Abstract 196: Modulation of Inflammatory Macrophage Phenotype Through Alternative Splicing
How alternative splicing (AS) regulates macrophage activation to the pro-inflammatory M1 phenotype, which is causally implicated in cardiometabolic diseases, remains unknown. Because AS is a major mechanism by which cell-specific functions are derived from the genome, we hypothesized that it modulates inflammatory macrophage activation. In an unbiased approach to detect AS, we performed deep RNA-seq (~130 million reads/sample) on non-activated M0, M1-activated (18 hours LPS / IFN-γ), and anti-inflammatory M2-activated (18 hours IL-4) macrophages of isogenic primary human monocyte derived macrophages from six individuals. At false detection rate (FDR)-adjusted P-value < 0.01, 1790 and 2072 genes were differentially expressed between M0 and M1, and M0 and M2 macrophages, respectively. Using DDS-Seq, a computational tool detecting AS using all rather than junction reads alone, 505 AS events for M1 and 511 events for M2 activation were detected at FDR P < 0.01. These events included cassette exons, alternative 3’ / 5’ splice sites, and tandem 3’ untranslated regions. Gene ontology analysis (Ingenuity, Qiagen) revealed differential enrichment for causal networks between M1 and M2 activation states (Table 1). AS genes in M1 activation, including PCR-validated ARRB1 and MAP4K4, were enriched for macrophage-specific and immune functions (Table 2). Also, 14 splicing factors, including CELF1 (3.68-fold increase, FDR P < 0.001), were upregulated between M0 and M1. 65% siRNA CELF1 knockdown decreased M1 marker CCL5 mRNA expression 48.5% compared to non-targeting control (P = 0.002). These findings suggest that during macrophage activation AS occurs in unique phenotype-specific patterns that modulate inflammatory phenotype.
Author Disclosures: J. Lin: None. H. Zhang: None. C. Xue: None. L. Qu: None. M. Li: None. M. Reilly: None.
- © 2015 by American Heart Association, Inc.