Arsenic causes distinct gene expression changes in macrophages polarizedin vitrowith either interferon-γ or interleukin-4

Abstract

Arsenic exposure is correlated with atherosclerosis in epidemiological studies and in animal models. We have previously shown that arsenic exposure enhanced atherosclerotic plaque size, increased plaque lipid content and decreased plaque smooth muscle cell and collagen contents in apoE-/- mice. However, the percentage of plaque-resident macrophages, the primary drivers of atherosclerosis remained unchanged. Therefore, we hypothesized that while arsenic does not change the quantity of macrophages, it alters macrophage transcriptome towards a pro-atherogenic state. To test this hypothesis, we used bone marrow-derived macrophages, polarized them to either IFN-ɣ stimulated, pro-inflammatory or IL-4 stimulated, alternatively-activated macrophages in the presence or absence of 0.67 µM (50 ppb) arsenic and performed RNA sequencing. Arsenic exposure altered the gene expression of the macrophages in a subtype-specific manner. Most differentially expressed genes (88%) were altered specifically in either IFN-ɣ- or IL-4-stimulated macrophages, while in the remaining 12% genes that changed in both cell types, did so in opposite directions. In IL-4-stimulated macrophages, arsenic significantly downregulated the genes involved in cholesterol biosynthesis and the chemokines, CCL17/CCL22. Whereas, in IFN-ɣ-stimulated macrophages, genes associated with liver X receptor (LXR) pathway were downregulated by arsenic. Using a bone marrow transplant experiment, we validated that deletion of LXRα from the hematopoietic compartment rescued arsenic-enhanced atherosclerosis in apoE-/- mouse model. Together, these data suggest that arsenic modulates subtype-specific transcriptomic changes in macrophages and further emphasize the need to define macrophage heterogeneity in atherosclerotic plaques in order to evaluate pro-atherogenic role of arsenic.

Unveiling the Impact of Arsenic Toxicity on Immune Cells in Atherosclerotic Plaques: Insights from Single-Cell Multi-Omics Profiling

“…Intriguingly, the increased granularity provided by scRNASeq demonstrated that arsenic alters the transcriptome of the resident-like and foam-like macrophages uniquely. Supporting this conclusion, previous in vitro data from our lab demonstrated that arsenic resulted in distinct gene expression changes depending upon whether IFN-ɣ or IL-4 was used to polarize macrophages (20). This is intriguing as macrophages typically respond to their microenvironment and adopt a polarized functional state to respond to stimuli (51).…”

“…Atherosclerotic plaques are complex microenvironments and recapitulating them in vitro is challenging. Therefore, we utilized data from our previously published bulk RNA-Seq comparison of IFN-ɣ or IL-4-activated bone marrow-derived macrophages with or without concomitant arsenic exposure (Figure 6G and Tables S16-S19) (20). Based on the expression of target genes, STAT6 is the only transcription factor significantly altered in IFN-ɣ -polarized macrophages (p-value: 3.63e-2), and the only transcription factor common between the two in vitro-acquired macrophages and the in vivo single cell sequencing datasets (p-value: 1.2e-3) (Figure 6H and Table S20).…”

“…While there is an arsenic-induced increase in plaque size and lipid content in this mouse model, the percentage of macrophages in these plaques remains unaltered (18, 19). To understand this, our lab investigated whether arsenic affects the gene expression of the plaque-resident macrophages using in vitro culture of bone-marrow-derived macrophages (20). To this end, we observed that the macrophages polarized with IFN-ɣ or IL-4 responded differently to arsenic exposure (20).…”

“…To understand this, our lab investigated whether arsenic affects the gene expression of the plaque-resident macrophages using in vitro culture of bone-marrow-derived macrophages (20). To this end, we observed that the macrophages polarized with IFN-ɣ or IL-4 responded differently to arsenic exposure (20). However, atherosclerotic plaque is a mesh of different immune cells, and their intercellular interaction makes the plaque microenvironment unique.…”