Transcriptome-Wide Analysis Reveals Modulation of Human Macrophage Inflammatory Phenotype Through Alternative Splicing

Abstract

Objective Human macrophages can shift phenotype across the inflammatory M1 and reparative M2 spectrum in response to environmental challenges, but the mechanisms promoting inflammatory and cardiometabolic disease-associated M1 phenotypes remain incompletely understood. Alternative splicing (AS) is emerging as an important regulator of cellular function, yet its role in macrophage activation is largely unknown. We investigated the extent to which AS occurs in M1 activation within the cardiometabolic disease context and validated a functional-genomic cell model for studying human macrophage-related AS events. Approach and Results From deep RNA-seq of resting, M1, and M2 primary human monocyte derived macrophages, we found 3860 differentially expressed genes in M1 activation and detected 233 M1-induced AS events; the majority of AS events were cell- and M1-specific with enrichment for pathways relevant to macrophage inflammation. Using genetic variant data for 10 cardiometabolic traits, we identified in M1 activation 28 trait-associated variants within the genomic loci of 21 alternatively spliced genes and 15 variants within seven differentially expressed regulatory splicing factors. Knockdown of one such splicing factor, CELF1, in primary human macrophages led to increased inflammatory response to M1 stimulation, demonstrating CELF1's potential modulation of the M1 phenotype. Finally, we demonstrated that an induced pluripotent stem cell derived macrophage system recapitulates M1-associated AS events and provides a high-fidelity macrophage AS model. Conclusions AS plays a role in defining macrophage phenotype in a cell- and stimulus-specific fashion. Alternatively spliced genes and splicing factors with trait-associated variants may reveal novel pathways and targets in cardiometabolic diseases.

Human Induced Pluripotent Stem Cell–Derived Macrophages for Unraveling Human Macrophage Biology

“…8 Further, more sophisticated functional characteristics, such as cholesterol efflux, cholesteryl ester hydrolysis, 14 and cytokine secretion profile in macrophages with M1 (lipopolysaccharide + interferon-gamma) and M2 (interleukin-4) activation, were compared between multiple HMDM and IPSDM lines and have demonstrated remarkable similarity. 8 Two additional studies from our group further elaborated that IPSDM recapitulate important alternative splicing events 15 and long non-coding RNA profiles (manuscript under review) of HMDM during macrophage activation, identifying IPSDM as uniquely suited to study human macrophage-specific transcriptome regulation. These protocols 8, 12, 13 utilized sequential exposure of iPSC to different cytokines that resemble hematopoietic specification, myeloid expansion, and harvested myelomonocytic cells for directed differentiation to macrophages with M-CSF.…”

Macrophages

“…There is no doubt that recent technological advances in cellular and molecular biology, such as precise genetic editing and manipulation of human iPS-derived macrophages, 71,90 coupled with improved in vivo detection, 91 tracking and manipulation 92,93 of selective monocyte and macrophage subsets, will accelerate the translation of this increasing knowledge into effective therapeutic strategies.…”

“…70 Our understanding of the molecular mechanisms that control M1/M2 responses is also progressing. Lin et al 71 developed new methodology to study those mechanisms in human macrophages. Their data revealed an important role for alternative splicing events, and more particularly splicing factor CELF1, in shaping gene expression of human monocyte-derived or iPS (inducible pluripotent stem cell)-derived macrophages in response to M1 stimulation.…”

“…Their data revealed an important role for alternative splicing events, and more particularly splicing factor CELF1, in shaping gene expression of human monocyte-derived or iPS (inducible pluripotent stem cell)-derived macrophages in response to M1 stimulation. 71 Interestingly, several cardiometabolic trait-associated variants occur within regulatory splicing factors that were differentially expressed in response to M1 activation, highlighting relevant novel targets. 71 The interactions between SMC and macrophages and the potential for SMC transition to macrophage-like cells in atherosclerosis are receiving increasing attention.…”

“…71 Interestingly, several cardiometabolic trait-associated variants occur within regulatory splicing factors that were differentially expressed in response to M1 activation, highlighting relevant novel targets. 71 The interactions between SMC and macrophages and the potential for SMC transition to macrophage-like cells in atherosclerosis are receiving increasing attention. NG2 (neural/ glial antigen 2) proteoglycan is shown to be important for very-low-density lipoprotein and low-density lipoprotein sequestration at the surface of synthetic SMCs, which then facilitate macrophage conversion to foam cells through direct cell-cell contact.…”