Abstract 157: Alternative Splicing of PLD1 and Its Role in Modulating Inflammatory Phenotypes in Primary Human Macrophages

Abstract

With roles in homeostasis and immunity, human macrophages can readily shift phenotype across the pro-inflammatory M1 and reparative M2 spectrum in response to environmental challenges, but the mechanistic underpinnings of this process remain incompletely understood. Recently we showed from RNA-seq that hundreds of alternative splicing events occur during primary human macrophage LPS / IFN-γ activation to the M1 phenotype associated with cardiometabolic diseases including atherosclerosis. From these events, we found that in M1 activation but not M2 activation, PLD1, which encodes an enzyme catalyzing the hydrolysis of phosphatidylcholine, undergoes increased inclusion of exon 16, with an increased percent spliced in (PSI) of 17.6% calculated from RNA-seq reads and validated by semi-quantitative PCR in human macrophages derived from 3 independent donors (A). Based on these observed differences, we hypothesized that PLD1 has important functional roles in M1 activation. To test this, we used siRNAs to knock down PLD1 in primary human macrophages (B), which induced an increased inflammatory response to LPS / IFN-γ stimulation. This increased inflammatory response was consistent with characteristics of a more robust M1 phenotype, including decreased zymosan phagocytosis in both resting and stimulated macrophages (C), and increased secretion of inflammatory cytokines such as IL-1β, IL-18, IL-6, and CCL5 (D, E). Morpholino induced skipping of PLD1’s exon 16 reduced PLD enzymatic activity to a similar degree seen in morpholino inhibition of PLD1 translation (F, G). These findings suggest that PLD1 may be a novel modulator of M1 activation in an isoform-dependent manner.