Transcriptomic Analysis of in-vitro foamy macrophages

Abstract

Macrophages provide a fundamental role in the immune system and atherosclerosis, reacting

immediately upon foreign pathogens through phagocytosis. Phagocytic cells such as monocytes or

macrophages uptake oxidized low-density lipoproteins in order to maintain cholesterol homeostasis

in the artery, and during this process, membrane-bound lipid droplets are accumulated in

macrophages, leading to formation of foam cells.

For in-vitro foam cell studies, macrophages are treated with oxLDL for foam cell formation.

Macrophages from various origins are used, and the most widely used cell systems are RAW 264.7

cells, bone marrow-derived macrophages (BMDMs) and Thioglycollate-medium elicited macrophages

(TGEMs). The problems associated with these in-vitro foam cell models are that they express different

cell surface markers or are inflammation-induced, leading to inconsistent in-vivo and in-vitro results.

By re-analyzing ChIP sequencing data of TGEMs and observing the main macrophage subset upon

Thioglycollate-medium injection provided clues that resident peritoneal macrophages without any

inflammatory stimuli may show similar phenotype compared to in-vivo foam cells.

In order to select the best in-vitro foam cell model, transcriptome analysis was done on foam

cells formed from RAW 264.7, BMDMs, TGEMs and resident peritoneal macrophages. Comparing

mRNA expression levels of various cell systems by RT-PCR with recent publication data (Kim et al.,

Circulation Research. 2018) and bulk RNA sequencing indicated resident peritoneal macrophages

show analogous mRNA expressions and cellular pathways compared to in-vivo foam cells. Observation

of upstream regulators showed key genes that are involved in the cellular changes upon oxLDL

stimulation.

We also observed gene expression changes in in-vitro foam cells representing various

atherosclerotic stages. By treating oxLDL for prolonged time points, we found rapid downregulation

of inflammatory gene expressions, and succeeding upregulation of cholesterol related gene

expressions. These collectively suggest in in-vitro induced foam cell studies by using resident

peritoneal macrophages, which is composed of tissue-resident large peritoneal macrophages, may be

the most adequate in-vitro foam cell model. In-vitro atherosclerosis by using this cell system may lead

to further future transcriptome studies regarding the effect of upstream regulators, resolution of IL-

1β-induced inflammation, LXR activation and SREBP signaling leading to cholesterol homeostasis in

hyperlipidemia.