eotaxin-3), TSLP (Thymic Stromal Lymphopoietin), Mpl (Thrombopoietin receptor), and CCL2 (Monocyte chemotactic protein-1) are direct miR-1 targets in human endothelium and co-regulated by this miRNA. These genes were also regulated by endothelial miR-1 in HDM-inflamed murine lungs. In CRS tissues, miR-1 levels were downregulated in highly eosinophilic biopsies and miR-1 targets showed an inverse correlation with the degree of eosinophilia. Conclusion: The initial stages of eosinophil recruitment in allergic inflammation is controlled through the co-expression of a group of endothelial-specific genes. This multi-gene circuit can be identified and targeted through miRNA studies.